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Workshop on
olive oil authentication
Madrid, Spain
10 & 11 June 2013
With the participation of the
International Olive Council
Organised by
European Commission
Directorate General
Agriculture and Rural Development
&
European Commission
Joint Research Centre
Institute for Reference Materials and Measurements
Poster Authors
Preparation of certified reference materials of
olive oil for physicochemical and sensory
characteristics
L. Cuadros-Rodríguez, A.
González-Casado, et al.
Chromatographic fingerprinting methodology for
olive oil authentication
A. González-Casado, L.
Cuadros-Rodríguez, et al.
Fatty Acid Composition and d13C of Bulk and
Individual Fatty Acids as Marker for Authen-
ticating Italian PDO/PGI Extra Virgin Olive Oils
A. Faberi, F. Fuselli, et al.
On-line HPLC-GC-FID for the evaluation of the
quality of olive oils though the methyl, ethyl, and
wax esters
M. Biedermann
Detection of plant oil DNA using molecular
markers and PCR analysis: a tool for disclosure of
olive oil adulteration
M. Vietina, C. Agrimonti,
N. Marmiroli
FT-MIR/ATR monitoring of virgin olive oil
oxidative stability under mild storage
conditions
M. Z. Tsimidou, N.
Nenadis , et al.
Challenges of U.S. Enforcement Increase with
Conflicting Standards & Methods
E. Balch
L. Cuadros-Rodríguez1, A. González-Casado1, A. Carrasco-Pancorbo1, C. Ruiz-Samblás1, J.A. García-Mesa2, F.P. Rodríguez-García3
1 Unit of Chemical metrology and Qualimetrics, Department of Analytical Chemistry, Faculty of Sciences, University of Granada, C/ Fuentenueva s/n, E-18071, Granada, Spain. E-mail: [email protected] 2 Centro "Venta del Llano", Agricultural and Fishery Research Institute (IFAPA), Consejería de Agricultura, Pesca y Medio Ambiente, Junta de Andalucía, Ctra. Bailén-Motril, km. 18.5, Apdo. correos nº 50, E-23620
Mengíbar, Jaén, Spain3 Agrofood Quality Control Service Consejería de Agricultura Pesca y Medio Ambiente Junta de Andalucía C/ Tabladilla s/n E 41071 Sevilla Spain
PREPARATION OF CERTIFIED REFERENCE MATERIALS OF OLIVE OIL PREPARATION OF CERTIFIED REFERENCE MATERIALS OF OLIVE OIL FOR PHYSICOCHEMICAL AND SENSORY CHARACTERISTICSFOR PHYSICOCHEMICAL AND SENSORY CHARACTERISTICS
3 Agrofood Quality Control Service, Consejería de Agricultura, Pesca y Medio Ambiente, Junta de Andalucía, C/ Tabladilla s/n, E-41071, Sevilla, Spain
A material, sufficiently homogeneous and stable with respect to one ormore specified properties, which has been established to be fit for itsintended use in a measurement process, and has been characterizedby a metrologically valid procedure for one or more specifiedproperties accompanied by a certificate that provides the value of the
WhatWhat´́s a Certified Reference Material (CRM)?s a Certified Reference Material (CRM)?
A CRM can:
-Improve the comparability of measurement results, and
Herewith we present the results of a series of studies for the elaboration, certification and distribution of
properties, accompanied by a certificate that provides the value of thespecified property, its associated uncertainty, and a statement ofmetrological traceability (ISO/IEC Guide 99:2007).
EverythingEverything waswas carriedcarried outout withinwithin thethe frameframeofof aa collaborationcollaboration betweenbetween::
•• AndalusianAndalusian Regional Government (Spain) Regional Government (Spain)
Aim of the studyAim of the study
-Be used for calibration, validation and/or quality control purposes
several CRMs of olive oil, which could be used in olive oil quality control laboratories
•• AndalusianAndalusian Agricultural and Fishery Agricultural and Fishery Research Institute (Research Institute (IFAPAIFAPA) )
•• University University of Granada of Granada
1st 1st stagestage: : InterOLEOInterOLEO‐‐CRM CRM 2nd 2nd stagestage: : SensOLEOSensOLEO‐‐CRM CRM
Structure of the projectStructure of the project
The certification of all the analytical parameters is related to the physicochemical characteristics
(updated Regulation EU 2568/91). We focused on the certification of
several sensory properties of olive oils.
Sensory Sensory Fruity, bitter and
pungent attributes
ANALYTICAL PARAMETERS TO BE CERTIFIED
Acidity Sterols• cholesterol• brassicasterol• 2,4-methylencholesterol
Fatty acid trans isomers
• t-oleic (t-C18:1)• t-linoleic (t-C18:2)• t-linolenic (t-C18:3)
Peroxide value
Determination of different Determination of different physicochemical parametersphysicochemical parameters
Determination of different Determination of different physicochemical parametersphysicochemical parameters
Physicochemical Physicochemical analysisanalysis
analysisanalysis
Panel TestPanel TestPanel TestPanel Test
pungent attributes and their levels; any negative attribute?...
• campesterol• campestanol• stigmasterol• ∆7-campesterol• ∆5,23-stigmastadienol• clerosterol• β-sitosterol• sitostanol• ∆5-avenasterol• ∆5,24-stigmastadienol• ∆7-stigmastenol• ∆7-avenasterol• apparent β-sitosterol• total sterols
t linolenic (t C18:3)• t-linoleic + t-linolenicFatty acids
• myristic (C14:0)• palmitic (C16:0)• palmitoleic (C16:1, n-7)• heptadecanoic (C17:0)• heptadecenoic (C17:1)• stearic (C18:0)• oleic (C18:1, n-9)• linoleic (C18:2, n-6)• linolenic (C18:3, n-3)• arachidic (C20:0)• eicosenoic (C20:1, n-11)• behenic (C22:0)• lignoceric (C24:0)
Waxes
• C36• C38• C40• C42• C44• total waxes
Aliphatic alcohols
• docosanol (C22)• tetracosanol (C24)• hexacosanol (C26)• octacosanol (C28)• total aliphatic alcohols
ECN42 triglycerides
• total ECN42 (HPLC)• theoretical ECN42 (GC)• Difference ECN42
Terpenic alcohols
• erytrodiol + uvaol
Alkyl esters
• methyl ester C16• ethyl ester C16• methyl ester C18• ethyl ester C18• total FAMEs (C16 + C18)• total FAEEs (C16 + C18)
UV spectrometry
• K232• K270• K
Steradienes
• 3,5-stigmastadiene
FurtherFurther stepssteps:: Once the materials have been produced, we consider as necessary:
- to assure the homogeneityassure the homogeneity of the units of the lot,
- to characterize the values of the analytical parameterscharacterize the values of the analytical parameters to be certified, and
t l t t bilit f th t i ll t t bilit f th t i l d th d d t diti
- L Cuadros-Rodríguez, J.M. Bosque-Sendra, A.P. de la Mata-Espinosa, A. González-Casado, P. Rodríguez-García, Elaboration of Four Olive Oil Certified ReferenceMaterials: InterOleo-CRM 2006 Certification Study, Food Anal. Methods (2008) 1, 259-269.- J.M. Bosque-Sendra, P. de la Mata-Espinosa, L. Cuadros-Rodríguez, A. González-Casado, F.P. Rodríguez-García, H. García-Toledo, Stability for olive oil controlmaterials, Food Chemistry (2011) 125, 1418-1422.
ReferencesReferences
- to evaluate stability of the materialsevaluate stability of the materials, under the recommended storage conditions.
CHROMATOGRAPHIC FINGERPRINTING METHODOLOGY FOR OLIVE OIL AUTHENTICATION
A. González‐Casado1, L. Cuadros‐Rodríguez1, C. Ruiz‐Samblás1, E. Pérez‐Castaño1, F.P. Rodríguez‐García2, M.G. Bagur‐González11Department of Analytical Chemistry, Faculty of Sciences, University of Granada, C/ Fuentenueva s/n, E‐18071, Granada, Spain.
E mail: agcasado@ugr es
INTRODUCTION
E‐mail: [email protected] Quality Control Service, Consejería de Agricultura y Pesca, Junta de Andalucía, C/Tabladilla s/n., E‐41071, Sevilla, Spain
There are several approaches that can be applied to the olive oil authentication. They differ in the scientific‐technical foundation,according to the information obtained. The most common techniques are: (i) chemical composition; (ii) stable isotopes; and (iii) DNA.The chemical‐based approach needs the accomplishment of chemical analyses to acquire either explicit or implicit information, aboutchemical constituents or chemical families related with characteristics to authenticate: geographical origin, botanical variety, category,differentiated quality, absence of foreign oils, etc.In order to practical consequence of the chemical‐based approach, it could be used three analytical methods: chemical markers,compositional profile, and instrumental fingerprinting.Our research focuses on fringerpringting methodologies. They consider the entire analytical signal, which is acquired and recorded bythe analytical instrument, directly from olive oil or from a previously isolated fraction, i.e. a spectrum or a chromatogram. The shapeand intensity of the recorded signal constitutes the instrumental fingerprint from the whole olive oil sample, or from the consideredfraction, because it is characteristic and reflects implicitly its chemical composition. Therefore, the methodology is based on theexistence of the chemical composition and they can be applied when the compositional methods are suitable.
FINGERPRINT APPLICATIONS
TGAs STs VOCsTGAs
LC GC
STs
LC GC
VOCs
GC PTR‐MS
TRIACYLGLYCEROLS STEROLS VOLATILE ORGANIC COMPOUNDS
(1), (2), (3) (3), (4), (5) (*) (6)(*) (*)
Edible oil
(mainly olive oil)
Signal acquisition
GC, LC, PTR‐MS
Pre‐processing ModelsChemometricsData mining
METHODOLOGY
• Baseline correction• Peak shifting• Mean Center• Autoescale
Exploratory data analysis, Classification, Prediction
GC‐MS chromatograms of vegetable oil blendssamples after peak shifting pretreatment
(iCoshift) (5)
PCA scores plot obtained from the data of theTAGs chromatographic profile of the olive oils:
th PC3 PC2 l (4)
OBJECTIVES
REFERENCES
(iCoshift) (5) scores on the PC3‐PC2 plane (4)
AUTHENTICATION
• Categories, varieties• Geographical origin• Identity, characterisation• Adulteration
(1) P.A. de la Mata Espinosa, J.M. Bosque Sendra, R. Bro, L. Cuadros Rodríguez. Discriminating olive and non‐olive oils using HPLC‐CAD and chemometrics. Anal. Bioanal. Chem., 399, 2083‐2092 (2011)(2) P.A. de la Mata Espinosa, J.M. Bosque Sendra, R. Bro, L. Cuadros Rodríguez. Olive oil quantification of edible vegetable oil blends using triacylglycerol chromatographic fingerprints and chemometric tools. Talanta, 85,177‐182
(2011)(3) C. Ruiz Samblás, C. Arrebola Pascual, A. Tres, S.M van Ruth, L. Cuadros Rodríguez. Authentication of geographical origin of palm oil by chromatographic fingerprinting of triacylglycerols and partial least square discriminant
analysis. J. Agric. Food Chem. (enviado, abr 2013)(4) C. Ruiz Samblás, L. Cuadros Rodríguez, A. González Casado, F.P. Rodríguez García, P. de la Mata Espinosa, J.M. Bosque Sendra. Multivariate analysis of HT/GC‐(IT)MS chromatographic profiles of triacylglycerol for classification
of olive oil varieties. Anal. Bioanal. Chem., 399, 2093‐2103 (2011)(5) C. Ruiz Samblás, F. Marini, L. Cuadros Rodríguez, A. González Casado. Quantification of blending of olive oils and edible vegetable oils by triacylglycerols fingerprint gas chromatographic and chemometrics tools. J. Chromatogr.
B 910, 71‐77 (2012)(6) C. Ruiz Samblás, A. Tres, A. Koot, S.M. van Ruth, L. Cuadros Rodríguez, A. González Casado. Proton transfer reaction‐mass spectrometry volatile organic compound fingerprinting for monovarietal extra virgin olive oil
identification. Food Chem., 134, 589‐596 (2012)(*) Currently under study
REFERENCES
Angelo Faberi*, Fabio Fuselli* Rosa Maria Marianella* and Manuel Sergi§
* Ministero delle Politiche Agricole Alimentari e Forestali – Dipartimento dell’Ispettorato Centrale della tutela della Qualità e Repressione Frodi dei Prodotti Agro-alimentari – Laboratorio Centrale di Roma, Via del Fornetto, 85 -
E-mail: [email protected]
§ Università degli Studi di Teramo – Facotà di Agraria, Via Carlo R. Lerici 1 - 64023 Mosciano Sant'Angelo (TE)
INTRODUCTION
Extra virgin olive oil (EVOO) is a fundamental landmark of the Mediterranean diet which has noticeable nutritional and organoleptic
characteristics.
European Regulation (EEC) 2568/91 has been setting the minimum requirements in order to allow labelling of an oil as extra virgin. These
general requirements, are based on physical-chemical and organoleptic parameters directly linked to freshness and quality of the product (free
acidity, peroxide index and ultraviolet absorption, panel test and recently alkyl esters) and other purity parameters, intended to prevent
fraudulent mixing with cheaper oils (refined, pomace or seed oils)
However EVOOs products exhibit great differences, either in organoleptic or nutritional or functional characteristics (e.g. polyphenols and
tocopherols content, aroma, etc.), which are related to cultivar of olives, production techniques and geographical origin.
The lack of official methods of analysis for assessing the origin implies that official control of special quality regulated production, such as PDO
or PGI, must rely only on checking the accompanying paper documentation.
The use of Isotope Ratio Mass Spectrometry (IRMS) has demonstrated as a tool that can improve geographical discrimination of unknown
samples, because this technique presents the advantage of giving results which are almost independent from cultivar employed and production
technique (1-3).
In this work the evaluation of the composition of Fatty Acids Methyl Esters (FAME) alongside with the determination of stable isotope ratio of C
in bulk oils and in main FAME constituents has been made
EXPERIMENTAL
Samples: Sampling of authentic extra virgin olive oil was made by ICQRF inspective personnel at oils mils. Selected olive oils of known origin
and designated to achieve the Protected Designation of Status. For each PDO/PGI three oil samples, related to three different maturation
stages, were collected at a distance of about 20-30 days from each other. For each sample additional information regarding agronomical and
technological features were also collected .
Oils samples were collected in 250 ml dark glass bottles covered with a black plastic envelope and then shipped to the laboratory. Upon receive
oils were filtered by means of a 0,45 mm barrel type nylon filter, in order to remove any sediment eventually formed which may deteriorate the
product. Samples were kept at 8°C until the analysis. An individual set of three samples was taken for each geographical mention for every
specific PDOs who has such distinction.
Fatty acid analysis by GC/FID : samples were transesterified with methanolic potassium hydroxide according to official method reported in
annex X(A) of Regulation (EC) 702/2007 - analysis by gas chromatography of methyl esters of fatty acids.
C isotope analysis of bulk oil by EA/IRMS: The carbon isotope ratio (13C/12C) of bulk oils was determined by flash combustion on a Thermo
(Bremen, Germany) elemental analyzer (EA) connected to a Thermo Fisher Delta V Plus (Bremen, Germany) isotope ratio mass spectrometer
(IRMS) by analyzing the ratio of ionic currents of m/z 44 (12C16O16O), m/z 45 (13C16O16O) ed m/z 46 coming from carbon dioxide arising from
sample combustion in the elementar analyzer.
The stable isotope composition of carbon, is calculated in delta (δ) notation as the per thousand (‰) deviations of the isotope ratio relative to
known standards (d% C = R sample - R standard / R standard) For 13C\12C ratio the standard utilized is Vienna Pee Dee Belemnite
limestone (VPDB).
Deterrmiantion of isotopic ratio of individual FAME: the determination was realized with a GC Isolink device constitued by a GC Thermo
Trace FID coupled to a ThermoFinnigan Delta V Plus isotopie ration mass spectrometer by means of a combustion interface GC/C/IRMS .
Results were corrected for the contribution of d 13C the methanol emplyoied for trans-esterification. Esterification procedure was the same as
for the determiantion of the fatty acid composition.
RESULTS
In order to carry out the statistical analysis, samples were divided into four categories defined according to the area of origin
The distribution of samples within each category was first evaluated by univariate analysis through representation of significant discriminant variables with box and whisker diagrams which show the dispersion of the samples within each class in function of the measured values for the isotopic ratio (Figure 1 - a, b, c)..
Samples were divided into the following four macro-regions (figure 2)
Nord : including samples produced in the regions of Lombardy, Veneto, Trentino, Liguria and Emilia Romagna
Center: including samples produced in Tuscany, Latium,. Abruzzo Umbria, Marche, Sardinia
South : including samples produced in Campania, Apulia, and Calabria
Sicily : including samples produced in Sicily
Composition data has demonstrated that there is a certain degree of correlation among main constituents fatty acids with geographical area of proven origin according to latitude. The values of d13C ‰ in both bulk and FAME also have indicated a similar evidence of correlation. However overlapping zones are very wide.
Fatty Acid Composition and d13C of Bulk and Individual Fatty
Acids as Marker for Authenticating Italian PDO/PGI Extra
Virgin Olive Oils
Figure 1a: box-and-whiskers plot of main
constituents fatty acids methyl esters as a
function of the geographical area of origin;
Figure 1b: box-and-whiskers plot of the
distribution of δ 13C/12C isotope ratios of
bulk oil as a function of the geographical area
of origin;
Figure 1c: box-and-whiskers plot of the δ 13C/12C
isotope ratio of the methyl esters of fatty acids as a
function of the geographical area of origin;
MULTIVARIATE ANALYSIS
The use of multivariate analysis represent a tool that can improve geographical discrimination of unknown samples
Principal component analysis (PCA), applied in the first stage of the data processing, represents one of the most frequently used chemometric tools, because allows to project in an easy way data from an higher to a lower dimensional space.
In the preliminary analysis of the olive oil data, 3 distinct preliminary PCA were performed to investigate clustering of samples on the basis of the area of provenience (Figure 3).
The PCA shows that the geographical discrimination of the samples improves by making of appropriate combinations of the parameters up to now considered individually. As one can see the introduction of the measure of the carbon isotope ratio on bulk oil and of the individual components fatty acids makes gives a better separation of the four classes. The diagrams have been built after an autoscaling pretreatment of each variable.
PLS-DA techniques as well shows (Figure-4) that when the whole set of data is considered (isotopic and composition), there is a substantial discrimination of the origin of the oil. As it can be seen, a clear separation between the data related to geographical origin can be observed.
Figure 3: graphs of the first and second
principal component obtained from (A)
compositional variables only; (B)
compositional variables and the δ
13C/12C of bulk oil; compositional
variables and the δ 13C/12C of bulk oil
and of the main constituent fatty acids
(C). Figure 2: macro-regions
where samples were taken
REFERENCES
1) Angerosa, F., Breas, O., Contento, S., Guillou, C., Reniero, F., Sada, E. (1999). Application of stable isotope ratio analysis to the characterization of the geographical origin of olive oils. Journal of Agricultural and .Food Chemistry , 47, 1013–1017.
2) Bontempo, L., Camin, F., Larcher, R., Nicolini, G., Perini, M., Rossmann, A. (2008). Discrimination of Tyrrhenian and Adriatic Italian olive oils using H, O, and C stable isotope ratios. Rapid Communications in Mass Spectrometry , 23, 1043–1048.
3) Camin, F., Larcher, R., Perini, M., Bontempo, L., Bertoldi, D., Gagliano, G., Nicolini, G., Versini, G. (2008). Characterization of authentic Italian extra-virgin olive oils by stable isotope ratios of C, O and H and mineral composition. Food Chemistry, 118, 901-909
Figure 4: Figure shows the PLS-DA score
plot of the first three latent variables using
d C13 data. In the plot about 61% of the
total variance of the data is represented.
.
- P
art
ially
concurr
ent
solv
ent
evap
ora
tion:
solv
ent
trappin
g r
eta
ins
vola
tile
sam
ple
com
ponents
during t
ransfe
r and s
olv
ent
dis
charg
e
via
the s
olv
ent
vapor
exit.
- T
he t
ransfe
r volu
me a
nd e
vapora
tion r
ate
has t
o b
e a
dju
ste
d t
o
meet
the c
apacity o
f th
e “
rete
ntion g
ap”
giv
en b
y t
he d
imensio
n o
f
the u
ncoate
d p
re-c
olu
mn (
7-1
5 m
, 0.5
3 m
m i.d
.).
- F
ully
concurr
ent
solv
ent
evap
ora
tion c
an b
e u
sed if
vola
tile
s a
re n
ot
analy
zed.
- It
is p
refe
rred w
henever
applic
able
, sin
ce it
requires s
hort
coate
d
pre
-colu
mns o
f 0.5
-1 m
(le
ss a
dsorp
tivity)
and t
he a
dju
stm
ent
of
the
conditio
ns is u
sually
uncritical.
- F
att
y a
cid
meth
yl este
rs a
re a
mong t
he m
ost
vola
tile
com
pounds t
o
be t
ransfe
rred w
ithout
losses u
sin
g t
his
techniq
ue.
- O
ptim
ization o
f th
e c
onditio
n m
ay b
e p
erf
orm
ed b
y a
mix
ture
of
n-a
lkanes (
fig.
3).
On
-lin
e H
PL
C-G
C-F
ID
for
the e
valu
ati
on
of
the q
uali
ty o
f o
live o
ils
tho
ug
h t
he m
eth
yl,
eth
yl,
an
d w
ax e
ste
rs
Mauru
s B
iederm
ann
Auth
ority
of
the
Intr
od
ucti
on
Incre
ased e
ste
r conte
nts
in o
live o
ils indic
ate
degra
ded
oliv
es:
meth
anol and e
thanol fo
rmed d
uring f
erm
enta
tion
are
tra
nseste
rified w
ith f
att
y a
cid
s f
rom
the t
rigly
cerides.
Wax e
ste
rs a
re m
ore
readily
extr
acte
d f
rom
the s
oft
skin
of
overr
ipe o
lives. A
pro
mis
ing c
orr
ela
tion o
f chem
ical analy
-
sis
and s
ensorial evalu
ation w
as c
onfirm
ed:
oils
with low
concentr
ation o
f th
ese e
ste
rs w
ere
of
hig
h s
ensory
qualit
y,
where
as m
any o
f th
e o
ils w
ith h
igh c
onte
nts
not
even m
et
the r
equirem
ents
for
extr
a v
irgin
oliv
e o
ils.
Lim
its o
n t
he
alk
yl and w
ax e
ste
r conte
nt
in o
live o
ils a
re s
pecifie
d in t
he
Com
mis
sio
n R
egula
tion 6
1/2
011.
An
aly
tical
meth
od
Dilu
ted o
ils a
re p
re-s
epara
ted b
y n
orm
al phase H
PLC
and t
he e
ste
r fr
action o
n-lin
e t
ransfe
rred into
GC
via
the
Y-inte
rface (
fig.
1)
by f
ully
concurr
ent
solv
ent
evapora
tion
(fig
2).
The m
eth
od inclu
des s
evera
l verification s
tandard
s
to m
onitor
pro
per
perf
orm
ance in t
he c
ritical aspects
for
each a
naly
sis
: w
ax a
nd m
eth
yl este
rs a
re e
lute
d a
t diffe
r-
ent
tim
es f
rom
HP
LC
; sta
ndard
s w
ere
intr
oduced t
o m
oni-
tor
the e
dges o
f th
e f
raction w
indow
. F
ully
concurr
ent
elu
ent
evapora
tion m
ay c
ause losses o
f th
e m
ost
vola
tile
fatt
y a
cid
meth
yl este
rs t
hro
ugh t
he v
apor
exit. A
vola
tile
verification s
tandard
was a
dded t
o o
ptim
ize t
he t
ransfe
r
conditio
ns [
1].
Resu
lts
100 o
live o
ils f
rom
the S
wis
s m
ark
ed s
old
as e
xtr
a v
irgin
were
analy
zed c
hem
ically
. A
sele
ction o
f th
ese (
with low
and
hig
h c
onte
nts
of
meth
yl and e
thyl este
rs)
were
als
o s
ensorial
evalu
ate
d.
Acco
rdin
g t
o p
revio
us w
ork
[2]
the b
est
oils
were
low
in
conte
nts
of
meth
yl and e
thyl ole
ate
. O
live o
ils w
ith e
levate
d
conte
nts
of
these e
ste
rs a
nd/o
r str
aig
ht
chain
wax e
ste
rs
were
devalu
ate
d a
s “
virgin
” or
“lam
pant”
oils
based o
n t
he
sensorial te
sting.
The t
wo t
ypes o
f qualit
y m
ark
ers
have little c
om
mon b
ack-
gro
und (
poor
corr
ela
tion,
fig.
8):
- A
hig
h w
ax e
ste
r conte
nt
is indic
ative o
f soft
, ripe o
r overr
ipe
oliv
es.
- F
att
y a
cid
meth
yl and e
thyl este
rs o
rigin
ate
fro
m a
lcohols
form
ed
by f
erm
enta
tion.
Refined o
live o
ils:
- T
he c
oncentr
ation o
f th
e w
ax e
ste
rs is h
igh,
how
ever
the c
on
-
centr
ation o
f m
eth
yl/eth
yl este
rs is low
. P
robably
deodora
tion o
f
the o
il re
moved a
larg
e p
roport
ion o
f th
ese m
ore
vola
tile
este
rs.
separa
tion
colu
mn
uncoate
dpre
-colu
mn
1-1
5 m
LC
pum
p
LC
colu
mn
bfv
tv
iv
UV
dete
cto
r
carr
ier
gas
Y-p
iece
vapor
exit
FID
iv: in
jectio
n v
alv
ebfv
: backflu
sh v
alv
etv
: tr
ansfe
r valv
e
waste
auto
sam
ple
r
Fig
ure
5:
HP
LC
-GC
-FID
chro
mato
gra
ms o
f este
rs o
f in
tere
st;
A e
xtr
a v
irgin
oliv
e
oil
with r
ath
er
hig
h c
oncentr
ations;
B e
xtr
a v
irgin
oil
with low
concentr
a-
tion o
f th
ese c
om
ponents
. IS
: in
tern
al sta
ndard
; V
S:
verificatio
n s
tan
-
dard
; P
: phyto
l este
rs
Tem
pera
ture
pro
gra
m 7
/m
in
140
C360
C
Me-17:0 (VS1)Me-18:1
Et-18:1
Et-20:0 (IS1)
Me-20:2 (VS2)
21-22:0 (IS2)
26-18:X
P-18:1
P18:0
P-20:0P-20:1
Squalene
Ste
rol
este
rs
28-18:X
A
Me-17:0 (VS1)
21-22:0 (IS2)
26-18:X
P-18:1
P-20:0
28-18:X
B
Et-20:0 (IS1)
Me-20:2 (VS2)
Me-18:1Et-18:1
Dilu
tion:
25 m
g e
dib
le o
il +
inte
rnal/verification s
tandard
mix
ture
+ 1
.5 m
l hexane
Inje
ction v
olu
me:
10 µ
l
LC
colu
mn:
250 x
2 m
m,
Spherisorb
Si 5 µ
m
Elu
ent:
4%
MT
BE
/penta
ne a
t 300 µ
l/m
in
Tra
nsfe
rred f
raction:
2-4
min
= 6
00 µ
l
Pre
-colu
mn:
40 c
m x
0.5
3 m
m i.d
., 0
.03 µ
m O
V-1
701-O
H,
connecte
d t
o
a m
eta
l T-p
iece
Separa
tion c
olu
mn:
20 m
x 0
.25 m
m i.d
., 0
.12 µ
m P
S-2
55 (
dim
eth
yl p
oly
silo
xane)
Carr
ier
gas:
Heliu
m a
t 60 k
Pa,
reduced t
o 4
0 k
Pa d
uring t
ransfe
r
Oven t
em
p.
pro
gra
m:
50 °
C (
3 m
in),
30 °
/min
to 1
30 °
C,
7 °
/min
to 3
60 °
C (
4 m
in)
Instr
um
enta
tion:
LC
-GC
9000,
Bre
chbühle
r A
G:
com
bi P
AL a
uto
sam
ple
r,
Phoenix
40 s
yringe p
um
p, T
herm
o T
race G
C
Sensorial te
sting w
as d
one b
y t
he S
wis
s O
live O
il P
anel (S
OP
) fr
om
the U
niv
ers
ity
of A
pplie
d S
cie
nces W
ädensw
il (Z
HA
W).
Fig
ure
8:
Sum
of
str
aig
ht
chain
wax e
ste
rs p
lott
ed a
gain
st
sum
of
meth
yl/eth
yl
ole
ate
. A
sele
ction o
f oils
were
sensorial evalu
ate
d.
Lig
ht
gre
en b
ox:
limits s
pecifie
d in t
he C
om
mis
sio
n R
egula
tion 6
1/2
011,
dark
gre
en
box:
sam
ple
s w
ithin
pro
posed s
tric
ter
limits.
Co
nclu
sio
ns
Meth
yl and e
thyl este
rs o
f fa
tty a
cid
s a
re u
sefu
l in
dic
ato
rs f
or
dete
rmin
ing t
he q
ualit
y o
f oliv
es a
nd t
he o
il pro
duced f
rom
these.
Onlin
e H
PLC
-GC
pro
vid
es a
larg
ely
auto
mate
d m
eth
od f
or
routine a
naly
sis
. T
he h
igh s
tabili
ty o
f F
ID a
s w
ell
as t
he larg
e
num
ber
of
sam
ple
s w
hic
h c
an b
e a
naly
zed d
aily
with a
min
imum
of
manpow
er
recom
mends t
he m
eth
od f
or
routine
use.
A m
ore
deta
iled a
naly
sis
of
the w
ax e
ste
r fr
action is d
e-
scribed in r
efe
rence [
3].
Oil
from
fre
sh a
nd p
art
ially
degra
ded o
lives (
fig.
6)
was isola
ted in t
he
labora
tory
and a
naly
zed f
or
there
este
r com
positio
n (
fig.
7):
- P
hyto
l este
rs (
P)
are
pre
dom
inating;
pre
sente
d a
fter
reduction b
y
facto
r 5.
- T
he c
oncentr
ation o
f th
e p
hyto
l, g
era
nyl-gera
nio
l (G
) and b
enzyl (B
z)
este
rs d
o n
ot
vary
sig
nific
antly.
- 4-5
tim
es m
ore
str
aig
ht
chain
palm
itate
s (
16:0
) and u
nsatu
rate
d C
18
acid
este
rs (
18:X
) of
C22-C
28 a
lcohols
were
extr
acte
d f
rom
the s
oft
er
skin
of
the d
egra
ded o
lives.
- D
egra
dation c
aused t
he c
oncentr
ation o
f m
eth
yl and e
thyl ole
ate
(18:1
-Me,
18:1
-Et)
to incre
ase b
y a
facto
r of
40-5
0.
0
20
40
60
80
100
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Peak areas (%)
C-A
tom
s
oc
32°C
50°C
70°C
90°C
0
30
60
90
120
150
16:0
18:X
P
G
B
z
18:1
-Me
18:1
-Et
Concentration (mg/kg)
Degraded
Fresh x5
0
20
40
60
80
100
120
140
160
180
200 0
50
100
150
200
250
300
Me-18:1 + Et-18:1 (mg/kg)
Str
aig
ht
ch
ain
wax e
ste
rs (
mg
/kg
)
Extr
a v
irgin
, not
evalu
ate
d
Extr
a v
irgin
Devalu
ate
d e
xtr
a v
irgin
Raffin
ate
carr
ier
gas
so
lven
t vap
or
+ l
ow
bo
ilers
hig
h b
oil
ers
syri
ng
e n
eed
leso
lven
t vap
or
low
bo
ilers
reta
ined
by s
olv
en
t tr
ap
pin
g
part
ially
concurr
ent
solv
ent
evap
ora
tion
fully
concurr
ent
solv
ent
evap
ora
tion
Larg
e v
olu
me o
n-c
olu
mn
tra
nsfe
r
Fig
ure
3:
n-a
lkane m
ixtu
re t
ransfe
rred a
t diffe
rent
oven t
em
pera
-
ture
s;
norm
aliz
ed p
eak a
reas.
Fig
ure
2:
Princip
le o
f part
ially
vs.
full
concurr
ent
solv
ent
evapora
tion
Veri
ficati
on
The p
erf
orm
ance o
f each a
naly
sis
is c
hecked b
y b
uilt
in t
ools
:
- Losses o
f fa
tty a
cid
este
rs b
y c
o-e
vapora
tion w
ith t
he e
luent
is c
ritical
apply
ing c
oncurr
ent
solv
ent
evapora
tion. T
he inte
rnal sta
ndard
(IS
1,
eth
yle
icosanoate
, E
t-20:0
) is
less v
ola
tile
than t
he m
eth
yl and
eth
yl
ole
ate
. A
more
vola
tile
verification s
tandard
is a
dded,
meth
yl hepta
de
-
canoate
(V
S1,
Me-1
7:0
). T
he r
atio M
e-1
7:0
/Et-
20:0
is m
onitore
d.
- A
noth
er
critical poin
t are
shifting r
ete
ntion t
imes
in N
PLC
, th
e a
naly
tes m
ay b
e e
lute
d o
ff t
he L
C
fraction w
indow
. T
he w
ax e
ste
r 21-2
2:0
(IS
2)
is
elu
ted a
t th
e b
egin
nin
g o
f th
e f
raction.
Meth
yl
eic
osadie
noate
(V
S2,
Me-2
0:2
) is
added a
s
second v
erification s
tandard
, its e
lution d
ete
r-
min
es t
he e
nd o
f th
e f
raction (
fig.
4).
The r
atios
betw
een I
S2,
VS
2 a
nd I
S1 is m
onitore
d.
wax esters, IS2
ethyl esters, IS1
methyl esters, VS2
LC
fra
ction
Fig
ure
4:
LC
elu
tion o
rder
of
analy
tes,
inte
rnal
and v
erification s
tandard
s
Fig
ure
6:
Oliv
es f
rom
the s
am
e t
ree:
good s
hape (
left
); d
am
aged o
lives (
righ
t)
Fig
ure
7:
fresh v
ers
us d
egra
ded o
lives
Refe
rences
Fig
ure
1:
Com
ponents
of
an o
n-lin
e H
PLC
-GC
-FID
instr
um
ent.
1
DETECTION OF PLANT OIL DNA USING MOLECULAR MARKERS AND PCR ANALYSIS: A TOOL FOR DISCLOSURE OF OLIVE OIL ADULTERATION
Michelangelo Vietina, Caterina Agrimonti, Nelson Marmiroli
Department of Life Sciences, University of Parma, Parco Area delle Scienze 11A, 43124 Parma, [email protected]
ABSTRACT
• Extra virgin olive oil is frequently subjected to adulterations withaddition of oils obtained from plants other than olive. DNA analysis isa fast and economic tool to identify plant components in oils.
• Extraction and amplification of DNA by PCR was tested in olives, inmilled seeds and in oils, to investigate its use in olive oil traceability.
•DNA was extracted from different oils made of hazelnut, maize,sunflower, peanut, sesame, soybean, rice and pumpkin. Comparingthe DNA melting profiles in reference plant materials and in the oils, itwas possible to identify any plant components in oils and mixtures ofoils.
•Real-Time PCR (RT-PCR) platform has been added of the newmethodology of High Resolution Melting (HRM), both were used toanalyze olive oils mixed with different percentage of other oils. Resultsshowed HRM a cost effective method for efficient detection ofadulterations in olive oils.
2
DNA EXTRACTION FROM OILS
Yields of DNA (ng/mL of starting volume of oil) after extraction based on
NucleoSpin Plant kit and CTAB. . The yields are expressed as averages
of three independent extractions, with standard deviation.
REAL-TIME PCR ANALYSIS OF OILS
Melting curves analysis of amplicons obtained by Real Time PCR conducted on DNA extracted from leaves (or seeds) and oils. (A) olive; (B) hazelnut; (C) maize; (D) sunflower; (E) sesame; (F) rice; (G) pumpkin; (H) peanut.
3
SEQUENCE CHARACTERIZED AMPLIFIED REGION
• The use of a SCAR marker, derived from multilocus markers, such as AFLPs or RAPDs, can allow to find the adulteration of an olive oil with a non olive oil, such as hazelnut oil. Moreover, a SCAR marker can be used in a high-throughput platform to assess and quantify the contribution of a single cultivar in commercial multivarietal oils. In Real-Time PCR, a SCAR marker, named CP-rpl16T, derived from an AFLP fingerprint of olive oil, was applied either on DNA extracted from (1) leaves and from a (2)100% olive oil and (3) 90% olive oil and 10% hazelnut oil.
1
32
DETECTION OF PLANT OIL DNA USING HIGH
RESOLUTION MELTING (HRM) POST PCR
ANALYSIS: A TOOL FOR DISCLOSURE OF OLIVE OIL
ADULTERATION
A. Red curve: HRM of DNA extracted from olive oil; Blue : HRM of DNA extractedfrom maize seed oil; Green : HRM of DNA extracted from an olive and maize oil mix (90%-10%).
B. B. Red : HRM of DNA extracted from olive oil; Blue : HRM of DNA extractedfrom sunflower seed oil; Green : HRM of DNA extracted from an olive and sunflower oils mix (90%-10%).
C. Red : HRM of DNA extracted from olive oil; Blue : HRM of DNA extracted from hazelnut seed oil; Green : HRM of DNA extracted from an olive and hazelnut oilsmix (90%-10%).
4
Acknowledgements
This study has been carried out with financial support from the Commission of the EuropeanCommunities, specific RTD programme “Quality of Life and Management of Living Resources”project, QLK1-CT-2002-02386, “Traceability of origin and authenticity of olive oil by combinedgenomic and metabolomic approaches (OLIV-TRACK)” coordinated by N. Marmiroli. Thecontent of this paper does not necessarily reflect the Commission of the EuropeanCommunities views and in no way anticipates the Commission’s future policy in this area. Thispaper had also the contribute of the Italian Minister of University and Research specialprogram PRIN “Rintracciabilità della composizione e dell’origine di oli d’oliva DOP, IGP e 100%Italiani attraverso metodiche genomiche, proteomiche e metabolomiche” coordinated alsoby N. Marmiroli and a contribute from the University of Parma (fund FIL 2002, 2003, 2004, 2005,2006). This work was also supported financially by Emilia-Romagna (IT) Regional project SIQUALwithin the research framework PRRIITT, Misura 3.4.
References
• Pafundo, S., Agrimonti, C., Marmiroli, N. (2005). Traceability of plant contribute in olive oil byAFLPs. Journal of Agricultural and Food Chemistry, 53, 6995-7002.
• Pafundo, S., Agrimonti, C., Maestri, E., Marmiroli, N. (2007). Applicability of SCAR marker tofood genomics: olive oil traceability. Journal of Agricultural and Food Chemistry, 55, 6052-6059.
• Vietina, M., Agrimonti, C., Marmiroli., N. (2013). Detection of plant oil DNA using HighResolution Melting (HRM) post PCR analysis: a tool for disclosure of olive oil adulteration.Food Chemistry, In press.
FT-MIR/ATR monitoring of virgin olive oil
oxidative stability under mild storage
conditions
Maria Z. Tsimidou, Nikolaos Nenadis, Ioannis Tsikouras
& Polidoros Xenikakis
School of Chemistry, Laboratory of Food Chemistry and
Technology, Aristotle University of Thessaloniki, 541 24
Thessaloniki, Greece,
e-mail: [email protected]
International Workshop in Bioactive compounds from Olea europaea:
Chemistry and Biology (EU IOC), Madrid 10-11 June 2013
Introduction Virgin olive oil (VOO) freshness has become a matter of concern among
consumers in relation to VOO exceptional nutritional and sensory
characteristics. As loss in the latter are observed in the oil mill
transportation or at sale points upon storage due to practices that
accelerate oxidation and hydrolysis1, scientific support is required in
cases of dispute.2 Discussions are on the way for updating the relevant
EU regulations on VOO quality characteristics. Toward this direction
modernization of methods of analysis is expected
Aim of the study The exploitation of the multiple information provided in
situ by Fourier Mid-IR spectroscopy equipped with an
Attenuated Total Reflectance (ATR) cell as a means to
extract information for loss of VOO freshness under mild
storage conditions for a period of 12 months
_______________________________________________
Results Table 1. Value ranges of quality indices determined for the test samples (n = 11) at different
storage periods in the dark
Storage period (months)
Quality Index t=0 t=6 t=12
Acidity (% oleic acid) 0.32-0.67 0.34-0.70 0.35-0.76
PV (meq O2/ kg oil) 6.5-8.6 9.0-13.1 13.3-20.0
K232 1.54-1.72 1.76-2.01 2.02-2.42
K270 0.10-0.12 0.12-0.15 0.13-0.20
Figure 1. Frequency distribution of A) peroxide and B) K232 values for EVOO samples
(n = 11) stored in the dark for 0, 6 and 12 months
Discussion
For all test samples, regardless storage time, the measured values for each
index (Table 1) were within the official limits (EU 2568/91 and
amendments) for extra virgin olive oil (EVOO)
Thus, upon storage up to 12 months, oxidation of samples was still at
early stages
Frequency distribution analysis for e.g. peroxide (Figure 1A) and K232
(Figure 1B) values showed a progressive increase which can be justified
by the presence of oxygen (10% headspace of the bottles)
Changes in acidity were slight
Figure 2. Typical FT-IR/ATR spectra of EVOO samples stored in the dark and frequencies
of bands of selected functional groups
Figure 3. Score plot of the first two PCs
obtained by PCA applied to the intensities of
selected wavenumbers of EVOO samples
stored in the dark for 0 (n=11), 6 (n=11), and
12 (n=11) months -3
-2
-1
0
1
2
3
4
-1 0 1 2
PC1 (98.1 %)
PC
2 (
1.2
%)
0 months
6 months
12 months
Changes in the intensity values at 3470 cm-1 (hydroperoxides) and in values
of intensity ratios (A3006/2924, A3006/2853, A3006/1746, A3006/1465, A3006/1163,
A1118/1097, A2853/1746, A2853/1417, A2853/1163, A2853/1118, A2853/1097, and A2853/723)
relevant to the degree of oil unsaturation did not offer more information
than the physicochemical criteria when examined one by one
Principal component analysis (PCA) applied to the intensity values of the
frequencies used in the above-mentioned ratios (Figure 3) showed that
samples stored for 1 year (excluding one as an outlier) clearly differed
from the rest. Those stored up to 6 months were grouped together with
fresh ones
Discriminant analysis (DA) after leave one out cross-validation gave a
81.3% correct classification: [6/11 (t=0), 10/11 (t=6), and 10/10 (t=12)]
Table 2 Number of principal components, percentage of total variance of PCA in three spectral regions
of the FT-IR spectra (2d derivative) of EVOO samples stored at t=0 (n=11), 6 (n=11), and 12 months
(n=11) and the relative classification results of DA
Spectral region Number
of PCs
Total variance
explained Classification
Original Cross validated
t=0 t=6 t=12 t=0 t=6 t=12
550-4000 cm-1 15 91.1% 10/11 10/11 11/11 8/11 10/11 11/11
715-2935 cm-1 14 91.3% 9/11 10/11 11/11 8/11 9/11 11/11
1020-1260 cm-1 3 92.1% 10/11 10/11 11/11 10/11 10/11 11/11
-4
-3
-2
-1
0
1
2
-1 0 1 2
PC1 (84.4 %)
PC
2 (
5.3
%)
0 months
6 months
12 months
-3
-2
-1
0
1
2
3
-1 0 1 2
PC1 (84.4 %)P
C3
(2
.4 %
)
0 months
6 months
12 months
Figure 4. Score plot of the first three PCs obtained by PCA applied to the second derivative of the
spectral region 1020–1260 cm-1
from the spectrum of EVOO samples stored in the dark for 0 (n= 11), 6
(n= 11), and 12 (n= 11) months
Examination of the discriminat activity of the 2d derivative of almost the
whole spectral region (550–4000 cm-1) and two narrower (715–2935 and
1020–1260 cm-1) recently used on acelerated oxidation studies of olive oil at
60 and 180 oC3,4 showed that using the narrowest region:
¨ Classification using a small number of components (only three PCs) was
achieved
¨ A better separation among oils stored at 0, 6, and 12 months was obtained
on the plane PC1-3
¨ Successful classification of samples by 94% was achieved even after
cross-validation
The frequencies contributing the most in PC3 according to scatter plot were
1229, 1175 (negative loadings), 1065, and 1053 cm-1 (positive loadings)
which should be related to –C–O group
Conclusions
¨ FT-MIR/ATR is a sensitive technique providing useful information for the early
stages of EVOO oxidative status even when physicochemical indices of oil remain
within the official limits
¨ Only the samples stored for 12 months were easily discriminated from the rest
¨ The most useful approach in checking freshness of an EVOO was the statistical
treatment of the 2d derivative of the spectral region 1020-1260 cm-1.
References Aknowledgements
1) Boskou, D. Olive Oil: Chemistry and Technology, AOCS Press, Champaign (Illinois)
2006.
2) Frankel, E. N., Mailer, R. J., Wang, S., Shoemaker, C. F.,
Flynn, D., INFORM –Int. News Fats Oils Related Mater. 2011, 22, 13–58.
3) Maggio, R. M., Valli, E., Bendini, A., Gomez-Caravaca, A. M. et al., Food Chem.
2011, 127, 216–221.
4) Mahesar, S. A., Bendini, A., Cerretani, L., Bonoli-Carbognin, M., Sherazi, S. T. H.
Eur.J. Lipid Sci. Technol. 2010, 112, 1356–1362.
N.N. thanks Ms A. Androulaki for tutorial in SPSS software use.
P.X. acknowledges the Union of Agricultural Cooperatives of
Sitia for financial support in terms of oil sampling, storage, and
physicochemical analyses that were carried out in its
installations
Present findings add to the usefulness of IR spectroscopy as a rigorous and low cost
technique for internal quality control in the olive oil industry but systematic inter-
laboratory studies are required before it can be considered as a robust tool in VOO
analysis
_____________________________________________________________________________________________________________________________________________________________________________
Experimental part
Oil samples and storage conditions
Extra virgin olive oil (EVOO) samples (500 mL) from Koroneiki cv olives and
representative of the production in the harvest year 2009-2010 were collected directly
form the three phase decanter of 11 olive oil mills in the regional union of Lassithi (Crete,
Greece). Portions were transferred in transparent glass bottles (10% headspace) and sealed
hermetically. Those at t=0 were immediately frozen and kept at -18oC till analysis. The
rest were stored in the dark at RT (23 ± 3oC) for t=6 and 12 months, respectively, and then
kept frozen till analysis.
Chemical and FT-IR analyses
Acidity, peroxide values and K232/270 indices were measured according to EU Regulation
2568/91 and amendments.
FT-IR spectra were acquired (64 scans/sample or background) in the range of 4000-400
cm-1 at a resolution of 4 cm-1 with the aid of an IRAffinity-1 spectrometer (Shimadzu
Corporation Kyoto Japan) using 0.8 ml of sample.
Data processing
The intensities of selected wavenumbers were collected from the untreated spectra
recorded for the samples. For chemometrics, all spectra were baseline corrected with the
aid of the IR-solution software __________________________________________________________________________________________________________________________________________________________________________________
Challenges of U.S. Enforcement Increase with Conflicting Standards & Methods
Authors: Eryn Balch North American Olive Oil Association, 3301 Route 66, Suite 205 Building C, Neptune, NJ 07753 USA E-mail: [email protected] As the world’s largest volume importer of olive oil and third-largest consuming country, U.S. regulators and importers historically relied on expertise from the IOC and producing supplier countries. Today, some rapidly growing domestic producers are exploiting the lack of public knowledge and limited in-country technical expertise and pursuing a mass media campaign aimed at discrediting all imported olive oil along with existing global standards. Enforcement in the U.S. is already a challenge because there is not a national mandatory standard for grade levels of olive oils and olive-pomace oils. If the FDA established a standard of identity it could be enforced anywhere along the supply chain by either government or private action. The USDA published grade standards in 2010, but compliance is voluntary. Mandatory compliance might be partially achieved through a USDA marketing order, although marketing orders are limited to testing only at time of local production or import. Enforcing marketing orders delays imports and is costly to both domestic and imported suppliers, and will not offer complete insurance against adulteration as ultimately there is no oversight to what is blended or sold after lot testing occurs. There are also four U.S. states that have established olive oil standards (CT, CA, NY and OR) but enforcement is limited within state borders. When the industry was unified in support of the IOC standard, progress toward a path to enforcement was happening – all of the state standards and the new USDA standard were implemented from 2008 – 2010. Progress began falling apart when the Australian AOA and U.S. UC Davis Olive Center began promoting research based on new methods claimed to be superior to the IOC methods. As the new methods were not being accepted into global standards, promotion campaigns based on these tests have been targeted to the consumer market instead and have become the basis for urging consumers to purchase only domestic olive oils. Of course, consumers (and even many industry personnel) don’t understand the meaning of the various authenticity and quality measures. They also don’t have access to confirm authenticity directly. The result is consumers that fear purchasing olive oil cut with olive-pomace oil or seed oils are given a false sense of security by certifications like the COOC Seal, which in reality doesn’t include authenticity analysis or even use the newly proposed testing methods. The disagreement on which methods or standard is appropriate has created major barriers in the advancement of enforcement opportunities in the U.S. Any effective standard will need to ensure both quality AND authenticity. In an attempt to reduce testing time and costs, divergent standards are now being proposed at various levels of state and federal government. Ultimately, divergent programs create trade issues and further confuse the marketplace. It is critical for the global industry to come together and agree on a common standard so it can be used as the basis for enforcement in countries like the U.S. References International Olive Council, (Nov. 2011). Trade Standard Applying to Olive Oils and Olive-Pomace Oils, COI/T.15/NC No 3/Rev. 6. USDA-AMS, (April 28, 2010). United States Standards for Grades of Olive Oil and Olive-Pomace Oil. California Olive Oil Council, (2012-2013). COOC Extra Virgin Certification Program 2012 -2013. http://www.cooc.com/seal-certification/ Frankel, E. N., Mailer, R. J., Shoemaker, C. F., Wang, S. C., Flynn, J. D. (2010). Report: Tests indicate that imported ”extra virgin” olive oil often fails international and USDA standards. UC Davis Olive Center. American Olive Oil Producers Association, (February 12, 2013). Written Submission on behalf of the U.S. Olive Oil Industry in Investigation No. 332-537, Olive Oil: Conditions of Competition between U.S. and Major Foreign Supplier Industries.
