Volatile profile and aroma potential of tropical Syrah wines elaborated in different maturation and maceration times using comprehensive two-dimensional gas chromatography and olfactometry

Journal Pre-proofs Volatile profile and aroma potential of tropical Syrah wines elaborated in different maturation and maceration times using comprehensive two-dimensional gas chromatography and olfactometry Janaína A. Barbará, Karine P. Nicolli, Érica A. Souza-Silva, Aline C.T. Biasoto, Juliane E. Welke, Cláudia A. Zini PII: DOI: Reference:

S0308-8146(19)31676-0 https://doi.org/10.1016/j.foodchem.2019.125552 FOCH 125552

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

20 May 2019 11 September 2019 16 September 2019

Please cite this article as: Barbará, J.A., Nicolli, K.P., Souza-Silva, E.A., Biasoto, A.C.T., Welke, J.E., Zini, C.A., Volatile profile and aroma potential of tropical Syrah wines elaborated in different maturation and maceration times using comprehensive two-dimensional gas chromatography and olfactometry, Food Chemistry (2019), doi: https:// doi.org/10.1016/j.foodchem.2019.125552

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Volatile profile and aroma potential of tropical Syrah wines elaborated in different maturation and maceration times using comprehensive two-dimensional gas chromatography and olfactometry

Running title: Odor & volatiles of tropical Syrah wines by GC×GC/MS & GColfactometry

Janaína A. Barbaráa, Karine P. Nicollia, Érica A. Souza-Silvab, Aline C. T. Biasotoc, Juliane E. Welked, Cláudia A. Zinia*

a Universidade

Federal do Rio Grande do Sul (UFRGS), Institute of Chemistry, zip code

91501970, Porto Alegre, Brazil b

Universidade Federal de São Paulo (UNIFESP). Institute of Environmental, Chemical

and Pharmaceutical Sciences, zip code 09913-030, Diadema, Brazil c Embrapa d

Semi-Arid, zip code 56302970, Petrolina, Brazil

UFRGS, Institute of Food Science and Technology, zip code 91501970, Porto Alegre,

Brazil

* Corresponding author. Phone: 55 51 33 08 72 17; Fax 55 51 33 37 04 42. Email address:

[email protected]

(C.A.

Zini)

1

Abstract

The influence of different combinations of Syrah grape maturation degree (19, 21 and 23 °Brix) and maceration times (10, 20 and 30 days) on the volatile profile and aroma potential was evaluated for the first time through different chromatographic platforms (GC×GC/TOFMS, GC-O-OSME, GC-FID and GC/MS). GC×GC/TOFMS analyses resulted in 145 identified compounds and among these 29 were determined to be the most important for wine differentiation. The aroma compounds allowed the discrimination of Syrah wines made with grapes macerated for a shorter time (ten days) due to the higher levels of volatile compounds. The evaluation of these wines through GC-O-OSME together with GC-FID, MS resulted in the designation of 19 °Brix as the most appropriate grape maturation degree to obtain a greater number of volatiles with pleasant odor and higher intensity and persistence. GC×GC/TOFMS allowed five and six co-elutions to be resolved, involving, respectively, ten and twelve important wine compounds.

Keywords: tropical wine; Syrah wine; GC×GC/TOFMS; GC-O-OSME; volatile compounds; odor; olfactometry.

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1. Introduction

Tropical wine regions such as the Northeast (NE) of Brazil, India, Thailand and Venezuela (Jogaiah et al., 2013) present the combination of high temperature and intense solar radiation on the grapevines, which allows ideal levels of grape maturation to be obtained (soluble solids ~ 20 °Brix) and acidity (titratable acidity of 6-8 g L-1). Syrah grapes have easily adapted to the wine region of São Francisco Valley (NE Brazil) due to climatic conditions and Syrah wines represent 30% of the production of this region. There is a great need for a better characterization of the wine quality of this region in all aspects in order to reach a deeper scientific and technical understanding about the real nature of these wines. There is a further need to achieve a Geographical Indication (GI process started in 2014) and, in the near future, a denomination of origin (DO) (Empresa Brasileira de Pesquisa Agropecuária, 2019) Grape maturation is associated with softening of tissues, decrease in acidity, accumulation of sugars, synthesis of anthocyanins in red varieties and formation of compounds with aroma potential. This physiological period begins in the veraisón (beginning of berry color change) and ends with the mature grape, lasting about 40 days, depending on grape variety, environmental conditions, as well as on the agricultural practices employed (Coelho, Rocha, Barros, Delgadillo, & Coimbra, 2007). The maceration period refers to the release of grape constituents (seeds, skin and pulp) after crushing, which is facilitated by the release and activation of hydrolytic enzymes from crushed cells (Jackson, 2014). During this step, the content of compounds related to aroma increases due to their extraction mainly from the skins of the grapes (Romano, Fiore, Paraggio, Caruso, & Capece, 2003).

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A harmonious combination between the periods of maturation and maceration is essential to reach wine pleasant aroma notes as well as to achieve a balanced wine astringency and color intensity. During grape ripening some volatiles and their precursors are synthesized from the catabolism of fatty acids, amino acids, sugars, pectin and carotenoids of berries, resulting in compounds of different chemical classes. In other words, ideally phenolic and aromatic maturation should match with technological maturation. However, this may or not happen and the status of both maturations should be verified to guarantee maximum overall wine quality. Volatile composition and its final sensory contribution are among the most important attributes of a high quality wine, directly influencing the consumer´s choice (Ribéreau-Gayon, Dubourie, Donèche, & Lonvaud, 2006). Hundreds of volatile compounds have been identified in wine, however only a few of these compounds are aroma active. The relationship between the volatile profile and the elucidation of aroma-active compounds can be achieved through the use of gas chromatography (GC) combined with different detectors including olfactometry (GC-O), mass spectrometry (GC/MS) and flame ionization (GC-FID). Nonetheless, the occurrence of co-elutions of wine volatiles in one-dimensional GC (1D-GC) has turned comprehensive two-dimensional gas chromatography (GC×GC) into a necessary tool when the elucidation of identity/aroma of each volatile compound is mandatory for the elucidation of wine aroma (Nicolli et al., 2018). The combined use of GC-O and GC×GC with mass spectrometric detector (TOFMS) helped to resolve co-elutions of compounds that were found in some potent odoriferous chromatographic regions of a commercial Merlot wine and also of a blend Sauvignon Blanc and Semillon wine from Australia, which were designated by a frequency detection (FD) sensory panel (Chin, Eyres, & Marriott, 2011). Later on, this same group investigated other Australian commercial Syrah wines

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(Chin, Eyres, & Marriott, 2015). Even though FD methods are simple, fast and do not require a trained panel of judges, their results provide only the intensity of the perceived odor when the compound is present in a given concentration, but it does not report the duration of the olfactometric event (Plutowska & Wardencki, 2008). Villiers et al. (2012) followed this same strategy and studied the odoriferous regions of two French ciders, but using OSME (, Greek word for odor) (McDaniel, Miranda-Lopez, Watson, & Libbey, 1990). This technique gives the intensity of any odor in a time scale, allowing to achieve reproducible, consistent and fast results, although it requires a trained sensory panel. One of the most significant aroma compounds for ciders (oct-1en-3-one) revealed by GC-O-OSME had its identity elucidated only with GC×GC/TOFMS. However, these research works have focused only in qualitative analysis

of

the

odoriferous

bands

shown

in

GC-O-OSME,

followed

by

GC×GC/TOFMS. Australian Syrah wines from Barossa Valley and Margaret River have been characterized by GC-O-AEDA (Aroma Extract Dilution Analysis) and GC/MS and only the most important odoriferous compounds have been quantified (Mayr et al., 2014). AEDA is a dilution to threshold method where the number of panel judges may be limited due to long time of analyses required and this can turn into lower precision and subjective results. It is based on the assumption that concentration and odor intensity increase in parallel for all odoriferous compounds in a sample, which is not true (Plutowska & Wardencki, 2008). In addition, the effect of grape shriveling on qualitative and quantitative volatile compounds of the same varietal wine of Australia was investigated for three different harvest periods, employing only GC/MS (Šuklje et al., 2016). A second publication of this same group showed some relation between qualitative volatile composition of Syrah wines and two different levels of maturity of

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the corresponding grapes, employing HS-SPME-GC×GC/TOFMS (Šuklje et al., 2019). Similar approach has been applied to the characterization of Chardonnay wines from Serra Gaúcha, although olfactometry was has not been employed in neither one of these reports (Welke, Zanus, Lazzarotto, & Zini, 2014). In a previous study of this group, the combination of GC×GC/TOFMS, GC-OOSME, GC-FID and GC/MS was found to be important to evaluate the influence of vine management on the quality of Merlot wines, taking into account all 220 volatile compounds (Nicolli et al., 2018). In addition, an investigation dealing with volatiles of Syrah wines of São Francisco Valley has shown a trend towards higher concentrations of some pleasant aroma volatiles, increased total phenolic content and color intensity with longer maturation (> 21 °Brix) and maceration periods (20 to 30 days), even though only GC/MS and odor activity value (OAV) were employed in this case (Barbará et al., 2019). OAV is obtained from the ratio between the concentration of an individual compound and its perception threshold. A volatile compound contributes to aroma when its concentration in wine is above the perception threshold. Therefore, odorants with OAV > 1 can be perceived (Guth, 1997). To the best of the authors’ knowledge, this is the first time that a more comprehensive and detailed study of a volatile profile as well as its odoriferous impact on Syrah wine aroma has been investigated, including different analytical techniques (GC×GC/TOFMS, GC-O-OSME, GC/FID and GC/MS). This study also aims to better characterize volatile compounds of São Francisco Valley Syrah wines in order to help achieving GI and DO for this emerging wine region, which is among the most important tropical wine regions of the world (Empresa Brasileira de Pesquisa Agropecuária, 2019) Moreover, the effect of different combinations of maturation degree and time of maceration of Syrah grapes on the wine volatile profile and aroma potential has been

6

investigated for the São Francisco Valley region, where the phenolic/aromatic maturation of the grapes may not be ideal at the time of harvest due to specific climatic characteristics.

2. Material and methods

2.1 Analytical reagents and supplies

Standard compounds including isobutanoic acid (2-methylpropanoic acid), isovaleric acid (3-methylbutanoic acid), 2-methyl valeric acid, hexanoic acid, octanoic acid, nonanoic acid, decanoic acid, dodecanoic acid, 1-propanol, 1-hexanol, 2-hexen-1ol, 2,3-butanedione, 1-nonanol, benzyl alcohol, 2-phenylethyl alcohol, 1-dodecanol, ethyl 3-methylbutanoate, ethyl hexanoate, hexyl acetate, ethyl octanoate, methyl decanoate, ethyl decanoate, diethyl succinate (diethyl butanedioate), 2-phenethyl acetate, ethyl dodecanoate, furfural, 2-furanmethanol, 2-heptanone, 2(5H)-furanone, 4ethylphenol, eucalyptol, α-terpineol, citronellol, β-damascenone, geraniol, guaiacol, 3mercaptohexanol with were purchased from Sigma-Aldrich (Steinheim, Germany) with purity higher than 98%. For each volatile compound a stock solution was prepared (10 mg L-1) in double-distilled ethanol purchased from Nuclear (São Paulo, Brazil). Standard solutions were diluted in a wine model solution, in order to obtain a matrix similar to wine with respect to ethanol (14%) and acidity (pH ranging from 3.3 to 3.5). The model wine was prepared with 6 g L-1 of (+) – tartaric acid (Synth, São Paulo, Brazil), ethanol 14% in MilliQ deionized water (purification system Millipore, Bedford, MA, USA) and the pH was adjusted to 3.5 with sodium hydroxide (Nuclear, São Paulo, Brazil), as previously reported (Welke, Zanus, Lazarotto, Schmitt, & Zini,

7

2012).

The

solid-phase

microextraction

(SPME)

fiber

used

was

Divinylbenzene/Carboxen/Polydimethylsiloxane (2 cm DVB/CAR/PDMS - 50/30µm StableFlex- Supelco, Bellefonte, PA, USA) and conditioned according to the manufacturer’s recommendations prior to its first use. Sodium chloride (NaCl) was purchased from Nuclear (São Paulo, Brazil) and was oven dried at 150 ºC for two hours.

2.2 Experimental designs, samples and wine production

The grapes used were obtained from an experimental area belonging to Fazenda Ouro Verde, located in Casa Nova, BA, Brazil (latitude: 9 ° 16'S; longitude: 40 ° 52'O; altitude: 413.5 m). The experimental design was in randomized blocks, with three replicates per treatment (referring to three stages of maturation), where 48 plants were used per repetition, reaching a total of 432 plants in the experiment, as detailed in a previous study (Barbará et al., 2019). Grapes were harvested in June and July 2013, at intervals of seven days, corresponding to (T1) before technological or industrial maturity, 113 days after pruning (DAP), with total soluble solids content of 19 ºBrix; (T2) ideal ripeness degree, 120 DAP with 21 ºBrix; and (T3) overripe grapes, 127 DAP and 23 ºBrix. These grapes were macerated (M) for three different times (10, 20 and 30 days and these maceration processes were named as M1, M2 and M3, respectively), resulting in eight combinations of experiments as follows: T1M1, T1M2, T2M1, T2M2, T2M3, T3M1, T3M2 and T3M3, as formerly described (Barbará et al., 2019). The combination T1M3 was not evaluated due to oenological problems related to the excessive astringency of this wine. There were less polymerized tannins in grapes harvested before technological maturity (T1) and the extension of maceration for 30

8

days resulted in unacceptable astringency in wine, which makes this combination of ripeness degree and maceration time an unfeasible industrial practice. Winemaking was performed in triplicate by the traditional method for young red wines. A more detailed explanation is given elsewhere (Barbará et al., 2019)

2.3 Determination of wine volatiles

Volatile compounds were extracted by headspace (HS) SPME as described in a previous study (Welke, Zanus, Lazzaroto, Schmitt, & Zini, 2012). Briefly, 1 mL of wine and 0.3 g of NaCl were placed in a 20 mL headspace glass vial and sampling of volatiles was performed at 55 ºC for 45 minutes without agitation, using a 2 cm DVB/CAR/PDMS fiber. Twenty-milliliter headspace vials with Teflon septa were purchased from Supelco (Bellefonte, PA, USA). The volatile compounds were desorbed in a GC inlet at 250 ºC for 5 min. A CTC Combi PAL autosampler (CTC Analytics, Zwingen, Switzerland) with SPME fiber conditioning station was used to extract the volatiles from the wine sample. The volatile profile of Syrah wines was elucidated using GC×GC/TOFMS and its odoriferous importance was verified by GC-O-OSME associated to GC/MS and GCFID to identify the aroma-active compounds.

2.3.1 Determination of volatile profile by GC×GC/TOFMS

The GC×GC system consisted of an Agilent 6890N (Agilent Technologies, Santa Clara, CA) was coupled to a Pegasus IV time-of-flight mass spectrometer (Leco Corporation, St. Joseph, MI, USA). The first capillary column employed in

9

chromatography system was a DB-WAX (polyethylene glycol, 30 m × 0.25mm × 0.25 mm) and the second column was a DB-17ms (50% phenyl-methylpolysiloxane; 1.70 m × 0.18 mm × 0.18 μm), (J&W Scientific Inc., Folson, CA, USA). The GC system was equipped with a secondary column oven and non-moving quad-jet dual stage thermal modulator. Helium (99.9999% purity, White Martins, Porto Alegre, RS, Brazil) was used as carrier gas at a constant flow of 1 mL min−1. The transfer line and injector temperature were respectively, 300 ºC and 250 ºC.

The MS parameters included

electron ionization at 70 eV with ion source temperature at 240 ºC and acquisition rate of 100 spectra s-1 and data were recorded between 45-450 m/z. Relative area of each compound tentatively and/or positively identified was calculated as follows: the sum of all areas of the detected peaks by GC×GC/TOFMS was considered 100%, and to each peak a percentage corresponding to its area was assigned. For quantification of the volatile compounds of Syrah wines, standard curves were acquired by a standard dilution solution series in a model wine. Validation parameters were performed according International Conference on Harmonization (ICH) guidelines (ICH, 2005). The analytical parameters were linearity, limits of detection (LOD), and quantification (LOQ) and precision (repeatability and intermediate precision) and recovery.

2.3.2 Determination of aroma-active compounds by GC-O-OSME, GC/MS and GC-FID

The samples were analyzed in an Agilent 7890 GC (Agilent Technologies, Santa Clara, CA) equipped with FID detector and also with a lab made olfactometer, each one installed in different detection ports. During the GC-FID analyses, the chromatographic, column was connected to the FID and during the GC-OSME analyses, the column was connected to the olfactometer port, which was kept at 280 ºC. A stainless steel tube was

10

hooked up to the top of the olfactometric port and was a support for a silanized glass tube (trimethylchlorosilane 98%, Acros Organics, Geel, Turnhout, Belgium). The chromatographic effluent would flow through this glass tube in order to reach the nose of the panelist, as reported elsewhere (Sampaio, Biasoto, & da Silva, 2015). A 3 L min-1 flow (FX010 fluxometer, Unitec, Jabaquara, SP, Brazil) of synthetic air (99.999 % purity, Air Products, Canoas, RS, Brazil) was employed to help the effluent achieve the end of the glass tube. The synthetic air was purified with charcoal (Synth, São Paulo, SP, Brazil) and humidified as it passed through a glass round bottomed flask. A GC/MS system (Shimadzu, Kyoto, Japan) equipped with a spectrometer QP 2010 was also used. Analyses were performed using a DB-Wax (100% polyethylene glycol, J&W Scientific Inc. Folsom, CA, USA) and a DB-5 (5% phenyl, 95% polydimethylsiloxane, J&W Scientific Inc. Folsom, CA, USA), both measuring 30 m × 0.25 mm × 0.25 μm. Desorption of volatile compounds occurred in the GC injection port at 250 °C for 5 min (FID and MS), in a splitless injection mode for 1 min. The temperature of the FID detector was kept at 280 °C. The mass spectrometric detector was operated in the full scan in the range of m/z 45-450 and in the electron ionization mode at 70 eV. Ion source and transfer line temperature was 280 °C. The odoriferous importance of the volatiles was determined using the OSME technique. GC effluent was directed to the sniffing port of GC-O and to panelist's nose by moistened synthetic air, as described in a previous work (Sampaio, Biasoto, & da Silva, 2015). Four panelists were trained to perceive odors such as "floral”, “sweet”, “pungent”, among others, in order to be able to register the type and intensity of the odoriferous stimuli. A 10 cm scale has been employed to measure intensity and the experiments were replicated four times for each wine. At its extreme left side, the scale reported "none" and at its right side, the scale provided "strong". A consensus

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aromagram for each wine sample was built with the average of all peaks detected at least twice by at least two judges. Comparisons among the aromagrams of GC-OOSME and the chromatograms obtained by GC-FID and CG/MS, using the same experimental parameters, allowed the identification of the odoriferous impact of the volatiles present in each wine sample. The area percentage of each peak related with GC-O-OSME was calculated considering the total area of the aromagram.

2.4 Identification of volatile compounds

The volatile compounds evaluated by both GC/MS and GC×GC/TOFMS were positively identified by comparison of the retention times and mass spectra with those of authentic standards listed in section 2.1. For the unavailable standards, tentative identification of wine aroma compounds in GC/MS and GC×GC/TOFMS analysis was performed by comparing their experimental retention indices (IRexp) with RI reported in the scientific literature (RIlit) (Welke, Manfroi, Zanus, Lazarotto, & Zini, 2012). A series of n-alkanes (C7-C24, Supelco, Bellefonte, PA), was injected in the GC following the same conditions employed for the wine samples and were used for experimental RIexp calculation according Van dan Dool and Kratz (Van Den Dool & Kratz, 1963). A compound was assumed to be tentatively identified only if the experimental and literature reported RI did not differed by more than 15 units. In addition, mass spectra similarity between the mass spectrometric information of each peak chromatography with those in the NIST 107 mass spectrum library version was at least 80% (National Institute of Standards and Technology, Gaithersburg, USA). Retention indices for GC-FID (RIFID) and GC-O-OSME (RIOSME) were calculated as mentioned above and a higher ΔRI between them were considered adequate due to acceptable deviation between the timing of the human nose detection and the moment 12

the olfactory judge reports the type of odor and its persistence (Sampaio, Biasoto, & da Silva, 2015).

2.5 Statistical Analysis

LECO Chroma TOF version 4.22 software (Leco Corporation, St. Joseph, MI, USA) was used for acquisition and data processing for GC×GC/TOFMS. Fisher ratios were calculated using Excel software as previously reported (Welke, Zanus, Lazzarotto, Pulgati, & Zini, 2014) and they were aimed to rank compounds that mostly contribute to the differentiation of the samples. The chromatographic areas of volatile compounds with higher Fisher ratio values were employed in principal component analysis (PCA) using Statistica for Windows program package (version 7.1; Statsoft, Tulsa, OK, 2005). Analysis of variance (ANOVA) followed by Tukey test (p < 0.05) was applied to verify the differences between the concentrations of volatile compounds obtained in the samples and quantified by GC×GC/TOFMS. Heat map were performed through XLSTAT2017 (Addinsoft, New York, USA) for Microsoft Excel using data of intensity and persistence of odor obtained in GC-O-OSME.

3. Results and Discussion

3.1 Influence of different combinations of maturation degree and time of maceration of the grapes in the volatile profile evaluated by GC×GC/TOFMS

Table S1 presents the experimental (RIexp) and literature RI (RIlit) of the 145 volatile compounds found in the Syrah wines, as well as the odoriferous notes reported

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in literature of some volatile compounds that were found important for the aroma of these wines. Table S2 shows the GC×GC/TOFMS relative area of each compound that presented a Fisher ratio higher than 5%. Among all the chemical groups found in the volatile profile of Syrah wines from São Francisco Valley, the esters had the highest number (46), followed by the alcohols (31), terpenes (19), acids (14), aldehydes (10), lactones (7), sulfur compounds (7), ketones (5), furans (4) and phenols (2). Šuklje et al. (2016) results agree with ours for wines from shriveled and non shriveled Syrah berries, investigated with GC/MS. A former work of this group that applied GC/MS to the same type of Syrah wines also pointed to the predominance of esters (12) and alcohols (14) in a total of 52 compounds identified (Barbará et al., 2019), but in the present work 145 volatiles were positively and/or tentatively identified. The 29 volatiles with the highest Fisher ratios (at least 5% of the Fisher ratio of the most discriminant compound) are listed in Table S2 and were considered the main responsible for the differences between the Syrah wines elaborated from grapes harvested at different maturation and maceration periods. This approach was successfully applied in the differentiation of other types of wine in previous studies (Welke, Zanus, Lazzarotto, Pulgati, et al., 2014) and was used to ensure all these 29 volatiles were considered in the quantification step, as shown in Table 1. Table S3 presents figures of merit of the quantitative analytical method. The lowest LOD (0.1 μg L-1) and LOQ (0.3 μg L-1) were found for 1-nonanol, while the highest LOD (9.9 μg L-1) and LOQ (29.9 μg L-1) values were verified for hexanoic acid. The RSD of repeatability and intermediate precision assays were lower than 13.3 and 14.7%, respectively and the average recovery for the compounds was 99%. Among these 29 volatiles, 7 compounds were found at levels lower than the LOQ of the method for all samples: 4-hepten-1-ol (#35), 1-nonanol (#39), ethyl dodecanoate (#93), ethyl-3-

14

phenyl-2-propenoate (#97), 5-ethoxydihydro-2(3H)-furanone (#112), 3-penten-2-one (#108) and 2-penten-1-ol (#26). Conversely, octanoic acid (#9) was found above the highest concentration used in analytical curves and for these reasons, these compounds were not included in PCA. Among the 29 compounds with the highest Fisher ratios (Table S2), seven were also detected by GC/MS: (Z)-3-hexen-1-ol (#29), decanoic acid (#11), octanoic acid (#9), (E)-nerolidol (#138), 1-octanol (#37), 1-nonanol (#39) and ethyl decanoate (#84) (Barbará et al., 2019). (Z)-3-Hexen-1-ol (#29) and decanoic acid (#11) were found to be important discriminating compounds in Syrah produced from unripe grapes and they also showed up with higher concentrations in T1 wines, as follows: (Z)-3-Hexen-1-ol with 35.7 g L-1 in T1M1 and decanoic acid with 1556 g L-1 in T1M1 and 1661 g L1

in T1M2 wines (Table 1). Ethyl decanoate (#84) has been found in higher

concentrations in M1 wines and GC×GC has shown that a co-elution of this ester occurred with ethyl methyl butanoate (#83) in

1D

(Co7, Figure S1). All

chromatographic co-elutions have been numbered from 1 to 15 and compounds involved in each one of them are designated between brackets as "Cox" after the name of the compound in the text and also in Tables S1, S2 and 3, where x is the number of the co-elution. The majority of compounds established as important by Fisher ratios have not been detected when GC/MS was employed and that might be attributed to lower sensitivity and/or to co-elution of compounds, taking into consideration that cryogenic modulation usually provides higher sensitivity and GC×GC, higher selectivity. It is well known that these drawbacks of 1D-GC/MS advises against its use as it might lead to an incomplete number of compounds, misidentification of some of them, in addition to

15

misinterpretation of the real contribution of each compound to concentration and to wine aroma. Principal component (PC) analysis provided five components (PC1, PC2 PC3, PC4 and PC5) that presented eigenvalues higher than one according to the Kaiser rule, which explained 98.7% of the total variance in the data and the variables that presented higher loadings for these PC are shown in Table S4. PC1 and PC2 were responsible for 73.4% of the total variation in the concentration of the volatiles shown in Table 1. The variables related to these components are positioned according to the loadings in Figure 1. Syrah wines elaborated with grapes macerated for shorter periods (T1M1, T2M1, T3M1, and T1M2) were clearly separated from the others (T2M2, T2M3, T3M2 and T3M3) (Figure 1A). T1M1 wine presented significantly higher concentration for the following eight compounds (esters, alcohols and sulfur compounds) that were responsible for the differentiation of these wines (Figure 1B, Tables 1, S4): ethyl 3(methylthio) propionate (#142), (Z)-3-hexen-1-ol (#29), 3-hexen-1-ol acetate (#70), 2methyl dihydro-3(2H)-thiophenone (#140), ethyl-2-hydroxy-benzoate (#91), 1-octanol (#37), methyl dodecanoate (#90) and hexyl acetate (#67). A similar general trend has been observed for wines made from red Vranec berries when the maceration period was extended from four and seven days to 14 and 30 days, as the relative amounts of esters and alcohols decreased with longer maceration. It is known that long maceration times might reduce the concentration of higher alcohols as they can be sorbed on macromolecules (Petropulos et al., 2014). Prolonged maceration may also block the Ehrlich mechanism that gives rise to higher alcohols originated from the catabolism of amino acids by the yeast action, which is the main route for the formation of these compounds (Yilmaztekin, Kocabey, & Hayaloglu, 2015).

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The second PC, responsible for 33.05% of data variability, allowed the separation of T1 wine samples (wines made from grapes harvested before technological maturation, 19 °Brix) on the negative part of the PC2 axis, while T2 and T3 wines remained in the positive part of PC2 (Figure 1, Table S4). T1M1 wines presented greater loadings on the negative part of PC2 with a significantly higher concentration of the following compounds: ethyl 3-(methylthio) propionate (#142), 2-methyl dihydro3(2H)-thiophenone (#140), ethyl-2-furancarboxylate (#104), methyl dodecanoate (#90), 1-octanol (#37), and decanoic acid (#11). 1-Octanol (#37) have already been reported in higher concentrations in Serradello red variety grapes of lower maturation degree (18.0 °Brix) than in grapes with 19.6 °Brix (Vilanova, Genisheva, Bescansa, Masa, & Oliveira, 2012). Carvone (#134) was the compound with the highest Fisher ratio and it achieved a maximum concentration in T2M3, being present in all wines under study. The reason why these wines have a higher concentration of carvone is at least interesting and deserves further investigation. Odor active compounds derived from limonene, such as carvone, menthol, menthone and pulegone have been pointed as responsible for mint notes in aged Bordeaux wines and they have been considered as potential markers of these wines (Picard, Tempere, de Revel, & Marchand, 2015). Among the 29 compounds responsible for differentiation of wines according PCA seven were tentatively identified for the first time in Syrah wines: methyl dodecanoate (#90), ethyl-2-hydroxy-benzoate (#91), ethyl furan-2-carboxylate (#104), 2-methyl dihydro-3(2H)-thiophenone (#140) diethyl pentanedioate (#89), 1-(2-furanyl) ethanone (#103), methylthio acetate (#139). However, they have already been reported in other varietal red wines, such as Brazilian Merlot (Nicolli et al., 2018), and Cabernet

17

Sauvignon wines, both from South Africa (Weldegergis, Crouch, Górecki, & Villiers, 2011).

3.2 Active aroma compounds analyzed by GC-O-OSME and quantified by GC×GC/TOFMS

Considering that GC×GC/TOFMS showed that grapes macerated for a shorter period (10 days, M1) presented the highest concentration levels of the 21 volatile compounds shown in the PCA (Figure 1), the samples T1M1, T2M1 and T3M1 were chosen for evaluation by the GC-O-OSME technique in order to verify their most important aroma active compounds. Table 2 shows the identification of 31 odoriferous regions in Syrah wines with the intensity (I) and persistence (P) of the odor of the chromatographic effluent, in addition to the respective aroma described by the judges of GC-O-OSME. Eleven esters (#56, 57, 58, 59, 60, 62, 65, 75, 84, 92, 96), ten alcohols (#17, 19, 20, 21, 28, 29, 34, 42, 43, 44), three acids (#1, 4, 14), two terpenes (#135, 137), two aldehydes (#47, 53), one ketone (#106), one lactone (#111) and one sulfur compound (#144) have been identified as the odoriferous compounds of Syrah wines. The major part of them contributed positively to the aroma of T1M1, T2M1 and T3M1 wines: 78, 71 and 75%, respectively (section 2.3.2), and their aroma notes were described as fruity, floral, sweet, refreshing, mint, among others, according to Tables 2, 3, and S1. Although, the influence of eight compounds [(acids #1, 4, 14), alcohols #20, 21, 28), -butyrolactone #111 and 3-(methylthio)-propanol #144] was considered detrimental to the odor of Syrah wines and their aromatic notes were described as vinegar, rancid, solvent, etc. Literature references for the odor descriptions in Table 3 are listed in Table S1. Taking into account that the 29 higher Fisher ratios were

18

calculated based on the chromatographic relative area of volatiles (Tables 1 and S2) and that Table 2 presents the results of GC-O-OSME, it was expected that the compounds in Tables 1 and 2 would be different. Ethyl decanoate (#84) is the only exception, as it has been found in GC×GC and in GC-O-OSME. Figure 2 shows the heat map performed with the data of odor intensity (I) and persistence (P) obtained with GC-O-OSME and listed in Table 2. T1M1 was differentiated from T2M1 and T3M1 by both I and P, as shown in dendrograms of Figure 2A and 2B, respectively. The compounds whose I and P were responsible for the differences among samples are listed in the vertical axis of the heat map. The T1M1 wines stood out in relation to the other samples due to the greater intensity of 12 compounds (red cells in T1M1 column of Figure 2A) that had a description of agreeable odor in GC-O-OSME analyses, including five esters, three alcohols, two aldehydes and two terpenes: ethyl acetate (#56), ethyl butanoate (#59), ethyl octanoate (#75), ethyl decanoate (#84), 2-phenethyl acetate (#92), 2-methyl-1propanol (#17), benzyl alcohol (#42), 1-dodecanol (#44), 4-ethyl benzaldehyde (#53), 3methyl-1-butanal (#47), citronellol (#135), and geraniol (#137). A general trend of decreasing concentrations of esters and alcohols has been discussed before, however establishing a relationship between variations in the concentration of esters during ripening and yeast sugar metabolism is challenging, as distinct esters behave differently. Metabolism of esters by yeasts depends on several factors and not only on yeast sugar metabolism. In addition, the link between sugar accumulation in grapes and aromatic maturity is not straightforward and it is often indirect and specific to each volatile compound (Antalick et al., 2015). In general, terpenes gradually increase their concentration as grapes mature and this trend was confirmed in the present work in T3M1. Although terpenes may have

19

their concentrations reduced when adequate levels of sugar are achieved. This reduction may be influenced by temperature and water availability during maturation of the grapes (Ribéreau- Gayon et al., 2006). In the case of Syrah wines, the patterns of two pairs of terpenes were different: concentrations of (E)-nerolidol (#138) and α-terpineol (#133) were higher for T3M1, while the highest amounts of citronelol (#135) and geraniol (#137) were observed in T1M1. Conversely, opposite evolution was reported to (E)nerolidol and terpineol in Spanish Serradello grapes, as their concentrations were reduced when maturation changed from 10.0 to 19.6 °Brix (Vilanova et al., 2012). Yuan & Qian (2016) presented increased concentrations of citronellol and geraniol with grape maturation (26 °Brix) if compared to less mature berries (19 °Brix) As mentioned before, ethyl decanoate (#84) was relevant in terms of Fisher ratio and also in relation to I. Its concentrations occurred in decreasing order in T2M1, T3M1 and T1M1 (67.3, 35.7 and 19.0 µg L-1, respectively, Table 3). Interestingly, its higher concentrations in T2M1 and T3M1 do not necessarily imply that its contribution to the whole Syrah wine aroma would be more significant for these wines. On the contrary, Figure 2A and Table 2 confirm that its I was higher in T1M1 wines. This is another example of the complexity of the volatile and odoriferous profiles of wines, showing that I may not be directly linked to concentration. In this case, a matrix effect may be responsible for these apparent contracdictory results. In fact, T2M1 and T3M1 presented higher alcoholic degrees (14.3 and 14.6%, respectively) than T1M1 (11.5%) and the higher concentration of alcohol might have suppressed the perception of some of the odoriferous compounds. Other two odoriferous esters presented similar behaviour: ethyl acetate (#56) and 2-phenethyl acetate (#92), as their aroma notes were more intense in T1M1, even though their concentrations were higher in T3M1. Benzyl alcohol (#42), 1-dodecanol (#44), and 3-methyl-1-butanal (#47) followed the same

20

pattern (Tables 2 and 3). The global aromatic intensity of Malbec wines was described as fruity when the ethanol content was lower (10.0-12.0%) and as herbaceous when ethanol was higher (14.5-17.2%). (Goldner, Zamora, di Leo, Gianninoto, & Bandoni, 2009). On the other hand, higher I and concentration may be found in T1M1 wines (Table 2), as in the case of ethyl butanoate (#59, I=4.93% and 34.6 µg L-1 in T1M1), isoamyl acetate (#62, I =2.04% and 438 µg L-1 in T1M1), and ethyl octanoate (#75, I=2.53% and 153 µg L-1 in T1M1). Results of Antalick et al. (2015) agree with this distinct evolution of concentrations of esters during maturation: ethyl octanoate and 2phenethyl acetate were higher in wines elaborated with less mature berries, although concentration of ethyl butanoate enhanced with longer maturation periods and the one of ethyl acetate remained constant between two different maturation stages (20.9 e 22.7 °Brix) for Cabernet Sauvignon berries. The importance of esters has been also mentioned by Condurso et al. (2016) in relation to Syrah wines from the Palermo region (Italy), where diethyl succinate, ethyl octanoate and decanoate, and isoamyl acetate have been highlighted due to their contribution to wine fruity and floral notes. A distinct trend was found for the fruity esters ethyl hexanoate (#65), ethyl-2methylbutanoate (#60), and the C6-alcohol (Z)-3-hexen-1-ol (#29) where their maximum concentrations were found in T1M1 wines, with berries that have not achieved technological maturity, while their highest I was achieved in T3M1 (Figure 2, Tables 2 and 3). 2-Phenylethyl alcohol (#43) has also shown its maximum aromatic intensity in T3M1. Higher concentrations of ethyl hexanoate (#65) were also reported in wines made with Cabernet Sauvignon grapes with 20.9 instead of 22.7 °Brix (Antalick et al., 2015). The diversity of olfactometric events that have been found in Syrah wines may be linked to different types of matrix effects. It is known that distinct

21

concentrations of phenolic compounds and polysaccharides in wine may suppress or increase the volatility and the perception of an odoriferous compound. Such phenomena depend also on the concentration and nature of the aroma compound (Villamor & Ross, 2013). No negative odor compounds were found at highest I in T1M1 sample. Dissimilarly, three compounds with the highest undesirable odor intensities were found in T2M1 sample: -butyrolactone (#111; pungent), 3-methylthio-1-propanol (#144; cooked green beans) and dodecanoic acid (#14; gas). T3M1 also presented three compounds with detrimental odors: 2-methyl-1-butanol (#20; fermented), 2methylpropanoic acid (#4; cheese) and 3-methyl-1-butanol (#21; solvent). Lower concentrations of esters in T2M1 and T3M1 may be related to high concentrations of acids, such as 2-methylpropanoic acid (#4, T2M1=510, I=6.21% and T3M1 = 506 µg L1,

I=7.69%) in these same wines (Table 3). Isovaleric acid (#6, 3-methyl butanoic acid)

and formic acid (#2) were also present in higher concentrations in T2M1 and T3M1. The presence of acids may reduce the activity of yeasts and this results in lower concentrations of esters (Saerens et al., 2008). This trend has been verified for the concentrations and for positive I of esters isobutyl acetate (#58), ethyl butanoate (#59), isoamyl acetate (#62), ethyl hexanoate (#65), ethyl octanoate (#75), ethyl propanoate (#57), and ethyl-2-methyl butanoate (#60) in Syrah wines made with grapes harvested with 21 (T2) and 23 °Brix (T3). The promising aroma potential of the wine T1M1 verified by the odor intensity was confirmed in the evaluation of the P of the volatile profile obtained by GC-OOSME (Figure 2B). Out of the 12 compounds with the highest intensity in T1M1 wines (Figure 2A), seven also have shown a long-term P of positive attributes: 4-ethyl benzaldehyde (#53), 3-methyl-1-butanal (#47), geraniol (#137), ethyl butanoate (#59),

22

citronellol (#135), benzyl alcohol (#42) and 2-phenethyl acetate (#92). 2-Phenylethyl alcohol (#43) that was more intense in T3M1 persisted for a longer time in T1M1. Among the compounds designated by GC-O-OSME, 2-phenethyl acetate (#92) and 2phenylethyl alcohol (#43) presented longer odoriferous P, as well as I. Alcohol #43 arises mainly during fermentation, having 2-phenylalanine as its precursor (Laminkanra, Grimm, & Inyang, 1996), while acetate #92 is formed formed from alcohol #43. In general, its contribution was related as positive to wine aroma (roses) (Li et al., 2014), while Mayr et al. (2014) reported 2-phenethyl acetate as having no influence on Australian Syrah wine aroma. In contrast, the same researchers described a positive influence of 2-phenylethyl alcohol (floral and roses) to the global wine aroma. In T3M1 wines the most persistent odorants were isobutyl acetate (#58; ripe fruit), ethyl hexanoate (#65, fruity), and (Z)-3-hexen-1-ol (#29, citrus). Among them, ethyl hexanoate has been already mentioned as important contributor to the aroma of Syrah wines (Mayr et al., 2014). Regarding the volatiles with undesirable odor, four compounds presented the highest P with an upward trend towards T3M1: 3-methylthio-1-propanol (#144; cooked green beans), 3-methyl-1-butanol (#21; solvent), 2-methylpropanoic acid (#4; cheese), and acetic acid (#1; vinegar). Other compounds presented a tendency to increased P in T1M1 or T2M1 wines: 1-hexanol (#28, cooked), -butyrolactone (#111, pungent), and dodecanoic acid (#14, gas). The

sulfur

compound

3-metilthio-1-propanol

(#144)

presented

higher

odoriferous intensity (T2M1) and persistence (T3M1) in Syrah wines made with berries with extended maturation degree if compared to T1M1, although its higher concentration has been related to T1M1 wines (Tables 2 and 3). Depending on planting conditions of the vines, grapes experience different conditions and may also undergo

23

distinct processing steps (solar radiation, temperature, yeast metabolism, thermal treatment or other non-enzymatic reactions). Consequently, the expression of fruity aromas can be diminished due to the reduction of the synthesis of carotenoids and terpenes, favoring the formation of unsaturated fatty acids and sulfur compounds that impart vegetable odors. In the case of sulfur compound #144, its concentration decreased with grape maturation, similar to what happened to some other compounds (Jackson, 2014). There were five Syrah wine compounds that presented odoriferous importance according to GC-O-OSME and were found only when GC×GC/TOFMS was employed (Table 2). Among them, four presented a positive contribution: 3-methyl-1-butanal (# 47, sweet), 4-ethyl-benzaldehyde (#53, sweet), ethyl propanoate (#57, refresh), ethyl-2methylbutanoate (#60, apple 5.6-9.5 µg L-1 in T1M1, T2M1, T3M1). This last ester coeluted (Co1) with methylthio acetate (#139, 5.6-10.5 µg L-1, sulfurous, Table 3) that imparts a detrimental odor to wine. Even though the concentration of these two coeluting compounds were found to be of the same order of magnitude, the ester positive odor predominated over the sulfur compound note and the final perception of the GC-OOSME judges was pleasant. The fifth compound that was detected only in 2D presented a negative contribution to wine odor: 2-methyl-1-butanol (#20, fermented, I=1.12-1.89, P=1.792.35). Despite the fact that these compounds were detected by the human nose due to their odorant potential, they were present in concentrations that were not sufficient to make them detectable by GC/MS. It is noteworthy that, for example, ethyl propanoate (#57, 7.4-12.4 µg L-1), ethyl-2-methylbutanoate (#60, 5.6-9.5 µg L-1), and 2-methyl-1butanol (# 20, 23.3-338 µg L-1) were found in lower levels in Syrah wines than in their Australian counterparts, where their concentrations ranged from, 318-723 µg L-1, 36-76

24

µg L-1 e 161250-208400 µg L-1, respectively (Mayr et al., 2014). An increase in the concentration of alcohol #20 in T3M1 can be observed (from 23.3/45.1-338 µg L-1). This is in accordance with Bindon et al. (2013) that have also found a higher concentration of 2-methyl-1-butanol, among other alcohols, in Cabernet Sauvignon wines when grapes were harvested with 26.0 instead of 20.3 °Brix. These results are in accordance with the fact that as grapes mature they may lose volatile compounds that impart positive notes to aroma, while other volatiles with detrimental effect to aroma may have their concentration enhanced. Among the odoriferous compounds detected by GC-O-OSME (Tables 2 and 3), six co-elutions (Co1, Co7, Co10, Co11, Co12, Co13, numbered according to Tables S1, S2) involving 12 important compounds for the Syrah wine aroma that were resolved only in the 2D of GC×GC/TOFMS, are shown in Figures S1 and S2. All of them encompassed one compound that positively contributed to wine aroma, while three also involved a component with negative description to odor. There was a predominance of the positive aroma in almost all coelutions, except for Co10, that involved 2-undecanol (#40, fruity) with 3-(methylthio)-1-propanol (#144, cooked green beans). In this case, concentration of 2-undecanol was lower than the one of 3-(methylthio)-1-propanol and the final aroma related by GC-O-OSME panel was negative and described also as cooked green beans. Co 12 encompassed geraniol (#137, geranium) and hexanoic acid (#7, cheese) and the OSME panel perceived a fresh, geranium aroma, despite the higher concentration of hexanoic acid (> 2160 µg L-1) compared to 12.8-14.2 µg L-1found for geraniol (Tables 2 and 3). The other coelutions and their specific characteristics have been already discussed throughout the text. Methylthio acetate (#139) and ethyl decanoate (#84) that were part of Co1 and Co 7 were among the 29 most relevant compounds in terms of Fisher ratio (Table 1).

25

Ethyl-2-methyl-butanoate (#60) was found to be relevant in the GC-O-OSME evaluation and it totally co-eluted with #139 (Co1). Finally, ethyl decanoate (#84) was also found to be important in olfactometry. 1-(2-Furanyl) ethanone (#103) and ethyl-2hydroxy-4-methylpentanoate (#79) were part of Co5 and 6, respectively and were among the 29 most relevant compounds pointed by Fisher ratios. In Co5, 1-(2-furanyl) ethanone (#103, caramel, coffee) co-eluted with camphor (#127, camphor, minty) and in Co6 ethyl-2-hydroxy-4-methyl pentanone (#79, fruit) co-eluted with propanoic acid (#3, rancid). These compounds may impart odoriferous notes to wine and their co-elutions are important to be reported, even though in these Syrah wines they have not been found relevant in GC-O-OSME. Three of the before mentioned co-elutions of Syrah volatiles are being reported for the first time in wines: Co1, Co7 and Co 12 (Figure S1). The others have already been reported in the headspace of Merlot wines: Co5, Co6, Co10, Co11, and Co13. Different chromatographic co-elutions have been found in Merlot wines (hexanoic acid with geranyl acetone; 1-propanol with 2-butenal; ethyl octanoate with 5-methyl-2(3H)furanone and p-cymenene, etc) which corroborate to show that co-elutions in 1D-GC may be different for distinct varieties resulting in higher complexity whenever a wine is submitted to investigation (Nicolli et al., 2018). A former research work of this group showed a general opposite trend, as concentration of volatile compounds were higher with longer maturation (>21 ºBrix) and maceration periods (20 to 30 days) for this same type of wines, but this is not surprising as this former work employed only GC/MS, where several co-elutions have been observed. Furthermore, various volatiles identified in GC×GC were not detected with GC/MS (Barbará et al., 2019). Conversely, GC×GC/TOFMS results obtained by Lago et al. (2017), concerning the same type of Syrah wines of the São Francisco

26

Valley reported higher levels of volatile toxic compounds when wines were produced from mature grapes (> 21 °Brix) and when macerated for longer periods of time (> 20 days). Interestingly, Cadot et al. (2012) have shown that Cabernet Franc wines made with less mature grapes (35 instead of 49 days after veraison) and vinified during shorter periods of time (9 instead of 15 days) have been highlighted due to their acidity, vegetal, humus and animal aromas, while presenting less intensity of bitterness, astringency, spicy aroma, and alcohol.

4. Conclusion

The combined use of GC×GC/TOFMS, GC-O-OSME, GC-FID and GC/MS allowed to verify that grapes harvested at 19 °Brix and macerated for 10 days were the most promising conditions to produce Syrah wines with high positive odor impact. Maintaining grapes in the vineyard (≥ 21 °Brix) and/or the prolongation of maceration time (≥ 20 days) were found to be unfavorable practices to the wine volatile profile and aroma potential. GC×GC/TOFMS proved to be efficient for solving cases of co-elutions of important wine aroma compounds presented by GC-O-OSME, to differentiate Syrah wines according to volatiles and also to unveil the presence of compounds that were found in Syrah headspace for the first time. In addition to scientific and technological innovations supplied by characterization of wine volatiles originated from different maturation and maceration periods of time, these results also provided support for the achievement of GI and DO labels for the Syrah wines of the São Francisco Valley and to improve their quality. The use of GC×GC/TOFMS, combined with GC-O-OSME, GC-FID and GC/MS seems to be not only important, but also essential for a complete and detailed characterization of wine volatiles and their olfactometric impact, even

27

though the high costs still associated with GC×GC/TOFMS have been preventing the wine industry from adopting this superior analytical 2D tool. This same type of analytical strategy may be applied to define vineyard management practices, vinification conditions, time of aging, etc, as well as to other challenges of the food and beverage industry.

Acknowledgments

The authors thank the National Council for Scientific and Technological Development (Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq), the Coordination for the Improvement of Higher Education Personnel (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, CAPES), and the Research Support Foundation of Rio Grande do Sul (Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul, FAPERGS) for financial support and scholarships: Janaína A. Barbará (CAPES AUX-PE-PROEX 587/2017), Érica A. Souza Silva (CNPq 300335/2015-6 and CNPq BJT 401581/2014-4) and Claudia A. Zini (CNPq Pq 1D 306067/2016-1).

We also acknowledge the supporting projects CNPq Universal

408625/2016-3, EMBRAPA SEG 03.13.06.017.00.00, FACEPE APQ-0921.5.07-14 and SIBRATEC/FINEP/FAPEG 01.13.0210.00, IP-Campanha.

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chromatography coupled to time-of-flight mass spectrometry for the detailed investigation of volatiles in South African red wines. Analytica Chimica Acta, 701, 98–111. http://doi.org/10.1016/j.aca.2011.06.006. Welke, J. E., Manfroi, V., Zanus, M., Lazarotto, M., & Zini, C. A. (2012). Characterization of the volatile profile of Brazilian Merlot wines through comprehensive two dimensional gas chromatography time-of-flight mass spectrometric detection. Journal of Chromatography A, 1226, 124–139. http://doi.org/10.1016/j.chroma.2012.01.002 Welke, J. E., Zanus, M., Lazarotto, M., Schmitt, K. G., & Zini, C. A. (2012). Volatile

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Characterization by Multivariate Optimization of Headspace-Solid Phase Microextraction and Sensorial Evaluation of Chardonnay Base Wines. Journal of Brazilian Chemical Society, 23(4), 678–687. Welke, J. E., Zanus, M., Lazzarotto, M., Pulgati, F. H., & Zini, C. A. (2014). Main differences between volatiles of sparkling and base wines accessed through comprehensive two dimensional gas chromatography with time-of-flight mass spectrometric detection and chemometric tools,. Food Chemistry, 164, 427–437. Welke, J. E., Zanus, M., Lazzarotto, M., & Zini, C. A. (2014). Quantitative analysis of headspace volatile compounds using comprehensive two-dimensional gas chromatography and their contribution to the aroma of Chardonnay wine. Food Research International, 59, 85–99. http://doi.org/10.1016/j.foodres.2014.02.002 Yilmaztekin, M., Kocabey, N., & Hayaloglu, A. A. (2015). Effect of Maceration Time on Free and Bound Volatiles of Red Wines from cv . Karaoglan ( Vitis vinifera L .) Grapes Grown in Arapgir , Turkey. Journal of Food Science, 80(3), C556–C563. http://doi.org/10.1111/1750-3841.12767 Yuan, F., & Qian, M. C. (2016). Development of C 13 -norisoprenoids , carotenoids and other volatile compounds in Vitis vinifera L . Cv . Pinot Noir grapes. Food Chemistry,

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34

Figure Captions

Figure 1: Plot of the two first principal components (PC1 and PC2) obtained in principal component analysis based on the 21 most discriminating volatile compounds of Syrah wines elaborated with grapes harvested at three different maturation degrees [T1: before technological or industrial maturity (19 ºBrix), T2: ideal ripeness degree (21 ºBrix), T3: overripe grapes (23 ºBrix)] and macerated during three different periods (M1: 10 days, M2: 20 days and M3: 30 days), resulting in the combinations T1M1, T1M2, T2M1, T2M2, T2M3, T3M1, T3M2 and T3M3. Figure 1(A) shows the distribution of wine samples and Figure 1(B) presents the distribution of volatile compounds in a PC1 and PC2 plot. Volatile compounds are designated by numbers in Figure 1(B), according to Table S1.

Figure 2: Heat map obtained using the intensity (A) and persistence (B) of odor mentioned in GC-O-OSME of Syrah wines elaborated with grapes harvested at three different maturation degrees [T1: before technological or industrial maturity (19 ºBrix), T2: ideal ripeness degree (21 ºBrix), T3: overripe grapes (23 ºBrix)] and macerated during 10 days (M1), resulting in the combinations T1M1, T2M1 and T3M1. Numbers between brackets are the ones designated to these same compounds in Tables S1. GCO-OSME experimental conditions are described in section 2.3.2.

Highlights

19°Brix was an appropriate maturation to obtain a higher number of pleasant Syrah wine volatiles 35

10 days of maceration was a promising condition to produce Syrah wines with positive odor impact

GC×GC/MS allowed resolving 5 and 6 co-elutions involving 10 and 12 important Syrah wine volatiles

GC×GC/MS, GC-O & GC/MS were essential for characterizing Syrah wine volatiles and their odor impact

Figure 1: Plot of the two first principal components (PC1 and PC2) obtained in principal component analysis based on the 21 most discriminating volatile compounds of Syrah wines elaborated with grapes harvested at three different maturation degrees [T1: before technological or industrial maturity (19 ºBrix), T2: ideal ripeness degree (21 ºBrix), T3: overripe grapes (23 ºBrix)] and macerated during three different periods (M1: 10 days, M2: 20 days and M3: 30 days), resulting in the combinations T1M1, T1M2, T2M1, T2M2, T2M3, T3M1, T3M2 and T3M3. Figure 1(A) shows the 36

distribution of wine samples and Figure 1(B) presents the distribution of volatile compounds in a PC1 and PC2 plot. Volatile compounds are designated by numbers in Figure 1(B), according to Table S1.

Figure 2: Heat map obtained using the intensity (A) and persistence (B) of odor mentioned in GC-O-OSME of Syrah wines elaborated with grapes harvested at three different maturation degrees [T1: before technological or industrial maturity (19 ºBrix), T2: ideal ripeness degree (21 ºBrix), T3: overripe grapes (23 ºBrix)] and macerated during 10 days (M1), resulting in the combinations T1M1, T2M1 and T3M1. Numbers between brackets are the ones designated to these same compounds in Tables S1. GCO-OSME experimental conditions are described in section 2.4.2

Table 1: Concentrations of 29 compounds indicated by Fisher ratios as the most important to differentiate Syrah wines elaborated using grapes harvested with three maturation degrees [T1: before technological or industrial maturity (19 ºBrix), T2: ideal ripeness degree (21 ºBrix), T3: overripe grapes (23 ºBrix)] and macerated during three different periods (M1: 10 days, M2: 20 days and M3: 30 days). Compounds are listed in decreasing order of Fisher ratios and designated by number according to Table S1. Compounds identified only after GC×GC/TOFMS analyses are in bold letters. GC×GC/TOFMS conditions are described in section 2.3.1 #

Compound Combinations of maturation degree and maceration time of grapes

Concentration (µg L-1) T1M1

T2M1

T3M1

T1M2 37

T2

134 carvone a

28.4±0.1d

27.5±0.04d

151±3b

28.6±0.2d

27.7

142 ethyl 3-(methylthio) propionate b

58.4±0.6a

10.6±0.4de

8.8±0.2f

42.1±1.0b

10.7±

< 7.1b

< 7.1b

< 7.1b

59.9±4.4a

<7

29 (Z)-3-hexen-1-ol d

35.7±0.1a

12.4±2.9b

8.6±0.6c

< 1.2d

<1

11 decanoic acid e

1556±52a

574±5b

302±21cd

1661±98a

70 3-hexen-1-ol acetate f

2.0±0.1a

< 0.7b

< 0.7b

< 0.7b

103 1-(2-furanyl) ethanone a

41.0±0.2c

45.2±1.1b

69.4±0.9a

38.2±0.7d

40.4

140 2-methyl dihydro-3(2 H)-thiophenone b

29.0±0.03a

20.2±0.6b

16.5±0.8c

28.4±1.0a

15.9±

10.1±1.3a

5.4±0.3b

1.9±0.4c

< 0.7c

<0

35 4-hepten-1-ol g

< 4.5

< 4.5

< 4.5

< 4.5

<

9 octanoic acid h

> 270

> 270

> 270

> 270

>2

3.6±0.1bc

3.8±0.5bc

21.7±2.5a

2.1±0.2bc

<1

142±7a

13.5±3.1de

18.8±2.2cd

67.2±0.1b

4.4±

15.4±0.3a

8.1±0.2b

6.0±0.2de

14.4±0.01a

6.7±

79 ethyl-2-hydroxy-4-methylpentanoate j

< 5.5d

22.7±1.4a

< 5.5d

24.6±0.4a

17.8

39 1-nonanol d

< 1.2

< 1.2

< 1.2

< 1.2

<

93 ethyl dodecanoate j

< 5.5

< 5.5

< 5.5

< 5.5

<

139 methylthio acetate j

7.9±0.4b

5.6±0.1c

10.5±0.3a

< 5.5c

<5

< 5.5

< 5.5

< 5.5

< 5.5

<

69.6±0.1b

22.3±8.4cde

145±7a

< 4.3

< 4.3

< 4.3

< 4.3

<

19.0±0.03c

67.3±7.9a

35.7±7.4b

< 5.7d

<5

< 4.3

< 4.3

< 4.3

< 4.3

<

< 1.2

< 1.2

< 1.2

< 1.2

<1

7.0±0.1a

< 5.5c

< 5.5c

6.6±0.1b

<5

83.0±4.0b

67.8±5.6b

169±15a

7.0±0.1d

1.8±

67 hexyl acetate f

17.9±0.7a

5.4±0.3bc

6.7±1.4b

1.5±0.04d

<0

110 acetophenone k

< 4.3

< 4.3

4.7±0.9

< 4.3

<

< 5.5c

< 5.5c

< 5.5c

14.2±0.5a

8.0±

18 2-pentanol c

91 ethyl-2-hydroxy-benzoate f

138 (E)-nerolidol i 37 1-octanol g 104 ethyl furan-2-carboxylate j

97 ethyl-3-phenyl-2-propenoate j 30 2-hexen-1-ol g 112 5-ethoxydihydro-2(3H)-furanone k 84 ethyl decanoate l 108 3-penten-2-one

k

26 2-penten-1-ol d 90 methyl dodecanoate j 133 α-terpineol i

89 diethyl pentanedioate j

366

<0

29.7±3.1cd 24.3±

SD: Standard Deviation; # Compounds are numbered according to Table 1. Quantification was performed with external calibration curves for the following compounds: a –β-damascenone, b – mercaptohexanol, c- benzyl alcohol, d -1-nonanol, e- hexanoic acid, f- isobutyl acetate, g-1-hexanol, h- octanoic acid, ieucalyptol, j- ethyl hexanoate, k – 2-heptanone, l- ethyl octanoate. The concentration ranges used in the calibration curves are shown in Table S2. When the amount of a compound was lower or higher than the concentration range used in the calibration curve, the concentration of that compound was expressed as “<” the lowest level of the curve and “>” than the highest level of the curve. When numbers are not followed by letters, there is no significant difference among them.

38

Table 2: Odoriferous compounds found in Syrah wines elaborated with grapes harvested at three different maturation degrees [T1: before technological or industrial maturity (19 ºBrix), T2: ideal ripeness degree (21 ºBrix), T3: overripe grapes (23 ºBrix)] and macerated during 10 days (M1), resulting in the combinations T1M1, T2M1 and T3M1, evaluated by gas chromatography with olfactometric, mass spectrometric and flame ionization detectors. Compounds identified only after GC×GC/TOFMS analyses are in bold letters. Compounds are listed in increasing order of elution and designated by number according to Table S1. GC-O-OSME experimental conditions are described in section 2.3.2. T1M1

T2M1

T3M1

RI OSMEa

RI FIDa

1068 1125 1391 1480 1905 1946 1973 960 1720 934

1094 1155 1366 1481 1882 1929 1976 957d 1714 900

5.80±0.56 1.35±0.05 ND ND 4.83±0.31 5.70±0.52 3.36±0.12 4.55±0.57 1.14±0.09 3.38±0.01

5.11±0.59 1.59±0.05 ND 2.33±0.07 2.85±0.08 4.08±0.31 2.27±0.09 ND ND 1.64±0.03

4.47±0.52 ND 1.22±0.02 ND 1.61±0.05 6.56±0.56 ND ND ND 2.84±0.09

noate

1009

955

ND

1.47±0.06

1.86±0.02

ate te hyl

980 1038

1001 1040

ND 4.93±0.30

3.95±0.49 2.94±0.32

1055

1037

5.09±0.08

ate ate te ate acetate anoate

1110 1239 1444 1642 1843 2063

1102 1227 1440 1633 1825 2013

one

969

ound

ribution propanol

(I) Odor intensity (cm) ± SD (%)b

T1M1 Odour GC-OOSME

T2M

(P) Odor persisten 5.75±0.68 1.00±0.21 ND ND 5.98 ± 0.08 13.58±0.95 2.53±0.27 5.91±0.48 0.62±0.44 2.99±0.16

5.69±0 1.22±0 ND 2.14±0 0.49±0 10.34± 2.02±0 ND ND 1.70±0

ND

1.39±0

3.93±0.39 2.90±0.25

fruity, sweet refreshing, sweet refreshing, citrus fruity fruity, sweet rose, honey floral sweet, fruity sweet, green fruity, red fruits refresh, green, fruity ripe fruit fruity, sweet

ND 5.76±0.39

2.56±0 3.56±0

5.76±0.10

6.11±0.34

strawberry, fruity

6.21±0.35

7.69±0

2.04±0.13 4.16±0.03 2.53±0.06 3.57±0.31 8.71±0.18 ND

1.99±0.06 4.43±0.35 2.17±0.06 1.94±0.09 5.86±0.60 1.00±0.01

ND 6.66±0.23 2.21±0.09 2.50±0.25 7.06±0.38 ND

1.70±0.20 4.81±0.33 1.82±0.23 2.29±0.34 14.52±1.91 ND

1.42±0 5.16±0 3.32±0 0.98±0 11.77± 1.05±0

980

3.08±0.12

3.04±0.10

2.29±0.20

2.41±0.08

3.40±0

1744 1887

1736 1857

6.05±0.02 3.90±0.40

3.64±0.35 0.67±0.01

3.79±0.39 ND

banana, fruit, fresh fruity, sweet fruity, sweet fruity, floral, sweet rose, floral, jasmine floral fruit, sweet, red fruits, fresh mint, citrus fresh, geranium

5.79±0.57 4.62±0.47

2.26±0 0.58±0

ne

1462 1687 2531 1214 1211 1329 1621

1460 1703 2492 1210 1215 1358 1617

6.55±0.12 5.59±0.53 ND ND 4.80±0.70 1.95±0.01 ND

5.12±0.50 6.21±0.49 4.21±0.30 1.12±0.02 4.44±0.10 2.66±0.08 1.04±0.01

7.50±0.32 7.69±0.70 ND 1.89±0.08 6.05±0.07 ND ND

5.78±0.09 6.23±1.07 ND ND 4.56±0.06 2.25±0.23 ND

5.62±0 6.86±0 3.71±0 2.35±0 4.86±0 1.55±0 0.58±0

o)1-propanol

1730

1733

2.23±0.01

3.38±0.09

3.02±0.09

vinegar cheese, rancid gas fermented, solvent pungent, solvent cooked, burnt pungent cooked green beans, wet bush, gas, green

1.50±0.12

2.50±0

1-ol xanol ol l alcohol

butanal aldehyde

ntribution

panoic acid cid butanol utanol

# Compounds numbered as in Table 1. a. Experimental retention index (RI) calculated using n-alkanes (C9-C24) on DB-Wax (100% polyethyleneglycol) for both GC-O-OSME (RIOSME) and GC-FID (RIFID) analyses. In GC-O-OSME, the retention time of the maximum intensity of the odor peak was used in RI calculation. b. Evaluated on a 10-cm scale

39

anchored at the left and right extremities by the intensity terms “none” and “strong”, respectively. It was obtained as an average intensity of the consensual aromagram constructed after the analyses of the samples by four (4) judges in four replicates. SD: standard deviation. c. Evaluated through % OSME area ± standard deviation: corresponds to the percentage of area of an odoriferous compound in relation to the sum of the area of all compounds detected when the OSME technique was used to obtain information on the volatiles determined by GC-O-OSME, through a sensory panel.

40

Table 3: Concentrations of the odoriferous compounds detected by GC-O-OSME and their respective co-elutions resolved through the GC×GC/TOFMS of Syrah wines elaborated using grapes harvested with maturation degrees [T1: before technological or industrial maturity (19 ºBrix), T2: ideal ripeness degree (21 ºBrix), T3: overripe grapes (23 ºBrix)] and macerated during 10 days (M1). GC×GC/TOFMS and GC-O-OSME experimental conditions are described in sections 2.3.1 and 2.3.2 #

Compound

C (µgL-1) T1M1

C (µgL-1) T2M1

C (µgL-1) T3M1

Odor GC-O-OSME

Odor from literature

ntribution 17

2-methyl-1-propanol c

359±10a

255±12b

249±3b

fruity, sweet

apple18

19

1-butanol c

41.1±0.3c

55.2±2.0b

74.1±4.2a

refreshing, sweet

sweet21

29

(Z)-3-hexen-1-ol d

35.7±0.1a

12.4±2.9b

8.6±0.6c

refreshing, citrus

green28

34

2-ethyl-1-hexanol e

27.1±0.9a

23.5±2.2a

25.3±3.4a

fruity

fruity18

42

benzyl alcohol f

90.8±0.4b

75.6±4.5c

113±7a

fruity, sweet

fruity, swe

43

2-phenylethyl alcohol f

>2840

>2840

>2840

rose, honey

rose28

114

γ-octalactone g

72.9±0.1a

45.5±4.3b

50.3±0.4b

44

1-dodecanol e

16.0±0.1b

9.3±0.4c

24.2±1.6a

floral

floral18

56

ethyl acetate h

1895±14b

1762±12c

2462±24a

fruity, red fruits

pineapple1

58

isobutyl acetate i

19.7±2.3a

7.8±0.2b

7.4±0.7b

ripe fruit

fruity39

59

ethyl butanoate j

34.6±0.7a

20.1±0.3b

20.5±4.1b

fruity, sweet

strawberry

62

isoamyl acetate h

438±8a

196±4c

236±38b

banana, fruit, fresh

banana, sw

65

ethyl hexanoate j

190±17a

123±4b

114±9b

fruity, sweet

green appl

75

ethyl octanoate k

153±14a

76.1±12.6b

66.6±10.8b

fruity, sweet

sweet, pea

84

ethyl decanoate k

19.0±0.03c

67.3±7.9a

35.7±7.4b

fruity, floral, sweet

Fruity10

83

ethyl methyl butanedioate

10.0±1.1b

15.2±0.7a

<0.7c

coconut, g

NF

i

92

2-phenethyl acetate j

7.4±0.4b

6.0±0.2c

8.4±0.5a

rose, floral, jasmine

rose-like, f honey44 honey, swe

136

β-damascenone g

104±5b

72.2±6.7c

173±16a

96

ethyl tetradecanoate j

<5.5

<5.5

<5.5

floral

flowery, fr

106

2,3-butanedione g

33.8 ±0.6b

37.1±2.1a

34.7±0.5ab

caramel2

17.1±0.8b

19.0±2.2b

26.7±1.6a

fruit, sweet, red fruits, fresh mint, citrus

135

citronellol l

137

geraniol l

12.8±0.5a

14.2±0.4a

14.0±0.8a

fresh, geranium

rose, geran

7

hexanoic acid

>2160

>2160

>2160

47

3-methyl-1-butanal c

4.7±0.01b

3.0±0.1b

27.3±0.1a

sweet, fruity

green, mal

57

ethyl propanoate j

12.4±0.2a

8.9±0.6b

7.4±0.1b

refresh, green, fruity

sweet, frui

60

ethyl-2-methyl butanoate

9.5±0.2a

7.1±0.1b

5.6±0.4b

strawberry, fruity

apple, swe

a

citric59

cheese, fat

j

139

methylthio acetate j

7.9±0.4b

5.6±0.1b

10.5±0.3a

53

4-ethyl benzaldehyde f

<7.1

<7.1

<7.1

sulfurous21 sweet, green

green, mal

ontribution

41

1

acetic acid a

>2160

>2160

>2160

vinegar

vinegar2

4

2-methylpropanoic acid a

391±11b

510±27a

506±19a

cheese, rancid

rancid, aci

14

dodecanoic acid b

<1.1

<1.1

<1.1

gas

dry, metall

20

2-methyl-1-butanol c

45.1±6.1b

23.3±2.0b

338±9a

fermented, solvent

earth, solv

21

3-methyl-1-butanol

>1800

>1800

>1800

pungent, solvent

solvent, ea

28

1-hexanol c

1114±34a

893±63a

1035±200a

cooked, burnt

herbaceous

111

γ-butyrolactone

371±6c

519±40b

601±22a

pungent

cheese-like

144

3-(methylthio)-1propanol e 2-undecanol d

50.5±2.6a

44.2±3.0b

11.0±0.1c

cooked green beans, wet bush, gas, green

cooked veg

<1.2

<1.2

<1.2

40

c

g

fruity2

* #co-elutions

and compounds are numbered according to Table S1. Quantification was performed with external calibration curves for the following compounds: a- hexanoic acid, b- octanoic acid, c- 1-hexanol, d- 1-nonanol, e- mercaptohexanol, f- benzyl alcohol, g- β-damascenone, h- diethyl butanedioate, iisobutyl acetate, j-ethyl hexanoate, k- ethyl octanoate, l-eucalyptol. Figures of merit are presented in Table S3. Odor from literature: Literature references of the odor descriptors of the compounds are given in Table S1 and the subscript numbers of Table S1 are the same referred here. NF. Aroma not found.

Declaration of interests

xThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

42