Microencapsulation of lipophilic bioactive compounds using prebiotic carbohydrates: Effect of the degree of inulin polymerization

This paper presents novel outcomes about the effect of degree of inulin polymerization (DP) on the technological properties of annatto seed oil powder obtained by freeze-drying. Inulins with two DP’s were evaluated: GR-inulin (DP ≥ 10) and HP-inulin (DP ≥ 23).

jo u r n 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 / c a r b p o l

Eric Keven Silvaa,∗, Giovani L Zabotb, Matheus A Bargasa, M Angela A Meirelesa

a LASEFI/DEA/FEA (School of Food Engineering)/UNICAMP (University of Campinas), Rua Monteiro Lobato, 80, Campinas, SP, CEP: 13083-862, Brazil

b Federal University of Santa Maria, UFSM, Rua Ernesto Barros, 1345, Cachoeira do Sul, RS, 96506-322, Brazil

Article history:

Received 18 May 2016

Received in revised form 5 July 2016

Accepted 17 July 2016

Available online 18 July 2016

Keywords:

Fructooligosaccharide

Functional carbohydrate

Glass transition temperature

Wall material

Water adsorption isotherms

Thispaperpresentsnoveloutcomesabouttheeffectofdegreeofinulinpolymerization(DP)onthe tech-nologicalpropertiesofannattoseedoilpowderobtainedbyfreeze-drying.InulinswithtwoDP’swere evaluated:GR-inulin(DP≥10)andHP-inulin(DP≥23).Micrographsobtainedbyconfocalmicroscopy wereanalyzedtoconfirmtheencapsulationofbioactivecompoundsusingbothinulins,especiallythe encapsulationofthenaturalfluorescentsubstance␦-tocotrienol.Microparticlesformedwithbothinulins presentedthesamecapacityforgeranylgeraniolretention(77%).Glasstransitionsofmicroparticles formedwithGR-inulinandHP-inulinsucceededat144◦Cand169◦C,respectively.Regardingwater adsorptionisotherms,microparticlesformedwithHP-inulinandGR-inulinpresentedbehaviorsofTypes

II(sigmoidal)andIII(non-sigmoidal),respectively.Reductionofwateradsorptioncapacityinthematrix

athighrelativemoistures(>70%)waspresentedwhenHP-inulinwasused.Atlowrelativemoistures (<30%),theoppositebehaviorwasobserved

©2016ElsevierLtd.Allrightsreserved

1 Introduction

The crescent interest of developing prebiotic substances is

aimedatnon-digestibleoligosaccharides.Oneoftheprebioticsis

inulin,a fructooligosaccharide generally extracted fromchicory

(Pandey et al., 1999), but alsofrom other sources asartichoke

(Ruiz-Aceituno, García-Sarrió,Alonso-Rodriguez,Ramos, &Sanz,

2016),andusedforstabilizingproteins(Mensink,Frijlink,vander

VoortMaarschalk,&Hinrichs,2015)orconvertedintoother

func-tionalingredientsbyinulinases(Mazuttietal.,2010).Longerchain

lengthsmakeinulinasdietaryfiberusefulforfoodand

pharmaceu-ticalapplications.Examplesofapplicationsincludeaslowcaloric

sweetener,assubstancethatprovidessoliddispersionfor

increas-ingdissolutionrate,asanagenttoformgelsandtoincreasethe

viscosityofsolutions,andasanon-digestiblefiber(Mensinketal.,

2015).Inulinalsopresentstheabilitytochangethegutflora

com-positionafterashortfeedingperiodbasedonresultsfrominvitro

studiesandhumansubjects(Kolida&Gibson,2007)

Theuseofinulindependsonitsdegreeofpolymerization(DP)

The molecularchain length can range someextent, commonly

between4and80(Mensinketal.,2015).Thedegreeofinulin

poly-∗ Corresponding author.

E-mail address: engerickeven@gmail.com (E.K Silva).

merizationdependsontheharvesttime,storagetime/temperature andgrowingconditions(Saengthongpinit&Sajjaanantakul,2005) Consequently,thedegreeofinulinpolymerizationdeterminesthe physicochemical characteristicstoa substantialextent,as mor-phology(i.e.,crystalmorphology,crystalstructureandstructurein solution),solubility,rheology(i.e.,viscosity,hydrodynamicshape and gelling),thermal characteristicsand physical stability(i e., glasstransitiontemperature,vaporsorptionandmelting temper-ature)andchemicalstability(Mensinketal.,2015)

Followingthescenarioofusinginulinasprebiotic substance (Zabot, Silva,Azevedo, &Meireles, 2016), bioactivecompounds extractedfromvegetalsourcesarealsoofrecentinterestbecauseof theirfunctionalproperties.Annattoseedoilisoneofthesourcesof value-addedbioactivecompounds,as␦-tocotrienol(Albuquerque

&Meireles,2012;Moraes,Zabot,&Meireles,2015)and geranyl-geraniol(Silva,Zabot,&Meireles,2015).Tocotrienol-richfractions obtainedfromannattoseedsactasnaturalantioxidantsby inhibit-inglipidoxidationoffishoiland lipid-basedformulaemulsions (Zou&Akoh,2015).In addition,geranylgeraniolmodulates the apoptosisofcarcinogencells(Marcuzzietal.,2012)

In this context, the objective of this study was to evaluate theinfluenceofthedegreeofinulinpolymerization(DP≥10and

DP≥23)onthephysicalpropertiesofannattoseedoil micropar-ticlesobtainedbyfreeze-drying.Inulinwasusedasencapsulating matrixandtheeffectofitsdegreeofpolymerizationonthe

recon-http://dx.doi.org/10.1016/j.carbpol.2016.07.066

0144-8617/© 2016 Elsevier Ltd All rights reserved.

the morphology and ␦-tocotrienol distribution in the particles

microstructures,geranylgeraniol retentionby theencapsulating

matrices,glasstransitionofmicroparticlesandphysicalstability

throughwateradsorptionisothermswerealsotheresponsestaken

intoaccountwhen evaluatingtheeffectof thedegreeofinulin

polymerization

2 Material and methods

2.1 Annattoseedoilextraction

Annattoseedoilused asactivematerial wasextractedwith

supercriticalCO2usingapilotscaleequipment(TharTechnologies,

Pittsburgh,USA)containinganextractionvesselof5Lwithinternal

diameterof10.2cm.Solventflowratewasequalto200gCO2/min

andthe bedwasmaintainedat 40◦Cand 20MPa,as described

indetailbya previousstudy(Silva,Gomes,Hubinger, Cunha,&

Meireles,2015)

2.2 Inulinsandmaterialsforchromatographicanalyses

Inulins used as active materials for encapsulating annatto

seedoilwerebothfromchicory:Orafti®GR(GR-inulin,DP≥10)

and Orafti®HP (HP-inulin, DP≥23) (BENEO-Orafti, São Paulo,

Brazil).Materialsforchromatographic analyseswere:methanol,

ethanolandhexane(Chemco,Hortolândia,Brazil);geranylgeraniol

(purity>85%,Sigma–Aldrich,Steinhein,Germany)andammonium

acetate(P.A.,Dinâmica,Campinas,Brazil)

2.3 Microencapsulationofannattoseedoil

Annattoseed oilwasencapsulatedwithGR-and HP-inulins

throughemulsificationassistedbyultrasoundwithnominalpower

of160Wduring3minusinga13mmdiameter,19kHzultrasonic

probe(Unique,Disruptor,800W,Indaiatuba,Brazil)foreach30mL

ofemulsion.Totalconcentrationofsolidsintheemulsion

(emulsi-fying+oil)wasequalto20g/100gofemulsion.Annattoseedoilwas

addedtothesuspensionscontainingGR-andHP-inulinsandthe

concentrationofoilwasmaintainedat20%relativetotheamount

ofsolids,thatis,4gofoilper100gofemulsion.Immediatelyafter

homogenization,theemulsionswerefrozeninaluminum plates

at−40◦C for 3handthen subjected tofreeze-drying(FD)

pro-cess.Dryingwasperformedina freeze-dryersystem(Liobras,L

101,SaoCarlos,Brazil).Thedriedemulsionswereconvertedinto

finepowdersthroughmaceration.Detaileddescriptionof

obtain-ingannattoseedoilmicroparticlesisreportedbyapreviousstudy

(Silva&Meireles,2015)

2.4 Reconstitutionpropertiesofemulsions

Thewettabilityofthepowderswasdeterminedasthemethod

reportedbyFuchsetal.(2006),withafewmodifications.The

sam-plesof powders (0.1g) weredispersed over water surface of a

Beckercontaining100mLofultrapurewaterunderstirringat25◦C

Thetimespentforimmersingorwettingthelastparticleofpowder

wasusedaswettabilityresponse

Theannattoseedoilmicroparticlesobtainedbyfreeze-drying

werereconstitutedinultrapurewater.Anamountof2gofpowder

wasmixedwith8gofultrapurewaterandthesolutionwasstirred

during30sinavortextypehomogenizer(PHOENIX,AP-56model,

Araraquara,SãoPaulo,Brazil)at25◦C

Dropletsizedistributionandmeandiameteroftheemulsion

droplets,afterreconstitutionandbeforedrying,weredetermined

bylightscatteringtechniqueusinglaserdiffraction(Mastersizer

2000MalvernInstrumentsLtd,Malvern,UK).Themeasurements

wereperformedat25◦C.Themeandiameterwascalculatedbased

onthemeandiameterofasphereofsimilararea,superficialmean diameter(D32),asEq.(1).Polydispersityindex(PDI)wascalculated

asEq.(2).Allsampleswereanalyzedthroughthewetmethod,with dispersioninwaterandrefractiveindexof1.52

D32=



nidi3



nidi2

(1)

PDI=(d90−d10)

d50

(2) Where:diisthemeandiameterofthedroplets;niisthenumber

ofdroplets;andd10,d50andd90arethediametersat10%,50%and 90%ofcumulativevolume,respectively

2.5 Scanningelectronmicroscopy(SEM) Micrographs were taken in a scanning electron microscope withEnergyDispersiveX-rayDetector(SEM)(Leo440i,EDS6070, SEM/EDS:LEOElectronMicroscopy/Oxford,Cambridge,England) Analyseswereperformedwith5kVacceleratingvoltageand50pA beamcurrentforobtainingthemicrographs

2.6 Confocalscanninglasermicroscopy(CSLM) CSLManalysiswasperformedusingaZeissLSM780-NLO con-focalonanAxioObserverZ.1microscope(CarlZeissAG,Germany) witha40×objective.Imagesweretakenbyexciting␦-tocotrienol moleculeswithlasersat488nmwavelength,withoutany previ-ouspreparationofthesamplesasaconsequenceofthefluorescent propertiesof␦-tocotrienol,similarlytotheproceduredescribedby

apreviousstudy(Silva,Zabot,Cazarin,MarósticaJr.,&Meireles, 2016)

2.7 Geranylgeraniolretention Geranylgeraniolcontentinannattoseedoil(beforeandafter encapsulation)wasdeterminedbyhigh-performanceliquid chro-matography(HPLC).Chromatographicanalyseswereaccomplished usingan HPLC-PDA(Waters,AllianceE2695, Milford,USA) sys-tem,consistingofaseparationmodulewithanintegratedcolumn heater,anautosamplerandaphotodiodearray(PDA)detector Sep-arationof geranylgeraniolwas fulfilled usinga fused-core type column (Kinetex, C18,100mm×4.6mm×2.6␮m; Phenomenex, Torrance, USA) An aliquot of 10␮L of each sample diluted to

500ppm (w/w)inhexane(Chemco,Hortolandia,Brazil)and fil-teredusingnylonmembrane(0.45␮m)wasinjected.Asolutionof methanol:ammoniumacetate50mM(90:10,v/v)wasthemobile phase.The columnwasmaintainedat40◦C Mobilephase flow ratewas1mL/minandtheanalyticalruntimewas7min.Detector wavelengthrangewas200–400nm.Geranylgeraniolwasdetected

at210nmand at2.4min,and itsquantificationwasperformed usingexternalstandardcalibrationcurve

Intending to quantify geranylgeraniol entrapped in the microparticlesafterusinginulinswithtwodegrees of polymer-ization,fourprocedureswerecarriedouttobreakthestructurefor releasingsuchcompound:

ICentrifugation: Approximately 0.1g of particles from each treatmentwasmixedwith4mLofultra-purewater.The sam-plesweremaintainedstaticduring24h;thereafter,theywere manuallyagitatedforreconstitutingtheemulsions.Aliquotsof 0.5mLweretransferredtoanEppendorftubeof2mL contain-ing1.3mLofhexane Themixtureswerethencentrifugedat

5000rpmfor20minandat10,000rpmfor5min.This proce-durewasperformedtobreaktheemulsionandtocapturethe

ger-anylgeraniolcontentwasanalyzedasfollowingtheanalytical

proceduresusedforpureannattoseedoil(oilbefore

encapsu-lation)

IIDiffusion in hexane and extraction assisted by ultrasound:

Approximately0.1gofparticlesfromeachtreatmentwasmixed

with20mLofhexane.Thesamplesweremaintainedstatic

dur-ing24hfor attaining thediffusionof geranylgeraniol.Inthe

sequence,thesamplesweresonicatedat600Wduring2min

usinga13mmdiameter,19kHzultrasonicprobe(Unique,

Dis-ruptor,800W,Indaiatuba,Brazil).Afterwards,analiquotwas

takenandsubjectedtochromatographicanalysesfollowingthe

sameproceduresdevelopedwithpureoil

IIIDiffusion in ethanol and extraction assisted by ultrasound:

ThisprocedurewasaccomplishedsimilarlytoprocedureII.The

onlyonedifferencewasthesubstitutionofhexanebyethanol

IVDiffusion in ethanol:water solution and extraction assisted by

ultrasound:Thisprocedurewasaccomplishedsimilarlyto

pro-ceduresIIandIII.Theonlyonedifferencewasthesubstitution

ofhexaneorethanolbyethanol:water(1:1,w/w)solution

2.8 Differentialscanningcalorimetry(DSC)

DSCanalysiswasperformedusingcalorimeterequipment(TA

60,ShimadzuCorporation,Kyoto,Japan)fordeterminingtheglass

transitiontemperature(Tg)ofmicroparticlesandinulinsusedas

wallmaterial.Approximately2mgofsample,underanatmosphere

with10mL/minofnitrogen,wassubmittedtotwo heating

gra-dientsforconstructingDSCcurves:25◦Cto110◦Cwithscanning

rateof10◦C/minandisothermalperiodof5min(firstrun);25◦C

to250◦C(secondrun).Tgwasobservedinthesecondrunatthe

midpointoftheglasstransitionrange(Botrel,deBarrosFernandes,

Borges,&Yoshida,2014)

2.9 Wateradsorptionisotherms

Water adsorption isotherms of microparticles containing

annatto seed oil, formed with both degrees of

polymeriza-tion, weredetermined by thegravimetric static method(Silva,

Borges, da Costa, & Queiroz, 2015; Silva, Fernandes, Borges,

Botrel,&Queiroz,2014).Thesampleswereconditionedin

phos-phorous pentoxide previously of performing the assays, until

constant weight Such procedure was fulfilled to assure that

onlytheadsorptionphenomenon couldbeobservedfor all

rel-ative moistures evaluated in the experiment Afterwards, the

microparticles were stored at 30◦C under some relative

mois-tureconditions(11.28%to90.20%)provided bysevensaturated

salinesolutions(lithiumchloride,magnesiumchloride,potassium

carbonate,sodiumbromide,sodiumchloride,potassiumchloride

andbarium chloride)(Greenspan, 1977).The parametersofthe

Guggenheim–Anderson–deBoer(GAB)model(Eq.(3))(Vanden

Berg,1984)werefittedusingtheexperimentaldataofequilibrium

moisture.TheQuasi-Newtonnonlinearregressionwasusedwitha

convergencecriterionof10−4ontheStatisticasoftware(Statsoft,

8.0,2007)

Xeq= XmCKaw

(1−Kaw) (1−Kaw+CKaw) (3)

Where:Xeqistheequilibriummoisture(gwater/100gdrysolids);

Xmisthemonolayermoisturecontent(gwater/100gdrysolids);aw

isthewateractivity(dimensionless);Cisaconstantrepresenting

theadsorptiononthefirstmonolayer;Kisaconstantrepresenting

theadsorptionofmoleculesofwateronmultilayers

Table 1

Reconstitution properties of annatto seed oil powders.

FE: fresh emulsion; RE: reconstituted emulsion.

The fit of themodel parameters wasassessed basedon the coefficientofdetermination(R2)andthemeanrelativepercentage deviationmodulus(E)(Eq.(4)):

E=100 n

n

 i=1



|Y− ˆY|

Y



(4)

Where:Yistheobservedvalue; ˆY isthemodelestimatedvalue;n

isthenumberofobserveddata

2.10 Statisticalanalysis Minitab16®softwarewasusedtoperformtheanalysisof vari-anceforverifyingtheeffectsofthedegreesofinulinpolymerization

onthemicroparticlescharacteristics.Differencesbetweenaverage valueswerecomparedusingTukey’stestwith5%ofsignificance (p-value<0.05).DSCresultswereanalyzedbydescriptiveapproach

3 Results and discussion

3.1 Reconstitutionpropertiesofannattoseedoilemulsions Theevaluationofreconstitutionpropertiesofparticulate sys-tems allows predicting the best conditions for applying and distributingencapsulatedactivecompounds.Inthissense,the wet-tabilityisoneofthemoreimportantphysicalpropertiestakeninto accountwhenthecharacteristicsof reconstitutedpowder prod-uctsareevaluated,becausethewettabilityexpressesthecapacity

oftheproducttoadsorbwater.Then,theresponsesof wettabil-ityofannattoseedoilmicroparticlesformedwithinulinsoftwo degreesofpolymerization(GR-inulin,DP≥10;HP-inulin,DP≥23)

asencapsulatingmatricesarepresented(Table1)

The use of GR-inulin (lower DP) enabled the formation of microparticleswithhigherinstantisationvelocity.Thisresultcould

beassociatedwiththedifferencesonthemolecularchain,because larger molecular chains could act as physical barrier against water penetration, thus reducing the velocityof water absorp-tion.Fernandes,Borges,andBotrel(2014)evaluatedthewettability

ofrosemaryessentialoilmicroparticlesobtainedbyspray-drying Theauthorsusedblendsofinulin/Arabicgumandinulin/modified starch, both with the same mass proportion of wall materials (1:1).Thefindingsindicatedtheinulin/Arabicgumblendpresented instantisationat93±9s,whileinulin/modifiedstarchblend pre-sentedinstantisationat131±8s

Dropletsizedistributionsofannattoseedoilemulsions stabi-lizedbyGR-andHP-inulinsinfreshstate(beforefreeze-drying) andreconstitutedstatearepresented(Fig.1).Forbothtreatments, thepatternsweredifferentwhenthefreshandreconstituted emul-sionsarecompared.Inaddition,bimodaldistributionsareseen.The responsesofD32andPDI(Table1)corroboratetheperformanceof GR-FDandHP-FDmicroparticles,whichtheresponseswerenot satisfactory,mainlywithrespecttothereconstitutionproperties Suchresponsesareassociatedwiththelackofsuperficialactivityof inulinmolecule,regardlessofitsdegreeofpolymerization.Inulin aloneaswallmaterialcannotforminterfacialfilmsovertheannatto seedoildroplets.Themainmechanismforemulsionsstabilization

Fig 1.Droplet size distributions of fresh and reconstituted emulsions and visual aspect of emulsions stored during 24 h after reconstitution.

islinkedtotheinulinactionasthickenerofcontinuousphase,

creat-ingaphysicalbarrieragainstthecoalescenceofoildroplets(Silva,

Gomes,etal.,2015).Thevisualaspectofemulsionsstored

dur-ing24hafterreconstitutionisalsopresented(Fig.1).Theemulsion

stabilizedwithHP-inulinshowedstabilityagainstphaseseparation

afterthereconstitution.However,theemulsionstabilizedwith

GR-inulinwasunstableduringthestorageperiod.Thefindingscould

beassociatedwiththedegreeofinulinpolymerization,because

thehighestdegreeledtotheformationofathickenercontinuous

phase,moreefficientforreducingthecoalescenceofoildroplets

(Silva&Meireles,2015)

3.2 Microstructuralanalysis

Themorphologyofannattoseedoilmicroparticlesformedwith

twodegreesofpolymerization(GR:DP≥10;HP:DP≥23)is

pre-sentedafterperformingSEMandCSLManalyses(Fig.2).SEMis

animportantanalysisusedforcharacterizingthemicrostructure

ofparticulateproducts.Withthemicrographs,theeffectsofthe

processonmicroparticlesformationandtheeffects of

composi-tionofwallmaterialsonthemorphologicalcharacteristicsofthe

powderproductscanbeassessed.Likewise,CSLManalysisisan

importanttoolforcharacterizingmicrostructuresbasedonthe

nat-uralfluorescentpropertiesofcompoundspresentinthestructure

orbasedontheadditionofsubstanceswithsuchproperties.The

mainadvantageofthetechniquereliesonthepossibilityof observ-ingtheinternalstructureofmicroparticleswithoutfragmenting thematerial.Theresultsobtainedafterperformingbothanalyses (SEMand CSLM)arecomplementary,whereas theycharacterize themorphologyanddistributionofthebioactivecompoundsonthe microstructureoftheparticles.Somescientificstudiesreportthese analysesasacombinedformofcharacterizationofpowder prod-uctscontainingencapsulatedbioactivecompounds(Carvalho,da CostaMachado,daSilva,Sartoratto,Rodrigues,&Hubinger,2016; Silva,Azevedo,Cunha,Hubinger,&Meireles,2016)

Theregionsinfluorescentgreen(Fig.2)indicatethe distribu-tionof␦-tocotrienolinthemicroparticlesstructure,whichmeans annattoseedoilwasdistributedthroughtheencapsulatingmatrix AlbuquerqueandMeireles(2012)reportedannattoseedoilisthe richestsourceof␦-tocotrienol,containingapproximately15g ␦-tocotrienol/100goil.Therefore,weinferbothinulinswereableto entrap␦-tocotrienolinsidethematrix(qualitativeanalysis) Regardlessthedegreeofinulinpolymerization,all micropar-ticles presented irregular morphology, typical of freeze-dried materials(Chranioti,Chanioti,&Tzia,2016).Themorphologyisa resultofbreakingthespongystructureformedafterremovingice crystalsintheencapsulatingsystemthroughsublimation Regard-ingthemicrostructure,nodifferenceswereobservedafterusing inulinswithdifferentmolecularchainlengths

Fig 2.SEM and CSLM micrographs of annatto seed oil microparticles.

3.3 Geranylgeraniolretention

Oneofthemainresponseswhenevaluatingtheefficiencyof

anencapsulatingmatrixistheabilityofretainingthetarget

bioac-tivecompoundintotheencapsulatingsystem.Thisabilityassures

theefficiencyofanexpectedactionafterapplyingthe

micropar-ticlesinadeterminedproduct.Inthissense,theinfluenceofthe

degreeofinulinpolymerizationongeranylgeraniolretentionwas evaluated,becausegeranylgeraniolpresentstherapeuticproperties associatedwithitsbiologicalactivitiesasanti-inflammatoryand anticanceragent.Annattoseedoil,beforeencapsulation,presented 25.0±0.6ggeranilgeraniol/100goil

Recoveringgeranilgeraniolfromannattoseedoilmicroparticles aimingtoperformitsquantificationwasfulfilledthroughtwo

dis-Fig 3.Effect of the degree of inulin polymerization on geranylgeraniol retention.

US: ultrasonication.

tinctmechanisms:i)emulsionswerereconstitutedbysuspending

themicroparticlesinwaterfollowedbycentrifugationtobreakthe

colloidalsystemandtocapturethecompound;ii)suspensionof

microparticleswasdoneindifferentorganicsolventsfordiffusing

thecompoundfollowedbybreakingtheparticlesmicrostructures

usingultrasonication.Theinfluenceofthedegreeofinulin

poly-merization ongeranylgeraniol retention could beseen (Fig.3

whereitrangedfrom40%to74%forGR-FDandfrom2%to77%

forHP-FD

Breaking the reconstituted emulsions by centrifugation for

extractingannattoseedoilwasnotanefficientmethodto

quan-tifygeranylgeraniol,becausehighviscositiesofemulsionsactedas

aneffectivephysicalbarriertoentraptheoil.Thelowestextraction

foundforHP-FDcorroboratesthisobservation,becausethe

viscos-ityoftheemulsionincreaseswithincreasingthedegreeofinulin

polymerization(Silva&Meireles,2015;Silva,Gomesetal.,2015)

Diffusingtheoilin organicsolventfollowedbysonicationof

thematrixforreleasingtheoilwasthemostefficientmethodfor

quantifyinggeranylgeraniolretainedinthemicroparticles

Diffu-sionin ethanol orethanol:water solutiondidnot influencethe

geranylgeraniolretention (p-value=0.725).Thelowest retention

foundafterusinghexaneasdiffusionmediumcanbeassociated

withthelowestpolarityofthissolventwhencomparingitwith

theotherdiffusionmedia.Furthermore,theinteractionofhexane

withtheencapsulatingmatrixislowandnotenoughtobreakthe

structureduringultrasonication

Microparticlespresentedthesamecapacityofgeranylgeraniol

retention(p-value=0.616).Ourresultscorroboratetheentrapment

efficienciesreported by Silva and Meireles (2015).The authors

reportedthat thedegreeofinulinpolymerizationdidnot

influ-encetheoilretentioninthemicroparticles,since GR-inulinand

HP-inulinresultedinthesameentrapmentefficiencyofannatto

seedoil

3.4 Glasstransitiontemperature(Tg)

DeterminingTgofdehydratedproductsisanimportantstepfor

characterizingparticulatesystems(GomesdaCosta,Silva,Toledo

Hijo,Azevedo,&Borges,2015).ConditionsoftemperatureaboveTg

providemoremolecularmobility,acceleratingthereactionrates

ConditionsoftemperaturebelowTgprovidehigherstabilityofthe

productsagainstdeteriorationduringstorage(Oikonomopoulou,

Fig 4.DSC curves of pure inulins and annatto seed oil microparticles.

Krokida,&Karathanos,2011).SilvaandMeireles(2015)evaluated X-raydiffractogramsofGR-andHP-inulins,aswellasGR-FDand HP-FDmicroparticles.GR-andHP-inulinswerecharacterizedas amorphousmaterialsandbothmicroparticleswerecharacterized

asamorphousmaterialswithcrystallineregions

Semicrystallinebiopolymerscanpresentthreethermal transi-tioncharacteristics:a)glasstransitiontoamorphousfraction;b) fusionofthecrystallinefraction;c)transitionasaconsequenceof crystallization(Teac˘a,Bodîrl˘au,&Spiridon,2013).When evaluat-ingencapsulatingsystems,themorerelevantphasetransitionis theglasstransition,becauseTgofamorphousmaterialsisdefined

asthetemperaturewhichthematerialchangesfromamorphous statetogummy(elastic)state.Therefore,afterthetransition,the microparticlescoatingstructurethatentrap(protect)theannatto seedoillooseitsefficiencyasencapsulatingmatrix,releasingthe activematerial

DSC curves of pure inulins and microparticles containing annattoseedoil(Fig.4)wereobtainedafterthecoolingperiodat secondscanningstage,aimingtoerasethethermalhistoryoverTg (Haque,Kawai,&Suzuki,2006).Overall,glasstransitionis eval-uated in terms of themoisture of thematerial, because water actsasplasticizing,thusreducingTgasaconsequenceofreducing interandintramacromolecularforces(Chuang,Panyoyai,Shanks,

&Kasapis,2015).Thesampleswereanalyzedattheconditionsof moistureandawfromFDprocess,asdescribedbySilvaandMeireles (2015),searchingforthecharacterizationofproductsobtainedon theirnaturalconditions

AccordingtoDSCcurves(Fig.4 thephenomenaofglass tran-sitionofGR,HP,GR-FDandHP-FDsamplessucceededat132◦C,

157◦C,144◦Cand 169◦C,respectively.Afteranalyzingthepure biopolymers,increasingthedegree ofpolymerizationincreased

Tgofinulin.Themolecularchainlengthofinulindidnot influ-enceTgofmicroparticles.Althoughdifferenceshigherthan25◦C

intheTgofpureinulinswereobserved,thepresenceofannatto seed oil approximated the physical properties of the systems Ronkart,Paquot,Fougnies,Deroanne,andBlecker(2009)evaluated themoisturecontentoverTgofinulin(meanDP=23).Theauthors concludedtheglasstransitionofinulinsucceededbetween150◦C and160◦Cformoisturecontentsbelow5wt.%(drymassbasis), similarlytoourfindings

Fig 5. Water adsorption isotherms of annatto seed oil microparticles.

3.5 Wateradsorptionisothermsofannattoseedoil

microparticles

Studyingtheisothermalbehaviorofwateradsorptionofannatto

seedoilmicroparticlesallowspredictingthebeststorage

condi-tionsformaintaininglongshelflifeofproducts.Wateradsorption

ofdehydratedproductsisthemainfactorofdegradationofsuch

products.Increasingmoisturecontent,afterwateradsorption,can

causeseveralchangesontheencapsulatingmatrix.Forinstance,

microparticlescanagglomerateorTgcanreduce,compromising

theefficiencyofthepolymericmatrix

Wateradsorption isotherms of annatto seed oilat 30◦C for

eachdegreeofinulinpolymerizationarepresented(Fig.5)

HP-FDsamplespresentedbehaviorofTypeII:sigmoidal.Accordingto

classificationofBrunauer,Deming,andTeller(1940),thisbehavior

istypicalofporousormacroporousmaterialswhichpresenthigh

adsorptionenergy.Otherwise,GR-FDsamplespresented

behav-iorofTypeIII:non-sigmoidal.Thisbehavioristypicalofporous

ormacroporousmaterialswhichpresentlowadsorptionenergy

Thebehaviorscorroboratethedifferencesontheequilibrium

mois-tures observedfor microparticles withrelative moisturehigher

Fig 6.Images of annatto seed oil microparticles after water adsorption at seven equilibrium relative moistures.

than30%.Inthesameconditionsofpressureandtemperature,

GR-FDsamplespresentedlargercapacityofwateradsorptionthanthat observedforHP-FDsamples.Then,wateradsorptionintheinulin withsmallermolecularchainexpendslowerenergy

Aftercomparingthebehaviorofthecurves,reductionofwater adsorptioncapacityinthematrixathighrelativemoistures(>70%) wasverifiedwhenthedegreeofinulinpolymerizationisincreased (highermolecularlength) However,theopposite behavior was observedatlowrelativemoistures(<30%).Similarfindingswere reportedbySchaller-Povolny,Smith,andLabuza(2000)when eval-uatingfourdegreesofinulinpolymerization(meanDPequalto5,

9,11and23)overwateradsorptionisothermsat23◦C.Thiseffect

iscoupledwiththemolecularlengthandarrangementofinulin,

Table 2

Parameters fitted for GAB model.

Microparticles X m (g water/100 g dry solids) C K R 2 E (%)

whichtheshortestchaincontainsmorehydroxylgroupsavailable

tobondwithwater

GABmodelwasusedtofittheparametersthatrepresentthe

experimentaldatabecausesuchmodelcansuccessfullydescribe

waterisothermaladsorptionofpowderproducts(Rao&Labuza,

2012;Zhou,Liu,Chen,Chen,&Labuza,2014).Thus,theparameters

ofGABmodelwerefittedandthefitwasevaluatedstatistically

(Table2).Accordingtohighvaluesofcoefficientofdetermination

(R2≥0.995)andlowvaluesofmeanrelativepercentagedeviation

modulus(E≤4.41),thecurvesfittedtotheadsorptionisotherms

weresuitabletodescribethebehavioroftheexperimentaldata

AfterfittingtheparametersofGABmodel,Xmwasobtained.This

parametercomprisesthecontentofwaterthatisstronglyadsorbed

tothebondingsitesonthemicroparticlessurface.Xmisimportant

forthephysicalstabilityofparticulatesystemsagainstlipid

oxida-tion,enzymaticactivity,non-enzymaticbrowningandchangesin

thestructuralcharacteristics.Therefore,Xmisconsideredtobethe

suitablemoisturecontentforextendingtheshelflifeofproducts

(Rao&Labuza,2012)

C and K constants of GAB model are associated with the

monolayer and monolayer properties, respectively Ranges of

5.67≤C<∞ and 0.24<K≤1 indicate that GAB model properly

describeswateradsorptionisotherms(Lewicki,1997).Increasing

Kbydecreasingthedegreeofinulinpolymerizationcorroborates

thechangeoftheshapeofisothermalcurvefromTypeIItoType

III

Afterthe equilibrium of microparticles with water vapor in

sevenrelative moistures,theimagesofthesamplesweretaken

(Fig.6).Novisiblephasetransition(i.e.,liquefaction)wasobserved

ineachmatrix,eventhoughathighrelativemoistures.However,

theGR-FDmicroparticlesdisplayedcakingaftersubmittedto

rel-ativemoisturehigherthan32%,whilstHP-FDmicroparticleswere

stableatallconditionsofrelativemoisture

4 Conclusion

Themicroparticlesformedandpresentedinthispaperare

high-lightedasaconsequenceoftheirsingularproperties,whichhave

beennotenoughstudiedonscientificcommunity.Such

micropar-ticles are totally functional, because the bioactive compounds

encapsulatedandtheencapsulatingmatrixaretherapeutic

sub-stancestobeappliedinfood-relatedandpharmaceutical-related

products Considering the technical approach, after comparing

inulins with two degrees of polymerization (GR: DP≥10; HP:

DP≥23)for encapsulatingannattoseed oil,theconclusionwas

thatthepolymericmatrixinfluencedthemicroparticlesproperties

Whentheemulsionswerereconstituted,theemulsionstabilized

with HP-inulin showed stability against phase separation 24h

afterthereconstitution.Bothinulins couldentrapannatto seed

oil;therefore,themicroparticlespresentedthesamecapacityof

geranylgeraniolretention(77%).Furthermore,forwateractivities

higherthan0.3,waterisothermaladsorptionwaslowerwhenusing

HP-inulin.Images of HP-FDmicroparticles indicated theywere

morestable atallconditions ofrelative moisture(11%to90%)

Therefore, HP-inulinis inferred tobe a potentialencapsulating

agentofbioactivecompoundsextractedfromvegetalsources,as

annattoseeds

Acknowledgements

Authors are grateful to CNPq (470916/2012-5) and FAPESP (2012/10685-8; 2015/13299-0) for the financial support Eric KevenSilvathanksCNPq(140275/2014-2)forthePh.D.scholarship andFAPESP(2015/22226-6)forthepostdoctoralscholarship Gio-vaniL.ZabotthanksFAPESP(2014/15685-1)forthepostdoctoral scholarship.M.AngelaA.MeirelesthanksCNPq(302423/2015-0) fortheproductivitygrant.Inaddition,theauthorswouldthankthe accesstoequipmentandassistanceprovidedbytheNational Insti-tuteofScienceandTechnologyonPhotonicsAppliedtoCellBiology (INFABIC)attheUniversityofCampinas;INFABICisco-fundedby FAPESP(08/57906-3)andCNPq(573913/2008-0)

References

Albuquerque, C L C., & Meireles, M A A (2012) Defatting of annatto seeds using supercritical carbon dioxide as a pretreatment for the production of bixin: Experimental, modeling and economic evaluation of the process Journal of Supercritical Fluids, 66, 86–94.

Botrel, D A., de Barros Fernandes, R V., Borges, S V., & Yoshida, M I (2014).

Influence of wall matrix systems on the properties of spray-dried microparticles containing fish oil Food Research International, 62, 344–352.

Brunauer, S., Deming, L S., & Teller, E (1940) On a theory of Van der Waals adsorption of gases Journal of the American Chemists Society, 62, 1723–1732.

Carvalho, A G S., da Costa Machado, M T., da Silva, V M., Sartoratto, A., Rodrigues,

R A F., & Hubinger, M D (2016) Physical properties and morphology of spray dried microparticles containing anthocyanins of jussara (Euterpe edulis Martius) extract Powder Technology, 294, 421–428.

Chranioti, C., Chanioti, S., & Tzia, C (2016) Comparison of spray, freeze and oven drying as a means of reducing bitter aftertaste of steviol glycosides (derived from Stevia rebaudiana Bertoni plant) – Evaluation of the final products Food Chemistry, 190, 1151–1158.

Chuang, L., Panyoyai, N., Shanks, R., & Kasapis, S (2015) Effect of sodium chloride

on the glass transition of condensed starch systems Food Chemistry, 184, 65–71.

Fernandes, R V d B., Borges, S V., & Botrel, D A (2014) Gum arabic/starch/maltodextrin/inulin as wall materials on the microencapsulation

of rosemary essential oil Carbohydrate Polymers, 101, 524–532.

Fuchs, M., Turchiuli, C., Bohin, M., Cuvelier, M E., Ordonnaud, C., Peyrat-Maillard,

M N., et al (2006) Encapsulation of oil in powder using spray drying and fluidised bed agglomeration Journal of Food Engineering, 75(1), 27–35.

Gomes da Costa, J M., Silva, E K., Toledo Hijo, A A C., Azevedo, V M., & Borges, S.

V (2015) Physical and thermal stability of spray-dried Swiss cheese bioaroma powder Drying Technology, 33(3), 346–354.

Greenspan, L (1977) Humidity fixed points of binary saturated aqueous solutions Journal of Research of the National Bureau of Standards A Physics and Chemistry, 81a, 89–112.

Haque, M K., Kawai, K., & Suzuki, T (2006) Glass transition and enthalpy relaxation

of amorphous lactose glass Carbohydrate Research, 341(11), 1884–1889.

Kolida, S., & Gibson, G R (2007) Prebiotic capacity of inulin-type fructans The Journal of Nutrition, 137(11), 2503S–2506S.

Lewicki, P P (1997) The applicability of the GAB model to food water sorption isotherms International Journal of Food Science and Technology, 32(6), 553–557.

Marcuzzi, A., Zanin, V., Piscianz, E., Tricarico, P M., Vuch, J., Girardelli, M., et al (2012) Lovastatin-induced apoptosis is modulated by geranylgeraniol in a neuroblastoma cell line International Journal of Developmental Neuroscience, 30(6), 451–456.

Mazutti, M A., Zabot, G., Boni, G., Skovronski, A., de Oliveira, D., Di Luccio, M., et al (2010) Optimization of inulinase production by solid-state fermentation in a packed-bed bioreactor Journal of Chemical Technology & Biotechnology, 85(1), 109–114.

Mensink, M A., Frijlink, H W., van der Voort Maarschalk, K., & Hinrichs, W L J (2015) Inulin, a flexible oligosaccharide I: Review of its physicochemical characteristics Carbohydrate Polymers, 130, 405–419.

Moraes, M N., Zabot, G L., & Meireles, M A A (2015) Extraction of tocotrienols from annatto seeds by a pseudo continuously operated SFE process integrated with low-pressure solvent extraction for bixin production The Journal of Supercritical Fluids, 96, 262–271.

Oikonomopoulou, V P., Krokida, M K., & Karathanos, V T (2011) Structural properties of freeze-dried rice Journal of Food Engineering, 107(3–4), 326–333.

Pandey, A., Soccol, C R., Selvakumar, P., Soccol, V T., Krieger, N., & Fontana, J D (1999) Recent developments in microbial inulinases Its production, properties, and industrial applications Applied Biochemistry and Biotechnology, 81(1), 35–52.

Rao, Q., & Labuza, T P (2012) Effect of moisture content on selected physicochemical properties of two commercial hen egg white powders Food Chemistry, 132(1), 373–384.

Ronkart, S N., Paquot, M., Fougnies, C., Deroanne, C., & Blecker, C S (2009) Effect

of water uptake on amorphous inulin properties Food Hydrocolloids, 23(3),

Ruiz-Aceituno, L., García-Sarrió, M J., Alonso-Rodriguez, B., Ramos, L., & Sanz, M L.

(2016) Extraction of bioactive carbohydrates from artichoke (Cynara scolymus

L.) external bracts using microwave assisted extraction and pressurized liquid

extraction Food Chemistry, 196, 1156–1162.

Saengthongpinit, W., & Sajjaanantakul, T (2005) Influence of harvest time and

storage temperature on characteristics of inulin from Jerusalem artichoke

(Helianthus tuberosus L.) tubers Postharvest Biology and Technology, 37(1),

93–100.

Schaller-Povolny, L A., Smith, D E., & Labuza, T P (2000) Effect of water content

and molecular weight on the moisture isotherms and glass transition

properties of inulin International Journal of Food Properties, 3(2), 173–192.

Silva, E K., & Meireles, M A A (2015) Influence of the degree of inulin

polymerization on the ultrasound-assisted encapsulation of annatto seed oil.

Carbohydrate Polymers, 133, 578–586.

Silva, E K., Fernandes, R V d B., Borges, S V., Botrel, D A., & Queiroz, F (2014).

Water adsorption in rosemary essential oil microparticles: Kinetics,

thermodynamics and storage conditions Journal of Food Engineering, 140,

39–45.

Silva, E K., Azevedo, V M., Cunha, R L., Hubinger, M D., & Meireles, M A A (2016).

Ultrasound-assisted encapsulation of annatto seed oil: Whey protein isolate

versus modified starch Food Hydrocolloids, 56, 71–83.

Silva, E K., Borges, S V., da Costa, J M G., & Queiroz, F (2015) Thermodynamic

properties, kinetics and adsorption mechanisms of Swiss cheese bioaroma

powder Powder Technology, 272, 181–188.

Silva, E K., Gomes, M T M S., Hubinger, M D., Cunha, R L., & Meireles, M A A.

(2015) Ultrasound-assisted formation of annatto seed oil emulsions stabilized

by biopolymers Food Hydrocolloids, 47, 1–13.

Silva, E K., Zabot, G L., & Meireles, M A A (2015) Ultrasound-assisted encapsulation of annatto seed oil: Retention and release of a bioactive compound with functional activities Food Research International, 78, 159–168.

Silva, E K., Zabot, G L., Cazarin, C B B., Maróstica, M R., Jr., & Meireles, M A A (2016) Biopolymer-prebiotic carbohydrate blends and their effects on the retention of bioactive compounds and maintenance of antioxidant activity Carbohydrate Polymers, 144, 149–158.

Teac˘a, C.-A., Bodîrl˘au, R., & Spiridon, I (2013) Effect of cellulose reinforcement on the properties of organic acid modified starch microparticles/plasticized starch bio-composite films Carbohydrate Polymers, 93(1), 307–315.

Van den Berg, C (1984) Description of water activity of foods for engineering purposes by means of the GAB model of sorption In B M McKenna (Ed.), Engineering and food (Vol 1) (pp 311–321) New York: Elsevier Applied Science.

Zabot, G., Silva, E K., Azevedo, V M., & Meireles, M A A (2016) Replacing modified starch by inulin as prebiotic encapsulant matrix of lipophilic bioactive compounds Food Research International, 85, 26–35.

Zhou, P., Liu, D., Chen, X., Chen, Y., & Labuza, T P (2014) Stability of whey protein hydrolysate powders: Effects of relative humidity and temperature Food Chemistry, 150, 457–462.

Zou, L., & Akoh, C C (2015) Antioxidant activities of annatto and palm tocotrienol-rich fractions in fish oil and structured lipid-based infant formula emulsion Food Chemistry, 168, 504–511.