Elucidation of the aroma compositions of Zhenjiang aromatic vinegar using comprehensive two dimensional gas chromatography coupled to time-of-flight mass spectrometry and gas chromatography-olfactometry

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Elucidation of the aroma compositions of Zhenjiang aromatic vinegar using comprehensive two dimensional gas chromatography coupled to time-of-flight mass spectrometry and gas chromatography-olfactometry Zhilei Zhou a,b , Shuangping Liu a,b , Xiangwei Kong a , Zhongwei Ji a,b , Xiao Han a,b , Jianfeng Wu c , Jian Mao a,b,∗ a National Engineering Laboratory for Cereal Fermentation Technology, Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi 214122, Jiangsu, China b National Engineering Research Center for Chinese Rice Wine, Shaoxing 312000, Zhejiang, China c Research and Development Center, Jiangsu King’s Luck Brewery Joint-Stock Company Limited, Huaian 223411, Jiangsu, China

a r t i c l e

i n f o

Article history: Received 13 October 2016 Received in revised form 5 January 2017 Accepted 5 January 2017 Available online xxx Keywords: Zhenjiang aromatic vinegar Aroma compounds Comprehensive two-dimensional gas chromatography Olfactometry

a b s t r a c t In this work, a method to characterize the aroma compounds of Zhenjiang aromatic vinegar (ZAV) was developed using comprehensive two dimensional gas chromatography (GC × GC) coupled with time-of-flight mass spectrometry (TOFMS) and gas chromatography olfactometry (GC-O). The column combination was optimized and good separation was achieved. Structured chromatograms of furans and pyrazines were obtained and discussed. A total of 360 compounds were tentatively identified based on mass spectrum match factors, structured chromatogram and linear retention indices comparison. The most abundant class in number was ketones. A large number of esters, furans and derivatives, aldehydes and alcohols were also detected. The odor-active components were identified by comparison of the reported odor of the identified compounds with the odor of corresponding GC-O region. The odorants of methanethiol, 2-methyl-propanal, 2-methyl-butanal/3-methyl-butanal, octanal, 1-octen-3-one, dimethyl trisulfide, trimethyl-pyrazine, acetic acid, 3-(methylthio)-propanal, furfural, benzeneacetaldehyde, 3-methyl-butanoic acid/2-methyl-butanoic acid and phenethyl acetate were suspected to be the most potent. About half of them were identified as significant aroma constituents in ZAV for the first time. Their contribution to specific sensory attribute of ZAJ was also studied. The results indicated that the presented method is suitable for characterization of ZAV aroma constituents. This study also enriches our knowledge on the components and aroma of ZAV. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Zhenjiang aromatic vinegar (ZAV) is a popular traditional seasoning in China. It is famous for the elegant and complex aroma. As a protected geographical indication of European Union (No. 501/2012), the unique flavor and manufacturing secret of ZAV has aroused the interest of many researchers. ZAV is a typical product of solid-state fermentation which is regarded as an efficient

∗ Corresponding author at: National Engineering Laboratory for Cereal Fermentation Technology, Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi 214122, Jiangsu, China. E-mail address: [email protected] (J. Mao).

process to improve the flavor, nutrition and healthy function of food [1]. It is mainly produced from sticky rice, koji (a specific fermented cereal containing moulds, yeasts and bacteria) and wheat bran. As the flowchart outlined in Fig. 1, the manufacturing process mainly includes liquid-state saccharification and alcohol fermentation, solid state acetic acid fermentation, leaching, decoction and aging [2]. Multitudinous microorganisms such as lactic acid bacteria, moulds, yeast and acetic acid bacteria are involved in the brewing process and produce large number of metabolites [1,3,4]. In addition, complex chemical reactions including hydrolysis of saccharides and amino acids under weakly acidic condition, Maillard reaction and esterification may occur during the high temperature (boiling) of decoction and long maturing time (several months or

http://dx.doi.org/10.1016/j.chroma.2017.01.014 0021-9673/© 2017 Elsevier B.V. All rights reserved.

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Fig. 1. The flowchart of Zhenjiang aromatic vinegar production.

longer) [5]. Therefore, like other fermentation products, the composition of ZAV is rather complex and results in the outstanding flavor. The aroma compound is an important factor affecting product quality and sensory acceptability of vinegar. Therefore, identification and quantitation of aroma compounds is a matter of particular interest for researchers. Traditional one dimensional gas chromatography is usually used in analyses of ZAV and typically dozens of compounds are determined in a single analysis. The extraction method for vinegar volatiles mainly includes head space solid-phase microextraction (HS-SPME) [6,7], solid-phase extraction (SPE) [8], stir bar sorptive extraction (SBSE) [9,10] and headspace sorptive extraction (HSSE) [11]. SPME, SBSE and HSSE are popular because of the ease of operation and solvent free nature. They are suitable for compounds with good volatility. The main advantage of SPE is high selectivity and sensitivity. It can be used to extract semi-volatile compounds and trace components [12,13]. However, due to the complexity of ZAV and the limited resolution power of traditional one dimensional GC, only several publications were related to analysis of ZAV volatile compounds [14–19] and no more than one hundred volatiles have been identified at present. Comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC × GC-TOFMS) has

shown to be a powerful and effective technique for analysis of complex samples resulted from its apparent advantages over traditional GC/MS: high peak capacity, high resolving power and structured chromatograms. It has been used to reveal the complex volatile composition of various foods [20] and amazing results were obtained. In this study, the volatile constituents of ZAV was revealed using GC × GC-TOFMS in combination with HS-SPME. The chromatographic conditions were optimized and a method suited for ZAV analysis was developed. The unknown peaks were tentatively identified with mass spectra comparison, structured chromatogram and linear retention index (LRI). Although every component is the potential contributor of food aroma, the odor-active compounds are usually regarded as the most significant. An essential tool for identification of odor-active constituents is the coupling of gas chromatography with olfactometry (GC-O). The ZAVs were analyzed with GC-O coupled with HS-SPME and some compounds were suggested as the key contributors [15,18]. In this paper, GCO analyses were also employed aiming at identification of new potent odorants with the help of the powerful resolving capability of GC × GC-TOFMS.

Please cite this article in press as: Z. Zhou, et al., Elucidation of the aroma compositions of Zhenjiang aromatic vinegar using comprehensive two dimensional gas chromatography coupled to time-of-flight mass spectrometry and gas chromatography-olfactometry, J. Chromatogr. A (2017), http://dx.doi.org/10.1016/j.chroma.2017.01.014

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2. Experimental 2.1. Samples and reagents Four bottled commercial Zhenjiang aromatic vinegars were purchased from local market and manufactured by Jiangsu Hengshun Vinegar-Industry Co., Ltd (Zhenjiang, China). Sample 1 and 2 belong to K type with a total acid content 5.50–5.99 g/100 mL; the other two (3 and 4) are B type, the total acid content is 5.00–5.49 g/100 mL. Sodium chloride was analytically pure and purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). The internal standard 4-methyl-2-pentanol and n-alkanes of C7-C26 used for linear retention index (LRI) determination were bought from Sigma-Aldrich China Co. (Shanghai, China). A SPME fiber of 75 ␮m Carboxen–polydimethylsiloxane (CAR/PDMS) from Supelco (Supelco, Belletonte, PA, USA) was used. The fiber was conditioned prior to use according to supplier’s instructions by inserting them into the GC injector. Pure water was obtained from a Milli-Q purification system (Millipore, Bedford, MA). 2.2. Sample preparation Sample preparation followed the method described by Natera el al. [6] with slight modification. A volume of 10 mL vinegar sample was added to a 20 mL vial. After saturated with sodium chloride, 30 ␮L internal standard solution of 4-methyl-2-pentanol (0.2 g/L in Milli-Q water containing 60 g/L of acetic acid) and a small magnetic stirring bar were added. The vial was tightly capped with a silicon septum. The sample was equilibrated at 70 ◦ C for 15 min and then extracted for 40 min at the same temperature. The fiber was desorbed in the injection port at 250 ◦ C for 6 min. Each sample was analyzed in duplicate. 2.3. GC × GC-TOFMS and GC-O analysis The GC × GC-TOFMS and GC-O system consisted of an Agilent 7890A GC (Agilent Technologies, Wilmington, DE, USA), a time-offlight mass spectrometer Pegasus 4D (Leco, St. Joseph, MI, USA), a sniff port (ODP 2, Gerstel, Germany) and a cold-jet modulator KT2001 retrofit prototype (Zoex, Lincoln, NE, USA). Two column sets were used. Their parameters and the temperature programs are shown in Table 1. The sniff port was connected to the first column before the modulator. The effluent from the 1 st column was split at a ratio of 4:1 between the sniff port and mass spectrometer. The carrier gas was high purity helium with a constant flow rate of 1.0 mL/min. The injection temperature was maintained at 250 ◦ C. The splitless mode was used. The time-of-flight mass spectrometer was operated at a spectrum storage rate of 100 Hz, using a mass range of m/z 35–400 and a multi channel plate voltage of 1600 V. The filament voltage was 70 eV. The ion source and transfer line were held at 240 ◦ C and 250 ◦ C, respectively. The modulation period was 5 s with a hot pulse time of 1.2 s. Intensity method was employed in GC-O analysis and five well trained panelists (three female and two male) were selected. The odor intensities were evaluated with a 6-point intensity scale from 0 to 5; “0” was none, “3” was moderate, and “5” was extreme. The retention time, intensity value, and odor descriptor were recorded. Each fraction was replicated two times by each panelist. The intensity scores from all assessors were averaged to yield the final odor intensity score. 2.4. Data processing The Leco Chroma TOF software (version V 4.32) was used to find all the peaks in the raw GC × GC chromatogram with the signal-tonoise (S/N) ratio higher than 20 and the similarity value greater than

3

500. The data processing including peak finding, deconvolution and library search was performed by this software fully automated without any user interaction, and the results were combined in a single peak table. The peaks were first screened with the mass spectra match factors and tentatively identified by structured chromatogram and comparison of experimental linear retention index (LRI) with those from literature or authentic compounds. 3. Results and discussion 3.1. Selection of GC × GC column system and chromatographic separation Preliminary experiments showed that the headspace of ZAV was rather complex and excessive peak co-eluting were observed in the analysis using traditional one-dimensional gas chromatography. In GC × GC analysis, an appropriate column set is vital to good separation. The combination of a non-polar first column and a polar or medium polar second column (usually called as normal column combination) is the most frequently used [21]. The ZAV sample was analyzed using this combination with a non-polar (TG-5MS) first column and a medium polar (Rtx-17) second column. Part A of Fig. 2 depicts the GC × GC-TOFMS contour plot (total ion chromatogram) of ZAV extracted with HS-SPME. Orthogonal separation was obtained and some compounds (furans, pyrazines and phenols) were found orderly eluted which is very helpful to the identification of unknown peaks. However, the peak shape of some polar compounds, such as acetic acid and 2-furfural, were not satisfactory. Acetic acid and 2-furfural were the most abundant volatiles observed in ZAV, they were not effectively retained by this column set and tailed badly, especially for acetic acid. This might interfere the separation and identification of small peaks. The so called “reversed type” column system with polar first column and non-polar or medium polar second column was reported more beneficial to the separation of polar compounds [21,22]. A HPINNOWax × Rtx-5MS column set was tested in this work. As shown in Fig. 2B, the peak shape of acetic acid and 2-furfural was better compared with that of TG-5MS × Rtx-17 column system. Orthogonal separation and structured chromatogram of furans, pyrazines and phenols were also obtained. In addition, the peaks in Fig. 2B distributed much wider in the whole two-dimensional plane and the resolution was more satisfactory compared with Fig. 2A. Therefore, the “reversed type” column system was selected for further analysis. 3.2. Identification of unknown peaks and volatile profile of ZAV In GC × GC analysis of complex samples, identification of unknown peaks could be effectively carried out by the combination of mass spectral match factors, structured chromatogram and retention index [23,24]. A peak table was obtained after the automatic processing of the software. The peaks resulting from stationary phase bleeding were removed manually. As a fermented product, ZAV was manufactured with complex processing and multiple microorganisms, the volatile constitutes were rather complex. More than 2000 peaks with similarity greater than 600 and S/N ratio higher than 50 were found. The number of peaks with similarity higher than 750 was over 800 hundred in each sample. Therefore, the peaks with similarity values lower than 700 and signal-to-noise ratio less than 50 were discarded to improve the identification credibility. Many homologues and derivatives, such as aldehydes, esters, alcohols, furans and phenols were found in ZAV. Ordered separation of some categories was obtained profiting from the chosen column system. The group-type separation of ethyl esters, 2-ketones, alcohols and organic acids were observed as expected and had been

Please cite this article in press as: Z. Zhou, et al., Elucidation of the aroma compositions of Zhenjiang aromatic vinegar using comprehensive two dimensional gas chromatography coupled to time-of-flight mass spectrometry and gas chromatography-olfactometry, J. Chromatogr. A (2017), http://dx.doi.org/10.1016/j.chroma.2017.01.014

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4 Table 1 The GC × GC column sets and temperature programs. Parameters set 1 Length (m) Diameter (mm) Stationary phase Film thickness (␮m) Temperature program

set 2 Length (m) Diameter (mm) Stationary phase Film thickness (␮m) Temperature program

a b c d

1st Column

2nd Column

30 0.25 TG-5MSa 0.25 40 ◦ C (0.2 min) → 3 ◦ C/min → 200 ◦ C → 10 ◦ C/min → 250 ◦ C(5 min)

2.0 0.1 Rtx−17b 0.1 50 ◦ C (0.2 min) → 3 ◦ C/min → 210 ◦ C→ 10 ◦ C/min → 260 ◦ C(5 min)

30 0.25 HP-INNOWaxc 0.25 40 ◦ C (0.2 min) → 3 ◦ C/min → 200 ◦ C → 10 ◦ C/min → 230 ◦ C(5 min)

1.9 0.1 Rtx-5MSd 0.1 50 ◦ C (0.2 min) → 3 ◦ C/min → 210 ◦ C → 10 ◦ C/min → 240 ◦ C(5 min)

TG-5MS (Thermo Fisher Scientific), a (5%-phenyl)-methylpolysiloxane. Rtx-17 (Restek), a (50%-phenyl)-methylpolysiloxane. HP-INNOWax (J&W Scientific, Folsom, CA), a poly(ethylene glycol). Rtx-5MS (Restek, Bellefonte, PA), a (5%-phenyl)-methylpolysiloxane.

Fig. 2. The GC × GC-TOFMS contour plot (total ion chromatogram, TIC) of Zhenjiang aromatic vinegar extracted by HS-SPME, A and B were analyzed with column set 1 and 2 in Table 1, respectively. The compounds identified as furans, pyrazines and phenols were mainly located in the region labeled as a, b and c, respectively.

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furans and derivatives, aldehydes and alcohols were also found. This is obviously different from the results from traditional one dimensional GC/MS [15,16,18]. The most abundant category is usually alcohols or acids and a small number of ketones and furans were identified in the analysis of one dimensional GC/MS. This mainly resulted from the high resolving power and sensitivity of GC × GCTOFMS over traditional GC/MS. Most of the ketones and furans are in trace level and could not be effectively separated and detected by one dimensional GC/MS. Furthermore, organic acid and furans occupied the highest area percentage, followed by esters, ketones and alcohols.

Fig. 3. Ordered chromatogram of alkyl furans and alkyl pyrazines, A and B were analyzed with column set 1 and 2 in Table 1, respectively.

discussed in GC × GC analyses of Chinese liquor Moutai [22] and Brazilian Merlot wines [25] using similar column system. A relatively large number of furans and pyrazines were observed in ZAV and were found ordered separated in this study. The apex plot of alkyl substituted furans and pyrazines is shown in Fig. 3. They obviously distribute along a straight line. The linearity and correlation coefficients are also showed in Fig. 3. The correlation coefficients of alkyl furans on both column systems were all higher than 0.98. As for alkyl pyrazines, good linearity (R2 = 0.956) was observed with the “reversed type” column set (set 2 in Table 1), but the linearity (R2 = 0.207) for the normal column combination (set 1 in Table 1) was very bad. This confirmed the superiority of the “reversed type” column set over the normal combination for analysis of ZAV. Using the linear relationship of two dimensional retention times of homologous members, their separation can be predicted or confirmed. This is very useful for credible identification. Furthermore, retention index was also employed to improve the identification reliability. The approximate one dimensional retention indices were calculated using the absolute retention time of n-alkanes from n-heptanes to n-hexacosane according to van den Dool and Kratz equation [26]. The obtained values were compared with those from the database [27]. The criterion of LRI difference between measured values and literature numbers for nonpolar and polar columns were set as 20 and 30, respectively. A total of 360 compounds were tentatively identified using this method and are listed in Supplementary Table S1. They were also classified into 10 categories and the compound number and area percentage of each group are summarized in Table 2. The most abundant class in number was ketones. A large number of esters,

3.2.1. Ketones and aldehydes A total of 67 ketones including 35 acyclic ketones and 32 cyclic ketones were identified. Among the acyclic ketones, 2and 3- substituted aliphatic ketones were abundant in number; many cyclopentanones and cyclohexanones were observed for cyclic ketones. The relative content of 3-pentanone, 3-hydroxy-2butanone (acetoin) and 1-(acetyloxy)-2-propanone are the highest (over 1%). Acetoin was initially produced in alcohol fermentation and further accumulated during acetic fermentation through transformation of ␣-acetolactate and 2,3-butanediol acted by acetic bacteria [28]. This compound could be further oxidized to form diacetyl which was identified as a key aroma compound of Sherry vinegar [29,30]. Among the 39 identified aldehydes, n-aliphatic aldehydes, 2and 3-substituted saturated and unsaturated aliphatic ketones and aromatic aldehyde are the dominant catagories. The homologous series of saturated straight-chain primary aldehydes from aldehyde to undecanal were all detected. 2-Methyl-butanal, 3-methylbutanal, benzaldehyde and benzeneacetaldehyde accounted for higher area percentage than other aldehydes in ZAV. Alcohol oxidation and raw material leakage might be responsible for aldehydes generation [28]. However, alcohol fermentation probably was also an important source because many aldehydes were detected in Chinese rice wine (the substrate of acetic acid fermentation) [31].

3.2.2. Organic acids, alcohols and esters Undoubtedly, acids are the most important flavor compounds in vinegars. They mainly derived from alcohol oxidation acted by acetic bacteria [3,28]. A total of 22 volatile organic acids were found in ZAV, most of which were saturated monocarboxylic fatty acids. The homologous series of straight-chain monocarboxylic acids from acetic acid to decanoic acid were all detected. Hexanoic acid, 3-methyl-butanoic acid and 2-methyl-butanoic acid made up higher area percentage than other volatile organic acids apart from acetic acid. Alcohols might mainly come from alcohol fermentation. The homologous series of saturated straight-chain primary alcohols from butanol to nonanol were all detected. Furthermore, some saturated branched alcohols, unsaturated alcohols, polyhydric alcohols and aromatic alcohols were identified. Esters are usually regarded as important contributor of the fruity and floral notes of vinegar. They might mainly derive from alcohol fermentation or esterification between alcohols and acids during aging [32,33]. Totally, 61 esters were identified in ZAV. Ethyl esters and acetic acid esters were the most prominent categories. The homologous series of saturated straight-chain fatty acids ethyl ester from C2-C8 and saturated straight-chain fatty alcohol acetates from C1-C8 were all identified. In addition to ethyl esters and acetic acid esters, esters derived from formic ethers, phenyl esters, dicarboxylic acid esters, polyol esters, oxo-fatty acid esters, unsaturated fatty acid esters and aromatic acid esters were also identified.

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Table 2 Summary of compound number and area percentage of different categories identified in ZAV. Categories

Number

Area percentage (%) Sample 1

Sample 2

Sample 3

Sample 4

Average

Aldehydes Alcohols Esters Organic acids Furans and derivatives Phenols Sulfur containing compounds Ketones Lactones Nitrogen containing compounds Pyrazines Others Total

39 33 61 22 41 11 27 67 18

2.0 4.0 5.8 14.6 9.7 0.24 0.39 5.0 0.17

2.1 3.8 6.2 15.1 10.2 0.21 0.41 5.1 0.15

2.0 4.1 5.6 13.4 9.5 0.25 0.39 5.3 0.17

1.9 4.2 5.8 13.7 8.9 0.22 0.38 5.0 0.19

2.0 4.0 5.9 14.2 9.6 0.23 0.39 5.1 0.17

19 22 360

0.56 0.51 –

0.61 0.53 –

0.57 0.51 –

0.52 0.49 –

0.57 0.51 –

3.2.3. Furans and derivatives, pyrazines and lactones One characteristic fragrance of ZAV is burnt and caramel-like odor [13]. Furans, pyrazines and lactones might be significant contributors because many of them impart this type of perfume [34–36]. A relatively large number of furanic compounds were detected in ZAV in our study. Alkyl substituted furans was the dominant category, many furan aldehydes and furanones were also detected. They might mainly result from sugar thermal degradation and Maillard reaction under acidic condition during the longtime high temperature decoction process (slightly boiling at about 102 ◦ C for at least 60 min) [37]. In addition, the addition of parched rice as colorant might also be responsible for furans generation because furanic compounds were important pyrolysis products of various saccharides under high temperature [38,39]. The peak of furfural was the largest among these furanic compounds. It accounted for more than half peak area of furan components. This compound, together with 5-hydroxymethylfurfural are usually regarded as an indicator of sugar degradation and Maillard reaction [40]. Also, they were found closely related to sugar and some odorants in sugar-rich wines [41,42]. Other compounds which were relevant to Maillard reaction were pyrazines [34,35,43]. A total of 19 pyrazines were identified and most of them were saturated alkyl pyrizines. Tetramethyl-pyrazine occupied the highest area percentage among them. Special attention has been put on this compound because of its potential health functions. It mainly generated during aging via synthesis of precursors [44]. In addition, 18 lactones were detected in ZAV. They might mainly form from cyclization of hydroxyl acids during alcohol and acetic fermentation and maturing. Although their relative content were not high (0.17% in total), they probably made positive contribution to the overall aroma of ZAV because many lactones have pleasant fragrance and low threshold [36]. 3.2.4. Sulfur containing compounds and phenols Considerable amount of sulfur compounds were detected in ZAV. The area percentage of 3-(methylthio)-propanal was the largest among them, followed by methanethiol and 3thiophenecarboxaldehyde. Volatile sulfur compounds might mainly generated from metabolism of sulfur sources and precursors acted by yeast [45]. Bacteria were probably also responsible for the formation of sulfur compounds [46]. Among the detected phenols, alkyl phenols and guaiacol derivatives were dominant. Their presence was likely relevant to materials leakage (rice, wheat Qu) and microbe metabolism [28]. 3.3. Identification of odor-active compounds using GC-O The odor-active components were further identified with GCO. In the present GC × GC analysis, elutes from the first column

were divided into two parts. One part was modulated into the second column and separated; the other part flowed directly into the sniff port and was evaluated by the assessors. Therefore, the odor from the olfactometer might arise from a zone consisted of several compounds. The identification of the odor-active components was performed based on the odor of each peak, the perceived odor descriptions of various peaks were compared with reported odor descriptors from literature [5,31,47–49]. Table 3 shows the odorants identified by GC-O. A total of 31 odor regions were found. Three odor regions corresponded to more than one compounds. These peaks co-eluted in one dimensional GC but well resolved in the second axis of GC × GC analysis. Three compounds (methanethiol, carbon disulfide and dimethyl sulfide) were found coincided with the odor (cooked cabbage) at RI (HP-INNOWax) around 760. The odor might result from their sensory mixture because they were reported to have a similar odor of cooked cabbage, rubber and putrefaction [48]. However, methanethiol might be the main contributor of this odor region if their threshold and relative content were taken into consideration (methanethiol, 0.02–2 ␮g/L in water, 0.043%; carbon disulfide, 0.05–0.5 ␮g/L in air, 0.015%; dimethyl sulfide, 0.3–10 ␮g/L in water, 0.006%). The perceived aroma at RI (HP-INNOWax) 910 was probably due to the isomer mixture of 2-methyl-butanal and 3-methyl-butanal because they both have a fruity, cocoa and malty aroma and their threshold were close to each other [50]. The odor described as rancid, acidic and cheese-like at RI (HP-INNOWax) 1655 was indicated by TOFMS as a mixture of 3-methyl-butanoic acid and 2-methyl-butanoic acid. The two peaks overlapped even in the GC × GC analysis with the polar × nonpolar column system. Their peak apexes were separated by only 0.03 s in the second dimension. However, they were recognized and identified by the deconvolution algorithm of TOFMS using the unique mass of each compound. These two compounds both impart an acidic and cheese-like odor and their odor threshold and relative content are also close to each other (3-methyl-butanoic acid, 0.033 mg/L [51], 0.38%; 2-methyl-butanoic acid, 0.01–0.1 mg/kg [52], 0.33%). This suggested that they both were important contributor of this odor zone. The identified odor compounds were mainly composed of acids, aldehydes, sulfides, furans, pyrizines, ketones, phenols and lactones. The most potent odorants (odor intensity higher than 4.0) were identified as: Methanethiol, 2-methyl-propanal, 2-methylbutanal/3-methyl-butanal, octanal, 1-octen-3-one, dimethyl trisulfide, trimethyl-pyrazine, acetic acid, 3-(methylthio)-propanal, furfural, benzeneacetaldehyde, 3-methyl-butanoic acid/2-methylbutanoic acid and phenethyl acetate. The odorants of 3-Methylbutanoic acid, furfural, trimethyl-pyrazine, dimethyl trisulfide, 2-methyl-butanal/3-methyl-butanal, acetic acid and phenethyl acetate were reported previously as the key aroma components

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Odor intensitya

RIb Sample 2

Sample 3

Sample 4

HP-INNOWax

TG-5MS

4.1

3.8

4.2

4.0

Vanilla, spicy Cocoa, fruity

4.8 5.0

4.8 5.0

4.6 4.8

4.4 5.0

Creamy, butter Vegetable, soil Fruity, fatty Mushroom, mouldy Creamy, roast Burnt sugar, peanut Onion, stewed meat Roasted potato Acid, vinegar Mouldy, mushroom Onion, soy sauce Burnt sugar, almond Peanut Almond Burnt sugar Acidic, smelly Floral, rose Floral Rancid, acidic, cheese-like

3.2 3.1 4.4 4.3 2.7 2.3 4.8 4.2 5.0 2.4 4.2 4.3 2.8 2.2 2.1 2.9 4.6 3.1 5.0

3.2 3.4 4.5 3.8 2.6 2.0 4.6 4.3 5.0 2.3 4.1 4.4 2.6 2.2 2.0 2.2 4.3 3.1 5.0

3.0 2.9 4.0 3.6 2.8 2.2 4.9 4.1 5.0 1.8 4.4 4.1 2.7 2.0 2.1 2.4 4.5 2.8 5.0

3.2 3.1 4.3 4.2 3.0 1.9 4.8 4.5 5.0 2.1 4.1 4.3 2.4 2.3 2.3 2.3 4.6 3.2 5.0

Rancid, acidic Sweet, honey, fruity Sweaty, rancid Spicy, clove Floral, rose Coconut Smoky Coconut, sweet Clove, smoky

1.6 4.2 3.5 3.3 3.6 3.7 2.7 3.3 2.6

1.8 4.3 3.5 3.5 3.5 3.4 2.8 3.5 2.1

1.5 4.5 3.2 3.2 3.6 3.6 2.6 3.3 2.4

1.6 4.2 3.4 3.7 3.2 3.5 2.8 3.2 2.7

748 758 766.9 804.3 908.8 913.7 972.2 1207.1 1272.2 1292.5 1294 1331.9 1351.7 1403 1414.7 1439.2 1445.1 1459.2 1461.7 1499.7 1554 1613.4 1620.7 1625.4 1655.5 1655.6 1729.5 1799 1836.8 1844.5 1898.1 1898.3 1942.6 2011.3 2182

492.3 nd 758.2 580.4 697.5 689.6 687.4 995.1 1008.4 nd 737.1 925.8 978.4 1011.8 661.2 986.6 915.8 839.8 1090.5 971.7 970 813.9 1051.4 nd 872.8 878.7 917.2 1261.2 1005.1 1095.6 1123 nd 1198.8 1369.8 1321.8

a b c d e

CAS

Referenced odor descriptione

Methanethiold Carbon disulfide Dimethyl sulfide 2-Methyl-propanald 2-Methyl-butanald 3-Methyl-butanald 2,3-Butanedioned 2-Pentyl-furand Octanald 1-Octen−3-oned 3-Hydroxy−2-butanoned 2,6-Dimethyl-pyrazined Dimethyl trisulfide Trimethyl-pyrazined Acetic acidd 1-Octen−3-old 3-(Methylthio)-propanald Furfurald Tetramethyl-pyrazined Benzaldehyded 5-Methylfurfurald Butanoic acidd Benzeneacetaldehyded Acetophenoned 3-Methyl-butanoic acidd 2-Methyl-butanoic acidd Pentanoic acidd Phenethyl acetated Hexanoic acidd Gulaiacold Phenylethyl alcohold -Octanolactoned 4-Methyl-gulaiacold -Nonalactoned 4-Vinyl-gulaiacold

74-93-1 75-15-0 75-18-3 78-84-2 96-17-3 590-86-3 431-03-8 3777-69-3 124-13-0 4312-99-6 513-86-0 108-50-9 3658-80-8 14667-55-1 64-19-7 3391-86-4 3268-49-3 98-01-1 1124-11-4 100-52-7 620-02-0 107-92-6 122-78-1 98-86-2 503-74-2 116-53-0 109-52-4 103-45-7 142-62-1 90-05-1 60-12-8 104-50-7 93-51-6 104-61-0 7786-61-0

Putrefaction, cooked cabbage [48] Rubber, cabbage [48] Cabbage, corn, molasses [48] Fresh, aldehydic, floral [49] Musty, chocolate, nutty [49] Malty, fruity, green, chocolate [31,49] Sweet, creamy, buttery [49] Fruity, green, earthy, vegetable [49] Aldehydic, waxy, citrus, fatty [49] Herbal, mushroom, earthy, musty[49] Sweet, buttery, creamy, fatty[49] Cocoa, roasted nuts, roast beef [49] Brothy, sulfury,cabbage [31,47] Nutty, roasted potato, peanut[5,49] Vinegar, sharp pungent, sour [31,49] Mushroom, earthy, oily, fungal [31,49] Onion, meat, mashed potatoes [48] Almond, burnt sugar [5,31] Peanut, musty, nutty, chocolate[31,49] Almond, cherry, fruity [31,49] Burnt sugar, caramel like, bready [31,49] Aged butter, acidic, cheese [31,47] Green, floral, rose, clover [31,49] Floral, sweet, hawthorn, cherry[31,49] Sour, sweaty, cheesy, smelly [31,49] Acidic, fruity, cheesy, fermented [31,49] Acidic, sweaty, rancid, cheesy [49] Floral, rose, sweet, honey, fruity [5,31] Cheesy, acidic, fatty, sweaty [31,49] Phenolic, smoky, spicy, vanilla [31,49] Floral, rose [31,49] Sweet coconut, waxy, creamy [31,49] Spicy, clove, vanilla, smoky [49] Sweet, creamy, coconut, fatty [31,49] Woody, spicy, clove [31,49]

The odor intensity values were the average of 10 analyses (five panelists × two times). RI, retention index detected on different stationary phases; nd, not detected. Identification based on mass spectrometry, RI comparison with the values from authentic compounds or NIST web RI database [27] and odor description. Compounds also identified by comparison with authentic standards. Odour description reported from previous literature.

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Sample 1 Cooked cabbage

Compound namec

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Ordor description

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Table 3 Odor compounds identified in ZAV by GC-O and GC × GC-TOFMS.

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in ZAV [15,18]. The other seven components were firstly identified as important odorants in ZAV in this work. Acidic aroma is unquestionably one of the most important sensory attributes of vinegar products. Acetic acid and 3-methylbutanoic acid/2-methyl-butanoic acid were identified as the most odor-active volatile acids in ZAV. They were also reported as the most potent odorants in strawberry vinegars [53], rabbiteye blueberry vinegars [54] and red wine vinegars [32]. Acetic acid is the most abundant acid in ZAV and imparts strong pungent and acidic odor, it is responsible for the acidic aroma of various vinegars. However, 3-methyl-butanoic acid/2-methyl-butanoic acid produce weak acidic and intense rancid and cheese-like odor [32]. They might be the main contributor of the fermentation fragrance of ZAV. Another typical sensory attribute of ZAV is caramel-like aroma [13]. It probably mainly arose from trimethyl-pyrazine and furfural among the most potent odorants because trimethylpyrazine has a roast potato, peanut and nut odor and furfural imparts almond and burnt sugar aroma [5]. Several aldehydes (2-methyl-propanal, 2-methyl-butanal/3-methyl-butanal, octanal and benzeneacetaldehyde) were identified as significant contributor of ZAV aroma. They all could produce some kind of fruity and floral fragrance. In addition, phenethyl acetate [5] imparts pleasant rose and sweet honey aroma. These compounds might interact with each other and contribute jointly to the sweet fruity and floral aroma of ZAV [13]. Two sulfur compounds, dimethyl trisulfide and 3-(methylthio)-propanal were found might be important odorants in ZAV. Sulfur containing compounds are usually regarded as offensive odorants because of the rotten egg, onion and cabbage smell. However, they produce cooked meat and soy sauce like fragrance and were thought responsible for the desirable wine aroma at low concentration [48]. Therefore, they might be important contributor of the soy sauce and fermentation like odor of ZAV. Although the potent odorants were identified and their odor characteristics were evaluated, quantitative analysis and aroma reconsitution would be necessary for a precise definition of the contribution of significant odorants to the overall aroma because of the impacts of non-volatile matrix and interactions among volatiles.

4. Conclusions The “reverse type” (polar × nonpolar) column set was proven to be superior to common column combination of nonpolar × medium polar because ZAV is rich in polar volatiles. A wide range of volatiles were successfully revealed by GC × GC-TOFMS and the odor active components were further identified with the help of GC-O. This indicated that rapid and informative identification of key odorants from complex matrices can be accomplished by coupling GC × GC-TOFMS and GC-O. Further work could be conducted on improvement of extraction selectivity and detection sensitivity on specific compounds, such as sulfur compounds to further improve this approach.

Acknowledgements The authors are grateful for the financial support of National Natural Science Foundation of China (No. 31571823), National High Technology Research and Development Program of China (863 Program, No. 2013AA102203), National Key Research and Development Program of China (No. 2016YFD0400503, 2016YFD0400504) and Postdoctoral Science Foundation of Jiangsu Province, China (No. 1501022B). The authors would also like to thank China Tobacco Anhui Industrial Co., LTD (Hefei, Anhui, China) for the supply of GC × GC-TOFMS and Mr. Zhiqiang Xu who is from this company for his helpful discussions and suggestions.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chroma.2017.01. 014.

References [1] Z.M. Wang, Z.M. Lu, Y.J. Yu, G.Q. Li, J.S. Shi, Z.H. Xu, Batch-to-batch uniformity of bacterial community succession and flavor formation in the fermentation of Zhenjiang aromatic vinegar, Food Microbiol. 50 (2015) 64–69. [2] D.R. Liu, Y. Zhu, R. Beeftink, L. Ooijkaas, A. Rinzema, J. Chen, J. Tramper, Chinese vinegar and its solid-state fermentation process, Food Rev. Int. 20 (2004) 407–424. [3] Z.M. Wang, Z.M. Lu, J.S. Shi, Z.H. Xu, Exploring flavour-producing core microbiota in multispecies solid-state fermentation of traditional Chinese vinegar, Sci. Rep. 6 (2016). [4] W. Xu, Z.Y. Huang, X.J. Zhang, Q. Li, Z.M. Lu, J.S. Shi, Z.H. Xu, Y.H. Ma, Monitoring the microbial community during solid-state acetic acid fermentation of Zhenjiang aromatic vinegar, Food Microbiol. 28 (2011) 1175–1181. [5] A.L. Wang, H.L. Song, C.Z. Ren, Z.G. Li, Key aroma compounds in Shanxi aged tartary buckwheat vinegar and changes during its thermal processing, Flavour Frag. J. 27 (2012) 47–53. [6] R. Natera Marı´ın, R. Castro Mejı´ıas, M. de Valme Garcı´ıa Moreno, F. Garcı´ıa Rowe, C. Garcı´ıa Barroso, Headspace solid-phase microextraction analysis of aroma compounds in vinegar: Validation study, J. Chromatogr. A 967 (2002) 261–267. [7] R. Castro Mejı´ıas, R. Natera Marı´ın, M. de Valme Garcı´ıa Moreno, C. Garcı´ıa Barroso, Optimisation of headspace solid-phase microextraction for analysis of aromatic compounds in vinegar, J. Chromatogr. A 953 (2002) 7–15. [8] E. Durán Guerrero, F. Chinnici, N. Natali, R.N. Marín, C. Riponi, Solid-phase extraction method for determination of volatile compounds in traditional balsamic vinegar, J. Sep. Sci. 31 (2008) 3030–3036. [9] E.D. Guerrero, R.N. Marín, R.C. Mejías, C.G. Barroso, Stir bar sorptive extraction of volatile compounds in vinegar: validation study and comparison with solid phase microextraction, J. Chromatogr. A 1167 (2007) 18–26. [10] A. Marrufo-Curtido, M.J. Cejudo-Bastante, E. Durán-Guerrero, R. Castro-Mejías, R. Natera-Marín, F. Chinnici, C. García-Barroso, Characterization and differentiation of high quality vinegars by stir bar sorptive extraction coupled to gas chromatography-mass spectrometry (SBSE-GC–MS), LWT Food Sci. Technol. 47 (2012) 332–341. [11] R.M. Callejón, A.G. González, A.M. Troncoso, M.L. Morales, Optimization and validation of headspace sorptive extraction for the analysis of volatile compounds in wine vinegars, J. Chromatogr. A 1204 (2008) 93–103. [12] B.T. Weldegergis, A.M. Crouch, T. Górecki, A. de Villiers, Solid phase extraction in combination with comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry for the detailed investigation of volatiles in South African red wines, Anal. Chim. Acta 701 (2011) 98–111. [13] R. Castro, R. Natera, E. Durán, C. García-Barroso, Application of solid phase extraction techniques to analyse volatile compounds in wines and other enological products, Eur. Food Res. Technol. 228 (2008) 1–18. [14] B.B. Guan, J.W. Zhao, M.J. Cai, H. Lin, L.Y. Yao, L.L. Sun, Analysis of volatile organic compounds from Chinese vinegar substrate during solid-state fermentation using a colorimetric sensor array, Anal. Methods 6 (2014) 9383–9391. [15] Z.B. Sun, Y.P. Fan, P. Ji, Comparative study on the flavor compounds of three brands of Zhenjiang aromatic vinegar analyzed by SPME-GC–MS-O, China Condiment 40 (2015) 26–29. [16] Z.B. Xiao, S.P. Dai, Y.W. Niu, H.Y. Yu, J.C. Zhu, H.X. Tian, Y.B. Gu, Discrimination of Chinese vinegars based on headspace solid-phase microextraction-gas chromatography mass spectrometry of volatile compounds and multivariate analysis, J. Food Sci. 76 (2011) C1125–C1135. [17] C. Xiong, Y.J. Zheng, Y.N. Xing, S.J. Chen, Y.T. Zeng, G.H. Ruan, Discrimination of two kinds of geographical origin protected Chinese vinegars using the characteristics of aroma compounds and multivariate statistical analysis, Food Anal. Methods 9 (2016) 768–776. [18] Y.k. Ma, J.K. Jiang, Y.Y. Wei, L.L. Sun, R. Xia, K.P. Xu, Establishment of fingerprint of aroma components of Zhenjiang frangrance vinegar based on GC–MS analysis and GC-olfactory, Food Sci. 28 (2007) 496–499. [19] Y.J. Yu, Z.M. Lu, N.H. Yu, W. Xu, G.Q. Li, J.S. Shi, Z.H. Xu, HS-SPME/GC–MS and chemometrics for volatile composition of Chinese traditional aromatic vinegar in the Zhenjiang region, J.I. Brewing 118 (2012) 133–141. [20] P.Q. Tranchida, G. Purcaro, M. Maimone, L. Mondello, Impact of comprehensive two-dimensional gas chromatography with mass spectrometry on food analysis, J. Sep. Sci. 39 (2016) 149–161. [21] M. Adahchour, J. Beens, U.A. Brinkman, Recent developments in the application of comprehensive two-dimensional gas chromatography, J. Chromatogr. A 1186 (2008) 67–108. [22] S.K. Zhu, X. Lu, K.L. Ji, K.L. Guo, Y.L. Li, C.Y. Wu, G.W. Xu, Characterization of flavor compounds in Chinese liquor Moutai by comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry, Anal. Chim. Acta 597 (2007) 340–348.

Please cite this article in press as: Z. Zhou, et al., Elucidation of the aroma compositions of Zhenjiang aromatic vinegar using comprehensive two dimensional gas chromatography coupled to time-of-flight mass spectrometry and gas chromatography-olfactometry, J. Chromatogr. A (2017), http://dx.doi.org/10.1016/j.chroma.2017.01.014

G Model CHROMA-358193; No. of Pages 9

ARTICLE IN PRESS Z. Zhou et al. / J. Chromatogr. A xxx (2017) xxx–xxx

[23] X. Lu, J.L. Cai, H.W. Kong, M. Wu, R.X. Hua, M.Y. Zhao, J.F. Liu, G.W. Xu, Analysis of cigarette smoke condensates by comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry I acidic fraction, Anal. Chem. 75 (2003) 4441–4451. [24] J. Dallüge, L.L.P. van Stee, X.B. Xu, J. Williams, J. Beens, R.J.J. Vreuls, U.A.T. Brinkman, Unravelling the composition of very complex samples by comprehensive gas chromatography coupled to time-of-flight mass spectrometry Cigarette smoke, J. Chromatogr. A 974 (2002) 169–184. [25] J.E. Welke, V. Manfroi, M. Zanus, M. Lazarotto, C. Alcaraz Zini, Characterization of the volatile profile of Brazilian Merlot wines through comprehensive two dimensional gas chromatography time-of-flight mass spectrometric detection, J. Chromatogr. A 1226 (2012) 124–139. [26] H. van Den Dool, P. Dec, Kratz, A generalization of the retention index system including linear temperature programmed gas-liquid partition chromatography, J. Chromatogr. A 11 (1963) 463–471. [27] NIST Chemistry Webbook, NIST Standard Reference Database Number 69, http://webbook.nist.gov/chemistry/. [28] F. Chinnici, E.D. Guerrero, F. Sonni, N. Natali, R.N. Marín, C. Riponi, Gas chromatography-mass spectrometry (GC–MS) characterization of volatile compounds in quality vinegars with protected european geographical indication, J. Agric. Food Chem. 57 (2009) 4784–4792. [29] R.M. Callejón, M.L. Morales, A.M. Troncoso, A.C. Silva Ferreira, Targeting key aromatic substances on the typical aroma of Sherry vinegar, J. Agric. Food Chem. 56 (2008) 6631–6639. [30] R.M. Callejón, M.L. Morales, A.C.S. Ferreira, A.M. Troncoso, Defining the typical aroma of Sherry vinegar: sensory and chemical approach, J. Agric. Food Chem. 56 (2008) 8086–8095. [31] S. Chen, Y. Xu, M.C. Qian, Aroma characterization of Chinese rice wine by gas chromatography-olfactometry, chemical quantitative analysis, and aroma reconstitution, J. Agric. Food Chem. 61 (2013) 11295–11302. [32] M. Charles, B. Martin, C. Ginies, P. Etievant, G. Coste, E. Guichard, Potent aroma compounds of two red wine vinegars, J. Agric. Food Chem. 48 (2000) 70–77. [33] M.L. Morales, W. Tesfaye, M.C. García-Parrilla, J.A. Casas, A.M. Troncoso, Evolution of the aroma profile of Sherry wine vinegars during an experimental aging in wood, J. Agric. Food Chem. 50 (2002) 3173–3178. [34] M.A.J.S. van Boekel, Formation of flavour compounds in the Maillard reaction, Biotechnol. Adv. 24 (2006) 230–233. [35] S. Fors, Sensory properties of volatile Maillard reaction products and related compounds, In: ACS symposium series-American Chemical Society (USA), (1983). [36] L. Dufossé, A. Latrasse, H.E. Spinnler, Importance of lactones in food flavorsstructure distribution, sensory properties and biosynthesis, Sci. Aliments 14 (1994) 17–50. [37] J.Y. Liu, J. Gan, Y.J. Yu, S.H. Zhu, L.J. Yin, Y.Q. Cheng, Effect of laboratory-scale decoction on the antioxidative activity of Zhenjiang aromatic vinegar: the contribution of melanoidins, J. Funct. Foods 21 (2016) 75–86.

9

[38] R. Talhout, A. Opperhuizen, J.G.C. van Amsterdam, Sugars as tobacco ingredient: effects on mainstream smoke composition, Food Chem. Toxicol. 44 (2006) 1789–1798. [39] E. Roemer, M.K. Schorp, J.J. Piadé, J.I. Seeman, D.E. Leyden, H.J. Haussmann, Scientific assessment of the use of sugars as cigarette tobacco ingredients: a review of published and other publicly available studies, Crit. Rev. Toxicol. 42 (2012) 244–278. [40] L. Ait Ameur, B. Rega, P. Giampaoli, G. Trystram, I. Birlouez-Aragon, The fate of furfurals and other volatile markers during the baking process of a model cookie, Food Chem. 111 (2008) 758–763. [41] J.S. Câmara, J.C. Marques, M.A. Alves, A. C, Silva ferreira 3-hydroxy-4,5-dimethyl-2(5H)-furanone levels in fortified madeira wines: relationship to sugar content, J. Agric. Food Chem. 52 (2004) 6765–6769. [42] A.C. Silva Ferreira, J.-C. Barbe, A. Bertrand, 3-hydroxy-4, 5-dimethyl-2(5H)-furanone: a key odorant of the typical aroma of oxidative aged Port wine, J. Agric. Food Chem. 51 (2003) 4356–4363. [43] F. Van Lancker, A. Adams, N. De Kimpe, Formation of pyrazines in Maillard model systems of lysine-containing dipeptides, J. Agric. Food Chem. 58 (2010) 2470–2478. [44] W. Xu, Q.P. Xu, J.H. Chen, Z.M. Lu, R. Xia, G.Q. Li, Z.H. Xu, Y.H. Ma, Ligustrazine formation in Zhenjiang aromatic vinegar: changes during fermentation and storing process, J. Sci. Food Agric. 91 (2011) 1612–1617. [45] J.H. Swiegers, I.S. Pretorius, Modulation of volatile sulfur compounds by wine yeast, Appl. Microbiol. Biot. 74 (2007) 954–960. [46] K.E. Seefeldt, B.C. Weimer, Diversity of sulfur compound production in lactic acid bacteria, J. Dairy Sci. 83 (2000) 2740–2746. [47] S.-T. Chin, G.T. Eyres, P.J. Marriott, Identification of potent odourants in wine and brewed coffee using gas chromatography-olfactometry and comprehensive two-dimensional gas chromatography, J. Chromatogr. A 1218 (2011) 7487–7498. [48] M. Mestres, O. Busto, J. Guasch, Analysis of organic sulfur compounds in wine aroma, J. Chromatogr. A 881 (2000) 569–581. [49] The Good Scents Company. http://www.thegoodscentscompany.com. [50] Y. Tan, K.J. Siebert, Quantitative structure-activity relationship modeling of alcohol ester, aldehyde, and ketone flavor thresholds in beer from molecular features, J. Agric. Food Chem. 52 (2004) 3057–3064. [51] V. Ferreira, R. López, J.F. Cacho, Quantitative determination of the odorants of young red wines from different grape varieties, J. Sci. Food Agric. 80 (2000) 1659–1667. [52] M. Larsen, L. Poll, Odour thresholds of some important aroma compounds in strawberries, Z. Lebensm Unters Forch 195 (1992) 120–123. ˇ M.L. [53] C. Ubeda, R.M. Callejón, A.M. Troncoso, J.M. Moreno-Rojas, F. Pena, Morales, Characterization of odour active compounds in strawberry vinegars, Flavour Frag. J. 27 (2012) 313–321. [54] M.S. Su, P.J. Chien, Aroma impact components of rabbiteye blueberry (Vaccinium ashei) vinegars, Food Chem. 119 (2010) 923–928.

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