Extractionofproteinmixturefromrapeseedforfoodapplications

MScThesisSemaYonsel

Supervisors:CostasNikiforidis,SridharanLakshminarasimhChairGroup:BiobasedChemistryandTechnology(BCT)September2017-March2018

2

3

ExtractionofproteinmixturefromrapeseedforfoodapplicationsCoursename: MScThesisBiobasedChemistryandTechnologyNumber: BCT80436StudyLoad: 36ECTSDate: 26-03-2018Student: SemaYonselRegistrationnumber: 940909-980-010Studyprogram: MBT(MasterBiotechnology)Specialization: ProcessTechnologySupervisor(s): CostasNikiforidis,

SridharanLakshminarasimhExaminer: ElinorScottChairgroup: BiobasedChemistryandTechnology

4

5

AcknowledgmentsItwasagreatprivilegeformetowriteathesisatBiobasedChemistryandTechnology.Itwasachallengingandagreatexperience;everydayIlearnedsomethingnew.Studyinginthisdepartmenthelpedmetoimprovemyresearchskills,tolearntosolveproblemsandtogainknowledgeonanalysistechniques.Firstofall,IwouldliketothankCostasNikiforidis,forhisguidance,patienceinansweringallmyquestions,supportandsharinghisknowledgewithmeduringthisproject.IwouldliketoexpressmygratitudetoSridharanLakshminarasimhforbeingalwaystheretogivemeadvices,moralsupportandhepwithmyexperimentalplanning.Moreover,IwouldliketothankElinorScottforacceptingtobemyexaminer.IowespecialthankstothetechniciansofFBRandFBEforgivingmeintroductionaboutthelaboratoryequipmentandhelpingmewhenIneed.Lastbutnotleast,IwouldliketothankmyfamilyandmyfriendsatWageningenandbackhomebyencouragingmetogofurthereachtime.

6

ABSTRACTIncreasing demand on protein due to increasing world population, prompts to search alternativesourcesandenvironmentalfriendlyprocesses.Rapeseedisapromisingproteinsourcecomposedofmostlyoilandprotein.Thecurrentconventionalmethodcontainsade-fattingstepusinghexaneasasolvent.Theobjectiveofthisthesisistodesignagreenprocess(aqueousextractionprocess)toextractrapeseedproteinmixtureswithoutde-fattingandtoinvestigatetheproductionofemulsionsusingthisproteinmixture.Intheaqueousextractionprocess,onlywaterwasusedasasolvent.Toextracttheproteinmixture,alkalineextractionandisoelectricprecipitationwereapplied.Asaresult,aproteinmixturewithaproteinpurityof60wt%wasobtained.TheproteincharacterizationwasdonebySDS-PAGE,whereall3majorrapeseedproteinfractionswereidentified.Theobtainedproteinmixturewasusedasemulsifierinasolutionwith0.5gprotein/100mL.pHoftheemulsionswereadjustedto7and3.8, to compare the resultswith dairy and dressing type applications. In food industry stability ofemulsionsareofprimaryimportance.Physicalstabilityofemulsionsaredeterminedbydropletsizedistribution,dropletcharge,stabilityagainstcreamingandrheologicalcharacterization.Thestabilitywasobservedduring5daysofstorageandtheemulsionformedatpH7wasfoundtobemorestablethanatpH3.8.Proteinmixturesobtainedbyaqueousextractionprocessmayhaveahighpotentialinfoodapplications.

7

TableofContents

ACKNOWLEDGMENTS.....................................................................................................................5

ABSTRACT.......................................................................................................................................6

1. INTRODUCTION.......................................................................................................................8

2. SCIENTIFICOBJECTIVE.............................................................................................................9

3. BACKGROUND.......................................................................................................................103.1. COMPOSITIONOFRAPESEED......................................................................................................103.2. RAPESEEDPROTEIN..................................................................................................................11

3.2.1. AminoAcidContent....................................................................................................113.2.2. ProteinFraction..........................................................................................................11

3.3. RAPESEEDMARKET..................................................................................................................133.4. CURRENTAPPLICATIONANDSTUDIES..........................................................................................133.5. FOODEMULSIONS....................................................................................................................14

4.MATERIALSANDMETHODS.......................................................................................................164.1.MATERIALS...................................................................................................................................164.2.AQUEOUSEXTRACTION...................................................................................................................16

4.2.1.Protocol...............................................................................................................................164.3.COMPOSITIONANALYSIS................................................................................................................18

Moisturecontent.........................................................................................................................18Oilcontent....................................................................................................................................19Proteincontent............................................................................................................................19ProteincharacterizationbySDS-PAGE........................................................................................19

4.4.PREPARATIONOFOILINWATEREMULSION.......................................................................................204.5.EMULSIONCHARACTERIZATION........................................................................................................20

Measurementofsizedistribution................................................................................................20ζ–Potentialmeasurementandtitrationcurve............................................................................21Emulsionstabilityagainstcreaming...........................................................................................21Emulsionrheology........................................................................................................................21

5.RESULTSANDDISCUSSION.........................................................................................................225.1.COMPOSITIONANDCHARACTERIZATIONBEFORETHEAQUEOUSEXTRACTIONPROCESS............................22

ζ-PotentialandTitrationCurve....................................................................................................225.2.COMPOSITIONANDCHARACTERIZATIONAFTERTHEAQUEOUSEXTRACTIONPROCESS..............................23

SDS-PAGE.....................................................................................................................................255.3.EMULSIONSTABILITY......................................................................................................................26

SizeDistribution...........................................................................................................................26CreamingBehaviour....................................................................................................................28RheologicalCharacterization.......................................................................................................30

6.CONCLUSION.............................................................................................................................33

7.RECOMMENDATIONS................................................................................................................34

REFERENCES..................................................................................................................................35

8

1. IntroductionIncreasingworld population accompanies scarcity among usable nutritional sources in addition toenvironmentalproblems.Proteinsareoneofthebuildingblocksforthehumandiet,animalfeed,andalsoindustrialapplications.Inparallelwithanincreasingworldpopulationandincreaseinwealth,thedemand forprotein rises,especiallyanimalbasedprotein.However, consumptionofanimalbasedproteinhasnegativeimpacts,suchasgreenhousegasemissions,excesswaterusage,airandwaterpollution,andhabitatloss11.Theseconditionsleadconsumerstoshifttheirdietmoretowardsaplantprotein rich diet, and to search for novel protein sources and sustainable processing strategies toextractthem14.One of the promising protein sources are agricultural crops, such as oilseeds. Oilseed crops arecultivatedfortheiroilandprotein.Theextractedoilisusedfornon-ediblepurposessuchasbiodieselorediblepurposes,suchasfooddressingsorcookingoil.Theremainingsolidlayerafteroilextractionis called de-fatted meal (cake), which is rich in protein and mostly used as feed than for foodapplications.Thethreemostpromisingoilseedcropsworldwidearesoy,rapeseedandsunflowerduetotheirhighnutritionalvalue9,10.AccordingtotheUnitedStatesDepartmentofAgriculture(USDA),rapeseed is third in worldwide oil seed production1 and according to the Food and AgricultureOrganizationoftheUnitedNations(FAO),itisthemostimportantsourceforvegetableoilsintheworldafter soybean2. Furthermore, in the last decade rapeseed production increased along with therapeseedoildemand17.ThereasonoffocusingonrapeseedratherthanotheroilseedcropsisthatitcangrowunderwiderclimateconditionsinNorthernandWesternEurope,hasgellingcharacteristicsatlowertemperatures,ahighfattyacidcontent,aconsistentthermalandstoragestabilityandawell-balancedaminoacidcomposition12,23,34.Incomparisontothemarketleadersoybean,rapeseedhasalowercarbonfootprint(521gC02-eq/kgrapeseedmealvs.578gC02-eq/kgsoybeanmeal)34.Rapeseedismainlycomposedofoil(37-50%)andproteins(15-26%)12,13,24,30.Currentproteinextractionprocessesarefocusedonproducinghighpurityproducts,namelyrapeseedproteinisolates(RPI)andrapeseed protein concentrates (RPC) from de-fatted rapeseed meal (cake) by aqueous-alcoholicproteinisolationrecoveryprocedures.Where,thedrivingforceisthesolubilitydifferencesofmajorrapeseed proteins due to pH or ionic strength12,24,26,29,30,32. Hexane usage in the de-fatting step toextractoilandintensivepHchangesbringalongpoorfunctionalproductsandlimitationsforfeedandfoodapplicationsbecauseofhealtheffectsandenvironmentalsafety.Toovercomethosemalfunctionsandtogainhighpurityproducts,non-sustainableandhighcostmethodsarebeingapplied.Accordingtostudiesforfoodapplicationsonoil-watersurfacerheologyandemulsionstability,mixtureshavesameorevenbetterfunctionalpropertiesthanthepureproducts43,44.Therefore,amildprocessthatextract amixture ofmain components has been proposed as an alternative to current extractionprocessesforfoodapplications,namelyAqueousExtractionProcess(AEP)43,43,50,51.InAEP,de-fattingandpurificationstepsareskippedandonlywaterisusedasasolventtoavoidharshconditions,hazardouschemicalsandproductdenaturation.Asaresultoftheextractionprocess,ahighamount of protein, a significant amount of oil and aminor amount of rest of the biomass canbeextractedasmixture.InAEP,oilisgatheredasoilbodies(OB’s)thatarereleasedintoaqueousmediaright after the protein diffusion44,50,51. On the contrary, in current conventional processes, proteinremaininthede-fattedmealwithinamixtureofcarbohydratesandfiberwhileoildissolvesintothesolvent. In this thesis, the obtained proteinmixture by theAEPwas used tomake an oil-in-wateremulsion tounderstand the functionalproperties for further foodapplications.Thewaste streamswerenot a concernof this thesis.However, they canbe consideredas a feed sourcedue to theirnutritionalvalue.

9

Emulsionbasedfoodproductsareoneofthefoodapplicationswheretheproteinfromoilseedcropscanbeused.Ingeneral,forallfoodproducts,stabilityisakeyfactor.Chemicalandphysicalstabilitydependsontheoilseedcompositionandtheextractionmethod45.Therefore,interfacialpropertiesandstructurecharacteristicsofspecificfractionsshouldbestudiedtounderstandtheapplicability.Incaseofemulsionfoodproducts, thecomponentmustbe incorporated intotheemulsionfoodproducts.Sincethede-fattingstepusinghexanewasomitted,thelikelihoodofdenaturationofsubstancesaredecreasedandprotein fractionscanbegathered in theirnative form43,44. Due tosimple,mildandgreenextraction,omittedpurificationtreatmentstepsandwaterusageassolvent,itisexpectedthatahighphysicalandchemicalstabilityandnutritionalvaluecanbeachieved.

2. ScientificObjective1. Todesignaprocessforextractionofproteinmixturesfromnon-defattedrapeseeds.2. Toinvestigatetheproductionofemulsionstabilizedbytheaqueouslyextractedproteinmixtures.

10

3. Background3.1. CompositionofRapeseedRapeseedisamemberoffamilyBrassica.TherearethreemajorspeciesnamelyB.napus(cultivatedinEuropeandCanada,havingablackorreddishcolor),B.rapa(cultivatedinCanada,havingablackoryellowcolor),B. juncea (cultivated inCanada,havingayellowcolor).Thedifference ismainly inoilcontent33.Dependingontheplantingseason,eitherfallorspring,rapeseediscalledwinterrapeseedor spring rapeseed, respectively22,27. It can grow with less sunlight and at lower temperaturescompared toothercrops22,27.Thereare two typesof rapeseed: industrialandedible (which isalsocalledCanola).Thedevelopmentoftheediblerapeseed,Canola,beganinCanadaandEurope,withtheaimtoimprovethequalityofextractedoilbyhavinglessthan2%oferucicacidandhavingalowlevelof glucosinate in thede-fatted rapeseedmeal (as30micromoles/gmeal)19,24. Erucicacidandglucosinatemakerapeseednon-edible.For instance,thenon-edible industrialrapeseedhas55%oferucicacidandhighlevelsofglycosinolate27.Thethreemajorpartsoftherapeseedare:embryo(composedofcotyledon,hypocotylandradicle),endospermandseedcoat(surroundstheembryoandtheendosperm)29.(Figure1).

The highest protein (approximately 25 %) and oil(approximately55%)contentareinthecotyledoncomparedtowholeseedandseedcoat29.Theweightoftheseedcoatis inverselyproportionaltotheseedquality.TheseedcoatcontainsN-basedcomponentswhichareproteinsandnon-proteins,wherethemajorcontributorsofnon-proteinsareglucosinolatesandtri-aminecompounds29.

Figure1.Acut-awaythree-dimensionalmodelofrapeseed35Rapeseedasawholeismainlycomposedofoilandproteins,followedbycarbohydrates,lignin,ashandsecondarymetabolites(phenoliccompounds).Studiesshowdifferentrangesforthecomposition(Table1).Thecontentdependsonthecultivar,temperatureandweatherchangesduringmaturationperiodandamountofsulphurwithintheseed12.Table3.1.AverageofReportedCompositionoftheEdibleRapeseed(Canola)*12,13,18,24,29

Oil(%) Protein(%) Carbohydrates(%) Lignin(%) Ash(%)43 21 14 8 5

*Componentcompositionsgivenindrymattercontent(dm%).Averagecontentcalculatedusingtheaverageofavailabledata,whichwillbeusedasacomparisonfortheresultsofthecompositionanalysis.Theoilcontentintherapeseedconsistsmostlyoffattyacids28.Theproteincontentconsistsofthreemajorproteinfractions:thetwostorageproteingroups(SSP’s):cruciferin(asglobulin)andnapin(asalbumin), which aremajor proteins of the cotyledon extract, and amembrane protein (structuralprotein),namelyoleosin8,12.Thefocusofthisresearchisontheproteincontentandproteinfractions,adetailedexplanationgiveninPart3.2.Thecarbohydratescompositionofrapeseedcanbedividedinto3groups:solublesugars,insolublecarbohydratesandfiber33.Sucroseisthepredominantsugar;cellulose,hemicelluloseandpectin30formtheinsolublecarbohydratefraction33.Fibersarefoundinthe seed coat, endosperm, and cell walls of the cotyledon cells. They contribute mostly to thecarbohydratefraction30.Phytatesareanothercomponentthatassociateas insolublecrystals inthe

Seed Coat Outer Cotyledon

Inner Cotyledon Radicle/

Hypocotyl

11

proteinstoragevacuoles(PSV)intheseed.Theyarerelatedtocarbohydratesintermsofstructure33.SaltsofCa,MgandKorphyticacidarereferredtophytates30.3.2. RapeseedProtein3.2.1. AminoAcidContentRapeseed,aplantbasedproteinsource,isapromisingalternativetoanimalbasedproteinsources.Inplantbasedproteins,proportionandvarietyoftheaminoacidsdependontypeoftheplant.Generally,plantproteinslackofoneormoreaminoacids,butplantbasedproteindietismoresustainableandhavehealthadvantages37.Rapeseed knownashaving abetter balanced aminoacid composition compared tootheroilseedscontaineightoutofthesenineessentialaminoacidsexcepttryptophan19.Comparedtosoy,rapeseedproteinhasahighercysteineandmethioninecontent,whicharesulphurcontainingaminoacids26.Itshouldbeconsideredthattherearelimitsforaminoacidconcentrationsinhumandiet.Surplusorlackofaminoacidscancausehealthissues.Theproteinextractionmethodinfluencestheconcentrationofamino acid compositions19,24. Therefore, protein extraction from rapeseed should be designedcarefullydependingontheapplicationoftheproductinfoodsector.3.2.2. ProteinFractionProteins in plant seeds can be located in the cell as protein bodies, in themembrane, or can bestructural.Toobtainproteinsthatareinthecell,thecellwallshouldberupturedbyapre-treatment.Toobtainmembraneproteins, lipid layersshouldbeseparated38.Rapeseedhasthreemainproteinfractions,namelycrucifein,napinandoleosin.Theprimary,secondaryandtertiarystructuresofthemdiffer highly, thus they have different functionalities and properties29. The predominant storageproteins, cruciferin andnapin, are located in protein storage vacuoles of embryonic tissues of theseed29.InEurope,theratioofcruciferintonapinvariesbetween0.6to0.2dependingonerucicacidandglucosinolatelevels30.Oleosinisastructuralproteinthatisfoundinthemembraneoftheorganellecalledoilbody(OB)whichistheoilstorageoftheseed24.CruciferinCruciferinisa12Sglobulinanditbelongstothecupinsuperfamily12,29.Itisthepredominantproteininrapeseed,responsiblefor60%oftotalproteincontent12,29.Cruciferinhasamolecularmassof300-360kDaandanisoelectricpointofpH7.2529.Itisaneutralprotein,whichcanactasgellingagentinits native form12,29. It is a salt soluble protein29. In despite of the limited information about theirbioactivity, it is stated that amino acids of cruciferin play an important role in gastro-intestinaldigestion30.

Thecruciferinhassixsubunitsstructuredintwotrimerunits(Figure2).Thetwo trimer units combine with each other by non-covalent bonds ashydrophobic,electrostatic,hydrogen,VanderWaalsorhydrogenbondedsalt bridges. Each protomer composed of two polypeptide chain linkedwithadisulfidebond,wherethepolypeptidesareacidica-(approximately40kDa,254to296aminoacids)andbasicb-(approximately20kDa,189to191)aminoacids24.

Figure2.Crystalstructureofcruciferin39(rcsb.org)

12

NapinNapinbelongstotheprolaminsuperfamily29.Itisa2Salbumin(or1.7S),whichisthesecondmajorcontributortotheproteincontent,comprising20%oftotalproteinintherapeseed12.Theamountofalbuminisrelatedtotheamountofsulphurcompoundspresentedintheseed,whicharerelatedtosulphurcontainingaminoacidssuchascysteineandmethionine12.Napinisknownasthenitrogenandsulphursupplieroftheseed,duetoitsabilitytomobilizeduringgermination;havinghighcontentofproteinbodiesandsubstantialaminoacidcomposition30.Furthermore,theyshowantifungalactivitiesinrapeseed30.

Napinhasamolecularmassof8-15kDa12. It has a strongalkalinity and canbesolubilizedinwater12.StudiesshowthatnapinhashighthermalstabilityandhighstructuralstabilityamongwidepHranges,whichcanbesolubleatlowpH’ssuchaspH2-429,30.AtpH11,napindoesnothaveanetelectriccharge29.

Figure3.Crystalstructureofnapin40(rcsb.org)Thematurenapinstructurecomposedofonesmall(4-6kDa)andonelarge(10-12kDa)polypeptidechainlinkedviafourdisulfidebonds(twointer-andtwointra-chaindisulfidebonds)(Figure3).Thelargepolypeptidechain is composedof two intra-chaindisulfidebondsbetweencysteine residues,whichstabilizethenapinmoleculewithfourdisulfidebridges24.OleosinOleosinisastructural(membrane)proteinthatcompromises2-8%ofthetotalproteininrapeseed.Itisanalkalineproteinwithalowmolecularweightaround15-20kDa.12,24,42.Oleosinisalsoknownasthedominantoilbodyprotein(OBP)inrapeseedwhichassociateswithoilbodies(OB)12,24.OBP’saresubcellularorganellesthatstabilizeOB’s38.Theyhavealonghydrophobicandahydrophilicdomain,whichcanassociatewiththelipidphaseandthesurfaceoftheOB,respectively24.

OB’s are subcellular hydrophobic organelles that store oil,which consist of storage triacylglycerols (TAG) and aresurrounded by phospholipids (PL) and oleosins (Figure 4)38.TAG, PL and oleosin content varies due to the size of OB’s,where it is in the rageof0.6-2.0µm.The sizeofOB’s variesdependingon the typeof theoilseed, environmental factorsandavailabilityofnutritionalrequirementssuchasnitrogenforoleosinsandphosphateforPL38.

Figure4.Proposedstructureofoilbodies41TheisoelectricpointofOB’sisdeterminedintherangeofpH5.7to6.6,whichmeansthatatneutralpH,thesurfacechargeisnegative.Theisoelectricpointvariesamongtheseedtypes38.TAGactsasacarbonandenergysourcefortheseedgerminationandgrowth38.TheoutersurfaceofTAG,phospholipid-proteincomplex,providesphysicalandchemicalprotectionagainstenvironmentalstresses,likemoistureandtemperaturechangesandpresenceofoxidativereagents.Oleosinactasasize-regulatorforoilbodies,whereOBsizeisinverselyproportionaltooleosincontent.Furthermore,it isresponsibleforfreezingtolerance,whichcreatesabenefit fortheseedthat itcangrowat lowtemperaturesandsurviveharshwinterconditions42.Oleosinhasanegativelychargedsurface.Thisis

13

why itcanstabilizeandpreventcoalescenceof theOB’sbycreatingsterichindranceandelectricalrepulsion24,42.IthasanN-terminal(N)amphipathicdomain(havingbothhydrophobicandhydrophiliccharacterization),acentralhydrophobicdomainandaC-terminal(C)amphipathica-helicaldomain.NandCdomainsareexpectedtoeasetheassociationwithphospholipidsurfaces38.3.3. RapeseedMarketWorldwide,thethreemostpromisingoilseedcropsare:soy,rapeseedandsunflowerduetotheirhighnutritionalvalue9,10.Globalrapeseedproductionin2017/2018isestimatedasapproximately70millionmetrictons(MMT)bytheUnitedStatesDepartmentofAgriculture(USDA)1.AccordingtoFoodandAgriculture Organization of the United Nations (FAO), rapeseed is the second leading source forvegetableoilsintheworld,aftersoybean2(Figure5).AccordingtoFAOSTAT,from1994to2016,Chinawasthetoprapeseedproducer(11.8millionMT),followedbyCanada(10.4millionMT)andIndia(6.3millionMT)asanaveragethroughyears16.IntheEuropeanUnion(EU),whererapeseedisusedmainlyasasourceforbiodiesel,GermanyandFrancearethemainproducerswithapproximately4millionMT16. Therefore, fromaneconomicalpointof view, rapeseed is apromisingalternativeasproteinsourcecomparedtoothervegetableoilseedsandanimalproducts.

Figure 5.Worldwide production of threemajor oilseed crops, data gathered fromOffice of GlobalAnalysis(OGA)153.4. CurrentApplicationandStudiesCurrentlyindustrialproductionwithrapeseedismainlyfocusedonoilextractionforedibleandnon-edibleusage.Theextract,which is calledde-fattedmeal (cake), is containingahighpercentageofvaluable protein which is utilized as a high protein biomass source in aquaculture and livestockindustries9,18,19.Highproductioncostspreventindustriestotakeadvantageoftheproteininrapeseedfor foodapplications rather than feed.Therefore, studiesmustbecontinued to reduceproductioncostsandincreaseyields.Inindustry,therapeseedoilisutilizedforbiodieselsinceitisarenewableenergysource,environmentfriendlywithlowcostextractionroute.Havinghighyieldsofoilperhectare(1,188to1,497litresperhectare),gellingcharacteristicat lowertemperatures,andhighfattyacidcontentcomparedtotheother vegetable oils make the rapeseed oil a competitive feedstock for biodiesel production. For

050

100150200250300350400450

2013 2014 2015 2016 2017 2018

MillionMetric

Ton

Year

Worldwide ProductionofThreeMajorOilseedCrops

Sunflower

Rapeseed

Soybean

14

example, in Europe, non-edible rapeseed is the most common used oilseed for biodieselproduction12,23.Furthermore,thelowsaturatedfatcontent,nutritionalvalueandconsistentthermalandstoragestabilityofrapeseedoilenablesinadditiontocookingoilawideapplicationrangeasafoodadditivesuchasfrommayonnaiseandsaladdressingstomargarinesandbakeryproducts20.Research for food application by using RPI and RPC demonstrated that the rapeseed protein canpotentiallybeusedinbakeryproducts,beverages,dairyandeggsubstitutes,processedmeatproducts,salad dressings and sauces flavourings12,19,24. The reason is that RPI and RPC show high nitrogensolubility at neutral pH26. As an example, RPC is a promising source for replacingmilk and othervegetableproteinsinsausage12,19,24.Moreover,accordingtoWHO,eventhedailyproteinintakecanbeachievedbyRPIandRPCsincede-fattedrapeseedmealcontainsrecommendedamountofessentialaminoacids3.However,de-fattedrapeseedmealalsocontainsglucosinolates,phenolics,phytatesandhighamountoffibers,whichhavenegativeeffectsondigestibility,colorandtasteoftheproduct19.Therefore,extensivestudiesareongoingforfoodapplications.Furthermore, studieswithRPI for filmswithwaterbarrierproperties,hydrogel as superabsorbent,protein based surfactants/foams/interface active molecules, protein-based plastics, protein-basedadhesivesandnanoparticlesforcontroldeliveryofbioactivesgavepositiveresults19,24.3.5. FoodEmulsionsEmulsionsconsistof two immiscible liquids,namelydispersedphaseandcontinuousphase (Figure6.a).Whereoneofthephasesisdispersedasdroplets intotheotheronebyshearingforce(Figure6.b)59.Themostcommonemulsiontypeisoil-in-water(o/w),suchasmayonnaiseandfooddressings.O/w emulsions comprise of the dispersion of small oil droplets into the aqueous phase byhomogenization.Homogenization isreferredtoasan intensemechanicalagitation infood industry(see Figure 6.c for emulsified state after homogenization) 45. Food emulsions have a large phaseboundaryorinterfacialareabetweencontinuousanddispersedphases,whichcontainsfreeenergy.Thesystemtriestoreducetheinterfacialfreeenergybyreducingtheinterface.Therefore,toincreasetheinterfacialareaanexternalforceisneeded59.

Emulsionsareunstablesystems;theirphysicochemicalpropertiescanchangeovertimeduetofurthertreatmentand/orstorage,sinceoildropletstendtoaggregateduetotheirrelativelyweakelectrostaticrepulsionforceswithrespecttoeachother.Thiscanleadtophenomenasuchaspartialseparation,flocculationandcoalescencewhichareundesirableforfoodapplications(Figure6.d).Stabilizersareused to prepare kinetically stable emulsions. Emulsifier is a type of stabilizer, which preventsaggregation of oil droplets due to its surface-active characteristics. Surface-active substances arecreatingaprotectivecoataroundthedropletbyadsorbingthesurfaceofit44,45.Emulsifierslowerthesurface free energy by adsorbing phase surface,which lowers surface tension and diminishes theinterfacialtensionbetweenthetwointerfacesofdispersedandcontinuousphases.Besides,reductionofinterfacialtensionandinterfacedeformationbecomeeasier59.

15

Figure 6. States of an emulsion. a: dispersed phase (oil) and continuous phase (water) beforeapplicationofshearingpower.b:Oildropletsaredispersingintothewaterphase.c:Emulsifiedstateafterhomogenization.d:Thermodynamicallyunstableemulsionafterstorageperiod.

Proteins are one of themost preferred emulsifiers, since they can enhance physical and chemicalstabilityofoildropletswithin theaqueousphase44,45. Inemulsions,proteinshave thecapability toenhancethestrengthofrepulsivestericforcesbetweenthedropletsthatareneartoeachother.Thisenhancement cause high stability against coalescence,which is needed for long storageperiods45.Furthermore, proteins are advantageous emulsifiers since they can rapidly absorb, change and re-arrangethenewlycreatedo/winterface.Duetotheirintermolecularinteractions,proteinscanformacohesiveandviscoelasticfilmoverthesurfaceoftheoildropletinanemulsion19.Toformaproteinemulsion, adsorption at the interface (usually occurring via hydrophobic interaction), reduction ofinterfacial energy and surface denaturation are required. Protein denaturation at the interfaceprovidesastableconformationandreduces interfacial freeenergy30.Therefore,AEPwillprovideastabilityadvantagesincetheproductwillconsistofamixtureofproteinfractionswhicharecruciferin,napinandoleosin.Furthermore,recentresearches67statedthatinemulsionsproteinmixturesprovidehigherstabilityoveroildropletsratherthanpureproteinisolates.

a b c d

16

4.MaterialsandMethods4.1.MaterialsFoodgraderapeseedwaspurchasedfromRagt(RagtSemences,France)andcanolaoilwasgathered(AlbertHeijn,TheNetherlands)Sodiumhydroxide(NaOH)waspurchasedfromSigmaAldrich(Sigma,USA).Hydrochloricacid(HCL,37%v/v)waspurchasedfromAnalaRNORMAPUR(France).Petroleumether40-60oCPECgradewasboughtfromACTU-ALL(ACTU-ALLChemicalsBV,TheNetherlands).Allchemicalswere analytical grade. In all experiments, demineralizedwaterwasusedexceptbuffers,whichwasMiliQwater obtained fromMerkMilipore (Merk, Germany). All the experimentswerecarriedoutatroomtemperature(23°C)andalltheanalyseswereperformedintriplicate.

4.2.AqueousExtractionStudiesregardingAEPismostlybasedonsoybeanforsimultaneousoilandproteinrecovery50,51.Forrapeseed,thefocusismostlyontheoilextractionorpureproteinisolationand/orconcentrationfromde-fattedrapeseedmeal.ThereisnootherstudyfortheproteinextractionfromwholerapeseedusingAEPmethod.Therefore, in this study, theextraction routewasbasedon theoil extractionbyAEP49,50,51.Severalmodificationsweredoneinordertogainaproductthatconsistsofmainlyproteins.Themodificationswereinfluencedbypreviousstudiesthatfocusonproteinisolationmethod12,19,29,32,48.Thede-hullingandde-fattingstepswereskippedtoinvestigatetheapplicabilityoffoodemulsionsinasimple,sustainableandmildway(Figure3.1).ThisstudydoesnotconcerntheoptimizationofAEPprocess.4.2.1.ProtocolWholeseedswerepulverizedusinganuniversalmill,IKAM20(IKA-Works,Germany),for70secondsat20,000rpm.Rapeseedflourwassoakedindemineralizedwaterwitharatioof1:7(w/v),atroomtemperature.pHwasadjustedto8using1.0MNaOH.pHwasadjustedevery15minutestosolubilizeproteinsinrapeseedasmuchaspossible.Meanwhilethemixturewascontinuouslystirredfor2hoursbyanoverheadstirrer(IKAEUROSTAR40digital,Germany),at150rpm.Themixturewasstoredinrefrigerator(4oC),overnight.Thenextday,themixturewassubjectedto intensiveagitationwithablender (Philips, The Netherlands) for 1 minute at maximum speed. After blending, the pH wasadjustedagainto8.Theblendedmixturewasfilteredthroughtwotimesfoldedcheesecloth(Localmarket, theNetherlands) to collect solubilizedprotein. The remaining solid phasewasmixedwithdemineralizedwaterwitharatioof1:7(w/v)andthemixturewasblendedandfiltratedagain.Thetwopermeates were mixed with an overhead stirrer at a speed of 150 rpm for 10 minutes to get ahomogeneous mixture and the pH was again adjusted to 8. The mixture was subjected to twocentrifugations(SorvallTMLegendTMXFR,ThermoScientific,USA).Thefirstcentrifugationwasrunat3,000 xg for 15 minutes at 4oC. The centrifugation process creates three streams: an oil fractioncontaining stabilized-oleosomes (supernatant/cream), an aqueous phase (subnatant), and a smallresidualsolidphase(pellet)thatprimarilyconsistsofcellulosicandhemicellulosicmaterialsfromthehulls. The pellet was removed and the cream and subnatant were collected and mixed with anoverheadstirrer.Themixturewasthensubjectedtoasecondcentrifugation,runat10,000xgfor30minutesat4oC.Thecreamandpelletwereremovedandthesubnatantwasrecoveredandstoredat4oC,untilfurtheruse.Toobtaintargetproteinfractions,acidicprecipitationwasappliedaccordingtotheisoelectricpoint(pI)oftherecovered liquidphase.To indicatethe isoelectricpoint,a ζ-potentialmeasurementwasexecutedandthepIwasdeterminedfromthetitrationcurve.

17

Figure3.1. Flowchartofproteinextractionprocess, the cross symbolizes thewaste streamand themagnifiersymbolizesfurtheranalyses

StorageOvernight,4

OC

Grinding2,000rpm,70s,

23oC

SoakinginalkalinemediapH8,1MNaOH,1:7(w/v),

2h,150rpm,23oC

Filtration2timesfolded

cheesecloth, 23oC

Mixing10min,150rpm,23

oC

AcidicprecipitationpHatpI (≅5),0.5MHCl,

1h,200rpm,23oC

Centrifugation3,000xg,15min,4

oC

Freezedrying3days

Rapeseed Rapeseedflour Mixture

Permeate(2)

Solidresidue

BlendingpH8,Maxspeed,1min,

23oC

Mixture

Mixture

Soakingindemineralizedwater

1:7(w/v),10min,150rpm,

23oC

BlendingMaxspeed,1min,23

oC

Mixture

Filtration2timesfolded

cheesecloth,23oC

Mixture

Solidresidue

Permeate(1)

MixingpH8,10min,150rpm,

23oC

1st Centrifugation3,000xg,15min,4

oC

Pellet

Creamand

subnatant2nd Centrifugation

10,000xg,30min,4oC

Permeate

mixture

Creamand

subnatant

mixture

Creamandpellet

Storage4

OC

Subnatant

AEP

StorageOvernight,-18

OC

Subnatant

Creamand

subnatant

PelletMixture

ProteinPrecipitation

Pellet

Pellet:proteinmixture

ProteinConcentration

18

ζ-PotentialmeasurementandtitrationcurveAnaliquot(10mL)ofsubnatantwasfirstfiltratedusingfilterpapercircles,604grade,25µmporesize(150mm)(WhatmanSchleicherSchullGmbH,Germany)andafunnel,togetaclearsolution.ThenthesubnatantwasdilutedwithMiliQwaterwitharatioof1:100(v/v).The ζ-potentialwasmeasured in order to determine themagnitude of the electrostatic or chargedependentrepulsionorattractionbetweenparticles.Measurementwasdoneusingadynamiclightscatteringinstrument(MalvernZetaSizer,NanoZS,MalvernInstrumentsLtd,UK).Thereflectiveindexwassetforprotein,at1.450.A titration curve was plotted to determine the isoelectric point (pI) of the subnatant. For pHadjustment, an auto titrationwasused (Malvern Instruments Ltd,UK). ThepHwas adjustedusingNaOH(0.1Mand0.5M)andHCl(0.5M)solutions.ThepHrangewasbetween2to12withincrementsof0.5.Eachmeasurementwasdone in12runswith3replications.ThedeterminedpIwasusedtoprecipitatethetargetproteinfractions.ProteinprecipitationTheliquidphasewascollectedfromtherefrigeratorandtheisoelectricprecipitationwasperformedusing0.5MHCl.ThepHadjustmentwasdoneaccordingtotheobtaineddatafromthetitrationcurve.AdjustmentwithHClcontinueduntil stabilization,meanwhile thesolutionwascontinuouslystirredwithanoverheadstirrer(IKAEUROSTAR40digital,Germany)at200rpm.AfterpHstabilization,thesampleswerecentrifuged(SorvallTMLegendTMXFR,ThermoScientific,USA)at3,000xgfor15minutes,at4oC.Thepelletwascollectedandspreadthroughinaplasticboxtogetamaximumsurfaceareaforthesubsequentfreeze-dryingstep.Thenthepelletwasstoredinafreezer(-18oC),overnight.FreezeDryingInordertopreservethestabilityofproteins,lyophilisationwasappliedbyusingafreezedryer(Christ,Germany).Thepelletwascollectedfromthefreezerafterastorageperiodandplacedintothefreezedryer.Theplasticboxwascoveredwithlaboratorytissuethatwastwotimesfoldedtoavoidmoistureduringtheprocess.Freezedryingtookthreedays:48hoursofmaindrying(0.011mbar)and24hoursoffinaldrying(0.0010mbar).Afterthreedays,thefreeze-driedpelletwascollectedandstoredinaScottbottlesealedwithpara-filmandalid,afterwhichitwasstoredat4oCuntilfurtheranalysisandproductionofemulsions.For the composition analysis, standardmethods of AOACwere used to indicatemoisture, oil andproteincontentswhichwereperformedintriplicate.4.3.CompositionAnalysisMoisturecontentThe moisture content of rapeseed was determined in rapeseed flour. One gram (1 g) of groundrapeseedwasdriedinanincubator(MemmertUniversalOven,TheNetherlands)at50oC,overnight.Driedsampleswereplacedintoadesiccator(DuranDN300,Germany)toavoidmoisturegenerationduringcoolingdown.Themoisturecontentinpercentagewasdeterminedgravimetrically,wherethedifferenceofsampleweightbeforeandafterthedryingwasdividedbythesampleweight.Analysiswasperformedintriplicate.(AOAC24.003).

19

OilcontentTheoilcontentofrapeseedwasdeterminedinrapeseedflourandinproteinmixtureusingaSoxhletapparatus,BehrotestEZ100H(BehrLaborTechnik,Germany).Theextractionrunfor12hours.Themodified standard method of AOAC57 was applied and the analysis was performed at roomtemperature,intriplicate.(AOAC7.060-7.062).

Samplesweredriedbeforetheoilextractiontoavoidmoisture,sincesomewater-solublematerialswouldbeextractedandgivefalsemeasurementintermsofoilcontent.Forrapeseedflour,ovendryingwasused,asexplainedinthemoisturecontentpart;forproteinmixture,thefreeze-driedproductwasused.Onegram(1g)ofeachsamplewasplacedintocelluloseextractionthimbles,30mmx80mm(GEHealthcareLifeSciencesWhatmanTM,UK).Oilwasextractedfromthesamplesbypetroleumether(200ml/sample).Tocontrolboilingand toavoidsuperheating,5boilingchips (Lamers&PleugerBV,TheNetherlands)wereaddedintoeachroundbottomflaskthatcontainedthesolvent.Heatersweresetat70%duringtheprocess.Aftertheoilextraction,oilcontainingroundbottomflasksweretransferredintoanincubator(MemmertUniversalOven,TheNetherlands)setat50oCfor1hourtoevaporatetheexcess solvent. The oil content (%) of the samples were determined gravimetrically, where thedifferenceofroundbottomflaskweightbeforeandaftertheoilextractionwasdividedbythesampleweight.ProteincontentThe Kjeldahlmethodwas used to determine the protein content of rapeseed flour and gatheredprotein mixture after freeze drying by using block digestion and fume suction systems (Gehardt,Germany).ThetitrationwasdonebyacompacttitratorforStatapplications(Metrohm,Switzerland).Ingeneral,theKjeldahlmethodisusedtomeasurenitrogencontentinasample.Toconvertnitrogentocrudeprotein,aconversionfactorofboth5.712,33,46,47and6.25wereused.Thefactor6.25wasusedtocomparethedatawithsimilarstudiesandwithsimilaroilseedcrops.Required sample amount was calculated according to the estimated protein content (1 mgprotein/sample). Samples were dried as stated in the moisture content part, to avoid faultymeasurementcausedbymoisture.TheanalysiswasdoneaccordingtotheFBRdepartmentprotocolin respect to standardmethods of AOAC57 (AOAC992.23), at room temperature, in triplicate. TheproteincontentofeachsamplewascalculatedusingEquation3.1.

Protein(mg)= ml-blanc ×0.1×14×KF (3.1)Where;0.1[M]istheHClconcentrationthatisusedintitration;14[g/mol]isthemolecularweightofnitrogen;KFistheKjeldahlfactor.ProteincharacterizationbySDS-PAGETheproteinprofileofrapeseedwasdeterminedbothforrapeseedflourandproteinmixture.Samplesde-fattedusingSoxhletapparatuswereusedtoavoidexcessoil.The solid samplepreparationwasdoneaccording to FoodQualityDepartment (WageningenU&R)protocol.Therequiredamountofsamplewascalculatedaccordingtotheproteincontentpercentage(1mgprotein/mL).Samplesweremixedwith250µlsamplebuffer(NuPAGELSDSampleBuffer),asstated in the protocol, in 1.5 mL Eppendorf tubes (Greiner bio-one, Germany). For the reducedcondition,samplesweredissolvedinthesamplebufferwithadditionof100µlreducingagent(NuPAGESampleReducingAgents),and650µlofMiliQwater(MerkMillipore);forthenon-reducedcondition,only750µlofMiliQwater(MerkMillipore)wasadded,asstatedintheprotocol.Preparedmixtureswereexposedtothreefreeze-thawcycles.Freeze-thawcyclewasappliedbyplacingthemixturesinto

20

afreezerat-18oCfor2hoursandthenmixingfor10mininaThermomixer(EppendorfAG,Germany)at90oC,300rpm.Afterthreefreeze-thawcycles,thesupernatantwasrecoveredbycentrifugation(VWR,EppendorfAG,Germany),whichperformedfor1minat2,000rpm,23oC.12µlsampleand10µlproteinmarker(PageRulerPlusPrestainedProteinLadder10-250kDa,ThermoScientific,Lithuania)wereloadedintoanelectrophoresisgel(NuPAGETM4-12%BisTrisGel,Invitrogen,USA).Thegelwasplacedinageltank(LifeTechnologiesMiniGelTank,ThermoScientific,TheNetherlands)andrunfor1hourat200V,45mAusinganelectrophoresispowersupply(VWRPowerSource300V,USA).ThenthegelwaswashedwithdemineralizedwaterandstainedwithSimplyBlueTMSafeStain(InvitrogenTM,USA).Thegelwasstoredatroomtemperatureunderslowshaking,overnight(Innova®44IncubatorShakerSeries,VWR,USA).Afterde-staining,gelwasphotographedwithaCanonEOS100D.4.4.PreparationofOilinWaterEmulsionEmulsion experiments were performed using the mixed protein mixtures, that were obtained byaqueousextractionmethod.Threedifferentbatchesofproteinmixtureswerecombinedinordertoguarantee an equal protein concentration of 1 g protein per sample.Oil inwater emulsionswerepreparedastriplicate.Theemulsionscontained10goil/100mLand0.5gprotein/100mL.First, thepHof theprotein-watersolutionwasadjusted to7,using0.5MNaOH.Thesolutionwasstirredwithamagneticstirrer(VWR,USA)for2hoursatminimumspeed,forpHstabilization.Then,the solutions were pre-mixed with a high-performance dispersion instrument (Ultra Turrax®, IKA,Germany)at8,000rpm,for1min.Pre-mixedsolutionsweresubjectedtohomogenizationbyahigh-pressurehomogeniserfor6runs(Lab-Homogeniser,DeltaInstruments,TheNetherlands).10mLfromeachsamplewerestoredinafridge(4oC)for3hours,whichwasrequiredforemulsionstabilization.Thestoredsampleswerethenusedforsizedistributionandζ–potentialmeasurements.Remainingemulsionsamplesweredividedintotwoportions.ThefirsthalfwaspreparedforemulsionstabilityanalysisandtheotherhalfwassubjectedtopHvaluechange.ThepHvalueofthesampleswasadjustedto3.867,byusing0.5MHClsolution.Emulsionswerestirredwithamagneticstirrer(VWR,USA)for20minatminimumspeed,tostabilizethepHvalue.10mLfromeachsamplewereagainstoredinafridge(4oC)forsizedistributionandζ–potentialmeasurements.Remainingemulsionsampleswerepreparedforemulsionstabilityanalysis.Thebriefexplanationoffurtheranalysiscanbefoundin4.5.EmulsionCharacterizationpart.4.5.EmulsionCharacterizationMeasurementofsizedistributionIn an emulsion, the size of the droplets have a strong effect on the stability45. Therefore, the sizedistributionoftheemulsionisanindicatorforfurtherapplications.Todeterminethesizedistributionoftheemulsions,alaserlightscatteringinstrumentwasused(MalvernMasterSizer3000,MalvernInstrumentsLtd,UK).Theanalysiswasappliedonfreshandaged(5days)emulsions,atbothpHvalues3.8and7.Thesampleswereanalysedintriplicateatroomtemperature.Beforetheanalysis,theemulsionsgatheredfromthefridgewerestirredwithaspatulafor2minutes,togetahomogenousdispersion.Then,smallaliquotswereaddedintoamixingchamber,forwhichtheobscurationratewassetat5to15%.Particlerefractiveindexwassetto1.46756(forcanolaoil)anddispersantrefractiveindexsetto1.3356(forwater).Thestirrerspeedwassetat1200rpm.Themeasurement cycle was repeated 5 times with a delay of 10 seconds. The measurements were

21

reported in respect to thesurfaceweighted(d32=∑nidi3/∑nidi

2,ni:numberofdropletspresent)andvolumeweighted(d43=∑nidi

4/∑nidi2)meandiameter.

ζ–PotentialmeasurementandtitrationcurveThe electrical characteristics of a droplet surface in an emulsion, indicates the tendency toaggregate33,45.Ingeneral,topreventaggregation,ionicemulsifiersareusedtoabsorbthesurfaceofthedroplet45,59.Tobeableto indicatetheelectricalcharacteristicsofadropletsurface,ζ–Potentialmeasurementwasdoneusing adynamic light scattering instrument (Malvern Zeta Sizer,NanoZS,MalvernInstrumentsLtd,UK).Fortheanalysis,theemulsionwasdilutedwithMiliQwaterwitharatioof1:100(v/v).Thereflectiveindexwassetforprotein,at1.450.Themeasurementtookplaceatroomtemperature.To determine the isoelectric point (pI) of the emulsion, a titration curve was plotted. For pHadjustment, an auto titrationwasused (Malvern Instruments Ltd,UK). ThepHwas adjustedusingNaOH(0.5M)andHCl(0.5M)solutions.ThepHrangewasbetween2to12withincrementsof0.5.Measurementwasdonein12runswith3replications.

EmulsionstabilityagainstcreamingToassessthephysicalstabilityoftheemulsionsatpHvaluesof3.8and7,avisualobservationwasdone.Theobservationtookplaceatroomtemperature.20mLofeachsamplewasplacedinacappedcylindricalcontainer.Theheightofthetotalemulsionand the serum that formed at the bottom of the container was measured with a ruler. Themeasurementsweredoneatthe1standthe5thdayofstorage.EmulsionrheologyRheologicalpropertiesoftheemulsionsweredeterminedbyRheometerPhysicaMCR301(AntonPaar301,Korea)withcontrolledshearstress.Concentriccylindergeometrywasusedtocharacterizethelowviscousemulsion(MeasuringCup,C-DG26.7/T200/Tiwithinternalandexternaldiameterof23.829mm,and27.600mm,respectively;MeasuringCylinder,DG26.7/Tiwithinternalandexternaldiameterof24.651mm,and26.677mm,respectively).Duringtheanalysistemperaturewaskeptconstantat25oCusingawaterpump(Waterpump,Tetra,USA).RHEOPLUS/32V3.62wasusedassoftware.Todetermine the linear viscoelastic regime, the amplitude sweep determination was done at anoscillationfrequencyof0.5Hz.Viscoelasticpropertiesoftheemulsionswereanalysedataconstantdeformationof0.002m,withfrequencyarangeof0.01to100Hz.Bychangingtheshearratesweepwitharangeof0.01to1000Hz,viscosityprofilewasdetermined.

22

5.ResultsandDiscussion5.1.CompositionandCharacterizationbeforetheAqueousExtractionProcessBefore AEP, the black coated, edible rapeseed was ground to gain rapeseed flour for furtherinvestigationoncompositionandcharacterization.Moisture,oilandproteincontentsofthisrapeseedflourweredetermined,andextractionyieldwascalculated.Thedeterminedcompositionofrapeseedflourwasfoundtohaveasimilarproteinandoilcontentcomparedtoliterature(Table5.1).ProteinwasmeasuredusingKjeldahlmethod.TheKjeldahlmethodmeasuresthetotalnitrogencontent,therefore,itcangiveafalseconclusion.Forinstance,inthisstudythede-hullingstepwasskipped.Therefore,apartfromprotein,non-proteincomponentsinthehullswere also measured during the analysis29. Both conversion factors 6.25 and 5.7 were used forcalculationofprotein.Theconversionfactor6.25isacommonlyusedfactortoconvertnitrogentocrudeprotein,andwasusedtocomparetheresultswiththecurrentliterature12,33,46,47.Factor5.7,usedforoilseedproteins46.Thecomparisonoftheresultswithliteraturedata(Table5.1)showadifferenceinoilcontent,whichcanbeaccepted,sinceoilcontentvarieswiththeseedtype12,28,33andthecoloroftheseedcoat.Blackseedcoatedrapeseedhasaloweroilcontentcomparedtoyellowseedcoated33.Table5.1.DeterminedCompositionofRapeseedFlour

Composition RapeseedFlour(dm%) AverageCompositionfromLiterature*(%)

Protein(N*6.25) 18.66±1.77 21Protein(N*5.7) 17.02±1.62

Oil 37.95±0.49 43*SeeTable3.1.intheBackgroundζ-PotentialandTitrationCurveToobtainthemajorproteinfractionsnamely,napinandcruciferin,precipitationatisoelectricpointwasapplied.BychangingthepHtowardsavaluewheretheproteinshavenetzerocharge(isoelectricpoint, pI), the repulsive forces between the proteins diminish and aggregates are formed58. Eachproteininthemixturecanhavedifferentisoelectricpoints.Therefore,thepIofthedissolvedrapeseedflourwasdeterminedtobeabletoprecipitateall rapeseedproteins.ThepIvaluewasfoundtobearoundpH5(Figure5.1).AtacidicpHvalues,between2to4.8,thesurfacechargeofrapeseedproteinswaspositivewhereasatslightlyacidicandbasicpHvalues,between5to12,thesurfacewasnegativelycharged. Besides, the degree of electrostatic repulsion between adjacent particles could bedeterminedfromtheFigure5.1.Athighζ-potentialvalues(eitherpositiveornegative),whichwerebetweenpH2to3and10to12,resistancetoaggregation32,58 ishigh.Onthecontrary,at lowzetapotential values,whichwerebetweenpH4 to 7, the tendency is aggregation, since the attractiveforcesbetweentheadjacentparticlesmayexceedrepulsiveforces32,58.

23

Figure5.1.TitrationcurveoftherapeseedproteinTheisoelectric-pointalsoconfirmedbystudies,whichdeterminedthelowestsolubilityoftherapeseedproteinatpHvaluesbetween3to5duetothenetzerochargeoftherapeseedproteins12,19,32,53,55.5.2.CompositionandCharacterizationaftertheAqueousExtractionProcessAftertheaqueousextractionprocess,freezedriedproteinmixturewassubjectedtomeasurementsfor determination of composition. The results showed that a high amount of protein, significantamountofoilandaminoramountoftherest-biomasswereobtainedasproduct(Table5.2).Theobtained results considering composition and extraction yield were compared with literature.However, since there are not published studies about aqueous rapeseed protein extraction, theconventionalrapeseedproteinextractionrouteandAEPforsoybeanweretakenintoconsideration.Therestofthebiomasswasexpectedtocontaincarbohydrates,ligninandimpuritiesasstatedintheliterature(Table3.1).However,thecomponentsotherthanoilandproteinwerenotofconcernofthisthesis.Table5.2.DeterminedCompositionofProteinMixture

Composition ProteinMixture(dm%)Protein(N*6.25) 65.46±6.46Protein(N*5.7) 59.70±5.02

Oil 16.90±0.88Afterdeterminationoftheproteincontent,proteinyieldoftheprocessandtheextractionyieldwerecalculatedusingequations5.1,5.2and5.3.ResultsweretabulatedonTable5.3.ProteincontentandextractionyielddependonthepHofextractionmedia,agitationrate,solidtowaterratio,particlesize,extraction time, number of extraction stages and temperature of the extraction media48,50,53,54,55.Therefore,toobtainhighpurityandhighextractionyield,moreresearchshouldbedoneinpropertiesofrapeseedproteinsandoptimalextractionconditions.

Proteinyield dm% =Protein% N*6.25 ×FreezeDriedProduct(g)Protein% N*6.25 ×DriedRapeseedFlour(g)

×100 (5.1)

Extractionyield % =CollectedPellet(g)RapeseedFlour(g)

×100 (5.2)

Extractionyield dm% =FreezeDriedProduct(g)DriedRapeseedFlour(g)

×100 (5.3)

-45

-25

-5

15

35

2 4 6 8 10 12

ZetaPoten

tial(mV)

pH

24

Table5.3.YieldoftheAqueousExtractionProcessProteinyield(dm%) Extractionyield(%) Extractionyield(dm%)

12.55±2.53 15.95±6.81 4.00±1.37Ingeneral,theconventionalrapeseedproteinextractionroutehasaproteincontentrangebetween70%to90%50,53,54,55.Thecontentdependsonselectedunitoperationsandconditions.Forinstance,Ghodsvalyetal.55obtained85%proteinpurity,withextractionyieldof15%andproteinyieldsof30%.Thisresultwassimilartoourfindingsinrespecttoextractionyieldbuttheextractionrouteshouldbeexaminedindepth.Tosolubilizetherapeseedproteinintheaqueousmedia,theextractionwascarriedoutdistantfromtheiso-electricpointtopreventprecipitationtheproteins.InthisthesispH8waschosen,thatwasdistant from isoelectric-pointof eachmajorprotein fraction,whichalso claimed tobe thegeneralreference pH for protein extractions50,51. Several studies show that increasing pH value in alkalinemediacanincreasetheextractionyield.Rosenthaletal.50andCampbelletal.51bothreportedthat,theextractionyieldhadaminor change frompH8 to10 (approx.80%yield) andhaddistinguishableincreaseafterpH10(approx.95%yield).AsitcanalsobeseeninFigure5.1,theproteinsolutionhadapproximately same zeta potential value from pH 8 to 10. Above pH 10, the zeta potential valueincreasedwhichalsoindicatestoslightchangesofprecipitation.However,proteinhydrolysisislikelyabovepH1032.Inrespecttotheextractabilityofproteins,pHofthealkalinemediaisalsocorrelatedwiththeproteinconcentrationinthealkalinemedia32,48,50.Gerzhovaetal.32highlightedthattheextractabilityincreaseswithincreasingconcentration,andinlowpHvaluestheextractabilityisaffectedmorewithincreasingconcentrationthaninhighpHvalues.DependingontheselectedpH8,solidtowaterratiowaschosenas1:7(concentration:12,5%).Todeterminetheoptimalsolidtowaterratiomorestudiesshouldbedone.Proteinlossescouldoccuratpossiblebottleneckslikegrinding,filtrationandcentrifugationsteps.Inthegrindingstep,thepowercouldnotbesufficienttoreleaseallthenutritionalcomponentsoutoftheseedsandhullsorcotyledon.Inthefiltrationstep,theretentatewasmostlyconsistedofhullsthatarealsoaproteinreserve.Inthecentrifugationstep,threestreamswerecreated,namely:anoilcreamfractioncontainingstabilized-OBs,anaqueousphasewithdissolvedproteins,andasmallresidualsolidphasethatwasexpectedtocontainprimarilycellulosicandhemicellulosicmaterialsfromthehulls.Asmentionedbefore,due topHof theextractionmedia,proteins interactwitheachother and formaggregateswhichresultsinprecipitation.Therefore,itispossiblethattherewasaproteinlossinthecentrifugationstepwheresomeproteinsaggregateandmergeinthepelletandcouldnotbeobtainedatsubnatantpart.Also,oilintheproteinmixturecouldbeexplainedbythesamemechanism.Duringtheexperimentsthecreamlayerthatcontainedoilwasscoped.However,duetothecomposition,itwaspossiblethatproteininteractedwithoil.Bysurroundingaroundoilparticles,theproteinmighthavepreventedoilparticlestomoveupwardsduetohigherdensityanddrewthemintosubnatantphase or into pellet65. Besides, several centrifugation steps could make them coalescence andprecipitate.Ontheotherhand,thesignificantamountofoilcontentwasrelevanttotheomittedde-fattingstep.Moreover,duetohighmobilityofsmallparticles indisruptedcells,smallparticlesizeaccompanieshighyieldonproteinandoilextraction50,51.Bygrinding,blendingandstirring,highshearforceshelptoreleasemorenutritionalcompoundsbutalsohelptoformmorestableemulsionsdependingontheoildropletsize.Thisargumentwillbediscussedindetailinthe5.3.EmulsionStability.

25

Temperatureisanotherparametereffectingtheyield.Atemperaturerangebetween20to30oCissuitablefortheextraction52.Temperaturesabove30oCincreasethesolidyield,whereasrevealproteindenaturationanddecreaseproteinandoilextractionyield50,63.Inthisthesis,extractionwasperformedatroomtemperature.Thiscouldbeoneofthereasonsoflowextractionyield(seeTable5.3).DuringthepHadjustmentforextractionmedia,anantagonisticbufferingeffectwasobservedduetorapidproteinandoilrelease.Toobtainallthenutritionalcompoundsintothemedia,stabilizationhadto be achieved. Campbel and Glatz 51 stated that the yield remained constant after 120 min ofextraction. It is however, depending on several parameters such as solid to liquid ratio andtemperature.Toincreasetherecoveryrateofproductscertainprocessstepscouldberepeated,likeincreasingofthenumberofextractionsbytheaqueousextractionprocess.AsNikiforidisandKiosseogluo49stated,byincreasingnumberofextractionsfrom1to3,extractionyieldincreasesinarangeof20%to60%dependingonpHoftheextractionmediaandsizeoftherawmaterial.SDS-PAGEToestimatetherecoveryoftherapeseedproteinfractions,theproteinprofilesofrapeseedflourandproteinmixturewerecompared;reducingandnon-reducingconditionswereapplied(Figure5.2).Theresultsforrapeseedproteinprofilewascoherentwithliteraturedata12,24,29,30,42.

Figure5.2.SDS-PAGEofrapeseedprotein.Mw:Proteinmolecularweightmarker,RF:Rapeseedflour,RFR: Rapeseed flour in reduced condition, PM: Protein mixture, PMR: Protein mixture in reducedcondition.Theproteinmixturewasobtainedattheisoelectricpointoftherapeseedprotein(approx.atpH5)IntheRFcolumn,therewere4majorbandswithhighintensity,150,30,23,15kDa,whichrepresentedcrucifern,oleosinandnapin(Figure5.2)24,29,49.Inthepresenceofareducingagent(Figure5.2,columnRFR)highmolecularweightedpolypeptidesat300and200kDadisappearedandthendissociatedintolower-molecularweightedpolypeptides,whichindicatedthepresenceofdisulphidebonds32,12,19,29,53,54(Figure5.2,columnRF,RFR).Besides,incolumnRFRlowcontrastbandswerealsoobservedwithinaline.Thisphenomenonmightbeeitherduetoproteinswithlowpurityoritwasthesignoffurtherproteindegradation(seeinFig.5.2theappearanceofdoublebandsataround50,23and15kDa).InthePMcolumn,fourmajorbandsweredeterminedwithmolecularweightsof16,18,30and53kDa,asalsostatedbyAlukoandMcIntosh53,thatidentifiedthe3majorproteinfractionsinrapeseed(Figure5.2).Compared to theRFcolumn, it couldbeseen thatbands identifiedascruciferinwereeitherdisappearedorhadaslightintensity(Figure5.2).Thisresultcouldbeaproofthatcruciferinwas

26

stronglyaffectedbytheprocessconditionsandtheremovalofthefractionwasincomplete.Cruciferinis a salt soluble protein29, whereas in this thesis salt was not added into the extraction media.Therefore, it was possible that cruciferin was not soluble enough in the extraction media. Thishypothesis was confirmed by Gerzhova et al.32 that high molecular weighted polypeptides wereobservedafterthesaltaddition.Anotheraspectisthat,purecruciferinhasanisoelectricpointatpH7.25.Thisvalue isclose topHof theextractionmedia,whichcouldcauseunwantedprecipitationsduringextraction.Proteinlosscouldhavebeenoccurredinthecentrifugationstep.InthePMRcolumn,thetwomajorbandsfromPMcolumnat50and29.5kDaandaminorbandat44kDadisappeared,which is also observed byWu andMuir64. This is an indication of presence of disulfide linkage ofcruciferin.Napinfractionwasidentifiedinbothrapeseedflourandproteinmixture,wherethebandswereat8and15kDa.TheintensityofthenapinbandswerelowerinthePMcolumncomparedtoRFcolumn(Figure5.2).ThepIofnapinisatpH11anditisexpectedthatnapincansolubilizeatlowerpHvalues.SincetheisoelectricprecipitationwasappliedataroundpH5,itwaspossiblethatsomeofnapinwassolubilizedinaqueousmediaandcouldnotbeprecipitatedandcouldnotbeobtainedinthepellet.ThishypothesisissupportedbyWanasundaraetal.29:SDPpageshowforextractionconditionsatlowacidicpH,suchas2and4,onlybandsofnapin(at8to15kDa)andatpHvalues7and10,bandsofcruciferinandnapin(at8,34and50kDa)areobserved.Thebandsat8and34kDacanalsobeseeninFigure5.2.InthecolumnPMR,lowermolecularweightedpolypeptidesataround8kDawereformedcomparedtoPMcolumn.Gerzovaetal.32foundsimilarresults,whichhighlightsthedisulfidelinkingofnapin.Oleosinwasdetectedat16and18kDa65,66.Thebandsweremore intenseatcolumnsforrapeseedmixturethaninrapeseedflour.Oleosinsaremembraneproteinswhicharealsoreferredasoilbodyproteins(OBP)12,24.AccordingtoNikiforidisetal.66oleosinwasseeninthecreamthatconsistsofoilbodies(OB).Therefore,thepresenceofoleosincouldberelatedtotheoilcontentintheextractionmixture.OleosinbandsbecamemoreintenseinthePMR,thaninPM,whichcouldbebecauseofthenewpolypeptideformationinsteadofdisulfidelinking32.5.3.EmulsionStabilityFood emulsions are micro-heterogenous materials they differ in size, shape and physicochemicalproperties.Thecharacteristicsofdropletsdominatethephysicochemicalpropertiesoftheemulsion.For emulsion stability, droplet size, droplet charge, interfacial properties and colloidal interactionsbetweenthedropletsdeterminethestabilityofanemulsion.DairyproductsarestableatpH7andsaladdressingtypefoodproductsarestableatpH3.867.InthisthesisstabilityofemulsionsatpH7and3.8 were compared, in order to estimate the suitable market for the rapeseedmixture that wasobtainedbyAEP.SizeDistributionIngeneral,foodemulsionscontainavarietyofdropletsize45.Therefore,thispolydispersesystemischaracterized in respect to the concentration of droplets in different size classes (particle sizedistribution).Intheanalysis,theparticlesizewasspecifiedwiththediameter.Twomeanparticlesizevalueswereusedbythedeterminationofthedistribution:surfaceweightedmeandiameter(d32)andvolumeweightedmeandiameter(d43).d32ismoresensitivetoindividualandthemajorityofparticles;d43ismoresensitivetolargeparticlesandthefewlargeaggregates45.Atypicalmeandropletdiameterofanemulsionisapproximately1µm,whichcanvarywithemulsificationconditionsandemulsifierconcentration59.

27

Figure5.3.Dropletsizedistributionof0.5%rapeseedproteinmixtureemulsion.a:EmulsionatpH7,b:EmulsionatpH3.8.Theemulsionsstoredat4oCfortotalof5days.Thesizeofthedropletwasplottedagainstthevolumedensity(%)(Figure5.3a,b).TwomajorpeakswereobservedinbothpHvaluesandstorageperiod(Figure5.3.a,b),thus,emulsionshavingabimodaldistribution.After1dayofstorage,emulsionatpH7hadasmallerindividualdropletsize(0.161µm)withahighdistributionandsmallaggregations.Ontheotherhand,emulsionatpH3.8hadabiggerindividual droplet size (8.759 µm)with a narrowdistribution andmore aggregations compared toemulsionatpH7.Comparedtothegeneralizedemulsiondropletsize,thedropletsizeatpH3.8wastoo large. For this specific pH value, the emulsification conditions and/or concentration of theemulsifier(proteinmixture)mightnotbecompatible59.After5daysofstorage,fortheemulsionatpH7, the size of the individual dropletswas shifted to larger droplets and the aggregation increased(Figure5.3.a).FortheemulsionatpH3.8therewasnotaremarkablechangebutthesizedistributionwasshiftedtoright,largersizerange(Figure5.3.b).Shiftingtohigherdiametersisausualphenomenonforemulsionsafterseveraldaysofstoragethatindicatesthedestabilizationprocess70.Asimilartrendfor sizedistributionunder storageperiodwasobtainedbyNikiforidis andKiosseogluo49,whousedmaize germ as emulsifier. Other authors have also reported the shift of size distribution andinvestigatedtheshiftatdifferentpHvalues(Mantovanietal70).

5.4.Schematicrepresentationofdropletsizechangeintheemulsion,basedonFigure5.3

b

0,01,02,03,04,05,06,07,08,0

0,01 0,1 1 10 100 1000 10000

VolumeDe

nsity

(%)

Size(µm)

pH7

24hstorage

120hstorage

0,01,02,03,04,05,06,07,08,0

0,01 0,1 1 10 100 1000 10000

VolumeDe

nsity

(%)

Size(µm)

pH3.8

24hstorage

120hstorage

Day D[3,2](µm) D[4,3](µm)1 0.161± 0.041 14.323± 7.2655 1.622± 0.080 30.136± 1.080

Day D[3,2](µm) D[4,3](µm)1 8.759± 0.478 34.557± 1.8595 8.138± 0.864 31.543± 2.522

15

Day1,pH7

Day1,pH3.8

Day5,pH7

Day5,pH3.8

28

Theseresultswereexpected,consideringthetitrationcurvefortheemulsionwithrapeseedproteinmixture(Figure5.4).Dropletchargeinemulsionsisanimportantparameterfordropletstabilityandphysicochemicalproperties.Italsoeffectstheinteractionwithotherchargedspecieslikeionsinwateretc.AccordingtotheFigure5.5,repulsiveforcesbetweendropletsdiminishattheiso-electricpointandcausedropletsclusterandformaggregates.Thus,atiso-electricpoint(pH5),theemulsionsystemhadtheleaststability.Theresultsaboutthesizedistributionwerecompatiblewiththetitrationcurve.AtpHvalues4to6thezetapotentialvaluewasremarkablylowwhichmeansthataggregationwaslikelytooccur.SincepH3.8isclosetotheiso-electricpoint,itwasexpectedtohaveaggregationinparallelwithbigdropletsize.Inaddition,thedropletsatpHvaluesbetween2to4and6to12hadsufficiently negative or positive charges to form amore stable emulsion. At those pH values, therepulsiveforcesbetweenadjacentproteinsdominateovertheattractiveforcesandthedropletsarelesslikelytoclusterandformaggregates.AsindicatedinFigure5.5,pH7wasfurtherawayfromtheiso-electricpointcompared topH3.8 that itwasexpected tohave lessaggregation.Besides, sinceproteinswereusedasemulsifier,hydrophobicinteractionsmightoccureventhough,repulsionisthenetresultduetothecorporationofstericandelectrostaticrepulsion.WhenthepHisclosertothepIofprotein,electrostaticattractionoccursbetweennegativeandpositivegroupsonthesurface.AtpI,hydrophobicattractionoccursandtheemulsiondropletstendtoaggregate(Figure5.5)59.

Figure5.5.Titrationcurveof0.5%rapeseedproteinmixtureemulsion.pHvaluesrangebetween2to12,withincrementof0.5.

CreamingBehaviourIngeneral,thedensityofthedropletsinanemulsionisdifferentfromtheliquidthatsurroundsthem.Intheoilinwateremulsion(o/w)systems,oilhasalowerdensitythantheaqueousphase.Whentheemulsionisstoredforfurtheruse,thenetgravitationalforceactsuponoildroplets.Duetodensitydifference,oildropletsstarttomovetothetopoftheemulsion.Thisinstabilityphenomenoniscalledcreaming.Creamingbehaviourisanimportantparameterforshelf-lifeoftheemulsionandforfurtherapplications. The rate of the creaming can be determined by Stokes Law for an isolated sphericalparticleinanideal(Newtonian)liquid(Equation5.4)45.AccordingtoStokeslaw,creamingbehaviourisaffected by the droplet size, density of dispersed and continuous phases and the viscosity of thecontinuousphase.

vStokes=-2gr2(ρ2-ρ1)

9η1 (5.4)

14

-35

-25

-15

-5

5

15

25

35

2 4 6 8 10 12

ZetaPoten

tial(mV)

pH

29

Where,vStokes:creamingvelocity,r:radiusofthedroplet,g:accelerationduetogravity,𝜌:density,𝜂:shearviscosity,1:continuousphase,2:dispersedphase.ThecreamingbehaviorwasmeasuredforemulsionsatpH7andpH3.8.AsshowninFigure5.6,after1dayofstorageatpH7therewasnoindicationofcreaming.However,atpH3.8,creaminghadalreadyoccurred.After5daysofstorageatbothpH7and3.8aserumlayeroccurredatthebottomoftheemulsionineachsample.TheserumlayeratpH3.8andafter5daysofstoragewasclearer,whichmeansthatthecreamlayerwaspackedmorecloselyonthetopoftheemulsion.

Figure5.6.Visualobservationforcreamingbehaviour.a-b:0.5%rapeseedproteinemulsionafter1dayofstorage,c-d:0.5%rapeseedproteinemulsionafter5daysofstorage.Where,samplesaandcwerehavingpHvalueof7andsamplesbanddwerehavingpHvalueof3.8.To get a better understanding about the rate of the creaming, the height (ratio of serum to totalemulsion)versustimewasplotted(Figure5.6).TheresultshowsthattherewasarapidcreamingatpH3.8andaslightincreaseafter5days.Ontheotherhand,emulsionatpH7hadlesscreamingwhere,itwasstableafter1dayofstorageandthenaremarkableincrease(Figure5.7).

Figure5.7.Creamingbehaviourof0.5% rapeseedproteinemulsion.Theemulsions storedat roomtemperaturefortotalof5days.Heightistheratioofserumtototalemulsion.

Itisknownthatrapidcreamingisaresultofaggregation45,59.Theaggregationformationisdependedonthecolloidalinteractionsbetweenemulsiondroplets,whichdeterminethephysicalstabilityoftheparticles.Theinteractiontypescanbegroupedasrepulsive(electrostatic,steric)andattractive(vanderWaals,hydrophobic,depletion)59forces.Attractiveinteractioncausesthetendencytoaggregateandrepulsiveinteractionprovidesdropletstobeindividual59.

a b

c d

0102030405060

0 20 40 60 80 100 120

Height,H

(%)

Time(h)

pH7 pH3.8

30

The reason of the fast creaming behaviour at pH 3.8was due to size distribution and the dropletcharge; bigger dropletsweremore likely to cluster (Figure 5.3, b).Due to thepH value, attractivecolloidal interactionsdominated,whichcausedtheaggregation.After5days,thedropletsizegrewlarger and theaggregation increased (Figure5.3, b). Thiswas theexplanationof themorepackedcreamlayerafter5daysofstorage(Figure5.6).AtpH7,atthefirstdayofstorage,individualdropletsweresmallandconsequentlymorestable(Figure5.3,a).Thiscouldberelatedtothenon-existenceofcream layer in the first day of storage (Figure 5.6). After 5 days of storage, at pH 7, therewas aremarkableincreaseincreamingbehaviour.Thereasoncouldbethatatday5theindividualdropletsizegrewlargerandmoreaggregationswereformed(Figure5.3,a).Thecoloroftheemulsionisalsoanindicatoraboutthesizeofthedroplets.Emulsionstendtobewhitewhenthedropletsizeisaround3µmandbyincreasingsizeupto30µm,thecolorchangestoyellow(colorofoil)59.These results indicated that smalldropletsmade theemulsionmore stable in respect to creamingbehaviour. Formation of aggregates with small droplets was less likely and the creaming slow incomparisonwithbigdroplets.Smallsizedropletsneededmoretimetogetoutofthedispersedphaseand tomove at the top of the emulsion.Nikiforidis and Kiosseogluo65 also stated that one of thereasonsforalowerstabilitymightduetothelargermeandropletsizewhichwasexpectedtohaverapidcreaming.RheologicalCharacterizationRheologicalcharacterizationaboutemulsionsgivesome informationabout (micro)structureof thesystem.ThecharacterizationmeasurementswerecarriedononlyfortheemulsionatpH7,becauseitwasmorestablethantheemulsionatpH3.8.Firstly,amplitudesweepandfrequencysweepmeasurementswereappliedtoindicatewhethertheemulsionsystemishavingaliquid-likeorgel-likestructure.Itisanimportantparameterforstorageandfurtherapplications.

Figure 5.8. a: Amplitude sweep determination at a constant oscillation frequency of 0.5 Hz; b:Frequencysweepdeterminationataconstantdeformationof0.002,withfrequencyrangeof0.01to100Hz.Themeasurementsweredoneatroomtemperature.Two parameters were recorded during the measurement to estimate the viscoelastic response:storagemodulus(G’)andlossmodulus(G’’),whereG’givesinformationaboutelasticresponseandG’’isaboutviscousresponse61.Amplitude sweep gives the rigidity and strength characteristics of the emulsion. Besides,with theamplitudesweepthelinearviscoelasticregion(LVER)isalsodetermined.Thisregionisimportantto

0,001

0,01

0,1

1

10

0,01 0,1 1 10 100

Mod

ulus(P

a)

Strain(%)

StorageModulusLossModulus

0,001

0,01

0,1

1

10

0,1 1 10

Mod

ulus(P

a)

Frequency(Hz)

StorageModulusLossModulus

a b

31

determinethestability.Inthismeasurement,LVERobservedfrom0,01to1%strain(Figure5.8,a).Thelongertheregionis,betterthepossibilityofastabilesystem62.Atlowstress,theemulsionpreserveditsstructurebutaround90%strain,disruptionofthestructureoccurred (Figure 5.8, a). Till reaching 90% strain, the storagemodulus (G’) is larger than the lossmodulus(G’’),whichmeanstheappliedforceisstillsmallerthantheintermolecularforcesbetweentheemulsiondroplets.Inthiscase,thematerialactsasanelasticsolid,wherethestoredenergycanstill be returnedwithout structuredeformation.However,when the force is higher than the intermolecularforces,G’’becomeslargerthanG’.Whenthestoragemodulus(G’)islargerthanthelossmodulus(G’’),whichmeanstheappliedforceisstillsmallerthantheintermolecularforcesbetweenemulsiondroplets.Inthiscasematerialactsasaweakelasticsolid,wherethestoredenergycanstillbereturnedwithoutstructuredeformation(network/gel-likestructure).However,whentheforceishigher than the intermolecular forces, G’’ becomes larger than G’. Thatmeans, the network-likestructuredisruptedandbecomeinline,whichmeansthatmaterialstartedtoflow.Inourcase,after90%theemulsionstructuredisruptedandthegivenenergystarttodispersed(Figure5.8,a).Frequencysweepshowsthestructuralresponseoftheemulsiontodeformationsinlongerorshortertimesclaes60. It isan importantparameter topredict thebehaviourof theemulsionat storageandapplication conditions. In Figure 5.8, b, emulsion was exposed to deformation oscillations. Thefrequency sweep force was kept constant and the frequency increased (Figure 5.8, b).When thefrequencyincreased,thedeformationwasfaster.AsimilarresultregardingthefrequencysweepwasobtainedbyNikforidisetal.67,whereemulsionsweremadeofoilbodiesandeggyolkblends.Insolidparticles, droplet interactions are higher, which means less frequency. It was observed thatapproximatelyupto9Hz,emulsionactedelasticandafterthatthestructurebecameviscous(Figure5.8,b).Inthatcase,thesystemseemedtoactasgelandcreamingwasunlikely.Thishypothesiswasconfirmedbyvisualobservationforcreamingbehaviour(Figure5.6,a).Creamingwasnotobservedin24hours,signifiedthattheemulsionhadagellikestructureandrigidnetwork.Secondly,viscosityprofileoftheemulsionwasanalysed(Figure5.9).Emulsionsarereferredasnon-Newtonianfluid.Theviscosityoftheemulsionsvariesdependingonshearratethattheyareexposedto(apparentviscosity)60.Non-Newtonianfluidstendtodecreasewithincreasingshearrate59,60.Thisphenomenoniscalledshear-thinning.Ingeneral,mostsystemscomeacrosstoshearthinning59,65,67,68.Theobservedviscosityatthatpointistheapparentviscosity.ForNewtonianfluids,apparentviscosityisconstant;fornon-Newtonianfluids,itisdependingontheshearrate59.Shearthinningprofilestartedataviscosityof5Pa.swithashearrateof0.012s-1anddecreasedto0.018Pa.swithashearrateof1000s-1.Whenshearwasapplied,thedropletsthathadanetwork-likestructurebecameinline,whichmeansthattheycouldfloweasily.Therefore,viscositywasdecreased.TheseresultsarecompatiblewithWhiteetal69,whoassessedtherheologyandstabilityofemulsionwhichformedbysunflowerseed oil bodies. The point at around 5 Pa.s, is called zero-shear viscosity60. This point is inverselyproportionaltocreamingvelocityofemulsiondroplets,thusanimportantindicatorfortheemulsionstability60.

32

Figure5.9.Viscosityversusshearratediagramfor0.5%rapeseedproteinemulsionafter24hofstorage,at4oC

0,001

0,01

0,1

1

10

0,001 0,01 0,1 1 10 100 1000

Viscosity

(Pa.s)

ShearRate(1/s)

33

6.CONCLUSIONInthisthesis,aninsightintorapeseedproteinfractionswasgainedandtheabilityeoftheaqueouslyextracted proteinmixture to stabilise emulsionswas investigated. The current literature does notdescribeanyaqueousextractionprocessforrapeseed.Ournon-optimizedprocessyieldedaproteinmixturewithlowpurityandyield.Betterresultsforyieldandpuritycouldbeobtainedbyoptimizationofprocessparameterslikeparticlesizeoftherawmaterial,pHoftheextractionmedia,agitationrate,concentrationofthemedia,durationandnumberofextractionstagesandtemperatureofthemedia.Moreover, protein characterization showed that all the major rapeseed protein fractions wereobtainedwhereas,cruciferinwasstronglyaffectedbytheprocess.Emulsions,producedusingtheobtainedproteinmixture,haddifferentstructuresandcharacteristicsatpH3.8and7.AtpH7,theindividualsizeofthedropletsintheemulsionwassmallerthanatpH3.8andtheaggregationswere lower.Theeffectof the interactionsbetweenthedropletsdirectedthestabilityoftheemulsionatstorageperiod.After5daysofstorage,thesizedistributionshiftedtohigherdiameterrangeatbothpHvalues.Combiningthesizedistributionresultswithcreamingbehaviourindicatedthatsmalldropletsizedemulsionswerelesslikelytofromaggregatesandthecreamingratewassmallerthaninlargedropletsizedemulsions.TheemulsionstructureatpH7wasfoundtodependonfrequency;itwasgel-likeatlowandbecamemoreviscousathighfrequencies.Theviscosityoftheemulsiondecreasedbyincreasingshearrate.Thestructuralcharacterizationoftheemulsionindicatedthatdropletshadinteractionswitheachotherandtheemulsioncouldbedisruptedbyapplicationofforce.These results proved that rapeseed proteinmixture could be an alternative for food applications,especiallyfordairyindustry.

34

7.RECOMMENDATIONS

The aqueous extraction process should be optimized to increase the extraction yield and proteinpurity.Herearesomepromptingsforfurtherstudiesatdifferentprocesssteps:

• De-hulling: inthisstudyde-hullingwasexcluded.Tohavehigheryields,de-hullingcouldbeapplied.

• Centrifugationandfiltration:Characterizationofproteinanddeterminationofproteinandoilcontents,shouldbedoneaftercentrifugationandfiltrationsteps.Proteinlosses,solubilityofeachfraction,effectofthemedia-pHoneachproteinfractioncouldbedetermined.Duringcentrifugation,thecreamlayerwasscopedandwasnotusedoranalyzedinthisstudy.Aftereachcentrifugationstep,thecreamlayershouldbeanalyzed.Compositionoftheproteinmixture and protein profile would give more insight into rapeseed protein fractions.Knowledgeaboutsolubilityofdifferentproteinfractionscouldleadtothereasonoflosses.

• Extraction:extractioncouldberepeatedatleast3timesforhigheryields.Saltwasnotaddedintoextractionmediuminthisstudy.However,cruciferinrecoverywasnothighenough.Saltadditioncouldbeanoptiontoincreasecruciferinrecovery.

• Emulsion: The concentrations of each protein fraction in the protein mixture should bedeterminedinordertounderstandtheeffectofeachfractionintheemulsificationprocess.Also,theconcentrationoftheproteinmixtureintheemulsionshouldbeoptimized.Proteinmixturesprovideabetterstabilitytoemulsions.Moreresearchshouldbedoneaboutthemechanismsofstabilization.Togetsmallerdropletsthus,morestableemulsions,pre-mixingandhomogenizationcouldberuninhigheragitationrateandpressure.ThiseffectshouldbeinvestigatedrelatingtootherparameterslikepH.To understandmore about changes in size distribution,microscopic examination could beappliedtoidentifywhethertheincreaseinsizeisduetoagingorcoalescence.Microscopicexaminationcouldalsohelptodeterminethemechanismbetweentheoilinsidetherapeseedproteinmixturewiththeaddedoiltofindoutwhethertheyclustertogetherornot.

• Foodapplications, studiesshouldbedoneaccording toemulsion formationatdifferentpHvalues.

35

REFERENCES[1]GlobalRapeseedProduction.(2017).RetrievedSeptember10,2017,fromhttp://www.globalrapeseedproduction.com/[2] OECD & FAO. (2015). Agricultural outlook 2015-2024. Retrieved September, 10, 2017, fromwww.Fao.Org/3/a-i4738e.Pdf[3]Haar,D.V.,Müller,K.,Bader-Mittermaier,S.,&Eisner,P. (2014).Rapeseedproteins-Productionmethodsandpossibleapplicationranges.Ocl,21(1).doi:10.1051/ocl/2013038[4]HeuzeV.,TranG.,SauvanD.,Lessirem.,andLebasF.,(2017).Rapeseedmeal.FeedipediaaprogrambyINRA,CIRAD,AFZandFAO.https://feedpedia.org/node/52.LastupdatedonJune21,2017.[5] Campbell L., Rempel C.B. & Wanasundara J.P.D. (2016). Canola/Rapeseed Protein: FutureOpportunitiesandDirections-WorkshopProceedingsofIRC2015.Plants(Basel).2016Apr13;5(2).doi:10.3390/plants5020017[6] Schweizer M., Segall K., Medina S., Willardsen R., Tergese J. (2006). Rapeseed/canola proteinisolatesforuseinthefoodindustry.ProcessTechnology.[7]NikiforidisC.Proteincomplexmixturesforfoodemulsions.(2016)[8]PereraS.P.,McIntoshT.C.andWanasundaraJ.P.D.(2016).StructuralPropertiesofCruciferinandNapinofBrassicanapus(Canola)ShowdistinctResponsestoChangesinpHandTemperature.Plants(Basel).2016Sep7;5(3).doi:10.3390/plants5030036[9]Fisher,E.(2015).AlternativeProteinstoClaimaThirdoftheMarketby2054.RetrievedSeptember20, 2017, from http://www.luxresearchinc.com/news-and-events/press-releases/read/alternative-proteins-claim-third-market-2054[10]Billing,S.(2016).ProteinChallenge2040:Sustainableproteinproductionforthefuture.RetrievedJanuary 10, 2018, from https://thefuturescentre.org/articles/8213/protein-challenge-2040-sustainable-protein-production-future[11]Henchion,M.,Hayes,M.,Mullen,A.,Fenelon,M.,&Tiwari,B.(2017).FutureProteinSupplyandDemand Strategies and Factors Influencing a Sustainable Equilibrium. Foods, 6(12), 53. doi:10.3390/foods6070053[12]AiderM.&BarbanaC. (2011).Canolaproteins:Composition,extraction, functionalproperties,bioactivity,applicationsasafoodingredientandallergenicity-Apracticalandcriticalreview.TrendsinFoodScience&Technology,22(1),21-39.doi:10.1016/j.tifs.2010.11.002[13]Eriksson,I.,Westerlund,E.&Aman,P.(1994).Chemicalcompositioninvarietiesofrapeseedandturniprapeseed,includingseveralsamplesofhullanddehulledseed.J.Sci.FoodAgric.66(2),233–240.doi:10.1002/jsfa.2740660219[14]Henchion,M.,Hayes,M.,Mullen,A.,Fenelon,M.,&Tiwari,B.(2017).FutureProteinSupplyandDemand: Strategies and Factors Influencing a Sustainable Equilibrium. Foods, 6(12), 53. doi:10.3390/foods6070053

36

[15]UnitedStatesDepartmentofAgriculture,WorldAgriculturalOutlookBoard,ForeignAgriculturalService,economicResearchService,&AgriculturalMarketingService. (2017).WorldRapeseedandProducts Supply and Distribution. Retrieved October 12, 2017 fromhttps://apps.fas.usda.gov/psdonline/reportHandler.ashx?fileName=Table17:World:RapeseedandProductsSupplandDistribution&reportId=716&templateId=13&format=html[16]FoodandAgricultureOrganizationoftheUnitedNations(2017).Crops.Retrieved,October12,2017,fromhttp://www.fao.org/faostat/en/#data/QC/visualize[17] Gustone, F. D. (2004). Rapeseed and canola oil: Production, processing, properties and uses.Oxford,EN:BlacwellPub.

[18]ÇulcuoğluE.,Ünay,E.,&Karaosmanoğlu,F. (2002).RapeseedCakeasBiomassSource.Energy

Sources,24:4,329-336,dOI:10.1080/00908310252888709

[19] Tan, S.H., Mailer, R. J., Blanchard C.L., & Agboola S. O. (2010). Canola Proteins for HumanConsumption: Extraction, Profile, and Functional Properties. Journal of Food Science, 76(1). doi:10.1111/j.1750-3841.2010.01930.x[20]AarhusKarIshamn.(2017).RapeseedOil.RetrievedOctober23,2017,fromhttp://www.aak.com/Global/Brochures/Rapeseedoil_from_AAK.pdf[21] AgriculturalMarketing Resource Center. (2017). Rapeseed. RetrievedOctober 23, 2017, fromhttps://www.agmrc.org/commodities-products/grains-oilseeds/rapeseed/[22]DworakowskaS.,Bednarz,S.,&Bodgal,D.(2011).Productionofbiodieselfromrapeseedoil.1stWorldSustainabilityForum(1-30November2011)[23]Herkes,J. (2014).RapeseedandCanolaforBiodieselProduction.RetrievedNovember4,2017,fromhttp://articles.extension.org/pages/26629/rapeseed-and-canola-for-biodiesel-production[24]Wanasundara, J.P.D.,McIntosh, T.C., Perera, S.P.,Withana-Gamage, T. S., &Mitra, P. (2016).Canola/rapeseedprotein-functionalityandnutrition.Ocl,23(4).doi:10.1051/ocl/2016028[25]NationalResearchCouncil(US)SubcommitteeontheTenthEditionoftheRecommendedDietaryAllowances.RecommendedDietaryAllowances:10thEdition.Washington(DC):NationalAcademiesPress (US); 1989. Available from: https://www.ncbi.nlm.nih.gov/books/NBK234932/doi:10.17226/1349[26]Zayas,J.F.(1997).Functionalityofproteinsinfood.Berlin:Springer[27]Johnson,D.L.,&Croissant,R.L.(1992).Rapeseed/CanolaProduction.no.0110.RetrievedOctober23,2017,fromhttps://dspace.library.colostate.edu/bitstream/handle/10217/183517/AEXT_ucsu2062201101992.pdf?sequence=1[28]Rad,A.H.S.,andZandi,P.(2012).ComparisonofWinterandSpringRapeseedCultivarsConsideringtheirOilContentandFattyacidscomposition.American-EurasianJ.Agric.&Environ.Sci.,12(2):243-248.

37

[29]Wanasundara,J.P.D.,Abeysekara,S.J.,McIntosh,T.C.,&Falk,K.C.(2011).SolubilityDifferencesofMajorStorageProteinsofBrassicaceaeOilseeds.JournaloftheAmericanOilChemistsSociety,89(5),869-881.doi:10.1007/s11746-011-1975-9.[30]WanasundraJ.P.D.(2011).ProteinsofBrassicaceaeOilseedsandtheirPotentialasaPlantProteinSource. Critical Reviews in Food Science and nutrition, 57(7), 635-677. doi:10.1080/10408391003749942[31]Khattab,R.,Eskin,M.,Aliani,M.&ThiyamU.(2009).DeterminationofSinapicAcidDerivativesinCanolaExtractsUsingHigh-PerformanceLiquidChromatography. Journalof theAmericanChemicalSociety,87(2),147-155.doi:10.1007/s11746-009-1486-0.

[32]Gerzhova,A.,Monor,M.,Benali,M.,&Aider,M.(2016).StudyoftotaldrymatterandproteinextractionfromcanolamealasaffectedbythepH,salteditionanduseofzeta-potential/turbidimetryanalysis to optimize the extraction conditions. Food Chemistry, 201, 243–252. doi:10.1016/j.foodchem.2016.01.074

[33] Daun, J. K., Eskin, N.A., & Hickling, D. (2011). Canola- Chemistry, Production, Processing andUtilization.AOCSPress.Pages119-162.

[34]KrimpenM.,Veldkamp,T.(2017)Newalternativeproteinsources:theirpotentialcontribution.LivestockResearch,WageningenUniversityandResearch.

[35]Borisjuk, L.,Neuberger,T., Schwender, J.,HeinzelN., Sunderhaus,S., Fuchs, J.,Rolletschek,H.(2013).SeedArchitectureShapesEmbryoMetabolism inOilseedRape.ThePlantCell,25(5),1625-1640.doi:10.1105/tpc.113.111740

[36] Hu, Z., Hua, W., Zhang, L., Deng, L., Wang, X., Liu, G., Wang, H. (2013). Seed StructureCharacteristics to Form Ultrahigh Oil Content in Rapeseed. PLoS ONE, 8(4). Doi:10.1371/journal.pone.0062099

[37] Campbell, T.C. (2015). Animal vs. Plant Protein. Retrieved December 14, 2017, fromhttp://nutritionstudies.org/animal-vs-plant-protein/

[38]Tzen,J.,Cao,Y.,Laurent,P.,RatnayakeC.,&HuangA.(1993).Lipids,Proteins,andStructureofSeedOilBodiesfromDiverseSpecies.PlantPhysiology,101(1),267-276.doi:10.1104/pp.101.1.267

[39]Tandang-Silvas,M.R., Fukuda,T., Fukuda,C., Prak,K.,Cabanos,C., K,mura,A.,Maruyama,N.(2010). Conservation and divergence on plant seed 11S globulins based on crystal structures.Biochimica Et Biophysica Acta (BBA) – Proteins and Proteomics, 1804(7), 1432-1442. Doi:10.1016/j.bbapap.2010.02.016[40]Rico,M.,Bruix,M.,Gonzalez,C.,Monsalve,R.I.,&Rodriguez,R.(1996)1HNMRAssignmentandGlobalFoldofNapinBnIB,aRepresentative2SAlbuminSeedProtein.Biochemistry,35(49),15672-15682.Doi:10.1021/bi961748q[41]Maurer,S.,Ghebremedhin,M.,Zielbauer,B.I.,Knorr,D;Vilgis,T.(2016).RetrievedJanuary11,2018,fromhttp://www.mpip-mainz.mpg.de/4120261/Oil_bodies_from_plant_seeds[42]Shimada,T.L.&Hara-Nishimura,I.(2010).Oil-Body-MembraneProteinsandTheirPhysiologicalFunctionsinPlants.Biological&.PharmaceuticalBuletin,33(3),360—363.doi:10.1248/bpb.33.360

38

[43]GeertsM.E.J.,Mienis,E.,Nikiforidis,C.V.,PadtA.,&Goot,A.J.(2017).Mildlyrefinedfractionsofyellow peas show rich behavior in thickened oil-in-water emulsions. Innovative Food Science &EmergingTechnologies,41,251–258.doi:10.1016/j.ifset.2017.03.009

[44]Nikiforidis,C.V.,Matsakidou,A.,&Kiosseoglou,V.(2014).Composition,propertiesandpotentialfood applications of natural emulsions and creammaterials based on oil bodies. RSC Adv., 4(48),25067-25078.doiI:10.1039/c4ra00903g

[45]Mcclements,D.J.(2007).CriticalReviewofTechniquesandMethodologiesforCharacterizationofEmulsion Stability. Critical Reviews in Food Science and Nutrition, 47(7), 611-649. Doi:10.1080/10408390701289292[46]Daun,J.K.(1999).Theeffectofglucosinolatesonthenitrogentoproteinratioforrapeseedandcanola;Bull.GCIRC1999,16,58-63.

[47]FoodandAgricultureOrganizationoftheUnitedNations(FAO).(1986).7.Foodanalysis:generaltechniques,additives,contaminantsandcomposition.ManualsofFoodQualityControl,FAOFoodandNutritionPaper14/7.[48] Rosental, A., Pyle, D.L., Niranjan, K. Aqueous and enzymatic process for edible oil extraction.(1996).DepartmentofFoodScienceandTechnology.EnzymeandMicrobialTechnology19,402-420.[49]Nikiforidis,C.V.&,Kiosseoglou,V.(2009).AqueousExtractionofOilbodiesfromMaizeGerm(Zeamays)andCharacterizationoftheResultingNaturalOil-inWaterEmulsion.JournalofAgriculturalandFoodChemistry,57(12),5591-5596.doi:10.1021/jf900771v[50]Rosental,A.,Pyle,D.L.,&Niranjan,K.(1998).SimultaneousAqueousExtractionofOilandProteinfromSoybean:MechanismsforProcessDesign.DepartmentofFoodandBioproductsProcessing,76(4),224-230.doi:10.1205/096030898532124[51]CampbellK.A.,&GlatzC.E.(2009).MechanismofAqueousExtractionofSoybeanOil.JournalofAgriculturalandFoodChemistry,57(22),10904-10912.doi:10.1021/j902298a[52]Wanasundara,J.,&McIntosh,T.(2008).U.S.PatentNo.US8557963.Washington,DC:U.S.PatentandTrademarkOffice.

[53]Aluko,R.E.,&McIntosh,T.(2001).Polypeptideprofileandfunctionalpropertiesofdefattedmealsand protein isolates of canola seeds. Journal of Food and Agriculture, 81(4):391–396. doi:10.1002/1097-0010(200103)81:43.0co;2-s

[54]Aluko,R.E.,McIntosh,T.,&Katepa-Mupondwa,F.(2005).Comparativestudyofthepolypeptideprofiles and functional properties of Sinapis alba and Brassica juncea seed meals and proteinconcentrates.JournaloftheScienceFoodandAgriculture,85(11),1931–1937.doi:10.1002/jsfa.2199

[55]Ghodsvali,A.,Khodaparast,M.H.,Vosoughi,M.,Diosady,L.(2005).Preparationofcanolaproteinmaterialsusingmembranetechnologyandevaluationofmealsfunctionalproperties.FoodResearchInternational,38(2),223–31.Doi:10.1016/j.foodres.2004.10.007

[56] Food and Agriculture Organization of The United Nations (FAO). (2015). Codex Alimentarius-InternationalFoodStandards,Standardfornamedvegetableoils.CODEXSTAN210-1999.

39

[57]FoodandAgricultureOrganizationofTheUnitedNations(FAO)FoodandNutritionPaper.(1986).Manualsof foodquality control. 7. Foodanalysis: general techniques, additives, contaminantsandcomposition.[58]Greenwood,R.(2003).Reviewofthemeasurementofzetapotentialsinconcentratedaqueoussuspensionsusingelectroacoustic.Advances inColloidand InterfaceScience,106 (1-3), 55-81.Doi:10.1016/s0001-8686(03)00105-2[59]Fennema,O.R.,Damodaran,S.,&Parkin,K.L. (2008).Fennema’sFoodChemistry4thEddition.BocaRaton:CRCPress.Page,784-843.[60]Cunningham,N. (2013). ExamplesofRheologyTestingTechniques.RetrievedMarch11,2018,fromhttp://www.rheologyschool.com/testing/testing-examples[61] Chaplin, M. (2018). Hydrocolloid Rheology. Retrieved March 11, 2018, fromhttp://www1.lsbu.ac.uk/water/rheology.html[62]Malvern Panalytical. (2018). Rheological Analysis of Dispersions by Frequency Sweep Testing.RetrievedMarch11,2018,fromhttps://www.azom.com/article.aspx?ArticleID=2884[63] Rustom, I. Y., Lopez-Leiva,M.,&Nair, B.M. (1991). A Study of Factors affecting extraction ofpeanut (ArachishypogaeaL.)solidswithwater.FoodChemistry42(2),153-165.doi:10.1016/0308-8146(91)90031-i[64]Wu, J.&Muir,A.D. (2008). Comparative Structural, Emulsifying andBiological Propertiesof 2Major Canola Proteins, Cruciferin and Napin. Journal of Food Science, 73(3). doi: 10.1111/j.1750-3841.2008.00675.x[65] Nikiforidis, C., & Kiosseoglou, V. (2010). Physicochemical Stability of Maize Germ Oil BodyEmulsionsasInfluencedbyOilBodySurface-XanthanGumInteractions.JournaloftheAgriculturalandFoodChemistry,58(1),527-532.doi:10.1021/jf902544j[66]Nikiforidis,C.,Kiosseoglou,V.,&Scholten,E.(2013).OilBodies:Aninsightontheirmicrostructure-maizegermvssunflowerseed.FoodResearchInternational52(1),136-141.doi:10.1016/j.foodres.2013.02.052[67] Nikiforidis, C.V., Biliaderis, C.G., & Kiosseogluo, V. (2012). Rheological characteristics andphysicochemical stability of dressing-type emulsions made of oil bodies-egg yolk blends. FoodChemistry,134(1),64-73.doi:10.1016/j.foodchem.2012.02.058[68]Nikiforidis,C.V.,Ampatzidis,C.,Lalou,S.,Scholten,E.,Karapantsios,T.D.,&Kiosseoglou,V.(2013).Purifiedoleosinsatair-waterinterfaces.SoftMatter,9(4),1354-1363.doi:10.1039/c2sm27118d[69]White,D.,Fisk,I.,MitchellJ.,Wolf,B.,Hill,S.,&GrayD.(2006).Sunflower-seedoilbodyemulsions:Rheologyandstabilityassessmentofanaturalemulsion.FoodHydrocolloids22(7),1224-1232.Doi:10.1016/j.foodhyd.2007.07.004[70]Mantovani,R.A.,FurtadoG.D.,Netto,F.M.,&Cunha,R.,L.(2018).Assessingthepotentialofwhey protein fibril as emulsifier. Journal of Food Engineering 223, 99-108. Doi:10.1016/j.jfoodeng.2017.12.006