Food Chemistry 136 (2013) 668–674

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Electronic nose and GC–MS analysis of volatile compounds in Tuber magnatum

Pico: Evaluation of different storage conditions
G. Pennazza a, C. Fanali b,⇑, M. Santonico c,a, L. Dugo b, L. Cucchiarini d, M. Dachà b, A. D’Amico a, R. Costa e,
P. Dugo e,b, L. Mondello e,b
a
Center for Integrated Research – CIR, Unit of Electronics for Sensor Systems, University Campus Bio-Medico, via Álvaro del Portillo 21, 00128 Rome, Italy
b
Center for Integrated Research – CIR, University ‘‘Campus Bio-Medico di Roma’’, via Álvaro del Portillo 21, 00128 Rome, Italy
c
Dipartimento di Ingegneria Elettronica, Università di Roma Tor Vergata, Via del Politecnico 1, 00133 Rome, Italy
d
Dipartimento di Scienze Biomolecolari, Università degli Studi di Urbino ‘‘Carlo Bo’’, Urbino, Italy
e
Dipartimento Farmaco-chimico, University of Messina, viale Annunziata, 98168 Messina, Italy

a r t i c l e i n f o a b s t r a c t

Article history: The characteristic aromatic composition of white truffles (Tuber magnatum Pico) determines its culinary
Received 30 April 2012 and commercial value. However modifications of truffle organoleptic proprieties occur during preserva-
Received in revised form 28 June 2012 tion. A study of headspace of white truffles by using Electronic nose (E-nose), gas chromatography–mass
Accepted 31 August 2012
spectrometry (GC–MS) and sensory analyses was performed. Truffles were stored at different conditions
Available online 13 September 2012
for 7 days: +4 and +8 °C wrapped in blotting paper or covered by rice or none of the above. Headspace E-
nose measurements and sensory analyses were performed each day. Statistical multivariate analysis of
Keywords:
the data showed the capability of E-nose to predict sensorial analysis scores and to monitor aroma profile
Truffles
Aroma
changes during storage. Truffle’s volatile molecules were also extracted by headspace solid phase mic-
Electronic nose roextraction technique and separated and identified by GC–MS. Partial Components Analysis of data
GC–MS was performed. E-nose and GC–MS results were in agreement and showed that truffle storage in paper
Shelf-life at +8 °C seemed to be the best storage condition.
Tuber magnatum Pico Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction the analysis of volatile compounds (Aprea et al., 2007; Bellesia,

Truffles are the fruiting bodies of hypogeous ascomycetous fun-
gi belonging to the genus Tuber, which form ectomycorrhizae on
the roots of angiosperms and gymnosperms. The truffles are appre-
ciated for two important features: for environmental and forestry
applications owing to the advantages that mycorrhizae provide
for host plant and for the economic value of some edible species.
Tuber magnatum Pico (the ‘‘white truffle’’), is one of the most
important species due to its culinary and commercial value. Italy
is one of the main site for T. magnatum production, where it repre-
sents a typical gastronomic specialty with a high economical value
in the food market.
White truffle aroma is very unique and characteristic, and its
complex composition has been the object of several studies over
the last 20 years, employing different technologies available for
Abbreviations: HS-SPME, headspace solid-phase microextraction; GC–MS, gas
chromatography–mass spectrometry; GC–FID, gas chromatography–flame ionisa-
tion detector; E-nose, electronic nose; PCA, Principal Component Analysis; PLS-DA,
Partial Least Square-Discriminant Analysis; PCs, Principal Components.
⇑ Corresponding author. Tel.: +39 06 225419123; fax: +39 06 225411972.
E-mail address: c.fanali@unicampus.it (C. Fanali).
Pinetti, Bianchi, & Tirillini, 1996; Gioacchini et al., 2008; Mauriello,
Marino, D’Auria, Cerone, & Rana, 2004; Pelusio et al., 1995).
The most used analytical techniques to concentrate the volatile
compounds of food aroma have been obviously those based on
headspace analysis (Sides, Robards, & Helliwell, 2000). For truffle
aroma, techniques such as dynamic headspace coupled to gas chro-
matography–mass spectrometry (GC–MS) (Talou, Delmas, & Gaset,
1989) and purge and trap GC–MS (Bellesia et al., 1996) have been
used to detect black perigord truffle and Italian white truffles aro-
mas, respectively. Headspace solid-phase microextraction (HS-
SPME) combined with GC–MS has been used to fully characterise
aroma of truffles of different species (Díaz, Ibáñez, Señoráns, &
Reglero, 2003; Díaz, Señoráns, Reglero, & Ibañez, 2002; Gioacchini
et al., 2005; Mauriello, Marino, D’Auria, Cerone, & Rana, 2004;
Zeppa et al., 2004). Some research has been devoted to the study
of the aroma quality of different truffle species and their changes
as a function of both species and preservation methods (Bellesia,
Pinetti, Bianchi, & Tirillini, 1998; Bellesia, Pinetti, Tirillini, &
Bianchi, 2001; Díaz et al., 2003; Falasconi et al., 2005; Gioacchini
et al., 2008).
Storage condition from the time of harvesting can deeply affect
the organoleptic characteristics of white truffle and is therefore

0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2012.08.086
 G. Pennazza et al. / Food Chemistry 136 (2013) 668–674
crucial for the shelf-life duration and, consequently, the determi-
nation of its value.
In this context, electronic nose has been widely used in the last
two decades in several studies oriented to food characterization
(Peris & Escuder-Gilabert, 2009). In fact, aroma is a key-informa-
tion for food evaluation in terms of quality (Di Natale et al.,
2006a), freshness (Alimelli et al., 2007) and safety (Santonico
et al., 2008).
A recent study explored the relative changes of the white truf-
fle’s aroma (T. magnatum Pico) in the days following the harvesting,
in order to determine the maximum preservation time for the
white truffles (Alba’s truffle), using two different analysis tech-
niques: SPME–GC–MS and the Pico2-electronic nose (Bellesia
et al., 1998). They demonstrated that Electronic Nose based on
metaloxide sensors is very sensitive for the detection of the truf-
fle’s relevant aroma molecules and that the results obtained about
truffle’s aging with both techniques are strongly correlated.
The object of this work was to monitor the composition of white
truffle aroma at different conditions and temperatures of storage,
in order to establish which storage condition allows the best main-
tenance of the aroma. Truffle’s aroma analyses were performed by
using a Quartz Microbalance Based Electronic nose, HS-SPME–GC–
MS technique and sensory analysis. A multivariate statistical anal-
ysis of obtained data was accomplished.
2. Materials and methods
2.1. Materials and reagents
All solvents were of GC grade and were used as received. Meth-
anol (MeOH) and formic acid were purchased from Farmitalia-Car-
lo Erba (Milan, Italy). Water (H2O), isopropanol (IPA), acetonitrile
(ACN), n-hexane (Hex), were supplied by Sigma–Aldrich (Milan,
Italy). For the measurement of Linear Retention Indices (LRIs) a
mix of n-alkanes ranging from decane to triacontane was used
(Supelco, USA).
2.2. Samples
Six white truffles (T. magnatum Pico) were harvested from nat-
ural truffle grounds near Urbino (Italy). All the samples were mea-
sured 1 day after gathering and the analysis was repeated each day
for 7 days storage using Electronic Nose. After the first analysis,
truffles were kept at different temperatures and storage materials:
+4 °C and +8 °C wrapped in blotting paper (P) or covered by rice (R)
or none of the above (N), all tightly closed in small glass bottles
(volume 100 mL).
2.3. Sensory analysis
A panelist group undertook truffles sensory analysis. The sen-
sory analysis took place in 7 sessions; in each session, six samples
(one for each storage condition) were evaluated by all panelists. A
profile of different sensory descriptors of truffles grouped for
appearance, odour and texture was assessed. A non-structured 3-
point scoring intensity scale was used by the panelists, where 0
means absence or very low intensity of the descriptors and 3
means very high intensity of the descriptors.
2.4. Electronic nose measurements
The functioning principle of the electronic nose used in this
study is based on an array of six Quartz Micro Balance (QMB) sen-
sors, covered by a set of metalloporphyrins, functionalized with six
different metals (Santonico et al., 2012). This is the electronic nose
669
designed and fabricated by the Sensors and Microsystems group of
the University of Rome ‘‘Tor Vergata’’. QMB sensors here used are
AT-cut plates oscillating in the thickness shear mode at the reso-
nance frequency of 20 MHz, driving electronic oscillators whose
frequency is barely similar to the mechanical resonance frequency.
QMB sensors are functionalized by solid state layers of metallopor-
phyrins. The electronic nose is endowed with a pump carrying gas-
eous samples in contact with the sensors. The sensors are
constantly exposed to a reference air and the exposure to the sam-
ple results in a negative shift of the resonant frequency. The differ-
ences between the frequency measured in the steady conditions
before and during the exposure are considered as the sensor main
feature and are underutilised in all the following data treatment.
Consequently, for each measured sample the electronic nose pro-
vides a pattern of six values.
A sampling protocol of truffles headspace was designed. Truffles
preserved at two different temperatures (+4 °C and +8 °C) were
placed in 100 mL flasks (used only for the measurement), sealed
and kept at 20 °C in order to allow a stable headspace composition.
The headspace was extracted by a constant flow of dry air and car-
ried into the electronic nose sensor cell (Fig. 1).
2.5. Headspace Solid-Phase Microextraction (HS-SPME) of aroma
compounds
Immediately before analysis truffles were cut with a sharp knife
and approximately 1 g was placed in a 20 mL crimped vial for com-
pounds extraction. SPME extraction was carried out in the head-
space mode by means of an AOC-5000 autosampler (Shimadzu)
hyphenated with the GC–MS system. A fused silica fibre coated
with a 50/30 lm layer of divinylbenzene/carboxen/poly-
dimethylsiloxane (Supelco, Milan, Italy), 1 cm long, was chosen
to extract the volatile components from the truffles. The fibre
was conditioned following the manufacturer’s instructions previ-
ous to its use. Samples were conditioned for 5 min at 50 °C, under
agitation (clockwise, anticlockwise rotation at 500 rpm), and then
underwent the extraction step for 20 min at 50 °C, still under agi-
tation. Analytes were then desorbed for 1 min at 260 °C in the GC
injector in splitless mode.
2.6. GC–FID analysis
A Shimadzu GC-2010 equipped with a programmed split/split-
less injector and a flame ionisation detector (FID) was used to per-
form GC analyses. All the analyses were carried out on a
30 m  0.25 mm i.d.  0.25 lm df Supelcowax-10 column (Supe-
lco, Milan, Italy). The split/splitless injector was held at a temper-
ature of 260 °C, and, after sampling time (1 min) in splitless mode,
a split ratio of 1:50 was applied. Helium at a constant linear veloc-
ity of 29.0 cm/s and a pressure of 94.3 kPa was used as carrier gas.
Temperature program was as follows: 40 °C at 3 °C/min to 250 °C,
at 10 °C/min to 270 °C, held 10 min. The FID temperature was set at
280 °C (sampling rate 40 ms) and gas flows were 40 mL/min for
hydrogen and 400 mL/min for air, respectively. Data were collected
by using GCsolution software (Shimadzu).
2.7. GC–MS analysis
GC–MS analysis was performed on a Shimadzu QP2010 instru-
ment, equipped with the same column used for GC analysis. Data
handling was supported by GCMSsolution (Shimadzu). Gas chro-
matographic parameters were as mentioned previously for GC–
FID analyses, except for: carrier gas (He) linear velocity was
30.0 cm/s, split ratio was 1:20, pressure 26.5 kPa. Other operating
conditions were as follows: ion source temperature, 200 °C; the
interface temperature, 250 °C. The acquisition took place in scan
670 G. Pennazza et al. / Food Chemistry 136 (2013) 668–674

Fig. 1. Experimental set-up used by the electronic nose: upper panel, cleaning phase, lower panel, measure phase. In both the panels the active phase is indicated by the
dashed arrows.

Fig. 2. The six-dimensional patterns registered for the electronic nose sensors. Along the x-axis the name of the six different sensors is reported and for each of these sensors
the three bars are referred to three truffles. Along the y-axis the frequency shift [Hz] registered is reported.

mode at a scan speed of 1666 within a mass range of 40–350 amu.
Compounds were identified by comparison of the spectra with
those in mass spectrometry libraries available in the market
(Adams, 2007; FFNSC 2, 2012; König, Joulain, & Hochmuth, 2001)
and with home-made libraries collecting spectra from food and fla-
vor matrices and standards supplied by Sigma–Aldrich (USA),
Firmenich (Switzerland), Givaudan (Switzerland), IFF (Netherland)
and Bedoukian (USA).
2.8. Statistical data analysis
E-nose and GC–MS data analysis was performed by a multivar-
iate approach. Gas sensor array is a mere mathematical
construction where the sensor outputs are arranged as compo-
nents of a vector resulting in a 6-dimensional patterns array. Prin-
cipal Component Analysis (PCA) was applied to both E-nose and
GC–MS data while and Partial Least Square-Discriminant Analysis
(PLS-DA) only to E-nose results.
3. Results and discussion
3.1. Electronic nose headspace analysis
E-nose analysis was performed on truffle samples at different
storage conditions to monitor shifts in aroma composition. E-nose
data analysis aimed to define the ability of the instrument to: (i)
 G. Pennazza et al. / Food Chemistry 136 (2013) 668–674
find a characteristic truffle’s aroma fingerprint; (ii) characterise the
quality of the samples; (iii) monitor the shelf-life; (iv) correlate re-
sults with the abundance of key-volatiles .
Each truffle headspace has given a characteristic fingerprint
identified by six numeric values, calculated as the frequency shifts
of each sensor respect to a zero-point referred to the carrier gas.
These fingerprints are reported in Fig. 2, where it can be observed
a common profile of the sensors response for three different truf-
fles. This suggests that the e-nose is able to identify a volatile sig-
nature which is distinctive of the truffle aroma. The differences in
magnitude evidenced for the truffles are related to their weight.
Thus data were normalised respect to truffles weight by linear nor-
malisation: each sensor response was divided by the sum of all the
sensor responses of the same sample (Santonico et al., 2012).
A PLS-DA has been performed on the E-nose normalised data
using the panelist evaluation as classification score of the analysed
samples. This model has been cross-validated via the Leave-One-
Out criterion. The results showed a good ability of the e-nose to
predict panelists scores with a RMSECV (Root Mean Square Error
Cross Validation) of 0.24, relative error of 8% respect to the total
panelist score range of variability (0–3).
The shelf-life of each truffle has been monitored by measuring
the daily E-nose shifts of each sample, compared to the measure
on the same sample taken at day 1. Fig. 3 shows the percentage
variation of the E-nose responses between the first and seventh
day.
The lowest E-nose shift differences have been registered for the
truffle samples stored in paper at +8 °C. These results suggest the
latter condition to be the one allowing the smallest aroma varia-
tion during storage. These results were confirmed by GC–MS data
as explained in the next paragraph.
3.2. GC–MS headspace analysis
To identify truffle headspace composition, analysis by HS-
SPME/GC–MS was performed on the seventh day of storage. By
using the described procedure, it was possible to identify 84 com-
pounds in the different truffle samples that are listed in Table 1.
Peak identification was obtained by matching the unknown spec-
tra with those contained in mass spectrometry libraries available
in the market, with MS library built in our laboratory using pure
standards and by comparison of experimental LRIs with those re-
trieved from literature. It must be emphasised that the GCMSsolu-
tion software greatly boosted the identification power through the
interactive use of LRIs. This software, along with the use of
Fig. 3. Variation (%) of the truffle volatile fingerprint registered by three out of six e-nose sensors (1, 3 and 8) between day 1st an day 7th of preservation in six different
conditions, paper, P, rice, R and nothing, N at 4 and 8 °C.
671
dedicated databases, allows discarding from the spectral matching
results those compounds having an LRI far from that experimen-
tally measured. However, as can be seen from Table 1, values re-
trieved from literature for this type of column stationary phase
(polyethyleneglycol) are shifted of around 20 units compared to
the experimental values. It is well known that LRIs acquired on po-
lar stationary phases are characterised by a much lower level of
both repeatability and availability in literature (D’Acampora Zell-
ner et al., 2008). The relative percentage was expressed as a mass
fraction of the total peaks area. Only semi-quantitative analysis
could be carried out, for both low availability of sample and scope
of the work which basically consisted of comparing data obtained
from samples stored in different conditions. The analytical method
was validated in terms of repeatability and precision. For repeat-
ability, three replicates of samples were extracted, analysed and
the RSD% values calculated for retention times (RSD% < 0.5%) and
peak area counts of compounds higher than 0.01% (RSD% = 1.4–
3.5%). To evaluate the precision, inter-day variations on the same
variables were determined, considering single runs carried out in
four consecutive days. In this case, RSD% was lower than 0.8% for
retention times, whereas it ranged from 4.5% to 18.0% for peak
areas.
Most of the compounds detected and tentatively identified have
been previously reported in other papers on the aroma of Tuber
species (Aprea et al., 2007; Díaz, Ibáñez, Reglero, & Señoráns,
2009; Díaz et al., 2002, 2003; Falasconi et al., 2005; Pelusio et al.,
1995).
The most represented peaks recorded on the gas chromato-
grams (relative abundance >1% in almost all the samples)
correspond to: bis-(methylthio)-methane, (methylthio)-methane,
3-methylbutanal, hexanal, pentanal, (2E)-heptanal, (2E)-octenal,
2-methyl-2-butenal, 1-cyclopentene-1-carboxaldehyde and 1-
octen-3-ol. In Fig. 4 is reported, as an example, a total ion current
(TIC) gas chromatogram of an analysed sample.
It is well known that bis-(methylthio)-methane is the major and
characteristic contributor to the aroma of white truffle T. magna-
tum (Aprea et al., 2007; Fiecchi, Kienle, Scala, & Cabella, 1967;
Splivallo, Ottonello, Mello, & Karlovsky, 2011). Its relative percent-
age is high in fresh truffle and decreases during storage (Aprea
et al., 2007). Differences were observed for its relative quantity
in analysed samples. The greatest percentage (36%) was measured
for truffle P stored at +8 °C, very low percentages (<1%) were mea-
sured for truffle R at +8 °C and truffle N at +4 °C. The result ob-
tained for truffle P at +8 °C correlates well with the small e-nose
response shift registered for the same sample.
672 G. Pennazza et al. / Food Chemistry 136 (2013) 668–674

Table 1
Relative percentages (percent normalised areas) and linear retention indices of volatiles determined in T. magnatum Pico samples extracted by HS-SPME and identified by GC–MS.

Peak Compound name LRI LRI lit Truffle N at Truffle R at Truffle P at Truffle N at Truffle R at Truffle P at
# exp +8 °C (%) +8 °C (%) +8 °C (%) +4 °C (%) +4 °C (%) +4 °C (%)
1 Pentane – 500 0.57 0.63 0.47 0.65 0.38 0.62
2 Hexane – 600 0.84 0.52 0.89 0.25 0.35 0.30
3 Methanethiol – 780b 0.05 n.d. 0.16 n.d. 0.06 0.05
4 Ethanal – 714b 0.48 0.73 0.31 2.56 0.77 0.68
5 (Methylthio)methane – 716b 0.39 0.36 3.17 2.31 7.29 1.62
6 Propanal – 571b 0.12 0.31 0.26 0.48 0.91 0.87
7 2-Methylpropanal – 821b n.d. n.d. n.d. n.d. 0.53 0.45
8 Acetone – 819c 0.20 1.17 2.19 n.d. 0.74 0.92
9 4-Hydroxybutanoic acid – 0.05 n.d. n.d. n.d. n.d. n.d.
10 2-Methylfuran – n.d. n.d. n.d. n.d. n.d. 0.10
11 Butanal – 832b 0.21 0.29 n.d. 0.12 0.10 n.d.
12 (2Z)-Butenal – n.d. 0.17 n.d. 0.17 0.15 0.24
13 2-Butanone – 945b n.d. 0.64 0.36 n.d. 0.19 0.19
14 2-Methylbutanal – 912b n.d. n.d. 1.55 0.94 1.65 1.38
15 3-Methylbutanal – 910b 0.25 0.30 11.80 4.01 13.51 14.71
16 Ethanol – 931c n.d. n.d. 2.13 n.d. n.d. n.d.
17 4-Methyl-3-penten-2-one – 1139b 0.36 n.d. n.d. n.d. n.d. n.d.
18 2-Propanol – – 0.07 0.19 n.d. n.d. n.d. n.d.
19 Dithiomethane – n.d. 0.57 n.d. n.d. n.d. n.d.
20 3-Buten-2-one – n.d. 0.63 n.d. n.d. n.d. n.d.
21 3-Methyl-2-butanone – 1214b 0.30 n.d. n.d. 0.71 n.d. n.d.
22 2,4-Dimethylfuran – n.d. n.d. n.d. 0.22 0.02 n.d.
23 (2E)-Butenal – 1123c 0.04 n.d. n.d. n.d. n.d. n.d.
24 Pentanal – 1040c 4.37 11.13 n.d. 2.09 0.92 0.51
25 2-Methylpentanal – 989a 0.38 n.d. 0.26 1.48 0.58 n.d.
26 2-tert-Butylthioacetic acid – – 0.14 n.d. n.d. n.d. n.d. n.d.
27 (2E)-Pentenal 1027 1047b n.d. n.d. n.d. 1.47 0.39 0.72
28 2,3-Pentanedione 1040 1054b n.d. n.d. n.d. n.d. n.d. n.d.
29 Dimethyl disulfide 1048 1071b n.d. n.d. 0.44 n.d. 0.06 0.09
30 Hexanal 1057 1056c 59.69 58.93 4.51 29.75 16.86 15.29
31 2-Methyl-2-butenal 1066 1101b n.d. n.d. 0.27 7.29 3.27 4.90
32 3-Penten-2-one 1093 1104a n.d. n.d. 0.21 n.d. n.d. n.d.
33 5-Hexen-2-one 1098 – n.d. 0.22 n.d. n.d. n.d. n.d.
34 2-Butylfuran 1098 1131a 0.32 n.d. n.d. 0.27 n.d. n.d.
35 Undecane 1100 1100b 0.04 n.d. n.d. n.d. n.d. n.d.
36 (2E)-Hexenyl butyrate 1107 – n.d. n.d. n.d. 1.64 0.14 0.32
37 Ethylbenzene 1109 1130c 0.30 n.d. n.d. n.d. n.d. n.d.
38 2-Methyl-2-pentenal 1124 – n.d. n.d. n.d. n.d. 0.17 0.55
39 2-Pentanone 1125 983b n.d. 1.06 n.d. n.d. n.d. n.d.
40 2-Heptanone 1145 1170b 0.69 n.d. n.d. 1.34 2.03 1.67
41 Heptanal 1148 1188c 0.79 0.58 2.24 n.d. n.d. n.d.
42 Limonene 1155 1197c 0.12 n.d. 0.46 n.d. n.d. n.d.
43 2-Methylbutanol 1167 1194a 0.07 n.d. 2.26 n.d. 0.63 0.53
44 3-Methylbutanol 1168 1184a n.d. n.d. n.d. n.d. 1.50 1.14
45 (2E)-Hexenal 1181 – 0.24 n.d. n.d. 0.32 0.69 1.01
46 2-Pentylfuran 1194 991a 1.19 n.d. 0.71 0.87 0.05 0.08
47 Pentanol 1213 1238c 0.43 1.35 n.d. 0.25 n.d. n.d.
48 3-Octanone 1220 1255a 0.16 n.d. 2.50 0.37 0.92 n.d.
49 bis-(Methylthio)-methane 1251 1217a 0.19 1.30 36.35 0.54 17.91 12.38
50 Octanal 1259 1289b 0.23 0.85 n.d. 0.17 0.07 n.d.
51 2-Octanone 1265 1285b n.d. n.d. n.d. 0.84 n.d. n.d.
52 1-Octen-3-one 1272 1313b 0.23 n.d. 0.03 0.37 0.36 1.65
53 (2E)-Heptenal 1297 1243b 1.62 0.55 0.47 2.80 1.32 1.10
54 6-Methyl-5-hepten-2-one 1312 1336b 0.14 n.d. 0.26 n.d. n.d. n.d.
55 Hexanol 1327 1365c 0.34 0.81 n.d. 0.84 n.d. 2.00
56 2-(1-Ethylpentyl)-1,3- 1333 1391a 0.06 n.d. n.d. n.d. 0.54 n.d.
dioxolane
57 Dimethyl trisulfide 1351 1377b n.d. n.d. 0.15 0.02 n.d. 0.03
58 2-Nonanone 1366 1388b 0.05 n.d. 0.17 0.05 0.03 0.03
59 Nonanal 1371 1387c 0.17 1.88 0.33 0.33 0.15 0.22
60 3-Octen-2-one 1384 1388b 3.05 0.76 0.23 0.74 0.64 0.43
61 1-Cyclopentene-1- 1391 n.d. n.d. 3.15 5.73 9.47 9.32
carboxaldehyde
62 (2E)-Octenal 1408 1345b 2.89 0.12 0.87 4.68 2.34 2.68
63 2-Heptylfuran 1414 1423a 0.05 n.d. n.d. n.d. n.d. n.d.
64 p-Cymenene 1415 1414b n.d. 0.05 n.d. n.d. n.d. n.d.
65 S,S- 1422 – n.d. n.d. n.d. n.d. 0.02 0.03
Dimethyldithiocarbonate
66 1-Octen-3-ol 1430 1394b 1.69 0.29 0.53 1.97 1.33 1.04
67 Heptanol 1434 1457c 0.15 n.d. n.d. n.d. n.d. n.d.
68 2-Ethylhexanol 1471 1487b n.d. n.d. 0.36 n.d. 0.09 0.12
69 Decanal 1481 1493c n.d. 0.38 0.25 0.16 n.d. 0.20
70 3-Nonen-2-one 1493 – n.d. n.d. 0.03 0.21 0.25 0.37
 G. Pennazza et al. / Food Chemistry 136 (2013) 668–674 673

Table 1 (continued)

Peak Compound name LRI LRI lit Truffle N at Truffle R at Truffle P at Truffle N at Truffle R at Truffle P at
# exp +8 °C (%) +8 °C (%) +8 °C (%) +4 °C (%) +4 °C (%) +4 °C (%)
71 Pyrrole 1498 1495c n.d. n.d. 0.04 n.d. n.d. n.d.
72 Benzaldehyde 1502 1507c 0.15 n.d. 1.32 0.36 0.76 0.36
73 (2E)-Nonenal 1518 1527b 0.10 n.d. 0.11 0.42 0.19 0.34
74 2-Undecanone 1585 1543b n.d. n.d. 0.28 n.d. n.d. n.d.
75 Sulfinylbismethane 1549 1576b 0.86 n.d. n.d. n.d. n.d. n.d.
76 3,5-Dimethyldihydro-2(3H)- 1550 – 2.04 n.d. n.d. 1.45 n.d. n.d.
furanone
77 2-Acetyl-5-methylfuran 1531 1588a 0.22 n.d. n.d. 1.09 0.09 0.49
78 (Z)-Dihydrocarvone 1591 1600c 0.10 n.d. n.d. n.d. n.d. n.d.
79 Hexadecane 1600 – n.d. n.d. n.d. 0.30 0.22 0.56
80 c-Butyrolactone 1613 1647b 0.06 n.d. n.d. n.d. n.d. n.d.
81 3-(Methylthio)- 1644 – n.d. n.d. 0.28 n.d. n.d. n.d.
acetaldehyde
82 (2E,4E)-Nonadienal 1693 1705c n.d. n.d. n.d. 0.66 0.34 0.60
83 2,4-Dimethyl-3-pentanone 1780 – 0.28 n.d. n.d. n.d. n.d. n.d.
84 Dimethylsulfone 1895 1934c n.d. n.d. 0.62 n.d. n.d. n.d.

TOTAL 87.53 86.74 82.96 83.26 90.97 82.90

LRI = Linear Retention Indices, exp = experimental, lit = literature.

a
from FFNSC 2 GC–MS library, Shimadzu.
b
from www.flavornet.com.
c
from http://www.odour.org.

Fig. 4. TIC gas chromatogram of headspace SPME adsorption and GC–MS analysis of T. magnatum Pico sample P stored a +4 °C. Peak assignment is in Table 1.

Among the other detected compounds, hexanal was the most
abundant one in almost all the samples, ranging from 4.5% to
59.7%. This has been reported as a degradation product deriving
from lipidic oxidation occurring during storage due to the activity
of lipoxygenase, some forms of which are active under nearly
anaerobic conditions (Aprea et al., 2007). High percentages of hex-
anal in our samples could be explained by the fact that the GC–MS
analyses were performed after 7 days of storage. Truffles R and N
stored at +8 °C showed the highest relative quantity of hexanal,
58.9% and 59.7%, respectively, indicating that, at this storage con-
ditions, degradation occurs at a major extent. On the contrary truf-
fle P stored at +8 °C showed the lowest percentage of hexanal
(4.5%) suggesting once more that this sample storage condition
better preserved the aroma composition.
Regarding the other major compounds, 3-methylbutanal and 1-
octen-3-ol have also been found in most truffle species. It has been
reported that 3-metylbutanal arises from Strecker amino acid deg-
radation while 1-octen-3-ol is typical of mushrooms and is synthe-
sized from linoleic acid (Splivallo et al., 2011). The relative
percentages ranged approximately form 0.2 (sample N at +8 °C)
to 14.7% (sample P at +4 °C) and from 0.3 (sample R at +8 °C) and
2.0% (sample N at +4 °C) for 3-methylbutanal and 1-octen-3-ol,
respectively.
The linear aldehydes like hexanal, heptanal and octanal comes
from lipid oxidation (Falasconi et al., 2005). To our knowledge
1-cyclopentene-1-carboxaldehyde has never been previously
reported. Its percentage ranged from 0% to 9.5% showing lower
values for truffles stored at +8 °C respect to the samples stored at
+4 °C.
2-Methyl-2-butenal has been reported as constituent of truffle
aroma (Díaz et al., 2002, 2003, 2009) and, in a similar way of 1-
cyclopentene-1-carboxaldehyde, its relative percentage showed
lower values for truffles stored at +8 °C respect to the samples
stored at +4 °C.
To perform multivariate analysis GC–MS data have been treated
as a multidimensional input: an array whose rows are the chro-
matograms and columns are the abundances of the 84 identified
compounds.
The results of the PCA data model are illustrated in Fig. 5, where
the scores plot of the first three Principal Components (PCs) is
674 G. Pennazza et al. / Food Chemistry 136 (2013) 668–674
Fig. 5. Scores plot of the first three PCs of the PCA model built on the GC–MS data.
Each score corresponds to the measure performed for each different storage
condition. Red dashed arrow indicates the direction of temperature increasing. Blue
dashed arrow indicates degradation direction. (For interpretation of the references
to colour in this figure legend, the reader is referred to the web version of this
article.)
reported. Temperature effect is explained along the PC1, sample
degradation is explained along PC2. The point on the scores plot
corresponding to the sample P at +8 °C is in the decreasing direc-
tion for both PC1 and PC2, thus this seems to be the best storage
condition. This evidence is corroborated by the correlation of these
directions with key-compounds reported in literature and here
detected as markers of the shelf-life shift evidenced in Fig. 5.
An increased percentage of hexanal and 2-methyl-2-butenal
correlates with the increasing temperature, while a higher percent-
age of bis-(methylthio)-methane is strongly correlated with the
typical ‘‘good’’ truffles aroma and it increases in the direction of
the samples preserved in paper at +8 °C. These considerations
about key volatile compounds increasing directions have been
extrapolated by the loadings analysis.
4. Conclusions
Concluding to our best knowledge this is the first study showing
the characterization of truffle aroma profile assessed with three
different approaches: electronic nose, GC–MS and panelists analy-
ses. Electronic nose and GC–MS analysis provide qualitative and
quantitative results while panelists analysis provides sensory eval-
uation which we report being well correlated with our instrumen-
tal data. Electronic nose showed the ability to determine a specific
truffle aroma pattern. A correlation was found between truffles
aging and the modifications of this characteristic aroma pattern.
Volatile molecules specific for storage conditions were identified
by headspace GC–MS analysis of truffles. These compounds identi-
fication casts the light over the useful but sometimes unclear qual-
itative discrimination assessed by electronic nose. In our
experimental conditions it seems that truffle storage in paper at
+8 °C causes the lowest aroma modification on a seven days time
frame.
The multidimensional assessment here exploited can open the
way to the novel technology design for the monitoring of special
food products such as truffles.
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