Nutritional quality assessment of extra virgin olive oil from the Italian retail market: Do natural antioxidants satisfy EFSA health claims?

Full text

Nutritional
quality
assessment
of
extra
virgin
olive
oil
from
the
Italian
retail
market:
Do
natural
antioxidants
satisfy
EFSA
health
claims?
Nicola
Caporaso
a
,
Maria
Savarese
b,
*,
Antonello
Paduano
a
,
Giuliano
Guidone
a
,
Elena
De
Marco
b
,
Raffaele
Sacchi
a,b
a
Department
of
Agriculture,
University
of
Naples
Federico
II,
Via
Universita
`100,
80055
Portici,
NA,
Italy
b
CRIOL,
Centro
Ricerche
per
l’Industria
Olearia,
c/o
Industria
Olearia
Biagio
Mataluni,
Via
Badia,
zona
industriale,
82016
Montesarchio,
BN,
Italy
1.
Introduction
Extra
virgin
olive
oil
(EVOO)
is
the
top
product
among
olive
oils
and
vegetable
oils.
It
is
produced
from
the
olive
fruit
by
mechanical
(physical)
methods
only,
and
no
additive
is
allowed
in
the
extraction
process.
EVOO
must
comply
with
strict
physico-
chemical
quality
requirements
and
organoleptic
characteristics
defined
by
national
and
European
laws
and
regulations.
A
distinction
is
made
between
the
different
grades
of
olive
oil
(extra
virgin,
virgin,
olive
oil)
on
the
basis
of
the
maximum
values
for
some
quality
indices
(acidity,
peroxide
value,
K
232
,
K
270
,
D
K
values,
alkyl
esters)
and
the
panel
test
score,
as
defined
by
EC
Regulation
2568/91
and
subsequent
modifications.
The
popularity
of
EVOO
is
linked
to
its
pleasant
aroma,
particularly
its
flavor
notes,
bitterness
and
pungency,
as
well
as
to
its
health
effects.
Extensive
scientific
research
has
demonstrated
that
its
beneficial
effects
on
human
health
are
related
to
its
balanced
fatty
acid
composition
(high
content
of
oleic
acid
and
ratio
between
omega-3
and
omega-6
fatty
acids)
and
vitamin
E
content
and
the
presence
of
phenolic
compounds,
also
called
‘‘biophenols’’
or
‘‘phenolics’’
(Servili
et
al.,
2009).
The
main
classes
of
hydrophilic
phenols
found
in
VOO
are
phenolic
alcohols
and
acids,
flavonoids,
lignans
and
secoiridoids.
These
compounds
have
been
reported
to
possess
many
health-promoting
functions,
such
as
antioxidant,
anti-inflammatory,
chemo-preventive
and
anti-
cancer
properties
(Servili
et
al.,
2009).
Some
authors
have
highlighted
specific
compounds
for
their
particularly
significant
impact
on
health,
e.g.
the
deacetoxy-ligstroside
aglycone,
also
known
as
oleocanthal,
which
has
been
proved
to
have
anti-
inflammatory
properties
similar
to
ibuprofen
(Inajeros-Garcı
´a
et
al.,
2010).
Olive-
and
olive
oil
polyphenols
are
recognized
as
Journal
of
Food
Composition
and
Analysis
40
(2015)
154–162
A
R
T
I
C
L
E
I
N
F
O
Article
history:
Received
3
November
2013
Received
in
revised
form
12
December
2014
Accepted
16
December
2014
Available
online
12
February
2015
Keywords:
Olive
oil
Italian
olive
Bioactive
non-nutrient
Food
labeling
Antioxidants
Polyphenols
Fatty
acids
Quality
indices
Nutritional
quality
EFSA
Food
analysis
Food
composition
A
B
S
T
R
A
C
T
Extra
virgin
olive
oil
(EVOO)
is
the
top
commercial
grade
of
olive
oil,
and
its
fatty
acid
composition
and
minor
compounds
have
many
documented
health
benefits.
The
European
Food
Safety
Authority
(EFSA)
has
recently
attributed
some
health
claims
to
EVOO.
Although
numerous
studies
have
been
carried
out
on
its
production
technology
and
nutritional
effects,
little
is
known
about
the
composition
and
quality
of
EVOO
from
the
retail
market.
Thus,
our
aim
was
to
evaluate
EVOOs
from
the
Italian
market
by
assessing
their
fatty
acid
composition,
quality
indices,
polyphenols,
tocopherol
content
and
antioxidant
activity
(ABTS
method)
with
a
view
to
the
possible
application
of
EFSA
health
claims.
High
variability
was
found
for
phenolic
compounds
and
tocopherols,
the
levels
of
which
were
significantly
higher
in
100%
Italian
labeled
oils
compared
with
European
Union
blends.
Consumption
of
the
recommended
daily
amount
of
EVOO
would
cover
about
50%
of
the
recommended
daily
allowance
(RDA)
of
tocopherols,
as
well
as
the
polyphenol
intake
recommended
by
EFSA.
Only
3
of
the
32
samples
had
a
phenolic
content
above
250
ppm.
Particularly
high
polyphenol
indices
were
found
in
the
samples
of
Italian
oils
covered
by
Protected
Designations
of
Origin
(PDOs).
In
conclusion,
the
food
industry
and
consumers
need
to
pay
close
attention
to
producing
and
choosing
the
best
EVOO
from
the
nutritional
viewpoint.
ß
2015
Elsevier
Inc.
All
rights
reserved.
Abbreviations:
ABTS,
2,20-azinobis(3-ethylenbenzothiazoline-6-sulfonic
acid);
Ty,
tyrosol;
OHTy,
hydroxytyrosol;
OHTy-DEDA,
dialdehydic
form
of
decarboxymethyl
oleuropein
aglycone;
TAA,
total
antioxidant
activity;
Ty-EDA,
dialdehydic
form
of
elenoic
acid
linked
to
tyrosol;
OHTy-EDA,
dialdehydic
form
of
elenoic
acid
linked
to
hydroxytyrosol;
Ty-EA,
aldehydic
form
of
elenoic
acid
linked
to
tyrosol;
OHTy-EA,
aldehydic
form
of
elenoic
acid
linked
to
hydroxytyrosol;
SPME,
solid-phase
microextraction;
HPLC–DAD,
high
performance
liquid
chromatography–diode
array
detector.
*Corresponding
author.
Tel.:
+39
0824
894141;
fax:
+39
0824
833771.
E-mail
address:
criol@mataluni.com
(M.
Savarese).
Contents
lists
available
at
ScienceDirect
Journal
of
Food
Composition
and
Analysis
jo
u
rn
al
ho
m
epag
e:
ww
w.els
evier
.c
om
/lo
cat
e/jfc
a
http://dx.doi.org/10.1016/j.jfca.2014.12.012
0889-1575/ß
2015
Elsevier
Inc.
All
rights
reserved.

potent
nutraceutical
compounds,
e.g.
their
high
antioxidant
activity
has
positive
effects
on
EVOO
shelf
life
and
on
the
reduction
of
in
vivo
oxidative
stress
in
humans
and
animals
(Baldioli
et
al.,
1996;
Servili
et
al.,
2009;
Martı
´n-Pela
´ez
et
al.,
2013).
Olive
oil
is
widely
known
as
the
main
source
of
fat
in
the
so-
called
Mediterranean
diet,
which
has
been
linked
to
reduced
risk
of
overall
mortality,
cardiovascular
mortality,
cancer
incidence
and
incidence
of
neurodegenerative
diseases
(Martı
´n-Pela
´ez
et
al.,
2013).
The
Mediterranean
diet
is
characterized
by
high
consump-
tion
of
vegetables,
legumes,
fruits
and
cereals
and
moderate
intake
of
wine,
fish,
white
meat
and
dairy
products.
Fat
consumption
is
relatively
high,
but
is
mainly
made
up
of
monounsaturated
fat
(oleic
acid),
due
to
the
extensive
use
of
olive
oil.
There
are
notable
differences
among
the
different
categories
of
olive
oils,
related
not
only
to
the
intensity
of
their
taste
and
aroma,
but
also
to
their
processing
conditions
and
nutritional
profile.
Thus,
the
information
on
the
label
of
this
kind
of
product
is
fundamental
to
understand
its
quality
and
health
benefits.
A
health
claim
is
defined
as
any
claim
that
states,
suggests
or
implies
that
a
relationship
exists
between
a
food
category,
a
food
or
one
of
its
constituents
and
health
(Reg.
EU
432/2012;
Martı
´n-Pela
´ez
et
al.,
2013).
The
European
Food
Safety
Authority
(EFSA)
has
approved
a
number
of
healthclaims
for
olive
oil
on
the
basis
of
generally
accepted
scientific
data,
e.g.
the
claim
that
‘‘olive
oil
polyphenols
contribute
to
the
protection
of
blood
lipids
from
oxidative
stress’’
(EFSA,
2010;
EFSA,
2011b).
In
recent
years
many
health
claims
have
been
considered
for
olives
and
olive
oil
and
their
minor
compounds,
particularly
phenolic
compounds
that
occur
naturally
in
olive
oil.
As
consumers
are
very
sensitive
to
health-related
notices,
it
is
important
to
understand
if
and
when
the
health
claims
authorized
by
EFSA
can
be
used
on
the
label
of
a
virgin
olive
oil.
Producers
or
industry
may
add
the
following
claim
in
the
olive
oil
label:
‘‘olive
oil
polyphenols
contribute
to
the
protection
of
blood
lipids
from
oxidative
stress’’,
when
the
product
contains
at
least
5
mg
of
hydroxytyrosol
and
its
derivatives
per
20
g
of
olive
oil
(Martı
´n-Pela
´ez
et
al.,
2013).
The
nutritional
profile
of
EVOO
is
widely
known
to
be
dependent
upon
the
conditions
of
olive
growing,
harvesting
and
processing
as
well
as
on
the
length
and
conditions
of
storage.
This
last
aspect
is
particularly
important
because
olive
oil
is
susceptible
to
lipid
oxidation
and
is
usually
bottled
in
transparent
glass
or
polyethylene
terephthalate
(PET)
containers,
which
are
exposed
to
light
throughout
storage.
As
olive
oil
contains
a
significant
amount
of
unsaturated
fatty
acids,
it
is
susceptible
to
lipid
oxidation
from
the
time
it
is
produced
until
it
is
cooked
and
consumed.
The
chemical
aspects
of
olive
oil
oxidative
stability
have
been
widely
studied
within
the
scientific
community
during
the
past
decades;
however
more
work
is
needed
to
find
other
ways
of
minimizing
lipid
oxidation.
As
previously
reported
by
a
recent
review
on
this
matter,
there
has
been
little
testing
of
shelf
life
in
the
retail
market,
probably
due
to
the
higher
oxidative
stability
of
VOO
compared
with
other
vegetable
oils
(Frankel,
2010).
Much
research
has
focused
on
analyzing
olive
oil
under
accelerated
oxidative
conditions,
e.g.
using
the
questionable
Rancimat
test,
while
other
studies
have
proposed
simple
but
ineffective
or
inaccurate
models
to
predict
the
future
stability
of
EVOO
(Frankel,
2010).
Some
compounds
found
at
low
concentrations,
particularly
polyphenols
and
tocopherols,
have
dramatic
effects
on
the
stability
and
nutritional
value
of
EVOO.
Tocopherols
and
other
natural
antioxidants
have
been
widely
correlated
with
the
oxidative
stability
of
VOO.
They
act
as
lipophilic
‘‘chain-breaking’’
antiox-
idants,
and
possess
free-radical
scavenging
effects
(Servili
et
al.,
2009;
Martı
´n-Pela
´ez
et
al.,
2013).
Among
tocopherols,
a
-tocoph-
erol
is
the
most
abundant
compound,
representing
about
95%
of
the
total
content
(Tasioula-Margar
and
Okogeri,
2001).
Rivera
del
A
´lamo
et
al.
(2004)
reported
the
characterization
of
a
large
number
of
EVOO
samples
obtained
exclusively
from
the
cultivar
‘Cornicabra’,
and
EVOOs
were
taken
directly
from
the
mill
and
stored
at
4
8C,
which
did
not
provide
information
about
the
quality
changes
at
room
temperatures.
In
previous
studies,
our
research
group
focused
on
the
shelf
life
and
stability
of
EVOOs
bottled
in
different
types
of
plastic
containers
(PET)
to
determine
the
effects
of
container
material
on
compositional
changes
during
12
months
storage
at
room
temperature
(Savarese
et
al.,
2013).
The
results
of
this
research,
like
other
findings
reported
in
the
literature,
showed
that
EVOO
undergoes
rapid
and
dramatic
changes
during
storage.
It
is
therefore
of
great
importance
to
assess
the
characteristics
of
this
food
product
at
retail,
due
to
the
major
implications
for
consumer
health
and
satisfaction.
Thus,
the
aim
of
this
work
was
to
evaluate
the
most
important
nutritional
parameters
for
defining
EVOO
quality
(fatty
acid
composition,
natural
antioxidant
concentration
and
antioxidant
activity)
in
samples
from
the
Italian
retail
market.
The
second
goal
was
to
assess
the
actual
possibility
for
producers
to
declare
EFSA
health
claims
on
EVOOs
based
on
their
composition,
particularly
their
antioxidant
concentration.
2.
Materials
and
methods
2.1.
Samples
Thirty
samples
of
EVOO
were
bought
from
different
retailers
and
supermarkets
located
in
Nola
(NA),
Benevento
and
Mon-
tesarchio
(BN),
Southern
Italy.
The
most
widely
distributed
olive
oil
brands
in
Italy
were
represented
(Cirio,
Farchioni,
Bertolli,
Desantis,
De
Cecco,
Dante,
Costa
D’oro,
Monini,
Carapelli,
Pietro
Coricelli,
Sagra).
Also,
samples
of
two
EVOOs
from
the
Cilento
PDO
–
Salella
and
Pisciottana
Pietrabianca
(Salerno,
Italy)
–
were
collected
directly
from
producers.
The
EVOOs
were
stored
in
a
cool,
dark
place
until
the
chemical
analyses
were
carried
out.
2.2.
Chemical
analyses
2.2.1.
Legal
quality
parameters
Olive
oil
acidity
(%
oleic
acid
per
100
g
olive
oil),
peroxide
value
(meq
O
2
kg
1
oil)
and
UV
determinations
(K
232
,
K
270
and
D
K)
were
carried
out
according
to
the
EC
Reg.
2568/1991
and
International
Olive
Council
(IOC)
standard
methods.
The
parameters
K
232
and
K
270
are
the
oil
absorbance
at
232
and
270
nm,
respectively,
and
D
K
was
calculated
from
the
absorbances
at
262,
268
and
274
nm.
Spectrophotometric
determinations,
K
232
,
K
232
and
D
K
analyses
were
carried
out
using
a
Shimadzu
UV-1601
spectrophotometer
(Shimadzu,
Kyoto,
Japan).
Sensory
analysis
was
carried
out
by
eight
assessors
who
were
fully
trained
in
the
evaluation
of
VOO
according
to
the
official
methods
of
the
IOC
(1996)
and
EC
Reg.
2568/1991.
2.2.2.
Fatty
acid
composition
GC
analysis
of
the
fatty
acid
methyl
esters
was
performed
as
described
by
Christie
(1982)
with
some
modifications.
The
olive
oil
was
diluted
in
hexane
(1%
oil)
and
0.4
mL
solution
was
added
to
0.2
mL
methanol
solution
with
2
N
KOH.
The
mixture
was
shaken
vigorously
for
1
min
and
1
m
L
of
the
hexane
organic
phase
was
collected
for
GC
injection.
A
Shimadzu
model
GC-17A
equipped
with
flame
ionization
detector
(FID)
(Shimadzu
Italia,
Milan,
Italy)
was
used
for
the
analysis.
The
acquisition
software
was
Class-VP
Chromatography
data
system
version
4.6.
(Shimadzu
Italia,
Milano,
Italy).
A
FAME
capillary
column,
60
m,
0.25
mm
i.d.
with
0.25
mm
50%
cyanopropyl-methyl
phenyl
silicone
was
used
(Quadrex
Corporation,
New
Heaven,
CT,
USA).
The
oven
tempera-
ture
was
held
at
170
8C
for
20
min
and
then
it
increased
at
a
rate
of
10
8C
min
1
until
220
8C,
held
for
5
min.
Injector
temperature
and
N.
Caporaso
et
al.
/
Journal
of
Food
Composition
and
Analysis
40
(2015)
154–162
155

FID
temperature:
250
8C.
Carrier
gas:
Helium.
Column
flow:
2
mL
min
1
.
Split
ratio:
1/60.
Injected
volume:
1
m
L.
Peak
identification
was
performed
by
comparing
the
retention
times
of
the
fatty
acids
with
those
of
pure
compounds
(mixture
of
pure
methyl
esters
of
fatty
acids;
Larodan,
Malmoe,
Sweden)
injected
under
the
same
conditions.
2.2.3.
Analysis
of
phenolic
compounds
The
phenolic
compounds
in
the
EVOOs
were
analyzed
as
described
by
Va
´zquez-Roncero
(1978),
with
slight
modifications.
The
oil
(10
g)
was
dissolved
in
10
mL
of
hexane
and
extracted
three
times
in
a
separating
funnel
with
7
mL
of
a
mixture
of
methanol:-
water
(60:40
v/v).
The
hydro-alcoholic
extract
was
washed
with
hexane
and
centrifuged
5
min
at
4000
rpm
using
a
PK
120
centrifuge
(ALC
International,
Milan,
Italy).
The
methanol
phase
was
collected
and
evaporated
in
a
vacuum
flask
using
a
rotary
evaporator
Mod.
Laborota
4000
efficient
(Heidolph
instruments,
Milan,
Italy)
at
40
8C.
The
residue
was
collected
using
2
mL
of
methanol
for
the
HPLC
injection.
HPLC
analysis
was
performed
using
an
HPLC
Shimadzu
mod.
LC-10ADVP
equipped
with
a
UV-Vis
(Photo)Diode
Array
detector
(Shimadzu
Italia,
Milan,
Italy),
using
a
reverse
phase
column
Spherisorb
S5
ODS3
250

4.6
mm
i.d.
(Phenomenex,
Castel
Maggiore,
BO,
Italy).
The
acquisition
software
was
Class-VP
Chromatography
data
system
vers.
4.6
(Shimadzu
Italia,
Milan,
Italy).
Eluent
A
was
water:trifluoroacetic
acid
(TFA)
97:3
v/v
and
eluent
B
was
methanol:acetonitrile
20:80
(v/v).
The
elution
gradient
started
from
5%
eluent
B
and
reached
60%
B
after
35
min
at
flow
rate
1
mL
min
1
.
The
injected
volume
was
20
m
L
and
chromatograms
were
recorded
at
wavelength
279
nm.
Tyrosol
was
used
as
the
external
standard
for
constructing
the
calibration
curve
(Tsimidou
et
al.,
1992).
Phenolic
compounds
were
identified
by
comparing
retention
times,
relative
elution
order
and
UV
absorbance
spectra
with
those
of
authentic
standards,
when
available,
or
with
those
reported
in
the
literature
(Mateos
et
al.,
2001;
Brenes
et
al.,
2000).
Identification
was
confirmed
by
LC/MS
analysis,
performed
on
a
LC-10AD
VP
(Shimadzu,
Kyoto,
Japan)
liquid
chromatograph
on-line
with
a
LCMS-2010EV
(Shimadzu,
Kyoto,
Japan)
mass
spectrometer,
equipped
with
an
electrospray
ionization
(ESI)
interface.
A
Discovery
HS
C18
column
(5
m
m,
150
mm

2.1
mm
i.d.,
Supelco,
Bellefonte
PA,
USA)
was
used
for
compound
separation,
using
a
flow
rate
of
0.35
mL
min
1
,
according
to
previously
published
literature
(Savarese
et
al.,
2007).
Tyrosol
and
hydroxytyrosol
were
used
as
pure
reference
compounds,
while
the
identification
of
the
other
compounds
was
carried
out
as
reported
above.
Quantification
of
individual
phenolic
compounds
was
carried
out
by
HPLC-UV
at
279
nm,
using
tyrosol
as
external
standard
(R
2
>
0.99,
five-point
calibration
curve).
2.2.4.
Tocopherol
quantification
An
aliquot
of
the
EVOO
samples
(0.20
g)
was
added
to
3
mL
ethyl
acetate
and
20
m
L
of
this
solution
was
injected
into
the
HPLC
apparatus
for
analysis
of
the
tocopherol
content.
The
elution
conditions
were
chosen
according
to
Tonolo
and
Marzo
(1989)
and
Napolitano
et
al.
(2004).
Eluent
A
was
a
solution
of
methanol/
water
+
acetonitrile
(73.2:1.8:25
v/v%);
eluent
B
was
ethyl
acetate.
Ethyl
acetate
was
used
for
washing
the
column
(15
min)
between
two
consecutive
sample
analyses.
A
gradient
elution
was
performed
for
19
min:
starting
from
100%
eluent
A
to
100%
eluent
B
solutions
within
1
min.
Subsequently,
starting
from
100%
eluent
B,
a
gradient
was
applied
to
reach
100%
A
in
15
min.
The
analysis
was
performed
using
a
Shimadzu
HPLC
mod.
LC-10ADVP
(Shimadzu
Italia,
Milan,
Italy)
with
a
Shimadzu
UV-Vis
Diode
Array
Detector
mod.
SPD-M10AVP
(Shimadzu
Italia,
Milan,
Italy).
A
reverse-phase
Spherisorb
S5
ODS3
column
was
used
(250

4.6
mm
i.d.).
Column
flow:
1.8
mL
min
1
.
The
wavelength
for
the
detector
was
set
at
290
nm.
Results
were
expressed
as
a
-tocopherol
concentration
(ppm
or
mg
kg
1
oil),
and
pure
a
-
tocopherol
was
used
as
the
external
standard
for
the
calibration
curve,
while
a
-tocopherol,
b
-tocopherol
and
g
-tocopherol
were
used
as
pure
reference
compounds
for
the
identification.
2.2.5.
Determination
of
antioxidant
activity
by
ABTS
+
method
Antioxidant
activity
of
EVOOs
was
analyzed
by
using
2.2
0
-
azino-bis(3-ethylbenzothiazoline-6-sulphonic)
acid
(ABTS
+
meth-
od)
according
to
Pellegrini
et
al.
(1999).
Then
88
m
L
of
a
140
mM
potassium
persulfate
solution
were
added
to
7
mM
ABTS
solution
previously
dissolved
in
water.
The
mixture
was
stored
in
the
dark
at
4
8C
for
at
least
6
h
before
use.
The
ABTS
+
stock
solution
was
diluted
with
ethanol
to
reach
an
absorbance
of
0.70

0.02
at
734
nm
and
at
25
8C.
A
five-point
calibration
curve
was
made
by
using
Trolox
solutions
diluted
in
ethanol
(18–120
mM).
The
analysis
was
carried
out
exactly
2.5
min
after
adding
the
sample
and
the
absorbance
was
read
at
734
nm
using
a
spectrophotometer
mod.
UV-1601
(Shimadzu
Italia,
Milan,
Italy).
The
inhibition
percentage
was
calculated
using
the
formula
A
734
%
=
(1

A
f
/A
0
)*100,
where
A
0
was
the
absorbance
of
the
blank
sample
and
A
f
was
the
absorbance
after
2.5
min.
The
inhibition
percentage
was
plotted
as
a
function
of
concentration.
The
TEAC
(Trolox
Equivalent
Antioxidant
Capacity)
was
calculated
from
the
ratio
of
the
linear
regression
coefficient
of
the
analyte
to
that
of
the
Trolox.
Results
were
expressed
as
mmol
Trolox
equivalents.
2.3.
Statistical
analysis
All
the
analytical
determinations
were
carried
out
at
least
in
triplicate.
Partial
least
squares
(PLS)
regression
analysis
was
performed
using
XLStat
2006
Version
6.6
software
(Addinsoft,
Paris,
France).
Differences
were
considered
significant
at
p
<
0.05.
3.
Results
and
discussion
3.1.
Quality
indices
and
fatty
acid
composition
As
reported
in
Table
1,
almost
all
the
samples
had
quality
parameters
within
the
legal
values
for
EVOOs,
with
some
exceptions.
Specifically,
the
alkyl
ester
value
and
ethyl
ester:-
methyl
ester
ratio
of
sample
1
were
slightly
higher
than
the
upper
limits.
Hence,
this
sample
should
be
classified
as
‘‘lampante’’
(i.e.
olive
oil
extracted
by
mechanical
methods
but
not
suitable
as
food,
except
after
refining)
according
to
the
modifications
made
to
the
alkyl
ester
limit
for
EVOO,
which
has
been
set
at
75
ppm
(EU
Reg.
61/2011).
The
average
alkyl
ester
content
of
the
commercial
bottled
EVOOs
was
about
31
ppm,
whereas
when
only
Italian
EVOOs
were
considered,
the
average
value
was
19
ppm.
Some
samples
(3,
8,
9,
12
16,
17,
18,
19)
recorded
very
low
values
for
these
compounds
(<15
ppm)
that
came
close
to
the
concentrations
in
the
PDO
samples,
thus
suggesting
a
higher
quality
of
raw
material.
This
was
confirmed
by
the
results
for
other
quality
indices,
as
these
samples
had
a
very
low
free
acidity
and
high
scores
for
the
organoleptic
assessment.
The
K
232
value
of
all
the
samples
was
within
the
upper
limit
fixed
for
this
parameter
by
EC
Regulation
n.
2568/91
and
further
modifications
for
EVOO
(K
232
2.50);
however,
the
K
270
value
of
two
of
the
samples
(6
and
16,
Table
1)
exceeded
the
limit
(K
270
0.22)
and
another
two
samples
recorded
a
value
very
close
to
the
legal
limit
(samples
15
and
27).
D
K
was
above
the
limit
in
sample
16
(
D
K
0.01).
According
to
the
organoleptic
assessment
results
(EU
Reg.
No
61/2011),
only
9
samples
of
the
32
analyzed
had
no
sensory
defects.
Fourteen
samples
were
slightly
defective
(mainly
rancid),
three
were
defective
(rancid,
fusty)
and
four
were
very
defective
(fusty,
musty,
earthy)
(data
not
shown).
The
samples
with
the
lowest
quality
indices
in
terms
of
acidity
and
peroxide
N.
Caporaso
et
al.
/
Journal
of
Food
Composition
and
Analysis
40
(2015)
154–162
156

value
(PV)
were
samples
1,
4
and
22
(for
acidity
value)
and
samples
5,
15
and
23
(for
PV).
Some
of
the
samples
also
had
the
lowest
sensory
score,
e.g.
sample
23
had
a
score
of
3.6
(data
not
shown).
The
highest
panel
test
scores
were
obtained
by
two
100%
Italian
EVOOs,
which
were
given
marks
of
7.0
and
6.7
for
samples
31
and
32,
respectively
(data
not
shown).
The
last
two
PDO
EVOOs
also
had
the
lowest
PV,
together
with
sample
29.
The
lowest
acidity
value
was
found
for
sample
18
(0.19),
followed
by
many
other
samples,
whose
acidity
was
in
the
range
0.22–0.30.
The
spectrophotometric
indices
(K
232
,
K
270
and
D
K)
were
also
evaluated,
as
reported
in
Table
1.
Samples
9
and
26
had
the
best
K
232
values
(lowest)
of
1.77
and
1.71,
respectively.
The
highest
value
for
this
parameter
was
2.24
in
sample
29.
K
270
varied
from
0.11
(sample
9)
to
0.20
(sample
16),
and
D
K
was
in
the
range
from
0.003
to
0.009.
The
best
D
K
values
were
for
sample
21,
followed
by
samples
28
and
32,
both
of
which
had
a
value
of
0.002.
Fatty
acid
composition
is
of
crucial
importance
in
determining
the
properties
and
health
benefits
of
a
food,
particularly
for
cardiovascular
health.
The
importance
of
fatty
acid
composition
has
also
been
established
by
EFSA.
The
replacement
of
saturated
fatty
acids
by
unsaturated
ones
(cis-monounsaturated
or
cis-
polyunsaturated)
helps
to
keep
LDL
cholesterol
at
normal
concentrations
in
blood
(EFSA,
2011a).
This
health
claim
can
be
used
for
foods
in
which
unsaturated
fatty
acids
account
for
at
least
70%
of
total
fatty
acid
content
and
represent
at
least
20%
of
energy
intake
(Reg.
EU
432/2012).
As
expected,
unsaturated
fatty
acids
accounted
for
more
than
80%
of
the
total
fatty
acid
content
of
all
the
samples
analyzed
and
oleic
acid
on
its
own
represented
more
than
70%
of
the
total
fatty
acids
in
almost
all
the
samples
(Table
2).
The
highest
concentration
of
this
fatty
acid
was
75.9%
(sample
22),
and
the
lowest
was
68.1%
(sample
11).
Despite
the
great
variability
of
fatty
acid
concentration,
which
has
been
attributed
to
multiple
factors,
the
EVOOs
had
high
contents
of
monounsatu-
rated
fatty
acids.
Regarding
the
concentration
of
unsaturated
fatty
acids,
no
significant
differences
were
generally
found
between
the
composition
of
the
Italian
and
EU
blends
of
olive
oils.
Only
in
the
case
of
C16:1
v
9
and
C20:1
a
statistical
difference
was
found
(p
<
0.05),
although
the
absolute
average
differences
were
very
small.
In
both
cases,
their
concentration
was
higher
in
the
EU
blends
than
in
the
100%
Italian
samples.
We
consider
that
these
slight
differences
are
due
to
the
differences
in
environmental
conditions
and/or
in
the
natural
variation
of
oil
composition
of
the
fruits,
i.e.
the
olive
cultivar.
The
unsaturated
to
saturated
fatty
acid
ratio
was
5.8
(average
value
for
all
the
samples),
with
no
difference
due
to
origin.
The
results
for
fatty
acid
composition
showed
no
correlations
with
the
oxidation
indices,
or
only
very
slight,
non-significant
ones
(data
not
shown).
This
finding
concurs
with
previous
work
(Fregapane
et
al.,
2013)
where
no
correlation
was
reported
between
fatty
acid
composition
and
oxidative
stability
or
quality
indices
in
EVOOs.
3.2.
Phenolic
compounds
and
tocopherols
As
reported
in
Table
3,
the
amount
of
phenolic
compounds
in
EVOOs
varied
greatly
according
to
sample.
Some
samples,
such
as
samples
31
and
32,
had
a
higher
content
of
phenolic
compounds,
particularly
complex
phenolics,
but
a
very
low
content
of
simple
phenolics
(tyrosol
and
hydroxytyrosol).
Conversely,
others
had
a
similar
content
of
simple
and
complex
phenolics
(samples
4,
5,
20,
23).
Table
1
Characteristics
and
quality
parameters
of
extra
virgin
olive
oils
sampled
on
the
Italian
retail
market.
a
Nr.
Origin
Price
(
s
)
PV
K
232
K
270
D
K
Acidity
FAAEs
(mg
kg
1
)
FAEEs/FAMEs
Panel
score
1
Blend
(EU)
4.90
13.8b
1.83a
0.12a
0.002a
0.49c
83.1d
2.3b,c
4.0
2
Blend
(EU)
5.58
12.9b
1.83a
0.15a,b
0.006a,b
0.41c
51.4c,d
2.7c
3.3
3
100%
Italian
6.82
7.9a
1.89a
0.16a,b
0.007b
0.26a
7.4a
0.8a,b
6.2
4
Blend
(EU)
2.99
12.5b
2.27b
0.14a,b
0.004a,b
0.47c
35.9c
0.5a
5.3
5
Blend
(EU)
3.99
16.1b
2.24b
0.14a,b
0.000a
0.38b
37.6c
2.1b,c
5.9
6
Blend
(EU)
3.89
8.9a
2.01a,b
0.20b
0.009b
0.38b
37.7c
1.1b
5.5
7
Blend
(EU)
2.89
10.5a,b
1.95a,b
0.14a
0.000a
0.38b
41.0c
1.4b
5.5
8
Blend
(EU)
5.59
7.4a
1.80a
0.13a
0.003a
0.35b
15.7b
1.4b
4.8
9
Blend
(EU)
5.50
7.4a
1.77a,c
0.11a
0.001a
0.31a,b
18.7b
1.9b,c
5.5
10
Blend
(EU)
3.59
10.6a,b
1.92a
0.13a
0.001a
0.36b
26.3b,c
0.6a
6.3
11
Blend
(EU)
3.59
8.3a,b
2.00a,b
0.12a
0.002a
0.43b
25.0b,c
1.8c
5.5
12
Blend
(EU)
5.59
7.3a
1.84a
0.15a,b
0.009b
0.22a
14.1a,b
0.8a
5.5
13
Blend
(EU)
4.03
9.3a,b
1.87a
0.15a,b
0.004a,b
0.30a,b
24.8c
2.1b,c
5.5
14
100%
Italian
4.65
11.9a,b
2.23b
0.18a,b
0.006a,b
0.37b
39.5c
0.6a
6.0
15
100%
Italian
4.99
16.7b
2.04b
0.19b
0.007b
0.40b,c
45.2c,d
2.4c
6.2
16
Blend
(EU)
7.29
7.1a
1.97a,b
0.20b
0.013b
0.35a,b
13.2a,b
1.3b
5.1
17
Blend
(EU)
4.99
7.4a
1.88a
0.16a
0.002a
0.36b
13.8a,b
1.1a,b
6.2
18
100%
Italian
6.27
8.1a
1.91a,b
0.13a
0.001a
0.19a
7.8a
0.8a,b
6.5
19
100%
Italian
8.59
12.9a,b
2.07a,b
0.13a
0.000a
0.25a,b
7.0a
1.2b
6.8
20
Blend
(EU)
5.70
9.0a,b
1.92a,b
0.18a
0.011b
0.37a,b
42.7c
0.5a
5.3
21
Blend
(EU)
3.99
9.2a,b
1.88a
0.14a
0.003a
0.35b,c
29.9c
2.0c
5.2
22
Blend
(EU)
5.45
9.7a,b
1.72a,c
0.15a
0.003a,b
0.47c
36.0c
1.6b
3.5
23
Blend
(EU)
5.35
15.1b
1.72a,c
0.17b
0.009b
0.44b,c
55.0
4.0d
3.6
24
Blend
(EU)
2.99
12.2a,b
2.12b
0.12a
0.001a
0.38b
32.1c
0.9a,b
6.0
25
Blend
(EU)
3.99
14.1b
2.14b
0.13a
0.000a
0.37b
17.8b
1.4b
5.7
26
Blend
(EU)
5.65
10.9a,b
1.71c
0.16a,b
0.009b
0.27a,b
22.0b
0.4a
5.4
27
Blend
(EU)
4.88
6.6a
1.88a
0.19b
0.011b
0.35a,b
38.6c
2.8c
4.3
28
Blend
(EU)
3.99
11.1a,b
1.88a
0.12a
0.002a
0.29a,b
55.0
1.5b
6.0
29
100%
Italian
4.99
5.7a
2.24b
0.18a,b
0.005a,b
0.43b
29.5c
2.0c
5.9
30
Blend
(EU)
3.99
8.7a,b
1.88a
0.15a,b
0.007b
0.28a,b
25.7b,c
1.9b,c
4.7
31
100%
Italian
20.00
7.8a
1.90a
0.12a
0.001a
0.40b
6.4a
2.2c
7.0
32
100%
Italian
20.00
6.3a
1.88a
0.11a
0.002a
0.30a
8.1a
1.7b
6.7
a
Values
are
averages
of
three
replicates
of
analysis,
with
the
exception
of
oil
prices.
PV:
peroxide
value,
expressed
as
meq
O
2
kg
1
oil;
FAAEs:
fatty
acids
alkyl
esters;
FAME:
fatty
acids
methyl
esters;
K
232
,
K
270
and
D
K
indicate
spectrophotometric
absorbances;
acidity
is
expressed
as
percentage
(%)
of
oleic
acid.
Price
was
expressed
in
Euro
per
bottle
(bottles
of
750
mL).
Panel
score
is
the
median
value
obtained
for
organoleptic
assessment
in
a
scale
ranging
from
0
to
10
(CVR
<
20%
for
panel
score
results).
Different
letters
in
the
same
column
indicate
significantly
different
values
(p
<
0.05).
The
coefficients
of
variation
(CVs)
for
PV,
spectrophotometric
indices
and
acidity
were
3.3%,
CV
for
FAAEs
was
6.7%.
N.
Caporaso
et
al.
/
Journal
of
Food
Composition
and
Analysis
40
(2015)
154–162
157

These
differences
between
samples
could
probably
be
explained
by
differing
storage
times.
It
is
known
that
during
olive
oil
storage,
complex
phenolic
compounds
undergo
degradation
phenomena
(mainly
hydrolysis)
that
lead
to
an
increase
in
simple
biophenols
such
as
tyrosol
and
hydroxytyrosol
(Montedoro
et
al.,
1993).
As
previously
reported
by
other
authors
(Fregapane
et
al.,
2013),
the
ratio
between
simple
(Ty
and
OHTy)
and
complex
polyphenols
could
be
considered
an
index
of
VOO
freshness.
Olive
cultivar
and
the
extraction
process
likewise
have
a
fundamental
influence
on
this
ratio;
therefore
it
is
necessary
to
have
an
accurate
evaluation
of
the
initial
phenolic
profile.
The
derivatives
of
hydroxytyrosol
(OHTy-EDA
and
OHTy-EA)
have
been
mainly
associated
with
the
positive
bitter
attribute
in
VOOs,
while
the
tyrosol
derivatives
(Ty-EDA
and
Ty-EA)
are
chiefly
associated
with
the
sensory
note
of
pungency
(Inajeros-Garcı
´a
et
al.,
2010).
The
most
abundant
phenolic
compounds
in
the
samples
analyzed
were
the
complex
polyphenols
Ty-EDA,
OHTy-EDA
and
OHTy-EA,
with
a
mean
content
of
40.1,
32.6
and
20.5
mg
kg
1
,
respectively.
The
concentrations
of
tyrosol
and
hydroxytyrosol
were
between
3.1–20.2
and
2.1–25.6
ppm,
respectively.
The
level
of
complex
phenolic
compounds
OHTy-EDA,
Ty-EDA
and
OHTy-EA,
varied
widely
in
the
ranges
2.2–174.4,
9.6–197.9
and
1.6–103.8
ppm,
respectively.
These
results
for
phenolic
compounds
are
in
accordance
with
previous
research
by
Bayram
et
al.
(2012)
reporting
the
testing
results
for
55
EVOOs
from
the
retail
market.
Despite
the
attention
paid
by
academic
literature
and
applied
research
to
the
phenolic
compounds
in
olives
and
virgin
olive
oils,
data
are
needed
on
the
actual
amount
of
these
compounds
in
retail
EVOOs,
due
to
their
importance
to
consumer
health.
It
has
been
proven,
and
recently
confirmed
by
the
European
Food
Safety
Authority
(EFSA),
that
when
consumed
at
a
daily
rate
of
5
mg,
expressed
as
hydroxytyr-
osol,
polyphenols
from
olive
oil
and
olive
leaves
protect
low-
density
lipoproteins
from
oxidative
damage.
This
intake
can
be
achieved
by
consuming
two
tablespoons
of
olive
oil
(20
g);
hence,
the
polyphenol
concentration
of
olive
oil
should
be
at
least
250
ppm.
The
EU
Reg.
432/2012
allows
the
following
health
claim
solely
for
olive
oils
with
a
minimum
polyphenol
concentration
of
250
ppm:
‘‘consumption
of
olive
oil
polyphenols
contributes
to
the
protection
of
blood
lipids
from
oxidative
damage’’.
This
concentration
is
justified
by
the
need
for
a
daily
intake
of
5
mg
of
hydroxytyrosol
or
its
derivatives,
through
the
daily
consumption
of
20
g
of
olive
oil.
Less
than
10%
of
all
the
samples
of
EVOOs
analyzed
had
a
polyphenol
concentration
above
250
ppm
(Table
3).
The
polyphenol
concentration
of
the
majority
of
the
bottled
EVOOs
from
the
retail
market
was
too
low
to
provide
the
minimum
intake
(5
mg)
required
to
have
an
antioxidant
effect
in
a
balanced
diet.
Another
point
to
bear
in
mind,
as
widely
reported
in
literature,
is
that
the
polyphenol
concentration
of
virgin
olive
oils
varies
greatly
depending
on
the
olive
cultivar,
agronomic
practices
and
degree
of
fruit
ripening,
as
well
as
on
the
conditions
of
processing
(type
of
olive
mill,
malaxation,
etc.)
and
fruit
and
oil
storage.
This
last
aspect
is
considered
to
be
one
among
the
most
important
factors
because
the
concentration
of
phenolic
compounds
decreases
during
storage,
especially
when
olive
oils
are
exposed
to
oxygen,
light
and
high
temperatures,
which
accelerate
lipid
oxidation.
As
shown
in
Fig.
1,
the
samples
labeled
as
100%
Italian
EVOO
had
a
higher
content
of
total
phenolic
compounds.
Tocopherols
are
important
constituents
of
foods.
Due
to
their
positive
effect
on
human
health,
the
recommended
daily
allow-
ance
(RDA)
of
tocopherols
has
been
fixed
at
12
mg.
a
-Tocopherol
is
Table
2
Fatty
acid
composition
(%)
of
bottled
extra
virgin
olive
oils
from
the
Italian
retail
market.
a
Nr.
C16:0
C16:1
v
9
C16:1
v
7
C17:0
C17:1
C18:0
C18:1
C18:1
v
7
C18:2
C20:0
C18:3
C20:1
C22:0
Squalene
C24:0
1
10.31
0.10
0.70
0.05
0.07
3.53
74.88
3.25
5.08
0.42
0.58
0.22
0.11
0.66
0.05
2
10.70
0.09
0.66
0.09
0.06
3.61
75.57
3.29
3.83
0.37
0.56
0.28
0.10
0.74
0.05
3
10.63
0.10
0.52
0.04
0.06
2.42
74.60
3.13
6.32
0.39
0.66
0.34
0.12
0.58
0.05
4
12.94
0.10
0.99
0.06
0.05
2.54
68.58
3.62
9.53
0.21
0.54
0.15
0.13
0.48
0.04
5
11.56
0.11
0.87
0.04
0.08
2.77
70.86
3.76
7.59
0.39
0.65
0.25
0.12
0.60
0.06
6
11.19
0.10
0.78
0.05
0.09
3.40
72.13
3.17
6.88
0.37
0.63
0.24
0.11
0.78
0.05
7
10.53
0.05
0.45
0.06
0.07
3.34
71.97
3.79
5.73
0.57
0.40
0.46
0.18
1.13
0.09
8
12.30
0.13
0.94
0.06
0.13
2.41
70.18
4.11
8.09
0.34
0.54
0.22
0.06
0.44
0.04
9
12.75
0.13
1.04
0.07
0.13
2.33
70.16
3.34
8.27
0.35
0.56
0.23
0.14
0.45
0.05
10
10.80
0.10
0.72
0.04
0.05
3.06
74.20
3.46
5.50
0.39
0.60
0.24
0.11
0.68
0.06
11
12.67
0.10
1.13
0.05
0.10
2.81
68.07
3.89
9.34
0.39
0.61
0.24
0.12
0.52
0.06
12
12.17
0.12
0.90
0.07
0.13
2.46
71.29
4.08
6.84
0.38
0.60
0.22
0.13
0.57
0.04
13
12.20
0.11
0.92
0.07
0.14
2.46
70.68
4.13
7.23
0.39
0.59
0.27
0.13
0.58
0.06
14
10.60
0.08
0.58
0.06
0.07
2.60
74.55
2.99
6.41
0.40
0.58
0.21
0.11
0.61
0.05
15
11.05
0.10
0.68
0.05
0.07
2.63
70.25
3.83
6.46
0.42
0.66
0.28
0.13
0.58
0.05
16
11.00
0.10
0.58
0.05
0.07
2.48
74.08
3.33
6.27
0.43
0.65
0.25
0.11
0.53
0.06
17
10.96
0.10
0.57
0.06
0.07
2.48
73.87
3.00
6.37
0.40
0.60
0.29
0.11
0.57
0.06
18
11.32
0.11
0.72
0.06
0.10
2.22
72.51
3.77
7.18
0.34
0.57
0.27
0.12
0.54
0.06
19
11.72
0.11
0.69
0.06
0.10
2.20
72.04
3.25
7.79
0.39
0.63
0.32
0.12
0.49
0.05
20
10.01
0.13
0.58
0.10
0.14
3.10
74.33
3.27
6.05
0.43
0.65
0.27
0.11
0.77
0.06
21
10.59
0.11
0.76
0.06
0.11
2.68
75.83
3.17
4.76
0.30
0.60
0.20
0.08
0.69
0.03
22
10.61
0.10
0.76
0.04
0.07
3.46
75.85
3.37
3.75
0.35
0.57
0.19
0.10
0.74
0.04
23
9.79
0.10
0.66
0.05
0.08
3.34
76.55
3.43
4.02
0.36
0.51
0.15
0.09
0.83
0.05
24
11.90
0.10
0.91
0.06
0.10
2.87
69.69
3.87
8.24
0.44
0.69
0.29
0.13
0.63
0.06
25
11.05
0.13
0.70
0.06
0.09
3.53
70.34
3.79
7.94
0.29
0.71
0.27
0.11
0.69
0.05
26
9.24
0.14
0.55
0.05
0.14
3.19
74.72
2.95
6.49
0.44
0.60
0.28
0.11
0.77
0.05
27
10.69
0.11
0.71
0.06
0.08
3.23
74.39
2.94
5.74
0.39
0.61
0.20
0.11
0.68
0.05
28
11.84
0.11
0.93
0.06
0.11
2.72
71.54
3.80
6.80
0.41
0.63
0.28
0.13
0.60
0.06
29
10.60
0.10
0.56
0.05
0.08
2.33
74.31
3.06
6.75
0.42
0.66
0.38
0.12
0.54
0.04
30
10.23
0.12
0.64
0.09
0.13
2.81
75.03
2.55
6.10
0.37
0.68
0.39
0.11
0.71
0.05
31
14.50
0.07
0.99
0.12
0.20
2.36
69.46
3.51
6.63
0.37
0.69
0.32
0.11
0.61
0.06
32
12.86
0.05
0.43
0.12
0.04
2.90
72.74
2.60
5.86
0.45
0.73
0.42
0.11
0.63
0.06
a
Values
are
averages
of
three
replicates.
Concentration
of
fatty
acid
methyl
esters,
expressed
in
percentage
(%)
of
total
fatty
acids
content,
according
to
official
IOC
method.
Precision:
CV

3.7%
for
fatty
acids
(FA)

1%,
CV

12.8%
for
FA
between
0.15
and
1%,
CV

65.9
for
FA

0.15%.
N.
Caporaso
et
al.
/
Journal
of
Food
Composition
and
Analysis
40
(2015)
154–162
158

Table
3
Phenolic
compounds,
tocopherols
and
antioxidant
activity
(ABTS
+
method)
in
extra
virgin
olive
oils
from
the
retail
market.
a
Sample
Phenolic
compounds
Antioxidant
activity
Tocopherols
OHTy
Ty
OHTy-EDA
Ty-EDA
A.P.
OHTy-EA
Ty-EA
TPC
a

b
+
g
1
8.9
13.7
10.9
19.1
4.8
16.6
9.0
101.8
14.5
121.1
3.9
2
3.0
8.0
2.8
13.2
5.7
7.9
4.2
62.1
29.3
153.4
0.9
3
8.3
9.0
73.9
77.5
41.1
37.4
16.1
281.1
22.7
167.8
ND
4
15.5
15.0
10.6
20.2
25.5
12.0
3.7
122.8
20.6
230.6
ND
5
10.9
13.1
14.1
15.9
29.5
9.7
1.7
117.3
22.7
214.4
0.3
6
11.7
13.7
34.8
42.5
18.8
19.3
6.5
171.3
17.9
164.4
1.3
7
6.4
7.0
22.9
30.7
17.6
11.8
3.8
122.4
50.3
267.5
7.8
8
10.3
7.9
16.1
23.5
32.6
1.6
ND
113.5
8.2
191.0
ND
9
2.5
3.1
12.2
22.9
20.6
4.2
1.1
88.7
24.3
215.6
ND
10
13.9
19.2
19.3
30.5
26.5
26.1
11.6
169.7
21.6
202.1
ND
11
11.0
9.7
26.1
25.9
19.7
19.5
4.7
136.9
38.5
218.0
ND
12
9.8
9.1
50.9
44.1
36.2
21.5
8.3
206.9
22.6
177.3
ND
13
8.6
8.5
35.5
39.5
32.0
22.9
12.2
184.6
25.2
210.0
ND
14
11.6
15.6
19.3
35.4
29.7
8.6
4.2
145.0
18.8
180.4
ND
15
16.9
15.1
34.5
36.9
24.5
19.4
7.4
174.2
45.9
174.7
ND
16
24.1
19.8
2.2
53.7
36.3
24.0
10.2
190.6
22.9
135.7
ND
17
16.5
16.8
33.8
42.6
31.2
20.4
8.2
191.6
41.4
220.9
ND
18
9.4
7.2
35.2
42.6
42.8
14.8
5.8
180.3
17.3
215.8
ND
19
11.4
9.0
37.8
38.7
43.4
21.3
9.6
194.7
49.7
219.1
ND
20
25.6
20.2
18.2
21.1
25.4
21.3
5.3
161.7
21.0
171.3
ND
21
13.8
11.7
47.6
44.8
14.4
51.1
16.8
227.3
59.6
223.1
3.9
22
2.1
8.6
10.2
27.8
8.8
16.6
7.7
105.8
8.9
196.4
5.7
23
2.5
6.6
5.2
9.6
3.7
7.6
2.3
54.8
25.6
119.2
4.8
24
12.9
12.6
11.6
19.3
23.6
16.8
6.6
123.6
19.0
255.2
ND
25
11.8
14.3
17.5
27.9
13.7
16.7
5.0
123.4
30.0
185.2
ND
26
2.6
7.1
17.2
27.6
11.3
15.5
6.1
104.5
28.5
108.8
ND
27
6.9
10.4
26.9
36.6
10.5
25.9
8.6
147.1
30.0
231.6
ND
28
1.1
3.9
14.2
23.8
18.2
12.6
4.0
89.4
18.5
162.1
ND
29
13.3
16.1
22.9
30.4
32.2
5.2
0.8
137.9