Food Chemistry 134 (2012) 662–668

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Food Chemistry

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Volatile fingerprinting of chestnut flours from traditionalEmilia Romagna (Italy) cultivars

M. Cirlini a, C. Dall’Asta a,⇑, A. Silvanini b, D. Beghè b, A. Fabbri b, G. Galaverna a, T. Ganino b

a Department of Organic and Industrial Chemistry, University of Parma, Parco Area delle Scienze, 17/a, I-43124 Parma, Italyb Department of Evolutionary and Functional Biology, Section of Plant Biology, Parco Area delle Scienze, 11/a, I-43124 Parma, Italy

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

Article history:Received 12 September 2011Received in revised form 10 January 2012Accepted 23 February 2012Available online 3 March 2012

Keywords:Castanea sativa Mill.HS-SPMEGC–MSVolatile fraction

0308-8146/$ - see front matter � 2012 Elsevier Ltd. Adoi:10.1016/j.foodchem.2012.02.151

⇑ Corresponding author.E-mail address: chiara.dallasta@unipr.it (C. Dall’As

The volatile profile of nine monocultivar chestnut flours, obtained from fruits grown in Italy (Parma prov-ince), was characterised by a head-space solid-phase microextraction (HS-SPME) coupled with GC–MStechnique. The volatile fraction was composed of 44 main compounds belonging to different classes,mainly aldehydes, ketones, alcohols, furans and terpenes. Aldehydes, in particular hexanal, are the mostabundant components. In order to better understand the origin of the different volatile compounds dur-ing the drying and milling processes, samples of fresh fruit were also analysed by the same technique andthe data obtained were statistically and critically compared in order to get a picture of the volatile evo-lution in chestnut from fresh fruit to flour. Finally, the nine monocultivar flours were chemometricallyclassified on the basis of the main odour descriptors associated with the volatile fingerprinting.

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1. Introduction cultivars (Martin et al., 2009; Pereira-Lorenzo et al., 2011). In addi-

Sweet chestnuts (Castanea sativa Mill.) have been cultivated andutilized for many centuries as one of the most important foodresources of the European mountain people (Neri, Dimitri, &Sacchetti, 2010).

After being, for centuries, the staple food for inhabitants ofEuropean mountain areas, in recent decades chestnut is regaininginterest among consumers due to its nutritional qualities andpotential health benefits, on account of a high amount of carbohy-drates and the presence of essential fatty acids, minerals, vitaminsand also fibre (Borges, Gonc-Alves, Soeiro de Carvalho, Correia, &Silva, 2008; Pereira-Lorenzo, Ramos-Cabrer, Diaz-Hernandez, Cior-dia-Ara, & Rios-Mesa, 2006). For this reason, chestnut fruits andderivatives seem to be suitable for human and animal food produc-tion and integration as substitutes of potato, wheat, bean and cornflours (Pires Borges, Soeiro Carvalho, Reis Correia, & Silva, 2007),especially for the preparation of food destined for people affectedby a number of allergies and ailments. The current chestnut marketin Europe, which is mainly based on Italian high-quality marronevarieties, thus offers new perspectives for growers. For this reason,commercial quality specifications, in terms of nutritional andorganoleptic characteristics, are now required.

In the past ten years, sweet chestnut has been extensively stud-ied from the genotypical and phenotypical point of view, with par-ticular emphasis on the molecular characterization of traditional

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ta).

tion, fresh and processed chestnut fruit nutritional and organolepticaspects have been addressed, mainly for cultivars from Spain andPortugal (Barreira, Casal, Ferreira, Oliveira, & Pereira, 2009; Bernardez,De la Montana, & Queijeiro, 2004; Goncalves et al., 2010; De VasconcelosMendes, Bennett, Rosa, & Cardoso, 2007). The chemical compositionof sweet chestnut has been recently reviewed, focussing on primaryand secondary metabolite occurrence in fruits, as well as on theirfate during processing (De Vasconcelos, Bennett, Rosa, & Ferreira-Cardoso, 2010). Surprisingly, the volatile compounds occurring inchestnut fruit and flour have not yet been extensively described,although their peculiar aroma is one of the most typical organolep-tic characteristic of chestnut-based products. To our knowledge,only one study (Krist, Unterweger, Bandion, & Buchbauer, 2004)reported information about the volatile compounds in Italianroasted chestnut, by application of a HS-SPME-GC/MS method. Inthis study, the main volatile compounds were c-butyrolactone(12.8%), c-terpinene (9.2%), furfural (6.3%), benzaldehyde (7.2%)and 4-methyl-2-pentanone (5.3%).

Italian chestnut germplasm includes hundreds of cultivars withspecific chemical and physical characteristics but, at present, onlysix chestnut cultivars, grown in various areas of the country, areregulated by PGI (Protected Geographical Indication) issued fromthe European Union. Chestnuts are commonly consumed unpro-cessed but, for industrial use, they can be transformed into flour,which also represents a viable storage method. The production ofchestnut flour is widely practised in Italy (Amorini, Manetti,Turchetti, Sansotta, & Villani, 2001; Sacchetti, Pinnavaia, Guidolin,& Dalla Rosa, 2004), by grinding dried chestnuts after stripping thepericarp and the episperm. This product can be employed in the

M. Cirlini et al. / Food Chemistry 134 (2012) 662–668 663

production of foodstuffs, such as cake, desserts or chips (Di Mona-co, Miele, Cavella, & Masi, 2010).

Chestnut fruits show a relatively short shelf-life because of theirsugar contents and high water activity (Pena-Mendez, Hernandez-Suarez, Diaz-Romero, & Rodriguez, 2008); for this reason drying isone of the traditional preservation and storage methods, and analternative to steaming and roasting (Correia, Leitao, & Beirao-da-Costa, 2009; Jermini et al., 2006).

Concerning Italian chestnut varieties, several studies have re-ported the characterisation of PGI cultivars, such as Marrone delMugello (Bellini, Giordani, Marinelli, Migliorini, & Funghini, 2009;Nazzaro, Barbarisi, La Cara, & Volpe, 2011) or the genetic improve-ment of ancient cultivars (Cutino, Marchese, Marra, & Caruso,2010; Martin, Mattioni, Cherubini, Taurchini, & Villani, 2010).Despite such studies, data regarding Italian varieties are stillscarce; in particular, there is a lack of data on flavour propertiesof chestnut fruit and flour and of other chestnut derivatives. Amore complete characterization of the different cultivars grownin Italy, for both their correct classification as well as to study theircompositional characteristics, is therefore now needed. The aim ofthis work was the characterisation of the volatile fraction of chest-nut flours deriving from nine Italian chestnut cultivars, grown inthe Parma province, in comparison with the aromatic fingerprintobtained for the chestnut fresh fruit, in order to study the variationof the volatile profile during the transformation process. In partic-ular, this work is part of an ongoing extensive characterisationstudy of the nine selected cultivars which is addressing both thechemical composition and the molecular characterisation of freshfruits and flours, in order to promote the accreditation of a pro-tected geographical indication (PGI).

2. Materials and methods

2.1. Plant material and sampling

Samples of chestnut fruits and flours were taken from differentplots located in the Emilia Romagna region (Italy), in particular inthe valleys of the two rivers Ceno and Taro in Parma province.Several local chestnut cultivars were considered, namely Luetta,Massese, Lusetta, Leccardina, Mondadì, Gursona, Ampollana, Preilaand Perticaccia, for a total of 9 cultivars. Each cultivar was grown indifferent zones of the valley, with different environmental condi-tions. For each cultivar, 4 plants, homogeneous as to age, vigourand cultivation conditions, were selected. Fruit drying was carriedout in traditional dry kilns by drying at least 10 kg of fruits for eachcultivar. These buildings, little two-floor cabins (metato), have asquare or rectangular plan, are built in local stone, and have a slabstone roof. In the ground, floor heating is produced by making a firewith wood and scrap chestnut. In the first floor, homogeneous lay-ers of chestnuts are laid for drying on a rack. During drying thefruit are turned over several times at a constant temperature(40 �C) for 30 d. Samples (10 kg for each cultivar) were milled,using a cereal mill at CRA (Council for the Research and Experi-mentation in Agriculture) – Experimental Institute for CerealCulture, Fiorenzuola d’Arda (Piacenza, IT).

To evaluate the origin of volatile compounds, endosperm sam-ples were also obtained from fresh fruit. For this analysis, only twocultivars were considered, Luetta and Leccardina, picking fruits fromthe same trees used to collect chestnuts for flour production.

2.2. Head space solid phase microextraction (HS-SPME) analysis

The volatile fraction of chestnut flour or of milled fresh fruit wasanalysed by headspace sampling, using the solid phase microex-traction technique (HS-SPME). For each SPME analysis, 1 g of the

sample (chestnut flour or fresh fruit) was placed in a 30 ml glassvial, adding 5 ml of distilled water. The vial was placed in a warmwater bath (40 �C) and stirred at 40 �C for 60 min. The fibre was in-serted and maintained in the sample head space for 60 min; it thenwas removed and immediately inserted into the GC–MS injector(220 �C for 2 min) for the desorption of compounds. All the analy-ses were performed in triplicate.

For the analyses, a silica fibre was used, coated with 50/30 lm of Divinylbenzene–Carboxen–Polymethylsiloxane (DVB/Carboxen/PDMS) (purchased from Supelco, Bellefonte, PA, USA).Before the analysis, the fibre was conditioned by insertion intothe GC–MS injector at 220 �C for 2 min, then, in order to testthe reproducibility of the experiment, it was exposed to theheadspace above a standard solution of toluene (concentration5 mg l�1 in methanol) in a 30 ml glass vial placed on a stirrerfor 30 s. Analogously, the desorption of compounds during injec-tion was accomplished by keeping the fibre in the injector for2 min at 220 �C. In particular, toluene was used as internal stan-dard (5 mg l�1) for all the analyses, in order to monitor the SPMEextraction performance, as well as the fibre performance duringthe analysis.

2.3. GC–MS analysis

Samples were analysed with an Agilent Technologies (SantaClara, CA) 6890 N gas-chromatograph coupled to an Agilent Tech-nologies 5973 mass spectrometer using a SUPELCOWAX 10 capil-lary column (Supelco, 30 m � 0.25 mm, f.t. 0.25 lm). Helium wasused as carrier gas, with a total flow of 18 ml min�1. The injectortemperature was set at 220 �C and the injection was performed inthe splitless mode (the valve was closed for two minutes). Oventemperature increased from 50 �C to 200 �C, at 5 �C per minuteafter an initial hold at 50 �C for 3 min. The final temperaturewas maintained for 18 min. The detector temperature was220 �C and the MS acquisition mode was full scan (from 40 m/zto 500 m/z) (Krist et al., 2004). Each sample was analyzed intriplicate.

Blank experiments were conducted in two different modalities:blank of the fibre and blank of the empty vial. These types of con-trol were carried out after every 25 analyses.

A mixture of n-alkanes (C8–C20) dissolved in n-hexane, whichwas employed for retention index determination, was suppliedby Supelco (Bellefonte, PA, USA). The retention indices werecalculated for components eluting under experimental conditionsbetween n-octane and n-eicosane. For those components withelution after C20, extrapolation using C18–C20 alkanes was used.

2.4. Data analysis

The main volatile compounds of the aromatic profiles wereidentified on the basis of their mass spectra compared with the ref-erence mass spectra libraries (Wiley275, NBS75K) and of their cal-culated retention indices through the application of the Kovats’formula, compared with those reported in the literature. When itwas not possible to find the retention index in the literature, a ten-tative identification was obtained by matching with mass spectralibraries’ data: a match quality of 98% minimum was used as acriterion.

For each analysis, the integrated peak areas were expressed asrelative percentages, taking the sum of total areas as 100%. All dataobtained were statistically analysed by one-way ANOVA, usingSPSS 17.0 (SPSS Inc., Chicago, IL). Means of relative percentagesof each compound detected were considered different at p < 0.05and significantly different at p < 0.01.

664 M. Cirlini et al. / Food Chemistry 134 (2012) 662–668

3. Results and discussion

3.1. General

A fibre with PDMS/Carboxen/DVB coating was used for HS-SPME analyses as it allowed us to simultaneously detect non-polar,medium-polar and polar compounds. The analyses of fruit andflour samples, performed in triplicate, and the blank analyses, wereperformed using one fibre without any significant change in sensi-tivity. The repeatability test was carried out by analysing the samesample five times, obtaining the RSD values of all compounds con-sidered between 0.85% and 9.35%. Repeatability of retention timeswas between 0.1% and 0.8%.

In total, 26 gas-chromatographic signals, obtained by the tri-phasic fibre, were identified for chestnut fresh fruit, while 44gas-chromatographic peaks were identified for chestnut flours.Among them, 14 peaks were found in both fresh fruits and flours.Concerning the fresh fruit volatile profile, all the compounds, withonly one exception, were unequivocally identified by means ofretention index calculation and comparison, as well as by matchingwith database fragmentation spectra. In the flour fingerprintingprofile, by contrast, 3 compounds were tentatively identified onthe basis of the correct matching of their fragmentation spectrawith the mass spectra database. Each gas-chromatographic peakwas manually integrated in order to obtain the relative percentageof any compound. All the identified volatile substances are listed inTable 1 (fresh fruit) and in Table 2 (flour), along with the corre-sponding odour description and the relative percentage obtainedas a mean value for the nine chestnut cultivars considered in thisstudy.

To our knowledge, only one study has reported the character-isation of the volatile fraction of roasted chestnut using GC–MS(Krist et al., 2004); also, in that case, the same SPME approachwas used for sample preparation. In that study, chestnut roastingwas done by placing the fruits in a 190 �C oven for 50 min whereas,

Table 1Compounds detected in chestnut fruits from different cultivars with odour description. Th

Compound Odour Method ofidentificationa

RT (min) LRIb L

Hexanal Herbal MS + LRI 6.14 1085 1b-Pinene Herbal MS + LRI 6.71 1109 1c-3-Carene Citrus MS + LRI 7.90 1155 1a-Phellandrene Terpenic MS + LRI 8.03 1160 1

D-limonene Citrus MS + LRI 8.84 1191 1

b-phellandrene Floral MS + LRI 9.24 1211 1c-Terpinene Terpenic MS + LRI 10.23 1243 1p-Cymene Terpenic MS + LRI 11.01 1271 1Octanal Aldehydic MS + LRI 11.55 1291 11-Hexanol Herbal MS + LRI 13.33 1357 12-Nonanone Fruity MS + LRI 14.45 1395 1Nonanal Aldehydic MS + LRI 14.72 1400 1a-Thujone Thujonic MS + LRI 15.24 1428 1b-Thujone Thujonic MS 15.74 1448 11-Heptanol Green MS + LRI 16.02 1459 13,5-Octadien-2-one Fatty MS + LRI 17.68 1524 1Benzaldehyde Fruity MS + LRI 17.96 1535 12-Nonenal Fatty MS + LRI 18.15 1545 1Linalool Floral MS + LRI 18.34 1551 1Linalyl acetate Herbal MS + LRI 18.58 1561 1c-Butyrolactone Bready MS + LRI 20.59 1648 1Nonan-1-ol Floral MS + LRI 21.08 1670 13-Nonen-1-ol Waxy MS + LRI 21.59 1694 1(E,E)-2,4-nonadienal Fatty MS + LRI 22.25 1719 12-Phenylethylalcohol Floral MS + LRI 26.79 1933 1Terpene compound – TI 33.16 2243 –

a MS + LRI, mass spectrum and LRI in agreement with the literature; TI, tentative idenb LRI: linear retention index on a Supelcowax 10 capillary column.# Only compounds showing DLRI lower than 10 have been considered as identified.

in our case, the drying process before milling was done in the tra-ditional dry kilns, allowing for a slow drying at 40 �C for 30 d.

3.1.1. Volatile fingerprinting of chestnut fresh fruit and flourThe compounds that contribute to the volatile profile of chest-

nut fruit were mainly terpenes (49.2% of total areas), followed bysaturated and unsaturated alcohols (24.3%), aldehydes (18.3%)and ketones (6.7%). These volatiles may have originated by the sec-ondary metabolism of the plant through the enzymatic oxidationof fatty acids, followed by b-scission rearrangements. Their occur-rence in chestnut fruit is thus influenced by both genetic and envi-ronmental factors. Two compounds were found to be the mostcharacteristic of the chestnut fruit GC fingerprint b-phellandrene(13.4%) and p-cymene (12.2%) with a herbal and terpenic note,respectively. Octanal and thujone isomers (about 8%) were alsopresent in medium percentages.

The main volatile compounds found in chestnut flours werealdehydes (60.8% of the total peak area) followed by alcohols(12.5%) and furans (10.7%). In comparison with fresh fruit, the alde-hydes increased significantly in flour and this fact can be ascribedto the technological treatment. In particular, aldehyde formation isprobably due to the mild but long lasting drying treatment towhich the chestnuts are subjected before flour production. Moriniand Maga (Morini & Maga, 1995) found these compounds in boiledchestnuts: the mild heating for a relatively long period under oxi-dizing conditions can be sufficient to induce lipid peroxidation, fol-lowed by degradation to aldehydes and ketones.

In particular, in the case of chestnut flour, all the aldehydes pre-senting a linear chain could result from degradative oxidation ofunsaturated fatty acids, especially oleic, linoleic and linolenic acids(Amorini et al., 2001) in which chestnuts are rich (Pires Borgeset al., 2007). Aromatic aldehydes, such as benzaldehyde (1.2%)and phenylacetaldehyde (0.15%), were also found in chestnut flour,probably as the result of the degradation of aromatic amino acidsunder drying conditions.

e unidentified substances are not listed.

RIliterature DLRI# Mean ± SD %(of total area)

080 De Vasconcelos Mendes et al. (2007) 5 3.28 ± 3.00107 Nazzaro et al. (2011) 2 3.13 ± 4.55148 Pires Borges et al. (2007) 7 0.30 ± 0.18160 De Vasconcelos Mendes et al. (2007) 0 0.23 ± 0.21194 De Vasconcelos Mendes et al. (2007) �3 4.89 ± 3.25

206 Bianchi, Careri, Mangia, and Musci (2007) 5 13.4 ± 4.13243 Nazzaro et al. (2011) 0 1.39 ± 1.00270 Bianchi et al. (2007) 1 12.2 ± 7.50286 De Vasconcelos Mendes et al. (2007) 5 8.86 ± 4.13354 Bianchi et al. (2007) 3 3.65 ± 3.33394 De Vasconcelos Mendes et al. (2007) 1 2.28 ± 0.62396 De Vasconcelos Mendes et al. (2007) 4 3.39 ± 1.15417 Nazzaro et al. (2011) 9 3.97 ± 1.09436 Nazzaro et al. (2011) 12 3.97 ± 1.09460 De Vasconcelos Mendes et al. (2007) �1 3.16 ± 1.54521 De Vasconcelos Mendes et al. (2007) 3 1.91 ± 1.01528 De Vasconcelos Mendes et al. (2007) 7 1.06 ± 0.17546 De Vasconcelos Mendes et al. (2007) �1 0.92 ± 0.98554 De Vasconcelos Mendes et al. (2007) �3 0.69 ± 0.29569 Yanagimoto, Ochi, Lee, and Shibamoto (2004) �8 1.23 ± 0.18647 Valim, Rouseff, and Lin (2003) 1 2.49 ± 0.68668 De Vasconcelos Mendes et al. (2007) 2 1.04 ± 0.29697 De Vasconcelos Mendes et al. (2007) �3 0.39 ± 0.21709 Pereira-Lorenzo et al. (2006) 10 0.83 ± 0.27925 Sacchetti et al. (2004) 8 0.45 ± 0.28

– 5.26 ± 3.82

tification by mass spectrum matching with those reported in the library.

Table 2Compounds detected in chestnut flours from different cultivars with odour description. The unidentified substances are not reported in the table.

Compound Odor type Identificationa RT (min) LRIb LRIliterature DLRI Mean ± SD %(of total area)

Hexanal Herbal MS + LRI 6.25 1085 1080 De Vasconcelos Mendes et al. (2007) 5 39.4 ± 3.49Heptanal Herbal MS + LRI 8.86 1192 1186 De Vasconcelos Mendes et al. (2007) 6 1.86 ± 0.14

D-limonene Citrus MS + LRI 9.13 1193 1194 De Vasconcelos Mendes et al. (2007) �1 0.59 ± 0.06

b-Phellandrene Herbal MS + LRI 9.37 1211 1206 Nazzaro et al. (2011) 5 0.69 ± 0.072-Pentylfuran Fruity MS + LRI 10.07 1237 1240 De Vasconcelos Mendes et al. (2007) �3 3.53 ± 0.021-Pentanol Fermented MS + LRI 10.55 1254 1256 De Vasconcelos Mendes et al. (2007) �2 1.05 ± 0.06Octanal Aldehydic MS + LRI 11.70 1291 1286 De Vasconcelos Mendes et al. (2007) 5 7.39 ± 0.312-Heptanol Citrus MS + LRI 12.41 1322 1326 De Vasconcelos Mendes et al. (2007) �4 0.14 ± 0.022-Heptenal Green MS + LRI 12.70 1333 1333 Miranda-Lopez, Libbey, Watson, and McDaniel (1992) 0 1.22 ± 0.131-Hexanol Herbal MS + LRI 13.33 1357 1354 De Vasconcelos Mendes et al. (2007) 3 3.48 ± 1.102-Nonanone Fruity MS + LRI 14.36 1395 1394 De Vasconcelos Mendes et al. (2007) 1 0.44 ± 0.03Nonanal Aldehydic MS + LRI 14.49 1400 1396 De Vasconcelos Mendes et al. (2007) 4 7.25 ± 0.343-Octen-2-one Fruity MS + LRI 14.89 1415 1419 Miranda-Lopez et al. (1992) �4 1.10 ± 0.06cis-linalool oxide – MS + LRI 15.14 1425 1423 Neri et al. (2010) 2 1.30 ± 0.032-Octenal Green MS + LRI 15.48 1438 1440 Miranda-Lopez et al. (1992) �2 2.48 ± 0.161-Octen-3-ol Earthy MS + LRI 15.90 1454 1456 De Vasconcelos Mendes et al. (2007) �2 3.56 ± 0.131-Heptanol Green MS + LRI 16.02 1459 1460 De Vasconcelos Mendes et al. (2007) �1 0.95 ± 0.07Furfural Bready MS + LRI 16.46 1476 1472 De Vasconcelos Mendes et al. (2007) 4 2.41 ± 0.224-Ethylcyclohexanol – MS 17.20 1504 – – 0.95 ± 0.22Benzofuran – MS + LRI 17.47 1515 1521 Pena-Mendez et al. (2008) �6 0.50 ± 0.103,5-Octadien-2-one Fatty MS + LRI 17.80 1524 1521 De Vasconcelos Mendes et al. (2007) 3 0.52 ± 0.06Benzaldehyde Fruity MS + LRI 17.95 1535 1539 Miranda-Lopez et al. (1992) �4 1.18 ± 0.122-Nonenal Green MS + LRI 18.19 1545 1546 De Vasconcelos Mendes et al. (2007) �1 0.77 ± 0.053,5-Dimethyl-cyclohexanol – MS 18.31 1550 – – 0.87 ± 0.181-Octanol Waxy MS + LRI 18.60 1561 1561 De Vasconcelos Mendes et al. (2007) 0 1.08 ± 0.075-Methylfurfural Bready MS + LRI 19.19 1585 1589 De Vasconcelos Mendes et al. (2007) �4 0.53 ± 0.152-Methylbenzofuran – MS + LRI 19.67 1606 1614 Miranda-Lopez et al. (1992) �8 0.46 ± 0.06c-Butyrolactone Bready MS + LRI 20.60 1648 1647 Valim et al. (2003) 1 1.59 ± 0.182-Decenal Waxy MS + LRI 20.76 1656 1652 De Vasconcelos Mendes et al. (2007) 4 0.24 ± 0.072-Phenylacetaldehyde Green MS + LRI 20.84 1659 1669 De Vasconcelos Mendes et al. (2007) �10 0.15 ± 0.07Acetophenone Floral MS + LRI 21.02 1668 1660 De Vasconcelos Mendes et al. (2007) 8 0.20 ± 0.062-Furfuryl alcohol Bready MS + LRI 21.18 1675 1669 Pena-Mendez et al. (2008) 6 1.63 ± 0.084-Methylthiazole Bready MS + LRI 21.27 1679 1681 Pereira-Lorenzo et al. (2011) �2 0.62 ± 0.06[E,E]-2,4-nonadienal Fatty MS + LRI 22.15 1719 1709 Pereira-Lorenzo et al. (2006) 10 0.16 ± 0.01Naphtalene – MS + LRI 22.25 1724 1718 Pires Borges et al. (2007) 6 1.69 ± 0.02Valerolactone Herbal MS 23.08 1762 – – 0.40 ± 0.07[E,E]-2,4-decadienal Fatty MS + LRI 24.56 1830 1832 Pereira-Lorenzo et al. (2006) �2 0.14 ± 0.02Caproic acid Fatty MS + LRI 25.60 1879 1872 Rychlik, Schieberle, and Grosch (1998) 7 1.92 ± 0.28Guaiacol Smoky MS + LRI 25.67 1882 1883 Rychlik et al. (1998) �1 3.53 ± 0.332-Phenylethylalcohol Floral MS + LRI 26.71 1933 1931 Sacchetti et al. (2004) 2 0.50 ± 0.10E-oak lactone Fruity, fatty MS + LRI 27.64 1979 1977 Sacchetti et al. (2004) 2 0.37 ± 0.08o-Cresol Smoky MS + LRI 28.58 2024 2017 Umano, Hagi, Nakahara, Shoji, and Shibamoto (2000) 7 0.29 ± 0.044-Ethyl guaiacol Smoky MS + LRI 28.65 2028 2031 Schieberle, Ehrmeier, and Grosch (1988) �3 0.54 ± 0.13m-Cresol Smoky MS + LRI 30.29 2106 2115 Sacchetti et al. (2004) �9 0.20 ± 0.03

a MS + LRI, mass spectrum and LRI in agreement with the literature; MS, mass spectrum in agreement with spectrum in Wiley275 and NBS75 K libraries.b LRI: linear retention index on a Supelcowax 10 capillary column.

M. Cirlini et al. / Food Chemistry 134 (2012) 662–668 665

The most abundant compound of the volatile fraction of chest-nut flour was hexanal (39.3%) with the characteristic green odour.Two other compounds were present in medium percentages: non-anal (7.2%) and octanal (7.4%), both with an aldehydic note.

Ketones were represented in both chestnut fresh fruit (6.7%)and flour (3.5%). In particular, 2-nonanone and 3,5-octadien-2-one were both detected in fresh fruit and in flour. Furthermore,flour was also characterised by the occurrence of 3-octen-2-one(1.15%) and acetophenone (0.20%), which are also produced byunsaturated fatty acid oxidative degradation. Beside aldehydesand ketones, only caproic acid (1.9%) and c-butyrolactone (2.5%)were found as the final products of lipid oxidation reaction.E-oak lactone was also found in chestnut flour, although at lowlevels (0.37%), probably deriving from thermal degradation ofchestnut bark during the drying step in kiln.

Furans, another important volatile group occurring in chestnutflour, could have originated from thermal degradation and rear-rangement of carbohydrates via the Maillard reaction. Moreover,furfural, 5-methylfurfural and furfuryl alcohol could also be as-cribed to the caramelization process under drying conditions,which may involve free sugars naturally occurring in freshchestnut fruits.

Among thiazoles, which may have originated by cysteine degra-dation during thermal processing, only 4-methylthiazole wasfound in the flour volatile fraction. Among volatile compounds,neither pyrazines nor other heterocyclic compounds were found,probably on account of the milder drying conditions applied inour study in comparison with those reported by Krist et al.(2004). On the other hand, the occurrence of a low amount ofnaphthalene (0.4%) in chestnut flours could be due to wood pyro-lysis and smoke deposition during the drying step in kiln.

Among phenolic compounds, guaiacol, 4-ethylguaiacol, o- andm-cresol were found in all the considered flours. These compoundsare formed during the thermal degradation of lignin, which canoccur under pyrolysis conditions. In particular, during dryingtreatment in kilns, chestnut fruits with barks are placed on a rack,located over a fire. The phenolic compounds can thus be ascribedto the smoke generated by burning chestnut wood or by thermaldegradation of barks. The occurrence of the same compounds hasbeen reported likewise also for malt dried in kilns under similarconditions (Gruber, 2010).

In order to better characterise the aroma profile of chestnutfruit and flour, the volatile compounds found in both samples weresplit into chemical classes and compared in order to magnify the

250 50 75peak area %

terpenes

linear aldehydes

ketones

alcohols

esters

furans

carboxilic acids

cyclic hydrocarbons

phenols

linear hydrocarbons

aromatic aldehydes

thiazoles

fresh fruit flour

Fig. 1. Comparison between volatile chemical groups found in chestnut fresh fruit and flour.

666 M. Cirlini et al. / Food Chemistry 134 (2012) 662–668

effect of the technological treatment on the volatile fraction, asshown in Fig. 1.

Comparing the different profiles, it appears that the fruit aro-matic fraction is mainly characterised by terpenes, which are prob-ably lost during the technological process used for flour production.In flour, indeed, only D-limonene and b-phellandrene were found ata significant percentages, those being 0.59% and 6.9%, respectively.The flour volatile profile appears to be characterised by a significantaldehyde content, probably due to the increase of lipid degradationoccurring during the drying process. A decrease in ketone and alco-hol contents also occurred after processing, while the technologicaltreatment gave rise to new compounds, such as furans, carboxylic

0

10

20

30

40

50

hexan

al

limonen

e

b-ocim

ene

octan

al

1-hex

anol

2-nona

none

nonanal

1-he

3

rela

tive

pea

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ea (

%)

fresh f

**

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**

β-ocim

ene

β-phell

andre

ne

Fig. 2. Changes in the chestnut volatile compounds found in both fresh fruit and flou

acids, thiazoles, phenols, aromatic aldehydes and linear as well ascyclic hydrocarbons.

Only 14 out of 25 compounds identified in fresh chestnut alsooccurred in chestnut flour, as shown in Fig. 2. Among them,statistical differences (ANOVA test, a = 0.05) were found only forhexanal and nonanal, both showing a significant increase duringthe technological treatment, and for D-limonene, b-phellandrene,2-nonanone, 1-heptanol, 3,5-octadien-2-one and c-butyrolactonewhich showed a slight decrease in flour due to the processing step.On the other hand, no significant change was found for 1-hexanol,octanal, benzaldehyde, 2-nonenal, 2,4-nonadienal or 2-phenyl-ethyl alcohol.

ptanol

,5-oct

adien

-2-o

ne

benza

ldeh

yde

2-nonen

al

g-butir

rolac

tone

2,4-n

onadien

ale

2-phen

yleth

ylalco

hol

ruit flour

** ** *

γ-buty

rolac

tone

r. Significant differences (ANOVA analysis) are shown (⁄⁄: p < 0.001; ⁄: p < 0.05).

M. Cirlini et al. / Food Chemistry 134 (2012) 662–668 667

3.2. Multivariate analysis

Since our study was also aimed at the aromatic description ofthe monovarietal flours obtained by drying and milling chestnutfruits from 9 local cultivars, the volatile compounds occurring inthe volatile fingerprint obtained were classified and grouped onthe basis of their odour descriptors, as shown in Table 2. Accordingto this classification, hexanal, heptanal, 2-heptenal, 2-octenal,1-heptanol, valerolactone, 1-hexanol, b-phellandrene and phenyl-acetaldehyde are responsible for the green and herbaceous aromaimpression of the considered chestnut flours. Aldehydic and citrusodour notes are due to limonene, 2-heptanol, octanal and nonanal,whereas floral notes should be ascribed to 2-phenylethyl alcoholand acetophenone. Fruity aromas are due to 2-pentylfuran, 2-nonanone, 3-octen-2-one, oak lactone and benzaldehyde, while1-pentanol, 3,5-octadien-2-one, 2,4-nonadienal, 2,4-decadienaland caproic acid are responsible for a fatty and fermented odourimpression. In addition, the earthy and waxy notes are mainlydue to 1-octen-3-ol, 1-octanol and 2-decenal. The characteristicbready-toasted notes are due to furfural, 5-methylfurfural, c-buty-rolactone, 2-furfuryl alcohol and 4-methylthiazole, while the phe-nolic compounds, guaiacol, 4-ethylguaiacol, o- and m-cresol, areresponsible for the smoky aroma impression in chestnut flour.

The obtained dataset was used for PCA analysis, carried out todifferentiate between the 9 cultivars for the 10 aromatic notes(see Fig. 3).

-2.5 -2 -1.5 -1 -0.5

PC1

-2.5

-2

-1.5

-1

-0.5

0

0.5

1

1.5

2

2.5

PC2:

19.

8%

Biplot ( sum of PC

Fig. 3. PCA analysis of volatile profiles for monovarietal

The analysis indicates that the first two principal components ex-plain 84.6% of the total variance among the monocultivar flours stud-ied. A comparison of scores and loadings for F1 and F2 allows theidentification of the aromatic notes having greater influence on theranking of flours. In particular, the green and the aldehydic notesshowed negative eigenvalues on the PC1 (eigenvalues of �0.929 and�0.671, respectively), while fruity, herbal and bready-toasted notesshowed high eigenvalues (>0.9) on the first component. Concerningthe PC2, the main positive contribution was due to the aldehydic andthe waxy notes (eigenvalues of 0.682 and 0.796, respectively), whilethe main negative contribution was ascribed to the fatty-fermentednote (eigenvalue of �0.546). The smoky note, although representedby 7% of the detected volatile compounds, does not provide a signifi-cant contribution to the flour classification, probably on account ofits homogeneity between samples. Smoky notes, in-deed, are due tophenolic compounds, which are formed by wood pyrolysis and smokedeposition in kiln: since all the chestnut cultivars were dried in sepa-rated racks at the same time and under the same conditions, theamount of phenolic compounds formed during the processing waslikely not cultivar-related.

The PCA analysis allowed for clustering of the 9 monocultivarflours into 4 groups according to their volatile profiles. In particu-lar, the flours obtained by Gursona and Preila chestnut cultivarsshowed a very peculiar aromatic profile, both obtaining positivescores on the PC1 but being separated by the different scores onthe PC2. Both flours, in particular, are characterised in their volatile

0 0.5 1 1.5 2 2.5

: 64.7%

1 and PC2: 84.6%)

flours obtained from 9 different chestnut cultivars.

668 M. Cirlini et al. / Food Chemistry 134 (2012) 662–668

profiles by a low amount of compounds having a herbal or an alde-hydic note. Moreover, the flour obtained by cultivar Gursona is alsocharacterised by a higher content of bready-toasted as well asearthy flavours in comparison to the cultivar Preila deriving flour.

Another group, showing negative scores on the PC1 and positivescores on the PC2, is composed of Lusetta, Massese and Leccardinaflours, which are characterised by a high amount of volatiles withan aldehydic note, while the last group, having negative scores onboth PC1 and PC2, is represented by Perticaccia, Mondadì andAmpollana flours, all of them showing a significant green note.

Thus, the characterization of the volatile profile of these floursnot only represent the basis for their correct identification from acommercial point of view but also, eventually, a potential for theiruse in pure form or as a mixture to impart particular sensorialnotes to the derived products.

4. Conclusions

This study represents one of the first attempts aimed at thecharacterization of the flavour of chestnut flour in comparison withthe corresponding fruits. In particular, nine chestnut cultivars(grown in a small area located in Parma province (Italy)) were con-sidered. Monocultivar flours were obtained by a traditional processcharacterised by a mild drying step in kiln, followed by milling. Theflour volatile fraction, obtained by a triphasic fibre SPME extrac-tion, was composed of 44 compounds, mainly aldehydes, alcoholsand furans. Compared to the fresh fruit volatile fraction, the flourshowed a significant decrease in terpenes and alcohols, coupledwith the formation of neogenic compounds due to thermal treat-ment, such as furans, thiazoles and phenols. The volatile compounddataset obtained for the nine monocultivar flours was then usedfor sample chemometric clustering on the basis of the associatedaromatic notes. PCA analysis allowed for identification of 4 groups,characterised by different aromatic impressions. Since the peculiararoma is one of the main organoleptic characteristics of chestnutflour, these results can be exploited, not only for commercial clas-sification, but also for the formulation of flour mixtures able to im-part a desired aromatic note to the final product.

Acknowledgement

We gratefully acknowledge Ivo Botti, Ivo Bertorelli and theC.A.P.O. association (Consorzio Castanicoltori Appennino ParmaOvest) for their excellent technical assistance.

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