Assessment of the stability of catechin-enriched extracts obtained from Arbutus unedo L. fruits: Kinetic mathematical modeling of pH and temperature properties on powder and solution systems

ContentslistsavailableatScienceDirect

Industrial

Crops

and

Products

j ourna l h o m e pa g e : w w w . e l s e v i e r . c o m / l o c a t e / i n d c r o p

Assessment

of

the

stability

of

catechin-enriched

extracts

obtained

from

Arbutus

unedo

L.

fruits:

Kinetic

mathematical

modeling

of

pH

and

temperature

properties

on

powder

and

solution

systems

Bianca

R.

Albuquerque

a,b

_,

_M.A.

_Prieto

a,c

_,

_Lillian

_Barros

a,d

_,

_Isabel

_C.F.R.

_Ferreira

a,∗

a_{CentrodeInvestigac¸ãodeMontanha(CIMO),ESA,InstitutoPolitécnicodeBraganc¸a,CampusdeSantaApolónia,1172,5300-253Braganc¸a,Portugal}

b_{DepartamentodeAlimentos,UniversidadeTecnológicaFederaldoParaná–CampusdeMedianeira,AvenidaBrasil,4232CEP85884-000,CaixaPostal}

271,Medianeira,Brazil

c_{NutritionandBromatologyGroup,FacultyofFoodScienceandTechnology,UniversityofVigo,OurenseCampus,E32004Ourense,Spain}

d_{LaboratoryofSeparationandReactionEngineering–LaboratoryofCatalysisandMaterials(LSRE-LCM),PolytechnicInstituteofBraganc¸a,Campusde}

SantaApolónia,1134,5301-857Braganc¸a,Portugal

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received4October2016

Receivedinrevisedform9January2017

Accepted1February2017

Availableonline10February2017

Keywords:

ArbutusunedoL.fruits

Catechin-enrichedextract

Physicalandchemicalstability

Reactionkineticsmodeling

pHandtemperatureeffects

a

b

s

t

r

a

c

t

ArbutusunedoL.(strawberry-tree)fruitscouldbeconsideredanalternativesourceofflavan-3-ols,in particularcatechinanditsderivatives.Thesecompoundsareknownforavarietyofapplicationsin food(especiallyasfoodadditives),pharmaceuticalandcosmeticindustry.Therefore,catechin-enriched extracts(60%flavan-3-olsand22%ofcatechin)wereobtainedfromA.unedofruitsandfurthersubmitted tophysicalandchemicalstabilitystudies,consideringthemainaffectingvariables(time,temperature andpH):i)astabilitystudyoftheextractsduringtheobtainingandstorageprocedures(powder sys-tem);andii)astabilitystudyoftheextractsinsimulatedfoodenvironment(aqueoussolutionsystem). Themeasuredresponsesweretheflavan-3-olsandcatechincontents,determinedbyHighperformance liquidchromatographycoupletoadiodearraydetector(HPLC-DAD),andtheantioxidantactivityofthe extractsevaluatedbyhydrophilicassays.Mechanisticandphenomenologicalequationswereusedto describetheresponses,andtheoptimalconditionsforflavan-3-ols(includingcatechin)stabilityas pow-derextractduringamonthwerepH=5.4andT=−20◦_{C;whileitsstabilityinaqueoussolutionremained}

duringthe24hofapplicationatpH<4andT<30◦_{C.Theseresultsprovideusefulinformationfor:i)}

poten-tialindustrialuseofA.unedofruitsasalternativesourcesofflavan-3-ols;andii)shelf-lifecalculations andcatechinlosspredictionsatspecificconditionsoftemperatureandpH.Finally,theresultsobtained showedacertainagreementwithpreviousreportsofcatechinstabilitystudiesinpowderandsolution systems,butprovidinganewalternativesource:A.unedofruits.

©2017ElsevierB.V.Allrightsreserved.

1. Introduction

Arbutus unedo L. (common name strawberry-tree, Ericaceae

family)isanornamentalspeciesnativefromtheMediterranean

regionthatproducesanediblereddishsweetberrythatisagood

sourceofhealthpromotingcompoundssuchastocopherols,dietary

fiber,carbohydrates,proteins,sugarsandflavonoids.Itisknownin

thepharmacology,foodandfeedsectorsforitsantiseptic,diuretic,

laxativeandcardiovascular positiveeffects (Ziyyatet al.,2002).

∗ Correspondingauthor.

E-mailaddress:[email protected](I.C.F.R.Ferreira).

However,apartfromsometraditionallyusesaspreservativesin

compotes,winesandliqueurs,thefruitsareconsiderasflavorless

havingareducenumberofapplicationsduetoitsdecomposition

ratesafterripening(Tardíoetal.,2006).Mostofthefruitproduction

isdiscarded,becauseitonlyreachesapleasantflavorduringashort

periodoftime,besidesthelackofcommercialplantations(Celikel

etal.,2008;Malesetal.,2006;Ruiz-Rodríguezetal.,2011).

There-fore,valorizingandproducingadded-valuefunctionalcompounds

fromthisunderusedfruitcouldbemoderninnovationapproaches,

highlydesirablebytheindustrialsectorandresearchcommunity

(Alarcão-E-Silvaetal.,2001;SekerandToplu,2010).

Most of the beneficial health effects and bioactive

proper-tiesofthestrawberry-treefruitarelinkedtoitshighcontentin

http://dx.doi.org/10.1016/j.indcrop.2017.02.002

B.R.Albuquerqueetal./IndustrialCropsandProducts99(2017)150–162 151

flavonoids(Dimitrios,2006;Heimetal.,2002;Pietta,2000).This

fruitisanexcellentsourceofflavan-3-ols,suchascatechinand

procyanidins(Barrosetal.,2010;Guimarãesetal.,2014).

Flavan-3-ols,andamongthemthecatechincompound,havebeenused

worldwidenamelybythenutraceuticalsectortoenrich

pharma-ceutical/cosmetic products, dietary supplements and functional

foods(Frauenetal.,2002;Nagaoetal.,2009;Shakaretal.,2008;

Wangetal.,2000;Zaveri,2006).Theycanbeaddedasplantextracts,

takingadvantageofthesynergisticeffectsbetweencompounds,or

asindividualmolecules,afterpurification(GadkariandBalaraman,

2015).Themostpopularsourceofcatechinisgreentea

present-ing∼1.5mg/gdw(Ananingsihetal.,2013;Zaveri,2006).Cocoais

thefruitknownwiththehighestcontentofcatechin(∼3mg/gdw),

followedbyblackgrape(∼0.2mg/gdw),apricot(∼0.15mg/gdw),

whitegrape(∼0.09mg/gdw),andbeingalsopresentinalower

con-tentinotherfruitssuchasapple,pear,strawberry,peach,among

others(GadkariandBalaraman,2015;Tsanova-Savovaetal.,2005;

Vinsonet al.,1999;Wangetal.,2011;Weietal.,2011).In this

scenario,strawberry-treefruitscouldbeconsideredasalternative

sourcesofmainlycatechinandotherflavan-3-olcompoundswith

reportedvaluesof∼1.4mg/goffruitdryweight(Albuquerqueetal.,

n.d.).

Extractionisacrucialstepbeforetheisolationofcompounds

fromplantmaterials(Ghasemzadehetal.,2014;Helenoetal.,2016;

Leeetal.,2013;Pinelaetal.,2016).However,flavan-3-olsand

cate-chinaresusceptibletodegradationbymanyenvironmentalfactors

andthestabilityofthesecompoundsduringstorageand

applica-tionprocessesisanessentialpreliminarycriterionthatneedstobe

examinedcautiously,beingasimportantastheclassicextraction

(techniqueandconditions)optimizationapproaches.

Understand-ingflavan-3-olsandcatechin stabilityisessential toensurethe

qualityanddeliveryofthesebioactivecomponents.Despitethe

existentstudiesaboutthestabilityofflavan-3-olsandcatechin

dur-ingstorageaspowderorinaqueoussystems(Komatsuetal.,2014;

Lietal.,2012),theresultscannotbedirectlyextrapolated,being

highlydependentontheplantmatrixsource.Thus,thestability

patternsofflavan-3-olsandcatechinfromstrawberry-treefruits

needtobeassessed.Thestabilityreactionofbothsystemsshare

thecentralaffectingvariables,whichhavebeenidentifiedastime

(t),temperature(T)andpH(Komatsuetal.,2014;Zhuetal.,2002).

Nonetheless,otherfactorssuchasconcentration,moisturecontent,

oxygenconcentration,etc.,havealsobeenpointedoutasrelatively

important(Lietal.,2011).

Thekeyissueliesdownbehindthefactthatflavan-3-olsand

catechin undergokinetic degradationand epimerization during

thermal processing and that the pH of the surrounding

envi-ronment accelerates/decelerates the kinetic degradations rates

(Komatsu etal.,2014; Ruiz-Rodríguez etal.,2011).The

analyt-icalsolutionof sucha systemthrough mathematical modelsis

importantandnecessary,butnotexemptofcomplexityduetothe

heterogeneousresponsesofthevariablesinvolved.Achievinga

suc-cessfulmathematicalmodelsolutionwouldallowtocontrolmost

factorsthataffectthesystem,helpingtostandardizethekey

vari-ablesforproducingstableplant-basedextractsandtherefore,to

optimizethecompleteextractionprocess.

Therefore, the aims of this study were to: 1) valorize the

underusedstrawberry-treefruitsasalternativesources of

func-tionalcompounds,byobtainingcatechin-enrichedextracts(CEE);

2)studythestabilityoftheextractsaspowder andinaqueous

solution(indicative of its potentialperformance in food

matri-ces)systems,accordingwiththemainaffectingvariables(t,Tand

pH);3)developkineticmathematicalmodelsforthesimultaneous

descriptionofflavan-3-olsdegradationunderdifferentrangesofT

andpH;and4)achievethebestoperatingconditionstoincrease

thephysicalandchemicalstabilityoftheCEE.

2. Materialandmethods

2.1. Overviewofthemethodology

Fig.1 shows a diagram for thewholeprocess, including: 1)

obtainingtheCEEfromA.unedofruits;2)stabilitystudyoftheCEE

aspowdersystemsalongstorage;3)stabilitystudyoftheCEEin

aqueoussolutionsystems;and4)analysesperformedineachstage

consideringthemainaffectingconditions(t,TandpH).

2.2. Standardsandreagents

HPLC-grade acetonitrile was from Fisher Scientific (Lisbon,

Portugal). Catechin was from Extrasynthese (Genay, France).

2,2-Diphenyl-1-picrylhydrazyl (DPPH) was obtained from

Alfa Aesar (Ward Hill, MA, USA). Trolox

(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) and formic acid were

fromSigma(St.Louis,MO,USA).Alltheotherchemicalswereof

analyticalgradeandpurchasedfromcommonsources.Waterwas

treated in a Milli-Qwater purification system(TGI Pure Water

Systems,USA).

2.3. Obtainingthecatechin-enrichedextracts

2.3.1. Sourcematerial

Thefruitsof ArbutusunedoL. (strawberry-treeberries) from

EricaceaeweregatheredintheNaturalParkofMontesinho

terri-tory,Trás-os-Montes,NortheasternPortugal,inNovember2008.

The botanicalidentificationwasconfirmedby thebiologist,Dr.

AnaMariaCarvalho,ofPolytechnicInstituteofBraganc¸a

(Trás-os-Montes,Portugal)accordingwithapreviousreportoftheauthors

(Guimarãesetal.,2013).Thefruitswerelyophilized(FreeZone4.5,

Labconco,KansasCity,MO,USA)andstoredinthedeep-freezerat

−20◦_{Cforsubsequentanalyses.}

2.3.2. Extractionprocedureandpurification

Each sample (1g) was extracted by maceration with30mL

of ethanol:water 20:80(v/v) at80◦C at 150rpm during90min

(Albuquerqueetal.,n.d.).Theextractwasfilteredthrough

What-manno.4paper.Theobtainedextractswereevaporatedat40◦C

(rotaryevaporatorBüchiR-210)toremoveethanol.For

purifica-tion,theaqueousphase wasdepositedontoa C-18solid phase

column (Chromabond sorbent C18 ec, Macherey-Nagel, Duren,

Germany),previouslyactivatedwithethanolfollowedbywater;

sugars and more polar substances were removed by passing

through50–80mLofwaterandthepurifiedextractwasfurther

elutedwith20–40mLofethanol.Thepurifiedextractwas

evapo-ratedat40◦Ctoremoveethanol.

2.4. Stabilitystudiesofthecatechin-enrichedextracts

2.4.1. Stabilityoftheextractsaspowdersystems

Thepurifiedextract(5mg)wasdissolvedin5mLofdistilled

water(control;pH=3)anddifferentpHvalueswereadjustedto2,4,

5,6,7,8and9withsolutionsofhydrochloricacidorsodium

hydrox-ide.The extractswithdifferentpH,includingthecontrol, were

lyophilized,and stored inEppendorfs (5mgof each lyophilized

extract)attemperaturesof−20,5,25and45◦Cforaperiodof0,

10,20and30days.Intheendofeachstorageperiod,thesamples

werekeptat−80◦_{Cforsubsequentanalysis.Atotalof112}

individ-ualexperimentalpoints(4t×4T×7pH)wereusedtounderstand

thepatternsbehindthecompoundstabilityoftheCEEpowder.As

responses,ateachperiodtheCEEpowderwasanalyzedinterms

ofitscontentinflavan-3-ols(includingcatechin)andthe

Fig.1. Diagramdescribingtheprocessextractionoftheenrichedextractsandthetwostabilitytestsperformed:(1)stabilityoftheextractsaspowdersystemsaccording

withthemainaffectingconditionsinvolvedinitsobtainingandstorageprocedures;and(2)stabilityoftheextractsinaqueoussolutionsystemsconsideringitspotential

B.R.Albuquerqueetal./IndustrialCropsandProducts99(2017)150–162 153

activityandironreducingpower)oftheextract.Allindependent

measureswereobtainedintriplicate(n=3).

2.4.2. Stabilityoftheextractsinaqueoussolutionsystems

simulatingfoodmatrices

The purified extract (1g) was dissolved in distilled water

(30mL).Fortheassays,1.5mLofthissolutionwaspouredinamber

vialsandthepHwasadjustedwiththeadditionof0.5mLof

Britton-RobinsonbuffersolutionpH3,5,7and9.Thesampleswerestored

inabathat25,40,55,70and80◦Cfor1,3,5,12and24h.Inthe

endofeachstorageperiod,thesampleswerekeptat−80◦_Cfor

subsequentanalysis.Atotalof120individualexperimentalpoints

(6t×5T×4pH)wereusedtounderstandthepatternsbehindthe

compoundstabilityoftheCEEinaqueoussolution.Asresponses,at

eachperiodthecontentinflavan-3-olsandcatechinwereanalyzed.

Allindependentmeasureswereobtainedintriplicate(n=3).

2.5. HPLC-DADmonitoringofflavan-3-ols,includingcatechin,

contentwithinthestabilitystudies

Thesamplesobtainedinthestabilitystudies(powderand

solu-tionsystems)wereanalyzedusinga Shimadzu20AseriesUFLC

(ShimadzuCorporation,Kyoto,Japan)withaquaternarypumpand

adiode arraydetector(DAD)coupledtoaLCsolutionsoftware

data-processingstation.SeparationwasachievedusingaWaters

SpherisorbS3ODS-2C18,(3␮m,4.6mm×150mm)column

ther-mostattedat35◦Candformicacidinwater0.1%(A),acetonitrile

(B)wereusedassolvents. Theelutiongradient establishedwas

15%Bfor5min,15%Bto20%Bover5min,20–25%Bover10min,

25–35%Bover10min,35–50%Bfor10min,andre-equilibrationof

thecolumn(15min),usingaflowrateof0.5mL/min.Detectionwas

carriedoutintheDADusing280nmaspreferredwavelength.

Cate-chinwasidentifiedbycomparingitsUVspectraandretentiontimes

withcommercialstandard,andcatechinderivativeswereidentified

usingpreviouslypublisheddataonA.arbutusfruit(Guimarãesetal.,

2013).Thequantitativeanalysiswasperformedusinga

calibra-tioncurveofcatechin(y=66243x–343411;R2_{=0.999).Thekinetic}

resultswereexpressedinmgofcatechinpergofextractandmg

offlavan-3-ols(catechinandderivatives- procyanidins)pergof

extract.

2.6. Antioxidantactivityoftheextractswithinthestability

studiesevaluatedbyhydrophilicassays

2.6.1. DPPHfree-radicalscavengingactivity

Different solution concentrations were prepared and were

(30␮L) mixed with a methanolic solution (270␮L) containing

DPPHfree-radicals(6×10−5mol/L)ina96-wellplate.The

reac-tionmixturewaslefttostandfor60mininthedark.Afterthat,

thereductionofDPPHfree-radicalswasdeterminedby

measur-ingtheabsorbanceat515nmusinganELX800MicroplateReader

(Bio-TekInstruments,Inc,Winooski,VT,USA)(Pinelaetal.,2012).

Thenonlineardose-responseoftheasymptoticend-pointvaluesof

thesolutionswascalculatedbytheWeibullmodelaspreviously

described(Prietoetal.,2015a,b),usingthefollowingequation:

DPPH•(A) =Nexp



−(ln2)1−˛



₂

_v

mA N˛



˛



(1)

inwhichAisthedoseofantioxidant.TheparameterNisthe

start-ingvalueofDPPHfree-radicals(30nM).The␣shapeparameteris

relatedtothemaximumslopeoftheresponse.Theparametervm

correspondstotheaveragenumberofDPPHmoleculesreducedper

gramofCEE(nMDPPH/g),whichisavalueofmaximal

predictabil-ityand,therefore,wasusedasresponse.

2.6.2. Ironreducingpower

Theiron reducingpowerassayevaluates thecapacityof the

samplestoconvertpotassiumferricyanide(Fe3+_{)intopotassium}

ferrocyanide (Fe2+_{), whichreacts withferricchloride toforma}

ferric-ferrouscomplexthatismonitoredspectrophotometrically.

Thedifferentsolutionconcentrations (0.5mL)weremixed with

sodiumphosphatebuffer(200mM,pH6.6,0.5mL)andpotassium

ferricyanide(1%w/v,0.5mL).Themixturewasincubatedat50◦C

for 20min,and then trichloroaceticacid(10% w/v,0.5mL) was

added.Themixture(0.8mL) waspouredin48-wellplates,with

deionisedwater(0.8mL)andferricchloride(0.1%w/v,0.16mL),

andtheabsorbancewasmeasuredat690nmusingthemicroplate

reader described above(Pinelaet al.,2012).Thedose-response

resultsshoweda linear relationand thefollowing linear

equa-tionwithoutinterceptwasusedtocomputetheaveragenumber

ofreducedmolecules:

RP (A) =mA (2)

inwhichAisthedoseofantioxidant.Theslopeparameterm

cor-responds totheaveragenumber of moleculesthat arereduced

pergramofCEE(nMFe2+_{/g)andisusedtocomputethepotential}

antioxidantactivityoftheextracts.

2.7. Mathematicalmodelsfortheanalysisoftheresponsestability

Bothstabilitytestswerestudiedasfunctionoffunctional

equa-tionstypicallyappliedinsimilarprocesses.

2.7.1. Individualmodelfortheanalysisofthestabilityvariable

effects

2.7.1.1. Effectofthetimeonthestabilityresponse.Fortheteffect,a

typicalexponentialfunctionwasapplied:

e (t) =kexp (−rt) (3)

wherekrepresentsthestartingpointandristhedecaydegradation

rateofthereaction.

2.7.1.2. Effectofthetemperatureonthestabilityresponse. The

Arrhe-nius equation establishes that the rate constant of a chemical

reactionisafunctionoftheabsoluteTaccordingtothefollowing

relation: e (T) =Aexp



−Ea RT



(4)

Wherethepre-exponentialfactor Arepresentsthefrequencyof

collisionsamongreactingmolecules,Ea istheactivationenergy

(kJ)andRtheconstantofgases(8.31kJ/mol.K).Incontext,AandEa

canbeconsideredasfittingparameters.

2.7.1.3. EffectofthepHonthestabilityresponse. Incontrastwith

theeffects ofthepreviousvariables, thepHhasmany possible

behaviorsandthereisnotanestablishedsingleequationthatcould

describeitseffects.Therefore,authorsareforcetotestseveral

phe-nomenologicalmodelstobeabletoreproduceitsconduct.Among

those typicalresponsesarethebelland theexponentialprofile

solution:

1)ThecharacteristicbellprofilesolutionforthepHeffectcanbe

describedbymanybellfunctions(DiMarcoandBombi,2001;Prieto

andMurado,2014).Afteranextensiveevaluation,inwhich

sev-eralequationsweretested,thegeneralizedexponentialfunction

function)wasfoundtobethemostsatisfactory,withleastnumber

ofparametersandhighestaccuracy:

e (pH) =km



i d



1−



pH pHopt

d +ln



pH pHopt

d



(5)

inwhichtheparameterdisrelatedtothedistancebetweenthetails

ofthefunction,iavaluerelatedtotheasymmetryofthebell

pro-file,kmthemaximumresponseeffectandpHopt thepHatwhich

kmtakesplace.Thismodelexplicitlyprovidesthecharacterizing

parameters(kmandpHopt)oftheresponse,andthereforetheir

sta-tisticalsignificancecanbetestedthroughthedeterminationofits

confidenceintervals.

2)AnothersolutionforthedescriptionofthepHeffectisthe

exponentialfunction,similartotheoneusedfortimeeffect,thatis

usuallyfoundinmanybiologicalsystemresponses(Komatsuetal.,

2014;Prietoetal.,2012b)andcanbeexpressedasfollows:

e (pH) =sexp (−bpH) (6)

wheresrepresentsthestartingpointandbisthedegradationrate

ofthereaction.

2.7.2. Multivariableanalysis

Thedevelopmentof atheoreticalmodelis greatlyfacilitated

bythepossibilityofcombiningallexperimentaldataintoa

sin-glemastercurvethatisabletoaccountfortheimportantvariables

simultaneously(Prietoetal.,2012a,b).Suchasolutionallowsto

controlmostfactorsthataffectthesystem,helpingtostandardize

thekeyvariablesforproducingreproducibleprotocolsand

there-fore,toobtainreproducibleresults.Despitetheexistenceofvery

rigorousresultsregardingthekineticsandthefactorsaffectingthe

kineticcurveofspecificmoleculesasfunctionofvariablessuchas

Tand pH(PrietoandVázquez, 2014;Prietoetal.,2015a;Rosso etal.,1995;Tijskensetal.,1997),simultaneoussolutionsarenot

performedalways,andbyapplyingthem,wecanprovideamore

extensiveknowledgetounderstandpartiallythegoverning

mech-anisms.

Tobeabletodevelopamultivariableanalysisofthesethree

variables,thelogicalapproachistoinserttheequationsthattake

controlofpHandTintoEq.(3)thatgovernsthetimevariableby

modifyingitsparameters,thestartingvalue(k)andthedegradation

rate(r).Evenifthethreevariablesarefullyindependent,anyevent

thatmayoccurinthesurroundingenvironmentofareactionalways

mustbereferredtothetimevariable.Therefore,aglobalpossible

descriptionofthestabilityatthemolecularlevelcouldbedescribed

bythefollowingapproach:

e(t,pH,T)=k•exp(−r•

t) where

k•(pH,T) =k×e (T) ×e (pH)

r•

(pH,T) =r×e (T) ×e (pH) (7)

wherek•representsthestartingpointandr•isthedegradationrate

ofthereactioncausedbytheeffectoftimeasdescribedEq.(3)but

modifiedbythegoverningequationsoftheeffectofT(Eq.(4))and

pH(Eqs.(5)or(6)).

2.8. Numericalmethodsandstatisticalanalysis

Allfittingprocedures,coefficientestimatesandstatistical

cal-culationswereperformedonaMicrosoftExcelspreadsheet.Fitting

andstatisticalanalysisoftheexperimentalresultstotheproposed

equationswerecarriedoutinfourphases:

-Coefficientsdetermination. Parametric estimateswereobtained

byminimization ofthesumof quadratic differencesbetween

observedandmodel-predictedvalues,usingthenonlinear

least-square(quasi-Newton)methodprovidedbythemacroSolverin

MicrosoftExcel2003(KemmerandKeller,2010),whichallows

quicktestingofahypothesesanditsconsequences.

-Coefficientssignificance.Thedeterminationoftheparametric

con-fidence intervals wascalculated using the‘SolverAid’ (Prikler,

2009).Themodelwassimplifiedbydroppingterms,whichwere

notstatisticallysignificantp-value(p)>0.05.

-Modelconsistency.TheFisherFtest(␣=0.05)testwasusedto

determinewhethertheconstructedmodelswereadequateto

describetheobserveddata(ShiandTsai,2002).

-Otherstatisticalassessmentcriteria.Tore-checktheuniformityof

themodelthefollowingcriteriawereapplied:a)The‘SolverStat’

macro(Comuzzietal.,2003),whichisusedfortheassessment

ofparameterandmodelpredictionuncertainties;b)R2_is

inter-preted as the proportion of the variability of the dependent

variableexplainedbythemodel;c)Adjustedcoefficientsof

mul-tipledetermination(R2

adj),whichisacorrectiontoR2takinginto

accountthenumberofvariablesusedinthemodel.

3. Resultsanddiscussion

3.1. Productionofacatechin-enrichedextractfrom

strawberry-treefruits

Thecatechin-enrichedextracts(CEE)obtainedfromA.unedo

fruitspresented60%offlavan-3-olsand22%ofcatechin(∼425and

∼150mgper100gdw,respectively)(Albuquerqueetal.,n.d.).

Com-poundswereidentifiedbycomparingitsUVspectraandretention

timewithacommercialstandardanditsderivativeswereidentified

usingapreviousreportoftheauthors(Guimarãesetal.,2013).The

flavan-3-olsvaluesareovertwentytimeshigherthanthosefound

inothertypicalplantsfromtheNortheasternPortugalsuchasdog

rose(RosacaninaL.,3.5mg/100gdw)andwildrose(R.micrantha

BorrerexSm.,2.9mg/100gdw)(Guimarãesetal.,2013),butmuch

lowerthanthosefoundincacao(3–5×103_{mg/100gdw),whichis}

consideredthemajorsource(Guetal.,2006;Vinsonetal.,1999).

Strawberry-treeberries(A.unedo) presentedsimilarcontentsof

catechintothosefoundingreentea(∼150mg/100gdw)andlower

thanthevaluesfoundincacao(∼300mg/100gdw)(Zaveri,2006),

whicharethemainindustrialsourcesofcatechin.Thegood

extrac-tionefficienciesofcatechin andderivativesreachedinthefruit

materialofA.unedoincomparisonwithothersourcesemphasizes

theneedtoperformmoredetailedevaluationsofthestabilityof

thecompoundsobtained.

Non-controllablevariablessuchassoilcultivation,sun

expo-sition, harvest time, etc., and controllable variables such as

theextraction conditions (time,solvent, temperature,etc.) and

techniques(ultrasound,maceration,microwave,etc.),mayaffect

compoundsconcentrationandcouldincrease/decreasetheyield

efficiencies.Thelatterparametershavebeenpreviouslyoptimized

bytheauthors(Albuquerqueetal.,n.d.)andthebestextraction

conditions(macerationfor90min,80◦C,20%ofethanoland30g/L)

wereusedinordertoobtaintheCEEforthestabilitystudies.

As reported previously, the stability of flavan-3-olsmay be

influencedby factorsincludingt(Komatsuetal.,2014), pH(Su

etal.,2003),T(Demeuleetal.,2002),oxygenlevel(Labbéetal.,

2008)andconcentrationofothercompoundssuchasantioxidants

level,metalionsandothercompositionalingredients(Zhuetal.,

1997).Although,beingalloftheseparametersrelevant,mostof

theauthorsagreethattheessentialonesaret,pHandT(Komatsu

etal.,2014;Lietal.,2012,2011),therefore,inthefollowingsections

thestabilityofflavan-3-olswillbemonitoredinfunctionoft,pH

B.R. Albuquerque et al. / Industrial Crops and Products 99 (2017) 150–162 155 Table1

Resultsoftheparametricandconfidenceintervalsoftheflavan-3-ols,catechinandantioxidantpropertiesfittedtothetimedependentmodelpresentedinEq.(3)atdifferentpHandTconditionswhenevaluatingthestability duringtheprocessforobtainingtheenrichedextract.

pH T FLAVAN-3-OLS Catechin DPPHSCAV.Activity IronReducingPower

k(t)(mg/g CEE) r(t)(day−1_{) R}2 _k(t)(mg/g CEE) r(t)(day−1_{) R}2 _k(t)(nM DPPH/g CEE) r(t)(nM DPPH/g CEE/day) R2 _k(t)(nM Fe2+_/gCEE) r(t)(nM Fe2+_/g CEE/day) R2 2.0 −20.0 25.43±2.37 0.0236±0.002 0.9311 9.58±0.76 0.0198±0.002 0.8897 87.8±8.8 0.019±0.002 0.9994 4.12±0.41 0.047±0.005 0.9944 2.0 5.0 25.12±2.46 0.0354±0.003 0.9790 9.58±0.81 0.0293±0.002 0.8485 90.0±8.0 0.038±0.003 0.8895 4.09±0.40 0.050±0.005 0.9662 2.0 25.0 25.31±2.48 0.0464±0.005 0.9794 9.58±0.78 0.0315±0.003 0.8097 87.4±8.4 0.044±0.004 0.9667 4.08±0.40 0.055±0.005 0.9796 2.0 45.0 23.50±2.16 0.0510±0.005 0.9177 9.58±0.83 0.0331±0.003 0.8630 87.8±8.1 0.063±0.006 0.9228 4.04±0.40 0.069±0.007 0.9836 4.0 −20.0 41.34±4.12 0.0265±0.003 0.9962 19.32±1.06 0.0083±0.000 0.8475 110.9±11.0 0.030±0.003 0.9933 4.82±0.48 0.044±0.004 0.9997 4.0 5.0 41.61±4.15 0.0304±0.003 0.9980 19.32±0.55 0.0074±0.000 0.8828 111.2±11.1 0.040±0.004 0.9940 4.81±0.48 0.050±0.005 0.9999 4.0 25.0 42.67±4.16 0.0325±0.003 0.9743 19.32±1.47 0.0177±0.001 0.8613 109.7±10.7 0.035±0.003 0.9789 4.74±0.47 0.059±0.006 0.9901 4.0 45.0 42.37±4.17 0.0354±0.003 0.9852 19.32±1.50 0.0132±0.001 0.8744 111.3±10.0 0.057±0.005 0.9001 4.80±0.48 0.085±0.008 0.9997 5.0 −20.0 58.51±5.79 0.0316±0.003 0.9896 30.25±2.56 0.0223±0.002 0.8464 122.5±12.0 0.044±0.004 0.9786 4.92±0.49 0.042±0.004 0.9942 5.0 5.0 59.29±5.92 0.0350±0.003 0.9991 30.25±2.79 0.0227±0.002 0.9236 123.5±11.9 0.031±0.003 0.9656 4.74±0.47 0.041±0.004 0.9986 5.0 25.0 58.23±5.72 0.0391±0.004 0.9820 30.25±2.46 0.0228±0.002 0.8947 119.2±11.9 0.054±0.005 0.9981 5.10±0.51 0.058±0.006 0.9996 5.0 45.0 58.97±5.88 0.0414±0.004 0.9966 30.25±2.53 0.0319±0.003 0.9362 118.8±11.8 0.094±0.009 0.9938 4.69±0.47 0.072±0.007 0.9982 6.0 −20.0 80.48±8.02 0.0377±0.004 0.9966 23.88±1.40 0.0151±0.001 0.8871 134.8±12.9 0.036±0.003 0.9554 5.54±0.55 0.040±0.004 0.9851 6.0 5.0 80.70±8.06 0.0409±0.004 0.9990 23.88±2.28 0.0146±0.001 0.9562 139.7±13.9 0.046±0.005 0.9945 5.55±0.55 0.047±0.005 0.9922 6.0 25.0 80.66±8.06 0.0442±0.004 0.9989 23.88±2.07 0.0193±0.002 0.8649 137.9±13.8 0.041±0.004 0.9992 5.56±0.55 0.054±0.005 0.9933 6.0 45.0 78.59±7.57 0.0509±0.005 0.9634 23.88±2.21 0.0217±0.002 0.9267 138.1±13.8 0.051±0.005 0.9986 5.55±0.55 0.061±0.006 0.9914 7.0 −20.0 78.79±7.83 0.0363±0.004 0.9939 24.82±1.81 0.0207±0.002 0.9275 189.1±18.4 0.042±0.004 0.9753 5.55±0.55 0.034±0.003 0.9958 7.0 5.0 79.16±7.91 0.0373±0.004 0.9996 24.82±1.74 0.0218±0.002 0.9015 191.6±17.9 0.038±0.004 0.9351 5.58±0.55 0.034±0.003 0.9871 7.0 25.0 79.26±7.92 0.0408±0.004 0.9998 24.82±1.80 0.0252±0.002 0.9250 191.6±18.8 0.047±0.005 0.9817 5.72±0.54 0.040±0.004 0.9475 7.0 45.0 77.22±7.56 0.0456±0.004 0.9785 24.82±2.21 0.0305±0.003 0.8921 194.4±18.7 0.068±0.007 0.9611 5.62±0.55 0.059±0.006 0.9720 8.0 −20.0 55.29±5.51 0.0282±0.003 0.9971 17.58±1.50 0.0149±0.001 0.8549 130.5±12.8 0.039±0.004 0.9803 4.62±0.46 0.042±0.004 0.9931 8.0 5.0 54.21±5.39 0.0328±0.003 0.9938 17.58±1.68 0.0174±0.002 0.9539 130.8±12.9 0.041±0.004 0.9845 4.74±0.45 0.038±0.004 0.9524 8.0 25.0 55.81±5.52 0.0347±0.003 0.9898 17.58±1.50 0.0148±0.001 0.8531 132.1±12.8 0.045±0.004 0.9682 4.47±0.43 0.050±0.005 0.9718 8.0 45.0 55.87±5.53 0.0372±0.004 0.9890 17.58±1.51 0.0164±0.001 0.8566 133.2±12.6 0.049±0.005 0.9473 4.67±0.46 0.052±0.005 0.9788 9.0 −20.0 27.60±2.72 0.0275±0.003 0.9844 15.78±1.42 0.0149±0.001 0.8986 99.7±10.0 0.039±0.004 1.0000 3.56±0.34 0.046±0.004 0.9682 9.0 5.0 27.59±2.73 0.0311±0.003 0.9882 15.78±1.41 0.0190±0.002 0.8947 100.4±10.0 0.039±0.004 0.9988 3.57±0.35 0.043±0.004 0.9931 9.0 25.0 27.61±2.73 0.0351±0.003 0.9893 15.78±0.96 0.0152±0.001 0.9113 100.1±10.0 0.044±0.004 0.9994 3.57±0.36 0.046±0.005 0.9975 9.0 45.0 27.64±2.74 0.0381±0.004 0.9907 15.78±1.38 0.0204±0.002 0.8739 100.2±9.8 0.040±0.004 0.9793 3.53±0.35 0.051±0.005 0.9773

3.2. Stabilityoftheextractsaspowdersystems

TheCEEpowderpreviouslyobtainedwasusedtoinvestigate

theeffectsoft(0–30days),pH(2–9)andT(−20to45◦C)onthe

stabilityofthemajorcompounds(flavan-3-olsandcatechin)and

ontheantioxidantactivity(measuredbyDPPHscavenging

activ-ity (DPPH) and iron reducing power (RP) assays).For all these

responses,theeffectoftwasdescribed individuallyby Eq.(3);

parametricresponsesandcorrelationcoefficientsarepresentedin

Table1.Then,themultivariableanalysesweretestedasdescribed

inEq.(7),byinsertingEqs.(4)and(5) intoEq.(3),substituting

bothtimedependentparametersofEq.(3)(kandr).Aftertesting

themultivariableapproachforallresponses(flavan-3-ols,catechin,

DPPHandRP),inallcasesthepHeffecthadaninfluenceonthe

asymptoticstartingkparameterandtheTeffecthadanexclusively

effectontherparameter.Therefore,aglobalmultivariablemodel

thatpredictstheeffectoft,pHandTonthestabilityofcompounds

contentoftheCEEonpowdersystemwasdescribedasfollows:

e (t,pH,T) =km



i d



1−



pH pHopt



d +ln



pH pHopt



d



exp



−Aexp



−Ea RT



t



(8)

AllparametricnotationsweredefinedintheMaterialand

meth-odssection.Theparametricsolutionsandcorrelationcoefficients

ofthemultivariableeffectsdescribed byEq.(8)forthestability

analysisof compounds content (flavan-3-ols and catechin) and

theantioxidantactivity (DPPHand RPassays)are presented in

Table2andFig.2,Fig.A1(supplementalmaterial),Fig.3andFig.

A2(Supplementalmaterial).Subsequent,inamoredetailmanner

theresultsoftheseanalysesweredescribedforalltheresponses.

3.2.1. Monitoringthestabilityofflavan-3-olsandcatechin

compoundsbyHPLC-DAD

ThegraphicalrepresentationoftheCEEpowderstabilityresults

oftheflavan-3-olsandcatechincontentasfunctionofthet,Tand

pHarepresentedinFig.2andFigureA1.Eachofthesefiguresis

dividedintotwosections(AandB).

SectionAshowstheindividualtime-dependentanalysis(during

30days)ofthedegradationeffectsof theflavan-3-olsand

cate-chincompoundsforeachofthepHstested(2,4,5,6,7,8and9).

Pointsaretheexperimentaldata(䊉25◦_C,5◦_{C, 25}◦_C,♦45◦_C)

andlines(—)themodeledresultsusingEq.(3).Theeffects

pro-ducedbythetandpHvariableshadagreatereffectonthestability

offlavan-3-olsandcatechincompoundsthantheTvariable.The

testedrangeofTwaslargeenoughbutwashighlyinfluencedbythe

pH.OnlybetweenpHs5and7thedegradationratescausedbytheT

effect(rparameter,Table1)showedawelldifferentiatedresponse.

AlthoughthepHsbetween5and7showedthehigherdegradation

ratesatanyT;theextractionperformanceandstabilityshowed

highercompoundscontentthananyotherpHasitcanbe

evalu-atedbytheparametervaluek(Error!Referencesourcenotfound.)

ofEq.(3).Themaximumstartingcontentobtainedwas80.70and

30.25mg/gCEEforflavan-3-olsandcatechin,respectively.The

sta-tisticalinformationofthefittingsperformedisillustratedusingtwo

basiccriteria,thebasicR2_{coefficientandtheparametricconfidence}

intervals.Inallcases,parameterswerehighlysignificant(␣=0.05)

andR2_{valueswerepredominantlyhigherthan0.95indicatingthe}

reliabilityoftheanalysisperformedwithEq.(3).Therefore,tofind

acompromiserangeofvaluesthatwillallowustodeterminethe

exactvariablerangethatwouldstabilizetheCEEpowder,the

mul-tivariableanalysismodelofEq.(8)wasappliedandpresentedin

sectionBofFig.2andFig.A1.

SectionBshows,forbothfigures(flavan-3-olsandcatechin

con-tent),theglobalmultivariablefittingprocedure(applyingEq.(8)

withparametricresultsinTable2)dividedinthreemain

subsec-tions:

B1:ShowsthepatternsofthekineticparameterskandrofEq.

(3)(pointsareindividualresultsandlinesmultivariableresponse)

asafunctionoftheirrespectiveaffectingvariables(pHaffecting

theparameterKandtheTaffectingtheparameterr).Inbothcases,

thepHresponseshowsabellprofilepatternwithaclearpeakat

6.7fortheflavan-3-olscontentand5.4forthecatechincontent.In

thecaseoftheTvariable,bothcasesshowedanincreasinglinear

profilewithanexpectedidenticalEavalues(2.87kJ)but

dissimi-larcollisionfrequency(0.13and0.08forflavan-3-olsandcatechin

content,respectively).

B2:Showsanillustrationofthe3Dresponseofthecombined

effectofthetimewiththeothertwovariables(pHandT)atthe

bestexperimentalconditionsfoundforstabilizingthecontentof

flavan-3-ols(T=−20◦_{CandpH=6.7)andcatechin(T=−20}◦_Cand

pH=5.4)oftheCEEinthepowdersystem.

B3:Showsthecorrelationbetweentheexperimentalvaluesand

thepredictedonesobtained withthemultivariablemodel

pre-sentedinEq.(8)forthepredictionofthecontentstabilityinCEE

powderofflavan-3-ols(R2_{=0.9741)andcatechin(R}2_=0.8741).

Thesevaluesareinagreementwiththosereportedpreviously.

Asanexample,Zhuetal.(2002)showedthatthebestshelflife

conditionsobtainedfor allflavan-3-olsfrom cocoa fruitwasat

apHvaluesbetween2and4independentlyofanystorage

tem-peraturebetween4and45◦C,andconcludedthatthestabilityis

fundamentallypH-dependent.

3.2.2. MonitoringtheremainingantioxidantactivityofCEE

compoundsbyhydrophilicassays

Thegraphicalrepresentationofthepowderstabilityresultsof

theDPPHscavengingactivityandironreducingpowerasfunction

ofthet,TandpHareshowninFig.A2andFig.3.Eachofthese

figuresisdividedintothesametwosections(AandB)and

ana-lyzedasthosedescribedinthepreviousheading.Theindividual

analysisofthetimeremainingactivitymeasuredinbothmethods

(SectionAofFig.A2andFig.3)showsastrongereffectofthepH

andtvariablethantheT,butwithaTeffectmorenotablethan

theonefoundin theanalysisofthestability ofthemain

com-poundsoftheCEE.ParametervaluesofEq.(3)ofallantioxidant

analysesaredisplayedin Table1. Thestatisticalinformation of

thefittingsshowedR2_{>0.95in almostallcasesandparametric}

confidenceintervalshighlysignificant(␣=0.05).Themultivariable

analysisbyEq.(8)isgraphicallyillustratedinsectionBofFig.A2

andFig.3,andtheparametricresultsarepresentedinTable2.The

patternsofthekineticparameterkshowabellprofileasafunction

ofpHwithaclearpeakat7.1and6.7forDPPHandRPmethods.

Inthecaseofthetemperature-dependentparameterr,theDPPH

scavengingactivityandironreducingpowershowedanincreasing

linearprofilewithsimilarEavalues(4.78and4.42kJ,respectively)

andcollisionfrequencyvalues(0.32and0.31,respectively).

There-fore,atlowertemperaturesandpHsrangingbetween6and7,the

timedegradationeffectsarereduced.Sucharelationisillustrated

bythe3Dresponseofthecombinedeffectofthetimewiththe

othertwovariables(pHandT)atthebeststabilizing

experimen-talconditionsfoundfortheantioxidantcompoundscontentinCEE

powder.Finally,subsectionB3showsthehighcorrelationvalues

betweentheexperimentalvalues andthepredictedonesbyEq.

(8),indicatingonceagaintheagreementofthedevelopedsolution.

3.3. Stabilityoftheextractsinaqueoussolutionsystems

simulatingfoodmatrices

Theinformationprovidedbytheantioxidantactivityanalysis

inthepowdersystemcannotbereplicatedintheaqueoussystem

B.R.Albuquerqueetal./IndustrialCropsandProducts99(2017)150–162 157

Fig.2.Powderstoragestabilityoftheflavan-3-olsasfunctionofthet,TandpH.SectionAshowstheindividualkineticanalysisofthedegradationeffectsoftheflavan-3-ols.

Pointsaretheexperimentaldata(䊉−25◦_C,5◦_C,25◦_C,♦45◦_{C)andlines(—)themodeledresultsusingEq.(3)(parametricresponsesandcorrelationcoefficientsin}

Table1).SectionBshowstheglobalmultivariable(trivariate)fittingprocedure:B1showstheparametricpatternsofthekineticparameterskandrofEq[3](pointsare

univariateresultsandlinesmultivariableresponse)asafunctionoftheirrespectiveaffectingvariables(pHaffectingtheparameterKandtheTaffectingther);B2showsan

illustrationofthe3Dresponseofthecombinedeffectofthethreevariablesatthebestexperimentalconditionsfound(T=−20◦_{CandpH=6.7);andB3showsthecorrelation}

Fig.3. Powderstoragestabilityoftheironreducingpower(RP)asfunctionofthet,TandpH.SectionAshowstheindividualkineticanalysisofthedegradationeffectsof

theantioxidantcompounds.Pointsaretheexperimentaldata(䊉−25◦_C,5◦_C,25◦_C,♦45◦_{C)andlines(—)themodeledresultsusingEq.(3)(parametricresponsesand}

correlationcoefficientsinTable1).SectionBshowstheglobalmultivariable(trivariate)fittingprocedure:B1showstheparametricpatternsofthekineticparameterskandr

ofEq[3](pointsareunivariateresultsandlinesmultivariableresponse)asafunctionoftheirrespectiveaffectingvariables(pHaffectingtheparameterKandtheTaffecting

ther);B2showsanillustrationofthe3Dresponseofthecombinedeffectofthethreevariablesatthebestexperimentalconditionsfound(T=−20◦_{CandpH=6.7);andB3}

B.R.Albuquerqueetal./IndustrialCropsandProducts99(2017)150–162 159

Table2

Resultsoftheparametricandconfidenceintervalsoftheflavan-3-ols,catechinandantioxidantpropertiesfittedtothemultivariable(t,TandpH)modelpresentedinEq.(7)

whenevaluatingthestabilityduringtheprocessforobtainingtheenrichedextract.

FLAVAN-3-OLS Catechin DPPHSCAV.Activity IronReducingPower

ParameterscontrollingtheeffectofthepHofthekineticparameterk(t)

Rmax(pH) (responseunits) 76.73±2.18 30.21±1.51 154.84±5.17 5.46±0.08

i(pH) 7.76±1.17 1.49±0.98 11.11±3.76 9.70±1.67

pHopt(pH) (pHunits) 6.70±0.14 5.43±0.26 7.16±0.31 6.73±0.19

d(pH) 2.21±0.15 3.87±1.66 1.55±0.09 1.33±0.04

ParameterscontrollingtheeffectsoftheTofthekineticparameterr(t)

A(T) (collisionfreq) 0.13±0.02 0.08±0.01 0.32±0.04 0.31±0.08

Ea(T) (kJ) 2.87±0.32 2.87±0.80 4.78±0.72 4.42±0.62

Statisticalinformationofthemultivariableanalysis(trivariate)

Obs 120 120 112 112

df 113 113 105 105

R2 _{0.9741 0.8792 0.8835 0.9578}

R2

adj 0.9318 0.8533 0.8648 0.9324

powdersystem,thetwotimeframeswerewidelyseparated.We

weremeasuringcompoundsdecayover30daysperiodandthe2h

periodofprocessingtheantioxidantresponsewasnotaninfluence

ontheoutputoftheresults.Incontrast,the24hperiodanalyzed

intheaqueoussystem,notonlyshowedextremelyhigh

degrada-tionrates comparedwiththepowdersystem,but alsothetwo

timeframesoverlaponetoeachothermakingtheanalysis

fruit-less.Althoughauthorsexpectedtobeabletocombinebothtime

framesinordertoovercomeanalyticallythissituation,theresults

obtainedshowedacomplexityhigherthanexpectedandwere

dis-cardedforfurtheranalysis.Therefore,theCEEpowder obtained

wasusedtoinvestigatetheeffectsoft,pHandTonthestability

ofcompoundscontent(flavan-3-olsandcatechin)inaqueous

solu-tionsystemsimulatingafoodenvironment.Foralltheseresponses,

thestabilityofthecompoundswasmonitoredbyHPLC-DADat

dif-ferenttimesequences(1,3,5,12and24h)andtheantioxidant

activitywasdiscarded.

Theindividualtime-dependentgraphicalanalysisofthe

aque-ous solution stability results of the flavan-3-ols and catechin

contentasfunctionoftheTandpHarepresentedinSectionAof

FigureA3(supplementalmaterial)andFig.4,respectively.Each

graphshowsthetimedegradationeffects(0–24h)ofeachTtested

(25,40,55,70and80◦C).Pointsaretheexperimentaldataofthe

differentpHs tested(䊉3, 5, 7and ♦9) andlines (—)the

resultspredictedbyEq.(3).Theparametricresultsandcorrelation

coefficientsarepresentedinTable3.Differentlythantheprevious

stabilitystudy,thecompoundscontent(flavan-3-olsandcatechin)

isidenticalinallcases,thustheparameterkofEq.(3)wouldnot

changeasfunctionofanyvariableandwillremainconstant(300␮g

flavan-3-ols/mLCEEand150␮gcatechin/mLCEE).Tobeableto

describethoseeffectsinamultivariateform,thedegradationrate

parameter(r)takesinallperturbationeffectscausedbytheTand

pHvariables.TheincreaseinTandpHunitscausesanexponential

increaseonthedegradationrateofCEEcompoundscontent.The

TeffectbehavesfollowingtheArrheniusconceptdefinition

pre-sentedinEq.(4).However,thepHeffectscannotbedescribedas

previouslywithabellprofilefunction,becauseasstatedbefore,

thegoverningmechanismsoftheeffectofpHonbasicchemical

reactionsorcomplexlivingorganismsaredissimilarand

hetero-geneous.FortheparticularcaseofthestabilityofCEEcompounds

inaqueoussystem,thepHeffectfollowsanexponentialrelationas

describedbyEq.(6).Therefore,aglobalmultivariablemodelthat

controlstheeffectoft,pHandTonthestabilityinaqueoussystem

canbedevelopedbysubstitutingtherparameterofEq.(3)with

theequationsgoverningtheT(Eq.(4))andthepHeffect(Eq.(6)),

asfollows: e (t,pH,T) =kexp



−pexp



−Ea RT+bpH



t



(9)

Whensubstitutingtherparameterbythemultiplicativeresult

of Eqs. (4) and (6), the new resulting expression share a

pre-exponentialfactor(Aands)andtheiruseinconjunctionwillbe

redundanttherefore,anewfactoritsdescribedandnotedasp.All

otherparametricnotationsasdefinedintheMaterialand

meth-ods section.SectionBof FigureA3 and Fig.4.showthe global

multivariablefittingresultsofapplyingEq.(9)todescribethefull

multivariabledata.Theparametric resultswerefor flavan-3-ols

(k=300.0±6.1;p=40.21×10−6;Ea=19.37;b=67.31)andfor

cat-echin(k=150.0±8.5;p=40.21×10−6;Ea=21.93;b=64.09).Inall

casesthestatisticaldescriptionwassignificantandthepredictionof

theCEEcompoundscontentstabilityinsolutionbyEq.(9)showed

highlyconsistentR2_{valuesforofflavan-3-olsand(R}2_=0.9629)and

catechin(R2_{=0.9585).SubsectionB1showstheparametricnet}

sur-facepatternofthekineticrasafunctionoftheirrespectiveaffecting

variables (pH and T). The conclusions arein concordancewith

previousresultsinpowdersystemsanddatapreviouslyreported

(Komatsuetal.,2014;Lietal.,2012,2011),findingthatatT<20◦C

andpH<4.5theCEEismorestableloweringasmuchaspossible

thedegradationrateofflavan-3-olsandcatechin.

Asinthepowdersystem,flavan-3-olsandcatechinofthe

straw-berrytreeinaqueoussolutionsystemprovedtobefundamentally

pH-dependent.AtpHvalueshigherthan7theflavan-3-olsand

cat-echincompoundsdecaycompletelyinafewminutesindependent

ofT.Thisphenomenonwasalsodetectedbyothersauthorsthat

studiedthestabilityofcatechinandderivativesfromothers

matri-cessuchasgreenteaandcacao(Komatsuetal.,2014;Lietal.,

2012;Zhuetal.,2002,1997).Insimilartermswithotherauthors

(Lietal.,2012),resultsindicatedthatcatechinenrichedextractsin

aqueoussolutionremainedstableinpHvalueslowerthan4and

temperaturebelow30◦Cforaperiodof24h.Evenathigh

ther-malprocessconditions(85◦C)atpHslowerthan4theflavan-3-ols

andcatechinaredetectedfor1hperiodwithoutgreatlosses.These

resultsmaylimittheCEEdirect application,favoring acidfoods

assomechesses,fruitjuice,vegetable/fruitproducts,mayonnaises

andyogurts(FoodIngredientsBrasil,2008).

4. Conclusions

Theanalysisoftheflavon-3-olsandcatechincompounds

sta-bilityfromthestrawberrytreefruitsiscrucialforpredictingthe

shelflifebehaviorofthecompoundsinvariousprocessing

situa-tions.Infact,foodprocessingorotherprocessesarefactorsthat

affectdirectlyontheintegrity ofthemolecules.Controlling the

conditionsoft,pHandTamongothersareessentialaspectsfor

keepingtheprocessefficiencyandforobtaininghighquality

prod-ucts.Theflavan-3-olsandcatechindegradationinpowdersystems

is differentfromthedegradation inaqueoussolutions, and the

solu-Fig.4.Catechinstabilityinaqueoussolutionsystemssimulatingfoodmatrices.SectionAshowstheindividualtime-dependentgraphicalanalysisofthestabilityresults

basedonthecatechincontentasfunctionoftheTandpH.Eachgraphshowsthetimedegradationeffects(0–24h)ofeachTtested(25,40,55,70and80◦C).Pointsarethe

experimentaldataofthedifferentpHstested(䊉3,5,7and♦9)andlines(—)theresultspredictedbyEq.(3).Theparametricresultsandcorrelationcoefficientsare

presentedinTable3.SectionBshowstheglobalmultivariablefittingresultsofapplyingEq.(9)todescribethefullmultivariabledata:B1showstheparametricnetsurface

patternofthekineticrasafunctionoftheirrespectiveaffectingvariables(pHandT);andB2showsthecorrelationbetweentheexperimentalvaluesandthepredictedones

B.R.Albuquerqueetal./IndustrialCropsandProducts99(2017)150–162 161

Table3

Resultsoftheparametricandconfidenceintervalsoftheflavan-3-olsandcatechinresponsesfittedtothetimedependentmodelpresentedinEq.(3)atdifferentpHandT

conditionswhenevaluatingtheapplicationstabilityoftheenrichedextract.

T pH FLAVAN-3-OLS Catechin k(t)(g/mL) r(t)(h−1) R2 _{k(t)(g/mL) r(t)(h}−1_{) R}2 25.0 3.0 300.0±29.06 0.011±0.003 0.9687 150.0±14.95 0.040±0.006 0.9965 25.0 5.0 300.0±29.78 0.081±0.024 0.9926 150.0±14.30 0.094±0.013 0.9536 25.0 7.0 300.0±30.00 3.138±0.941 0.9888 150.0±15.00 2.598±0.390 0.9989 25.0 9.0 300.0±30.00 7.952±2.386 0.9981 150.0±15.00 6.094±0.914 0.9990 40.0 3.0 300.0±26.60 0.002±0.001 0.8866 150.0±14.51 0.015±0.002 0.9671 40.0 5.0 300.0±29.35 0.058±0.017 0.9783 150.0±13.92 0.043±0.006 0.9280 40.0 7.0 300.0±30.00 2.543±0.763 0.9961 150.0±14.93 1.555±0.232 0.9955 40.0 9.0 300.0±30.00 6.156±1.847 0.9977 150.0±15.00 7.828±1.174 0.9997 55.0 3.0 300.0±30.00 0.001±0.000 0.9988 150.0±14.31 0.008±0.001 0.9541 55.0 5.0 300.0±29.55 0.045±0.013 0.9849 150.0±14.65 0.029±0.004 0.9767 55.0 7.0 300.0±30.00 1.820±0.546 0.9979 150.0±14.64 1.211±0.177 0.9758 55.0 9.0 300.0±30.00 5.156±1.547 0.9989 150.0±15.00 7.828±1.174 0.9998 70.0 3.0 300.0±12.98 0.002±0.000 0.4328 150.0±8.09 0.001±0.001 0.9397 70.0 5.0 300.0±28.98 0.038±0.011 0.9659 150.0±13.37 0.023±0.003 0.8916 70.0 7.0 300.0±30.00 1.374±0.412 0.9999 150.0±14.24 1.004±0.143 0.9492 70.0 9.0 300.0±30.00 4.156±1.247 0.9989 150.0±15.00 7.828±1.174 0.9991 85.0 3.0 300.0±6.42 0.000±0.000 0.2140 150.0±0.45 0.002±0.001 0.9301 85.0 5.0 300.0±28.70 0.033±0.009 0.9566 150.0±10.71 0.016±0.002 0.9141 85.0 7.0 300.0±29.99 1.085±0.326 0.9997 150.0±13.78 0.840±0.116 0.9190 85.0 9.0 300.0±30.00 3.545±1.064 0.9991 150.0±15.00 6.131±0.920 0.9991

tions.Mathematicalmodelsweredevelopedandmultiplegraphical

plotswereconductedtoestablishandillustratetheoptimum

val-uesoftheindependentvariablesstudied.Thus,thekineticmodels

couldbeusedforcalculatingshelf-lifeandpredictingcompounds

stabilityatgivenTandpHconditionsforpowderandaqueous

sys-tems.Theoptimalstabilityconditionsforflavan-3-ols(including

catechin)inpowdersystemswashigheratpH=5.4andT=−20◦_C

duringamonth,whileinaqueoussolutionremainedintactatpH<4

andT<30◦Cfor aperiod ofatleast24h. Highcorrelations and

significantparameterconfidenceintervalswerealwaysobtained.

Therefore,itisproposedthatthesesemi-empiricalkineticmodels

couldbeappliedtopredicttheeffectsofTandpHontheshelflife

ofcompoundsinpowderandaqueoussystem.Moreover,usingthe

optimalprocessingconditions,itispossibletoproducefunctional

extractswithhighpotentialasnutraceuticalsorasactive

ingredi-entsinthedesignoffunctionalfoods,whichcanbealsoextended

tootherindustrialfieldssuchaspharmaceuticalandcosmeceutical

industries.

Acknowledgements

The authors thank the Foundation for Science and

Tech-nology (FCT, Portugal) and FEDER under Program PT2020 for

financial supporttoCIMO (UID/AGR/00690/2013) and L.Barros

(SFRH/BPD/107855/2015)grant.ToPOCI-01-0145-FEDER-006984

(LALSRE-LCM),fundedbyFEDER,throughPOCI-COMPETE2020and

FCT.ToXuntadeGaliciaforfinancialsupportforthepost-doctoral

researcherofM.A.Prieto.B.AlbuquerquethanksCeleidePereira

(UTFPR,Brazil)forhermasterco-supervision.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in

theonlineversion,athttp://dx.doi.org/10.1016/j.indcrop.2017.02.

002.

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