Using a gas chromatographic method (GC-MSanalysis), it was possible to determine the volatile constituent of an odorous flower from Vitis vinifera varieties growing in Sicily. More than 50 compounds were identified and the technique allowed us to determine that sesquiterpenes, as well as monoterpenes such as limonene and cymene, were the principal components. The odour-profiles allowed us to distinguish between variety groups or even single varieties.

Barbagallo, M. G.

Pisciotta, A.

Saiano, F.

English

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Vitis 53 (2), 111–113 (2014)Identification of aroma compounds of Vitis vinifera L. flowers by SPME GC-MS analysis M. G. BARBAGALLO, A. PISCIOTTA and F. SAIANODepartment of Agricultural and Forest Sciences, Università degli Studi di Palermo, Palermo, ItalyCorrespondence to: Dr. A. PISCIOTTA, Department of Agricultural and Forest Sciences, Università degli Studi di Palermo, Viale delle Scienze, Edificio 4, Ing. H, 90128 Palermo, Italy. E-mail: antonino.pisciotta@unipa.itSummaryUsing a gas chromatographic method (GC-MS-analysis), it was possible to determine the volatile con-stituent of an odorous flower from Vitis vinifera varie-ties growing in Sicily. More than 50 compounds were identified and the technique allowed us to determine that sesquiterpenes, as well as monoterpenes such as limonene and cymene, were the principal components. The odour-profiles allowed us to distinguish between variety groups or even single varieties. K e y  w o r d s :  grapevine, flowering, sesquiterpenes, monoterpenes.IntroductionWine aroma chemistry has been meticulously studied over the last few years, and several researchers have identi-fied many volatile compounds that are found in grapes and wines of different cultivars. A previous report focused on the olfactory properties and on the identification of character impact odorants attributable to the cultivar (SÁNCHEZ-PAL-OMO et al. 2010). Although the release of volatiles, includ-ing aliphatics, terpenoids, and phenylpropanoids, from an-thers and/or pollen has been reported (DOBSON et al. 2000), the chemical composition of the flower volatiles of Vitis vinifera, which are responsible for its odor, has not been substantially investigated until recently (BUCHBAUER et al. 1994a, 1994b, 1995, LÜCKER et al. 2004). Low molecular-weight terpenoids, which include a large array of monoter-penes, sesquiterpenes, and norisoprenoids, are commonly found as volatiles emitted from the flowers, fruits, and leaves of plants (SCHRADER and BERGER 2001). Grapevine flowers produce numerous sesquiterpenoid volatiles [e.g. (E)-β-caryophyllene, α-humulene, (+)-valencene, (E,E)-α-farnesene, and (-)-7-epi-α-selinene], presumably as attract-ants for pollinators (BUCHBAUER et al. 1994a, 1995, MARTIN et al. 2009). The sesquiterpene valencene is a major vola-tile emitted from the flowers of white and red varieties of the grapevine Vitis vinifera L. (BUCHBAUER et al. 1994a, 1994b, 1995). In this study, we have provided qualitative “odour profiles” in order to obtain more information about the aroma of grape flowers. We report the results of a head space SPME study as a useful tool (short sampling time, small sample amount, and no use of solvents) for an early and preliminary characterisation of volatile compounds.Material and MethodsGrape inflorescences from eight varieties were col-lected in 2012 from vineyards located in the Marsala area (West Sicily 100 m a.s.l.). All vines were subjected to the same trellis system, pruning method, and cultivation prac-tices. The eight selected varieties were six white: ‘Inzolia’, ‘Catarratto Comune’, ‘Grillo’, ‘Zibibbo’ (syn. ‘Muscat of Alexandria’), ‘Sauvignon Blanc’ and ‘Fiano’; and two red: ‘Frappato’ and ‘Cabernet Sauvignon’. At full flowering de-velopment stage (BBCH 67), three whole inflorescences per replicate (three) and per variety (eight), each with the same light condition and shoot position, were collected from nine different vines. The selected inflorescences were placed in 250 mL vials with silicon septa and stored at 4 °C until analysis.Preliminary experiments (data not shown) were car-ried out to select the coating of the fiber and its thickness, as well as to identify the most appropriate conditions, such as the length and temperature of the exposure in the head space over the sample. These experiments were mainly de-signed to enhance the volatilisation of compounds from the matrix while preventing their decomposition, and to op-timise the gas-fiber equilibrium. Before use, the selected SPME fiber, which was coated with divinylbenzene/car-boxen/polydimethylsiloxane (DVB/CAR/PDMS, 50 μm, Supelco), was conditioned for 2 h at 250 °C in the inlet of a gas chromatograph. Analyses were performed on the sampled whole inflorescences that were placed in 250 mL glass vials sealed with a silicon septum. After 60 min of equilibration at 25 °C, the SPME fiber was inserted in the silicon septum with the help of a manual holder system. After 5 min at 25 °C, the SPME fiber was recovered and immediately inserted into the injector port of the gas chro-matograph, allowing for 1 min desorption at 250 °C. Three replicates of each sample were analysed. C h r o m a t o g r a p h i c  p r o c e d u r e s :  A GC in-strument (Agilent 6890), equipped with a mass selective detector (Agilent 5975), was used for the chromatographic analyses. A fused silica capillary column Carbowax (30 m length, 0.25 mm internal diameter, and 0.25 μm film thick-ness; Supelco) served as the stationary phase. The injector was used in splitless mode and had a temperature of 250 °C. Experimental chromatographic conditions were as fol-lows: Helium carrier gas at 1 mL∙min-1; and an oven tem-perature program with a 5 min isotherm at 40 °C followed by a linear temperature increase of 4 °C∙min-1 up to 200 °C,  112 M. G. BARBAGALLO et al.where it was held for 2 min. The MS scan conditions were: source temperature 230 °C, interface temperature 280 °C, EI at 70 eV, and mass scan range 33-350 amu. A commer-cial library (NIST05) was used interactively with the MS data for compound identification. Standards, required to confirm some assignments, were obtained from Fluka and used without further purification. The relative proportions of the essential oil constituents were expressed as percent-ages obtained by GC-MS peak area normalisation, with all relative response factors being taken as one.A hierarchical cluster analysis based on Euclidean dis-tance matrices with the Unweighted Pair Group Method with Arithmetic Averages (UPGMA) was performed using R version 3.0.1 (R Core Team, 2013).Results and DiscussionThe Table shows the principal constituents found as the average of the three replicates. The percentage of the relative standard deviation was always < 10 %. It is well known that different extraction techniques could result in different quali-quantitative extract compositions and that, because of the equilibrium nature of the SPME method, the GC-MS profiles do not immediately correspond to the composition of the detected substances in the sample or in the head space. Our goal, however, was to take advantage of the ability of the SPME method to extract/concentrate both low and high-eluting components, allowing for com-parisons of the chromatographic profiles for different sam-ples under similar experimental conditions.The Figure shows the clear identification of four groups of varieties according to their different aromatic compounds: 1 s t  G r o u p  ( ‘ G r i l l o ’ - ’ Z i b i b b o ’  ( s y n .  ‘ M u s c a t  o f  A l e x a n d r i a ’ ) ) :  ‘Grillo’ and ‘Zibib-bo’ are characterised by the prevalence of (E)-β-farnesene (exceeding 40 %), low values of (-)-β-caryophyllene (less than 10 %), and modest values of valencene and 7-epi-α-selinene (Table).2 n d  G r o u p  ( ‘ C a b e r n e t  S a u v i g n o n ’ -’ F r a p p a t o ’ - ’ S a u v i g n o n  B l a n c ’ - ) :  ‘Cabernet Sauvignon’ and ‘Frappato’ show similar profile. They are characterised by the presence of (in order of increasing value): (EE)-α-farnesene, 7-epi-α-selinene, (-)-β-caryo-phyllene, and (+)-valencene. These account for 78.5 % and 82.3 % of the total compounds in the two varieties, respectively. ‘Sauvignon Blanc’ has more 7-epi-α-selinene and (+)-valencene than ‘Cabernet Sauvignon’. Even if ‘Ca-bernet Sauvignon’ is located in the same group, it shows additional compounds, such as 6-methyl-5-hepten-2-one, p-cymene, limonene, and α-trans-bergamotene (Table). However, a prevalence of (+)-valencene has previously been reported (MARTIN et al. 2009). T a b l e Percentage of compounds divided in sesquiterpenes, monoterpenes, ketones and estersCompoundsGrillo%Zibibbo%Cabernet S.%Frappato%Sauvignon B.%Catarratto C.%Fiano%Inzolia%Sesquiterpenes   (-)-β-caryophyllene 9.0 7.7 21.0 21.1 18.5 36.1 14.2 8.4   (E.E)-α-farnesene 10.4 7.7 14.6 9.0 12.1 15.7 27.2 45.2   (E)-β-farnesene 41.0 45.2 2.5 7.4 6.7 13.9 12.1 17.8   α-humulene 3.1 4.5 3.7 3.3 4.2 5.0 5.1 2.9   α-selinene 4.5 4.4 0.0 4.3 3.6 8.7 10.5 3.9   7-epi-α-selinene 11.4 11.3 17.6 20.2 19.2 3.1 0.0 4.4   (+)-valencene 15.6 16.0 25.3 32.0 27.9 4.3 0.7 6.1Monoterpenes   α-trans-bergamotene 1.2 1.2 3.0 0.0 2.0 2.2 5.2 5.0   p-cymene 0.8 0.6 4.8 0.2 2.4 4.4 7.2 2.3   limonene 1.7 1.1 3.5 0.7 1.7 3.5 5.8 1.0Ketones   6-methyl-5-hepten-2-one 0.7 0.2 3.2 1.4 1.0 2.5 8.8 1.3Esters   ethyl benzoate 0.6 0.0 0.6 0.3 0.5 0.6 3.3 1.5Figure: Hierarchical cluster analysis dendrogram on the Eucli-dean distance matrix, UPGMA aggregation method. Identification of aroma compounds of flowers by SPME GC-MS 1133 r d  G r o u p  ( ‘ C a t a r r a t t o  C . ’ ) :  ‘Catarratto Comune’ is represented by the prevalence of (-)-β-caryo-phyllene (36.1 %), (EE)-α-farnesene (15.7 %), (E)-β-far-nesene (13.9 %), α-selinene, and α-humulene. Its profile was qualitatively close to the 4th group, but it differed quantitatively (Table).4 t h  G r o u p  ( ‘ F i a n o ’ - ’ I n z o l i a ’ ) :  ‘Fiano’ and ‘Inzolia’ are both characterised by the dominance of (EE)-α-farnesene, though at different percentages. Particu-larly, ‘Inzolia’ is characterised by the prevalence of (EE)-α-farnesene (45.2 %) and (E)-β-farnesene (17.8 %), followed by (-)-β-caryophyllene, (+)-valencene and α-trans-berga-motene. ‘Fiano’ is the variety with the greater diversity of compounds, having (EE)-α-farnesene (27.2 %), (-)-β-cary-ophyllene (14.2 %), (E)-β-farnesene (12.1 %), α-selinene (10.5 %), p-cymene, limonene, α-trans-bergamotene, α-hu-mulene and the highest presence of 6-methyl-5-hepten-2-one (8.8 %) than the other varieties (Table).ConclusionsThe technique we used (SPME GC-MS analysis) was useful in distinguishing between different odorous profiles and, therefore, enabled the discrimination of the grape va-rieties we examined. The results showed that the predomi-nant flower aroma compounds can be used to distinguish between variety groups, or even single varieties, grow-ing under identical conditions. It is interesting to note the similar aroma profiles between ‘Cabernet Sauvignon’ and ‘Sauvignon Blanc’ (BOWERS and MEREDITH 1997), and be-tween ‘Grillo’ and ‘Zibibbo’ (syn. ‘Muscat of Alexandria’) (DI VECCHI STARAZ et al. 2007). Further studies are needed to expand both the varietal data set and the list of known aroma compounds to additionally contribute to our knowl-edge of variety behaviours and how external factors (such as climate, vineyard management, etc.) can impact on the composition of flower aroma profiles.   ReferencesBOWERS J. E.; MEREDITH C. P.; 1997: The parentage of a classic wine grape, Cabernet Sauvignon. Nat. Genet. 16, 84-7.BUCHBAUER, G.; JIROVETZ, L.; WASICKY, M.; HERLITSCHKA, A.; NIKIFOROV, A.; 1994a: Aroma of white wine flowers. Z. Lebensm. Unters. For-sch. 199, 1-4.BUCHBAUER, G.; JIROVETZ, L.; WASICKY, M.; HERLITSCHKA, A.; 1994b: Headspace analysis of Vitis vinifera (Vitaceae) flowers. J. Essent. Oil Res. 6, 313-314.BUCHBAUER, G.; JIROVETZ, L.; WASICKY, M.; NIKIFOROV, A.; 1995: Aroma from red wine flowers. Z. Lebensm. Unters. Forsch. 200, 443-446. DI VECCHI STARAZ, M.; BANDINELLI, R.; BOSELLI, M.; THIS, P.; BOURSIQ-UOT, J. M.; LAUCOU, V.; 2007; Genetic structuring and parentage analysis for evolutionary studies in grapevine: Kin group and origin of the cultivar Sangiovese revealed. J. Am. Soc. Hortic. Sci. 132, 514-524.DOBSON, H. E. M.; BERGSTROEM, G.; 2000: The ecology and evolution of pollen odors. Plant Syst. Evol. 222, 63-87.LÜKER, J.; BOWEN, P.; BOHLMANN J.; 2004: Vitis vinifera terpenoid cycla-ses: functional identification of two sesquiterpene synthase cDNAs encoding (+)-valencene synthase and (-)-germacrene D synthase and expression of mono- and sesquiterpene synthase in grapevine flowers and berries. Phytochemistry 65, 2649-2659.MARTIN, D. M; TOUB, O.; CHIANG, A.; LO, B. C; OHSE, S.; LUND, S. T.; BOHLMANN, J.; 2009: The bouquet of grapevine (Vitis vinifera L. cv. Cabernet Sauvignon) flowers arises from the biosynthesis of sesquiterpene volatiles in pollen grains. Proc. Nat. Acad. Sci. 106, 7245-7250.R CORE TEAM; 2013: R: A language and environment for statistical com-puting. R Foundation for Statistical Computing, Vienna, Austria (http://www.R-project.org/).SÁNCHEZ-PALOMO, E.; GÓMEZ GARCÍA-CARPINTERO, E.; ALONSO-VILLEGAS, R.; GONZÁLEZ-VIÑAS, M. A.; 2010: Characterization of aroma compounds of Verdejo white wines from the La Mancha re-gion by odour activity values. Flavour Fragr. J. 25, 456-462.SCHRADER, J.; BERGER, R.G.; 2001: Biotechnological production of terpe-noid flavor and fragrance coumpounds. In: H. J. REHM (Ed.): Bio-technology, 10, 373-422, Wiley-VCH, Weinheim.Received June 12, 2013

Identification of aroma compounds of Vitis vinifera L. flowers by SPME GC-MS analysis

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Identification of aroma compounds of Vitis vinifera L. flowers by SPME GC-MS analysis

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