3/19/14UCDAVISMethodstoDetermineOxidationExtraVirginOliveOilSensoryMethodsFreeFattyAcidsPeroxideValueConjugated DienesCarbonylCompounds·TBAValueGasChromatographicMethodsOLIVEFluorescenceMethodsCENTEPERCENTAGEOFSAMPLESFAILINGSENSORYTheInternational OliveCommittee'sPANELSTHATWERECONFIRMEDBYCHEMICALTESTS(IOC)olive oil standardsFree fatty acidity values provide an indication of how the fruit washandled prior to processing and the length of time from harvest to milling.Free acidity is also an early indicator of the potential longevity of the oil.LUONI200000It may be no higher than 0.35% in extra virgin olive oils (EVOO),0733FILIPPOBERIO00STAR1164Peraxide Values It should shown a value no greater thatn 20meq/ig.Experience indicates that highquality, recently miled oils exhibit peroxide62BERTOLLI130values below 12 meq/kg- Truly excellent oils may have PV as low as 7 meq/kgCOLAVITA10070(9 meq/kg for.organic oil).17POMPEIANas1000AVE,ALL68001265OUV abs0rbance, the standards callfor K232 absorbance values below2.5BRANDSLow values correlate with high-quality oil, as UV absorbance detects early andlater states of oxidation.1
3/19/14& 1& Extra Virgin Olive Oil . • Sensory Methods • Free Fatty Acids • Peroxide Value • Conjugated Dienes • Carbonyl Compounds • TBA Value • Gas Chromatographic Methods • Fluorescence Methods Methods to Determine Oxidation The International Olive Committee’s (IOC) olive oil standards Free$fa'y$acidity$values provide&an&indica1on&of&how&the&fruit&was& handled&prior&to&processing&and&the&length&of&1me&from&harvest&to&milling.& Free&acidity&is&also&an&early&indicator&of&the&poten1al&longevity&of&the&oil.&& It&may&be&no&higher&than&0.35%&in&extra&virgin&olive&oils&(EVOO).&& Peroxide$Values$It&should&shown&a&value&no&greater&thatn&20&meq/kg.& Experience&indicates&that&highLquality,&recently&milled&oils&exhibit&peroxide& values&below&12&meq/kg.&Truly&excellent&oils&may&have&PV&as&low&as&7&meq/kg& (9&meq/kg&for&organic&oil).&& UV$absorbance,&the&standards&call&for&K232&absorbance&values&below&2.5.& Low&values&correlate&with&highLquality&oil,&as&UV&absorbance&detects&early&and& later&states&of&oxida1on.& PERCENTAGE$OF$SAMPLES$FAILING$SENSORY$ PANELS$THAT$WERE$CONFIRMED$BY$CHEMICAL$TESTS BRAND$ #$OF$ SAMPLES$ FAILING$ SENSORY$ PANELS$ Free$ Fa'y$ Acids$ Fa'y$ acid$$ Profile$ Peroxide$ Value$ UV$ K232$ DAGs$ LUCINI& 2& 0& 0& 0& 0& 0& FILIPPO&BERIO& 15& 0& 0& 0& 7& 33& STAR& 11& 0& 0& 0& 0& 64& BERTOLLI& 13& 0& 0& 0& 8& 62& COLAVITA& 10& 0& 0& 0& 0& 70& POMPEIAN& 17& 0& 0& 0& 35& 100& AVE,&ALL& BRANDS& 68& 0& 0& 0& 12& 65&
3/19/14MethodsofOliveOilAnalysisAcidityfromLipolysis:Acidity Free Fatty AcidsFactors Acidity ValueNC-OH MicrobialHoa·OxidationORCOOH+ EnzymesP1-Peroxide ValueTemperaturedroer-UVAbsorption Moisture:CompositionofFattyAcidsPreparation of Methyl Esters-DeterminationbyCapillaryGLCKiritsakis, 1998AcidityLimits(IOOC)I.FreeFattyAcidsFreeFaty Acids (%olelc acid)Category.Percentagebyweight ofa specifiedfatty acid0.9ExtraVirginOlive Oil(e.g.,%oleicacid)3.3oils,marineoils,andanimalfatsRefined Oil<0.4Olive Oll<.1Crude Olive Residue OiNoLimitRefined Olive Residue Oll<0.4Olive Residue Oll<.1AOCS Official Method Ca 5a-40Kiritsakis, 19982
3/19/14& 2& Methods&of&Olive&Oil&Analysis& • Acidity& – Free&Fa^y&Acids& – Acidity&Value& • Oxida1on& – Peroxide&Value& – UV&Absorp1on& • Composi1on&of&Fa^y&Acids& – Prepara1on&of&Methyl&Esters& – Determina1on&by&Capillary&GLC& Acidity&from&Lipolysis& • Factors& – Microbial& – Enzymes& – Temperature& – Moisture& Kiritsakis,&1998& Acidity&Limits&(IOOC)& Category$ Free$Fa'y$Acids$(%$oleic$acid)$ Extra&Virgin&Olive&Oil& 3.3& Refined&Oil& <0.4& Olive&Oil& <1.1& Crude&Olive&Residue&Oil& No&Limit& Refined&Olive&Residue&Oil& <0.4& Olive&Residue&Oil& <1.1& Kiritsakis,&1998& I.&&&Free&Fa^y&Acids& • Percentage&by&weight&of&a&specified&fa^y&acid& (e.g.,&%&oleic&acid)& • Applicable&to&all&crude&and&refined&vegetable& oils,&marine&oils,&and&animal&fats& AOCS&Official&Method&Ca&5aL40&
3/19/141I.FreeFattyAcidsProcedureAcidValueWeigh the designated sample size into an oil sampleAmountof KOH (mg)neededto neutralize thebottleorErlenmeyerflask.(Capbottleandshakefree fatty acids in 1g of samplevigorouslyfor1min if oil has been blanketed withcarbon dioxide gas.)Applicableto crude and refined animal,.:Add the specified amount ofhot neutralized alcoholvegetable, and marine fats and oils, andand 2mL of indicator.various productsderivedfrom them.Titrate with standard sodium hydroxide, shakingvigorouslyuntil the appearance of the first permanentpink color of the same intensity as that of theneutralized alcohol before the addition of the sample.The color must persist for 30 seconds.AOCS Official Method Ca 5a-40AOCS Official Method Cd 3d-631.UVAbsorbanceK2a2: Measure conjugated dienes.K2o: Measure conjugated trienes Determines conjugated hydroperoxides formed as primary:Detects substances formed during earth treatment in refiningoxidation productsprocess(Rss+Rga)A=K,_(++kmdAKRzK.isthespecificextinctionatwavelengthwithmaximumabsorbance (270 nm)..Lowabsorbancevaluescorrespondtogoodquality olive oil.AOAC Official Method Ch 5-913
3/19/14& 3& Free&Fa^y&Acids&Procedure& • Weigh&the&designated&sample&size&into&an&oil&sample& bo^le&or&Erlenmeyer&flask.&(Cap&bo^le&and&shake& vigorously&for&1&min&if&oil&has&been&blanketed&with& carbon&dioxide&gas.)& • Add&the&specified&amount&of&hot&neutralized&alcohol& and&2&mL&of&indicator.& • Titrate&with&standard&sodium&hydroxide,&shaking& vigorously&un1l&the&appearance&of&the&first&permanent& pink&color&of&the&same&intensity&as&that&of&the& neutralized&alcohol&before&the&addi1on&of&the&sample.&& The&color&must&persist&for&30&seconds.&& AOCS&Official&Method&Ca&5aL40& II.&&&&Acid&Value& • Amount&of&KOH&(mg)&needed&to&neutralize&the& free&fa^y&acids&in&1&g&of&sample& • Applicable&to&crude&and&refined&animal,& vegetable,&and&marine&fats&and&oils,&and& various&products&derived&from&them& AOCS&Official&Method&Cd&3dL63& • K232:&&Measure&conjugated&dienes& • Determines&conjugated&hydroperoxides&formed&as&primary& oxida1on&products& I.&UV&Absorbance& AOAC&Official&Method&Ch&5L91& • K270:&&Measure&conjugated&trienes& • Detects&substances&formed&during&earth&treatment&in&refining& process& – Kmax&is&the&specific&ex1nc1on&at&wavelength&with& maximum&absorbance&(270&nm).&& • Low&absorbance&values&correspond&to&good& quality&olive&oil.&
3/19/14Calculations:UltravioletAbsorptionBeer s Law::A=ebc0.25g oil +25rExPure isooctane/Ka= Pure cyclohexaneACXS=bXcK, = specific extinction at,E, = extinction measured at,A=absorbance-S = thickness of the cuvetteRecord Abs @ 232nm, 268nm, 262nm, 272nme =molar absorbtivity (mor’ cm-)(cm)b =path length (cm):C =concentration of thec =concentration of the compoundsolution(g/100mL)in solution (mol L-1)K=Abs/Conc.AOACOfficialMethodCh5-91AOACOfficialMethodCh5-91Procedures-BackTitrationIl.PeroxideValueOll sample50ml 3:2 acetic acid + isooctane solutionExtentofoxidationPrimarvproduct-Earlvstageofoxidation250mlBlueDtsapceared0.1M Na,5,0,0.5ml Sat. KISit for 1 minYellow Disappeared(keep shaking)0.5ml 10% SDS0.5ml starch indicator8OmI Distilled H,CAOAC Official Method Cd Sb-90a
3/19/14& 4& Ultraviolet&Absorp1on& 0.25g&Oil&&+&&& Pure&isooctane/& Pure&cyclohexane& Record&Abs&@&232nm,&268nm,&262nm,&272nm& 25ml& AOAC&Official&Method&Ch&5L91& • Kλ&=&specific&ex1nc1on&at&λ • E&λ&=&ex1nc1on&measured&at&λ • S&=&thickness&of&the&cuve^e,& (cm)& • C&=concentra1on&of&the& solu1on&(g/100mL)&& • K=Abs/Conc.$ Calcula1ons:& • Beers&Law&:& • A&=ebc& • A&=absorbance&& • e&=molar&absorb1vity&(molL1&cmL1)& • b&=path&length&(cm)& • c&=concentra1on&of&the&compound& in&solu1on&(mol&LL1)& AOAC&Official&Method&Ch&5L91& II.&Peroxide&Value& • Extent&of&oxida1on& • Primary&product&–&Early&stage&of&oxida1on& ProceduresL&Back&Titra1on& Oil&sample& 50ml&3:2&ace1c&acid&+&isooctane&solu1on& 0.5ml&Sat.&KI& Sit&for&1&min& (keep&shaking)& 30ml&Dis1lled&H2O& 0.1M&Na2S2O3& 0.5ml&10%&SDS& 0.5ml&starch&indicator& Yellow&Disappeared& Blue&Disappeared& 250ml& AOAC&Official&Method&Cd&8bL90&
3/19/14GasChromatographyCCmethodsInjectorComputerorDetectorInjectorHanjiang ZhuovenovenTypicalyTprogranmingiscohurequired, whichprovides a basis forthe separation ofColumn ovensample componentsbased onBP.OuterwailofcolumnCapillarycolumns0.10.53mminner.diameteStationary phase(0.15 μm mick)(a)ars000Column.wallWal-coatedSuppon-coatedPorousapentubusntubulartubularCoucolumncolumn(SCOT)(WCOT)(PLOT)(e)5
3/19/14& 5& GC Methods Hanjiang Zhu Gas$Chromatography$ Typically&T& programming&is& required,&which& provides&a&basis&for& the&separa1on&of& sample&components& based&on&BP.& Capillary$columns$
3/19/14FlamelonizationDetector(FID)FattyacidProfile.Fatty acid?Sensitive towards organicsMaincomponent inedibleoil?Analyte is burned in H2/air,CaCFicewhich produces CH and CHO*--Indicator of PurityTniglmid"pw"Fah4niHytogFigure 24-14nAPoAhCA]FattyacidFattyacid constituent of soybean olFattyacid constituent of corn ollLLLLFattyacid constituent of olive oilaOlelc acidC18:1n
3/19/14& 6& Flame&Ioniza1on&Detector&(FID)& •&Sensi1ve&towards&organics& •&Analyte&is&burned&in&H2/air,&&&&&&&&& which&produces&CH&and&CHO+& Fa'y$acid$Profile$ • Fa'y$acid$ – Main$component$in$edible$oil$ – Indicator$of$Purity$ Fa'y$acid$ 0& 10& 20& 30& 40& 50& 60& 70& C16:0& C16:1& C18:0& C18:1& C18:2& C18:3& Others& %$Fa'y$acid$composiXon$ Fa'y$acids Fa'y$acid$consXtuent$of$olive$oil$ OH O Oleic$acid$ C18:1$ 0.0& 10.0& 20.0& 30.0& 40.0& 50.0& 60.0& C16:0& C18:0& C18:1& C18:2& C18:3& Others& %$Fa'y$acid$composiXon$ Fa'y$acids Fa'y$acid$consXtuent$of$soybean$oil$ soybean& 0& 10& 20& 30& 40& 50& 60& C16:0& C18:0& C18:1& C18:2& C18:3& Others& %$Fa'y$acid$composiXon$ Fa'y$acids Fa'y$acid$consXtuent$of$corn$oil$ corn& 0& 10& 20& 30& 40& 50& 60& C16:0& C18:0& C18:1& C18:2& C18:3& C20:1& Others& %$Fa'y$acid$composiXon$ Fa'y$acids Fa'y$acid$consXtuent$of$canola$oil$ Canola&
3/19/14FattyacidFattyAcids-methodsIOC limitsFattyacidprofile.Myristic acidC14:0≤0.05Palmitic acidC16:07.5-20.0-Preparation of fatty acid methyl esters (FAMEs)C16:10.3-3.5Palmitoleic acid?Methyl esterizationHeptadecanoic acidC170503Heptadecenoic acidC17:≤0.3Gas Liquid ChromatographyStearicacidC18:00.5-5.0olelc acidC18:155.0 - 83.0Unoleic acidC18:23.5-21.0Unolenic acidC18:3<1.0arashidic asidC20:0≤0.6Eicosenolc acidC20:1≤0.4BehenicacidC22:0≤0.250.2Lignoceric acidC24:0FAMEStandardAcid methyl esterization0.05g of oilfneblocks0.6ml HCI-MeOHVortexOvernight ( 14h)40C1813.0ml of MeOH1.0ml hexaneNazsO1.0mtHzCStandfor15mlRemctheuppVorterC16:C18:2C18:0c1a:
3/19/14& 7& Fa'y$acid && IOC&&limits& Myris1c&acid& C14:0& <&0.05 Palmi1c&acid& C16:0& 7.5L&20.0& Palmitoleic&acid& C16:1& 0.3L3.5& Heptadecanoic&acid& C17:0& <&0.3 Heptadecenoic&acid& C17:1& <&0.3 Stearic&acid& C18:0& 0.5&L&5.0& Oleic&acid& C18:1& 55.0&L&83.0& Linoleic&acid& C18:2& 3.5&L&21.0& Linolenic&acid& C18:3& <&1.0 Arachidic&acid& C20:0& <&0.6 Eicosenoic&acid& C20:1& <&0.4 Behenic&acid& C22:0& <&0.2 Lignoceric&acid& C24:0& <&0.2 Fa'y$Acids$\$methods$ • Fa'y$acid$profile – PreparaXon$of$fa'y$acid$methyl$esters$(FAMEs)$ • Methyl&esteriza1on – Gas$Liquid$Chromatography$$ Acid&methyl&esteriza1on 0.05g&of&oil&& 3.0ml&of&MeOH& Hea1ng&block&@& 40 Remove&the&upper&layer& Vortex& Overnight&(~&14h) Na2SO4 1.0mlH2O& 1.0ml&hexane& Stand&for&15&min& &&Vortex& 0.6ml&HClLMeOH& FAME$Standard$ C18:1$ C18:0$ C18:2$ C18:3$ C16:0$ C16:1$
3/19/14UCDAVISOliveOilIdentificationofDiacylglycerolIsomersUsingGC-MSCLNYERWhatareDAGs?Olive OilC16:0CH,OCOR,CH,OCOR,C18:1C18:1OHCHOHOHCHOCORC18:1CH,OCOR3C16:0CH,OH1,3-diacylalycerol1,2-diacylglycerolDHC18:CIB:1FreshOliveOil:1,2-DAG/totalDAG>40%8
3/19/14& 8& Olive$Oil$ C18:1$ C18:0$ C16:1$ C16:0$ C18:2$ C18:3$ Identification of Diacylglycerol Isomers Using GC-MS What are DAGs? ! Fresh Olive Oil: 1,2-DAG/total DAG > 40% Olive Oil
3/19/14SensoryMethodsPeroxideFormation and DecompositionAnalyticalpanel5-20 subjects trained to differentiate and rank samplesusing numerical scale: 0 to 10 and reference standardConsumer panel50 untrained subjects to indicate preference and acceptabilityoeoPV.orSensitivityConjugatedability to detect sensory characteristicsdiennsThreshold value: lowest concentration of volatile detected by50% of the panelistsAldehydesorAdvantages;CarboryisSensitive: some flavors/odors can only be detected by sensorymethods, most relevant to consumer perception of qualityDisadvantages:Time Not reproducible, costly, difficult to train panel judgesVolatileCompoundsGCAldehydes, carboxylic acid, alcohol, hydrocarbons...FlavorsLipolysisOff-flavorsOxidationVolatileDdeSensoryPreperbeshelCompoundTheeiEVOOOsidized-EVOOHesanalGreen,appleFatty.powerful,oly.grassy13-L0OH0.322-Heu12/13-Ln00HBimer,dgreenOxidzedtallorOctanal11-000H0.32Fattyshare,citrus2-OctenalFruity,soapPowerful,faty,ctrus11-L00H9/10-000H13.9FattywaxPenetratine9/10-L00H3.2C
3/19/14& 9& Analy1cal&panel:&&& &&5L20&subjects&trained&to&differen1ate&and&rank&samples& &&using&numerical&scale:&0&to&10&and&reference&standard& Consumer&panel:& &&50&untrained&subjects&to&indicate&preference&and&acceptability& Sensi1vity:&& &&ability&to&detect&sensory&characteris1cs& &&Threshold)value: lowest&concentra1on&of&vola1le&detected&by& &&&50%&of&the&panelists& Advantages:& &&Sensi1ve:&some&flavors/odors&can&only&be&detected&by&sensory& &&&&methods,&most&relevant&to&consumer&percep1on&of&quality& Disadvantages:& &&Not&reproducible,&costly,&difficult&to&train&panel&judges& Sensory Methods Peroxide&Forma1on&and&Decomposi1on& Volatile Compounds ! Aldehydes, carboxylic acid, alcohol, hydrocarbons. ! Flavors ← Lipolysis ! Off-flavors ← Oxidation VolaXle$ Compound Sensory$ProperXes Precursor Odor$ Threshold EVOO OxidizedLEVOO Hexanal Green,&apple Fa^y,&powerful,&oily,&grassy 13LLOOH 0.32 2LHexenal Bi^er,&almond,&green Oxidized,&tallow,&pungent 12/13LLnOOH 10 Octanal Fa^y&sharp,&citrus 11LOOOH 0.32 2LOctenal Fruity,&soap Powerful,&fa^y,&citrus 11LLOOH Nonanal Fa^y,&waxy 9/10LOOOH 13.5 2LNonenal Penetra1ng,&fa^y,&waxy,& beany,&rancid 9/10LLOOH 3.2 Morales, M.T., Rios, J.J., Aparicio, R., 1997. Changes in the volatile composition of virgin olive oil during oxidation: Flavors and off-flavors. J Agri. Food Chem. 45, 2666-2673. Peak Identification of Oil Sample (GC)
3/19/14MainCleavageMechanismsforMain Cleavage Mechanisms forLinolenateLinoleate HydroperoxidesHydroperoxidesMethvtg-oxononanoate_OOH2,4,7R24-Decadienal.MethytoctanoateOOHHeA7020mLipid Oxidation MethodsMethodsSensitivityPrecisionInformationDetermination ofSteroisin Olive Oils.fromDifferentVarietiesSensoryHighvGaschromatoerapnyLowHighVolatiles (GC)HighLowHighUVanalysisHighHighLowThe extra virgin olive oils (California, USA) produced from seven different oliveCarbonylsLowHighLowvarieties,Arbequina, Ascolano, Frantoio, Koronelki, Manzanillo, Mission andAnisidine valueHighLowSerillano, were bought in local grocery stores.LowPeroxidevalueHighLowLowOxygenuptakeHighLowSaponification was achieved by heating EVOO in 2M KOHEthanol at 80°C forLowonehour.The unsaponifiables wereextracted by diethyl ether.Then1000mg/TBALowHighLowGmL silia SPE was chosen for all sample preparafions due to higher recoveryVolatile acidsLowLowrate. The steps of the SPE clean-up for purification are shown in Fig. 2. SaimplesLowwere derivatized with silylation reagent before the GC-MS determination.(Rancimat)10
3/19/14& 10& OOH R 2,4-Decadienal Methyl 9-oxononanoate Methyl octanoate 9 R Hexanal OOH 13 Pentane Main$Cleavage$Mechanisms$for$ Linoleate Hydroperoxides OOH R 2,4,7-Decatrienal Methyl 9-oxononanoate Methyl octanoate 9 R 2,4-Heptadienal OOH 12 R 2- or 3-Hexenal OOH 13 OOH Propanal 16 R Main$Cleavage$Mechanisms$for$Linolenate Hydroperoxides The extra virgin olive oils (California, USA) produced from seven different olive varieties, Arbequina, Ascolano, Frantoio, Koronelki, Manzanillo, Mission and Serillano, were bought in local grocery stores. Saponification was achieved by heating EVOO in 2M KOH/Ethanol at 80°C for one hour. The unsaponifiables were extracted by diethyl ether. Then 1000mg/ 6mL silia SPE was chosen for all sample preparations due to higher recovery rate. The steps of the SPE clean-up for purification are shown in Fig. 2. Samples were derivatized with silylation reagent before the GC-MS determination. DeterminaXon$of$Sterols$in$Olive$Oils$from$Different$VarieXes$ by$Gas$Chromatography$