《食品化学》课程教学课件（国外讲稿）08 Lipids part 03

3/19/14 UCDAVIS Methods to Determine Oxidation Extra Virgin Olive Oil Sensory Methods ·Free Fatty Acids ·Peroxide Value ·Conjugated Dienes ·Carbonyl Compounds .TBA Value Gas Chromatographic Methods Fluorescence Methods The International Olive Committee's PERCENTAGE OF SAMPLES FAILING SENSORY PANELS THAT WERE CONFIRMED BY CHEMICAL TESTS (IOC)olive oil standards 15 0. 07 33 Peraxide Values It should shown a value no greater thatn 20 meq/kg. STAR 64 BERTOLLI 0 0 0 8 62 COLAVITA 0 17 0 00 35 100 UV absorbance,the standards call for K232 absorbance values below 2.5. 680001265 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&

3/19/14 Methods of Olive Oil Analysis Acidity from Lipolysis ·Acidity -Free Fatty Acids -Acidity Value Factors -060-9 HC-DH ·Oxidation +e-0m -Microbial -Peroxide Value EC0-R N H ME-Or -Enzymes -Temperature -UV Absorption -Moisture Composition of Fatty Acids -Preparation of Methyl Esters Determination by Capillary GLC Kintsakk,1998 Acidity Limits(IOOC) I.Free Fatty Acids Free Fatty Acids () Percentage by weight of a specified fatty acid (e.g.,oleic acid) Applicable to all crude and refined vegetable oils,marine oils,and animal fats c Oi AOCS Offclal Method Ca 5a-40

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&

3/19/14 Free Fatty Acids Procedure ll.Acid Value Weigh the designated sample size into an oil sample Amount of KOH(mg)needed to neutralize the bottle or Erlenmeyer flask.(Cap bottle and shake vigorously for 1 min if oil has been blanketed with free fatty acids in 1g of sample carbon dioxide gas.) Applicable to crude and refined animal, Add the specified amount of hot neutralized alcohol and 2 mL of indicator. vegetable,and marine fats and oils,and various products derived from them Titrate with standard sodium hydroxide,shaking vigorously until the appearance of the first permanent pink color of the same intensity as that of the neutralized alcohol before the addition of the sample. The color must persist for 30 seconds. AOC5 Offidal Method Ca 5a-40 I.UV Absorbance K232:Measure conjugated dienes ated hydroperoxides formed as primary AR-Raesgs) Low absorbance values correspond to good quality olive oil. AOAC Offcial Method Ch5-91

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.&

3/19/14 Ultraviolet Absorption Calculations: ·Beer'sLaw ·A=ebc bxc A 、A=absorbance Record Abs 232nm.268nm,262nm,272nm S=thickness of the cuvette, ntration of the solution (g/100mL) ·K=Abs/Conc. AQAC Official Method Ch5-91 ll.Peroxide Value Procedures-Back Titration ·Extent of oxidation Primarv product-Earlv stage of oxidation 人人 0.1M Na,S.O 0.Sm Sat.Kl Yellow Dsappeared 30ml Distilled H.O AOAC Official Method Cd 8b-90

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:& • Beer
s&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&

3/19/14 Gas Chromatography GC ethods Hanjiang Zhu Column oven ased on P. Capillary columns

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$columns$

3/19/14 Flame lonization Detector(FID) Fatty acid Profile Sensitive towards organics Fatty acid .Analyte is burned in H2/air, -Main component in edible oil which produces CH and CHO -Indicator of Purity nemd Fatty acid f soybean o rn o Fatty ad constituent of ollve oll : 8u 6

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&

3/19/14 Fatty acid Fatty Acids-methods IOC limits C140 ≤005 Fatty acid profile tic add 75200 -Preparation of fatty acid methyl esters(FAMEs) ·Methyl esterization 055.0 -Gas Liquid Chromatography 55.083.0 e c20 chenic add C220 02 C24:D FAME Standard ght (14h) c181 Bemove the C16:0 182

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$

3/19/14 UCDAVIS Olive Oil a81 Identification of Diacylglycerol Isomers Using GC-MS 8 What are DAGs? Olive Oil CH2OCOR CH,OCOR CHOCOR2 HOH -OH 0H CH2OH CH2OCOR -c15:0 -C18:1 1,2-diacylglycerol 1.3-diacylglycerol 一C18:1 C18:1 Fresh Olive Oil:1,2-DAG/total DAG 40% Betertion Time (min 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

3/19/14 Sensory Methods Peroxide Formation and Decomposition Analytical panel: 5-20 subjects trained to differentiate and rank samples using numerical scale:0 to 10 and reference standard Consumer panel 50 untrained subjects to indicate preference and acceptability PV.or Sensitvity: ability to detect sensory characteristics Conjugated Threshold volue:lowest concentration of volatile detected by 50%of the panelists dvantages: Sensitive:some flavors/odors can only be detected by sensory methods,most relevant to consumer perception of quality Not reproducible,costly,difficult to train panel judges Time Volatile Compounds Peak ldentification of Oil Sample(GC) .Aldehydes.carboxylic acid,alcohol,hydrocarbons. .Off-vors 3-L00H 032 9/1000 32

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)

3/19/14 Main Cleavage Mechanisms for Main Cleavage Mechanisms for Linolenate Linoleate Hydroperoxides Hydroperoxides Methyl g-oxononanoate 900H R 2,4-Decadienal Methyl octanoate -er Lipid Oxidation Methods 7 Methods Sensitivity Precision Information Sensory High Low High Volatiles (GC) High Low UV analysis Carbonyls Low Anisidine value Low High Peroxide value Low Oxygen uptake Low 目 TBA Low Low en for all ga Volatile acids Low Low Low (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$