MinorMinor ComponentsComponents asas markersmarkers ofof oliveolive oiloil authenticityauthenticity andand qualityquality
V. Van Hoed, R. Verhé, M. Andjelkovic
Ghent University - Faculty of Bioscience Engineering - Department of Organic Chemistry
OverviewOverview
I. Importance of analysis and authentification of olive oil II. Methods for determination of 1. Quality 2. Genuineness III. Authentication issues IV. Olive oil components used in this study V. Examples 1. Harvest time 2. Cultivar and geographic origin VI. Conclusions Importance of Analysis and Authentication of olive oils
EVOO : mechanical extraction : unique composition and delicate aroma Lampante OO : needs refining Consumers preference: high quality + unchanged aroma and natural elements
Higher price than other vegetable oils
Danger of adulteration with cheaper ingredients = economical fraud = risk for consumers’ health
Importance of Analysis and Authentication of olive oils
Detect adulteration with cheaper ingredients
Need for analysis and authentication methods Codex Alimentarius CA Commission Draft, 2003 European Commission (EC) EC Reg No 2568/1991 and amendment 1989/2003 International Olive Oil Council (IOOC) IOOC Trade Standards, 2003 = Official methods
BUT sophisticated refining/adulteration Need for continual research = new methods, not (yet) evaluated by standardizing bodies, but proposed by researchers Methods for olive oil quality verification a) Methods included in the international Standards
• Olive oil sampling and laboratory sample preparation • FFA • PV • Absorbances in the UV region • Organoleptic assessment of VOO • Volatile halogenated solvents in OO • Metals Addressed in α • -tocopherol this lecture • Moisture and volatile matter content • Insoluble impurities in petroleum ether b) Methods not included in the international Standards • Phenolic compounds • Volatile Compounds • Partial Glycerides: MG, DG • Accelerated oxidation tests • Pigments (and derivatives) • Contaminants
Methods for olive oil genuineness verification Do the olive oil parameters correspond to the class it is said to be? Genuineness: actually processing the alleged or apparent character a) Methods included in the international Standards • FA composition • t-unsaturated FA • FA in the 2-position of TAG • ∆ECN42 values • Sterol composition • Erythrodiol and uvaol • Wax content • Aliphatic alcohol content • Stigmastadienes • Spectrophotometric analysis in the UV region
b) Methods not included in the international Standards • Triacylglycerols • Alcoholic fraction • Other abosorptions in the visible and UV spectra • Hydrocarbons Authentication issues ~ Traceability Adulteration with other vegetable oils Olive Variety Geographical Origin
Adulteration Current Problems • Addition of hazelnut oil • Olive oil subjected to forbidden deodorization in mild conditions • mixtures of virgin olive oil with olive oil obtained by second centrifugation of the olive pastes (Remolido) Varietal Characterization Characterization of virgin olive oil by geographical origin
Influencing factors Oil composition
olive Fatty Acids variety composition geographical location ripeness Triacylglycerol of olives composition agricultural Quality practices Tocopherols (α-tocopherol) Authenticity storage Authenticity conditions of olives Phytosterols processing
Polyphenols storage conditions … … of oils Fatty acids composition
FA composition and Olive oil genuineness (evoo) C18:1 : 55-83 % typical for olive oil : quality (stability, health) + authenticity
C18:3 ≤ 1 % (highest in Moroccan genuine oo) Minor FA prominent in seed oils: C20:1 ≤ 0.4 % <> adulteration with SFO, RSO C22:0 ≤ 0.2 % <> adulteration with SFO, RSO C22:1 ND <> adulteration with RSO C24:0 ND <> adulteration with peanut oil ! Limits not useful if adulteration with ≤ 5% of seed oil FA composition similar for hazelnut oil, high oleic sunflower oil other analyses needed Analysis: GC of FAME
Triacylglycerol composition
TAG in authenticity assessment : evidence mixing with Re-esterified (olive) oils Hazelnut oil LLL cfr. supra: useful BUT addition canola oil till 7.5% not detected ∆ECN42 value (~ ∆ LLL) Desterolized seed oils on the market with similar FA composition as olive oil adulteration not discernible from FAME analysis Falsification: seed oils: high content in ECN42 TAG (LLL) Detection of falsification: ∆ECN42 = | LLLreal - LLLtheor | ≤ 0.2 (EVOO) LLLreal = LLL content from HPLC analysis of TAG LLLtheor = LLL content, theoretical, calculated from FAME Analysis : Oil dilution; RP-HPLC + RI or ELSD GC (phenyl-methyl-silicone phase) Tocopherols ( mainly α-tocopherol)
In oil: Antioxidant Human health: vitamin E
content : 100-300 ppm (good quality olive oils) composition : 90% α–tocopherol ↓↓ during refining Added to refined (seed) oils to ↑ ox.stab. (allowed)
Analysis Oil dilution in hexane Separation/Quantification: normal phase HPLC + fluorescence detection
Phytosterols
in plant: part of cell membranes; very characteristic for each botanical species Human health: Antihypercholesterolemic
Content in olive oil : 1000-2000 mg/kg Refining ↓ sterols (dehydration) formation of steradienes Adulteration with seed oils content > 2000 mg/kg
Composition : characteristic content and profile in olive oils desmethylsterols, 4-methylsterols, 4-dimethylsterols
In this study: 24-methylene cycloartanol β-sitosterol cycloartenol Campesterol citrostadienol Stigmasterol
Analysis Saponification Extraction unsaponifiables Gas chromatograpy of unsaps Phenolic compounds
In oil; human health: antioxidant
Content : 50-1000 mg/kg (usual: 100-300)
Composition : Characteristic content and profile in olive oils Simple phenols, phenolic acids, lignans, flavonoids In this study: - Hydroxytyrosol - Vanillin - Tyrosol - Lutein - Vanillic acid - Apigenin - p-coumaric acid
Analysis SPE Extraction + clean-up Total phenols content: Folin-Ciocalteu colorimetric method Individual phenols : Separation on HPLC: RP-HPLC-UV (280 nm) + MS
Examples
Oils from European and non-European countries
Analysis of characteristic composition
Principal Components analysis of results Clustering of similar olive oils Cultivar
Origin PCA analysis on olive oils Examples : Oils from
Spain South-France Corsica Italy Syria
Analyses
Fatty Acids
Triacylglycerols Tocopherols (α-tocopherol)
Phytosterols Polyphenols
http://www.drakecorp.com/s_distribution/ee-map.gif
PCA analysis on olive oils
Geographical Origin Cultivars
Spain Picual, Verdial, Cornicabra
South-France Aglandou, Cailletier, Tanche, PDO Aix en Provence & Vallée des Baux
Corsica Ghjermana, Zinzala, Sabina
Many different cultivars, each in 1 repetition: Italy Pisciottana, Tonda Iblea, Ottobratica, Gentile di Chieti, Carolea, Ogliarola Leccese, Cellina di Nardo, Nocellara del Belice, Coratina, Taggiasca, Bosana, Dritta, Tonda di Cagliari, Cima di Mola, Peranzana, Sinopolese
Syria Zayti, Sourani, Nibali, Commercial
http://www.drakecorp.com/s_distribution/ee-map.gif 1. FAME and TAG
-40 -20 0 20 40
Oils of 2 countries: I •Italian I Higher PUFA
•Spanish S I I I S Higher MUFA I I POO Sat S C16.0 S C18.1n7C16.1n7PoOPPoOOPOPPOSI S PLO PLLC18.3n3C20.1n9PLPPLnOSqaleneOLnOOLnLC17.1C17.0C24.0C16.1C20.0C22.0PLnLSLOLLLPPSC18.0SOO S MonoC18.1n9 LOL OOO
In this case: Comp.2 C18.2n6 Poly LOO S S I Separation based on I country S No cultivars separation I S -40 -20 0 20 40 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6
-0.6 -0.4 -0.2 0.0 0.2 0.4 0.6
Table:statistiek-oo-2009-FATAG.xls Comp.1
File: FATAG.sdd
1. FAME and TAG -100 -50 0 50
I Pisciottana S Oils of 3 countries: S
I S FFF F FFFFFI C FFFF S LOO FFFF C C18.2n6Poly FFFF C S OOO •Italian I PLO LOLOLnOI F C MonoC18.1n9S C18.1n7C16.1n7C18.3n3C20.1n9PLLPLnOPoOOPoOPPLPC20.0C18.0POSSLOPPSSOO S POPFFFF S Higher PUFA I C16.0I Sat FF NA I CFFF S CFFFPOOFF CCFFF •Spanish S I FFF F C F
Higher MUFA Comp.2
I
•Corsican C •French F -100 -50 0 50 -0.6 -0.4 -0.2 0.0 0.2 Both: intermediate
-0.6 -0.4 -0.2 0.0 0.2 Comp.1
Table:statistiek-oo-2009-FATAG.xls File: FATAG4.sdd 2. FAME and TAG + sterols + tocols
-200 0 200 400 Oils of 2 countries: I I •Italian I I totster All different cultivars: scattered; I From Lecce (It) TotComSter betasito Slight distinction for oils from Cornicabra S I
Lecce Comp.2 m24cyclo S citrostadC18.2n6d5avenaPolyPLOcycloart m24.totC20.1n9C18.3n3C16.1n7C18.1n7Sqalenecitro.totbeta.totd5a.totC18.0PoOOC16.1C17.0C17.1C20.0C22.0C24.0OLnOPoOPPLnOstigmOLnLcyclo.totSOOca.totPLnLC16.0st.totPOScampSLOPPSPOPLOOPOOLOLPLPPLLLLLSat C18.1n9Mono S S OOO S S Picual, Verdial (S)SS •Spanish S S tot.toco I I Picual & Verdial: separated from -0.2 0.0 0.2 0.4 0.6 I
Cornicabra (mainly: sterols -200 0 200 400 composition) I
-0.2 0.0 0.2 0.4 0.6 Comp.1
2. FAME and TAG + sterols + tocols
-200 0 200 400
Oils of 3 countries: •Italian I I Many different cultivars totster I (and regions) scattered I TotComSter C I I betasito Zinzala (C) •Spanish S Cornicabra S C From Lecce (It) Picual, Verdial : close C Ghjermana m24cyclo C Comp.2 SCcitrostadcycloart C C18.2n6d5avenaPoly Cornicabra: further m24.totC18.1n7C18.3n3C20.1n9C16.1n7citro.totbeta.totd5a.totPoOOOLnOC18.0C20.0PoOPPLnOstigmcyclo.totca.totC16.0SOOcampst.totPOSSLOPOPLOOPPSLOLPOOPLPPLOPLLSat S C18.1n9OOOMono S SS I •Corsican C SS S tot.toco I I Picual, Verdial (S) Ghjermana, Zinzala, I Sabina (poorest in sterols) -200 0 200 400 cultivars: separated C Sabina (c) C C -0.4 -0.2 0.0 0.2 0.4 -0.4 -0.2 0.0 0.2 0.4 Comp.1 3. Sterols + tocols + phenols -2 0 2 4
From Imperia Sy Syrian - South TotComStertotsterbetasito Example: I
Oils of 3 countries: Syrian - Middle J Sy VanA cycloart stigm VanI •Italian I camp m24cyclo d5avena S citrostad Apig •Spanish S Cornicabra (S) (Huelva) Com tot.toco Sy TotPhenP.co From Cosensa Sy Tyr •Syrian Sy I I Fer Sy HyTyr S Comp.2 Syrian - North •(Jordan: J) S Lut Sy Picual, Verdial (S) S Huelva SSSSS Picual, Verdial (S) S -2 0 2 4 Torres De Albanches, Orcera Commercial
-0.4 -0.2 0.0 0.2 0.4 Sy
-0.4 -0.2 0.0 0.2 0.4
Comp.1
4. FAME, TAG + sterols + tocols + phenols Example: Spanish extra virgin olive oils
Cultivar Region (Spain) Subregion Cornicabra Castilla Toledo Verdial Picual Huelva, Villarrasa Verdial Picual Huelva, Bartolina: 50 km from Verdial Andalucia Villarasa Picual Jaen, Puerta de Segura Picual Jaen, Torres de Albanchez Picual Jaen, Orcera Picual
Oils: Kindly provided by: Prof. A. Martin & P. Hernandez, CSIC, Spain Toledo
Ja én Huelva
4. FAME, TAG + sterols + tocols + phenols -10 -5 0 5 10 FAME + TAG V
Jaen P Villarasa (3 Verdial, 1 P: Picual P Picual) C: Cornicabra V: Verdial P POO C16.0Sat C18.1n7POP SOOC16.1n7PoOOC20.1n9C18.3n3C18.0POSC20.0PoOPPLnOPPSPLLPLPSLOLOL Mono OLnO PLO C18.2n6Poly C18.1n9 PV Comp.2 LOO Region OOO P Cultivar PPicual in Villarasa
C Toledo -10 -5 0 5 10
V -0.6 -0.4 -0.2 0.0 0.2 0.4
-0.6 -0.4 -0.2 0.0 0.2 0.4 Comp.1 4. FAME, TAG + sterols + tocols + phenols
FAME + TAG + -60 -40 -20 0 20 40 sterols P m24cyclo Puerta P DS P: Picual C: Cornicabra totster V: Verdial V cycloart V d5avenaC18.2n6LOO C18.1n9OOOcampC16.1n7C18.3n3C20.1n9C18.1n7MonoPoOOC18.0OLnOC20.0PoOPPLnOSOOC16.0POOstigmPOSPPSPOPSLOPLPPLLLOLPLOSatPoly P citrostad VP
Region Comp.2 TotComSterbetasito P Subregion P C Cultivar Orcera -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 -60 -40 -20 0 20 40
-0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6
Comp.1
4. FAME, TAG + sterols + tocols + phenols
FAME + TAG + -50 0 50 sterols + toco C
totster P: Picual TotComSter C: Cornicabra Puerta betasito V: Verdial DS P
cycloartcitrostad d5avenaC18.1n9OOOMonocamp m24cyclo C18.1n7C16.1n7C18.3n3C20.1n9C18.2n6PoOOC18.0C16.0OLnOC20.0PoOPPLnOPOOSOOstigmPOSPOPPPSSLOPLPPLOLOLPLLSatPolyLOO V
Comp.2 P tot.toco P PP V Region V P Subregion
Cultivar -0.5 0.0 0.5 -50 0 50
-0.5 0.0 0.5 Comp.1 4. FAME, TAG + sterols + toco + phenols
FAME + TAG + sterols + -150 -100 -50 0 50 toco + phenols Toledo C Folin Huelva Jaen PP Jaen
JaenJaen TotComSterbetasitotot.toco P: Picual totster OOO cycloartApigC18.1n9MonoPOO d5avenaC18.3n3C20.1n9C16.1n7C18.1n7campPoOOOLnOC18.0C20.0VanAPoOPPLnOC16.0stigmVanISOOPOSPPSPOPSLOPLPPLLSat Lutm24cycloC18.2n6P.coPLOPolyLOL C: Cornicabra HyTyr citrostadLOO V: Verdial
Comp.2 Tyr Huelva TotPhen Huelva V Huelva Region V V Subregion Cultivar -0.6 -0.4 -0.2 0.0 0.2 0.4 V Huelva -150 -100 -50 0 50
-0.6 -0.4 -0.2 0.0 0.2 0.4 Comp.1
ConclusionsConclusions
Extra virgin olive oil: high quality, high price adulteration Lower quality olive oils QUALITY Other vegetable oils AUTHENTICITY
Detection : chemical analyses: FA, TAG, minor components Clustering of oils in groups ~ Cultivar ; Region ; Subregion FA + TAG main groups + minor compounds: Sterols, tocopherols, polyphenols
more detailed separation: Region, subregion, cultivar, agricultural practices Improving models combination of various components Extension number of samples New tools in determination EVOO quality and authenticity ThankThank youyou
forfor youryour attentionattention !!