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Journal of Essential Oil Research

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Effectiveness of electronic nose systems to detect

bergamot (Citrus bergamia Risso et Poiteau) essential
oil quality and genuineness
a b b b b
Mariateresa Russo , Demetrio Serra , Francesca Suraci & Santo Postorino
a
Department STAFA, Laboratory of Food Chemistry , University of Reggio Calabria , Reggio
Calabria , Italy
b
Mediterranean Foundation of Research Terina , Lamezia Terme (CZ) , Italy
Published online: 20 Mar 2012.

To cite this article: Mariateresa Russo , Demetrio Serra , Francesca Suraci & Santo Postorino (2012) Effectiveness of
electronic nose systems to detect bergamot (Citrus bergamia Risso et Poiteau) essential oil quality and genuineness, Journal
of Essential Oil Research, 24:2, 137-151, DOI: 10.1080/10412905.2012.659530

To link to this article: http://dx.doi.org/10.1080/10412905.2012.659530

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 The Journal of Essential Oil Research
Vol. 24, No. 2, April 2012, 137–151

Effectiveness of electronic nose systems to detect bergamot (Citrus bergamia Risso

et Poiteau) essential oil quality and genuineness
Mariateresa Russoa,b*, Demetrio Serrab, Francesca Suracib and Santo Postorinob
a
Department STAFA, Laboratory of Food Chemistry, University of Reggio Calabria, Reggio Calabria, Italy; bMediterranean Foun-
dation of Research Terina, Lamezia Terme (CZ), Italy
(Received 16 November 2011; final form 9 January 2012)

The quality of an essential oil is determined by its composition as well as by the complex aroma features. The aim
of this work was to analyze the effectiveness of electronic nose (e-nose) systems to detect quality and genuineness of
bergamot essential oils (BEOs). Tested methods included volatile analysis with the e-nose based on metal-oxide semi-
conductor sensors and analysis of chemical composition of essential oil by chromatography (GC/FID, GC/MS). The
Downloaded by [Umeå University Library] at 11:11 22 April 2014

paper reports preliminary results on the potential of an e-nose to discriminate natural cold-pressed bergamot oil (BEO
C-P) from those deterpenated and bergapten-free; between samples obtained from different cultivars; from samples
obtained from fruit grown in the Protected Denomination of Origin (PDO) area and those outside the PDO; and
finally from synthetic essential oils (SEO). This feasibility study has confirmed that an e-nose demonstrates the ability
to supply a concrete support to BEO quality control. The e-noses, with subsequent DFA treatment of data, offer the
advantages of rapidity and reliability. Further validation studies are currently under way with the aim of enriching
and updating the databases of reference for BEOs. In order to broaden the range of e-nose use, tests on sensors chiral
comportment are in progress, in order to bring this same approach in the study of adulteration of BEOs.
Keywords: Citrus bergamia Risso et Poiteau; bergamot essential oil; artificial olfactory system; electronic nose;
metal oxide semiconductor

Introduction the most valuable and is therefore mainly employed in

Bergamot is the common name of the fruit and plant of
Citrus bergamia Risso and Poiteau, an endemic species
grown almost exclusively in the southern Italian region
of Calabria in the area of Reggio Calabria and here
limited to a narrow strip of the coast, along the Ionian
and the Thyrrenian seas, of about 150 km (Figure 1),
which presents particular pedoclimatic conditions for its
cultivation (1, 2).
Currently three different cultivars are cultivated in
Calabria: cv. ‘Femminello’, with slender branches and
smooth fruits; cv. ‘Castagnaro’, more vigorous with
less spherical fruit and a little wrinkled; and mainly cv.
‘Fantastico’, characterized by a good yield and essential
oils quality, which represents about 75% of the whole
production (3).
Up until now, bergamot fruits have been used
mostly for the extraction of its essential oil from the
peel, a clear yellow-green liquid. The oil was originally
extracted by hand, but today it is extracted by a
mechanical cold-pressing procedure. Bergamot essential
oil (BEO) is the most profitable product of bergamot
industrial processing.
Among the Citrus peel oils, because of its unique fra-
grance and freshness related to its composition, BEO is
the perfumery and cosmetic industries. It is also used in
food and confectionery industries as liquors, teas and
sweets aromas and, because of its antiseptic and antibac-
terial properties, is inserted in the pharmacopoeia of sev-
eral countries in the pharmaceutical industry (2).
The aroma profile of BEO, characterized by top
note: rich, sweet-fruity, citrus; middle note: herbaceous,
floral-citrus, sweet, slightly oily and dry-out: light,
sweet-balsamic, tea-like, mainly depending on the dif-
ferent bio-pedo-climatic habitat and cultivar, degree of
ripeness of fruits, and extraction processes. BEO con-
sists of a volatile fraction (93–96%), whose main com-
ponents are, in approximate decreasing percentage,
limonene, linalyl acetate, linalool and a non-volatile
fraction (4–7%), essentially represented by coumarins
and psoralens (that is bergamottin, citroptene, bergap-
ten, and so on). The main phototoxic activity of fur-
ocoumarins present in bergamot oil is ascribed just to
bergapten, so that furocoumarin-free extracts and dis-
tilled extracts are often used, instead of the natural oil,
to prepare commercial products (2).
The quality of essential oils, for their use in differ-
ent industrial sectors, is closely related to the specific
aroma.

*Corresponding author. Email: mariateresa.russo@unirc.it

ISSN 1041-2905 print/ISSN 2163-8152 online

Ó 2012 Taylor & Francis
http://dx.doi.org/10.1080/10412905.2012.659530
http://www.tandfonline.com
 138 M. Russo et al.
Therefore, the main aims of the aroma research
were to discern and identify the compounds that are
responsible for the different aromas, consisting of a
complex mixture of thousands of different chemical
volatile species. In addition, the odor-active compounds
have to be isolated and characterized from a multitude
of volatiles (4, 5).
A large number of studies have been conducted to
develop analytical techniques and methods aimed at
determining the quality of the essential oils through the
analysis of chemical composition as well as their odor
activities (5).
Chromatographic and spectroscopic analytical tech-
niques and their combination and sensory analysis are
commonly used, all available to give rise to more
detailed data for qualitative and quantitative determina-
tion of chemical essential oil composition (5–11). Addi-
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tional information in aroma research has been obtained
with gas chromatographic (GC) olfactomeric methods.
The GC-sniffing or GC-olfactometry (GC-O) method
has contributed to the classification of volatiles into
odorants with a high aroma relevance and volatiles
with secondary importance, presumably non-odor-active
(4, 12).
Analytical techniques can be complex, expensive,
time consuming and they require a well-equipped ana-
lytical laboratory and a skilled staff. On the other hand,
human sensory analysis conducted with a panel or a
‘nez’ provides the evaluation of a different matrix as a
whole, resulting from the impact of its odors on human
senses. A skilled sensory panel is very costly and there
is a limit to the number of replicate samples, which can
be evaluated because of olfactory adaptation to odors.
New trends are occurring in aroma research assess-
ment techniques, based on the use of non-destructive
techniques as the artificial olfactory system (AOS), bet-
ter known as electronic noses (e-noses) (13, 14).
This approach would complete, but not replace,
conventional analyses of volatile compounds by sen-
sory methods and by traditional analytical techniques.
E-noses have been designed for automated detection
and recognition of odors, vapors and gases (15). It does
not decompose the matrix but supplies a global
evaluation of aroma, miming the human olfactory sys-
tem, in an attempt to give the senses, which are proper
to humans, instrumental objectivity. The first pioneering
studies on the concept of an artificial nose system able
to measure odors were reported for first time in 1982,
by Persaud and Dodd of the University of Warwick,
Coventry (15). To Gardner and Bartlett is ascribed the
classical definition of an e-nose as: ‘an instrument,
which comprises an array of electronic chemical
sensors with partial specificity and an appropriate pat-
tern recognition system, capable of recognizing simple
or complex odors’ (16, 17).
An e-nose multi-sensor assay recognizes odor pat-
terns as a whole, without decomposing the odor, as it
occurs during chromatographic analysis, and identifies
the odor profile of the sample in the same way as the
mammalian olfactory system does. Then, an e-nose
gives a simple pattern of sensorial responses as an out-
put form of signal response. The signal response of
each sensor comprises a pattern, equatin to a specific
‘fingerprint’ associated with the different samples in
analysis (16, 17).
Odor databases must be built up and a multivariate
data analysis is performed in order to discriminate the
different samples. These patterns are compiled into a
database for further analysis using statistical routines
like pattern recognition methods, cluster analysis and
artificial neural networks (16–18).
Despite the limitations regarding the chemical infor-
mation of the matrix aroma composition, the e-nose
offers some advantages, such as simple operation pro-
cedures and rapid analysis suitable for on-line monitor-
ing.
E-noses find mainly applications in the food indus-
try for quality control, process monitoring, freshness
evaluation, shelf-life investigation and authenticity
assessment (18). A large amount of work has already
been carried out on oils (19–21), wine and liquor (22–
26), meat (27), fish (28, 29), eggs (30, 31), milk, dairy
products and cheese (32–35), cereals (36, 37), coffee,
and on many other foodstuffs and beverages, as well as
on the odor quality evaluation of food packaging (38–
42), but also in classification of chemotypes and/or of
essential oils extracted from different species and/or
plant clones and perfumes (43–45).
For each application, it is necessary to develop a
specific protocol with individual sensor set-up for the
best selectivity/sensitivity compromise, the generation
of a congruent and specific database, and the selection
of appropriate pattern recognition techniques for the
final data elaboration (19, 46).
The approach based on the use of an e-nose is
described in this work, for the first time, with refer-
ence to the quality control and authentication of
BEO. This paper reports preliminary results on the
potential of an e-nose based on metal-oxide semicon-
ductor (MOS) sensor technology in the solution of
discrimination between: (a) a cold-pressed BEO
(BEO C-P) obtained from the three different culti-
vars; (b) a BEO C-P from different areas of Pro-
tected Designation of Origin (PDO); (c) a BEO C-P
from these areas and those from cultivations placed
outside the PDO area; (d) BEO C-P, BEO C-P ber-
gapten-free, BEO deterpenated and BEO ‘Torchiato’.
Finally, the effectiveness of the e-nose has been
tested to discriminate between natural BEO C-P and
synthetic essential oils (SEO).
 The Journal of Essential Oil Research
The fingerprint responses of the sensor array of
BEO aromas were correlated with the results obtained
by standard chromatographic techniques.
Experimental
Samples
The analyses were carried out on genuine BEO C-Ps,
produced during the 2007–2008 season with the most
commonly used extraction technique (Pelatrice
machine), on laboratory-extracted bergamot oils (BEO
L-Es), on Torchiati oils obtained from the residue of
the extraction of the cold-pressed oils and on different
commercial kinds of BEOs.
The BEO L-E samples were obtained by applying
manual pressure on the fruit epicarp, to cause the
breaking of the utricles and the release of the oil itself,
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which was collected and centrifuged.
BEO C-P samples have been supplied by a local
industry, samples of BEO C-P (A), BEO bergapten-free
(B) and BEO deterpenated (C) were obtained from the
same lot of fruits, and BEO Torchiato (D) from the res-
idue of the extraction.
Figure 1. Protected Denomination of Origin (PDO) area of bergamot (Citrus bergamia).
139
Industrial BEO C-P from fruit of the Tyrrhenian
coast PDO cultivation area (E), synthetic bergamot
essential oil (SEO) (F) and BEO C-P from fruit of
Ionian PDO area (G) were supplied from another local
industry.
The essential oils from fruits of cv. Fantastico from
three different areas were obtained in the laboratory fol-
lowing the procedure indicated above: BEO L-E of the
Tyrrhenian PDO area (Re); BEO L-E Ionian PDO area
(Ba); BEO L-E outside the PDO bergamot area (La)
(Figure 1).
Finally, industrial BEO samples were extracted
from fruits cultivated in the same habitat but from dif-
ferent cultivars: Castagnaro (Ca), Femminello (Fe) and
Fantastico (Fa). All the samples were stored at 4°C
before analysis.
E-nose equipment and protocol
E-nose measurements were performed using an e-nose
model ISE Nose 2000 (ISE, Pisa, Italia), equipped with
a sensor box of twelve metal-oxide sensors based on
SnO2 (TGS type from Figaro Inc.), whose electrical
resistance is affected by reducing gases. In addition, the
 140
e-nose is equipped with two sensors for humidity and
temperature control.
A controlled airflow, coming from a chromato-
graphic air generator, is continuously maintained on the
sensors to ensure a constant oxygen feed. The sample
headspace is extracted from the vial, with a fifteen-
position autosampling unit, combined with a constant
flow of pure air to sensors box. The raw e-nose
response consists of twelve resistance versus time
curves, one for each sensor. Different operational
parameters influence the performance of the e-nose sys-
tems, such as flow, temperature and sample preparation
condition. Then, in order to define the protocol of anal-
ysis and examine the discriminative capability of the e-
nose, a series of samples were first evaluated and dif-
ferent sample preparation conditions were tested. As a
result, the e-nose protocol was optimized under the
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conditions described below.
In order to release volatile organic compounds into
the headspace, an aliquot of 20 μL of BEO essential
oils was placed in a 22-mL vial and sealed. To ensure
reproducibility, each vial was kept at the temperature of
30°C by a thermostatic bath for 15 minutes before
injection. Transport gas humidity was also controlled.
A sample headspace volume of 5 mL was drawn from
the vial at 5 mL/second, injected into the e-nose and
Figure 2. Digital ‘fingerprint’ of bergamot essential oil cold-pressed bergamot oil. The response ΔR/R0 × 100 vs time (s) has a
different color for each sensor and temperature and humidity of the sensors box traces.
M. Russo et al.
delivered to the sensors with a pure air carrier gas
(chromatographic air) at a flow rate of 300 mL/minute.
Sensor resistances were recorded for 700 seconds
(using a 1-second sampling interval). A delay of 300
seconds was used to allow the sensors to return to their
baseline value before the next injection. After injection,
the line that carries the sample was flushed for 100 sec-
onds with pure air before subsequent samples were pro-
cessed. The experimental conditions developed ensured
that a correct baseline was established, allowing the
recovery of sensors between samples.
Feature extraction and statistical analysis
The signals from the e-nose sensor array are processed
by means of pattern recognition methods, which,
exploiting the cross-correlations between the sensor
responses, extract information contained in all sensor
outputs. In particular, a pattern recognition procedure
performs a qualitative analysis of the environment and
evaluates the multidimensional data set from gas sensor
array, seeking the underlying main relationships in the
data set itself in order to analyze the data structure and
discriminate between different data classes belonging to
different aroma chemical patterns. Different aromas
require the building up of a database of patterns. This
database of labeled patterns is used to train the pattern
 Downloaded by [Umeå University Library] at 11:11 22 April 2014

Table 1. Percentage composition of bergamot essential oils.

BEO BEO BEO BEO BEO BEO Synthetic BEO BEO BEO BEO C-P BEO C-P BEO C-P
C-P Defurocum. Deterpenated Torchiato C-P C-P BEO L-E L-E L-E Castagnaro Fantastico Femminello

LRI
expa LRI litb (A) (B) (C) (D) (E) (G) (F) (Re) (Ba) (La) (Ca) (Fa) (Fe)
Triciclene 923 923 – – – – tr – – – tr – tr – –
a-Thujene 925 927 0.31 0.28 – 0.27 0.29 0.26 – 0.23 0.20 0.18 0.21 0.19 0.17
a-Pinene 932 933 1.16 1.06 0.01 1.02 1.14 1.01 0.15 0.93 0.75 0.68 0.85 0.68 0.68
Camphene 953 953 0.03 0.03 0.01 0.04 0.04 0.04 – 0.03 0.02 0.02 0.03 0.02 0.03
Sabinene 972 972 1.01 0.93 0.01 0.93 1.20 0.88 0.09 0.83 0.76 0.67 0.99 0.67 0.68
b-Pinene 976 978 5.75 5.12 0.04 5.61 7.28 5.35 tr 4.31 4.13 3.68 5.54 3.68 3.64
6-Methyl-5-hepten- 982 986 0.01 0.04 – 0.05 0.02 0.02 0.02 – – – – – –
2-one
b-Myrcene 990 991 1.04 1.02 0.05 1.00 0.88 0.92 0.61 1.22 0.98 0.74 0.96 0.74 0.75
Octanal + a-phel- 1004 1006–1007 0.06 0.06 – 0.04 0.06 0.06 0.05 0.07 0.08 0.07 0.20 0.07 0.05
landrene
δ-3-Carene 1010 1009 – – – 0.01 – 0.01 0.04 – – – tr tr –
a-Terpinene 1016 1017 0.16 0.15 tr 0.16 0.16 0.14 0.02 0.15 0.15 0.13 0.32 0.13 0.11
p-Cimene 1023 1025 0.36 0.22 0.43 0.41 0.08 0.08 0.02 0.07 0.08 0.01 0.02 tr 0.13
Limonene 1029 1030 42.44 41.06 1.42 41.36 32.81 35.64 30.12 39.40 37.59 39.29 35.84 28.29 29.74
Z-b-Ocimene 1036 1036 0.03 0.02 0.02 0.04 0.04 0.04 0.02 0.03 0.02 0.03 0.09 0.03 0.03
E-b-Ocimene 1046 1046 0.23 0.22 0.10 0.28 0.25 0.21 0.05 0.22 0.16 0.18 0.27 0.18 0.16
γ-Terpinene 1057 1058 7.20 6.66 3.23 7.78 7.29 6.17 0.02 7.07 7.04 5.87 7.70 5.87 4.59
trans-Sabinene 1065 1069 0.03 0.03 0.06 0.02 0.04 0.03 0.01 0.05 0.05 0.05 0.01 0.05 0.04
hydrate
Octanol 1071 1073 tr tr 0.05 0.03 tr tr tr – tr – tr tr tr
Terpinolene 1087 1086 0.32 0.30 0.38 0.35 0.31 0.27 0.24 0.34 0.33 0.28 0.52 0.28 0.22
Linalool 1102 1101 9.98 11.40 23.16 10.18 7.12 11.05 10.12 14.30 13.18 23.49 17.14 23.49 25.98
The Journal of Essential Oil Research

Nonanal 1104 1107 0.03 0.03 0.04 0.02 0.05 0.06 – 0.03 0.05 – – – –
cis-Limonene oxide 1132 1137 tr – tr – – – 0.02 – – – tr – –
trans-Limonene 1137 1140 tr – tr tr tr tr tr – tr tr – tr tr
oxide
Isopulegol 1141 1145 – – tr – – – 0.03 – – – tr – –
Citronellal 1152 1152 – tr 0.03 tr – – 0.02 – – tr 0.02 tr tr
Terpinen-4-ol 1179 1180 0.03 0.02 0.10 0.06 0.02 0.03 tr 0.02 0.03 0.03 0.10 0.03 0.04
a-Terpineol 1194 1195 0.09 0.10 0.25 0.49 0.06 0.08 0.10 0.09 0.12 0.15 0.62 0.15 0.12
Decanal 1204 1208 0.05 0.05 0.11 0.02 0.06 0.06 0.03 0.04 0.04 0.06 0.07 0.06 0.03
Octyl acetate 1212 1210 0.10 0.08 0.24 0.08 0.14 0.11 – 0.07 0.07 0.10 0.12 0.10 0.06
Nerol 1228 1229 0.08 0.10 0.19 0.10 0.05 0.05 0.02 0.11 0.09 0.21 0.11 0.21 0.22
Neral 1236 1238 0.19 0.22 0.45 0.12 0.17 0.22 0.04 0.31 0.32 0.44 0.30 0.44 0.30
cis-Sabinene hydrate 1253 1246d 0.08 0.07 0.11 0.05 0.08 0.06 tr 0.09 0.09 0.08 0.08 0.08 0.06
acetate
Linalyl acetate 1255 1251d 26.30 28.00 63.45 26.41 37.75 34.51 44.75 26.28 30.84 20.65 24.76 31.65 29.93
(Continued)
141
 Downloaded by [Umeå University Library] at 11:11 22 April 2014

Table 1. (Continued).
142

BEO BEO BEO BEO BEO BEO Synthetic BEO BEO BEO BEO C-P BEO C-P BEO C-P
C-P Defurocum. Deterpenated Torchiato C-P C-P BEO L-E L-E L-E Castagnaro Fantastico Femminello

LRI
expa LRI litb (A) (B) (C) (D) (E) (G) (F) (Re) (Ba) (La) (Ca) (Fa) (Fe)
Geranial 1270 1268 0.28 0.32 0.66 0.18 0.26 0.33 0.03 0.47 0.49 0.66 0.48 0.66 0.47
Bornyl acetate 1285 1285 tr 0.01 0.04 0.02 0.02 tr 1.41 0.02 0.02 tr 0.02 0.01 0.01
Undecanal 1307 1309 – 0.01 0.01 – – tr – – – – tr – –
Nonyl acetate 1312 1308d tr 0.02 0.04 – tr 0.02 – tr tr tr tr tr tr
Methyl geranoate 1320 1320 tr 0.01 tr – tr – tr tr tr 0.02 0.03 0.02 0.02
Linalyl propanonate 1334 1333d 0.04 – – 0.05 0.05 0.04 – 0.04 0.04 0.05 0.05 0.05 0.06
d-Elemene 1337 1335 – 0.05 0.08 tr – – 0.05 – – tr – tr tr
a-Terpinyl acetate 1349 1349 0.15 0.13 0.35 0.20 0.19 0.13 11.60 0.14 0.11 0.08 0.12 0.08 0.05
Citronellyl acetate 1354 1353 0.03 0.02 0.06 0.03 0.02 0.03 0.08 0.03 0.02 tr 0.02 tr tr
Neryl acetate 1359 1361 0.42 0.34 0.93 0.41 0.42 0.32 0.03 0.58 0.29 0.37 0.28 0.37 0.33
Geranyl acetate 1378 1381 0.40 0.27 0.83 0.35 0.26 0.30 0.07 0.38 0.24 0.30 0.39 0.30 0.14
Dodecanal + decyl 1410 1412 0.05 0.04 0.09 0.05 0.06 0.05 0.01 0.05 0.05 0.05 0.06 0.05 0.04
acetetate
b-Caryophyllene 1421 1424 0.39 0.32 0.89 0.40 0.28 0.35 0.03 0.46 0.32 0.28 0.37 0.28 0.27
trans-a-Bergamoten- 1432 1434 0.32 0.28 0.73 0.39 0.27 0.29 tr 0.40 0.33 0.31 0.37 0.31 0.23
e
a-Humulene 1448 1454 0.03 0.02 0.06 0.03 0.02 0.03 – 0.04 0.02 0.02 0.03 0.02 0.02
cis-b-Farnesenec + 1452 1452d 0.07 0.07 0.14 0.08 0.07 0.07 tr 0.07 0.08 0.09 0.09 0.09 0.08
b-santalene
M. Russo et al.

Germacrene D 1478 1480 tr tr – – – – – – tr – tr – –

Sesquiterpene 1483 – 0.05 0.08 0.10 0.07 0.05 0.04 0.02 0.07 0.05 0.04 0.06 0.05 0.05
Sesquiterpene 1486 – 0.02 0.02 0.04 0.02 tr 0.02 tr 0.02 0.02 0.02 0.02 0.02 tr
(E,E)-a-Farnesene + 1503 1501 0.03 0.03 0.07 0.05 0.03 0.03 – 0.05 0.04 0.04 0.04 0.04 0.03
sesquiterpene
b-Bisabolene 1510 1508 0.46 0.41 0.83 0.58 0.40 0.43 tr 0.56 0.49 0.44 – 0.44 0.34
Tridecanal 1519 1518e – – – tr tr tr tr 0.17 tr – – – –
b-Sesquiphelland- 1524 1523 0.02 0.02 tr 0.03 0.02 tr – tr – 0.02 0.02 0.02 tr
rene
Germacrene B 1536 1567 tr tr 0.02 0.02 tr 0.02 Tr 0.02 tr tr 0.02 0.01 tr
(E)-Nerolidol 1559 1556d tr tr 0.02 tr tr 0.02 – 0.00 0.07 – – – tr
Tetradecanal 1615 1614 0.02 – – – – tr – tr tr tr tr tr tr
Norbornanold 1640 1638d 0.02 tr – 0.02 0.02 tr – 0.02 0.02 0.02 0.04 0.02 tr
Canferenol 1649 1654d 0.02 0.16 tr 0.03 0.02 0.02 0.02 0.03 0.03 0.02 0.02 0.02 tr
a-Bisabolol 1689 1685 0.02 0.02 – 0.03 0.02 0.02 – 0.03 0.02 0.02 0.02 0.02 0.02
Nootkatone 1783 1780d – 0.05 – 0.02 tr – – tr – – tr tr –

Notes: LRI, linear retention indices; BEO C-P, cold-pressed bergamot essential oil; BEO L-E, laboratory-extracted bergamot essential oil; tr, traces (<0.01); aIUPAC 1997; bBonaccorsi et al., 2011 (50);
d
Verzera et al., 2002 (51); eMadruga, 1993 (52); cCorrect isomer not characterized; d 2,3-dimethyl-3-(4 methyl-3-pentenyl)-2 norbornanol.
 The Journal of Essential Oil Research 143

recognition system and to configure the recognition sys-

tem in order to classify different aromas (19, 46, 47).
In each e-nose cycle, the digital ‘fingerprint’
(Figure 2) ΔR/R0 × 100, where ΔR is the difference
R R0 (R is the instant resistance and R0 is the resis-
tance at the beginning of the acquisition, is recorded
as a function of time for each sensor. In order to
reduce the size of data set produced by the twelve
sensors’ responses of time curves and make it consis-
tent with the pattern recognition system, a more com-
pact form has been necessary. For this reason, the
number of sensors used must be reduced, selecting
those which, with reference to the specificity of the
sample, show the most discriminating response, and
extract descriptive features from these curves. The
most common approach is to represent a time series
with one or two values for each sensor (46, 47). In
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this work, for all classifications, the module of the

first two Fourier components of functions ΔR/R0 ver-
sus time for the five sensors that provide the best
result has been employed, with exception of data set
cluster 5 classification, for which we use only the
module of the first Fourier components. The pattern
recognition technique used was the canonical DFA
(Duggan/French approach), coupled with a ‘cross-vali-
dation’ process, in which the minimum accepted rec-
Figure 4. Dendrogram obtained by cluster analysis using the
quantitative essential oil data matrix in Table 1.
ognition percentage was set at 85%. The
classification related to data over-fitting was avoided
by keeping the ratio samples to variables above three
in all data treatments, as suggested in the literature
(19, 46).

Figure 3. Gas chromatogram of the cold-pressed bergamot oil (sample A) showing the major components: 1, a-thujene; 2, a-
pinene; 3, sabinene; 4, b-pinene; 5, b-myrcene; 6, octanal + a-phellandrene; 7, a-terpinene; 8, p-cimene; 9, limonene; 10, (Z)-b-
ocimene; 11, (E)-b-ocimene; 12, γ-terpinene; 13, trans-sabinene hydrate; 14, terpinolene; 15, linalool; 16, terpinen-4-ol; 17, a-ter-
pineol; 18, decanal; 19, octyl acetate; 20, nerol; 21, neral; 22, cis-sabinene hydrate acetate; 23, linalyl acetate; 24, geranial; 25,
linalyl propionate; 26, a-erpinyl acetate; 27, neryl acetate; 28, geranyl acetate; 29, b-caryophyllene; 30, trans-a-bergamotene; 31,
cis-b-farnesene + b-santalene; 32, germacrene D; 33, b-bisabolene.
 144 M. Russo et al.

GC analysis GC/MS analysis

The quantitative GC analysis were performed on a Shi-
madzu GC 17A (Shimadzu, Kyoto, Japan) operating
with a split/splitless injector. Column: SE52 (5% diphe-
nyl, 95% polydimethylsiloxane) 30 m × 0.25 mm inner
diameter (i.d.) × 0.25 μm film thickness (Mega, Leg-
nano, Italy). Temperature program: from 60°C (8 min-
utes) to 100°C at 3°C/minute, from 100°C to 130°C at
2.5°C/minute, from 130°C to 180°C at 3°C/minute (8
minutes). Injector temperature: 250°C. Injection vol-
ume:1 μL. Inlet pressure: 102.3 kPa. Carrier gas: He,
linear velocity (u): 30.2 cm/second. Injection mode:
split (60:1). Flame ionization detector (FID) (280°C):
H2 flow: 40 mL/minute; airflow: 400 mL/minute; make
up flow (He): 30 mL/minute. Sampling rate: 40 milli-
seconds. Data handling was carried out by means of
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GC/MS analyses were performed on a Shimadzu GC-
17A coupled to a Shimadzu QP5050A quadrupole
mass-selective spectrometer (Shimadzu, Kyoto, Japan).
Column: SE52-MS (5% diphenyl + 95% poly-
dimethylsiloxane) 30 m × 0.25 mm I.D. × 0.25 μm
film thickness (Mega). Injection volume: 1 μL. Inlet
pressure: 56.7 kPa. Carrier gas: He, linear velocity (u):
36.5 cm/second. Injection mode: split (60:1). MS inter-
face temp.: 280°C; MS mode: EI; mass range 40–350
u; scan speed: 1000 u/second; interval: 0.50 seconds.
Data handling was made through GC/MS solution 1.10
(Shimadzu).
The essential oil components were identified by
comparison of retention indices, mass published spectra
data (48) and the National Institute of Standards and

GC Solution 2.10 (Shimadzu). Three independent anal-
yses were carried out, and data on the average value
and coefficient of variation (CV%) were calculated.
The relative percentages of the components are shown
in Table 1.
Technology (NIST 21 and NIST 107) mass spectral
library.
The linear retention indices (LRIs) were calculated
for all the volatile constituents using the retention data
of the homologous series of saturated aliphatic hydro-

Figure 5. E-nose discrimination of three groups: (D) ‘Torchiato’ BEO, (G) industrial cold-pressed bergamot oil from Ionian PDO
area, (F) synthetic bergamot essential oil (SEO). Data set (sample): 112 measurements. Variables: five sensors, two features, three
classes to be discriminated. Ratio sample/variables = 3.73. Cross-validation: test 95.5%, DFA recognition 97.3%.
 The Journal of Essential Oil Research 145
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Figure 6. E-nose discrimination of: (A) Industrial cold-pressed bergamot essential oil; (B) bergamot essential oil (BEO)
bergapten-free; (C) BEO deterpenated. All samples has been obtained from the same lot of BEO. Data set (sample): 136
measurements. Variables: five sensors, two features, three classes to be discriminated. Ratio sample/variables = 4.53. Cross-
validation: test 89.7%, DFA recognition 94.1%.

Results and discussion

carbons within C8 to C24 performed in the same col-
umn and at the same conditions used in the GC analy-
sis for the essential oils, in according to the IUPAC
Gold Book (49). Table 1 shows the values of LRI
experimentally obtained compared with reference LRI
values from the literature (50–52).
Statistical analysis on BEOs
The percentage composition of the essential oil sam-
ples was used to determine the relationship between
the different samples by cluster analysis using the
Kyplot vers. 2.0 software. The quantitative data set
(thirteen samples and sixty-four compounds) were ana-
lyzed using standard clustering algorithms. The cluster
analysis was performed to identify relatively homoge-
neous groups based on the percentage composition of
essential oils and Euclidean distance was selected as a
measure of similarity.
Essential oil composition
All BEO samples were analyzed by GC and GC/MS to
characterize the volatile fraction to explain the differ-
ences between samples as highlighted by the e-nose.
The composition of the BEOs in single components
expressed as relative percent of peak area (average val-
ues determined out of triplicates) determined by GC-
FID is reported in Table 1. In the same table are
reported the LRI experimentally determined compared
with those reported in literature. The CV% determined
for the peak area of each component, out of triplicates,
was never above 10.32%; thus the repeatability of the
method was good.
The GC chromatogram of a BEO C-P (sample A)
is also reported in Figure 3.
A total of sixty-four compounds were identified in
the BEOs essential oils. The essential oils were found
to contain mainly, in decreasing amount order: limo-
 146 M. Russo et al.
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Figure 7. E-nose discrimination of four groups: (A) Industrial cold-pressed bergamot essential oil (BEO C-P); (E) industrial BEO
C-P from Tyrrhenian PDO area; (F) synthetic bergamot essential oil; (G) industrial BEO C-P from Ionian PDO area. Data set
(samples): 162 measurements. Variables: five sensors, two features, four classes to be discriminated. Ratio sample/variables =
4.05. Cross-validation: test 90.1%. DFA recognition 94.4%.

nene, linalyl acetate, linalool, γ-terpinene, b-pinene, a-
pinene, b-myrcene and sabinene.
The cluster analysis is widely used to identify rela-
tively homogeneous groups based on the percentage
composition of essential oils (53, 54). On the basis of
similarity, BEO samples are united into clusters, as
graphically represented in Figure 4. It can be seen that
the whole spectral set splits into main clusters: the first
two are represented by BEO deterpenated and SEO,
and another groups all the BEOs C-P but separated into
sub-clusters which grouped, respectively, BEOs of cv.
Castagnaro and Femminello with Fantastico and BEO–
La obtained from the fruit of cv. Fantastico cultivated
out of PDO area, which was similar to BEO-Fa
obtained from fruits of the same cultivar collected in
PDO area with obvious differences in composition to
justify belonging to different sub-clusters.
The other sub-clusters group, in a single class to
demonstrate the existing differences, shows the samples
of BEO bergapten-free and BEOs from a different
growing area, distinct from the industrial samples
obtained from fruit masses consisting of a different cul-
tivar with a prevalence of Fantastico.
E-nose
The whole data set cluster for e-nose analysis was
structured as follows:
• Data set cluster 1 – 112 measures were used to
discriminate three different essential oils, BEO
C-P ‘Torchiato (D), industrial BEO (G), SEOs
(F) widely available on the market (Figure 5);
• Data set cluster 2 – 136 measures were used to dis-
criminate: BEO C-P (A), BEO bergapten-free (B)
and BEO deterpenated (C) the last two were
obtained from the same initial matrix (A) (Figure 6);
• Data set cluster 3 – 162 measures have been used
to discriminate between different industrial BEO
C-P samples produced from fruit cultivated in
different PDO cultivation areas and SEO (F)
(Figure 7);
 The Journal of Essential Oil Research 147
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Figure 8. E-nose discrimination of three groups: Fa, cold-pressed bergamot essential oil (BEO C-P) cv. Fantastico; Ca, BEO C-P
Castagnaro; Fe, BEO C-P cv. Femminello cv. Data set (sample): 131 measurements. Variables: five sensors, two features, three
classes to be discriminated. Ratio sample/variables = 4.36. Cross-validation: test 100%, DFA recognition 100%.

• Data set cluster 4 – 131 measures were used to
discriminate BEO C-P samples obtained from
different cultivars: Castagnaro (Ca), Femminello
(Fe) and Fantastico (Fa) (Figure 8);
• Data set cluster 5 – 55 measures were used to dis-
criminate between BEO L-E) from fruits of the
same cv. Fantastico collected in three different
areas: BEO L-E from the Tyrrhenian PDO area
(Re); BEO L-E from the Ionian PDO area (Ba);
BEO L-E outside the PDO area (La) (Figure 9).
The first data set cluster discrimination (Figure 5)
was carried out on samples very different from each
other, to test the macro-discriminating capacity of the
e-nose. As expected, the e-nose was able to discrimi-
nate with excellent performance between the three
types of essential oil as demonstrated by the classifica-
tion with a 95.5% cross-validated recognition rate and
a DFA recognition of 97.3%. GC analysis clearly dem-
onstrates the qualitative differences between the sam-
ples and confirms the e-nose results.
Figure 4 shows the DFA obtained from the analysis
of three kinds of industrial essential oils obtained from
the same lot of cold-pressed essential oil: BEO C-P
(A), BEO bergapten-free (B), and BEO deterpenated
(C); the last two samples were obtained from BEO C-P
matrix (A).
The e-nose system discriminates significantly the
BEO deterpenated samples from the other two samples,
with an 89.7% cross-validated recognition rate. The
discrimination rate between BEO C-P and BEO bergap-
ten-free is less marked, because of the similarity of the
composition of the volatile fraction of the two samples.
The system discriminates sharply deterpenated BEO
samples from all the other oils and can differentiate
BEO C-P from bergapten-free oils. In this last case,
however, discrimination is less clear, although statisti-
cally significant, because of the degree of similarity of
their chemical composition, as shown in Table 1. BEO
bergapten-free has been obtained by separation of ber-
gapten from BEO C-P with the addition of an alkali
solution. The process, if properly conducted, allows the
 148 M. Russo et al.
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Figure 9. E-nose discrimination of three groups: Re, laboratory-extracted bergamot essential oil (BEO L-E) from Reggio Cal
(Tyrrhenian PDO area); Ba, BEO L-E. from Ionian PDO area; La, BEO L-E from Lamezia Terme (outside PDO bergamot
cultivation area). Data set (sample): 55 measurements. Variables: five sensors, one feature, three classes to be discriminated. Ratio
sample/variables = 3.66. Cross-validation: test 100%, DFA recognition 100%.

separation of a high percentage of bergapten and citrop-
ten without damaging BEO aroma quality because it
eliminates components of the so-called non-volatile
fraction, which has no effect on the overall bouquet of
the essential oil. Chromatographic analysis data con-
firmed that the defurocoumarinization process does not
cause important changes of the volatile aromatic frac-
tion but the e-nose was still able to discriminate
between very similar samples.
Figure 5 shows the comparison between three
cold-pressed BEOs from different PDO areas and a
synthetic oil. Numerous studies have shown that in
the PDO area of bergamot cultivation (Figure 1),
there are homogeneous sub-areas, which give rise to
qualitatively different essential oils because of slight
differences in concentration of some chemical compo-
nents (55, 56). Chromatographic data show that the
chemical compositions of cold-pressed oils are simi-
lar, with particular regard to samples E and G. The
high DFA cross-validated recognition rate (90.1%)
confirms that the e-nose is capabile of discriminating
between very similar samples, also confirming the
ability to discriminate between all the samples of nat-
ural essential oil and synthetic with a DFA recogni-
tion of 94.4%.
Given the economic importance of bergamot and its
value closely linked to the quality of the essential oil,
we considered it of interest to verify the ability of the
e-nose to recognize essential oils obtained from berga-
mot fruit, harvested in the same geographic area in the
same cultivation conditions, but obtained from fruits of
three cultivars still widely cultivated in Calabria: ‘Cas-
tagnaro’, ‘Femminello’ and ‘Fantastico’. The essential
oils have been produced from a local manufacture by a
cold-pressing extraction process. In Figure 6 are shown
the results. The e-nose analysis showed a sharp dis-
crimination with a good cross-validated recognition rate
(100%). Again GC data confirmed e-nose results
(Table 1).
Finally, it is well established that the essential
oils produced in Calabria and, in particular, in the
province of Reggio Calabria, are considered, on the
 The Journal of Essential Oil Research
international market, the best in the world, so the
European Community, in 2001, recognized the PDO
for this product.
In order to further illustrate the potential use of
e-nose technology as rapid, inexpensive and effective
in BEOs quality control, we considered it interesting
to conduct a further experiment using three different
essential oils obtained from fruits, of the same culti-
var and from plants grafted on the same rootstock
(sour orange), but collected in different areas: two
located in the PDO area and one outside PDO area,
specifically in the north of the province of Reggio
Calabria, on the Tyrrhenian coast, called Lamezia
Terme (Figure 1).
The data set cluster is shown in Figure 7. The
graph was created with the only one discriminant func-
tion, as this was sufficient to obtain a good discrimina-
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tion between the three categories. The cross-validation
test clearly demonstrates the applicability of the model.
The e-nose significantly discriminates the two
macro-classes, outside the PDO area and within,
including those from fruits collected in two sub-PDO
areas Ionian (Ba) and Tyrrhenian (Re).
In conclusion, this feasibility study has confirmed
that the e-nose demonstrates the ability to supply con-
crete support to BEOs quality control. Moreover, fur-
ther validation studies are currently under way with the
aim of enriching and updating the databases of refer-
ence for the BEOs.
We can envision that the method here described
may represent the basis for future approaches to be
used by operators in the essential oil production indus-
tries, as well as in the perfume industry as a technical
completion of those classically used.
In order to broaden the range of e-nose applica-
tions, tests on sensors chiral comportment are in pro-
gress. These would allow application of the same
approach used in the present study to reveal the adulte-
ration of BEOs commonly spiked with bitter orange or
other Citrus oils cheaper than natural BEO and espe-
cially with SEO.
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