COBISS Code 1.01 DOI: 10.14720/aas.2015.105.1.01 Agrovoc descriptors: wines, red wines, volatile compounds, provenance, indigenous organisms, aromatic compounds, esters, smell, extraction Agris category code: f60, q04 Volatile profile of wine Teran PTP Helena BAŠA ČESNIK1, Dejan BAVČAR1, Klemen LISJAK1* Received November 19, 2014; accepted December 18, 2014. Delo je prispelo 19. novembra 2014, sprejeto 18. decembra 2014. ABSTRACT Teran PTP is a protected wine with a recognized traditional denomination produced from a grapevine variety 'Refosk' in winegrowing district Kras in Slovenia (European Union, 2009; Pravilnik, 2008). The aromatic profile of 82 Teran PTP wines produced in years 2011, 2012 and 2013 was monitored. In total the content of 16 volatile compounds was determined. The volatile compounds from wine were extracted following the liquid-liquid extraction and determined with a GC-MS method. The odour activity values and relative odour contributions were calculated for each volatile compound identified. Among sensorial important volatiles the highest odour activity values were determined for ethyl octanoate, ethyl hexanoate, isoamyl acetate and ethyl butyrate. Other research papers also showed, that all red wines investigated except one contained ethyl octanoate, ethyl hexanoate, isoamyl acetate and ethyl butyrate above sensory thresholds. Key words: Teran PTP, wine, volatile compounds, esters, aroma, GC/MS IZVLEČEK AROMATIČNE SPOJINE VINA TERAN PTP Vino Teran PTP je zaščiteno vino s priznanim tradicionalnim poimenovanjem, ki ga pridelujejo iz grozdja sorte 'Refošk' v vinorodnem okolišu Kras v Sloveniji (European Union, 2009; Pravilnik, 2008). Aromatični profil vina Teran PTP smo spremljali tri leta v 82 vzorcih iz letnikov 2011, 2012 in 2013. Določevali smo vsebnost 16 hlapnih spojin. Za določitev hlapnih spojin smo uporabili ekstrakcijo tekoče-tekoče, kateri je sledila določitev z GC-MS. Za vseh 16 spojin smo izračunali aktivne vonjalne vrednosti in njihove relativne prispevke. Med senzorično pomembnimi hlapnimi spojinami smo največje aktivne vonjalne vrednosti določili za etil oktanoat, etil heksanoat, izoamil acetat in etil butirat. Drugi raziskovalni članki so tudi pokazali, da so vsa preiskovana rdeča vina z izjemo enega vsebovala etil oktanoat, etil heksanoat, isoamil acetat in etil butirat nad pragom zaznave. Ključne besede: Teran PTP, vino, hlapne spojine, estri, aroma, GC/MS 1 INTRODUCTION Teran PTP (Recognised Traditional Denomination) is a typical wine from the Kras winegrowing district of the Primorska wine growing region in Slovenia, where it is usually called just »Teran«. The production of this unique red wine has been known since the first century AD (Vodopivec, 1999). Teran PTP is a wine produced from a variety 'Refosk' and known for its deep reddish-violet colour, caused by an abundance of anthocyanins and its medium tannin content (Vanzo et al., 2012). Due to its favorable phenolic compounds (anthocyanins), it is known for having positive health effects and good nutritional value for consumers (Fornasario et al., 2012). Taste of wine teran is both astringent and sour, derived from its high phenolic content and high total acidity. The unique feature of this wine is its fruity odour, reminiscent of raspberries, strawberries and cherries. Fruity odour of Teran PTP is considered most pronounced in the first year after vinification (Vodopivec, 1999). The volatile fraction of wine determines to a great extent its aroma, which is one of the most Agricultural Institute of Slovenia, Hacquetova ulica 17, SI-1000 Ljubljana, Slovenia, PhD,* corresponding author: klemen.lisjak@kis.si Acta agriculturae Slovenica, 105 - 1, marec 2015 str. 40 - 123 Helena BASA CESNIK et al. important characteristics influencing wine quality and consumer preferences (García-Carpintero et al., 2012b). However, the wine volatile fraction is extremely complex, where more than 1000 aromatic compounds have been identified, originating from different chemical groups, mostly higher alcohols, aldehydes, ethyl esters of fatty acids, fatty acids, ketones, monoterpenes and volatile phenols (Andujar-Ortiz et al., 2009). A content of listed aromatic compounds in wines range from a few ng l-1 to hundreds of mg l-1 (Andujar-Ortiz et al., 2009). The presence, abundance and various combinations of volatile compounds can be greatly affected by viticultural practices (variety, cultivation, grape thinning etc.), environmental conditions (climate, soil) and oenological measures (fermentation, yeasts, postfermentation treatments etc.) (Ribéreau Gayon et al., 2006; Welke et al., 2014). Not all volatile compounds present in wine contribute to aroma (Welke et al., 2014). The influence of volatile compounds on the final aroma depends on their content and the specific perception threshold of each compound. The threshold of olfactory perception is defined as the lowest content capable of producing an olfactory sensation detected by the human nose, and that can be identified by at least 50 % of the judges on a sensory evaluation panel (Welke et al., 2014). In our research, esters were of particular interest as they are usually responsible for the typical aroma of most not-aromatic varieties of wine (Etievant 1993; Ferreira et al, 1995). One factor that supports the use of this approach is the typical fruity smell of Teran PTP wines, which is correlated to abundant contents of esters. The results of the present study are particularly interesting, since the aromatic profile of Teran PTP wine has never been investigated before and even less over a multi-year period. Previous studies of Teran PTP were mostly concentrated on phenolic compounds and their influence on wine technologies or wine quality (Novak, 2011; Vrscaj Vodosek and Kosmerl, 2004). To determine the volatile compounds in the wine, different analytical techniques were used: discontinuous or continuous liquid-liquid extraction (LLE) (Andujar-Ortiz et al., 2009), solid phase extraction (SPE) (García-Carpintero et al., 2012a; García-Carpintero et al., 2014), solid phase microextraction (SPME) (Revi et al., 2014) and stir bar sorptive extraction (SBSE) (Martínez-Gil et al., 2012; Kosmerl and Zlatic et al., 2009). It should be mentioned that the LLE technique is slowly being replaced by more manageable and solvent-free techniques. However, this type of extraction is still a reference technique used for wine aromatic compounds extraction. The main advantages of this technique are its capacity to extract a wide range of compounds of different volatilities (as long as they have an affinity to the solvent), its high repeatability level and the possibility of carrying out simultaneous extractions (Andujar-Ortiz et al., 2009). For determination, gas chromatography (GC) was used, coupled to a flame ionization detector (FID) (Pino and Queris, 2011; Moreno-Pérez et al., 2013) or a mass spectrometer (MS) (Callejón et al., 2009; Pino and Queris, 2011). The mass spectrometer is the most widely used, because it enables unequivocal qualitative and quantitative detection of substances. Quantitative detrermination of the impact of aromatic compounds on the overall aroma of wine can be done by calculating the odor activity value (OAV) and relative odour contribution (ROC). On the other hand, a qualitative evaluation can be done based on the odor descriptors of each component e.g., floral, fruity, green, solvent, plastic, toasted and others (Welke et al., 2014). The aims of the present work are to present a volatile profile of Teran PTP wine produced in the Primorska winegrowing region (Kras district) in vintages 2011, 2012 and 2013 and a differentiation of an obtained volatile profile from other red wines. We focused our research on the different groups of volatile compounds formed during alcoholic fermentation together with C6 compounds deriving from the grapes. Some wine physico-chemical characteristics were also determined for all samples and presented in the article. Acta agriculturae Slovenica, 105 - 1, marec 2015 Volatile profile of wine Teran PTP 2 MATERIALS AND METHODS 2.1 Samples The Teran PTP wine samples were collected from the winegrowing district Kras wine producers directly from stainless steel tanks and/or wooden barrels. During a three-year monitoring period, 82 wines were sampled from different producers; 39 samples from the 2011 vintage, 22 samples from the 2012 vintage and 21 samples from the 2013 vintage. The wines were sampled 9 months after fermentation, after the completion of malolactic fermentation and before bottling. Analyses of the volatile compounds and standard wine parameters were performed one month after sampling at the Central Laboratories of the Agricultural Institute of Slovenia. 2.2 Materials used for determining the volatile compounds The standard volatile compounds, with the highest available purity (minimum of 98 %), were obtained from Merck, Sigma Aldrich, Fluka and SAFC. Stock solutions of each of the standard volatile compounds in pure dichloromethane were prepared with contents ranging from 1.8 to 2.5 g l-1. From the stock solutions, one mixed solution of all the minor volatile compounds was prepared. The final standards were prepared with proper dilutions from this flask. Internal standard 4-nonanol (0.12 g dissolved in 100 ml dichloromethane) was added using a 0.05 ml Hamilton syringe to 10 ml of dichloromethane standard solution and mixed. 2.3 Extraction and determination of volatile compounds For the extraction of minor volatile compounds (^g l-1), discontinuous liquid-liquid extraction (LLE) with dichloromethane (Sigma-Aldrich) was used. The wine (100 ml) was then transferred into a 250 ml Erlenmeyer flask. To this, 23 ^g of 4-nonanol was added as an internal standard, using a 0.05 ml Hamilton syringe from the corresponding ethanol solution. Dichloromethane (40 ml) was added and the mixture was stirred at 350 rpm for 20 min. Then the mixture was centrifuged (8500 g, 10 min) and the organic phase was recovered. The aqueous phase was re-extracted twice, using the same method. Finally, the organic phases were combined and dried over sodium sulphate. They were concentrated to a final volume of 1 ml with a rotary evaporator and nitrogen gas flow prior to GC-MS analysis. This method is described in detail in other articles (Bavčar et al., 2011a; Bavčar et al., 2011b; Bavčar and Baša Česnik, 2011). 2.4 Calculation of odour activity value and relative odour contribution The OAV is a quantitative approach for determining the impact that volatile compounds have on the aroma of wine. The OAV is obtained from the ratio between the content of an individual compound and its perception threshold. A volatile compound contributes to aroma when its content in wine is above the perception threshold. Therefore, only compounds with an OAV>1 can be perceived. (Pino and Queris, 2011; Welke et al., 2014). Another quantitative factor is the relative odor contribution (ROC), which is the percentage of the impact of a particular aroma compound. It is the ratio of the OAV percentage of each individual compound and the sum of the OAV of compounds that showed OAV>1 (Welke et al., 2014). 2.5 Statistical analysis Data were collected and edited using Excel (Microsoft Office Professional Plus 2010) and analysis of variance (one-way ANOVA) was performed on physico-chemical characteristics and aromatic compounds data using Statgraphics® Centurion XVI statistical software package (StatPoint Technologies). Acta agriculturae Slovenica, 105 - 1, marec 2015 Helena BASA CESNIK et al. 3 RESULTS AND DISCUSSION Over a three-year period, we also monitored the basic parameters of Teran PTP wine. The results of the wine physico-chemical characteristics were obtained by adhering to standard EEC methods (European Union, 1990). These results are presented in Table 1. Most of statistically different physico-chemical characteristics were found in wines from 2011 vintage. But differences between the 2011, 2012 and 2013 vintages are not extensive and most probably caused only by different climatic conditions (Ribereau Gayon et al., 2006; Vodopivec, 1999). We can confirm that Teran PTP wines contain moderate levels of alcohol (12 vol %), are high in total dry extract and acidity levels, and have surprisingly low contents of both free and total sulphur dioxide in correlation with regulation's demands (Pravilnik, 2004). Table 1: Average physico-chemical characteristics of Teran PTP wines for the 2011, 2012 and 2013 vintages Preglednica 1: Povprečne fizikalno-kemijske značilnosti vin Teran PTP letnikov 2011, 2012 in 2013 Vintage 2011 Vintage 2012 Vintage 2013 n=39 n=22 n=21 Alcohol (vol. %) 12.01±0.60 A 11.95±0.58 A 12.06±0.46 A Extract (g l-1) 30.0±2.4 B 27.3±1.7 A 27.1±2.6 A Total acidity (g l-1 as tartaric) 7.5±0.7 A 8.0±0.8 B 7.5±0.8 AB Volatile acids (g l-1as acetic) 0.62±0.17 B 0.45±0.11 A 0.73±0.13 C Free SO2 (mg l-1) 13±3 B 12±1 A 12±4 A Total SO2 (mg l-1) 43±6 B 40±9 B 35±7 A pH 3.37±13 B 3.26±0.12 A 3.33±0.14 AB Relative density 0.9958±0.0001 B 0.9948±0.0007 A 0.9946±0.0008 A Lactic acid (g l-1) 2.1±0.4 B 1.5±0.4 A 2.5±0.6 C Reducing sugar (g l-1) 2.5±0.7 B 1.2±0.3 A 1.3±0.7 A n = number of samples all data present mean value ± standard deviation significant differences between vintages are indicated A, B, C at p <0.05 The focus of our research was to determine the presence of certain volatile compounds in Teran PTP wines from the 2011, 2012 and 2013 vintages. The results are presented in Table 2. We found that Teran PTP wine contains high amounts of 1-hexanol (the mean value of the three vintages was 1292 ^g l-1) and y-butyrolactone (the mean value of the three vintages was 12920 ^g l-1). We also found significant amounts of 2-phenyl-ethyl-acetate (the mean value of the three vintages was 49 ^g l-1), isoamyl acetate, benzaldehyde, benzyl alcohol, c7's-3-hexen-1-ol, ethyl butyrate, ethyl decanoate, ethyl dodecanoate, ethyl hexadecanoate, ethyl hexanoate and ethyl octanoate. Acta agriculturae Slovenica, 105 - 1, marec 2015 Volatile profile of wine Teran PTP Table 2: Contents (^g l-1) of volatile compounds in Teran PTP wines from years 2011, 2012 and 2013 and their threshold values according to ((a) Li et al., 2008; (b) Duarte et al., 2010; (c) García-Carpintero et al., 2012a; (d) Rocha et al., 2004, 2005, (e) Sánchez-Palomo et al., 2012; (f) García-Carpintero et al., 2014; (g) Welke et al., 2014) Preglednica 2: Vsebnosti (^g l-1) hlapnih spojin v vinih Teran PTP letnikov 2011, 2012 in 2013 in njihovi pragovi zaznave kot navedeno v ((a) Li et al., 2008; (b) Duarte et al., 2010; (c) García-Carpintero et al., 2012a; (d) Rocha et al., 2004, 2005, (e) Sánchez-Palomo et al., 2012; (f) García-Carpintero et al., 2014; (g) Welke et al., 2014) Vintage 2011 Vinta ge 2012 Vintage 20i3 Sensory treshold min - max average±std min - max average±std min - max average±std ALDEHYDES n-Hexaldehide (Capronaldehide) n.d. n.d. n.d. n.d. n.d. n.d. Benzaldehyde n.d. - 91 8±18 A 3.0.CO;2-Q Fornasario S., Tramer F., Žiberna L., Passamonti S. 2012. Biološka uporabnost in aktivnost pigmentov grozdja pri živalih: implikacije za zdravje ljudi, Simpozij AGROTUR, Ljubljana, 28. November 2012, pp. 7-13 García-Carpintero E. G., Gómez Gallego M. A., Sánchez-Palomo E., González Viñas M. A. 2012a. Impact of alternative technique to ageing using oak chips in alcoholic or in malolactic fermentation on volatile and sensory composition of red wines. Food Chemistry, 134: 851-863, DOI: 10.1016/j.foodchem.2012.02.194 Acta agriculturae Slovenica, iG5 - i, marec 2Gi5 Helena BASA CESNIK et al. García-Carpintero E.G., Sánchez-Palomo E., Gómez Gallego M. A., González-Viñas M. A. 2012b. Free and bound volatile compounds as markers of aromatic typicalness of Moravia Dulce, Rojal and Tortosí red wines. Food Chemistry, 131: 90-98, DOI: 10.1016/j.foodchem.2011.08.035 García-Carpintero E. G., Sánchez-Palomo E., Oliveria González Viñas M. A. 2014. Volatile composition of Bobal red wines subjected to alcoholic/malolactic fermentation with oak chips. Food Science and Technology, 55: 586-594, DOI: 10.1016/j.lwt.2013.10.024 Imre S. P., Kilmartin P. A., Rutan T., Mauk J. L., Nicolau L. 2012. Influence of soil geochemistry on the chemical and aroma profiles of Pinot noir wines. Journal of Food, Agriculture and Environment, 10: 280-288 Li H., Tao Y. S., Wang H., Zhang L. 2008. Impact odorants of Chardonnay dry white wine from Changli County (China). European Food Research and Technology, 227: 287-292, DOI: 10.1007/s00217-007-0722-9 Kosmerl T., Zlatic E. 2009. Determination of 2-aminoacetophenone in wines using the stir bar sorptive extraction method coupled with GC-MS and GC-NPD. Mitteilungen Klosterneuburg, 59: 121-126 Martínez-Gil A. M., Garde-Cerdán T., Zalacain A., Pardo-García A. I., Rosario Salinas M. 2012. Applications of an oak extract on Petit Verdot grapevines. Influence on grape and wine volatile compounds. Food Chemistry, 132: 1836-1845, DOI: 10.1016/j.foodchem.2011.12.016 Moreno-Pérez A., Vila-López R., Fernández-Fernández J. I., Martínez-Cutillas A., Gil-Muñoz R. 2013. Influence of cold pre-fermentation treatments on the major volatile compounds of three wine varieties. Food Chemistry, 139: 770-776,DOI: 10.1016/j.foodchem.2013.01.052 Novak S. 2011. The influence of oenological tannins on quality of wine Teran PTP. Graduathion thesis, Biotehnical Faculty, Ljubljana 2011, 65 str. Pino J. A., Queris O. 2011. Analysis of volatile compounds of mango wine. Food Chemistry, 125: 1141-1146, DOI: 10.1016/j.foodchem.2010.09.056 Pravilnik o vinu z oznako priznanega tradicionalnega poimenovanja - teran, Uradni list Republike Slovenije 16/2008 z dne 15.2.2008, str 1166 Ramey D., Ough C.S. 1980. Volatile ester hydrolysis or formation during storage of model solutions and wines. Journal of Agricultural and Food Chemistry, 28, 5:28-934; DOI: 10.1021/j f60231a021 Revi M., Badeka A., Kontakos S., Kontominas M.G. 2014. Effect of packaging material on enological parameters and volatile compounds of dry white wine. Food Chemistry, 152: 331-339, DOI: 10.1016/j.foodchem.2013.11.136 Ribéreau Gayon, P., Dubourdieu, D., Doneche, B., Lonvaud, A. 2006 (2nd ed). Handbook of Enology. Volume 1. The Microbiology of Wine and Vinifications. John Wiley & Sons, Chichester Rocha S.M., Coutinho P., Delgadillo I., Cardoso A.D., Coimbra M. A. 2005. Effect of enzymatic aroma release on the volatile compounds of white wines presenting different aroma potentials. Journal of the Science of Food and Agriculture, 85, 2: 199-205; DOI: 10.1002/jsfa.1937 Rocha S.M., Rodrigues F., Coutinho P., Delgadillo I., Cardoso A.D., Coimbra M. A. 2004. Volatile composition of Baga red wine. Assessment of the identification of the would-be impact odorants. Analytica Chimica Acta, 513, 1: 257-262; DOI: 10.1016/j.aca.2003.10.009 Sánchez-Palomo E., Gómez García-Carpintero E., Gómez Gallego M. A., González Viñas M. A. 2012. Gas Chromatography in Plant Science, Wine Technology, Toxicology and Some Specific Applications. The Aroma of Rojal Red Wines from La ManchaRegion -Determination of Key Odorants. Salih B., Çelikbiçak O. (eds.). Published online, InTech: 147-170, DOI: 10.5772/2517 Vanzo A., Šuklje K., Jenko M., Čuš F., Bavčar D., Lisjak K. 2012. Polifenolni potencial terana. Bioaktivne spojine Terana, Simpozij AGROTUR, Ljubljana, 28. November 2012, pp. 29-50 Vodopivec M. 1999. Kraški teran, Ljubljana, ČZP Kmečki glas: 162 str. Vrščaj Vodošek T., Košmerl T. 2004. Določanje fenolnih spojin in barvnih parametrov v vinih refošk in teran različnega geografskega porekla. V: Jubilejni 10.slovenski kemijski dnevi 2004, Maribor, 23. in 24. september 2004. Maribor 1-12 Welke J. E., Zanus M., Lazzarotto M., Alcaraz Zini C. 2014. Quantitative analysis of headspace volatile compounds using comprehensive two-dimensional gas chromatography and their contribution to the aroma of Chardonnay wine. Food Research International, 59: 85-99, DOI: 10.1016/j.foodres.2014.02.002 Acta agriculturae Slovenica, 105 - 1, marec 2015