Determination of Selected Aromas in Marquette and Frontenac Wine

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3-19-2018 Determination of Selected Aromas in Marquette and Frontenac Wine Using Headspace-SPME Coupled with GC-MS and Simultaneous Olfactometry Somchai Rice Iowa State University, [email protected]

Nanticha Lutt University of California at Berkeley

Jacek A. Koziel Iowa State University, [email protected]

Murlidhar Dharmadhikari Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/abe_eng_pubs AnnePa Frte ofnnel thel Agriculture Commons, Bioresource and Agricultural Engineering Commons, and the CSoheuthmical Dakot aE Sntgatinee Uenrivinergsit Cy ommons The ompc lete bibliographic information for this item can be found at https://lib.dr.iastate.edu/ abe_eng_pubs/888. For information on how to cite this item, please visit http://lib.dr.iastate.edu/ howtocite.html.

This Article is brought to you for free and open access by the Agricultural and Biosystems Engineering at Iowa State University Digital Repository. It has been accepted for inclusion in Agricultural and Biosystems Engineering Publications by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Determination of Selected Aromas in Marquette and Frontenac Wine Using Headspace-SPME Coupled with GC-MS and Simultaneous Olfactometry

Abstract 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.

Keywords aroma dilution analysis, wine, odor activity value, principal component analysis, solid-phase microextraction, GC-MS-Olfactometry

Disciplines Agriculture | Bioresource and Agricultural Engineering | Chemical Engineering

Comments This article is published as Somchai Rice, Nanticha Lutt, Jacek A Koziel, Murlidhar Dharmadhikari, Anne Fennell, "Determination of Selected Aromas in Marquette and Frontenac Wine Using Headspace-SPME Coupled with GC-MS and Simultaneous Olfactometry," Separations 5 (2018): 20. DOI: 10.3390/ separations5010020. Posted with permission.

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

Somchai Rice 1,2,3 ID , Nanticha Lutt 4, Jacek A. Koziel 1,2,5,* ID , Murlidhar Dharmadhikari 3,5 and Anne Fennell 6

1 Department of Agricultural and Biosystems Engineering, Iowa State University, Ames, IA 50011, USA; [email protected] 2 Interdepartmental Toxicology Graduate Program, Iowa State University, Ames, IA 50011, USA 3 Midwest Grape and Wine Industry Institute, Iowa State University, Ames, IA 50011, USA; [email protected] 4 Genetics and Plant Biology Program, University of California at Berkeley, Berkeley, CA 94704, USA; [email protected] 5 Department of Food Science and Human Nutrition, Iowa State University, Ames, IA 50011, USA 6 Plant Science Department, South Dakota State University and BioSNTR, Brookings, SD 57007, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-515-294-4206

Received: 30 December 2017; Accepted: 8 March 2018; Published: 19 March 2018

Abstract: 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.

Keywords: aroma dilution analysis; wine; odor activity value; principal component analysis; solid-phase microextraction; GC-MS-Olfactometry

1. 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.

Separations 2018, 5, 20; doi:10.3390/separations5010020 www.mdpi.com/journal/separations Separations 2018, 5, 20 2 of 15

Flavor and aroma compounds are complex. Although frequently subjected to sensory analysis, flavor and aroma compounds have limited objective measures specifically identifying the chemical and aroma links. Wines from Albillo and Muscat grape varieties (V. vinifera) have been shown to exhibit more “fruity” aromas and less “vegetal” and “floral” aromas at higher Brix during harvest [2]. Isobutyl methoxypyrazine, C6 alcohols, and hexyl acetate were shown to decrease in wine as Cabernet Sauvignon (V. vinifera) grape maturity developed [3]. The research by Bindon et al. [3] also demonstrated how higher sugar levels led to higher levels of volatile esters, dimethyl sulfide, and glycerol. Many studies have investigated the aroma profile of wines [4–7], with only a few that are focused on cold hardy grapes V. labrusca [8] or hybrids of V. vinifera, V. labrusca, and V. riparia [9,10]. In addition to TSS, pH and titratable acidity of the grapes is monitored before harvest. Some varieties of wine have a distinct varietal aroma, and these can be attributed to a few compounds. Examples include 2-methoxy-3-isobutyl pyrazine (a green bell pepper aroma) reported in Sauvignon blanc [11] or 4-vinylguaiacol (spicy, clove aromas) found in Gewurztraminer [12]. Some varieties such as Chardonnay or Seyval do not have characteristic aromas originating from one or two specific compounds [13,14]. Understanding the volatile organic compounds (VOCs) that can contribute to the overall aroma profile of wine is important in making high quality, aromatic wines. Use of solid-phase microextraction (SPME) and GC-MS is common for characterization of VOCs and other compounds in wine [15,16] and food grade alcohols [17,18]. VOC partitioning into headspace is suppressed with increasing concentration of ethanol in wine samples; ethanol influenced matrix-VOC partitioning more than glucose levels [19], possibly affecting SPME extraction efficiency of flavor and aroma compounds. SPME is an equilibrium extraction method and if extraction conditions such as extraction temperature, agitation, sampling time can be controlled in the laboratory quantitation using SPME is possible [20]. A previously developed method, optimizing the efficiency of SPME [21], is used in this research. Aroma dilution analysis (ADA) is helpful in determining the major contributor to the aroma profile of the wine. ADA has been used to evaluate aroma character of Pinot noir grapes [7], Carmenere red wine [22], and Grenache rose wines [23]. It is difficult to dilute extracts while using SPME, so an ADA approach using successive sample dilutions of wine has been demonstrated [24]. Use of SPME in place of ADA has an advantage of speed due to eliminating sample preparation steps. Perceived aroma is not only based on its chemical concentration. Another consideration is the odor detection threshold of a compound (ODT), or the lowest concentration (mass/volume) needed for detection by a human nose [25]. It is critical to recognize that any odor threshold found is valid only at the conditions the test was run. For example, the odor compound will have a different threshold in water, air, and wine and is influenced by environmental conditions during the test. Odor activity value (OAV) is the ratio of the concentration of a compound to its odor detection threshold (ODT), and has been previously discussed [26–29]. It is important to recognize that for the same compound, odor threshold and taste threshold usually have very different values. Recognition thresholds and difference detection thresholds are also used in sensory evaluation of wines, but only ODT is considered in this research. Frontenac (UMN 89 × Landot 4511) was introduced in 1996 and Marquette (MN 1094 × Ravat 262) was introduced in 2006 by University of Minnesota. These cold-climate cultivars are of importance because Marquette parentage includes Pinot Noir (grandparent), Cabernet Sauvignon and Merlot (great-great grandparents), Cabernet Franc, and Sauvignon Blanc [30]. Similar aroma profiles are expected between Marquette and Frontenac wines because the cultivars share 25% parentage from Landot Noir [31,32]. Evaluating the total aroma compounds present in headspace of Frontenac and Marquette wines is important to identify potential varietal aroma character. This information can then be used to compare the aroma of Frontenac and Marquette wines to the more recognizable vinifera wines (Figure1). Separations 2018, 5, 20 3 of 15 Separations 2018, 5, x FOR PEER REVIEW 3 of 15

Figure 1. 1. PedigreePedigree of of Frontenac Frontenac and and Marquette Marquette cultivars. cultivars. Parentage Parentage of Marquette of Marquette includes includes Pinot PinotNoir, CabernetNoir, Cabernet Sauvignon, Sauvignon, Merlot, Merlot, Cabernet Cabernet Franc, Franc, and Sauvignon and Sauvignon Blanc. Blanc.Marque Marquettette and Frontenac and Frontenac share commonshare common parentage parentage (about (about70%). This 70%). figure This is ﬁgureadapted is from adapted pedigree from maps pedigree from maps Chateau from Stripmine Chateau [31,32].Stripmine [31,32].

The objectives of this study were to: (1) determine the relative concentrations of VOCs in the 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 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 samples to characterize the most odorous compounds; and (3) calculate OAV of key aroma compounds. compounds. Effects of increased berry “hang time”, i.e., the time allowed to remain on the vine before Effects of increased berry “hang time”, i.e., the time allowed to remain on the vine before harvest, on harvest, on wine aroma will help enologists use viticultural practices to enhance desired winemaking wine aroma will help enologists use viticultural practices to enhance desired winemaking styles. styles. 2. Materials and Methods 2. Materials and Methods 2.1. Samples, Standards, and Matrix Blank 2.1. Samples, Standards, and Matrix Blank Marquette and Frontenac grapes from the 2014 growing season were harvested at 22◦ and 24◦ Brix at SouthMarquette Dakota and State Frontenac University grapes (Brookings, from the South 2014 Dakota—44.3114 growing season◦ wereN, 93.7984 harvested◦ W). at The 22° vineyard and 24° wasBrix partat South of an NE1020Dakota evaluationState University trial and (Brookings, was a randomized South Dakota—44.3114° complete block design. N, 93.7984° There wereW). fourThe vineyardreplicates was for eachpart of cultivar an NE1020 (1 replicate evaluation per blocktrial and with was six a vines randomized in each complete replicate). block Vines design. were grown There wereas high four cordon replicates in standard for each NE1020 cultivar viticulture (1 replicate protocol. per block Three with clusters six vines were in each taken replicate). from each Vines vine weeklywere grown to monitor as high Brix cordon and in for standard other sample NE1020 aliquots. viticulture All berriesprotocol. were Three removed clusters from were clusters taken from and eachaliquots vine were weekly taken to from monitor the poolBrix ofand berries for other for each sample replicate. aliquots. Marquette All berries at 22 ◦wereBrix removed was harvested from clusterson 9 September and aliquots 2013 were and taken 24◦ Brix from on the 21 pool September of berries 2013. for Frontenaceach replicate. at 22 Marquette◦ Brix was at harvested 22° Brix was on 13harvested September on 20139 September and 24◦ Brix 2013 on and 21 September24° Brix on 2013. 21 September 2013. Frontenac at 22° Brix was harvestedA single on 13 batch September of red wine 2013 was and made 24° Brix from on each21 September harvest time 2013. point using the same winemaking protocol.A single Brieﬂy, batch 5 gallonof red wine fermenters was made were from used each for harvest each fermentation, time point using 3 vines the from same each winemaking replicate (12protocol. vines total).Briefly, Single 5 gallon fermentations fermenters werewere usedused asfor the each fruit fermentation, is from the NE1020 3 vines coordinatedfrom each replicate evaluation (12 vinestrial, nottotal). a commercial Single fermentations grower. were used as the fruit is from the NE1020 coordinated evaluation trial, Winesnot a commercial were produced grower. according to a standard lab protocol at Tucker’s Walk Vineyard and Farm WineryWines (Garretson, were produced SD, USA). according Red grapes to a standard were mechanically lab protocol crushed/destemmed, at Tucker’s Walk Vineyard treated and with Farm SO2 Wineryto 25 ppm, (Garretson, and the must SD, USA). inoculated Red grapes with Pasteur were mechanically Red (Red star) crushed/destemmed, yeast for fermentation treated on the with skins SO at2 to70 25◦F ppm, (21.1 ◦andC). Afterthe must 5 days, inoculated must was with dejuiced Pasteur and Re fermentationd (Red star) yeast continued for fermentation in glass carboys on the at skins 70 ◦F (21.1at 70 ◦°FC) (21.1 until °C). dry. After Malolactic 5 days, culture must was was dejuiced added during and fermentation the last third continued of the alcoholic in glass fermentation, carboys at 70 as °Fdetermined (21.1 °C) until by sugar dry. Malolactic content. culture was added during the last third of the alcoholic fermentation, as determined by sugar content.

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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.

2.2. 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. Table1 outlines dilution factors and weighting factors used in this research.

Table 1. Dilution and weighting factors of successive wine sample dilutions used in headspace solid phase microextraction gas chromatography-mass spectrometry-olfactometry (HS-SPME GC-MS-O) aroma dilution analysis.

Sample Model Wine Total Volume Dilution Factor Weighting Factor Volume (mL) Volume (mL) (mL) 0 1.000 4.000 0.000 4 2 0.500 2.000 2.000 4 4 0.250 1.000 3.000 4 8 0.125 0.500 3.500 4 16 0.063 0.250 3.750 4 32 0.031 0.125 3.875 4

2.3. 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 Separations 2018, 5, 20 5 of 15

30 m length × 0.53 mm ID × 0.5 µm film thickness (SGE, Austin, TX, USA). The second polar column was SOLGEL-Wax stationary phase with dimensions of 30 m length × 0.53 mm ID × 0.5 µm film thickness (SGE, Austin, TX, USA). A constant pressure of 5.7 psi was maintained at the midpoint between the first and second column using MultiTrax™ V.10.1 (MOCON, Round Rock, TX, USA) system automation and MSD ChemStation™ D.02.02.275 data acquisition software (Agilent, Santa Clara, CA, USA). Additional analysis was done using MassHunter Workstation (Agilent, Santa Clara, CA, USA) with NIST11 mass spectral database and BenchTop/PBM with Wiley Registry of mass spectral data, 7th edition (Palisade Corporation, New York, NY, USA). Flow from the analytical column was directed to the single quadrupole mass selective detector and the olfactometry port in an open-split interface via fixed restrictor tubing. 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.

3. Results

3.1. 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 (Table2). Compounds including isoamyl alcohol, ethyl octanoate, 2,4-di-tert-butylphenol, ethyl acetate, ethyl hexanoate, ethyl decanoate, isocyanatomethane, and ethyl Separations 2018, 5, 20 6 of 15 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.

Table 2. Compounds identiﬁed in the headspace of Marquette and Frontenac wines using AMDIS and a lab developed mass spectral library.

No. Compound Relative Retention 1 1 Acetaldehyde 0.18 2 Methyl acetate 0.22 3 2-nitropropane 0.22 4 Isobutyraldehyde 0.22 5 Ethanol 0.25 6 Ethyl acetate 0.27 7 Acetic acid ethenyl ester 0.30 8 Methylbutanal 0.31 9 1,1-dimethyl-hydrazine 0.33 10 n-Propyl acetate 0.38 11 Isobutanol 0.39 12 Ethyl isobutyrate 0.42 13 1-butanol 0.46 14 Isobutyl acetate 0.46 15 Ethyl butyrate 0.51 16 Isoamyl alcohol 0.54 17 3-methylpentane 0.55 18 Isocyanatomethane 0.55 19 Ethyl methylbutyrate 0.58 20 Ethyl 3-methylbutanoate 0.59 21 Isoamyl acetate 0.65 22 Ethyl lactate 0.72 23 Styrene 0.73 24 1-hexanol 0.76 25 Acetic acid 0.79 26 1,2-dimethyl hydrazine 0.79 27 Ethyl hexanoate 0.85 28 1-Heptanol 0.92 29 2,3,4-trimethylpentane 0.94 30 Isoamyl butyrate 0.94 31 Undecane 0.97 32 Benzaldehyde 0.97 33 3-Nonanone (IS) 1.00 34 Methyl octanoate 1.06 35 1-Octanol 1.06 36 Octyl formate 1.06 37 Linalool 1.08 38 Phenylethanal 1.11 39 Ethyl octanoate 1.16 40 1-Nonanol 1.20 41 Pentanoic acid 1.25 42 Propyl octanoate 1.28 43 Ethyl nonanoate 1.30 44 Methyl salicylate 1.30 45 Methyl acetylsalicylate 1.30 46 Phenylethyl alcohol 1.34 47 Phenethyl isobutyrate 1.37 48 Phenethyl phenyl acetate 1.37 49 Ethyl decanoate 1.42 50 Octanoic Acid 1.47 51 β-damascenone 1.47 52 Isoamyl octanoate 1.49 53 Ethyl laurate 1.66 54 2,4-di-tert-butylphenol 1.76 55 Diethyl phthalate 1.85 56 Ethyl tetradecanoate 1.89 57 Ethyl hexadecanoate 2.14 58 Dibutyl phthalate 2.37 1 Relative retention is the ratio of the compound retention time to the retention time of the internal standard 3-nonanone, in minutes.

Principal component analysis (PCA) was performed on the 58 VOCs found in all wine samples (Figure2). Please refer to Table2 for compound identiﬁcation. Wine produced from Frontenac berries harvested at 22◦ Brix (F22-1, F22-2, and F22-3) is associated with higher levels of 1-octanol (compound 35), ethyl laurate (compound 53), methylbutanal (compound 8), undecane (compound 31), and ethyl Separations 2018, 5, x FOR PEER REVIEW 7 of 15

1 Relative retention is the ratio of the compound retention time to the retention time of the internal standard 3-nonanone, in minutes.

Principal component analysis (PCA) was performed on the 58 VOCs found in all wine samples (Figure 2). Please refer to Table 2 for compound identification. Wine produced from Frontenac berries Separationsharvested2018 at ,22°5, 20 Brix (F22-1, F22-2, and F22-3) is associated with higher levels of 1-octanol (compound7 of 15 35), ethyl laurate (compound 53), methylbutanal (compound 8), undecane (compound 31), and ethyl tetradecanoate (compound 56). Wine produced from Frontenac berries harvested at 24° Brix (F24-1, ◦ tetradecanoateF24-2, and F24-3) (compound is associated56 ).with Wine higher produced levels fromof ethyl Frontenac isobutyrate, berries ethyl-3-methylbutanoate, harvested at 24 Brix (F24-1, ethyl F24-2,lactate, and and F24-3) 1-hexanol. is associated Wine produced with higher from levels Marquette of ethyl berries isobutyrate, harvested ethyl-3-methylbutanoate, at 22° Brix (M22-1, M22-2, ethyl ◦ lactate,and M22-3) and 1-hexanol. is associated Wine with produced higher from levels Marquette of acetaldehyde, berries harvested ethyl atbuty 22rate,Brix 1-hexanol, (M22-1, M22-2, isoamyl and M22-3)butyrate, is and associated 1-nonanal. with Wine higher produced levels of from acetaldehyde, Marquette ethyl berries butyrate, harvested 1-hexanol, at 24° Brix isoamyl (M24-1, butyrate, M24- ◦ and2, and 1-nonanal. M24-3) is Wine associated produced with from higher Marquette levels of berries ethyl hexanoate, harvested atpropyl 24 Brix octanoate, (M24-1, and M24-2, isobutyl and M24-3)acetate. is associated with higher levels of ethyl hexanoate, propyl octanoate, and isobutyl acetate.

Figure 2. Results of a PCA on wines produced from Frontenac and Marquette berries harvested at 24 Figure 2. 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 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 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 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 indicate the compound identiﬁed, and are listed in Table2. An example of sample naming convention is F22-1 used to represent wines produced from Frontenac berries, harvested at 22° Brix, analysis 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 Figure3. 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. Separations 2018, 5, x FOR PEER REVIEW 8 of 15

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 SeparationsFigure 3. 2018The, 5cluster, 20 containing wine made from Frontenac berries harvested a 22° Brix is the 8most of 15 dissimilar to the other clusters representing the rest of the wines.

Figure 3. A constellation plot showing similarities in aroma compounds in Marquette and Frontenac Figure 3. A constellation plot showing similarities in aroma compounds in Marquette and Frontenac wines produced from berries harvested at 22° and 24° Brix. The wine samples are arranged as wines produced from berries harvested at 22◦ and 24◦ Brix. The wine samples are arranged as endpoints endpoints and each cluster joins as a new point, with lines drawn representing membership. The and each cluster joins as a new point, with lines drawn representing membership. The longer the line, longer the line, the greater the difference. the greater the difference.

3.2. Aroma Dilution Analysis 3.2. Aroma Dilution Analysis Results of the full scan total ion chromatogram (TIC) for each 4 mL sample of wine was overlaid 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 with the panelist generated aromagram (Supplementary Materials, Figures S1–S4). Olfactometry results including aroma descriptors, intensity, GC column retention time, and aroma event (“peak”) 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 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, 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 and 24◦ Brix Marquette, respectively. These results were used as the master results to compare aromas from the diluted samples. from the diluted samples. Each wine sample was diluted and analyzed again with the same methodology using dilution 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 factors of 2, 4, 8, 16, and 32. The OD of each aroma event (detection) corresponds to the dilution of the the wine in which the event was no longer present (not detected). The compound with the highest wine in which the event was no longer present (not detected). The compound with the highest OD OD was contributing significantly more to the total aroma profile of the wine. After weighing the was contributing significantly more to the total aroma profile of the wine. After weighing the aroma aroma notes by the dilution factor and intensities, these new values were shown in an ADA plot. 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 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 at 3.9 min) in the undiluted sample. After ADA (Figure4), 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 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 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 discussion. These two aroma events corresponded to retention times of compounds simultaneously identified by mass spectrometer: (1) ethyl hexanoate; and (2) ethyl isobutyrate. identified by mass spectrometer: (1) ethyl hexanoate; and (2) ethyl isobutyrate.

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Figure 4. Aroma dilution analysis plot of wine made from Marquette grapes harvested at 22° Brix. Figure 4. Aroma dilution analysis plot of wine made from Marquette grapes harvested at 22◦ Brix. FigureThe compound 4. Aroma most dilution impactful analysis in the plot total of winearoma made of this from sample Marquette occurred grapes at 13.3 harvested min (light at blue 22° Brix.bar), The compound most impactful in the total aroma of this sample occurred at 13.3 min (light blue Theassigned compound odor dilution most impactful number in8, theand total simultaneous aroma of thisly identified sample occurred using a maat 13.3ss spectrometer min (light blue as ethylbar), bar), assigned odor dilution number 8, and simultaneously identiﬁed using a mass spectrometer as assigned odor dilution number 8, and simultaneously identified using a mass spectrometer as ethyl ethylhexanoate. hexanoate. hexanoate.

Marquette wine made from grapes harvested at 24°◦ Brix had five aroma notes (excluding ethanol at 3.9Marquette min) in the wine undiluted made from sample. grapes After harvested ADA at(Figure 2424° Brix 5), had two five fiveevents aroma aroma were notes calculated (excluding to beethanol most atimpactful 3.9 min) to in the thethe total undilutedundiluted aroma profile sample.sample. of After the wine: ADA (1) (Figure 22.0 min, 55),), two twoOD events events8 and (2) werewere 8.3 calculatedmin,calculated OD 2. to toThese bebe mostmost two impactfularoma events to to the the correspond total total aroma aroma to profile profileretention of of the the times wine: wine: of(1) (1) compounds22.0 22.0 min, min, OD ODsimultaneously 8 and 8 and (2) (2)8.3 8.3min, identified min, OD OD2. These 2.by Thesemass two twoaromaspectrometer: aroma events events (1)correspond ethyl correspond decanoate; to retention to retentionand (2) times ethyl times butyrate.of ofcompounds compounds simultaneously simultaneously identified identified by by mass spectrometer: (1) ethyl decanoate; and (2) ethyl butyrate.

Figure 5. Aroma dilution analysis plot of wine made from Marquette grapes harvested at 24° Brix. Figure 5. Aroma dilution analysis plot of wine made from Marquette grapes harvested at 24° Brix. FigureThe compounds 5. Aroma dilutionmost impactful analysis in plot the of total wine aroma made of from this Marquette sample occurred grapes harvestedat: (1) 22.0 at min, 24◦ Brix.assigned The The compounds most impactful in the total aroma of this sample occurred at: (1) 22.0 min, assigned compoundsodor dilution most number impactful 8; and in (2) the 8.3 total min, aroma assigned of this odor sample dilution occurred number at: (1) 2, 22.0 while min, simultaneously assigned odor dilutionodoridentified dilution number using number a 8; mass and 8; (2)spectrometer and 8.3 min,(2) 8.3 assigned as:min, (1 )assigned ethyl odor decanoate; dilution odor numberdilution and (2) 2,number ethyl while butyrate. simultaneously2, while simultaneously identified usingidentified a mass using spectrometer a mass spectrometer as: (1) ethyl as: decanoate; (1) ethyl decanoate; and (2) ethyl and butyrate. (2) ethyl butyrate. Frontenac wine made from grapes harvested at 22° Brix had six aroma notes (excluding ethanol at 3.9Frontenac min) in the wine undiluted made from sample. grapes After harvested ADA (Figureat 22° Brix 6), hadtwo sixevents aroma were no tescalculated (excluding to beethanol most Frontenac wine made from grapes harvested at 22◦ Brix had six aroma notes (excluding ethanol atimpactful 3.9 min) to in the the total undiluted aroma profilesample. of After the wine: ADA (1) (Figure 13.3 min, 6), two OD events16; and were (2) 18.0 calculated min, OD to 16. be Thesemost at 3.9 min) in the undiluted sample. After ADA (Figure6), two events were calculated to be most impactfultwo aroma to events the total correspond aroma profile to retention of the wine: times (1) of 13.3 compounds min, OD simultaneously16; and (2) 18.0 identifiedmin, OD 16. by These mass impactful to the total aroma profile of the wine: (1) 13.3 min, OD 16; and (2) 18.0 min, OD 16. These twospectrometer: aroma events (1) ethyl correspond hexanoate; to retention and (2) ethyl times octanoate. of compounds simultaneously identified by mass two aroma events correspond to retention times of compounds simultaneously identified by mass spectrometer: (1) ethyl hexanoate; and (2) ethyl octanoate. spectrometer: (1) ethyl hexanoate; and (2) ethyl octanoate.

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Figure 6. Aroma dilution analysis plot of wine made from Frontenac grapes harvested at 22° Brix. The Figure 6. Aroma dilution analysis plot of wine made from Frontenac grapes harvested at 22◦ Brix. Figure 6. Aroma dilution analysis plot of wine made from Frontenac grapes harvested at 22° Brix. The Thecompounds compounds most most impactful impactful in the in total the totalaroma aroma of this of sample this sample occurred occurred at: (1) at: 13.3 (1) min, 13.3 min,assigned assigned odor compounds most impactful in the total aroma of this sample occurred at: (1) 13.3 min, assigned odor odordilution dilution number number 16; and 16; and(2) (2)18.0 18.0 min, min, assigned assigned odor odor dilution dilution number number 16, 16, while while simultaneously dilution number 16; and (2) 18.0 min, assigned odor dilution number 16, while simultaneously identifiedidentified using a massmass spectrometerspectrometer as:as: (1)(1) ethyl hexanoate;hexanoate; andand (2)(2) ethylethyl octanoate.octanoate. identified using a mass spectrometer as: (1) ethyl hexanoate; and (2) ethyl octanoate. Frontenac wine made from grapes harvested at 24° Brix had six aroma notes (excluding ethanol Frontenac wine made from grapes harvested at 24◦ Brix had six aroma notes (excluding ethanol at 3.9Frontenac min) in the wine undiluted made from sample. grapes After harvested ADA (Figure at 24° Brix 7), fourhad sixevents aroma were no tescalculated (excluding to beethanol most at 3.9 min) in the undiluted sample. After ADA (Figure7), four events were calculated to be most atimpactful 3.9 min) to in the the total undiluted aroma sample.profile of After the wine:ADA (1)(Figure 18.0 min,7), four OD events 8; (2) 22.3were min, calculated OD 8; (3)to 8.6be mostmin, impactful to the total aroma profile of the wine: (1) 18.0 min, OD 8; (2) 22.3 min, OD 8; (3) 8.6 min, impactfulOD 4; and to (4) the 6.3 total min, aroma OD 4. profileThese fourof the aroma wine: events (1) 18.0 correspond min, OD 8;to (2) retention 22.3 min, times OD of 8; compounds(3) 8.6 min, OD 4; and (4) 6.3 min, OD 4. These four aroma events correspond to retention times of compounds ODsimultaneously 4; and (4) 6.3 identified min, OD 4.by These mass fourspectrometer: aroma events (1) correspondethyl octano toate; retention (2) ethyl times decanoate; of compounds (3) 1- simultaneously identified by mass spectrometer: (1) ethyl octanoate; (2) ethyl decanoate; (3) 1-pentanol; simultaneouslypentanol; and (4) identified ethyl isobutyrate. by mass Ethyl spectrometer: butyrate was(1) ethylalso present octano butate; not(2) discussedethyl decanoate; because (3)OD 1- = and (4) ethyl isobutyrate. Ethyl butyrate was also present but not discussed because OD = 1 for this pentanol;1 for this event.and (4) The ethyl aroma isobutyrate. event at Ethyl 13.0 minbutyrate also hadwas analso OD present = 1 and but was not not discussed detected because by the ODmass = event. The aroma event at 13.0 min also had an OD = 1 and was not detected by the mass spectrometer. 1spectrometer. for this event. It isThe possible aroma that event not at enough 13.0 min mass also of had analyte an OD was = 1directed and was to notthe detectedmass spectrometer by the mass at It is possible that not enough mass of analyte was directed to the mass spectrometer at retention time spectrometer.retention time It 13.0 is possible min, via that the not open-split enough massinterf oface analyte to sniff was port, directed and therefore to the mass did spectrometer not generate ata 13.0 min, via the open-split interface to sniff port, and therefore did not generate a chemical signal. retentionchemical signal.time 13.0 It is min, likely via that the this open-split compound interf hasace a very to sniff low port,ODT, and and thereforethe human did nose not was generate a better a It is likely that this compound has a very low ODT, and the human nose was a better detector for chemicaldetector for signal. this Itcompound. is likely that this compound has a very low ODT, and the human nose was a better detectorthis compound. for this compound.

Figure 7. Aroma dilution analysis plot of wine made from Frontenac grapes harvested at 24° Brix. The Figure 7. Aroma dilution analysis plot of wine made from Frontenac grapes harvested at 24° Brix. The Figurecompounds 7. Aroma most dilutionimpactful analysis in the total plot aroma of wine of madethis sample from Frontenacoccurred at: grapes (1) 18.0 harvested min, assigned at 24◦ odorBrix. compounds most impactful in the total aroma of this sample occurred at: (1) 18.0 min, assigned odor Thedilution compounds number most 8; (2) impactful 22.3 min, in assigned the total odor aroma dilution of this samplenumber occurred 8; (3) 8.6 at: min, (1) 18.0assigned min, assignedodor dilution odor dilutionnumber 4;number and (4) 8; 6.3 (2) min, 22.3 assignedmin, assigned odor odor dilution dilution number number 4, while 8; (3) simultaneously 8.6 min, assigned identified odor dilution using a numbermass spectrometer 4; and (4) 6.3 as: min,(1) ethyl assigned octanoate; odor (2)dilution ethyl denumber canoate; 4, while (3) 1-pentanol; simultaneously and (4) identified identiﬁedethyl isobutyrate. using a mass spectrometer as: (1) ethyl octanoate; (2) ethyl de decanoate;canoate; (3) 1-pentanol; an andd (4) ethyl isobutyrate. Five compounds are identified as key aromas in Marquette and Frontenac wine samples from ADA.Five These compounds compounds are are identified ethyl butyrate, as key aromas ethyl deca in Marquettenoate, ethyl and hexanoate, Frontenac ethyl wine isobutyrate, samples from and ADA.ethyl hexanoate. These compounds Concentrations are ethyl of butyrate, the five ethylcompou decandsnoate, in wine ethyl samples hexanoate, are calculatedethyl isobutyrate, by external and ethyl hexanoate. Concentrations of the five compounds in wine samples are calculated by external

Separations 2018, 5, 20 11 of 15

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 Separations 2018, 5, x FOR PEER REVIEW 11 of 15 ethyl hexanoate. Concentrations of the five compounds in wine samples are calculated by external 2 calibration. Quantitation Quantitation range range an andd coefficients coefficients of determination of determination (R2) (Rfrom) from the linear the linear model model are given are givenin Table in Table3. Ethyl3. Ethylbutyrate butyrate and et andhyl ethyldecanoate decanoate concentrations concentrations are lo arewer lower than thanthe quantitation the quantitation limit limitfor this for method. this method. Calculated Calculated concentrations concentrations of ethyl ofhexanoate, ethyl hexanoate, ethyl isobutyr ethylate, isobutyrate, and ethyl andoctanoate ethyl ◦ ◦ octanoatein Frontenac in Frontenac and Marquette and Marquette wines winesproduced produced from fromberries berries harvested harvested at 22° at 22 andand 24° 24 BrixBrix are summarized inin Figure Figure8. Generally,8. Genera theselly, these compounds compounds have decreased have decreased concentrations concentrations in the headspace in the ofheadspace wine samples of wine as samples sugar content as suga ofr content the grapes of the at grapes harvest at increased.harvest increased. Marquette Marquette wines had wines higher had concentrationshigher concentrations of ethyl of hexanoate ethyl hexanoate (9.5 and (9.5 1.3 and ppm), 1.3 ppm), ethyl isobutyrateethyl isobutyrate (0.7 and (0.7 0.5 and ppm), 0.5 ppm), and ethyl and octanoateethyl octanoate (41.9 and(41.9 17.9 and ppm). 17.9 Analyteppm). Analyte concentrations concentrations in Frontenac in Frontenac wines were wines 1.3 were ppm 1.3 (not ppm detected (not ◦ indetected wine producedin wine produced from 24 fromBrix) 24 of° Brix) ethyl of hexanoate, ethyl hexanoate, 0.5 ppm 0.5 ofppm ethyl of ethyl isobutyrate, isobutyrate, and 21.5and 21.5 and 15.6and ppm15.6 ppm of ethyl of ethyl octanoate. octanoate.

Table 3.3. QuantitationQuantitation range range and and linear linear coefficients coefﬁcients of of determination determination for for 5 key 5 key aroma aroma compounds compounds in inwine. wine.

CompoundCompound Quantitation Quantitation Range (ppm)(ppm)R R2 2 −2 2 EthylEthyl hexanoate hexanoate 2.89 × × 10−2–1.16–1.16 × 10102 0.9980.998 EthylEthyl isobutyrate isobutyrate 2.86 × × 10−−22–1.15–1.15 × 10102 0.9990.999 EthylEthyl octanoate octanoate 2.88 × × 10−−22–1.15–1.15 × 10102 0.9980.998 −2 2 EthylEthyl decanoate decanoate 2.79 × × 10 −2–1.12–1.12 × 10102 0.9970.997 Ethyl butyrate 2.93 × 10−2–1.17 × 102 0.999 Ethyl butyrate 2.93 × 10−2–1.17 × 102 0.999

Figure 8. Concentrations of target analytes in headspace of Marquette and Frontenac wines made Figure 8. Concentrations of target analytes in headspace of Marquette and Frontenac wines made from from berries harvested at 22° and 24° Brix. A five-point external calibration was performed using berries harvested at 22◦ and 24◦ Brix. A ﬁve-point external calibration was performed using standards standards spiked into model wine. Data represent the mean of replicate analysis (n = 3) of each wine spiked into model wine. Data represent the mean of replicate analysis (n = 3) of each wine produced. produced. F22 represents Frontenac wine produced from berries harvested at 22° Brix, F24 represents F22 represents Frontenac wine produced from berries harvested at 22◦ Brix, F24 represents Frontenac wineFrontenac produced wine fromproduced berries from harvested berries atharvested 24◦ Brix, at etc. 24° Brix, etc.

3.3. Calculation of OAV 3.3. Calculation of OAV The OAV of the three quantified compounds are calculated from published odor detection 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 thresholds and are reported as >1000 (Figure9). The key aroma compounds impart fruity characters to the wines as determined by a single human panelist. The dominant aroma compound in all wine 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. samples of this study is ethyl isobutyrate (OAV > 50,000), which imparts a fruity aroma.

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FigureFigure 9. Odor9. Odor (aroma) (aroma) activity activity values values ofof targettarget analytesanalytes in in headspace headspace of of Marquette Marquette and and Frontenac Frontenac wineswines made made from from berries berries harvested harvested at at 22 22°◦ and and 2424°◦ BrBrix.ix. Odor Odor detection detection thresholds thresholds for for each each analyte analyte in in waterwater (and (and wine wine when when available) available) from from Leffingwell Leffingwell and an Associatesd Associates [33 [33]] were were used used to to calculate calculate OAV. OAV. OAV OAV is the ratio of analyte concentration to its odor detection threshold. Ethyl octanoate is the is the ratio of analyte concentration to its odor detection threshold. Ethyl octanoate is the dominate dominate aroma compound present in headspace of Marquette and Frontenac wine samples aroma compound present in headspace of Marquette and Frontenac wine samples (excluding alcohol), (excluding alcohol), followed by ethyl hexanoate and ethyl octanoate. These compounds impart fruity followed by ethyl hexanoate and ethyl octanoate. These compounds impart fruity aromas to the aromas to the wine samples. wine samples. 4. Discussion and Conclusions 4. Discussion and Conclusions In this research, headspace sampling using SPME, GC-MS and simultaneous olfactometry was usedIn thisto investigate research, headspacethe key volatile sampling aroma usingcompounds SPME, in GC-MS Frontenac and and simultaneous Marquette wines olfactometry harvested was usedat totwo investigate maturity time the keypoints. volatile ADA aroma was performed compounds to determine in Frontenac the andkey compounds Marquette winespresent harvested in these at twowine maturity samples, time and points. detected ADA by was human performed nose. Fifty- to determineeight VOCs the were key compoundsdetected in headspace present in of these wines wine samples,made from and detectedFrontenac by and human Marquette nose. Fifty-eightgrapes harvested VOCs at were 22° detectedand 24° Brix in headspace by GC-MS, of excluding wines made fromethanol Frontenac and andIS. In Marquette this study, grapes the harvesteduse of an at in 22ternal◦ and standard 24◦ Brix bywas GC-MS, used to excluding estimate ethanol relative and IS. Inchromatographic this study, the useretention of an internaltime and standard relative wasconcen usedtrations, to estimate assuming relative equal chromatographic response factor retentionacross timeall and analytes. relative A range concentrations, of VOCs was assuming detected equal by ma responsess spectrometry factor across including all analytes. 9 alcohols, A 5 range aldehydes, of VOCs was5 detectedalkanes, 29 by esters, mass 2 spectrometry each of ketones, including nitrogenous 9 alcohols, compounds, 5 aldehydes, phenolic 5 compounds alkanes, 29 and esters, others. 2 each Yeast metabolism produces important wine volatiles such as higher alcohols, fatty acids, esters, and of ketones, nitrogenous compounds, phenolic compounds and others. Yeast metabolism produces aldehydes. The reported results correspond to expected VOCs with the exception of diethy phthalate important wine volatiles such as higher alcohols, fatty acids, esters, and aldehydes. The reported and dibutyl phthalate. Phalates in the wine could be an issue of contamination from packaging [34]. results correspond to expected VOCs with the exception of diethy phthalate and dibutyl phthalate. Marquette wines made from grapes harvested at 22° Brix had nine more aroma notes than grapes Phalates in the wine could be an issue of contamination from packaging [34]. harvested at 24° Brix. The most impactful compounds as◦ determined by ADA were ethyl hexanoate andMarquette ethyl isobutyrate wines made (both from from grapes 22° Brix harvested grapes); and at 22 ethylBrix decanoate had nine and more ethyl aroma butyrate, notes both than from grapes ◦ harvested24° Brix atgrapes. 24 Brix. Published The most aroma impactful descriptors compounds for ethyl hexanoate as determined are apple by ADA peel and were fruit, ethyl sweet hexanoate and ◦ andrubber ethyl for isobutyrate ethyl isobutyrate, (both from grape 22 forBrix ethyl grapes); decano andate, ethyl and apple decanoate for ethyl and butyrate ethyl butyrate, [35]. Frontenac both from ◦ 24 winesBrix grapes.made from Published both harvest aroma points descriptors had six for aroma ethyl notes hexanoate each. The are most apple impactful peel and compounds fruit, sweet as and rubberdetermined for ethyl by isobutyrate, ADA were ethyl grape hexanoate for ethyl and decanoate, ethyl octanoate and apple (both forfrom ethyl 22° butyrateBrix grapes); [35 ].and Frontenac ethyl winesoctanoate, made fromethyl bothdecanoate, harvest 1-pentanol, points had and six ethyl aroma isobutyrate notes each. from The 24° Brix most grapes. impactful Published compounds aroma as determineddescriptors by for ADA ethyl were octanoate ethyl hexanoateare fruit and and fat, ethyl balsamic octanoate for 1-pentanol (both from [35]. 22 An◦ Brix external grapes); calibration and ethyl octanoate,was used ethyl to quantify decanoate, ethyl 1-pentanol, hexanoate, and ethyl ethyl isobutyrate, isobutyrate and from ethyl 24◦ octanoate,Brix grapes. and Published OAVs were aroma descriptorscalculated. for The ethyl most octanoate impactful are aroma fruit compound and fat, balsamic with calculated for 1-pentanol OAV >50,000 [35]. was An externalethyl isobutyrate calibration wascontributes used to quantify a fruity ethyl aroma. hexanoate, ethyl isobutyrate, and ethyl octanoate, and OAVs were calculated. The mostA impactfulprevious study aroma reported compound β-damascenone, with calculated phenylethyl OAV >50,000 alcohol, was acetic ethyl acid, isobutyrate linalool, and contributes ethyl a fruityhexanoate aroma. 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 A previous study reported β-damascenone, phenylethyl alcohol, acetic acid, linalool, and ethyl growing degree days and differed by location in Pednault et al., 2013 [9]. In the wines in this study, hexanoate quantified in Frontenac and Marquette grapes from veraision to harvest [9]. The compound no difference was found in ethyl hexanoate; however, these were taken from fruit with a greater level 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 Separations 2018, 5, 20 13 of 15 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 Table2) 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.

Supplementary Materials: The following are available online at http://www.mdpi.com/2297-8739/5/1/20/s1. Acknowledgments: This study was funded by the USDA’s Specialty Crops Research Initiative Program of the National Institute for Food and Agriculture, Project #2011-5118130850. Author Contributions: S.R. and J.A.K. conceived and designed the experiments; N.L. and S.R. performed the experiments; S.R. analyzed the data; J.A.K., A.F. and M.D. contributed reagents/materials/analysis tools; and S.R., A.F., M.D., and J.A.K. wrote the paper. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

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