Inactivation, Aggregation and Conformational Changes of Polyphenol Oxidase from Quince

15 Pages·2017·2.58 MB·English

molecules
Article
Inactivation, Aggregation and Conformational
Changes of Polyphenol Oxidase from Quince
(Cydonia oblonga Miller) Juice Subjected to Thermal
and High-Pressure Carbon Dioxide Treatment
AamirIqbal1,AyeshaMurtaza1,ZafarullahMuhammad1,AbdeenE.Elkhedir1,MingfangTao1
andXiaoyunXu1,2,*
1 KeyLaboratoryofEnvironmentCorrelativeDietology,HuazhongAgriculturalUniversity,
MinistryofEducation,Wuhan430070,China;[email protected](A.I.);
[email protected](A.M.);[email protected](Z.M.);
[email protected](A.E.E.);[email protected](M.T.)
2 CollegeofFoodScienceandTechnology,HuazhongAgriculturalUniversity,No.1,ShizishanRoad,
Wuhan430070,China
* Correspondence:[email protected];Tel.:[phone]
(cid:1)(cid:2)(cid:3)(cid:1)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:1)
(cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)
Received:19June2018;Accepted:12July2018;Published:17July2018
Abstract: Polyphenol oxidase (PPO) causes the browning reaction in fruits and vegetables and
deterioratesthequality. Thermaltreatmentforenzymeinactivationmayresultindefectsasopposed
to high pressure CO (HPCD) processing. In this study, the changes in activity, dissociation,
2
aggregation and conformation of purified PPO from thermal and HPCD treated juice were
investigated. HPCDexhibitedinactivationofPPOat55–65◦Cwhereasthermalprocessingalone
atthesametemperatureresultedinPPOstillshowingactivity. Underthermaltreatmentat25and
65◦C,thebrowningdegreewashigher(0.39and0.24)thanforHPCD-treatedjuice(0.23and0.12).
FluorescenceandcirculardichroismspectralresultsindicatedthatHPCDinducedlargedecreases
inintensities,revealingarearrangementofthesecondarystructureanddestructionofthenative
configurationofthePPOmolecule. Theparticlesizedistribution(PSD)patternrevealedstructural
modificationleadingtoinitialdissociationandsubsequentaggregationofPPOafterHPCDtreatment.
Polyacrylamide gel electrophoresis (PAGE) analysis exhibited that molecular size of protein was
40kDa. Inconclusion,theHPCDmethodwasfoundtobemoreeffectivethanthermaltreatmentto
inactivatePPO.Structuralmodificationsprovidedbetterinsightsintothephenomenaofactivation
andinactivationofPPO.
Keywords: polyphenoloxidase;highpressurecarbondioxide;aggregation;λmax;conformation
1. Introduction
Quince(CydoniaoblongaMiller)isthefruitoftheMaloideae,asub-familyoftheRosaceaefamily[1].
Commerciallysignificantfruitsincludingapplesandpearsalsobelongtothisfamily[2]. Quinceis
consideredafunctionalfruitduetoitsmedicinalandnutritionalvaluesandismostlyusedinthe
productionofjam,marmalades,jelly,liquorandcakes. Quincefruithasgainedmuchattentioninthe
lastdecadeduetoitshighantioxidantcapacityresultingfromphytochemicalsincludingphenolic
compounds such as hydroxycinnamic acid and proanthocyanidins [1–3]. Storage conditions and
further processing cause enzymatic browning of fruit, leading to physical and chemical damages
resultinginundesirablequalitydepletion[4]. Thebrowningphenomenonismainlyduetothehigh
activityofenzymesfromtheoxidoreductasegroupsuchaspolyphenoloxidase(PPO,EC1.14.18.1)
Molecules2018,23,1743;doi:10.3390/molecules23071743 www.mdpi.com/journal/molecules
Molecules2018,23,1743 2of15
andperoxidase(POD,EC1.11.1.7)[5]. Withintheoxidoreductasegroup,PPOismainlyresponsible
fortheformationofbrownpigmentbyoxidationofpolyphenol. ThePPOisconsideredasacopper
protein. The active site of the PPO enzyme contains two copper ions, which are surrounded by
6 histidine residues and a single cysteine residue. It also serves as structural support for enzyme
molecule, thereby affecting the activity of protein. Copper ions present in the active site of PPO
are responsible for oxidation-reduction processes that lead to the transition of active sites among
met-,oxy-,anddeoxy-forms. Thus,copperplaysasignificantroleinthestructureandactivityofthe
PPOenzyme[6]. ThePPOisanenzymecomprisedoftwosubunits(HandL).ThesubunitHhasa
binuclearcopper-bindingsiteinthedeoxy-statewhileLsubunitscoordinateeachcopperiontocatalyze
twodifferentreactionsinvolvingmolecularoxygen;oxidationofmonophenols(O-hydroxylation)to
O-diphenolsandO-diphenolstoO-quinones,whicharefurtherresponsibleforthepolymerizationof
brownpigments[7,8].
Thermal treatment has been the traditional technology for the inactivation of enzymes. Heat
alsocausesdetrimentalchangesinthefoodproperties,i.e.,lossofflavor,color,nutrientsandtexture.
Variousinnovativetechnologiesincludinghighpressurecarbondioxide(HPCD),high-hydrostatic
pressure(HHP),ultra-sonication,andirradiationhavebeenusedtoreducethedetrimentalchanges
in various fruit and vegetables caused by thermal technology [4,9]. HPCD is considered an
innovative and emerging non-thermal technology, which is an alternate to traditional heating or
high-pressureprocessingoffruitjuicesandisdeemedcomparativelysafeforthosecompoundswhich
areheat-liable[10,11]. TheliteraturesuggeststhatHPCDisefficientintheinactivationofPPO[12,13].
HPCDutilizesCO propertieswhichcaninactivateenzymesbypHlowering, celldisruption, cell
2
membranemodificationandcellularcomponentextraction[14]. TheinactivationofPPOinprocessed
fruitsandvegetablesisthemainindexofquality. NumerousstudiesonthePPOenzymefromseveral
sources,i.e.,apple,banana,pear,carrot,celeryandloquat,havereportedenzymecharacterization,
therebycontrollingtheiractions[15,16].
TheenzymaticinactivationofPPO,qualityparametersandstructuralanalysisofpurifiedPPO
from quince juice treated with thermal and non-thermal technology (HPCD) are addressed in the
present paper. To our knowledge, no work has been done on fruit quality attributes, enzymatic
inactivationandstructuralmodificationofPPOpurifiedfromjuiceafterthermalandHPCDtreatments.
QuincejuicewasexposedtovaryingheatandHPCDtreatmentsofvaryingtemperaturesandpressure.
StructuralanalysiswasconductedtoexplorethemechanismofthermalandHPCDtechnologyon
theaggregationanddeformationoftheproteinafterpurificationwiththeactivityofthepolyphenol
oxidaseenzyme.
2. MaterialsandMethods
2.1. ExtractionofQuinceJuice
Freshquince(CydoniaoblongaMiller)atoptimummaturitywasboughtfromanativemarketin
Xinjiang,China. Freshquincewaswashed,peeledandcutintoslices. Sliceswerecrushedwithajuice
extractorwith0.1%(w/w)ascorbicacidaddedtoinhibitundesirableenzymaticbrowning. Thejuice
wasfilteredthroughacheeseclothtoremovecoarseparticlesandthencentrifugedat4000rpmfor
5min. TheresultingjuicewassubjectedtothermalandHPCDtreatments.
2.2. ThermalandHPCDTreatments
2.2.1. UntreatedQuinceJuice
Foreachexperiment,30mLoffreshlysqueezedjuicewithoutheatorHPCDtreatmentwasused
asthecontrol.
Molecules2018,23,1743 3of15
2.2.2. ThermalProcessingofJuice
Thermaltreatmentwascarriedoutinawaterbathatvaryingtemperatures(25–65◦C)witha
10◦Cincreasefor20min. Quincejuice(30mL)waspouredinto50mLplastictubes. Thetreatment
timewasstartedwhenthedesiredtemperatureofthequincejuiceasmeasuredbydigitalthermometer
(Wuhan,China)wasreached. Afterthethermaltreatment,thesampleswereimmediatelyplacedina
refrigeratorforfurtheranalysis. Enzymeactivityandotherqualityparameterswerethendetermined.
2.2.3. HPCDProcessingofJuice
HPCD treatment followed the method of Liao et al. [17] in which 99.5% pure CO obtained
2
fromWuhanCo. (Wuhan,China)wasintroducedintothepressurevessel. Quincejuice(30mL)was
transferredintoa50mLsterileplasticbottle. Foreachexperiment,theHPCDpressurevesselwas
sanitized with ethanol and preheated to the experimental temperature before juice samples were
placedinside. Afterthedesiredtemperaturesof25,35,45,55and65◦Cwerereached,thechamber
vesselwaspressurizedto20MPawithaplungerpump. Thequincejuiceinthechambervesselwas
heldatconstanttemperatureandpressurefor20min.Thepressurerampusedwas20MPa/30s,andat
theendofthetreatment,thechambervesselwasgraduallydepressurizedfor1.5–2min. Pressurization
anddepressurizationtimewasnotincludedintreatmenttime. FollowingtheHPCDtreatment,quality
parametersofthequincejuiceswereevaluated.
2.3. QualityAttributesofQuinceJuice
2.3.1. ColorimetricMeasurement
Colorimetric measurement of quince juice was performed at ambient temperature with a
chromometer(CR-410;KonicaMinoltaCorporation,Osaka,Japan). ThecolorimetricvaluesofL*,a*,
andb*weremeasured,whereL*indicatedlightness,a*indicatedredness,andb*indicatedyellowness.
2.3.2. BrowningDetermination
ThebrowningdegreeofquincejuicewasevaluatedusingaMultiskanspectrophotometer(Thermo
Scientific,Waltham,MA,USA)[14]. Thesamplesolutionwascentrifuged(10,000rpm)for20min,
andtheBDwasmeasuredafteradding100µLofsampletotheenzyme-linkedimmunosorbentassay
(ELISA)plateandtestedimmediatelyatλ=420nmbyusingasimplekineticmethod.
2.3.3. Physico-ChemicalAnalysis
ThejuicepHwasdeterminedusingadigitalThermopHmeter(ThermoFisher,Waltham,MA,
USA).Totalsolublesolid(TSS)contentsweremeasuredbyadigitalAbberefractionmeter(Shanghai
PrecisionandScientificInstrumentCo.,Shanghai,China).
2.4. PPOExtractionandPurification
Quince juice was collected after treatments (thermal and HPCD) and PPO extraction and
purificationmethodsfollowedourformerstudy[9]inwhich25%(NH4) SO saturationwasused
2 4
tofractionatethejuicetoremoveimpurities,followedby80%saturatedsolutionfor1hforprotein
precipitation. Juicewasthensubjectedtocentrifugationat15,000rpmfor20min. Proteinprecipitates
wereresuspendedin0.5mol/LTris-HClbuffer(pH7.0)andweredialyzedagainstTris-HClbufferfor
24handconcentratedbyultra-filtrationandpurifiedbyusingDEAEsepharosefastflowcolumns
(1.0×10cm).FractionscontaininghigherPPOactivitywereselectedforfurtheranalysis.Thefractions
withthehighestPPOactivitywereconcentratedandstoredforfurtheranalysis.
Molecules2018,23,1743 4of15
2.5. PPOActivity
The relative activity of crude and purified enzyme was measured by following the method
presented by Liu et al. [11] with slight modifications Crude enzyme was made by adding 0.1%
polyvinylpolypyrrolidone(PVPP)and(0.1%)TritonX-100to2mLquincejuice. Afterastoragetime
of1hat4◦C,thejuicewascentrifugedat10,000rpmfor20min. Thiscrudeenzymewasusedfor
PPO activity. An aliquot of 50 µL supernatant was mixed with 0.1 M catechol substrate solution
(200µL)andwith0.05Mphosphatebuffer(pH7.2). Absorbanceofthesolutionwasdeterminedata
wavelengthof420nmat30sintervalsfor3min(30◦Cincubation)inaspectrophotometer(Multiskan
FC-ThermoScientific,Waltham,MA,USA).ThePPOspecificactivity(Abs/min)wastakenasthefirst
linearpartoftheslopefromthereactioncurve. Therelativeactivity(RA)wasdeterminedusingthe
followingequation:
ActivityofjuiceafterthermalorHPCDtreatment
Relativeactivity= − × 100
Activityofuntreatedjuice
2.6. ProteinDetermination
ProteincontentswereanalyzedaccordingtotheBradfordmethodatawavelengthof595nm.
Bovineserumalbumin(BSA)wasusedasthestandardforprotein[18].
2.7. ElectrophoreticAnalysisofThermalandHPCD-TreatedPPOEnzymes
Sodium dodecyl sulphate (SDS) and non-denaturing polyacrylamide gel electrophoresis
(Native-PAGE)wasusedtoidentifiedthepurificationprocessbyusingtheMini-proten4cellsystem
(Bio-Rad,Hercules,CA,USA)todeterminethemolecularmassofpurifiedPPO[15]. Gelswereused
toevaluatethepurityandmolecularweightofpurifiedPPOprotein.
2.8. StructuralAnalysisofUntreatedandTreated(ThermalandHPCD)PPO
2.8.1. CircularDichroismSpectralMeasurement
Circular dichroism analysis was conducted according to earlier studies [19,20] with a JASCO
spectropolarimeter (JASCO, Tokyo, Japan). CD measurements were recorded by using optical
path-length 0.009 cm quartz: 0.2 mg/mL protein sample was dissolved in 50 mmol/L Tris-HCl
buffer(pH7.0),usingTrisbufferasablanksample. CDparameterswereobservedintheultraviolet
rangeof190–240nmfor2sand1nmbandwidthat100nm/min. TheCDdatawereexpressedas
mdeg(molarextinctioncoefficientdifference)andtheproteinsecondarystructurewasestimatedfrom
Yang’sequation.
2.8.2. FluorescenceSpectralMeasurement
Intrinsicfluorescencespectralanalysiswasconductedusingafluorescencespectrophotometer
(F-4600,Hitachi,Tokyo,Japan). Proteinsamples(0.2mg/mL)weresubjectedtospectrophotometer
analysistodeterminethemaximumexcitationwavelength. Proteinsampleswerescannedforemission
spectraatλem300–400nmofprotein.Theemissionandexcitationslitwidth(emandex)wereadjusted
at5nm. Thespeedwassetas240nm/min,anda0.1sresponsetimewasused[15].
2.8.3. PSDMeasurement
Particle size distribution (PSD) was measured by a Zetasizer Nano-ZS device (Malvern Co.,
Malvern,UK)accordingtothedescriptionofHuetal.[21]. ThepurifiedproteinsamplesfromHPCD
andthermaltreatedjuicewereadjustedto0.3mg/mLwithphosphatebuffer(50mM,pH7.0)for
determinationofproteinPSD.Awavelengthlaserof532nmoflightscatteringwitha15◦ reflection
anglewasappliedtothesamplesolution. ThesizemeasurementresultscontainingPSDanalysiswere
takenasthemeanfromfourreadings.
Molecules 2018, 23, x FOR PEER REVIEW 6 of 15
(mature, immature, latent and active forms) isoforms, and its increased activity at 25 °C (TP) may be
due to the presence of latent PPO [15,26]. In comparison, the results of HPCD treated quince juice
showed that the RA decreased to 78.26%, 69.55%, 59.45% and 34.18% with increased temperatures of
25, 35, 45 and 55 °C respectively. Nearly complete inactivation occurred at 65 °C (Figure 1A). HPCD-
treated juice significantly lowered the PPO activity as compared to thermal-treated juice at same
temperature. Research reports suggest that PPO inactivation of juice under HPCD treatment was
improved with increased pressure, temperature and treatment time [22,27]. Enzymatic inactivation
under HPCD conditions may be due to changes in the secondary structure of PPO, which might be
Molecules2018,23,1743 5of15
the result of pH lowering after CO2 dissolved in solution. Li et al. also reported that enzymatic activity
of apple PPO under HPCD treatment showed biphasic changes of initial activation and then sudden
2in.9a.cStitvataitsitoicna laAt n3a5l–y5si5s °C for 15 min [20]. Similar results were found in banana pulp, where PPO
activity under HPCD gradually decreased from 40.7% to 11.6% with treatment at 45–60 °C for 30 min
DatawereanalysedbyANOVA(analysisofvariance),andmeancomparisonsweremadeby
at 20 MPa [22]. The results of relative activity of PPO after purification are shown in Table 2.
Tukey’s test. The mean was taken at a significantly different 95% confidence interval (p ≤ 0.05).
Compared to crude PPO activity (116.22%), the lower activity (46%) was observed for purified PPO
ThegraphswereconstructedusingOrigin9.0software(OriginLab,Northampton,MA,USA).
using 25 °C under thermal treatment. The lowest relative activity (7.40%) was observed for purified
3P.PROe ssuulbtjseactnedd Dtoi s6c5u °sCsi ounnder HPCD treatment. The activity of the crude PPO enzyme was found to
be higher than purified enzyme in our study. This might be due to the reason of ammonium sulphate
3.1. ChangesinColor,pHandTSSofQuinceJuice
saturation during enzyme extraction, which decreases the level of PPO activity. Dialysis of protein
preciTphiteactoe ldourprianrga mpuetreifriscaotfijounic aelsaoft ecrauHsPeCs Dlosasn odft choeprmpearl itorenast,m wehnitc(h2 5a–ls6o5 d◦Cec)raeraesseh PoPwOn ainctFivigituyr e[218B]..
TIth ies rgeednneersasllayn bdeylieelvloewd ntheasst lionsdsi coaft ePdPOby aac*tiavnitdy bm*avya lbuee sdoufet htoe PqPuOin cfoerjmuiicnega fstoemrteh ienramcatilvter epahtmeneonlts
w[2e9r]e alnedss atlhsoan bethcaeucsoen otrfo al ,gaonodd gcroardreulaaltliyond ebcertewaeseend twheit hα-ihneclriexa csoinngtetnetm apnedr aretusirdeu. aTlh aectciovliotyr uvnaldueer
(Ha*PaCnDd btr*e)aotfmHePnCt. DT-htere iartreedveqrusiinbclee djueiccoemwpaosssiitgionnifi ocfa nαt-lhyelloixw oecrccuormrepda raefdtetro HthPeCtDhe. rTmotaal-l tirneaactetidvajutiicoen.
Aofn PinPcOre laesaedisn tbor sigtrhutcnteusrsailn mdiocdatifeidcabtyiotnh eanLd* vdaelnuaetuwraastioonbs oefr vthede pwriothteaint.e Tmhpeesrea ptuhreenionmcreenasae sdhuorwinegd
bthoatht PHPPOC aDctaivnidtyt hcaenrm bea lirtrreevatemrseibnltys .inAacstiimvailtaerdfi bnyd HinPgCwDa [s2r4e]p. Iotr itse gdebnyerYaullye tbaell.ie[2v2e]d, it.hea.,t ibnrboawnnainnag
jiusi cdeu,eb rtoow PnPiOng-mdeevdeialotepdm oexnitdwataisonsl oowf eprhiennHolPs.C IDn jouuicre satusdcyom, ipt awreads tdoettehremrminaelldy tthreaat tiendaactnidvactoionntr oolf
jPuPicOe. iCn oqluorincchea jnugicees ionccquurirnecde ujunidceerc oHuPldCDre struelattfmroemntb, rsouwggneisntgincga uthsaetd HbyPCPDPO p,ewrfhoircmhsl ebaedttserto ththaen
dtheeterrmioarla ttrieoantmofejnuti caeloqnuea. lity.

FFiigguurree 11.. ((AA)) RReellaattiivvee aaccttiivviittyy ooff PPPPOO;; ((BB)) CCoolloorr ppaarraammeetteerrss ((LL**,, aa** aanndd bb**)) ooff qquuiinnccee jjuuiiccee ttrreeaatteedd wwiitthh
tthheerrmmaall aanndd HHPPCCDD ttrreeaattmmeenntt aattd diiffffeerreennttt teemmppeerraattuurreess..

TheinfluenceofHPCDandthermaltreatmentonpHandTSSofquincejuiceispresentedin
Table1. ThepHvalueofuntreatedjuicewas4.74,butafterHPCDtreatment,decreasedto4.23,4.20
and4.11at25,45and65◦Crespectively.ThermallytreatedjuiceshowedonlyaslightdecreaseinpHas
comparedtothecontrol. ThepHdeclineafterHPCDtreatmentcouldbeattributedtoCO dissolving
2
i nthejuice,whichfurtherdistributescarbonate,H+ionsandbicarbonates,thusincreasingtheacidity
andloweringthepHvalue. Thisfindingwasinaccordancewithstudiesofcarrot,watermelonand
applejuiceinwhichpHwasreducedsignificantlyafterHPCDtreatment[16,23]. pHisimportantin
thatitcanbeassociatedwithenzymeinactivation. AtlowerpH,CO maymoreeasilyinteractwith
2
theenzyme-boundarginine, resultinginaproteincomplexwhichcancausethelossofenzymatic
activity[24]. Accordingtostructuralanalysis,ithasbeenprovedthatbasicaminoacidslikearginine,
lysineandhistidinearethosemostcommonlyfoundinCO -proteinbindingsites. HPCDtreatment
2
couldcausetheformationofcomplexcovalentcarbamateswithfreeaminoacidgroupsontheenzyme
Molecules2018,23,1743 6of15
surface that were potentially responsible for reducing enzyme activity [10,25]. Untreated quince
juice showed a TSS value of 11.55 ◦Brix. After HPCD and thermal treatment, TSS values did not
alter significantly (Table 1). During storage, TSS also did not change significantly, indicating that
theprocessingtreatmentsdidnotimpactsolublecarbohydratessuchasglucoseandsucrosethatare
consideredthemajorcontributorstoTSS.
Table1.pHandbrixofquincejuicefromthermalandHPCDtreatments.
Treatment Temp pH ◦Brix
Control(Untreated) 4.74±0.23a 11.55±0.22a
25 4.75±0.18a 11.54±0.18a
35 4.73±0.24a 11.56±0.2a
Thermal-treated(20min) 45 4.73±0.13a 11.54±0.2a
55 4.70±0.22a 11.51±0.19a
65 4.69±0.17a 11.49±0.19a
25 4.23±0.21ab 11.25±0.37a
35 4.23±0.23ab 11.19±0.16a
HPCD-treated(20MPa,20min) 45 4.20±0.19ab 11.10±0.15a
55 4.18±0.14ab 10.98±0.17a
65 4.11±0.11b 10.92±0.21a
Datapresentedasthemean±SD(standarddeviation). Differentlettersrepresentsignificantdifferenceamong
means(p≤0.05).
3.2. Activation&InactivationofCrudePPOEnzymebyThermalandHPCDTreatments
TheRAofquincecrudePPOduringthermalandHPCD(20MPa)treatmentsat25to65◦Cfor
20 min is shown in Figure 1A. PPO after thermal treatment showed the highest RA (116.22% and
98.09%)at25and35◦C.respectively. RAgraduallydecreasedto81.46%at45◦Candshowedamore
rapid decrease to 61.64% and 45.14% at 55 and at 65 ◦C respectively. PPO is present in numerous
(mature,immature,latentandactiveforms)isoforms,anditsincreasedactivityat25◦C(TP)maybe
duetothepresenceoflatentPPO[15,26]. Incomparison,theresultsofHPCDtreatedquincejuice
showedthattheRAdecreasedto78.26%,69.55%,59.45%and34.18%withincreasedtemperatures
of 25, 35, 45 and 55 ◦C respectively. Nearly complete inactivation occurred at 65 ◦C (Figure 1A).
HPCD-treatedjuicesignificantlyloweredthePPOactivityascomparedtothermal-treatedjuiceat
sametemperature. ResearchreportssuggestthatPPOinactivationofjuiceunderHPCDtreatmentwas
improvedwithincreasedpressure,temperatureandtreatmenttime[22,27]. Enzymaticinactivation
under HPCD conditions may be due to changes in the secondary structure of PPO, which might
betheresultofpHloweringafterCO dissolvedinsolution. Lietal. alsoreportedthatenzymatic
2
activityofapplePPOunderHPCDtreatmentshowedbiphasicchangesofinitialactivationandthen
suddeninactivationat35–55◦Cfor15min[20]. Similarresultswerefoundinbananapulp,where
PPOactivityunderHPCDgraduallydecreasedfrom40.7%to11.6%withtreatmentat45–60◦Cfor
30minat20MPa[22]. TheresultsofrelativeactivityofPPOafterpurificationareshowninTable2.
ComparedtocrudePPOactivity(116.22%),theloweractivity(46%)wasobservedforpurifiedPPO
using25◦Cunderthermaltreatment. Thelowestrelativeactivity(7.40%)wasobservedforpurified
PPOsubjectedto65◦CunderHPCDtreatment. TheactivityofthecrudePPOenzymewasfoundto
behigherthanpurifiedenzymeinourstudy. Thismightbeduetothereasonofammoniumsulphate
saturationduringenzymeextraction,whichdecreasesthelevelofPPOactivity. Dialysisofprotein
precipitateduringpurificationalsocauseslossofcopperions,whichalsodecreasePPOactivity[28].
ItisgenerallybelievedthatlossofPPOactivitymaybeduetoPPOformingsomeinactivephenols[29]
andalsobecauseofagoodcorrelationbetweentheα-helixcontentandresidualactivityunderHPCD
treatment. Theirreversibledecompositionofα-helixoccurredafterHPCD.TotalinactivationofPPO
leadstostructuralmodificationanddenaturationoftheprotein. ThesephenomenashowedthatPPO
activitycanbeirreversiblyinactivatedbyHPCD[24]. Itisgenerallybelievedthatbrowningisdue
Molecules2018,23,1743 7of15
toPPO-mediatedoxidationofphenols. Inourstudy,itwasdeterminedthatinactivationofPPOin
Molecules 2018, 23, x FOR PEER REVIEW 7 of 15
quincejuiceoccurredunderHPCDtreatment,suggestingthatHPCDperformsbetterthanthermal
treatmentalone.
Table 2. Secondary structure contents of thermal- and HPCD-treated PPO.
Table2.Secondarystructurecontentsofthermal-andHPCD-treatedPPO.
Secondary Structure Contents
Treatments Random Coil PPO Activity Concentration
Treatments α-Helix (%) β-Sheet (%) β-STeucornnd (a%ry) StructureC(%on)t ents (%) (%)
Native α-3H9e.l3i4x (±% 1).42a β-S1h7e.e9t ±(% 1).13d β-T2u6rn.41(% ±) 0.59aR ando1m6.C35o i±l (0%.7)5d PPO24A.2ct3i v±i t0y.7(%6d) Con8c.e5n7t r±a 0ti.o6n7b(% )
25 N°Ca ttihveermal 39.3348.±961 ±.4 02a.96b 17.918±.41 ±.1 03d.78cd 26.4214±.480. 5±9 0a.82ab 16.3158±.160 .7±5 1d.12cd 244.223.5±8 ±0 .716.0d2a 182.5.778± ±0 0.6.97b3a
2525◦ C°Cth HerPmCaDl 38.9365.±260 ±.9 60b.78b 18.42±0.05. 7±8 0cd.53b 24.482±3.80.58 2±a 0b.77b 18.162±0.319.1 2±c 0d.82c 423.658.8±8 ±1 .00.26a4b 142..0784 ±± 00..1953ac
6255 ◦°CC HthPeCrmDal 35.2360.±810 .±7 81b.77c 20.52±3.40 .±5 30b.35b 23.852±1.201.7 ±7 b1.11c 20.3924±.508. 8±2 c0.66b 36.2898.5±3 0±.6 14.b2c 43..0941± ± 00..1059cc
65◦Cthermal 30.81±1.77c 23.4±0.35b 21.21±1.11c 24.58±0.66b 29.53±1.2c 3.91±0.09c
6655◦ °CCH HPPCCDD 24.1214.±110 ±.9 70d.97d 27.72±7.70 .±7 70a.77a 18.41±8.14.1 ±5 d1.15d 29.7929±.709. 9±2 a0.92a 070.470.4±0 ±1 .133.3e3e 00..2277± ± 00..001155dd
Data presented as the mean ± SD (standard deviation). Different letters represent significant
Datapresentedasthemean±SD(standarddeviation). Differentlettersrepresentsignificantdifferenceamong
mdiefafenrse(npc≤e 0a.m05o).ng means (P ≤ 0.05).
33..33.. EEffffeecctt ooff TThheerrmmaall aanndd HHPPCCDD PPrroocceessssiinngg oonn BBrroowwnniinngg DDeeggrreeee ooff QQuuiinnccee JJuuiiccee
TThhee BBDD ooff qquuiinnccee jjuuiiccee dduurriinngg ssttoorraaggee aatt 44 ◦°CC aafftteerr HHPPCCDD aanndd tthheerrmmaall ttrreeaattmmeenntt iiss sshhoowwnn iinn
FFiigguurree 22.. UUnnttrreeaatteedd jjuuiiccee iinniittiiaallllyy hhaadd aa bbrroowwnniinngg ddeeggrreeee ooff 00..4455 bbuutt iinnccrreeaasseedd ttoo 00..9988 aafftteerr 88 ddaayyss ooff
ssttoorraaggee.. HHPPCCDD rreedduucceedd tthhee BBDD vvaalluuee ccoommppaarreedd ttoo tthheerrmmaallllyy ttrreeaatteedd jjuuiiccee ((FFiigguurree 22AA)).. BBDD ddeeccrreeaasseedd
wwiitthh iinnccrreeaassiinngg tteemmppeerraattuurree aanndd wwitihth dadyasy osfo sftosrtaograeg. Te.heT BhDe BoDf thoefrtmhaerllmy atrlleyatterde aqtuedincqeu jiuniccee jauti 2ce5
a°Ct 2w5a◦sC 0w.39a san0.d3 9it adnedcrietadseedcr etoa s0e.d27t oan0d.2 07.2a4n das0 t.h2e4 taesmthpeertaetmurpee irnactrueraeseindc rtoea 5s5e dantod 5655 a°Cnd (F6i5gu◦rCe
(2FAig).u Irne a2dAd).itIinona,d HdPitCioDn, tHrePaCtmDentrte watamse fnotuwnda stofo buen mdotoreb eeffmecotriveee finfe ccotinvteroinllicnogn tbrroolwlinnginbgr,o aws nHinPgC,Das-
HtrePaCteDd- tqreuaintecde qjuuiicnec oenju dicaey o1n sdhoayw1eds hBoDw vedaluBeDs voaf l0u.e2s3 oafn0d. 203.1a2n adt 02.51 2anadt2 655a °nCd, r6e5s◦pCec,trievseplyec, twivheillye,
wthheirlmetahl-etrrmeaatle-dtr ejuaitceed ejxuhiciebietexdh ivbaitleudesv oalfu 0e.s39o fan0.d3 90.a2n4d (F0i.g2u4r(eF i2gBu)r. e2B).

FFiigguurree 22.. BBrroowwnniinngg ddeeggrreeee dduurriinngg ssttoorraaggee aatt 44 ◦°CC ((AA)) TThheerrmmaall ttrreeaattmmeenntt;; ((BB)) HHPPCCDD ttrreeaattmmeenntt ooff
quince juice at different temperatures.
quincejuiceatdifferenttemperatures.
Similar results for the reduction of BD were reported for HPCD-treated apple and water melon
SimilarresultsforthereductionofBDwerereportedforHPCD-treatedappleandwatermelon
juice in comparison to thermally treated juice [27,30]. The BD of carrot juice was also reported to
juice in comparison to thermally treated juice [27,30]. The BD of carrot juice was also reported to
decrease from increased pressure and treatment time of HPCD. Under HPCD treatment at 20 MPa
decreasefromincreasedpressureandtreatmenttimeofHPCD.UnderHPCDtreatmentat20MPa
for 15 min (25 °C), the BD of carrot juice significantly decreased to 0.272, whereas untreated juice
for 15 min (25 ◦C), the BD of carrot juice significantly decreased to 0.272, whereas untreated juice
showed a higher value of 0.779 [31]. BD is closely related to enzyme actions, especially PPO. HPCD
showedahighervalueof0.779[31]. BDiscloselyrelatedtoenzymeactions,especiallyPPO.HPCD
treatment can inactivate enzymes more efficiently as compared to thermal treatment for prevention
treatmentcaninactivateenzymesmoreefficientlyascomparedtothermaltreatmentforpreventionof
of the browning of quince juice.
thebrowningofquincejuice.
3.4. Particle Size Distribution of Thermal- and HPCD-Treated PPO
3.4. ParticleSizeDistributionofThermal-andHPCD-TreatedPPO
Thermal and HPCD treatments affected the PSD pattern of purified PPO as shown in Figure 3.
ThermalandHPCDtreatmentsaffectedthePSDpatternofpurifiedPPOasshowninFigure3.
The native PPO exhibited a wider span with two Sections (I and II); Section (I) revealed that native
ThenativePPOexhibitedawiderspanwithtwoSections(IandII);Section(I)revealedthatnative
protein partially aggregated at 58.8 nm with the fraction containing 16.76%, while mostly PPO
aggregates were distributed in section II, which exhibited a particle pattern ranging from 165 to 615
nm with the highest fraction (24.59%) at 342 nm. Peak diameter results suggest that the small sized
proteins tend to aggregate to form large accumulates of PPO. For thermally treated PPO at 25 °C,
Molecules2018,23,1743 8of15
protein partially aggregated at 58.8 nm with the fraction containing 16.76%, while mostly PPO
aggregates were distributed in section II, which exhibited a particle pattern ranging from 165 to
6M15olencumlesw 20i1t8h, 2t3h, ex FhOigRh PeEsEtRf rRaEcVtIiEoWn ( 24.59%)at342nm. Peakdiameterresultssuggestthatthesm8 oaf l1l5
sizedproteinstendtoaggregatetoformlargeaccumulatesofPPO.ForthermallytreatedPPOat25◦C,
lolowweerri nintetennssititiieessw witithht twwoos seeccttioionnssw weerreeo obbsseerrvveedd:: SSeeccttiioonn IIa att3 377.8.8n nmmw witihtht htheef rfaracctitoionno occccuuppyyiningg
1144.0.099%%,,a annddS Seecctitoionn( I(II)I)w witihths soommeep paartritaialllylya aggggrreeggaateteddp prrooteteinin( 2(29955n nmm))w witihthf rfaracctitoionnsso occccuuppyyiningg
2200.9.922%%..A Att6 655◦ °CC,,t thheeP PSSDDp paatttteerrnns shhoowweeddo onnees seeccttioionnw witihtha af rfraacctitoionnv vaalulueeo off2 299.6.6a att3 34422n nmm..H HPPCCDD
aatt2 255◦ °CCe exxhhiibbiitteedda ah hiigghheerri inntteennssiittyyt thhaannt thheet thheerrmmaallt trreeaattmmeenntt( (2255◦ °CC)) wwiitthh ttwwoo sseeccttiioonnss;; SSeeccttiioonn ((II))::
6688.1.1n nmmw witihtha a2 200.7.777%%n nuummbbeerrf rfraacctitoionna annddS Seecctitoionn( I(II)I:):2 266.1.199%%a att2 29955n nmm..A Att6 655◦ °CCw witihthH HPPCCDD,,t hthee
ssppaanno offt htheep paarrtitcicleled diaiammeeteterrf ufurtrhtheerri ninccrereaasesedda annddP PPPOOw waasss tsrtoronngglylyi ninaactcitvivataetdeda nanddd denenatauturerded. .

Figure 3. Particle size distributions of native PPO in comparison to thermal- and HPCD-treated PPO.
Figure3.ParticlesizedistributionsofnativePPOincomparisontothermal-andHPCD-treatedPPO.
Several studies have reported that HPCD treatment resulted in aggregation and dissociation of
Several studies have reported that HPCD treatment resulted in aggregation and dissociation
protein. HPCD at high temperature caused dissociation of large aggregates with high particle
of protein. HPCD at high temperature caused dissociation of large aggregates with high particle
diameter, indicating small aggregates with small particle diameters [20,32]. It has also been shown
diameter,indicatingsmallaggregateswithsmallparticlediameters[20,32]. Ithasalsobeenshown
that intermediates might exhibit size differences from the native structure, resulting in different
that intermediates might exhibit size differences from the native structure, resulting in different
properties [23]. High temperature and HPCD treatment induced small particle aggregation and
properties [23]. High temperature and HPCD treatment induced small particle aggregation and
larger particle dissociation [20]. The PSD pattern of purified PPO from quince after HPCD treatment
largerparticledissociation[20]. ThePSDpatternofpurifiedPPOfromquinceafterHPCDtreatment
showed dissociation and formation of aggregates. Enhanced interactions among protein molecules
showeddissociationandformationofaggregates. Enhancedinteractionsamongproteinmolecules
resulting from the catalytic site increased the inactivation rate by reduced particle size, resulting in
resultingfromthecatalyticsiteincreasedtheinactivationratebyreducedparticlesize,resultingin
dissociation and disintegration of the protein structure [15,33]. PPO with a small size may associate
dissociationanddisintegrationoftheproteinstructure[15,33]. PPOwithasmallsizemayassociate
to form aggregates at high temperature with further aggregation leading to significant accumulation,
toformaggregatesathightemperaturewithfurtheraggregationleadingtosignificantaccumulation,
thereby enhancing the distribution span [11]. High temperature under HPCD treatment enhanced
the homogenization. The aggregation induced by HPCD was probably due to a reduction in pH. CO2
reacts with water to form carbonic acid, which reduces the environmental pH. Hence, reduced pH
caused folding and unfolding of protein chains, which lead to aggregation. Furthermore, carbonic
acid may react with copper atoms and the histidine residues present at the catalytic site, destroying
the active catalytic centre and causing inactivation of PPO [21].
Molecules2018,23,1743 9of15
therebyenhancingthedistributionspan[11]. HightemperatureunderHPCDtreatmentenhancedthe
homogenization. TheaggregationinducedbyHPCDwasprobablyduetoareductioninpH.CO
2
reactswithwatertoformcarbonicacid,whichreducestheenvironmentalpH.Hence,reducedpH
causedfoldingandunfoldingofproteinchains,whichleadtoaggregation. Furthermore,carbonicacid
mayreactwithcopperatomsandthehistidineresiduespresentatthecatalyticsite,destroyingthe
activecatalyticcentreandcausinginactivationofPPO[21].
Additionally,depressurizationofCO afterHPCDtreatmentcausedtheformationofagasliquid
2
interface. Nonpolarresiduesoccurredinthegasphasewhilepolarparticlesorientedtotheliquid
phase,causingunfoldingofproteinchains. Thus,unfoldedmoleculeswillundergoaggregationof
particles. Meanwhile,depressurizationcausesrapidgasexpansionfromenzymes. Thisgasexpansion
inducedanexplosiveeffectandahomogenizationeffectofaggregates[20,21].
Inthisstudy,comparedtothermaltreatment,thePSDpatternofHPCD-treatedpurifiedPPO
showedsignificantvariationswithbroaderspansoflargerparticlediametersresultinginmassive
changesinPPOconformation. Thesestructuralchangesincludingfragmentation,deformationand
aggregationaffectedtheactivecatalyticsiteofPPOmoleculesandalteredthesecondarystructure,
resultinginPPOactivationandinactivation. PSDpatternofHPCD-treatedPPOfromquinceshowed
markedsignificantvariationswithbroaderspansoflargerparticlediametersresultinginmassive
changesinPPOconformation.
3.5. EffectofThermalandHPCDTreatmentsontheSecondaryStructureofPPO
CDspectraareavaluablespectroscopictechniquetoinvestigatechangesinsecondarystructure
including the α-helix, β sheet and β-turn of PPO protein. The α-helix contents were calculated
for negative elliptical peaks of 222 and 208 nm while the 214 nm peak is a characteristic of a
β-sheet [15,34,35]. The CD spectra of native PPO in comparison to HPCD and thermally treated
PPOareshowninFigure4.ThenativepurifiedPPOexhibitedanegativepeakat218nmandapositive
peak at 194 nm (Figure 4). The data suggest thatnative PPO possesses an α-helix arrangement in
secondaryconstellation. TheCDspectraofthermallytreatedPPOat25◦Cshowedminormodification
from native PPO, indicating no structural changes. However, at 65 ◦C, considerable changes in
PPOsecondarystructureresulted. ComparedtothenativePPO,thepositivepeakvalueat194nm
changedslightlyat25◦Cbutgraduallyincreasedaspressurewasappliedfor20min. Thenegative
ellipticityincreasedandthepositivepeakvaluedecreasedbyincreasingtemperatureunderHPCD,
suggestingthelossintheα-helixcontentandenhancedstructuredisorder. Moreover,theCDspectra
ofHPCD-treatedPPOproteinshowedmajorchangesinsecondarystructureascomparedtothermally
treatedPPO.
TheestimationofthesecondarystructureshowedthatthermalandHPCDtreatmentscauseda
markedreductioninthecontentsoftheα-helixandβ-turnandenhancedthecontentsoftheβ-sheet
andrandomcoil(Table2). NativePPOconsistedof39.34%α-helix,17.9%β-sheet,26.41%β-turnand
16.35%randomcoil. ThesecondarystructureofthermallytreatedpurifiedPPOshowedlittlechange
at25◦C,butat65◦C,theα-helixdecreasedto38.1%whiletheβ-sheetandrandomcoilincreased
to 23.40 and 24.58% respectively. Thus, loss of α-helix contents disorganized the native structure
of PPO and eventually enhanced the negative ellipticity of CD spectra. Moreover, when pressure
wasappliedincombinationwithtemperatureat65◦C,theα-helixandβ-turncontentdecreasedto
24.11%and18.40%whilethecontentsoftheβ-sheetandrandomcoilincreasedto27.70%and29.79%,
respectively(Table2). Hightemperaturedamagedthenativestructureoftheprotein,leadingtothe
reorganizationofsecondarystructure. Theβ-sheetcontentofPPOincreasedafterHPCDtreatment,
facilitatingaggregation. ThismightbeduetoasuddenreleaseofCO causingrapidgasexpansion,
2
inducingexplosiveandhomogenizationimpactsontheproteinaggregates[20].
Molecules 2018, 23, x FOR PEER REVIEW 9 of 15
Additionally, depressurization of CO2 after HPCD treatment caused the formation of a gas liquid
interface. Nonpolar residues occurred in the gas phase while polar particles oriented to the liquid
phase, causing unfolding of protein chains. Thus, unfolded molecules will undergo aggregation of
particles. Meanwhile, depressurization causes rapid gas expansion from enzymes. This gas
expansion induced an explosive effect and a homogenization effect of aggregates [20,21].
In this study, compared to thermal treatment, the PSD pattern of HPCD-treated purified PPO
showed significant variations with broader spans of larger particle diameters resulting in massive
changes in PPO conformation. These structural changes including fragmentation, deformation and
aggregation affected the active catalytic site of PPO molecules and altered the secondary structure,
resulting in PPO activation and inactivation. PSD pattern of HPCD-treated PPO from quince showed
marked significant variations with broader spans of larger particle diameters resulting in massive
changes in PPO conformation.
3.5. Effect of Thermal and HPCD Treatments on the Secondary Structure of PPO
CD spectra are a valuable spectroscopic technique to investigate changes in secondary structure
including the α-helix, β sheet and β-turn of PPO protein. The α-helix contents were calculated for
negative elliptical peaks of 222 and 208 nm while the 214 nm peak is a characteristic of a β-sheet
[15,34,35]. The CD spectra of native PPO in comparison to HPCD and thermally treated PPO are
shown in Figure 4. The native purified PPO exhibited a negative peak at 218 nm and a positive peak
at 194 nm (Figure 4). The data suggest that native PPO possesses an α-helix arrangement in secondary
constellation. The CD spectra of thermally treated PPO at 25 °C showed minor modification from
native PPO, indicating no structural changes. However, at 65 °C, considerable changes in PPO
secondary structure resulted. Compared to the native PPO, the positive peak value at 194 nm changed
slightly at 25 °C but gradually increased as pressure was applied for 20 min. The negative ellipticity
increased and the positive peak value decreased by increasing temperature under HPCD, suggesting
the loss in the α-helix content and enhanced structure disorder. Moreover, the CD spectra of HPCD-
treated PPO protein showed major changes in secondary structure as compared to thermally treated
Molecules2018,23,1743 10of15
PPO.

FFiigguurree 44.. CCDD ssppeeccttrraa ooff nnaattiivvee PPPPOO iinn ccoommppaarriissoonn ttoo tthheerrmmaall-- aanndd HHPPCCDD--ttrreeaatteedd( (2255& &6 655◦ °CC)) PPPPOO..

A previous study stated that thermal treatment resulted in a reduction of the α-helix content
in mushroom PPO, while β-sheet and random coil contents increased. Temperature increase also
influencedthestructuralchangesinPPO[35,36]. OurHPCDresultsareconsistentwithLietal.[20]
in which a decline in α-helix contents followed by decrease in PPO activity was observed in
amyloid-formingaggregates. CDspectrarevealedthatpurifiedPPOiscorrelatedwithPPOactivity.
AccordingtoourresultsofCDandPPOactivity,theincreasesinβ-sheetandβ-turncontributedto
thegreaterchangeinPPOconformation,indicatingthecollapseofthecatalyticcenter. TheHPCD
treatmentcausedchangesintheα-helixcontent,whichalteredtheinteractionbetweenthecopper
centreandtheα-helixsite,collapsingtheactivesite,andthusresultinginPPOinactivation[24].
HPCDmaychangehydrogenbondinginteractionswithinPPO,leadingtoconformationalchanges
ofthesecondarystructureandultimatelyresultinginPPOinactivation.
3.6. FluorescenceSpectroscopyAnalysis
Aspreviouslydescribed,variationsinenzymetertiarystructurerelatetothechangesinintrinsic
fluorescenceemission. Thepolarityofproteinresiduesincludingtryptophanandtyrosineprovidesa
valuabletechniquetocharacterizeproteinconformation[15]. Thermal-andHPCD-treatedpurified
PPO was examined by fluorescence spectroscopy (Figure 5). Native PPO displayed the largest
emission peaks at 331.2 nm (λmax) with fluorescence intensity of 990 a.u. The data indicate that
tryptophan remnants were located in the nonpolar environment [20]. Under thermal treatment
(25◦C),λmaxwasslightlyred-shiftedto332.6nmanditsintensitydecreasedto933.2a.u.,whilethe
λmaxofHPCD-treatedPPOatthesametemperatureincreasedrapidlyto334.8nmanddisplayed
3.6 nm red shifts as compared with native PPO. However, when the temperature increased from
25–65◦Cat20MPa,theflorescencespectrumexhibitedblueshiftsatλmax314.2nmanditsintensity
decreased from 761.6 to 255.7, indicating the loss of fluorescence intensity of PPO when 20 MPa
pressure was applied [29]. Hence, high temperature showed enormous changes with blue shifts
andreducedfluorescenceintensityinfluorescencespectra,causinginactivationofPPO.Apossible