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Article
Aroma Constituents in Shanxi Aged Vinegar before and after Aging
Jingjing Liang, Jianchun Xie, Li Hou, Mengyao Zhao, Jian Zhao, Jie Cheng, Shi Wang, and Bao-Guo Sun
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03019 • Publication Date (Web): 17 Sep 2016
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Page 1 of 34 Journal of Agricultural and Food Chemistry

Aroma Constituents in Shanxi Aged Vinegar before and after Aging

1
Jingjing Liang, *1Jianchun Xie, 1Li Hou, 1Mengyao, Zhao, 1Jian Zhao, 2Jie Cheng, 2Shi

Wang, and 1Bao-Guo Sun

1
Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Key

Laboratory of Flavor Chemistry, Beijing Technology and Business University (BTBU),

2
Beijing 100048, China. Institute of Quality Standard and Testing Technology for

Agro-products of CAAS, Beijing 100081, China.

*
Corresponding author: Jianchun Xie, School of Food Science and Chemical Engineering, Beijing Technology and Business

University, No. 33 Fucheng Road, Haidian District, Beijing 100048, China. Phone and Fax: (+86) -10-68984859. E-mail:

xjchun@th.btbu.edu.cn.

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1 ABSTRACT: Shanxi aged vinegar is one of the most famous China traditional cereal

2 vinegars produced by spontaneous solid-state fermentation. However, aroma composition of

3 Shanxi aged vinegar was still ambiguous. The Shanxi vinegars before and after aging were

4 both analyzed by solvent-assisted flavor evaporation (SAFE) combined with GC-MS as well

5 as GC-O in aroma extract dilution analysis (AEDA). Total of eighty-seven odor-active

6 regions were found by GC-O, among which eighty odor-active compounds were identified.

7 By GC-MS/MS in selected reaction monitoring (SRM) mode, thirty important identifications

8 were quantitated using authentic standards. In comparison, the aroma molecules for the

9 vinegars before and after aging were almost the same, only their levels were altered with

10 mostly the esters and some compounds of pungent smells lost and those from the maillard

11 reaction especially the pyrazines (e.g. tetramethylpyrazine) greatly increased. As for the aged

12 vinegar, the compounds found in high FD factors (> 128) were 3-(methylthio)propanal,

13 vanillin, 2,3-butanedione, tetramethylpyrazine, 3-methylbutanoic acid, γ-nonalactone, guaiacol,

14 3-(methylthio)propyl acetate, dimethyl trisulfide, phenylacetaldehyde, 2-ethyl-6-methylpyrazine,

15 2-acetylpyrazine, 2,3-dimethylpyrazine, furfural, and 3-hydroxy-2-butanone. However, the

16 aroma compounds found in high concentrations (> 25 µg/L) in the aged vinegar were acetic

17 acid, followed by 2,3-butanedione, furfural, 3-hydroxy-2-butanone, tetramethylpyrazine,

18 furfuryl alcohol, and 3-methylbutanoic acid.

19 KEYWORDS: Shanxi aged vinegar, solvent-assisted flavor evaporation, GC-O, quantitation,

20 aroma extract dilution analysis, selected reaction monitoring

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21
INTRODUCTION

22 Vinegar is one of the most favorite acidic seasonings. In most Asian and European

23 countries, it is the necessary seasoning in every dinner. Traditional Chinese vinegars, also

24 called cereal vinegars, are important seasonings in Chinese daily life, which have a long

25 history of more than 3000 years. There are four famous China-style cereal vinegars,

26 Shanxi aged vinegar, Zhenjiang aromatic vinegar, Sichuan bran vinegar and Fujian

27 Monascus vinegar in China. Different from most European countries, in China the

28 spontaneous solid-state fermentation rather than the submerged fermentation is used

29 during acetic acid fermentation.1-3

30 Up to now, there have been many studies on flavor composition of China-style

31 cereal vinegars. 1, 3-6 For example, 2,3-butanediol, 2,3-butanedione,3-hydroxy-2-butanone,

32 furfural, acetic acid, and butanedioic acid diethyl ester had been found as volatiles from

33 Zhenjiang aromatic vinegar by headspace solid phase micro extraction (HS-SPME) and

34 gas chromatography-mass spectrometry (GC-MS).4 However, most of the studies on

35 China-style cereal vinegars had been focused on the volatile compounds by GC-MS

36 rather than the odor-active compounds, which in fact are only a small fraction of the

37 volatile compounds that actually contribute to overall aroma. GC-O is an effective

38 approach to screen the compounds of aroma-activities in foods. The time-intensity

39 analysis, aroma extract dilution analysis (AEDA), Charm analysis, and frequency

40 detection are the usual techniques utilized in GC-O analysis to detect and evaluate the

41 odorants sniffed.7-10 As for AEDA, the highest dilution, in which the odor of a compound

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42 can no longer be detected, is defined as its flavor dilution (FD) factor. The greater the FD

43 factor of a compound, the greater the compound is thought to contribute to overall aroma.

44 Thus by AEDA not only those of odor-activities but also those of potent odor-activities,

45 also called key aroma compounds, can be acknowledged. 10

46 Flavor of the China-style cereal vinegars is highly related to their types, fermentation

47 raw materials, and production areas.1 Shanxi aged vinegar is one of the most important

48 traditional Chinese cereal vinegars in China. It originated from Shanxi province in

49 northern China. Now Shanxi still is the main manufacturing location for Shanxi aged

50 vinegar. Different from Zhenjiang aromatic vinegar whose primary raw material is

51 sticky rice mainly planted in southern China, the primary raw material for Shanxi aged

52 vinegar is sorghum mainly planted in northern China. Shanxi aged vinegar tastes thicker

53 and tarter than Zhenjiang aromatic vinegar. The production of Shanxi aged vinegar

54 mainly goes through four manufacturing processes, that is, alcoholic fermentation, acetic

55 fermentation, thermal processing, and aging. The aroma of Shanxi aged vinegar formed

56 during the fermentation process consists of a large number of esters, aldehydes, ketones,

57 alcohols, and heterocyclic compounds.11-12 Thermal processing can produce lots of

58 pyrazines, aldehydes, ketones, and other aroma compounds, resulting from the associated

59 chemical reactions such as hydrolysis and Maillard reaction.13-14 Aging processing, where

60 water is evaporated or frozen, is also rich in chemical reactions and chemical changes.

61 Through aging processing, the flavor of Shanxi aged vinegar is to be soft, round,

62 continuous, and of no precipitation.2

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63 The key aroma compounds in Shanxi aged vinegar made from tartary buckwheat

14
64 had been characterized by Wang et al in dynamic headspace sampling and

65 AEDA/GC-O analysis. Yet, for the aforementioned common sorghum Shanxi aged

66 vinegar, how about its aroma composition and which compounds are of significance to

67 overall aroma is still ambiguous. At present, headspace techniques in particular

68 HS-SPME have been primarily used in the analysis of volatile flavor compounds of

69 China cereal vinegars.1,3,4,6,14 In comparison with the HS-SPME,6 solvent-assisted flavor

70 evaporation (SAFE) is not only a mild treatment but also in the nature of no matrix effect

71 from acetic acid and other components such as sugars, organic acids, amino acids,

72 phenolic acids, proteins, and inorganic ions. 15-17 However, as far as we know, the SAFE

73 technique had never been adopted in the analysis of China cereal vinegars.

74 Therefore, the aims of the present work were (1) to analyze the odor-active

75 compounds in Shanxi aged vinegar before and after aging by SAFE and AEDA/GC-O; (2)

76 to quantify the odorants by GC-MS/MS in selected reaction monitoring mode (SRM)

77 using the authentic standards; (3) to expose the aroma composition of the Shanxi aged

78 vinegar and know how it was changed through aging, since the aging process is so long a

79 period and critical to flavor and output of Shanxi aged vinegar. The research results may

80 provide guidance to improve the technology and flavor quality of Shanxi aged vinegar.

81
MATERIALS and METHODS

82 Materials. The Shanxi vinegars before or after aging, were obtained from Dingzhou

83 Branch of Shanxi Aged Vinegar Group Co. Ltd., Baoding City, Hebei Province, China.

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84 For the sake of representativeness, the samples of different batches well mixed were used

85 in the study. The main raw materials of the vinegars were sorghum, wheat bran, and

86 water. The preparation procedures were as described in the literatures,2,3,14,18 including

87 steaming of raw materials for about 1 h, addition of Da Qu, alcoholic fermentation for 18

88 days, acetic acid fermentation for 15 days, thermal processing at 85°C for 3 days, and

89 finally aging for about 1 year. The Da Qu was made from barley and pea, which was used

90 as starter culture. The aging of the vinegars was conducted spontaneously in 500-L

91 open-top ceramic jars under ambient temperatures placed in a big glass house with a

92 curtain type door, which is just the traditional folk technology called as “insolating in

93 summer and taking out ice in winter.”

94 Reagents and Chemicals. Total of fifty-eight authentic chemicals were used in the

95 identification or quantitation of the odor-active compounds. They were all in a high

96 purity ≥ 95% (GC), purchased from J&K Chemical Ltd. (Beijing, China): dimethyl

97 disulfide (98%), 3-(methylthio)propanal (98%), dimethyl trisulfide (98%), 3-(methylthio)

98 propyl acetate (98%), methylpyrazine (98%), trimethylpyrazine (98%), ethylpyrazine

99 (99%), 2,3-dimethylpyrazine (98%), 2-ethyl-6-methylpyrazine (98%), 2-acetylpyrazine

100 (99%), 2-acetylpyrrole (98%), tetramethylpyrazine (98%),

101 2,3,5-trimethyl-6-ethylpyrazine (98%), 2-acetyl-3,5-dimethylpyrazine (98%); furfuryl

102 alcohol (98%); furfural (98%), 2-acetylfuran (98%), 5-methylfurfural (98%), 2-furfuryl

103 acetate (98%), 2-acetyl-5-methylfuran (98%), 3-methylbutanal (98%), heptanal (95%),

104 benzaldehyde (98%), phenylacetaldehyde (98%), (Z)-2-nonenal (95%), vanillin (99%),

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105 2,3-butanedione (98%), 3-hydroxy-2-butanone (95%), acetophenone (98.5%),

106 β-damascenone (98%), benzophenone (99%), phenylethylalcohol (98%),

107 3-methylbutanoic acid (98%), pentanoic acid (98%), hexanoic acid (98%), octanoic acid

108 (98%), benzoic acid (98%), propanoic acid, ethyl ester (98%), isoamyl formate (98%),

109 lactic acid, ethyl ester (98%),γ-pentalactone (98%), succinic acid, diethyl ester (98%),

110 acetic acid, 2-phenylethyl ester (98%), γ-octalactone (98%), γ-nonalactone (98%),

111 ethyl caprate (98%),γ-decalactone (98%), methyl tetradecanoate (98%), hexadecanoic

112 acid, ethyl ester (98%), p-cresol (99%) guaiacol (98%), 4-ethylphenol (98%),

113 4-methylguaiacol (98%), 4-ethylguaiacol (98%), acetic acid (98%), propanoic acid (98%),

114 butanoic acid (98%), and ethyl acetate (98%). Serial n-alkanes (C6~C23) were purchased

115 from Beijing Chemical Reagents Co. Ltd. (Beijing, China). The internal standard,

116 1,2-dichlorobenzene (99%), was purchased from Sigma-Aldrich Co. Ltd (Shanghai,

117 China).

118 Solvent-Assisted Flavor Evaporation (SAFE). The head and the legs of the SAFE

119 unit (Deutsche Forschungsanstalt für Lebensmittelchemie, Freising, Germany) were

120 thermostated at 25 °C. Two 500 mL flasks were carefully fixed onto the two legs of the

121 unit, where one was called sample flask to receive the dropping sample and the

122 distillation residue, another was called distillate flask to receive the distillate. The

123 vacuum system in a range of 10-4~10-5 mbar was achieved by an Edwards EXT 75DX

124 vacuum pump (China Edwards Technologies Trading (Shanghai) Co Ltd ). Liquid

125 nitrogen was poured into the cooling trap and also into a beaker surrounding the 500 mL

126 distillate flask. A sample of 100 mL vinegar was handled in one run for about 1 h. The

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127 distillate was extracted by freshly redistilled dichloromethane (3×50 mL). The combined

128 extracts were dried over anhydrous sodium sulphate and concentrated to 0.5 mL using a

129 Vigreux column (50 cm×1 cm i.d.). Two replicates were performed and subjected to the

130 following analyses.

131 Gas Chromatography and Mass Spectrometry (GC-MS). An Agilent 7890B gas

132 chromatograph coupled with a 5977C mass spectrometer (Agilent Technologies, USA)

133 was used. The carrier gas was helium in 1 mL/min. The separation was on both a HP-5

134 MS (30 m × 0.25 mm × 0.25 µm) and a DB-Wax (30 m × 0.25 mm × 0.25 µm) columns

135 (Agilent Technologies, USA). For the HP-5 column, the initial oven temperature was

136 35 °C, holding for 2 min; then ramped to 170 °C at 3 °C /min; and finally ramped to

137 250 °C at 5 °C /min, holding for 2 min. For the DB-Wax column, the initial oven

138 temperature was 35 °C, then ramped to 170 °C at 3 °C /min; and finally ramped to 230 °C

139 at 5 °C /min, holding for 1 min. The sample injected was 1 µL at 250 °C in split mode.

140 The split ratio was 20 : 1.

141 The mass detector was operated at 150 °C in electron impact mode at 70 eV. The

142 solvent delay was 3 min. The ion source was at 230 °C. The transfer was at 250 °C. The

143 chromatograms were recorded by full scan in the 40~450 mass range.

144 Gas Chromatography and Olfactometry (GC-O). An Agilent 7890A gas

145 chromatograph (Agilent Technologies, USA) equipped with a FID detector and a

146 DATU 2000 high-resolution olfactometer system (DATU Inc. USA.) was used.8 The

147 column was HP-5 30 m × 0.25 mm × 0.25 µm. The carrier gas was nitrogen at 1.0

148 mL/min. The initial oven temperature was 40 °C, then ramped to 250 °C at 5 °C /min. 1

149 µL of sample was injected in splitless mode at 250 °C. The GC effluent to the odor port

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150 was enclosed with a stream of humidified air of 16 L/min and transferred by one length

151 of stainless steel tube (10 mm i.d) to the Teflon detection cone.

152 The samples of the concentrates were stepwise diluted using dichloromethane to

153 obtain dilutions (1:1, 1:2, 1:4, 1:8, 1:16,. . . , 1: 1024, and so on) of the original

154 solutions. Each dilution was submitted to GC-O analysis. The time to effuse

155 dichloromethane was figured out in advance to avoid harm. The odor characteristics

156 sniffed were recorded and each odorant was finally assigned a FD factor representing the

157 highest dilution. Three trained sniffers performed the AEDA/GC-O analyses. Retention

158 times of the odor responses were converted into retention index (RI) values relative to the

159 series of n-alkanes (C6-C23).

160 Identification of Compounds. The identification of the odor-active compounds was

161 based on mass spectra in GC-MS, retention indices (RI) relative to C6-C23 n-alkanes in

162 both GC-MS and GC-O analyses, odor characteristics sniffed in GC-O, and the

163 comparison of the above parameters with those of the available authentic chemicals. In

164 GC-MS analyses, both a polar column (DB-Wax) and a weak polar column (HP-5) were

165 employed to separate and identify the compounds as far as possible. The mass spectra of

166 the peaks and the corresponding RIs on both the columns were used for the identification.

167 For mass spectra, compounds were identified according to NIST 2011 mass spectra

168 libraries installed in the GC-MS equipment together with manual interpretation.
169 Quantitative Determination of the Odorants. The Trace 1310-TSR-8000

170 GC-MS/MS with a TG-5 MS 30 m × 0.25 mm × 0.25 µm column (Thermo Fisher

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171 Scientific, USA) was used. The programmed oven temperatures were identical to those in

172 the above HP-5 column GC-MS analysis. The mass spectrometry was detected in selected

173 reaction monitoring (SRM) mode. The sample injected was 1 µL at 250 °C in splitless

174 mode.

175 The odor-active compounds which were authentic chemicals available and in high

176 concentrations in the vinegars or of high FD factors were mainly quantitated. The

177 analytes were the concentrates mentioned above by the SAFE treatment. Calibration for

178 each compound was performed using the relative area to internal standard. The internal

179 standard used was 1, 2-dichlorobenzene (200 µg/mL in dichloromethane). The parent

180 ions to the daughter ions, the collision energies, the concentrations of the standard

181 solutions prepared in dichloromethane, and the calibration curves obtained for each of

182 the odorants were presented in Table 1. The quantity of an odor-active compound in a

183 concentrate was calculated by the calibration equation, and then taking into account the

184 extraction yield of the concentrate converted to micrograms in per liter vinegar (µg/L).

185 The results were the averages of two replicates.

186
RESULTS and DISCUSSION.

187 The odor descriptors, the flavor dilution factors, the identified compounds, and the

188 concentrations of the quantitated compounds for the vinegars before and after aging were

189 presented in Table 2. Total of eighty-seven odor-active regions were found, of which

190 eighty were identified but seven remained unknown. For the eighty compounds, fifty-four

191 were positively identified by RI, MS, odor descriptors, and authentic compounds;

192 whereas nineteen were identified without authentic compounds, four were identified

193 without MS, and three were identified without MS as well as authentic compounds. The

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194 seven identifications without using MS were because they were undetected in GC-MS on

195 either the HP or the DB-Wax column. Total of thirty odor-active compounds were

196 quantitated, which mostly showed a high FD factor or a high concentration in the

197 vinegars. Otherwise, a couple of compounds though of a low concentration or a small FD

198 factor e.g. methyl pyrazine also were quantitated as the authentic chemicals were

199 obtainable in our laboratory. For the quantative determination, rather than GC-MS, SRM

200 detection in tandem mass spectrometry (GC-MS/MS) was adopted, since the latter is

201 advantageous in specifity, sensitivity, and detection limit. As could be seen in Table 2,

202 γ-decalactone which was undetectable in the GC-MS analysis was also quantitively

203 determined.

204 (1)The Odor-active Compounds before Aging. In Table 2, before aging, total of

205 eighty odor-active regions were detected in the GC-O analysis. The typical odor

206 characteristics sniffed were sour, fruity, floral, nutty, roasted, cheesy, buttery, grassy, spicy,

207 ect. Among them seventy-five compounds were identified, including four sulfur-containing

208 compounds, eight nitrogen-containing heterocyclic compounds, twelve oxygen-containing

209 heterocyclic compounds, nine aldehydes, nine ketones, one alcohol, eight acids, sixteen

210 esters, six phenols, and two ethers. The compounds (eleven) 3-(methylthio)propanal,

211 vanillin, 2,3-butanedione, 3-methylbutanoic acid, acetic acid, tetramethylpyrazine, guaiacol,

212 γ-nonalactone, 3-(methylthio)propyl acetate, butanoic acid and 2,3-dimethylpyrazine were

213 of high FD factors (log2FD﹥5), suggesting they were of importance to overall aroma of

214 the Shanxi vinegar before aging. The compounds in high concentrations (>25 µg/L) were

215 acetic acid, followed by 2,3-butanedione, furfural, 3-hydroxy-2-butanone, octanoic acid,

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216 tetramethylpyrazine, furfuryl alcohol, and 3-methylbutanoic acid. The compounds in both

217 high concentrations and high FD values were acetic acid, followed by 2,3-butanedione,

218 tetramethylpyrazine, and 3-methylbutanoic acid.

219 The sulfur-containing compounds sniffed were with onion, meaty, fruity, and cooked

220 potato odors. They can be formed during the thermal processing via Maillard reaction. The

221 Strecker degradation of sulfur amino acids such as cysteine and methionine in Maillard

222 reaction can produce the sulfur compounds. However, sulfur compounds can also be

223 released during the fermentation process. 3-(methylthio)propanal, also called methional,

224 with boiled potato odors, can be synthesized by Saccharomyces cerevisiae in different

19
225 fermented foods. 3-(methylthio)propanal here probably arose from the alcoholic

226 fermentation, which subsequently remained in the vinegars. 20 So did 3-(methylthio) propyl

227 acetate. Due to the nature of low odor thresholds, the four sulfur compounds discovered

228 were all of a high FD factor ( log2FD ) ranging from 4 to 14, but in a rather low

229 concentration. Among them, 3-(methylthio)propanal (0.19 µg/L, log2FD=14) was of the

230 highest FD factor, followed by 3-(methylthio) propyl acetate, dimethyl trisulfide, and

231 dimethyl disulfide. Noticeably, so far, the presence of sulfur flavor compounds in the

232 common sorghum Shanxi vinegar had been rarely reported. 3-(methylthio)propanal and

233 3-(methylthio) propyl acetate were found for the first time in Shanxi vinegar.

234 3-(methylthio)propanal had been found in red wine vinegar 21 and other fermented foods. 19

235 Otherwise, the occurrences of dimethyl trisulfide in Shanxi aged tartary buckwheat vinegar

14
236 and Sherry vinegar, 22 and dimethyl disulfide in Zhenjiang vinegar, Beijing rice vinegar,

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237 and Shanxi aged vinegar 23 , had been revealed.

238 The number of nitrogen-containing heterocyclic compounds found was more than that

239 of the sulfur-containing compounds. The FD values (log2FD) of the nitrogen-containing

240 heterocyclic compounds ranging from 1 to 10, lower than those of the sulfur-containing

241 compounds. Except for trimethyl oxazole and 2-acetylpyrrole, the nitrogen-containing

242 heterocyclic compounds all were attributed to pyrazine and its derivatives, whose typical

243 odor characteristics were nutty and roasted. Among them, tetramethylpyrazine showed both

244 a high concentration and a high FD factor, followed by 2,3-dimethylpyrazine,

245 trimethylpyrazine, and ethylpyrazine. Pyrazine and its derivatives in Shanxi vinegar are

246 mainly formed by the maillard reaction during thermal processing, as there is an abundance

247 of sugar and nitrogen source in the solid fermentation substrate of the vinegar. 13-14

248 The oxygen-containing heterocyclic compounds in Table 2 all belonged to the furan

249 derivatives. They had FD factors (log2FD) from 1 to 4. Most of them gave sweet and

250 caramel odors. Overall, the concentrations of the oxygen-containing heterocyclic

251 compounds were higher than those of the nitrogen-containing heterocyclic compounds.

252 Among them, furfural (248.01 µg/L) was the richest, followed by furfuryl alcohol (35.08

253 µg/L) and 5-methylfurfural (16.81 µg/L). Also, the furan derivatives were mainly generated

254 by the maillard reaction in thermal processing. It is well known that furfural can be yielded

255 from pentose while 5-methylfurfural can be yielded from hexose.24 The presence of furfural

256 had been reported in a wide variety of vinegars e.g. Zhenjiang aromatic vinegar,4 and

257 Balsamic vinegar. 25 Worth mentioning, furfural had a high concentration but was of a low

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258 FD factor (log2FD=4) in the vinegar, probably due to its nature of high odor threshold. As

259 was reported, the odor threshold of furfural in the young red wines was found to be as

260 high as 14100 µg/L. 26

261 The structures of the aldehydes in Table 2 were miscellaneous with FD values

262 (log2FD) ranging from 0 to 14. They were typically of green, herbal, sweet, and floral odors.

263 Benzaldehyde, 3,5-dihydroxybenzaldehyde, vanillin, 5-methyl-2-phenyl-2-hexenal, and

264 piperonal, which belong to aromatic aldehydes, can originate from the plant-based raw

265 materials of sorghum, barley and so on. Benzaldehyde was proved to increase during the

266 thermal processing.13-14 Phenylacetaldehyde and 3-methylbutanal can produce from the

267 degradation of phenylalanine and isoleucine, respectively. Heptanal and (Z)-2-nonenal can

268 produce from the degradation of fatty acids. Among the aldehydes, vanillin with sweet and

269 chocolate odors was of a high FD factor (log2FD=14) and in the highest concentration

270 (20.29 µg/L), followed by phenylacetaldehyde, 5-methyl-2-phenyl-2-hexenal and

271 benzaldehyde. The other aldehydes were all in both low concentrations and small FD

272 values (log2FD < 4). Vanillin is present in the Sherry vinegars10, 27 and the strawberry

273 vinegar. 20 It was found for the first time as an odorant in Shanxi vinegar.

274 The ketones were mostly with fruity, buttery, and floral odors. Among the ketones

275 found, 2,3-butanedione (328.29 µg/L, log2FD =13) was in the highest concentration and of

276 a high FD factor, followed by 3-hydroxy-2-butanone or β-damascenone.

277 3-Hydroxy-2-butanone and 2,3-butanedione usually are present in fermented foods and

278 beverages such as wine , which can be produced in alcoholic fermentation by the action of

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279 microorganisms. 21 They can also be formed from sugar degradation via Maillard reaction,

280 and are the precursors of tetramethylpyrazine in Shanxi vinegars in the maillard reaction. 5

281 β-Damascenone and the aromatic ketones e.g. benzophenone should come from the

282 plant-based raw materials used in the fermentation process. Here, β-damascenone

283 (log2FD=5) was for the first time found in Shanxi vinegar, but it proved in too low a level

284 in the vinegars to exactly quantitate in the present study. β-damascenone had been found in

285 Sherry vinegar by HS-SPME and gas chromatography-olfactometry. 22

286 Only one alcohol of 2-phenylethanol (16.51 µg/L) was found. It was in a low FD

287 value (log2FD =3) with fruity and floral notes. It might derive from the reduction of

288 phenylacetaldehyde in alcoholic fermentation or origin from the raw materials used.

289 The acids mainly contribute to the tart and sour flavor of vinegars. They generated in

290 the acetic fermentation process. The acids identified from the vinegar before aging

291 included seven aliphatic acids of two to eight carbon atoms, and benzoic acid. Among

292 them, 3-methylbutanoic acid showed the highest FD factor (log2FD =13), followed by

293 acetic acid (log2FD =10) and butanoic acid (log2FD =6); while in view of concentration

294 acetic acid (581 µg/L) was the highest, followed by octanoic acid, and 3-methylbutanoic

295 acid.

296 The esters usually are considered most important to flavor of vinegars. A lot of esters

297 have been found from Zhenjiang aromatic vinegar 4 and Balsamic vinegar. 25 The esters are

298 of typical fruity aroma characteristics. They can be formed by the reaction of alcohols with

299 organic acids under the effect of the esterase, yeast or mold. From this un-aged Shanxi

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300 vinegar, total of sixteen esters were identified, with FD values from 1 to 9, including six

301 lactones (e.g. γ-hexalactone and γ-decalactone) and ten aliphatic esters. Lactic acid, ethyl

302 ester (23.77 µg/L) was in the highest concentration, followed by succinic acid diethyl ester

303 (7.75 µg/L), and γ-decalactone (6.99 µg/L). While for the FD values, γ-nonalactone

304 (log2FD =9) ranked the highest, followed by lactic acid ethyl ester (log2FD =4) and

305 γ-hexalactone (log2FD =4). The other esters shown in Table 2 were in quite a low FD value

306 (log2FD <4). Worth mentioning, according to the FD values found in the present work, it

307 seemed that the esters were not as potent as said to overall aroma for the Shanxi vinegar.

308 The phenols possessed woody, spicy and herbal odors with log2FD values ranging

309 from 0 to 9. They can be produced from the degradation of lignin in the solid fermentation

310 substrate during the smoking thermal processing.1 In sherry vinegars, wine vinegars,

311 balsamic vinegars, and strawberry vinegars also are found the phenols, which might

20
312 originate from fruit contamination caused by different microorganisms. Among the

313 phenols, guaiacol was in the highest concentration (11.34 µg/L) as well as FD value

314 (log2FD=9) followed by 4-ethylguaiacol (log2FD=3) and 4-methylguaiacol (log2FD=3).

315 Compared to all the aforementioned literatures on volatile flavor constituents of China

316 cereal vinegars, the largest number of phenols (six) were found together for the present

317 work. From eleven types of China cereal vinegars, Xiao et al 1 only found four phenols as

318 volatiles by SPME and GC-MS; while in the tartary buckwheat vinegars, only phenol was

319 found as an odorant by headspace purge and trap and GC-MS-O.14 As we know, the

320 phenols are polar compounds of low volatilities and soluble in water. The discovery of

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321 more number of phenols probably resulted from the efficient SAFE extraction method used

322 in the present analysis.

323 The aromatic ethers identified from the vinegar were estragole and anethole, which

324 were in very low FD values. They probably originated from the fermented raw materials of

1
325 the vinegars. The presence of anethole had been reported by Xiao et al in Shanxi aged

326 vinegar.

327 Above all, there were five odor active regions not yet identified from this Shanxi

328 vinegar before aging. These mainly gave spicy, herbal and earthy smells, which might arise

329 from the microbial fermentation, and the sorghum or other raw materials. They should have

330 minimal effects on overall aroma due to low FD values (log2FD < 4).

331 (2) The Odor-active Compounds after Aging and Changes. From Table 2, it could

332 be seen that after aging, GC-O only revealed sixty-one odor-active regions, among which

333 fifty-six were identified with five unknown. The identifications were composed of four

334 sulfur-containing compounds, thirteen nitrogen-containing heterocycles, seven

335 oxygen-containing heterocycles, seven aldehydes, five ketones, one alcohol, seven acids,

336 six esters, four phenols, and two ethers. Similar to those before aging, the unknown

337 odor-active regions were of low FD values.

338 In comparison with those before aging, the odor-active molecules listed were mostly

339 the same, however, their quantities were changed. On the one hand, the concentrations of

340 most of the compounds e.g. 2-phenylethanol, acetic acid, and 3-methylbutanoic acid in the

341 Shanxi vinegar after aging were lowered. And even twenty-four compounds were not there

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342 again, including ten esters, five oxygen-containing heterocyclic compounds, four ketones,

343 two aromatic aldehydes, two phenols, and one acid (Table 2). In fact, all these lost

344 compounds were in low FD values (log2FD ≤ 4 ) and of low concentrations before aging.

345 After aging, the number of compounds in high concentrations (> 25 ug/L) was reduced

346 from eight to seven. However, conversely the concentrations of some compounds e.g

347 dimethyl trisulfide, trimethylpyrazine, and phenylacetaldehyde were increased. Especially,

348 tetramethylpyrazine was increased markedly from 35.7 to 82.36 µg/L, while there were

349 five new pyrazine compounds found after aging. It could be seen from Table 2 that the

350 new attendees 2-ethyl-6-methylpyrazine and 2-acetylpyrazine each had a high FD value

351 (log 2 FD ) of 8, indicating they were of importance to overall aroma. Besides, similar to

352 tetramethylpyrazine, its precursor 2,3-butanedione again remained in both a high

353 concentration and a high FD value.

354 As shown in Figure. 1, after aging, the top fifteen compounds of high FD factors

355 (log2FD﹥7) were 3-(methylthio)propanal, vanillin, 2,3-butanedione, tetramethylpyrazine,

356 3-methylbutanoic acid, γ-nonalactone, guaiacol, 3-(methylthio)propyl acetate, dimethyl

357 trisulfide, phenylacetaldehyde, 2-ethyl-6-methylpyrazine, 2-acetylpyrazine,

358 2,3-dimethylpyrazine, furfural, and 3-hydroxy-2-butanone. The compounds in high

359 concentrations ( > 25 µg/L) were acetic acid, followed by 2,3-butanedione, furfural,

360 3-hydroxy-2-butanone, tetramethylpyrazine, furfuryl alcohol, and 3-methylbutanoic acid.

361 The compounds in both high concentrations and high FD values were 2,3-butanedione,

362 followed by tetramethylpyrazine, and 3-methylbutanoic acid. Compared to before aging,

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363 the most obvious changes were that more pyrazine compounds in a high FD value were

364 found and acetic acid was not so potent to overall aroma of the vinegar as before. However,

365 after aging the Shanxi vinegar often tastes more acidic, as the non-volatile acids such as

366 lactic acid and succinic acid in the vinegar tend to be concentrated with aging time. 3

367 During the aging processing, water in the vinegar is evaporated or frozen in an open

368 top jar, where volatile compounds are liable to be lost. In the meantime, there are various

369 complex chemical reactions taking place. However, the level increase of the aroma

370 compounds e.g. dimethyl trisulfide, tetramethylpyrazine, and phenylacetaldehyde, and the

371 emergence of five new pyrazine compounds indicated that the maillard reaction was of

372 significance in the aging process due to the fermented cereal vinegars rich in proteins,

373 amino acids, and reducing sugars. In addition, during the aging, the contents of amino

374 acids and reducing sugars in the vinegar would increase with time.3 It could be seen from

375 Figure. 2, even for the same compounds in the Shanxi vinegars, after the aging process,

376 the changes of their levels within the composition formula would lead to the alteration of

377 aroma profile of the vinegar. Anyway, the increase in the pyrazine compounds, and the

378 decrease in acetic acid and some other compounds of pungent smells contributed greater

379 to the development of the unique flavor of Shanxi aged vinegar, which was thicker, round,

380 and gentle compared to that before aging.

381 In summary, total of eighty odor-active compounds were identified from the Shanxi

382 vinegars before and after aging, where 3-(methylthio)propanal, 3-(methylthio)propyl

383 acetate, vanillin, and β-damascenone were found for the first time as odorants in Shanxi

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384 vinegar. The aroma compounds of high FD values before aging mainly were attributed to

385 sulfur-containing compounds, nitrogen-containing heterocyclic compounds, fatty acids,

386 lactones, ketones, aromatic aldehydes, and phenols, which originated from alcoholic

387 fermentation, the mailard reaction, acetic acid fermentation, or the raw materials used.

388 After aging process the aroma molecules were mostly unchanged but their concentrations

389 were being modified. The obvious changes after the aging process were that the

390 compounds with pungent smells e.g. acetic acid and p-cresol were partly or entirely

391 evaporated with water. Instead, the sulfur compounds and especially the pyrazine

392 compounds that give nutty, and roasted notes to the vinegar were further highlighted.

393 These demonstrated during the aging processing, Maillard reaction again played

394 important roles in the development of the unique flavor of Shanxi aged vinegar. The

395 results would be helpful to improve the flavor quality and processing technology of

396 Shanxi aged vinegar.

397
AUTHOR INFORMATION

398 Corresponding Author

399 *Jianchun Xie, School of Food Science and Chemical Engineering, Beijing Technology

400 and Business University, No. 33 Fucheng Road, Haidian District, Beijing 100048, China.

401 Phone and Fax: (+86) -10-68984859. E-mail: xjchun@th.btbu.edu.cn

402 Funding

403 The present work was supported by National Natural Science Foundation of China.

404 (NSFC. 31371838, 31671895 )

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405 Notes

406 The authors declare no competing financial interest.

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407
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Table 1 The analysis conditions, the standard solutions, and the calibration equations for the thirty odor-active compounds in the Shanxi

vinegars before and after aging determined quantitatively by GC-MS/MS in selected ion monitoring (SRM) mode
1 2 3
Compounds Retention indices SRM (m/z) Calibration equations Concentrations (mg/L)
2,3-butanedione 617 86>43(15) ;43>15(5) y=7.3378x+0.0963;R2=0.9979 5, 10, 20, 40, 80, 160
2
acetic acid 687 60>45(10) y=9.7240x-0.0416; R =0.9983 5, 10, 20, 40, 80, 160
2
3-hydroxy-2-butanone 710 88>45(5);45>27(15) y=6.2881x+0.0119;R =0.9972 5, 10, 20, 40, 80, 160
methylpyrazine 812 94>67(10) ; 67>26(10) y=4.9176x-0.0549;R2=0.9983 0.5, 1, 2, 4, 8, 16
2
lactic acid, ethyl ester 825 75>45(5);45>15(15) y=4.4224x+0.0269;R =0.9968 0.5, 1, 2, 4, 8, 16
2
3-methylbutanoic acid 847 60>42(10);60>45(15) y=5.5701 x+0.0271;R =0.9981 0.5, 1, 2, 4, 8, 16
furfural 856 96>39(15);67>39(10) y=7.2172x-0.0581;R2=0.9985 5, 10, 20, 40, 80, 160
2
furfuryl alcohol 860 98>42(10) y=3.6339x+0.0209;R =0.9982 0.5, 1, 2, 4, 8, 16
2
heptanal 887 70>55(5);57>29(10) y=3.2869x+0.0188;r =0.9983 0.5, 1, 2, 4, 8, 16
3-(methylthio)propanal 904 104>48(10);76>61(5) y=1.2669x-0.0558;R2=0.9980 0.025, 0.05, 0.1, 0.2, 0.4, 0.8
2
ethylpyrazine 907 108>80(10) y=5.9747x+0.0693;R =0.9987 0.5, 1, 2, 4, 8, 16
2
2,3-dimethylpyrazine 915 108>67(10) y=8.2647x+0.0106;R =0.9988 0.5, 1, 2, 4, 8, 16
benzaldehyde 952 105>77(10) y=0.8833x+0.0082;R2=0.9992 0.5, 1, 2, 4, 8, 16
2
5-methylfurfural 957 109>53(10) y=5.5419x+0.0104;R =0.9989 0.5, 1, 2, 4, 8, 16
2
dimethyl trisulfide 960 126>79(15) ;79>64(15) y=0.2719x+0.0065;R =0.9984 0.05, 0.1, 0.2, 0.4, 0.8, 1.6
trimethylpyrazine 997 122>81(10) ; 122>42(15) y=1.4940x-0.0184;R2=0.9990 0.5, 1, 2, 4, 8, 16
2
phenylacetaldehyde 1038 120>91(10) y=2.1670x+0.0324;R =0.9984 0.5, 1, 2, 4, 8, 16

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2-acetylpyrrole 1061 109>94(10);94>66(10) y=1.6197x+0.0252;R2=0.9983 0.5, 1, 2, 4, 8, 16

2
guaiacol 1084 124>109(10) ;109>81(10) y=0.9122x-0.0029;R =0.9981 0.5, 1, 2, 4, 8, 16
tetramethylpyrazine 1085 136>54(15) ;54>39(5) y=3.3813x+0.0341;R2=0.9985 1, 2, 4, 8, 16, 32
2
(Z)-2-nonenal 1142 70>55(5) ;57>29(10) y=1.1257x-0.0191;R =0.9866 0.5, 1, 2, 4, 8, 16
2
2-phenylethanol 1118 122>91(15) ; 91>65(15) y=3.7422x-0.0171;R =0.9981 0.5, 1, 2, 4, 8, 16
3-(methylthio)propyl acetate 1123 73>43(10) y=0.1623x+0.0045;R2=0.9982 0.05, 0.1, 0.2, 0.4, 0.8, 1.6
2
4-methylguaiacol 1188 138>123(10);123>95(5) y=0.6450x+0.0065;R =0.9992 0.05, 0.1, 0.2, 0.4, 0.8, 1.6
2
succinic acid, diethyl ester 1189 129>101(5) ;101>73(5) y=5.0559x+0.0633;R =0.9986 0.5, 1, 2, 4, 8, 16
octanoic acid 1197 73>55(10) ;60>42(10) y=3.5916x+0.0797;R2=0.9980 0.5, 1, 2, 4, 8, 16
2
4-ethylguaiacol 1273 152>137(10) ;137>122(10) y=0.2319x+0.0023;R =0.9970 0.05, 0.1, 0.2, 0.4, 0.8, 1.6
2
γ-nonalactone 1377 85>57(5);85>29(10) y=1.8042x+0.0987;R =0.9983 0.5, 1, 2, 4, 8, 16
vanillin 1420 151>123(10) y=1.3211x+0.0955;R2=0.9965 0.5, 1, 2, 4, 8,16
2
γ-decalactone 1468 85>29(10); 57>29(10) y=1.9583x+0.0123;R =0.9989 0.5, 1, 2, 4, 8,16
1,2-dichlorobenzene 1024 146>111(15) ; 111>75(10) - 2

1
parent ion > daughter ion (collision energy) (eV) used for the calibration. 2 x was the peak area relative to that of the internal standard

3
1,2-dichlorobenzene, y was the concentration in the extract of a vinegar by solvent assisted flavor distillation (SAFE). The concentrations of

the standard solutions prepared in dichloromethane.

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Table 2 The retention indices, odor descriptors, and flavor dilution factors ( log2FD) in GC-O analysis and the compounds identified

with some of their concentrations determined in the Shanxi vinegars before and after aging
2 3 4
1 1
log2FD Concentrations (µg/L) Identification
RI Odor descriptors Compounds
a b a b Methods
547 spicy, sweet unknown - 1
598 fruity, sweet 2-butanone 0 - - - MS/RI/Odor
608 buttery, sweet 2,3-butanedione 13 14 328.29 ± 2.11 322.64 ± 1.82 MS/RI/Odor/S
621 fruity ethyl acetate 3 - - - MS/RI/Odor/S
668 pungent, fruity 3-methylbutanal 1 2 - - MS/RI/Odor/S
680 pungent, sour acetic acid 10 6 581 ± 2.83 440.66 ± 1.92 MS/RI/Odor/S
684 fruity propanoic acid, ethyl ester 2 - - - MS/RI/Odor/S
699 buttery 3-hydroxy-2-butanone 4 8 96.62 ± 0.45 93.06 ± 0.37 MS/RI/Odor/S
707 onion, cabbage dimethyl disulfide 4 7 - - MS/RI/Odor/S
730 sour, fruity propanoic acid 2 7 - - MS/RI/Odor/S
736 nutty, roasted pyrazine - 1 - - MS/RI/Odor
787 roasted, nutty methyl pyrazine 1 1 5.87 ± 0.10 5.74 ± 0.09 MS/RI/Odor/S
788 fruity isoamyl formate 2 - - - MS/RI/Odor/S
791 rancid, cheesy butanoic acid 6 5 - - MS/RI/Odor/S
801 caramel dihydro-2-methyl-3(2H)-furanone 1 0 - - MS/RI/Odor
822 yogurt lactic acid, ethyl ester 4 2 23.77 ± 0.17 - MS/RI/Odor/S
842 rancid, cheesy 3-methylbutanoic acid 13 12 29.23 ± 0.18 27.23 ± 0.16 MS/RI/Odor/S
855 caramel furfuryl alcohol 4 6 35.08 ± 0.18 40.40 ± 0.14 MS/RI/Odor/S
861 roasted, earthy trimethyl oxazole 2 3 - - MS/RI/Odor
868 caramel furfural 4 8 248.01 ± 1.15 225.53 ± 0.72 MS/RI/Odor/S
881 green, sweet heptanal 2 1 1.28 ± 0.03 - MS/RI/Odor/S

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891 sweet 2-acetoxy-3-butanone 2 4 - - MS/RI/Odor

897 sour, cheesy pentanoic acid 3 1 - - MS/RI/Odor/S
906 nutty, sweet 2-acetylfuran 1 0 - - MS/RI/Odor/S
909 boiled potato 3-(methylthio)propanal 14 15 0.19 ± 0.01 0.23 ± 0.01 MS/RI/Odor/S
917 nutty ethyl pyrazine 3 3 7.61 ± 0.04 7.74 ± 0.03 MS/RI/Odor/S
921 roasted, popcorn 2,3-dimethyl pyrazine 6 8 8.66 ± 0.14 8.79 ± 0.10 MS/RI/Odor/S
954 fruity γ-pentalactone 1 - - - MS/RI/Odor/S
959 almond benzaldehyde 3 7 12.36 ± 0.09 9.76 ± 0.06 MS/RI/Odor/S
964 caramel 5-methylfurfural 3 0 16.81 ± 0.16 10.82 ± 0.10 MS/RI/Odor/S
972 roasted meat dimethyl trisulfide 5 9 0.61 ± 0.01 1.01 ± 0.02 MS/RI/Odor/S
986 earthy, musty trimethyl pyrazine 3 3 9.9 ± 0.21 13.04 ± 0.27 MS/RI/Odor/S
990 earthy, green 2-pentylfuran 3 - - - MS/RI/Odor
1005 nutty, musty 2-ethyl-6-methyl pyrazine - 8 - - MS/RI/Odor/S
1008 fruity, caramel 2-furfuryl acetate 2 3 - - MS/RI/Odor/S
butanedioic acid, monomethyl
1012 fruity, sweet 3 - - - MS/RI/Odor
ester
1026 nutty, roasted 2-acetylpyrazine - 8 - - MS/RI/Odor/S
1026 rancid, creamy hexanoic acid 3 3 - - MS/RI/Odor/S
1040 sweet, burnt sugar 2-acetyl-5-methylfuran 3 - - - MS/RI/Odor/S
1044 herbal, sweet γ-hexalactone 4 - - - MS/RI/Odor
1047 rose phenylacetaldehyde 5 9 6.27 ± 0.04 20.46 ± 0.13 MS/RI/Odor/S
1060 sweet, fruity acetophenone 2 - - - MS/RI/Odor/S
1064 licorice 2-acetylpyrrole 2 0 8.89 ± 0.13 8.28 ± 0.11 MS/RI/Odor/S
1077 phenol, smoke p-cresol 1 - - - MS/RI/Odor/S
1085 spicy, sweet (3E)-4-(2-furyl)-3-buten-2-one 3 - - - MS/RI/Odor
1085 woody guaiacol 9 11 11.34 ± 0.06 10.81 ± 0.04 MS/RI/Odor/S
1090 nutty, roasted tetramethyl pyrazine 10 13 35.7 ± 0.28 82.36 ± 0.44 MS/RI/Odor/S

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1098 fruity, floral 2-phenylethanol 3 0 16.51 ± 0.13 2.28 ± 0.01 MS/RI/Odor/S

1102 cinnamon, caramel 3-(2-furanyl)-2-propenal 4 5 - - MS/RI/Odor
1120 fruity 3-(methylthio)propyl acetate 6 9 1.25 ± 0.03 1.40 ± 0.03 MS/RI/Odor/S
1125 fruity, sweet benzyl methyl ketone 2 - - - MS/RI/Odor
1126 nutty, musty 2,5-dimethyl-3-isopropylpyrazine - 1 - - MS/RI/Odor
1145 fruity, sweet pantolactone 2 - - - MS/RI/Odor
1152 green, fatty (Z)-2-nonenal 1 2 10.66 ± 0.09 9.69 ± 0.06 MS/RI/Odor/S
1159 musty 2,3,5-trimethyl-6-ethylpyrazine 1 0 - - MS/RI/Odor/S
1169 woody, sweet 4-ethylphenol 2 5 - - MS/RI/Odor/S
1175 rancid octanoic acid 3 1 44.82 ± 0.81 - MS/RI/Odor/S
1181 sweet, fruity succinic acid, diethyl ester 3 0 7.75 ± 0.07 6.26 ± 0.04 MS/RI/Odor/S
1192 balsam benzoic acid 3 - - - MS/RI/Odor/S
1195 woody, herbal 4-methylguaiacol 3 4 0.53 ± 0.01 0.51 ± 0.01 MS/RI/Odor/S
1201 licorice, anise estragole 0 0 - - RI/Odor
1206 nutty, roasted 2-acetyl-3,5-dimethylpyrazine - 2 - - MS/RI/Odor/S
1222 sweet, burnt sugar 5-hydroxymethylfurfural 3 - - - MS/RI/Odor
1237 rancid, spicy 1-phenyl-1,2-propanedione 0 - - - MS/RI/Odor
1246 spicy, green unknown 3 4
1250 fruity, floral acetic acid, 2-phenylethyl ester 1 - - - MS/RI/Odor/S
1260 coconut, sweet γ-octalactone 2 - - - MS/RI/Odor/S
1271 sweet, spicy 4-ethylguaiacol 3 4 0.84 ± 0.02 0.50 ±0 .01 MS/RI/Odor/S
1292 spicy, herbal unknown 2 1
1294 fennel, herbal anethole 1 1 - - RI/Odor
1320 earthy unknown 3 -
1338 herbal, sweet piperonal 2 3 - - RI/Odor
1363 coconut γ-nonalactone 9 11 3.06 ± 0.06 3.57 ± 0.06 MS/RI/Odor/S
1380 fruity ethyl caprate 2 5 - - RI/Odor/S

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1382 smoky, woody 4-propylguaiacol 0 - - - MS/RI/Odor

1390 fruity, apple β-damascenone 5 7 - - RI/Odor/S
1398 herbal, sweet 3,5-dihydroxybenzaldehyde 0 - - - MS/RI/Odor
1402 sweet, chocolate vanillin 14 14 20.29 ± 0.13 16.51 ± 0.10 MS/RI/Odor/S
1466 fruity γ-decalactone 3 1 6.99 ± 0.07 - RI/Odor/S
1485 green, rancid 5-methyl-2-phenyl-2-hexenal 4 - - - MS/RI/Odor
1514 spicy unknown 0 -
1520 earthy unknown - 1
1616 sweet, floral benzophenone 2 2 - - MS/RI/Odor/S
1626 musty, herbal unknown 1 1
1675 herbal, rancid 2-phenyl-3-(2-furyl)- propenal 2 - - - MS/RI/Odor
1720 fruity methyl tetradecanoate 1 2 - - RI/Odor/S
1988 fruity hexadecanoic acid, ethyl ester 2 - - - MS/RI/Odor/S

1
Kovats retention indices, and odor characteristics sniffed in GC-O analysis. 2 the Shanxi vinegars before aging (a) and after aging (b);

“-”, undetected in GC-O analysis. 3 the concentrations were expressed as means ± standard deviations (two replicates) (µg) in per liter of

vinegar; “-”，not quantitated. 4 MS, identified by NIST 11 mass spectral database; RI, agreed with the published retention indices; Odor,

agreed with the published odor descriptions; S, the analytical parameters of MS, RI, and Odors all agreed with those of the authentic

standards used.

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3-(Methylthio)propanal
2,3-Dimethyl pyrazine γ-Nonalactone
Dimethyl trisulfide
Vanillin
2-ethyl-6-methylpyrazine
2-acetylpyrazine

（Retention index）
）

3-(Methylthio)propyl acetate
Benzaldehyde
Tetramethyl pyrazine
Guaiacol
Furfural Phenylacetaldehyde
Furfuryl alcohol
3-Methylbutanoic acid
3-Hydroxy-2-butanone
Acetic acid

2,3-Butanedione

Figure 1. The comparison of flavor dilution factors (Log2FD) and concentrations

(µg/L) for the odor-active compounds in the Shanxi vinegars before and after aging.

The retention indices were corresponding to the compounds listed in Table 2, among

which the quantitated or the new pyrazines found in the aged vinegar of potent

odor-activities were shown. The compounds of log2FD≥ 6 in the vinegar either before

or after aging were labeled.

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Figure 2. Spider-web for the odorants of high flavor dilution factors (log2FD≥ 5), the

full distance on the scale was defined to be 5, distance from the origin for a compound

is 5 times the ratio of its log2FD value divided by the highest log2FD value among the

presented compounds of the vinegar.

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Graph for table of contents

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