Natural inhibitors of enzymatic browning

156 Pages·2003·6.3 MB·English

by  Yoruk, Ruhiye

NATURALINHIBITORSOFENZYMATICBROWNING
By
RUHIYEYORUK
ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL
OFTHEUNIVERSITYOFFLORIDAINPARTIALFULFILLMENT
OFTHEREQUIREMENTSFORTHEDEGREEOF
DOCTOROFPHILOSOPHY
UNIVERSITYOFFLORIDA
2003
Copyright2003
by
RuhiyeYoruk
Thisworkisdedicatedtothoseintheworldwhocaremost:Myparents,Munewerand
NizamettinYoruk;andmysisterandbrother,SeviyeandFahri.Youryearsofselfless
loveandunwaveringsupport,nearandafar,alwaysfoundawaytoreachme.Itis
throughyouthatIamabletosucceedinthisendeavor.Thankyou.
ACKNOWLEDGMENTS
Iwouldliketotakethisopportunitytothankmyadvisor,Dr.Marshall,forhis
enthusiasmandcontinuedsupportthroughoutmygraduatestudieshereattheUniversity
ofFlorida.Ialsoappreciateandamindebtedtohimforhispersonalguidanceand
friendshipduringmygraduateprogram.Fiehasalwaysimpressedmewithhisabilityto
adaptpubliclytoanyaudience.Ihadtremendousscientificanddecisiveprofessional
experiencesathislaboratory.Forthis,Iamtrulygrateful.
IwouldliketoexpressmysincereappreciationtoDr.Balaban,Dr.Hogsette,Dr.
Gregory,andDr.Prestonforservingonmysupervisorycommittee.Iextendspecial
thankstoDr.BalabanandDr.Hogsettefortheirpersonalfriendshipandprofessional
guidance.IwouldliketothankDr.Butlerforhisguidanceinrearinghousefliesatthe
beginningofthisresearchandforhiscontinuedencouragement.Iextendspecialthanks
toDr.Gregory,andDr.Prestonfortheirhelpinmakingthisprojectapositive
experience.IwouldalsoliketothankDr.Gregory,Dr.Preston,Dr.Sims,Dr.Talcott,
andDr.Kristinssonfortheirinvaluablecommentsandsuggestionsonthemanuscripts.
IwouldliketorememberDiana,whopassedawaysuddenlyin2001,andthankher
forassistingmeinthehouseflyproductionandformakinganenjoyablework
environmentintheDepartmentofEntomology.Iextendspecialthankstothepersonnel
oftheU.S.DepartmentofAgricultureinGainesvillefortheirassistanceinthe
productionofhouseflypupae.IappreciateandamindebtedtoMr.Hughesforkeeping
mycomputerrunningandforthefreestrawberriesandoranges.
IV
Iwouldalsoliketotakethisopportunitytothankmyparents,Munewerand
NizamettinYoruk(thebestadvocateonecouldeverhave)foralwaysbelievinginme.
Theirunwaveringsupportandcaringprovidesthedrivetoachievemygoals.Ialso
appreciateandamindebtedtomybrother,Fahri,forhisundyingencouragement.His
selflessloveandeternalsupportalwaysfoundawaytoreachmefromthousandsofmiles
away.Tomysister,Seviye,Ioweallmysuccess.Sheisdrivenbypersonalexcellence,
andismybestfriendandrolemodelinstrengthandcourage.IwouldliketothankGod
forgivingmethestrengthandaptitudetowithstandthetribulationsofthisendeavor.
ThisresearchwassupportedinpartbyfundingfromAgro-IndustriesandPost-
HarvestManagementService,AgriculturalSupportSystemsDivisionintheFoodand
AgriculturalOrganizationoftheUnitedNations.IwouldalsoliketothanktheU.S.
DepartmentofAgriculture,CenterforMedical,Agricultural,andVeterinaryEntomology
inGainesvilleforprovidinghouseflypupae.
v
TABLEOFCONTENTS
Page
ACKNOWLEDGMENTS iv
LISTOFTABLES ix
LISTOFFIGURES x
ABSTRACT xii
INTRODUCTION 1
LITERATUREREVIEW 5
ImportanceofEnzymaticBrowningintheFoodindustry 5
Problem 5
BiochemicalFoundationoftheProblem 6
PolyphenolOxidase(PPO) 9
MolecularMechanismofAction 9
PlantPPO 12
GeneralPropertiesofAppleandBananaPPO 17
Substratespecificity 18
EffectofpHandtemperature 20
InsectPPO 23
Inhibition/PreventionofEnzymaticBrowning 27
OxalicAcid 32
MODEOFINHIBITIONOFPOLYPHENOLOXIDASEBYOXALICACID 34
Introduction 34
MaterialsandMethods 36
PreparationofPPO 36
PreparationofOxalicAcidSolutions 37
AssayofPPOActivityandInhibition 37
PreincubationofPPOwithOxalicAcid 38
TreatmentofOxalicAcidwithCupricAcetate 38
RestorationofEnzymaticActivityofOxalicAcid-TreatedPPO 39
ScanningStudies 39
InhibitoryEffectofDicarboxylicAcids 40
ResultsandDiscussion 40
ThepHActivityProfileofPPO 40
vi
Concentration-andpH-dependentInhibitoryEffectofOxalicAcid 40
EffectofPreincubationonInhibitionbyOxalicAcid 43
InhibitionKinetics 45
ChelatingEffectofOxalicAcidontheActive-SiteCopper 46
ScanningStudies 50
ComparisonofInhibitoryEffectivenessofDicarboxylicAcids 53
MACHINE-VISIONANALYSISOFANTIBROWNINGPOTENCYFOROXALIC
ACID:ACOMPARATIVEINVESTIGATIONONBANANAANDAPPLE 55
Introduction 55
MaterialsandMethods 57
PreparationofFruitSlicesandTheirTreatmentswithOxalicAcid 57
ComparisonofAntibrowningEffectivenessofDicarboxylicAcids 58
ColorDataAnalysis 58
ColorSpectra 59
StatisticalAnalysis 59
DeterminationofpHandOxalicAcidConcentrationintheTreatedTissues 59
ResultsandDiscussion 60
VisualObservation 60
EffectofOxalicAcidandStorageTime 62
ChangesinColorSpectra 69
ThepHandOxalicAcidConcentration 74
ComparisonofAntibrowningEffectivenessofDicarboxylicAcids 76
INVESTIGATIONSONPOTENTIALOFOXALICANDASCORBICACIDSTO
CONTROLBROWNINGOFMINIMALLYPROCESSEDAPPLEAND
BANANAUSINGMACHINEVISION 81
Introduction 81
MaterialsandMethods 82
PreparationofFruitSlicesandTheirTreatmentswithOxalicandAscorbic
Acids 82
ColorDataAnalysis 83
StatisticalAnalysis 84
DeterminationofOxalicAcidConcentrationintheTreatedTissues 84
ResultsandDiscussion 84
EffectofAscorbicAcidonBrowningInhibition 84
AntibrowningPotencyofOxalicAcidinConjunctionwithAscorbicAcidon
AppleSlices 85
AntibrowningPotencyofOxalicAcidinConjunctionwithAscorbicAcidon
BananaSlices 98
APPLEPOLYPHENOLOXIDASEINHIBITOR(S)FROMCOMMONHOUSEFLY
(MuscadomesticaL.) 102
Introduction 102
vii
018
MaterialsandMethods 104
HouseFlyPupae 104
PreparationofInhibitorExtract 104
PreparationofApplePPO 106
AssayofPPOActivityandInhibition 108
ThepHOptimumofApplePPO 108
EffectofHeatingandFreezingandThawingonInhibitor(s) 109
ThepHStabilityofInhibitor(s) 109
GammaIrradiation 109
Ultrafiltration 11
ProteinDetermination 110
PreliminaryPurificationStudies 110
Acetoneprecipitation 110
Ammoniumsulfateprecipitationandcolumnchromatography 11
ResultsandDiscussion 112
OptimizationofpHConditionsforPPOActivityandInhibition 112
InhibitorActivityoftheHouseFlyDuringMetamorphosis 115
CharacterizationofCrudeInhibitor(s)FromHouseFlyPupae 117
Effectofinhibitorconcentration 117
Temperaturestability 11
ThepHstability 119
Gammairradiation 120
Ultrafiltration 120
PreliminaryStudiesonPurificationofHouseFlyInhibitors 122
Solventfractionation 122
Ammoniumsulfatefractionationandcolumnchromatography 122
CONCLUSIONS 125
LISTOFREFERENCES 127
BIOGRAPHICALSKETCH 141
viii
8
2- LISTOFTABLES
T3-able ESge
4-
2-1. RelativesubstratespecificityofPPOsfromappleandbanana 19
2. Estimatedoxalicacidcontentofsomecommonfoodsofplantorigin 33
1. ComparisonofinhibitoryeffectofdicarboxylicacidsonPPOactivity 54
1. ChangeinpHwithoxalicacidconcentrationinthetreatedtissues 74
6-1. Heatingeffectoninhibitoractivity 11
6-2. Ultrafiltrationofinhibitorpreparation 121
IX
LISTOFFIGURES
Figure page
22--1.Enzymaticandsubsequentnonenzymaticcondensationprocessesforbrowning 7
3-
2-2.ProposedmonophenolanddiphenoloxidationpathwaysofNeurosporacrassa
PPO 11
2-3.Enzymaticoxidationofthephenolicaminoacidtyrosinetomelanin 13
4.Mechanismofpreventionofenzymaticbrowningbyascorbicacid 29
1.EffectofpHonthecatalyticactivityofPPO 41
3-2.EffectofpHandoxalicacidconcentrationontheinhibitionofPPOactivity 41
3-
34--3.EfvfaelcutesofpreincubationontheinhibitionbyoxalicacidforPPOatdifferentpH 44
3-4.Kineticdeterminationofmodeofinhibition 45
3-5.Effectofcupricacetatetreated-oxalicacidonPPOactivity 47
3-6.Effectofaddedcopper,ataconstantenzymeconcentration,ontherestorationof
enzymaticactivitytooxalicacid-treatedPPO 49
3-7.Effectofoxalicacidontheabsorptionspectrumofthefinalproduct(s)of
PPO-catecholreaction 50
8.Incubationofoxalicacidwiththefinalproduct(s)ofPPO-catecholreaction 52
1.Videoimagesofoxalicacidtreatedbananaandappleslicesduringstorage 61
4-2.Changesinbrowning(Lo*-Lt*)oncutsurfacesofbananaasafunctionofoxalic
acidconcentrationandstoragetime 63
4-3.Browningindicesoncutsurfacesofoxalicacidtreatedbanana 65
4-4.Changesinbrowning(Lo*-Lt*)oncutsurfacesofappleasafunctionofoxalic
acidconcentrationandstoragetime 67
4-5.Browningindicesoncutsurfacesofoxalicacidtreatedapple 68
x