Abstract
Five free volatile monoterpenes (linalool, α-terpineol, citronellol, nerol, and geraniol; FVmT) in free-run musts obtained from crushed grapes of white cv. Zilavka in two consecutive vintages (2011 and 2012) were quantified with headspace solid phase microextraction gas chromatography-mass spectrometry. Unexpectedly high concentrations of the analyzed FVmT were found in free-run musts of Zilavka, a variety of grapes (and wines) not previously described as having high monoterpene content. The most abundant monoterpenes were linalool, α-terpineol, and geraniol, with tens of μg/L of nerol. Citronellol was detectable, but its low concentration was quantified in free-run musts in only one experimental trial. Higher concentrations of the analyzed FVmT were found in the free-run musts obtained in 2012 after hot and dry summer months and an early grape harvest. Increasing maceration temperatures from 6 to 12°C, and then to 20°C during the 20-hr treatment resulted in increased extraction of FVmT. However, only the increases in nerol concentrations were statistically significant with increases found with higher maceration temperatures in both years. The calculated odor activity values for linalool, α-terpineol, and geraniol were relatively high (1.6 to 8). The results indicated monoterpenic potentials of Zilavka. Thus, there is an opportunity for wine producers to consider technological modifications directed to the extraction of FVmT and monoterpene profiling of Zilavka wines.
Vitis vinifera cv. Zilavka (Žilavka) is an autochthonous Herzegovinian white grape variety of commercial importance for wine production in Bosnia and Herzegovina. This cultivar comprises ~70% of the winegrape varieties in Herzegovina (FAO 2012). While Zilavka has been grown in Herzegovina for more than 600 years, there is no “standard” Zilavka genotype (Tomić et al. 2012). Zilavka wines have a good reputation in the Balkans and some European countries, and its export and prices have increased during the last decade (FAO 2012). According to available literature and prevailing winemaking practices (grape crushing, fast pressing, relatively high fermentation temperatures), Zilavka has not been considered to be an aromatic grape variety regardless of odor attributes (fruity, citrus, honey, aromatic plants, floral, and grassy) assigned to its wines by producers and merchants. To date, there have been no published studies on cv. Zilavka grape and wine aroma compounds or systematic wine sensory analyses.
Among the precursors and substances found in grapes that contribute varietal aromas to wines, the free volatile monoterpenes (FVmT) are the most studied. Terpenes are usually linked to the varietal aromas of Muscat varieties (Macaulay and Morris 1993, Belancic et al. 1997, Cabaroglu and Canbas 2002, Sánchez Palomo et al. 2006, Selli et al. 2006, del Caro et al. 2012). Several studies explored FVmT in non-Muscat variety grapes and wines, including aromatic varieties such as Gewürztraminer and Riesling (McCarthy and Coombe 1985, Marais and Rapp 1988, Reynolds and Wardle 1989, Girard et al. 2002, Skinkis et al. 2008). Free or bound terpenes (as glycosides) have been found in grapes and wines of terpene aroma neutral grapes, including Zelen, Ribolla Gialla, Malvasia Istriana, Boal, Sercial, Verdelho, Vinho Verde, Chardonnay, and Pinot blanc (Câmara et al. 2004, Genisheva and Oliveira 2009, Bavčar et al. 2011, Vilanova et al. 2013).
Researchers and practitioners seek enological techniques to increase the release of FVmT from grapes into wine or for their release from the odorless bound form into free forms with an aroma that can be perceived. Attention has been focused on prefermentative skin contact (maceration of pomace) of various times and different temperatures (Marais and van Wyk 1986, Ramey et al. 1986, Marais and Rapp 1988, Macaulay and Morris 1993, Piñeiro et al. 2006, Selli et al. 2006, Sánchez Palomo et al. 2006). Other treatments with the same purpose, such as prefermentative freezing of pomace or grapes (Bavčar et al. 2011) or heating of pomace, have been explored with variable results (Marais and van Wyk 1986, Macaulay and Morris 1993).
Herzegovinian Zilavka wine producers apply conventional technology of white winemaking with quick removal of juice from the skins (Herjavec et al. 2008). Although prefermentative maceration may threaten the quality of white wine by increased extraction of phenolic compounds, it could be a relatively simple means of increasing the aromatic potential of grapes in quality wines (Piñeiro et al. 2006, Sánchez Palomo et al. 2006, Selli et al. 2006). To date, results from only one study on prefermentative maceration of cv. Zilavka pomace have been published (Herjavec et al. 2008). This study did not address the quantitative analysis of aromatic substances in grapes and wine, but did stress the positive impact of moderate prefermentative maceration (10°C/10 hr) on the sensory properties of Zilavka wine.
The aim of this study was to quantify linalool, α-terpineol, citronellol, nerol, and geraniol in the free-run must from cv. Zilavka pomace macerated for 20 hr at three different temperatures in two consecutive vintages.
Materials and Methods
Vitis vinifera L. cv. Zilavka grapes (planted in 1975) grown in 2011 and 2012 at a commercial vineyard at Domanovici were used in this research (lat. 43°08′23″N; long. 17°46′59″E). Regular Zilavka viticultural practices were applied in the vineyard, including Moser cordon; winter and summer pruning; slightly late June defoliation in the cluster zone; annual mineral fertilization; organic fertilization once in five years; full disease, pest, and weed control; and no irrigation. In 2011, the grapes were harvested on 12 Sept (the usual harvest time for Zilavka in Herzegovina), and in 2012, the harvest was on 14 Aug. According to local winegrowers, the most probable reasons for the unusually fast ripening and early harvest of grapes in 2012 were the very high temperatures and lack of rainfall during June, July, and August of that year (Table 1). The calculated yields of grapes were ~7,500 kg/ha in 2011 and ~7,000 kg/ha in 2012, which are considered to be typical yields for Zilavka grapes in Herzegovina. Sugar (Oechsle hydrometer), total acid content (TA, as tartaric acid, OIV-MA-AS313–01; International Organisation of Vine and Wine 2014), and pH (pH-meter HI98129; Hanna Instruments, Szeged, Hungary) of grapes were measured in triplicate and the average values were calculated.
Healthy grapes were manually harvested and transported in plastic bags for processing (at a small, private winery in Stolac). Grapes were crushed using the Grifo DMC (Grifo Macchine Enologiche s.n.c., Piadena, Italy) crusher-destemmer with minimal destruction of berry skins (rollers at the widest setting). The pomace obtained was sulfurized with 100 mg/L K2S2O5 and distributed into 5-L polyethylene terephthalate plastic containers for the planned maceration treatments. The experimental prefermentative macerations of pomace were carried out at 6, 12, and 20°C, each lasting for 20 hr and each in five repetitions. After maceration, free-run musts were separated on an improvised gravity dejuicer with average yields of 550 mL/kg in 2011 and 480 mL/kg in 2012. Free-run musts were allowed to settle (4°C/4 hr) and samples for FVmT quantification were collected. Must samples were obtained in 20-mL sample vials, sealed, frozen, and stored at −18°C. All the frozen samples from one year were transported to the laboratory and analyzed in April of the next year.
Headspace solid phase microextraction was used for the extraction of FVmT, whereas identification and quantification of linalool, α-terpineol, citronellol, nerol, and geraniol was performed by gas chromatography (Agilent Technologies 7890A, Shanghai, China) coupled with a mass spectroscopy detector (Agilent Technologies 5975C). Automatic sampling was used (MPS 2, Gerstel, Germany), and a capillary column (INNOWax, 30 m × 0.25 mm; film thickness, 0.25 μm; Agilent Technologies), precolumn (fused silica deactivated, 2 m × 0.25 mm; Agilent Technologies), and liner (part number 18740–80200; Agilent Technologies) were attached on the chromatograph. A mixture of the five analyzed FVmT was used for calibration. Linearity was verified by dilution of the FVmT standards (Sigma-Aldrich, Buchs, Switzerland) and determined by linear regression using the F-test. The linear model was applied from the lowest (0.1 to 5 μg/L) to the highest (25 to 50 μg/L). From the calibration curve (R2; 0.983 to 0.996), limits of detection (0.4 to 3.0 μg/L) and limits of quantification (1.5 to 10.1 μg/L) were calculated. Both extraction and quantification were performed in the laboratory and by the methods described in Bavčar et al. (2011).
Statistical analyses of the data (variability, ANOVA, Tukey’s HSD test) were performed using OpenStat software (version Dec 2014).
Results and Discussion
Zilavka grapes grown in 2011 had 21.2% (w/v) sugar, pH 3.3, and 7.1 g/L TA, and the same grape variety grown in 2012 had 23.8% sugar, pH 3.3, and 6.1 g/L TA. The following two important points may be emphasized: (1) there were relatively high concentrations of both total FVmT and some of the five measured FVmT in free-run musts of aroma neutral Zilavka, and (2) there were higher concentrations of FVmT in free-run musts originating from the harvest in 2012 than in free-run musts originating from the harvest in 2011 (Table 2).
The total concentration of the five measured FVmT in free-run musts of Zilavka were higher than those concentrations reported for some other aroma neutral grape varieties (Câmara et al. 2004: 13 to 73 μg/L in musts of Boal, Malvasia, Sercial, and Verdelho; Genisheva and Oliveira 2009: up to 165 μg/L in the grapes of seven varieties in the appellation Vinhos Verde; Bavčar et al. 2011: 25 to 170 μg/L in wines of Zelen, Ribolla Gialla, and Malvasia Istriana). Doneva-Šapčeska et al. (2006) reported moderate concentrations of FVmT for Italian Riesling (290 μg/L) and Chardonnay grapes (280 μg/L). The total concentration of the five FVmT in the experimental free-run musts were comparable to those reported for Gewürztraminer (Girard et al. 2002, Vilanova et al. 2013) and to the concentrations reported for some Muscat varieties (Belancic et al. 1997, Cabaroglu and Canbas 2002, Doneva-Šapčeska et al. 2006, del Caro et al. 2012). Concentrations of individual monoterpenes undergo changes with grape ripening, but the total concentration usually increases from veraison to full maturity, with possible decreases if grapes are overripe (Marais and Van Wyk 1986, Reynolds and Wardle 1997, Fenoll et al. 2009). Maturing and fully mature grapes contain higher amounts of glycosidic-bonded monoterpenes than free monoterpenes (Girard et al. 2002, Doneva-Šapčeska et al. 2006). The types of FVmT and more favorable conditions for enzymatic and acidic hydrolysis of terpene conjugates with liberation of monoterpenes are both increased after the crushing of grapes (Marais and van Wyk 1986, Castro Vázquez et al. 2002, Sánchez Palomo et al. 2006). Thus, the aromatic terpene profile of wine could include free terpenes from grapes and terpenes released from glycosides.
The concentrations of single monoterpenes (linalool, α-terpineol, and geraniol) in cv. Zilavka free-run musts from the 2012 harvest were approximately twice as high as those measured in the 2011 harvest. Extremely warm and dry conditions during the grape maturation in 2012 caused forced ripening and an early harvest of grapes rich in FVmT. There are not many recent studies on the impact of temperature and humidity during ripening on the accumulation of FVmT in grapes. McCarthy and Coombe (1985) reported that unirrigated cv. Riesling vines produced grapes with more potentially volatile terpenes than grapes from irrigated vines. Some studies found positive effects of sun exposure on grape terpene concentrations (Reynolds and Wardle 1989, Macaulay and Morris 1993, Belancic et al. 1997, Reynolds and Wardle 1997). Accordingly, higher concentrations of FVmT in Zilavka grapes in 2012 than in 2011 could be partially explained by 100 hr more sunlight during June, July, and August 2012 than in the same months in 2011. The dynamics of FVmT concentrations during grape ripening was not examined in this study. Thus, the reason for the higher FVmT concentrations found in rapidly matured Zilavka grapes from the 2012 harvest remains to be explained.
Increasing temperatures during the 20-hr prefermentative maceration treatment of pomace from 6 to 12°C and from 12 to 20°C resulted in increases in total and single FVmT in Zilavka free-run musts. This is consistent with the results of studies that indicated increases in the extraction of terpenes with increasing temperatures and durations of prefermentative skin contact in the production of white wines (Marais and Rapp 1988, Cabaroglu and Canbas 2002, Piñeiro et al. 2006, Sánchez Palomo et al. 2006, Selli et al. 2006). Increased maceration temperatures were relatively more efficient in 2011 (a 9.7% increase in total FVmT with an increase in temperature from 6 to 12°C and a 25.9% increase in total FVmT with an increase in temperature from 12 to 20°C) than in 2012 (corresponding increases in total FVmT were 5.2 and 7.5% in 2012, respectively). The difference was caused by better extraction of geraniol with the temperature shift from 12 to 20°C in 2011, which was characterized by moderate weather conditions during grape ripening. This indicated that extractability of individual FVmTs depends upon the conditions of their biosynthesis. The increase in the temperature of maceration did not result in a statistically significant increase in the concentrations of any of the FVmT analyzed in the free-run musts produced in either year. In fact, only the relatively low concentrations of nerol were higher with the increase in temperature from 12 to 20°C in either year. The same increase in temperature led to a statistically significant increased extraction of linalool and geraniol in 2011, but not in 2012. Increased temperatures of maceration caused increases of the concentrations of α-terpineol in the free-run musts, but this was not statistically significant in either 2011 or 2012. Causes of the moderate increase in some FVmT concentrations with increasing maceration temperatures could be due to the thermal intensification of their rearrangements and degradation (Williams et al. 1980) or equilibrium in solution.
High concentrations of FVmT in cv. Zilavka free-run musts were due to high concentrations of linalool, α-terpineol, and geraniol, with moderate concentrations of nerol. Citronellol was quantified in one of six experimental trials. Although different data for sensory thresholds of the analyzed FVmT summarized from various sources or established for individual monoterpenes could be found (Guth 1997, Ferreira et al. 2000, Song et al. 2015), the odor activity values (OAV) for linalool, α-terpineol, and geraniol were statistically significant. Thus, using conservative thresholds of 25 μg/L for linalool (Song et al. 2015), 250 μg/L for α-terpineol (Ferreira et al. 2000), and 30 μg/L for geraniol (Guth 1997), the calculated OAV were: for linalool, 4 in 2011 and 8 in 2012; for α-terpineol, 1.6 in 2012; and for geraniol, 4 in 2011 and 7 in 2012. Some of the prefermentative maceration treatments applied yielded concentrations of FVmT comparable to those of some Muscat and esteemed aromatic non-Muscat varieties, such as Gewürztraminer. The likely additional release of FVmT from glycosides during winemaking suggests the significant potential for Zilavka wine monoterpene profiling.
Conclusion
High concentrations of FVmT in free-run musts from the macerated pomace of the autochthonous Herzegovina white variety Zilavka in two consecutive vintages were found. FVmT content of grapes was considerably higher in 2012, which was characterized by warm and dry conditions during the grape ripening and forced grape maturation. Increased temperatures and 20-hr maceration of the pomace caused increased extraction of FVmT, but the differences were not statistically significant for the FVmT quantified in either year. High OAV values for linalool and geraniol in both years were calculated. This research did not assess the possible negative effects of pomace maceration, such as the reported extraction of undesirable phenolic compounds. With regard to the possible side effects of long-lasting maceration at relatively high temperatures, the results obtained suggest 20-hr prefermentation maceration of Zilavka pomace at temperatures up to 12°C or possibly shorter maceration at 20°C would be optimal. Future studies could focus on modifications of wine technology, including fermentation regimes, which could increase the concentration of monoterpenes in wines made from cv. Zilavka grapes.
Acknowledgments
The research was funded in part by the Ministry of Education and Science of Federation of Bosnia and Herzegovina through the project No. 1261-11/12. The authors thank Hercegovina Produkt d.o.o Citluk for providing the grapes.
- Received June 2015.
- Revision received September 2015.
- Accepted September 2015.
- Published online December 1969
- ©2016 by the American Society for Enology and Viticulture
Literature Cited
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