Determination of Selected Aromas in Marquette and Frontenac Wine Using Headspace-SPME Coupled with GC-MS and Simultaneous Olfactometry

Understanding the aroma profile of wines made from cold climate grapes is needed to help winemakers produce quality aromatic wines. The current study aimed to add to the very limited knowledge of aroma-imparting compounds in wines made from the lesser-known Frontenac and Marquette cultivars. Headspace solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) with simultaneous olfactometry was used to identify and quantify selected, aroma-imparting volatile organic compounds (VOC) in wines made from grapes harvested at two sugar levels (22◦ Brix and 24◦ Brix). Aroma-imparting compounds were determined by aroma dilution analysis (ADA). Odor activity values (OAV) were also used to aid the selection of aroma-imparting compounds. Principal component analysis and hierarchical clustering analysis indicated that VOCs in wines produced from both sugar levels of Marquette grapes are similar to each other, and more similar to wines produced from Frontenac grapes harvested at 24◦ Brix. Selected key aroma compounds in Frontenac and Marquette wines were ethyl hexanoate, ethyl isobutyrate, ethyl octanoate, and ethyl butyrate. OAVs >1000 were reported for three aroma compounds that impart fruity aromas to the wines. This study provides evidence that aroma profiles in Frontenac wines can be influenced by timing of harvesting the berries at different Brix. Future research should focus on whether this is because of berry development or accumulation of aroma precursors and sugar due to late summer dehydration. Simultaneous chemical and sensory analyses can be useful for the understanding development of aroma profile perceptions for wines produced from cold-climate grapes.


Introduction
The grape berry undergoes significant changes during ripening, including acid catabolism and the accumulation of sugar, anthocyanins, flavor and aroma compounds [1]. Brix measurements correspond to the percent total soluble solids (TSS), i.e., sugar, in a given weight of grape juice. Sugar content increases throughout berry ripening and is often monitored as a function of maturity.  [31,32].
The objectives of this study were to: (1) determine the relative concentrations of VOCs in the headspace of Marquette and Frontenac wines to an internal standard; (2) perform ADA on the wine samples to characterize the most odorous compounds; and (3) calculate OAV of key aroma compounds. Effects of increased berry "hang time", i.e., the time allowed to remain on the vine before harvest, on wine aroma will help enologists use viticultural practices to enhance desired winemaking styles.

Samples, Standards, and Matrix Blank
Marquette and Frontenac grapes from the 2014 growing season were harvested at 22° and 24° Brix at South Dakota State University (Brookings, South Dakota-44.3114° N, 93.7984° W). The vineyard was part of an NE1020 evaluation trial and was a randomized complete block design. There were four replicates for each cultivar (1 replicate per block with six vines in each replicate). Vines were grown as high cordon in standard NE1020 viticulture protocol. Three clusters were taken from each vine weekly to monitor Brix and for other sample aliquots. All berries were removed from clusters and aliquots were taken from the pool of berries for each replicate. Marquette at 22° Brix was harvested on 9 September 2013 and 24° Brix on 21 September 2013. Frontenac at 22° Brix was harvested on 13 September 2013 and 24° Brix on 21 September 2013.
A single batch of red wine was made from each harvest time point using the same winemaking protocol. Briefly, 5 gallon fermenters were used for each fermentation, 3 vines from each replicate (12 vines total). Single fermentations were used as the fruit is from the NE1020 coordinated evaluation trial, not a commercial grower.
Wines were produced according to a standard lab protocol at Tucker's Walk Vineyard and Farm Winery (Garretson, SD, USA). Red grapes were mechanically crushed/destemmed, treated with SO2 to 25 ppm, and the must inoculated with Pasteur Red (Red star) yeast for fermentation on the skins at 70 °F (21.1 °C). After 5 days, must was dejuiced and fermentation continued in glass carboys at 70 °F (21.1 °C) until dry. Malolactic culture was added during the last third of the alcoholic fermentation, as determined by sugar content.  [31,32].
The objectives of this study were to: (1) determine the relative concentrations of VOCs in the headspace of Marquette and Frontenac wines to an internal standard; (2) perform ADA on the wine samples to characterize the most odorous compounds; and (3) calculate OAV of key aroma compounds. Effects of increased berry "hang time", i.e., the time allowed to remain on the vine before harvest, on wine aroma will help enologists use viticultural practices to enhance desired winemaking styles.

Samples, Standards, and Matrix Blank
Marquette and Frontenac grapes from the 2014 growing season were harvested at 22 • and 24 • Brix at South Dakota State University (Brookings, South Dakota-44.3114 • N, 93.7984 • W). The vineyard was part of an NE1020 evaluation trial and was a randomized complete block design. There were four replicates for each cultivar (1 replicate per block with six vines in each replicate). Vines were grown as high cordon in standard NE1020 viticulture protocol. Three clusters were taken from each vine weekly to monitor Brix and for other sample aliquots. All berries were removed from clusters and aliquots were taken from the pool of berries for each replicate. A single batch of red wine was made from each harvest time point using the same winemaking protocol. Briefly, 5 gallon fermenters were used for each fermentation, 3 vines from each replicate (12 vines total). Single fermentations were used as the fruit is from the NE1020 coordinated evaluation trial, not a commercial grower.
Wines were produced according to a standard lab protocol at Tucker's Walk Vineyard and Farm Winery (Garretson, SD, USA). Red grapes were mechanically crushed/destemmed, treated with SO 2 to 25 ppm, and the must inoculated with Pasteur Red (Red star) yeast for fermentation on the skins at 70 • F (21.1 • C). After 5 days, must was dejuiced and fermentation continued in glass carboys at 70 • F (21.1 • C) until dry. Malolactic culture was added during the last third of the alcoholic fermentation, as determined by sugar content. This wine was shipped to Iowa State University (Ames, IA, USA) for chemical and sensory analysis. Wine samples were aliquoted into 40 mL, pre-cleaned, glass amber vials with a PTFE lined screw cap. These vials were purged with helium to prevent oxidation of the wine samples and stored in a refrigerator before analysis.
A 5 mg/mL potassium bitartrate in 12.5% ethanol, pH 3.3 model wine was prepared by dissolving 5 g of potassium bitartrate (Fischer Scientific, Waltham, MA, USA) in 120 mL absolute ethanol (Fischer Scientific, Waltham, MA, USA) and q.s. to 1000 mL with deionized water in a 1000 mL volumetric flask. The solution was stirred for 10 min at room temperature, then filtered to remove any solids. The pH was adjusted with a 3.3 N hydrogen chloride. This model wine was used for all successive dilutions of wine samples and verified with the analytical method to be a suitable aroma and matrix blank.
For analysis of the undiluted sample, 4 mL of wine was pipetted into a cleaned 10 mL glass amber vial with metal screw top lid fitted with a PTFE-lined septum. The 10 mL vial also contained 2 g of sodium chloride, CAS 7440-23-5 (Sigma-Aldrich, St. Louis, MO, USA). 3-nonanone (99%), CAS 925-78-0 (Sigma-Aldrich, St. Louis, MO, USA), was used as an internal standard (IS) for semi-quantification of aroma compounds. The final concentration of IS in wine (0.205 mg/L) was achieved by adding 10 µL of an 81.9 mg/L IS in ethanol (w/v) to each 4 mL of wine. Triplicate samples were analyzed.

Aroma Dilution Analysis
A simple ADA was performed by analyzing successive dilutions of the wine sample, until the odor response from each compound or chromatographic column elution time region of interest was no longer noted at the olfactory detector. The odor dilution (OD) was assigned to the value of the sample dilution that results in odor extinction (i.e., not detected) at the olfactory detector (sniff port of GC). The higher the OD, the more significant that compound was in the overall aroma profile of the sample. Triplicate samples were analyzed, by a single trained panelist. Table 1 outlines dilution factors and weighting factors used in this research.

Automated GC-MS-Olfactometry System
A CTC CombiPal™ autosampler with a heated agitator (LEAP Technologies, Inc., Carrboro, NC, USA) was used during the entire experiment. The optimized sampling parameters are described elsewhere [18]. Briefly, a 1 cm 50/30 µm divinylbenzene (DVB)/carboxen (CAR)/polydimethylsiloxane (PDMS) SPME was used for headspace sampling. Extraction time was 10 min at 50 • C, after 10 min incubation at 50 • C. Agitation speed was 500 rpm. The fiber was thermally desorbed in a 260 • C GC inlet for 2 min before exposure in sample headspace. Analytes were desorbed into the GC inlet for 2 min at 260 • C.
The analysis was performed on a 6890N GC/5973 Network mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). The instrument allowed for heartcutting with a Dean's switch, cryogenic focusing, and was equipped with a FID and an olfactometry port. The GC contains two columns connected in series. The first non-polar column was a BPX-5 stationary phase with dimensions For this research, full heartcut was utilized from 0.05 to 35.00 min. An advantage of using two-dimensional gas chromatography is that separation is based on different and independent physico-chemical interactions (i.e., boiling point on the first column vs. polarity on the second column) for the entire chromatographic run. This serves as a fast screening method for VOCs, and can help select target VOCs for subsequent analysis. Sample flow was first directed through the non-polar column, then immediately to the polar column, therefore known retention indices for either column phase were used for identification. The following instrument parameters were used: injector, 260 • C; column, 40 • C initial, 3.0 min hold, 7 • C/min ramp, 220 • C final, 11.29 min hold; carrier gas, UHP helium (99.999%) with combination oxygen and moisture in-line gas trap. The mass detector was operated in electron ionization (EI) mode with an ionization energy of 70 eV. The mass detector ion source and quadrupole were held at 230 • C and 150 • C, respectively. Full spectrum scans were collected with the mass filter set from m/z 33 to m/z 450. The MS was auto-tuned daily before analysis. Use of full scan for data acquisition allowed for library search techniques using NIST11, and Wiley 6th edition mass spectral databases, and AMDIS with the accompanying food and flavor targeted mass spectral library.
Olfactometry data were generated using AromaTrax™ V.10.1 software (MOCON, Round Rock, TX, USA). Recorded parameters included an aroma descriptor ("note") and perceived intensity. The area under the peak of each aroma note in the aromagram is calculated as width × intensity × 100, where the width is the length of time (min) that the aroma persisted, in minutes. ADA was performed by analyzing successive dilutions of the same sample on the AromaTrax system V 10.1 (MOCON, Round Rock, TX, USA). The results files created have information about each aroma note such as elution time, intensity, and aroma descriptors. This information was combined into an ADA aromagram using a dilution and intensity weighting model. In this model, each peak in the results files that match a peak in the master file (Dilution Factor 0-Neat) is added to the ADA file based upon the intensity, multiplied by the dilution factor. For example, for a peak found in all 5 dilutions with intensities 90, 75, 45, 35, 5 and dilution factors of 1/2, 1/4, 1/8, 1/16, 1/32, the final intensity would be: (90 × 0.5) + (75 × 0.25) + (45 × 0.125) + (35 × 0.063) + (5 × 0.031) = 72. These values were then plotted in an analog manner, with % full-scale intensity vs. time.

Semi-Quantitative Analysis of Volatiles in Wine
Ethanol was present in all samples, including the matrix blank (model wine). The chromatographic peak at RT 3.9 min is ethanol in all total ion chromatograms (TIC). Since ethanol is present in all samples, it is not included in the further discussion. The signal-to-noise ratio of total ion chromatograms of ≥10 was considered acceptable. Percent spectral match of sample spectra to library spectra was acceptable at 65% or higher.
58 unique VOCs are detected in the headspace of Marquette and Frontenac wines produced from grapes harvested at 22 • and 24 • Brix ( lactate are present in wine samples at higher concentrations relative to 0.205 ppm of 3-nonanone IS and assuming equal detector response for all analytes. A full summary of retention times, published aroma descriptors, and relative concentrations of these 58 analytes detected in all samples are given in the Supplementary Materials, Table S1. Principal component analysis (PCA) was performed on the 58 VOCs found in all wine samples ( Figure 2). Please refer to Table 2  tetradecanoate (compound 56). Wine produced from Frontenac berries harvested at 24 • Brix (F24-1, F24-2, and F24-3) is associated with higher levels of ethyl isobutyrate, ethyl-3-methylbutanoate, ethyl lactate, and 1-hexanol. Wine produced from Marquette berries harvested at 22 • Brix (M22-1, M22-2, and M22-3) is associated with higher levels of acetaldehyde, ethyl butyrate, 1-hexanol, isoamyl butyrate, and 1-nonanal. Wine produced from Marquette berries harvested at 24 • Brix (M24-1, M24-2, and M24-3) is associated with higher levels of ethyl hexanoate, propyl octanoate, and isobutyl acetate.
Results of a PCA on wines produced from Frontenac and Marquette berries harvested at 24 and 24° Brix. This biplot shows both the relationships of the wines to each other and the associations among the VOCs detected in the headspace. Vector arrows of the VOCs are not shown, but can be assumed to originate from the origin and ending at the numbered markers. The numbered markers indicate the compound identified, and are listed in Table 2. An example of sample naming convention is F22-1 used to represent wines produced from Frontenac berries, harvested at 22° Brix, analysis replicate 1.  Table 2. An example of sample naming convention is F22-1 used to represent wines produced from Frontenac berries, harvested at 22 • Brix, analysis replicate 1.
A hierarchical cluster analysis was performed to identify the grouping of wine samples based on the degree of similarity in aroma compounds in headspace, and the constellation plot is shown in Figure 3. The cluster containing wine made from Frontenac berries harvested a 22 • Brix is the most dissimilar to the other clusters representing the rest of the wines.
A hierarchical cluster analysis was performed to identify the grouping of wine samples based on the degree of similarity in aroma compounds in headspace, and the constellation plot is shown in Figure 3. The cluster containing wine made from Frontenac berries harvested a 22° Brix is the most dissimilar to the other clusters representing the rest of the wines.

Aroma Dilution Analysis
Results of the full scan total ion chromatogram (TIC) for each 4 mL sample of wine was overlaid with the panelist generated aromagram (Supplementary Materials, Figures S1-S4). Olfactometry results including aroma descriptors, intensity, GC column retention time, and aroma event ("peak") areas were collected simultaneously with chemical analysis (Tables S1-S5). There were 15, 6, 7, and 7 aroma notes found in wines made from 22° Brix Frontenac, 24° Brix Frontenac, 22° Brix Marquette, and 24° Brix Marquette, respectively. These results were used as the master results to compare aromas from the diluted samples.
Each wine sample was diluted and analyzed again with the same methodology using dilution factors of 2, 4, 8, 16, and 32. The OD of each aroma event (detection) corresponds to the dilution of the wine in which the event was no longer present (not detected). The compound with the highest OD was contributing significantly more to the total aroma profile of the wine. After weighing the aroma notes by the dilution factor and intensities, these new values were shown in an ADA plot.
Marquette wine made from grapes harvested at 22° Brix had 14 aroma notes (excluding ethanol at 3.9 min) in the undiluted sample. After ADA (Figure 4), two events were calculated and identified as the most impactful to the total aroma profile of the wine: (1) 13.3 min, OD 8; and (2) 6.6 min, OD 2. The event at 3.9 min is ethanol, present in all samples, and therefore not included in the further discussion. These two aroma events corresponded to retention times of compounds simultaneously identified by mass spectrometer: (1) ethyl hexanoate; and (2) ethyl isobutyrate.

Aroma Dilution Analysis
Results of the full scan total ion chromatogram (TIC) for each 4 mL sample of wine was overlaid with the panelist generated aromagram (Supplementary Materials, Figures S1-S4). Olfactometry results including aroma descriptors, intensity, GC column retention time, and aroma event ("peak") areas were collected simultaneously with chemical analysis (Tables S1-S5). There were 15, 6, 7, and 7 aroma notes found in wines made from 22 • Brix Frontenac, 24 • Brix Frontenac, 22 • Brix Marquette, and 24 • Brix Marquette, respectively. These results were used as the master results to compare aromas from the diluted samples.
Each wine sample was diluted and analyzed again with the same methodology using dilution factors of 2, 4, 8, 16, and 32. The OD of each aroma event (detection) corresponds to the dilution of the wine in which the event was no longer present (not detected). The compound with the highest OD was contributing significantly more to the total aroma profile of the wine. After weighing the aroma notes by the dilution factor and intensities, these new values were shown in an ADA plot.
Marquette wine made from grapes harvested at 22 • Brix had 14 aroma notes (excluding ethanol at 3.9 min) in the undiluted sample. After ADA (Figure 4), two events were calculated and identified as the most impactful to the total aroma profile of the wine: (1) 13.3 min, OD 8; and (2) 6.6 min, OD 2. The event at 3.9 min is ethanol, present in all samples, and therefore not included in the further discussion. These two aroma events corresponded to retention times of compounds simultaneously identified by mass spectrometer: (1) ethyl hexanoate; and (2) ethyl isobutyrate.      Frontenac wine made from grapes harvested at 24° Brix had six aroma notes (excluding ethanol at 3.9 min) in the undiluted sample. After ADA (Figure 7), four events were calculated to be most impactful to the total aroma profile of the wine: (1)  (3) 1pentanol; and (4) ethyl isobutyrate. Ethyl butyrate was also present but not discussed because OD = 1 for this event. The aroma event at 13.0 min also had an OD = 1 and was not detected by the mass spectrometer. It is possible that not enough mass of analyte was directed to the mass spectrometer at retention time 13.0 min, via the open-split interface to sniff port, and therefore did not generate a chemical signal. It is likely that this compound has a very low ODT, and the human nose was a better detector for this compound.  and (4) ethyl isobutyrate. Ethyl butyrate was also present but not discussed because OD = 1 for this event. The aroma event at 13.0 min also had an OD = 1 and was not detected by the mass spectrometer. It is possible that not enough mass of analyte was directed to the mass spectrometer at retention time 13.0 min, via the open-split interface to sniff port, and therefore did not generate a chemical signal. It is likely that this compound has a very low ODT, and the human nose was a better detector for this compound. Frontenac wine made from grapes harvested at 24° Brix had six aroma notes (excluding ethanol at 3.9 min) in the undiluted sample. After ADA (Figure 7), four events were calculated to be most impactful to the total aroma profile of the wine: (1)  (3) 1pentanol; and (4) ethyl isobutyrate. Ethyl butyrate was also present but not discussed because OD = 1 for this event. The aroma event at 13.0 min also had an OD = 1 and was not detected by the mass spectrometer. It is possible that not enough mass of analyte was directed to the mass spectrometer at retention time 13.0 min, via the open-split interface to sniff port, and therefore did not generate a chemical signal. It is likely that this compound has a very low ODT, and the human nose was a better detector for this compound.  Five compounds are identified as key aromas in Marquette and Frontenac wine samples from ADA. These compounds are ethyl butyrate, ethyl decanoate, ethyl hexanoate, ethyl isobutyrate, and ethyl hexanoate. Concentrations of the five compounds in wine samples are calculated by external calibration. Quantitation range and coefficients of determination (R 2 ) from the linear model are given in Table 3. Ethyl butyrate and ethyl decanoate concentrations are lower than the quantitation limit for this method. Calculated concentrations of ethyl hexanoate, ethyl isobutyrate, and ethyl octanoate in Frontenac and Marquette wines produced from berries harvested at 22 • and 24 • Brix are summarized in Figure 8. Generally, these compounds have decreased concentrations in the headspace of wine samples as sugar content of the grapes at harvest increased. Marquette wines had higher concentrations of ethyl hexanoate (9.5 and 1.3 ppm), ethyl isobutyrate (0.7 and 0.5 ppm), and ethyl octanoate (41.9 and 17.9 ppm). Analyte concentrations in Frontenac wines were 1.3 ppm (not detected in wine produced from 24 • Brix) of ethyl hexanoate, 0.5 ppm of ethyl isobutyrate, and 21.5 and 15.6 ppm of ethyl octanoate. calibration. Quantitation range and coefficients of determination (R 2 ) from the linear model are given in Table 3. Ethyl butyrate and ethyl decanoate concentrations are lower than the quantitation limit for this method. Calculated concentrations of ethyl hexanoate, ethyl isobutyrate, and ethyl octanoate in Frontenac and Marquette wines produced from berries harvested at 22° and 24° Brix are summarized in Figure 8.

Calculation of OAV
The OAV of the three quantified compounds are calculated from published odor detection thresholds and are reported as >1000 (Figure 9). The key aroma compounds impart fruity characters to the wines as determined by a single human panelist. The dominant aroma compound in all wine samples of this study is ethyl isobutyrate (OAV > 50,000), which imparts a fruity aroma.

Calculation of OAV
The OAV of the three quantified compounds are calculated from published odor detection thresholds and are reported as >1000 (Figure 9). The key aroma compounds impart fruity characters to the wines as determined by a single human panelist. The dominant aroma compound in all wine samples of this study is ethyl isobutyrate (OAV > 50,000), which imparts a fruity aroma.

Discussion and Conclusions
In this research, headspace sampling using SPME, GC-MS and simultaneous olfactometry was used to investigate the key volatile aroma compounds in Frontenac and Marquette wines harvested at two maturity time points. ADA was performed to determine the key compounds present in these wine samples, and detected by human nose. Fifty-eight VOCs were detected in headspace of wines made from Frontenac and Marquette grapes harvested at 22° and 24° Brix by GC-MS, excluding ethanol and IS. In this study, the use of an internal standard was used to estimate relative chromatographic retention time and relative concentrations, assuming equal response factor across all analytes. A range of VOCs was detected by mass spectrometry including 9 alcohols, 5 aldehydes, 5 alkanes, 29 esters, 2 each of ketones, nitrogenous compounds, phenolic compounds and others. Yeast metabolism produces important wine volatiles such as higher alcohols, fatty acids, esters, and aldehydes. The reported results correspond to expected VOCs with the exception of diethy phthalate and dibutyl phthalate. Phalates in the wine could be an issue of contamination from packaging [34].
Marquette wines made from grapes harvested at 22° Brix had nine more aroma notes than grapes harvested at 24° Brix. The most impactful compounds as determined by ADA were ethyl hexanoate and ethyl isobutyrate (both from 22° Brix grapes); and ethyl decanoate and ethyl butyrate, both from 24° Brix grapes. Published aroma descriptors for ethyl hexanoate are apple peel and fruit, sweet and rubber for ethyl isobutyrate, grape for ethyl decanoate, and apple for ethyl butyrate [35]. Frontenac wines made from both harvest points had six aroma notes each. The most impactful compounds as determined by ADA were ethyl hexanoate and ethyl octanoate (both from 22° Brix grapes); and ethyl octanoate, ethyl decanoate, 1-pentanol, and ethyl isobutyrate from 24° Brix grapes. Published aroma descriptors for ethyl octanoate are fruit and fat, balsamic for 1-pentanol [35]. An external calibration was used to quantify ethyl hexanoate, ethyl isobutyrate, and ethyl octanoate, and OAVs were calculated. The most impactful aroma compound with calculated OAV >50,000 was ethyl isobutyrate contributes a fruity aroma.
A previous study reported β-damascenone, phenylethyl alcohol, acetic acid, linalool, and ethyl hexanoate quantified in Frontenac and Marquette grapes from veraision to harvest [9]. The compound ethyl hexanoate, responsible for fruity aromas increased in berry juice with increased growing degree days and differed by location in Pednault et al., 2013 [9]. In the wines in this study, no difference was found in ethyl hexanoate; however, these were taken from fruit with a greater level Odor detection thresholds for each analyte in water (and wine when available) from Leffingwell and Associates [33] were used to calculate OAV. OAV is the ratio of analyte concentration to its odor detection threshold. Ethyl octanoate is the dominate aroma compound present in headspace of Marquette and Frontenac wine samples (excluding alcohol), followed by ethyl hexanoate and ethyl octanoate. These compounds impart fruity aromas to the wine samples.

Discussion and Conclusions
In this research, headspace sampling using SPME, GC-MS and simultaneous olfactometry was used to investigate the key volatile aroma compounds in Frontenac and Marquette wines harvested at two maturity time points. ADA was performed to determine the key compounds present in these wine samples, and detected by human nose. Fifty-eight VOCs were detected in headspace of wines made from Frontenac and Marquette grapes harvested at 22 • and 24 • Brix by GC-MS, excluding ethanol and IS. In this study, the use of an internal standard was used to estimate relative chromatographic retention time and relative concentrations, assuming equal response factor across all analytes. A range of VOCs was detected by mass spectrometry including 9 alcohols, 5 aldehydes, 5 alkanes, 29 esters, 2 each of ketones, nitrogenous compounds, phenolic compounds and others. Yeast metabolism produces important wine volatiles such as higher alcohols, fatty acids, esters, and aldehydes. The reported results correspond to expected VOCs with the exception of diethy phthalate and dibutyl phthalate. Phalates in the wine could be an issue of contamination from packaging [34].
Marquette wines made from grapes harvested at 22 • Brix had nine more aroma notes than grapes harvested at 24 • Brix. The most impactful compounds as determined by ADA were ethyl hexanoate and ethyl isobutyrate (both from 22 • Brix grapes); and ethyl decanoate and ethyl butyrate, both from 24 • Brix grapes. Published aroma descriptors for ethyl hexanoate are apple peel and fruit, sweet and rubber for ethyl isobutyrate, grape for ethyl decanoate, and apple for ethyl butyrate [35]. Frontenac wines made from both harvest points had six aroma notes each. The most impactful compounds as determined by ADA were ethyl hexanoate and ethyl octanoate (both from 22 • Brix grapes); and ethyl octanoate, ethyl decanoate, 1-pentanol, and ethyl isobutyrate from 24 • Brix grapes. Published aroma descriptors for ethyl octanoate are fruit and fat, balsamic for 1-pentanol [35]. An external calibration was used to quantify ethyl hexanoate, ethyl isobutyrate, and ethyl octanoate, and OAVs were calculated. The most impactful aroma compound with calculated OAV >50,000 was ethyl isobutyrate contributes a fruity aroma.
A previous study reported β-damascenone, phenylethyl alcohol, acetic acid, linalool, and ethyl hexanoate quantified in Frontenac and Marquette grapes from veraision to harvest [9]. The compound ethyl hexanoate, responsible for fruity aromas increased in berry juice with increased growing degree days and differed by location in Pednault et al., 2013 [9]. In the wines in this study, no difference was found in ethyl hexanoate; however, these were taken from fruit with a greater level of ripening. However, β-damascenone, phenylethyl alcohol, acetic acid, and linalool are present in the wines from this study. Metabolic action of yeasts an influence wine aroma; specifically, precursors bound to glycosides or by decarboxylation of hydroxycinnamic acids to the equivalent vinylphenols [1]. Another study on Frontenac wine aroma reports 15 VOCs using a stir bar coated with PDMS [36]; 13 of these compounds were also detected in this study using headspace DVB/Carboxen/PDMS SPME. Similar VOCs detected were (from Table 2) compound numbers 12, 16, 19, 21, 22, 24, 27, 37, 39, 44,  46, 49, and 50. The optimized headspace SPME method yielded more compounds detected when compared to PDMS stir bar coated sample preparation method. In a previous study of commercial Frontenac wines, using a trained panel, aroma descriptors of the most impactful characters were black currant, cherry, and cooked vegetable [37]. These aromas correspond to methoxymethylbutanethiol or mercaptomethylpentanone (black currant) [35], and acrolein, butyrophenone, or cyclohexyl cinnamate (cherry) [38]. Other significant aromas in the sensory study included black berry, jammy, cooked vegetable, fresh green, cedar, floral, geranium, tamari, and earthy [37]. There is no consensus on the mechanism for explaining olfactory perception. Mixtures of odorants, even at concentrations below the detection thresholds, act in a synergistic manner [39]. This might be the key to understanding wine aroma, where hundreds of compounds are present.
A limitation to this study is the small sample size. Only one five-gallon vinification could be completed per treatment (22 • Brix and 24 • Brix) because only 12 vines were available. PCA analysis, in this study, only accounted for 48% of the variance between vinifications and is more reflective of the analytical method. More replications, repeated over several years would yield more data to draw direct conclusions about the aroma of Frontenac and Marquette wines. The olfactometry portions of this study allows for a single panelist at a time. The use of more panelists, n > 10, could help account for variation within wine consumers due to age, sex, recognition and detection thresholds, etc.
This research adds to the growing body of knowledge about lesser-known wines from cold-hardy grapes grown in Midwest U.S. states. More research is needed to understand the aroma potential of these new cold-hardy grapes to produce quality wines that can stand on the same stage as traditional Old World wines. New cold-hardy grape cultivars that are complex hybrids of Vitis vinifera and native Vitis riparia have created a cold climate wine industry in North America. Frontenac and Marquette are vigorous, disease resistant and can grow sustainably in regions with low winter temperatures [30]. While there is significant information available for the aroma of V. vinifera wines, very little is known about the aroma profile of wines from cold-hardy grapes. Research is warranted to identify potential signature aromas in these new hybrid varieties as this information can advance viticultural practices, improve marketing and bolster the local economies of cold climate vineyards and wineries.