Synthesis of structured triacylglycerols enriched in n-3 fatty acids by immobilized microbial lipase

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

Food

Microbiology

Synthesis

of

structured

triacylglycerols

enriched

in

n-3

fatty

acids

by

immobilized

microbial

lipase

Maria

Elisa

Melo

Branco

de

Araújo

_,

_Paula

_Renata

_Bueno

_Campos

_,

Thiago

Grando

Alberto

_,

_Fabiano

_Jares

_Contesini

_,

_Patrícia

_de

_Oliveira

_Carvalho

a_{UniversidadeSãoFrancisco,LaboratoryofMultidisciplinaryResearch,Braganc¸aPaulista,SP,Brazil}

b_{UniversidadeEstadualdeCampinas,CollegeofFoodEngineering,Campinas,SP,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received10June2015 Accepted4April2016 Availableonline19July2016 AssociateEditor:JorgeGonzalo FariasAvendano

Keywords:

Lipase Immobilization Soybeanoil

Polyunsaturatedfattyacids

a

b

s

t

r

a

c

t

Thesearchfornewbiocatalystshasarousedgreatinterestduetothevarietyof micro-organismsandtheirroleasenzymeproducers.NativelipasesfromAspergillusnigerand

Rhizopusjavanicuswereusedtoenrichthen-3long-chainpolyunsaturatedfattyacids

con-tentinthetriacylglycerolsofsoybeanoilbyacidolysiswithfreefattyacidsfromsardine oilinsolvent-freemedia.Fortheimmobilizationprocess,thebestlipase/supportratios were1:3(w/w)forAspergillusnigerlipaseand1:5(w/w)forRhizopusjavanicuslipaseusing AmberliteMB-1.Bothlipasesmaintainedconstantactivityfor6monthsat4◦C.Reaction time,sardine-freefattyacids:soybeanoilmoleratioandinitialwatercontentofthelipase wereinvestigatedtodeterminetheireffectsonn-3long-chainpolyunsaturatedfattyacids incorporationintosoybean oil.Structured triacylglycerolswith11.7and7.2%of eicosa-pentaenoicacid+docosahexaenoicacidwereobtainedusingAspergillusnigerlipaseand Rhizopusjavanicuslipase,decreasingthen-6/n-3fattyacidsratioofsoybeanoil(11:1to 3.5:1and4.7:1,respectively).Thebestreactionconditionswere:initialwatercontentof lipaseof0.86%(w/w),sardine-freefatyacids:soybeanoilmoleratioof3:1andreactiontime of36h,at40◦C.Thesignificantfactorsfortheacidolysisreactionwerethesardine-free fattyacids:soybeanoilmoleratioandreactiontime.Thecharacterizationofstructured tri-acylglycerolswasobtainedusingeasyambientsonic-sprayionizationmassspectrometry. Theenzymaticreactionledtotheformationofmanystructuredtriacylglycerolscontaining eicosapentaenoicacid,docosahexaenoicacidorbothpolyunsaturatedfattyacids.

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).

Introduction

Lipases (acylglycerolacylhydrolases, EC 3.1.1.3) are biocata-lystswithmanyindustrialapplications,naturallyactingon

∗ _{Correspondingauthor.}

E-mail:[email protected](M.E.Araújo).

carboxylicester bondstocatalyze thehydrolysis of triacyl-glycerols (TAGs). In non-aqueousmedia, they can catalyze esterification, alcoholysis,acidolysis and transesterification reactions.Thescreening,kineticcharacterizationand immo-bilizationofmicrobiallipasesforuseinbiocatalysishasbeen

http://dx.doi.org/10.1016/j.bjm.2016.07.003

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

animportantfocusofresearchinourlaboratory.Previouslywe havedescribedtheselectionbyscreeningtechniquesonsolid andliquidmediaoflipase-producingmicroorganismsandthe characterizationofthecatalyticperformanceoffungallipase preparations for hydrolysis and esterification reactions.1–4 Among these strains, Aspergillus niger, Rhizopus javanicus,

Fusarium oxysporum and Penicillium solitumare described as

lipase-producers for the first time.3,5 _{The chemo-,} regio-andenantiospecificbehavioroftheseenzymesarethegreat industrialinterest.Mostmicrobiallipasessuchasthosefrom

A.niger,Rhizopusdelemar,Rhizopusmiehei,Mucorjavanicus,R.

javanicusandYerrowialipolyticashow1,3-positionalspecificity

releasing2-monoacylglyceroland1,2-and2,3-diacylglycerol asproductsfromthesubstrate.2_{LipasefromA.nigergavethe} best yields and enantioselectivities in the esterification of raceminibuprofen.1,6

Lipasescanbeemployedinthemodificationofthe chemi-calstructureofnaturalTAGstoimprovetheirphysicochemical properties and/or their health benefits. For instance, long chainpolyunsaturatedfattyacidssuchasthen-3family

(n-3LCPUFAs)maybeincorporatedintoTAGsfromoilsandfats

vialipase-catalyzedreactions.Thesen-3LCPUFAs,especially eicosapentaenoicacid(EPA)anddocosahexaenoicacid(DHA), maypromotespecificeffectsrelatedtometabolismandhealth andtheyshowgreatpromisefornutraceuticalapplications.

Theconceptofstructuredtriacylglycerols(STAGs)implies modificationofthefattyacidscompositionand/orfattyacids locationontheglycerolbackbone,andimprovementofthe physical and/or physiological properties of dietary lipids. Studies have also shown that enzymatically or chemically modified TAGs exhibit similar or, in some cases, greater benefits compared to blended oils with similar fatty acid compositions.7–11

Soybeanoil(SO)isacheapandabundantcommodityin Brazil,representinganimportantsourceoffattyacidsfor reg-ular consumption. SO presents ann-6/n-3 fatty acids ratio rangingbetween7.1:1and 11:1.12,13 _{Basedon experimental} andclinicalstudies,themostfavorablen-6/n-3ratioforhuman nutritionisproposedtorangebetween2:1and4:1.14

In a previous study, we reported that TAGs from SO enrichedinEPAandDHAcanbeobtainedbyacidolysis cat-alyzed by a commercial R. miehei lipaseimmobilized on a macroporousanion-exchangeresin(LipozymeRMIM®_).13_We alsoreportedthatsomelipases,derivedfromnative microor-ganisms,providegoodresultsforobtainingPUFAconcentrates asacylglycerolsbyhydrolysisofborageoil,salmonoiland sar-dineoil.3,5,15_{Therearesomestudiesreportingtheproduction} ofTAGsenrichedwithn-3 PUFAbyenzymatic acidolysis.16 However, most of them have used commercially-available lipasesinreactionmediacontainingorganicsolventsandfew studiesexploringnative lipaseshavebeenconducted. This facthighlightstheimportanceofexploitingthecapacitiesof novelmicrobiallipasestoincreasethecontentofn-3LCPUFAs invegetableoils.

Immobilizationofthenativelipasesisanimportantpart oftheprocess insofar asthe immobilizedenzymes can be easilyrecoveredandreusedtherebymakingtheprocess eco-nomicallyviable. Onaccount ofthe relativelyhigh surface hydrophobicityoflipases,the processofsimpleadsorption ontosuitablyhydrophobicsupportshasbeenthemoreused

strategyforlipaseimmobilization.Therefore,this workhas threemainobjectives:(i)toimmobilizethenativelipasesfrom

A. niger(AN) and R.javanicus(RJ) onAmberlite MB-1 using

differentlipase/supportratios;(ii)todeterminetheactivity, stabilityanddegreeofincorporationofn-3LCPUFAsby acidol-ysisofSOwithamixtureoffreefattyacidsfromsardineoil (sardine-FFA)insolvent-freemedia,byfreeandimmobilized lipases;and (iii)toinvestigatesomeofthevariables which may influence the incorporationof n-3LCPUFA into SO by enzymaticacidolysis.

Materials

and

methods

Chemicals

Amberlite MB-1 and chemical and culture media reagents wereobtainedfromMerck&Co.,Inc.(Darmstadt,Hesse, Ger-many)andfromSigma–AldrichCo.(St.Louis,MO,USA).Olive oil(Carbonell®_{) (Deoleo,Madrid, Spain),soybean oil(Liza}®₎

(Cargill,Inc.,SãoPaulo,SãoPaulo,Brazil)andsardineoil (Catal-entPharmaSolutions,Inc.,Sorocaba,SãoPaulo,Brazil)were purchasedinthelocalmarket.Thepreparationoffreefatty acidsfromsardineoil(sardine-FFA)wascarriedoutusing alka-line hydrolysis followed by acidification withacetic acid.17 Itsmajorcomponentfatty acidswere stearic(5.7%), myris-tic(7.4%),palmitoleic(8.1%),palmitic(16.5%)andoleic(15.3%) acids,EPA(19.8%)andDHA(11.4%).

Productionanddeterminationoflipasesactivities

ThisstudywasperformedusingANandRJlipasesproducedin ourlaboratoryaspreviouslydescribed.6_{Theproductionwas} carriedoutinabasalmediumwithaninitialpHvalueof6.0 thatconsistedof2%peptone,0.5%yeastextract,0.1%NaNO3,

0.1%, KH2PO4 0.05% MgSO4. 7H2Oand 2%ofolive oil.

Cul-tureswerecarriedoutinErlenmeyerflasks(500mL)containing 120mLofthegrowthmedium.Thecultureswereinoculated with1mLofsporessuspension(105_–106_{spores/mL)andthe}

flaskswereagitatedonarotaryshaker(130rpm)at35◦Cfor 72h.Afterthat,thecultureswerefilteredandclearedfiltrates weretreatedwithammoniumsulphate(80%saturation).The precipitates were dialyzed overnight in sodium phosphate buffer,pH7.0,andlyophilizedforuseasacrudelipase prepa-rationinpowderform.Theresidualwaterofthelyophilized lipaseswas0.84%(w/w).Hydrolyticactivitywasdetermined bytriolein,witholiveoilasthe substrateand employinga standardoleic acidcurve.Oneactivityunit (U)wasdefined as1␮molofoleicacidreleasedperminuteofreaction.The specificactivitywasexpressedasU/mgoftotalprotein.The amountofproteinwasdeterminedbythemethodofLowry etal.18 _{ANlipasewith7.5andRJlipasewith6.8U/mgwere} used,withoutfurtherpurification.

Immobilizationbyadsorption

The crude lipases were immobilized by adsorption onto Amberlite MB-1 support at different lipase/support ratios (w/w).Thecrudelipasepreparation(0.07–0.17g)wasdissolved in5mLofsodiumphosphatebuffer (50mM,pH7.0),mixed

with500mgofdriedsupportandshakenat200rpmfor12h usingaTecnalorbitalshakerTE-421(Tecnal,Ltd.,Piracicaba, SãoPaulo,Brazil). Aftervacuumfiltration, themixturewas washed threetimes with deionized waterand rinsed with sodiumphosphatebuffer.Thelipasepreparationswerefreeze driedandstoredat4◦Cuntilfurtheruse.

Immobilizationyield

Immobilizationyieldwasdefinedasfollows: Immobilizationyield (%)=



afree



×100

where aimm is the total hydrolytic activity of immobilized

enzyme (U/mg of protein) and afree is the total hydrolytic

activityofinitialenzymepreparation(U/mgofprotein).The amountofproteinboundtothesupportwascalculatedby sub-tractingthe amountofproteinrecoveredafterwashingthe AmberliteMB-1-lipasecomplexfromtheamountofprotein usedforimmobilization.

Acidolysisreaction

Thefreeandimmobilizedlipases(10%,w/w)wereaddedtothe reactionmedium(13g)composedofsardine-FFAandSOina molarratioof3:1,in50mLconicalflaskswithsilicone-capped stoppers.Thereactionwascarriedoutundernitrogen atmo-sphereand 0.001%BHTwasaddedtothereactionmedium toavoiddegradationofthePUFAs.Thereactionmixturewas incubated at40◦C using aTecnalorbital shakerTE-421, at 160rpmfor12h,andthereactionwasstoppedbyfilteringthe lipase.Thereactionproductwasflushedoutwithastreamof nitrogenandstoredat−20◦Cuntilanalyzed.TheSTAGswere purifiedandanalyzedbyGCandEASI-MSasdescribedbelow. Thereactionsandanalyseswerecarriedoutintriplicate.

Experimentaldesign

Responsesurfacemethodology(RSM)wasadoptedasit pro-videsasetofmathematicalandstatisticalmethodsthatmake it possibleto model phenomenaand find combinations of experimentalvariables thatwillleadtooptimalresponses. A23 _{fullfactorialdesignwith4centerpointstodetermine}

theresidualvariance(Table2)wasusedinthestatistical opti-mizationassays.Thisprocessmadeitpossibletodetermine thebestconditionsfortheacidolysisreactionforeachlipase. Theindependentvariables(orfactors)studiedwere:reaction time(hours, X1), sardine-FFA:SO mole ratio (X2)and initial

watercontentoflipase(%w/w,X3).Thedependentvariable

investigatedwasthen-6/n-3FAsratiooftheSTAGs.The exper-imental matrix was designed using Statistica 9.0 software (StatSoft,Inc.)(Tulsa,OK,USA),andthesignificanceofdata wasaccessedbytheanalysisofvariance(ANOVA)statistical test.

PurificationoftheSTAGs

TheSTAGs and FFAs obtainedafterthe acidolysisreaction were separated using a previouslydescribed method, with modifications:thereactionmixturewasdissolvedinn-hexane

to200mL,150mLof0.8NKOH(hydro-alcoholicsolution,30% ethanol) were added, and the mixturewas agitated.19 _The hydro-alcoholicphase(containingFFAs aspotassiumsalts), andthehexanephase(containingtheTAGs)weredecanted, then the hydro-alcoholic phase was extracted twice more

with20mLofn-hexane.Bothn-hexanesolutionsweremixed

together,hexanewasevaporatedoff,theSTAGswereweighed andtherecoverypercentagewascalculated.Afterthe purifi-cationstep,itwaspossibletorecoverabout80%oftheSTAGs. Athin-layerchromatography(TLC)ofthehexaneextractwas performedinordertoconfirmtheefficiencyoftheprocess. ThepresenceofacylglycerolsandFFAswasidentifiedbyTLC onplates ofsilica-gel (TLCplates,SILG-25;Aldrich Chemi-calCo.,Milwaukee,WI,USA)activatedbyheatingat105◦C for20min.Thesampleswerespottedontheplate(dissolved inhexane,7%,v/v)withauthenticstandardsandthen devel-opedinchloroform/acetone/methanol(95:4.5:0.5,v/v/v).Spots ofeachlipidwerevisualizedbysprayingtheplatewithiodine vaporinanitrogenstream.

Gaschromatography(GC)andeasyambientsonic-spray

ionizationmassspectrometry(EASI-MS)

The STAG and FAs profiles were obtained by direct mass spectrometryusingtheeasyambientsonic-sprayionization (EASI-MS) and by gas chromatography (GC) as previously described.13_{TheFAsfromSTAGswereconvertedintoFAME} (fattyacid methylester)bytreatmentwithBF3-methanol.20

Theanalysiswereperformedbygaschromatographywitha flameionizationdetectorusingaChrompackGas Chromato-graph(Chrompack,SãoPaulo,SãoPaulo,Brazil)anda50-m fused silica capillarycolumn(WCOTCP-Sil88,Chrompack, Chromtech, MN, USA). The fatty acids were identified by comparison of the relative retention times with authentic standardsfromSigmaChemicalCompanyandexpressedas arelativepercentage(%totalfattyacids).Asingle-quadrupole LCMS2010massspectrometer(ShimadzuCorp.,Kyoto,Japan) withahomemadeEASIsourcewasusedtoobtainthespectra inthepositiveionmode.Theanalysisconditionswere:N2

neb-ulizinggasat3Lmin−1,surfaceangleofca.30◦andmethanol flowrateof20␮Lmin−1.Thesamples(2␮L)were placedon thepapersurface(brownKraftpaper).Themassspectrawere accumulatedover60sandscannedinthe50–1200m/zrange.

Results

and

discussion

Immobilizationoflipases:bestlipase/supportratioand

stabilityofenzymes

Thecapacityofincorporationofn-3LCPUFAs(EPA+DHA)was dependentonthelipasesourceandlipase/supportratiosfor immobilization(Table1).Whenthelipaseswereusedinthe freeform,insignificantincorporationofn-3LCPUFAsintoSO wasachievedin12hofreaction.Thetotaln-3PUFAscontent in the acidolysisproductswas 4.0–7.0% whenimmobilized lipases were used, except for a lipase/support ratio of1:7 (<1.0%)whichgavelessthan50%ofimmobilizationyieldfor bothlipases.Thehighercontentwas7.0%whenimmobilized ANwasemployed(1:3lipase/supportratio).Largeramounts

Table1–Influenceofthelipase/supportratiosonthe percentageofn-3PUFAsinsoybeanoilafteracidolysis reactionscatalyzedbyfreeandimmobilizedlipases. Lipase Lipase/support ratio(w/w) Immobilization yield(%) n-3PUFAsin soybeanoil (%totalfatty acids) ANa _{Nosupport – 1.9} 1:3 54.5±8.9 7.0 1:4 61.5±2.4 5.7 1:5 58.5±7.6 5.8 1:7 36.1±4.2 <1.0 RJb _{Nosupport – <1.0} 1:3 61.4±4.9 4.2 1:4 60.8±7.1 4.0 1:5 69.3±3.7 5.9 1:7 40.3±7.5 <1.0

a _{LipasefromAspergillusniger.}

b _{LipasefromRhizopusjavanicus.}

ofenzymedidnotfurtherimprovetheincorporationofn-3

LCPUFAs. This may be due to enzyme aggregation, which leadstoanincreaseinsubstratediffusioninhibition,causing the saturation of the reaction rate. These results indicate thatimmobilizedlipaseshavemoreaccesstothesubstrate molecules than free lipases and, thus, the immobilized systemshowedhigherincorporationofn-3LCPUFAsintoSO. Duringtheimmobilizationprocess,multiplebindingsitesare createdastheproteinmolecules,initiallyfoldedandableto contactthesurfaceononlyoneside,begintounfoldfollowing contact.21

Storageoffreeandimmobilizedlipases

Fig.1showsthestoragestabilityoftheimmobilizedlipases comparedtothatoffreelipases.After6monthsofstorageat 4◦C,theresidualhydrolyticactivityofimmobilizedANand RJlipaseswas75and73%,respectively,whilethatofthefree lipaseswas35and 28%,respectively.Thesedatashowthat theimmobilizationproceduremayincreasethestorage sta-bilityandthatprovesthesignificantadvantageofAmberlite asanenzymeimmobilizationresin.Furthermore,the capac-ityofincorporationofn-3LCPUFAsintoSOwaskeptconstant after6monthsofstorageat4◦C.Theincorporationofn-3 LCP-UFAsintoSOshowedlittlevariation(5–8%)foreitherlipaseas shownontheright-handyaxisofthegraph.

In addition to increasing storage stability, the immobi-lizationoftenprovidestheextraadvantageofincreasingthe catalytic activity of the lipasecompared with that of free enzymes. An immobilization procedure for A. niger lipase bycovalentbindingonchitosan-coatedmagnetic nanoparti-cleswasrecentlyreported.22_{Immobilizedderivativesretained} above80%oftheir initialactivity after15 hydrolyticcycles, andhighstoragestabilityduring50dayswasobserved.Also, lipasefromRhizomucormieheiand M. javanicusimmobilized inmicroemulsion-basedorganogelsmaintainedgood enan-tioselectivitiesinthehydrolysisofketoprofenvinylesterand theiractivitiesincreased12.8-foldand7.8-fold,respectively, comparedwiththeirfreeforms.23

Fig.1–Stabilityoflipasesduring6monthsofstorageat 4◦C.freeANlipase;immobilizedANlipase;䊉freeRJ lipase;immobilizedRJlipase.andrepresentthetotal

n-3PUFAscontentonsoybeanoilafteracidolysisusing immobilizedANandRJlipases.Eachdatapointrepresents theaverageofthreeexperimentsandtheerrorbarsshow thestandarddeviation.Operationalconditions:13gof sardine-FFA+soybeanoil(3:1),166mgofANlipaseor 100mgofRJlipase,12h.AN,Aspergillusniger;RJ,Rhizopus javanicus.

Responsesurfacemethodology(RSM)

The highest n-3 LCPUFA incorporations (11.7 and 7.2% of EPA+DHAforANandRJ,respectively)intoSObybothlipases wereobtainedinrun6(Tables2and3),usingasardine-FFA:SO moleratioof3:1,aninitialwatercontentoflipaseof0.84% (w/w)andafter36hofreaction.Table3showstheFAs com-positionofSO(beforehydrolysis)andofthepurifiedSTAGs obtainedbyacidolysis.ThemajorFAsinSOwerelinoleicacid (18:2n-6,56.5%ofthetotalFAs),oleicacid(18:1n-9,25.8%)and palmiticacid(16:0,11.4%).Theenzymaticacidolysisshoweda positivechangeinthen-6/n-3FAsratio,increasingthen-3 fam-ilyanddecreasingthen-6FAsduetotheincorporationofn-3

LCPUFAsinto SO.Then-6/n-3FAsratiodecreasedfrom 11:1 intheoriginalSOtoapproximately3.5:1and4.7:1after aci-dolysiscatalyzedbyANandRJlipases,respectively.Thedata showedthatintheconditionsofrun6theTAGsshowlower oleicacid(18:1n-9)andlinoleicacid(18:2n-6)contentwhen comparedtotheoriginalSO.Therefore,theincorporationof EPAandDHAincreasedpalmitic acid(16:0)andstearicacid (18:0)content.Forbothlipases,EPAincorporationwashigher thanDHAincorporation,whichmaybeduetotheEPA con-tentofthefishoilusedintheacidolysisreaction(19.8%)being higherthanitsDHAcontent(11.4%).

Somelipaseshavebeenfrequently usedtodiscriminate between EPA and DHA in concentratescontaining both of thesefattyacids,thusprovidingthepossibilityofproducing

n-3PUFAconcentrateswithdominanceofeitherEPAorDHA. Theselectivityoflipasesisinfluencedbythepositional dis-tributionofthefattyacidsandtheglyceridestructuredueto the regiospecificityandtriglyceridespecificity ofthelipase.

A.nigerlipaseincreasedDHAand EPAconcentrationincod

Table2–Experimentalmodelandlevelsofthevariablesusedforoptimizingtheacidolysisofsoybeanoilby immobilizedANandRJlipases.

Runs Variables EPA+DHA(%totalfattyacids)

Reactiontime (X1,hour)

Sardine-FFA:(X2, soybeanoilmoleratio)

Initialwatercontentof lipase(X3,%w/w) ANa _RJb 1 −1 12 −1 3:1 −1 0.45 2.6 2.2 2 +1 36 −1 3:1 −1 0.45 3.4 2.9 3 −1 12 +1 1:3 −1 0.45 1.6 1.4 4 +1 36 +1 1:3 −1 0.45 2.0 1.3 5 −1 12 −1 3:1 +1 0.84 6.8 5.3 6 +1 36 −1 3:1 +1 0.84 11.7 7.2 7 −1 12 +1 1:3 +1 0.84 1.8 1.8 8 +1 36 +1 1:3 +1 0.84 3.0 2.5 9 0 24 0 1:1 0 0.62 3.5 3.1 10 0 24 0 1:1 0 0.62 3.5 2.3 11 0 24 0 1:1 0 0.62 3.0 3.0 12 0 24 0 1:1 0 0.62 3.2 2.7

a _{LipasefromAspergillusniger.}

b _{LipasefromRhizopusjavanicus.}

Table3–Fattyacidcomposition(%ofthetotalfatty acids)ofthepurifiedSTAGsobtainedbyacidolysisof soybeanoilunderdifferentconditions.

Fattyacids Beforereaction Afterreaction(run6) Soybeanoil ANa _RJb SFAsc _{12.6 13.5 17.3} C16:0 11.4 12.5 13.8 C18:0 1.2 2.0 3.5 MUFAsd _{25.8 18.2 20.4} C16:1n-7 ndf _{0.6 1.4} C18:1n-9 25.8 17.6 19.0 PUFAse _{61.5 67.9 59.7} C18:2n-6 56.5 52.7 49.3 C18:3n-3 5.0 3.5 3.2 EPAn-3 ndf _{6.4 4.6} DHAn-3 ndf _{5.3 2.6} EPA+DHA ndf _{11.7 7.2} n-3 5.0 15.2 10.4 n-6 56.5 52.7 49.3 n-6/n-3 11:1 3.5:1 4.7:1

a _{LipasefromAspergillusniger.}

b _{LipasefromRhizopusjavanicus.}

c _{Saturatedfattyacids.}

d_{Monounsaturatedfattyacids.}

e _{Polyunsaturatedfattyacids.}

f _Notdetected.

themodelsystem,thelipasefromCandidarugosashowedthe highestdiscriminationagainstDHA,whilethelipasesfrom

PseudomonasfluorescensandPseudomonascepaciadiscriminated

againstEPAthemost.Intheoils,thePseudomonaslipasesalso discriminatedagainstEPAthemost,whileDHAwasinitially discriminatedthemostbythelipasefromThermomyces

lanug-inosus.However,afterlongerreactiontimestheenrichmentof

DHAintheglyceridefractionoftheoilswasgreatestforthe lipasefromC.rugosa.26

Thestatisticalanalysisshowedthatonlytheeffectsofthe variablessardine-FFA:SOmoleratioandinitialwatercontent oflipasewerestatisticallysignificantwitha95%confidence level(p<0.05).Theestimatedregressioncoefficientsforboth lipasesareshowninTable4andtheANOVAforbothlipases areshowninTable5.Theresultswerestatisticallyvalidated bymeansoftheFtest.TheR2_{valueswereequalto0.77and}

0.64.ThemodelsaredescribedinEqs.(1)and(2).Withthese models,itispossibletopredicttheoptimalresponse (percent-ageofEPAandDHA)oftheprocessinanyconditionscovered bytherangeoftheexperiment(Table2).

EPA+DHA (%)=3.84−2.01X2+1.71X3 (AN) (1)

EPA+DHA (%)=2.97−1.32X2+1.12X3 (RJ) (2)

Table4–RegressioncoefficientsfortheanalysisoftheacidolysisreactionsusingANandRJlipases. Factor Regression

coefficientsa

Standard

errora

p-Valuea _t-Valuea _Regression

coefficientsb Standard errorb p-Valueb _t-Valueb Mean 3.84 0.53 0.000053 7.16 2.97 0.25 0.000001 11.57 Substrates moleratio −2.01 0.65 0.013446 −3.06 −1.32 0.31 0.002273 −4.20 Initialwater contentof lipase 1.71 0.65 0.028330 2.60 1.12 0.31 0.005983 3.57

a _{ResultsforAN(Aspergillusniger)lipase.}

Fig.2–TAGprofilesobtainedbyEASI(+)-MSfororiginalsoybeanoil(A)andsoybeanoilmodifiedbytheacidolysisreactions catalyzedbyAspergillusniger(B)andRhizopusjavanicus(C)lipases.

EASI-MSprovidesfasteranalysiswithnosample prepa-ration, using a more simple apparatus compared to other techniques.Fig.2showsTAGprofilesobtainedbyEASI-MSfor SObefore(A)andafteracidolysiscatalyzedbyAN(B)andRJ (C)lipases.CharacteristicTAGofSOwhich,containingmostly linoleic(L),oleic(O),linolenic(Ln),palmitic(P)andstearic(S) acids,weredetectedmainlyas[TAG+Na]+_{ionsandattributed}

toC52:4(PLL, m/z877),C52:3(PLO, m/z879),C54:7(LLLnor OLnLn,m/z899),C54:6(LLLorOLLn,m/z901),C54:5(OLLor OOLn,m/z903),C54:4(OOL,m/z905),C54:3 (OOO,m/z907),

C54:2(SOO,m/z909)andC54:1(SSO,m/z911)(Fig.2A). Corre-spondent[TAG+K]+_{ionsofm/z893(PLL),m/z895(PLO),m/z915}

(LLLnorOLnLn),m/z917(LLLorOLLn),m/z919(OLLorOOLn),

m/z 921 (OOL) and m/z 923 (OOO) were also detected.The lipase-catalyzed acidolysisofSO with sardine-FFAresulted in asubstantiallymodified TAG profilemainlyin theform ofhigherm/z(Fig.2BandC). New,highlyunsaturatedTAG specieswere detected,showingapossibleadditionofDHA andEPAintheTAGmoleculesofSO,suchasC52:6ofm/z873 [MPDHA+Na]+_{,C54:6ofm/z878[POEPA+Na]}+_{,C52:6ofm/z889}

Table5–Analysisofvariance(ANOVA)forEPA+DHAincorporation(%totalfattyacids)afteracidolysisreactionsusing ANandRJlipases.

Sourceofvariationa _{Sumofsquares Degreesoffreedom Meansquare F}

Regression 55.86 2 27.93125 8.1021

Residues 31.02 9 3.44741

Lackoffit 30.84 6 5.14111 85.6852

Pureerror 0.18 3 0.06000

Total 86.88 11

Sourceofvariationb _{Sumofsquares Degreesoffreedom Meansquare F}

Regression 24.17000 2 12.08500 15.2492

Residues 7.13250 9 0.79250

Lackoffit 6.74500 6 1.12417 8.7032

Pureerror 0.38750 3 0.12917

Total 31.30250 11

a _{ResultsforAN(Aspergillusniger)lipase,regressioncoefficient:R}2_=0.64,F

0.05;2;9=4.26.

b _{ResultsforRJ(Rhizopusjavanicus)lipase;regressioncoefficient:R}2_=0.77,F

0.05;2;9=4.26.

[MPDHA+K]+_{,C54:6 ofm/z917[LLLor OLLnorPOEPA+K]}+_,

C54:5ofm/z919[PSEPA+K)+_{,C58:12ofm/z945[LEPAEPAor}

MDHADHA+Na]+_{,C60:14ofm/z969[LnEPADHA+Na)}+_,C60:12

ofm/z 973 [PDHADHA+Na]+ _{and C60:11 ofm/z 975}

(SEPA-DHA+Na]+_{.ThedataindicateasignificantamountofLC-PUFA}

esterifiedintoSTAGs,whichmayberelevantinthe develop-mentofalternativePUFA-formulationsforclinicalnutritionor incorporationintofoodproducts.

TAGscontainingn-3LCPUFAscanbeobtainedby lipase-catalyzedacidolysisorinteresterificationwithfattyacidethyl esters,eitherinorganicorinsolvent-freemedia,asreported inseveral studies.19,27–29 _{STAGs enrichedwith unsaturated} fattyacidsandlowinpalmiticacidcontentviaacidolysisof microalgaeoil (Schizochytriumsp) with oleic acid were pro-ducedusingLipozymeRMIM.30_{Theunsaturatedfattyacids} content increasedfrom 70.20% to90.9% at the sn-1,3 pos-itionsintheSTAGsundertheoptimalcondition(i.e.,lipase loadof7%,molarratio ofmicroalgaeoilTAGstooleicacid of 1:3, and temperature of 65◦C). In most of these stud-ies,high-costsn-1,3,regioselective,immobilized,commercial, microbiallipaseshavebeenused.Thehighcostof commer-ciallipasesandthelowoperationalstabilityofsomeofthem havebeenrecognizedasthemainconstraintstotheuseof lipase-catalyzedprocessesforSTAGsproduction.Therefore, thesearchfornovelbiocatalysts,presentingbothhighactivity and operationalstability, isa waytomakeenzymatic pro-cessescompetitive.

Conclusion

STAGscontainingEPAandDHA,showingan-3/n-6ratiowithin theproportionsrecommendedasidealforclinicalnutrition, weresuccessfullyobtainedbyacidolysiscatalyzedbynative AN and RJlipases immobilized on Amberlite MB-1. Under the conditions optimized by RSM, the acidolysis reactions usingbothlipasesledtoanadvantageousexchangebetween the acylradicals from TAGsofSOand FFAs obtainedfrom Braziliansardineoil.TheimmobilizationonAmberliteMB-1 probably exerts a significant influence on the stabilization of the conformation of lipases and on their resistance to

denaturation, favoringtheirstoragetime, especiallyforAN lipase.Oneofthegreatestadvantagesofimmobilizationisto facilitateanenzyme’sreusability,whichisessentialtorender the process technologically and economically viable. Little informationisavailableonthepotentialhealthbenefitsofthe STAGmoleculesthatarerichinn-3LCPUFAs.Furtherworkis neededtogettoknowthephysiologicaleffectsoftheuseof STAGsproducedbyincorporationofEPAand/orDHAintoSO forclinicalnutrition.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

The authors wish to thank the Brazilian Financing Agen-cies: Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo (FAPESP)andConselhoNacionalde Desenvolvimento CientíficoeTecnológico(CNPq).Theauthorsalsothankthe LaboratórioThoMSondeEspectrometriadeMassas,Instituto deQuímica(UNICAMP)andtheCentrodeMicologiada Univer-sidadedeLisboa(Portugal)forthetaxonomicidentificationof isolates.

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