Volatile Compounds of Young Wines from Cabernet Sauvignon, Cabernet Gernischet and Chardonnay Varieties Grown in the Loess Plateau Region of China

In order to elucidate the aroma components of wine produced in the Loess Plateau region of China, volatile compounds of young wines from Cabernet Sauvignon, Cabernet Gernischet and Chardonnay varieties grown in the new ecological region were investigated for the first time in this research. Among the volatile compounds analyzed by HS-SPME with GC-MS, a total of 45, 44 and 42 volatile compounds were identified and quantified in Cabernet Sauvignon, Cabernet Gernischet and Chardonnay wines, respectively. In the volatiles detected, alcohols formed the most abundant group in the aroma compounds of the three wines, followed by esters and fatty acids. According to their odor active values (OAVs), 18 volatile compounds were always present in the three wines at concentrations higher than their threshold values, but ethyl octanoate, ethyl hexanoate, and isoamyl acetate were found to jointly contribute to 92.9%, 93.3%, and 98.7%, of the global aroma of Cabernet Sauvignon, Cabernet Gernischet and Chardonnay wines, respectively. These odorants are associated with “fruity’’ and ‘‘ripe fruit’’ odor descriptors.

create an especially healthy environment for vines in the Loess Plateau region, but to date, there has been no published research on the volatile characteristics of wines produced in the Loess Plateau region of China, and their study could help winemakers optimize operational conditions (harvest parameters, juice preparation, fermentation techniques, use of yeasts, bacteria and enzymes, etc.) in order to emphasize one or more aromas in the final wines. In this study, we selected three representative varieties, Cabernet Sauvignon, Cabernet Gernischet and Chardonnay, which are separately cultivated at Rongzi Chateau in Xiangning County, and investigated the volatile compounds of three monovarietial wines, with volatiles being extracted by solid-phase microextraction (SPME) and detected by GC-MS.

Alcohols
Alcohols represented the largest group in terms of the number and concentration of aroma compounds identified in all three wines, followed by esters and fatty acids. The subtotal concentrations of alcohols in the three wines were in the 130,900.2-256,309.2 μg/L range, being 46.6-64.7% of the total volatile compounds detected.
Alcohols are formed from the degradation of amino acids, carbohydrates, and lipids [22]. The composition of alcohols differed both qualitatively and quantitatively among the three wines. This volatile fraction was mainly composed of isoamyl alcohol, 2-octanol, isobutyl alcohol and 2-phenylethanol; these four alcohols had concentrations >9,000 μg/L, (and existed in at least one of the wines studied). Isoamyl alcohol was the most abundant alcohol, accounting for >72% of the total alcohols in all three wines studied. Cabernet Sauvignon and Cabernet Gernischet wines contained 20 and 18 types of alcohols, respectively, and the alcohol profiles of the two wines were more diverse than that of Chardonnay wine which only contained 15 types of alcohols. (E)-2-Hexen-1-ol, 2-octanol and 2-ethyl-1-hexanol were absent in the wine made from Cabernet Gernischet and Chardonnay. Other missing alcohols in Chardonnay wine were 1-pentanol, 2-heptanol, 3-(methylthio)-1-propanol, benzyl alcohol and 1-dodecanol, and in Cabernet Gernischet wine they were (Z)-2-hexen-1-ol and 2-nonanol. In Cabernet Sauvignon wine, 1-propanol and (Z)-2-hexen-1-ol were absent, and 2-ethyl-1-hexanol was only present in trace amounts.

Esters
There were also significant differences in the type and amount of esters present in the three wines. In general, the number and proportion of esters in Chardonnay wine (34.2%) were higher than those of Cabernet Gernischet (31.0%) and Cabernet Sauvignon (26.3%), but the total ester contents in the three wines exhibited the opposite order. Although their amounts differed in the three wines, ethyl acetate, isoamyl acetate, ethyl lactate and ethyl octanoate were the major esters found in the aroma compounds. Phenethyl acetate and heptyl acetate were absent in Cabernet Sauvignon and Cabernet Gernischet wines, respectively, while isobutyl acetate was unique to Chardonnay wine. Acetate esters are the result of the reaction of acetyl-CoA with higher alcohols formed from degradation of amino acids or carbohydrates [23]. On the other hand, fatty acid ethyl esters are produced enzymatically during yeast fermentation and from ethanolysis of acyl-CoA formed during fatty acid synthesis or degradation. Their concentration depends on several factors: yeast strain, fermentation temperature, aeration degree and sugar content [23]. Ethyl lactate, a product of malolactic fermentation during wine vinification [24], was lower in Chardonnay wine than in Cabernet Sauvignon and Cabernet Gernischet wines.

Fatty acids
The production of fatty acids has been reported to be dependent on the composition of the must and fermentation conditions [25]. Acetic acid was the major fatty acid found, constituting 45.4% to 59.4% of the total fatty acid content of the wines. Acetic acid is produced during alcoholic and malolactic fermentation. At low levels this compound lifts wine flavors; however, at high levels, it is detrimental to the taste of wine by leaving the wine tasting sour and thin [26]. Heptanoic acid could only be identified as a trace component in Cabernet Sauvignon wine. Isovaleric acid and propanoic acid were absent in Cabernet Sauvignon and Chardonnay wines, respectively. Isobutyric acid, hexanoic acid, octanoic acid, and decanoic acid were found in all three wines. These C 6 to C 10 fatty acids at concentrations of 4 to 10 mg/L impart mild and pleasant aroma to wine; however, at levels beyond 20 mg/L, their impact on wine becomes negative [27]. The C 6 to C 10 fatty acids might have a positive impact on the aroma of the three wines examined in the current study since their levels were all far below 20 mg/L.

Terpenes
Numerous studies have reported that the terpenoid compounds could be used analytically for varietal characterization. It is known that terpene compounds are secondary plant constituents, whose biosynthesis begins with acetyl-CoA [28]. The formation of terpenes by Saccharomyces cerevisiae has not previously been observed and terpenes are not changed by the yeast metabolism during fermentation [29]. In the present study, three terpenes were detected in the wines. They were citronellol, linalool and limonene, and their concentrations were very low. They made up of less than 0.1% of the total volatile compounds in all three wines. Linalool was found solely in Chardonnay wine, the limonene could only be detected in Cabernet Gernischet and Chardonnay wines. Hence, these terpenyl compounds could serve as potential indicators to distinguish wine derived from Chardonnay from those from Cabernet Sauvignon and Cabernet Gernischet [8].

Aldehydes and ketones
The composition of aldehydes and ketones varied greatly between the wines. Nonanal, benzaldehyde, geranylactone and acetoin were found in all three, whereas β-ionone and decanal were unique in the aroma compounds of Cabernet Gernischet wine, and furfural existed only in Cabernet Sauvignon and Chardonnay wines.

Odor activity values (OAVs)
Though dozens of volatiles were detected in each wine sample, not all of the components have the same impact on the overall aroma character of a wine. Of all the compounds analyzed, only those displaying OAVs greater than 1 were deemed to contribute to wine aroma [30]. The contribution of each volatile compound with OAVs above 1 to the aroma of each wine can be evaluated qualitatively by means of its associate descriptor, and quantitatively by means of its OAVs.  Table 2 listed the OAVs for the 18 odor-active compounds (OAV > 1 in at least one of the wines studied), Chardonnay and Cabernet Sauvignon wines had the same odorants with OAVs above 1, and in contrast, Cabernet Gernischet wine contained the most odor-active compounds, suggesting that the Cabernet Gernischet wine presented more complex flavor than the others. In addition, the OAVs for ethyl octanoate, ethyl hexanoate and isoamyl acetate in Chardonnay wine were close to two times higher than those of Cabernet Sauvignon and Cabernet Gernishcet wines.
The computation of relative odor contribution (ROC) proposed by Ohloff [31] is a useful index for determining the important aroma components in a complex system. Based on the ROC values of single compound, we found that the global aroma of all three wines was dominated by fermentative aromas, namely, the ethyl esters of fatty acids that conferred fruity notes to all the wines. Specifically, ethyl octanoate, ethyl hexanoate, and isoamyl acetate jointly accounted for 92.9%, 93.3%, and 98.7%, of the global aroma of Cabernet Sauvignon, Cabernet Gernischet and Chardonnay wines, respectively ( Table 2). The results in this study were partially consistent with the results in a previous study [8] showing that the three aroma compounds mentioned above jointly accounted for 97% and 99%, respectively, of the global aroma of Cabernet Sauvignon and Cabernet Gernischet wines from Huailai County of China. In our study, alcohols were the predominant groups which constituted the aroma compounds rather than acids as in the previous study [8]. Thirteen aroma compounds whose concentrations are higher than threshold values from the Chardonnay wine produced in Changli County of China were identified [9]. In contrast, 12 aroma compounds whose concentrations are higher than threshold values were idenified in present study, among which seven aroma compounds, namely ethyl octanoate, ethyl hexanoate, isoamyl acetate, ethyl decanoate, octanoic acid, isoamyl alcohol, and phenethyl acetate overlapped with ones in the study above. The characteristics of local climate and soil could lead to the discrepancy between our results and others. Based on the OAVs and the ROC values of aroma compounds, ethyl octanoate, ethyl hexanoate, and isoamyl acetate are able to exert a strong influence on wine aroma: they are responsible for a major part of the aroma characteristics of young wines, but it does not mean they are the most significant aroma compounds in terms of sensory evaluation, and sensory studies are necessary to further confirm the impact of the odor-active compounds already identified. In contrast, the contributions of phenylethyl acetate and 2-phenylethanol were minimal. Based on their ROC, phenylethyl acetate and 2-phenylethanol each accounted for less than 1% of the global aroma perceptions of the three wines. According to Escudero et al. [32], even if they were present at a concentration higher than their threshold values, compounds such as fusel alcohols, acids, esters and volatile phenols are not able to affect individually the flavor of the wines. This is because these compounds are common in any kind of fermented alcoholic beverages that share similar aromatic properties. Moreover, the aromatic buffer would be caused by the presence in wine of relatively high concentrations of ethanol, ethyl esters, fusel alcohols, volatile phenols, β-damascenone, and fatty acids and can be broken only by the presence of an aroma with very different aroma properties, such as 4-methyl-4-mercaptopentan-2-one.

Vineyard conditions and vinification
All vineyards are located in the Rongzi Chateau of Xiangning County in the Loess Plateau region, which is situated approximately between 35°59′-36°02′ N, 110°47′-110°50′ E with an altitude of 1,200-1,300 m, and where the loess depth exceeds 200 m. In the collection all the vineyards have similar characteristics (age and cultivation management). All the vines were cultivated in Spring 2007, in a seedling root system with multiple main vine fan-training, and 2.5 × 1.0 m (row × vine) spacing.
All grape berries were harvested manually at optimum technological maturity, as judged by indices of sugar and acid content in 2009. Pre-fermentation treatments and winemaking were performed as described by Li [33]. Briefly, grapes were crushed on an experimental destemmer-crusher and then transferred to stainless-steel containers. Thirty L of each treatment wine were produced in three replicates. Fifty mg/L of SO 2 and 30 mg/L of pectinase (Lallzyme Ex) were added to the musts and the contents were mixed by hand. After maceration of the musts for 24 h, 200 mg/L of dried active yeast (Saccharomyces cerevisiae strain, Lallemand, Danstar Ferment AG, Switzerland) was added to the musts, according to commercial specifications. Alcoholic fermentation was carried out at 20 to 25 °C to dryness (reducing sugar < 4 g/L) which took place over a 6-8 days period and density controls were maintained during this period. At the end of alcoholic fermentation the wines were separated from pomace, and then added 50 mg/L of SO 2 . After fermentation, the wine samples were bottled and stored at 5 °C prior to analysis. All the samples were five months old at the time of analysis. Total sugar, total acidity, pH, total phenolics, total tannins, reducing sugar and ethanol were analyzed [34] (Table 3).

Reagents
All standards were purchased from Aldrich (Milwaukee, WI, USA) and Fluka (Buchs, Switzerland). Purity of all standards was above 99%. Model solutions were prepared using the methods reported by Howard et al. [35]. 4-Methyl-2-pentanol was used as the internal standard. For quantification, 8-point calibration curves for each compound were prepared using the method described by Ferreira et al. [7], which was also used as a reference to determine the concentration range of standard solutions. The regression coefficients of calibration curves were above 98%.

HS-SPME procedure
Aroma compounds of the wine samples were extracted by HS-SPME and analyzed using gas chromatography/mass spectrometry as described by Zhang et al. [8]. Five milliliters of wine sample and 1 g NaCl were placed in a 15-mL sample vial. The vial was tightly capped with a PTFE-silicon septum and heated at 40 °C for 30 min on a heating platform agitation at 400 rpm. The SPME (50/30-μm DVB/Carboxen/PDMS, Supelco, Bellefonte, PA, USA), preconditioned according to manufacturer's instruction, was then inserted into the headspace, where extraction was allowed to occur for 30 min with continued heating and agitation by a magnetic stirrer. The fiber was subsequently desorbed in the GC injector for 25 min.

GC-MS analysis
The GC-MS system used was an Agilent 6890 GC equipped with an Agilent 5975 mass spectrometer. The column used was a 60 m × 0.25 mm HP-INNOWAX capillary with 0.25 μm film thickness (J & W Scientific, Folsom, CA, USA). The carrier gas was helium at a flow rate of 1 mL/min. Samples were injected by placing the SPME fiber at the GC inlet for 25 min with the splitless mode. The oven's starting temperature was 50 °C, which was held for 1 min, then raised to 220 °C at a rate of 3 °C /min and held at 220 °C for 5 min. The mass spectrometry in the electron impact mode (MS/EI) at 70 eV was recorded in the range m/z 20 to 450 U. The mass spectrophotometer was operated in the selective ion mode under autotune conditions and the area of each peak was determined by ChemStation software (Agilent Technologies). Analyses were carried out in triplicate.

Statistical analysis
Statistical analysis was conducted by SPSS 16.0 for Windows with three replications of the same sample.

Conclusion
This work shows the first study on volatile compounds of young wines from Cabernet Sauvignon, Cabernet Gernischet and Chardonnay varieties grown in the Loess Plateau region of China. In this study, a total of 45, 44 and 42 volatile compounds were identified and quantified in Cabernet Sauvignon, Cabernet Gernischet and Chardonnay wines, respectively. The three young wines analyzed were characterized by the presence of higher levels of higher alcohols, esters and fatty acids. According to the OAVs and ROC values, 18 volatile compounds were always present in the three wines at concentrations higher than their threshold values, but ethyl octanoate, ethyl hexanoate and isoamyl acetate were the most characteristic aroma-active compounds of the three young wines.