Characterization of Key Odorants in Chinese Chixiang Aroma-Type

Mar 23, 2015 - Liquor by Gas Chromatography−Olfactometry, Quantitative. Measurements, Aroma Recombination, and Omission Studies. Haiyan Fan, Wenlai ...
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Characterization of Key Odorants in Chinese Chixiang Aroma-Type Liquor by Gas Chromatography−Olfactometry, Quantitative Measurements, Aroma Recombination, and Omission Studies Haiyan Fan, Wenlai Fan,* and Yan Xu Synergetic Innovation Center of Food Safety and Nutrition , Key Laboratory of Industrial Biotechnology, Ministry of Education, and Laboratory of Brewing Microbiology and Applied Enzymology, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, China ABSTRACT: Chixiang aroma-type liquor is extensively welcomed by consumers owing to its typical fatty aroma, particularly in southern China. To our knowledge, no comprehensive characterization of aroma and flavor from chixiang aroma-type liquor has been published. It is still a confused question which components are the most important in characterizing its unique aroma. A total of 56 odorants were identified in chixiang aroma-type liquor by aroma extract dilution analysis (AEDA), and in different quantitative measurements, 34 aroma compounds were further demonstrated as important odorants according to odor activity values (OAVs). Furthermore, this research suggested that the aroma of chixiang aroma-type finished liquor could be successfully reconstituted by mixing 34 aroma compounds in the concentrations measured. Omission experiments further confirmed (E)-2nonenal as the key odorant and revealed the significance of (E)-2-octenal and 2-phenylethanol for the overall aroma of chixiang aroma-type liquor. 3-(Methylthio)-1-propanol (methionol), diethyl 1,7-heptanedioate (diethyl pimelate), diethyl 1,8octanedioate (diethyl suberate), and diethyl 1,9-nonanedioate (diethyl azelate), identified as the characteristic aromas of chixiang aroma-type liquor in 1995, had no effects on aroma based on omission/addition experiments. KEYWORDS: Chinese chixiang aroma-type liquor, AEDA, quantitative measurements, OAV, aroma recombination, omission/addition experiments, (E)-2-nonenal

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weight). After soaked for 30 days, base distillate is filtered out and matured in a stainless steel tank for 20 days. The aged liquor is adjusted to a target ethanol concentration and blended for constant quality in the finished products.3 These specific maturation procedures could generate the typical fatty aroma of chixiang aroma-type liquor. The volatile composition of chixiang aroma-type liquor is quite complex. Feng and Qiu5 first determined the volatile compounds in chixiang aroma-type liquor. A total of 66 volatiles were identified by gas chromatography−mass spectrometry (GC−MS), including 25 esters, 21 alcohols, 9 carbonyl compounds, 8 acids, and 4 acetals. The authors thought that 2-phenylethanol, diethyl pimelate, diethyl suberate, diethyl azelate, and methionol were the characteristic aromas of chixiang aroma-type liquor. In fact, 2-phenylethanol elicits intense rose- or honey-lik -aroma, and methionol gives a cooked potato aroma.6 Diethyl pimelate, diethyl suberate, and diethyl azelate are odorless compounds. In 2010, Zhang and He7 detected 118 volatile compounds in chixiang aroma-type liquor by headspace solid-phase microextraction (HS-SPME) followed by GC−MS. Up to now, most of studies were focused on the identification and quantification of volatile compounds. It is still a puzzling question which ones are the most important or unique aroma compounds in chixiang aroma-type liquor.

INTRODUCTION Chinese liquor is a traditional indigenous distilled spirit and the most popular among alcoholic beverages in China, with a production exceeding 12 million kL in 2013. Based on aroma characteristics, they are generally classified into the following categories: soy sauce, strong, light, sweet and honey, roastedsesame-like, chixiang, complex, herblike, fenxiang, laobaiganxiang, and texiang aroma- and flavor-type liquors.1 Among them, chixiang aroma-type liquor has gained popularity due to its fatty aroma, particularly in southern China. One of the most important quality markers for distilled beverages is the aroma, which is clearly influenced by raw materials, fermentation process, and maturation.2 In general, chixiang aroma-type liquor is typically fermented from rice.3 First, fermentation mash is prepared by mixing cooked rice, xiaoqu, and water in a fermenter. Xiaoqu is a kind of mold culture, which contains Rhizopus, Aspergillus, yeast, bacteria, and complex enzymes spontaneously proliferated on cooked rice and soybean. It was usually used as saccharifying agent and fermentation starter to initiate the fermentation process.4 The semi-solid-state fermentation is typically carried out at 28−32 °C for 20 days under anaerobic conditions in a 50-kL fermenter. In the fermentation process, the saccharification of starch and fermentation of sugars are carried out simultaneously. After fermentation, the liquor is distilled out with steam, commonly known as zhaijiu or base distillate,3 then aged in a sealed pottery jar at ambient temperature. At the beginning of the storage, a whole piece of cooked pork meat is soaked in base distillate for removing its unpleasant off-flavor compounds. The pork meat accounts for about 20% of the base distillate (by © 2015 American Chemical Society

Received: Revised: Accepted: Published: 3660

December March 23, March 23, March 23,

23, 2014 2015 2015 2015 DOI: 10.1021/jf506238f J. Agric. Food Chem. 2015, 63, 3660−3668

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Journal of Agricultural and Food Chemistry Table 1. Detailed Information on Liquors aroma type

full name

abbreviation

alcohol content (% vol)

geographic origin (province of China)

soy sauce strong light complex laobaiganxiang roasted-sesame-like herblike fenxiang texiang chixiang chixiang

soy sauce finished liquor strong finished liquor light finished liquor complex finished liquor laobaiganxiang finished liquor roasted-sesame-like finished liquor herblike finished liquor fenxiang finished liquor texiang finished liquor chixiang base distillate chixiang finished liquor

SSFL SFL LFL CFL LBGXFL RSLFL HLFL FXFL TXFL CXBD CXFL

53 52 53 50 43 46 54 52 45 38 38

Guizhou Sichuan Shanxi Anhui Hebei Shandong Guizhou Shaanxi Jiangxi Guangdong Guangdong

naphthalene were supplied by Alfa Aesar (Tianjin, China). Diethyl pimelate, diethyl suberate, and diethyl azelate were provided by ANPEL Scientific Instrument Co., Ltd. (Shanghai, China). Pentyl acetate (internal standard, IS1), 2,2-dimethylpropanoic acid (IS2), ethyl octanoate-d15 (IS3), n-hexyl-d13 alcohol (IS4), ethyl 2-phenylacetate-d3 (IS5), and p-fluorobenzaldehyde (IS6) were purchased from ANPEL Scientific Instrument Co., Ltd. (Shanghai, China). IS1 was used as internal standard for compounds with high concentrations (ethyl acetate, ethyl lactate, 1-propanol, 2-methylpropanol, 3methylbutanol, and 2-phenylethanol). IS2−IS6 were used as internal standards for fatty acids, esters, alcohols, benzoic compounds, and carbonyl compounds, respectively. The remaining compounds (1,1,3-triethoxypropane, γ-octalactone, and γ-nonalactone) were quantified with IS3. A C5−C30 nalkane mixture (Sigma−Aldrich, Shanghai, China) was used for determination of linear retention indices (RIs). Sodium chloride, sodium bicarbonate, sulfuric acid, anhydrous sodium sulfate, and absolute ethanol were from China National Pharmaceutical Group Corp. (Shanghai, China). Diethyl ether and pentane obtained from ANPEL Scientific Instrument Co., Ltd. (Shanghai, China) were freshly distilled before use. Sample Preparation. According to literature,10 a total of 50 mL of liquor sample was diluted to 10% ethanol by volume with Milli-Q water (Millipore, Bedford, MA), boiled for 5 min, and then cooled to room temperature in a 1 L flask, saturated with NaCl. The diluted liquor sample was extracted 3 times with 20 mL of a mixture of pentane/diethyl ether (1:1 by volume). The combined extracts were further separated into acidic/water-soluble fraction (AF) and neutral/basic fraction (NBF). Both fractions were dried with 5 g of anhydrous Na2SO4 overnight, concentrated to a final volume of 200 μL under a gentle stream of nitrogen, and then stored at −20 °C until analysis. Gas Chromatographic−Olfactometric and −Mass Spectrometric Analysis. GC−O and GC−MS analysis were performed on an Agilent 6890 gas chromatograph equipped with an Agilent 5975 mass-selective detector (MSD) and an olfactometer (ODP 2, Gerstel, Germany). Samples were analyzed on both a DB-FFAP column (60 m × 0.25 mm i.d., 0.25 μm film thickness, J&W Scientific) and a DB-5 column (30 m × 0.25 mm i.d., 0.25 μm film thickness, J&W Scientific). The column carrier gas was helium at a constant flow rate of 2 mL/min. Each concentrated fraction (1 μL) was injected into the GC and the sniffing port with a split ratio of 1:1. The injector temperature was set at 250 °C. The oven temperature was held at 50 °C for 2 min, then programmed to 230 °C at a rate of 6 °C/min, and finally

Gas chromatography−olfactometry (GC−O), often using aroma extract dilution analysis (AEDA) and Osme, coupled with careful quantitative analysis of the odor-active compounds and calculation of odor activity values (OAVs), has enabled significant advances in understanding of important odorants in alcoholic beverages. Aroma recombination and omission experiments are being extensively and successfully used to confirm the key aroma compounds of alcoholic beverages, such as beer,8 brandy,2 Chinese rice wine,9 Chinese liquor,10 whisky,11 and wine.12 To our knowledge, no studies have reported on the contribution of aroma compounds to the characteristic aroma of chixiang aroma-type liquor. The aims of the present study were (i) to identify important odorants in chixiang aroma-type liquor by GC−O, (ii) to quantify these compounds by different pretreatment methods and various advanced analytic approaches, and (iii) to perform aroma recombination and omission/addition experiments to confirm the key aroma compounds of chixiang aroma-type liquor.

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MATERIALS AND METHODS Liquors. Detailed sample information is listed in Table 1. Only chixiang aroma-type finished liquor (CXFL) and its base distillate (CXBD) were used for GC−O analysis. All liquors (500 mL for each bottle) were stored at 4 °C until analysis. Chemicals. All chemicals were of analytical reagent grade, with at least 97% purity. Ethyl acetate, ethyl 2-methylpropanoate, ethyl butanoate, ethyl 2-methylbutanoate, ethyl 3methylbutanoate, 3-methylbutyl acetate, ethyl pentanoate, ethyl hexanoate, ethyl heptanoate, ethyl 2-hydroxypropanoate (ethyl lactate), ethyl octanoate, 3-methylbutyl hexanoate, ethyl nonanoate, ethyl decanoate, diethylbutanedioate, 1-propanol, 2methylpropanol, 1-butanol, 3-methylbutanol, 1-pentanol, 1hexanol, 1-heptanol, 1-octanol, propanoic acid, 3-methylbutanoic acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, hexanal, heptanal, octanal, 1,1,3-triethoxypropane, 3-hydroxy-2-butanone, nonanal, 2-nonanone, (E)-2octenal, 2-furancarboxaldehyde (furfural), 2-decanone, (E)-2nonenal, (E)-2-decenal, (E)-2-undecenal, (E,E)-2,4-decadienal, styrene, benzeneacetaldehyde, ethyl benzoate, ethyl 2-phenylacetate, 2-phenylethyl acetate, benzyl alcohol, ethyl 3-phenylpropanoate, (E)-cinnamaldehyde, 3-(methylthio)-1-propanal (methional), 3-(methylthio)-1-propanol (methionol), 2-phenylethanol, γ-octalactone, γ-nonalactone, 4-methylguaiacol, phenol, 4-ethylguaiacol, 4-ethylphenol, and O-(2,3,4,5,6pentafluorobenzyl)hydroxylamine hydrochloride (PFBHA) were purchased from Sigma−Aldrich Co., Ltd. (Shanghai, China). Acetic acid, 2-methylpropanoic acid, butanoic acid, and 3661

DOI: 10.1021/jf506238f J. Agric. Food Chem. 2015, 63, 3660−3668

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Journal of Agricultural and Food Chemistry held at 230 °C for 10 min. The temperature of the olfactory port was kept at 280 °C. Mass spectra in the electron ionization mode (EI) were recorded at 70 eV ionization energy. The temperature of the ion source was 230 °C, and the mass range was from 30 to 350 amu. Four trained panelists (two females and two males), three graduate students and one teacher from the Laboratory of Brewing Microbiology and Applied Enzymology at Jiangnan University, were selected for the GC−O study. During a GC run, a panelist placed his/her nose close to and above the top of the sniffing port, recorded the odor of the chromatographic effluent as well as retention time. Analyses were repeated in duplicate by each panelist. Aroma Extract Dilution Analysis. For AEDA, each concentrated fraction, containing NBF and AF, was diluted stepwise with diethyl ether as the solvent in a series of 1:3, 1:9, 1:27, ..., 1:729 dilution. Each dilution was submitted to GC−O analysis under the same GC conditions described above until no odorant could be detected. The flavor dilution (FD) factor of each compound represented the maximum dilution in which the odorant could be perceived. Identification of each aroma compound was carried out by comparing with their odors, retention index (RI), and mass spectra with those of pure standards. Quantitative Analysis of Aroma Compounds. Gas Chromatography with Flame Ionization Detector. Ethyl acetate, ethyl lactate, 1-propanol, 2-methylpropanol, 3-methylbutanol, and 2-phenylethanol (Table 3) were quantified by gas chromatography with flame ionization detector (GC-FID) according to ref 13. Liquor sample was spiked with IS1 to final concentration 176 mg/L. One microliter of disposed sample was injected into the GC in split mode 37:1. The correction factors calculated in 38% ethanol by volume are showed in Table 3. Nitrogen was used as carrier gas at a constant flow rate of 1 mL/min. The separations were performed on a DB-Wax column (30 m × 0.25 mm i.d. × 0.25 μm film thickness; J&W Scientific). The oven temperature was initially set at 60 °C for 3 min, ramped at 5 °C/min to 150 °C for 5 min, and then increased to 230 °C at 10 °C/min for 5 min. The injector and detector temperatures were set at 250 °C. All analyses were repeated in triplicate. Liquid−Liquid Microextraction. Fatty acids were quantified by liquid−liquid microextraction (LLME) according to the method of Wang et al.14 Diluted liquor sample (18 mL) with 6 μL of IS2 solution (3.41 mg/L final concentration) was saturated with NaCl and then extracted for 3 min with 1 mL of redistilled diethyl ether. The GC−MS conditions were set as for GC−O analysis on a DB-FFAP column (60 m × 0.25 mm i.d., 0.25 μm film thickness, J&W Scientific) described above. Selective ion monitoring (SIM) mass spectrometry was used to quantify the odorants. The ion monitored for IS2 was m/z 57. The standard curve for fatty acids was carried out by plotting the response ratio of standard compounds and IS2 against the concentration ratio (Table 3). The limits of detection (LOD) was calculated as the analyte concentration of a standard that produced a signal-to-noise ratio of 3. The recovery of target compounds was estimated according to ref 14. Headspace Solid-Phase Microextraction−Gas Chromatography−Mass Spectrometry. As the methods reported,10 each liquor sample was diluted with Milli-Q water (Millipore, Bedford, MA) to a final concentration of 10% ethanol by volume. A total of 8 mL of diluted solution with 10 μL of IS solution (IS3, IS4, and IS5 with concentrations of 87.8, 163, and 207 mg/L in ethanol, respectively) was put into a 20 mL

screw-capped vial and then saturated with NaCl. An automatic headspace sampling system (MultiPurposeSample MPS 2 with a SPME adapter, from Gerstel Inc., Mülheim, Ruhr, Germany) with a 50/30 μm divinylbenzene/carboxen/poly(dimethylsiloxane) (DVB/CAR/PDMS) fiber (2 cm, Supelco Inc., Bellefonte, PA) was used to extract volatile odorants. The conditions of HS-SPME remained the same as in ref 10. The GC−MS conditions were set as for GC−O analysis on a DBFFAP column (60 m × 0.25 mm i.d., 0.25 μm film thickness, J&W Scientific) described above. The ions monitored for IS3, IS4, and IS5 were m/z 91 and 142, 64 and 78, and 104 and 46, respectively. The LOD and recovery of target compounds were detected by the method described above. Quantitation of Carbonyl Compounds by Headspace Solid-Phase Microextraction−Gas Chromatography−Mass Spectrometry after Derivatization with O-(2,3,4,5,6Pentafluorobenzyl)hydroxylamine Hydrochloride. Quantitation of carbonyl compounds after derivatization with PFBHA was performed by GC−MS on a DB-FFAP column (60 m × 0.25 mm i.d., 0.25 μm film thickness, J&W Scientific) modified from literature.15 The liquor sample was diluted to 10% ethanol by volume. Eight milliliters of dilution was saturated with NaCl and added to a 20 mL standard headspace vial, and then 10 μL of a solution of IS6 (1.0724 mg/L in ethanol) and 120 μL of PFBHA (50 g/L in water) were added. The fiber used was three-phase fiber as described above. The sample was incubated at 65 °C for 10 min and extracted for 45 min at the identical temperature under stirring at a rotation speed of 250g, then transferred the fiber to the injector for desorption at 250 °C for 300 s. During the GC run (in splitless mode), a constant flow rate (1 mL/min) of the carrier gas (helium) was maintained. The injector temperature was held at 280 °C. The oven temperature was programed to 50 °C for 2 min and raised to 100 °C at a rate of 6 °C/min for 0.1 min, then to 160 °C at a rate of 2 °C/min for 0.1 min, and finally at 5 °C/min to 230 °C for 10 min. The mass spectrometer was operated in electron ionizationI mode at 70 eV with SIM. The ion monitored for IS6 after derivatization was m/z 319. Monitored ions of carbonyl compounds after derivatization are listed in Table 3. The standard curve and LOD, as well as recovery of carbonyl compounds, were measured by the method mentioned above. Determination of Odor Thresholds. For the calculation of OAVs, odor thresholds of some aromas were determined in 46% ethanol/water solution by triangle tests based on ref 16. Aroma Recombination of Chixiang Finished Liquor. Ethanol/water solution (38% ethanol by volume) was used as the matrix for recombination. The aroma compounds with OAVs ≥ 1 (Table 4) of CXFL were spiked into the matrix according to their occurring concentrations (Table 3). The overall aroma profiles of the CXFL liquor and reconstituted models were evaluated by 10 well-trained assessors (five males and five females, 30 years old on average), eight graduate students and two teachers from the Laboratory of Brewing Microbiology and Applied Enzymology at Jiangnan University. All of the assessors were trained for 2 months (45 min/day) to describe and recognize the odor qualities of approximately 30 odorants. Testing samples (20 mL) were presented to assessors in four-digit-coded, covered glasses in a sensory panel room at 20 ± 1 °C. The assessors rated eight aroma attributes to describe the overall aroma: fatty, grassy, sweet, alcoholic, floral, fruity, cheesy, and acidic. The intensity of each aroma attribute was rated on a 6-point scale, where 0 is none, 1 is very weak, 2 is weak, 3 is moderate, 4 is strong, and 5 is very strong. The 3662

DOI: 10.1021/jf506238f J. Agric. Food Chem. 2015, 63, 3660−3668

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Journal of Agricultural and Food Chemistry

Table 2. Aroma Compounds Identified by Gas Chromatography−Olfactometry in Chixiang Aroma-type Finished Liquor and Its Base Distillate RI

FD factor

no.

FFAP column

DB-5 column

aroma compd

descriptor

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

892 961 1035 1038 1045 1060 1071 1087 1102 1128 1137 1199 1232 1235 1255 1256 1280 1271 1304 1310 1334 1354 1369 1376 1427 1437 1459 1453 1442 1454 1466 1479 1499 1516 1530 1539 1555 1602 1610 1603 1620 1634 1640 1655 1695 1727 1742 1744 1786 1801 1822 1825 1858 1872 1906 1886 1955 1959

589 750 530 817 860 856 803 620 878 913 669 902 789 1028 892 763 1003 1128 717 1115 823 892 1121 1089 1203 1070 601 903 978 1238 840 1190 1289 1160 675 1090 792 802 1398 1273 1052 837 1165 1170 977 879 1218 1365 1254 1271 1327 971 1037 1348 1132 1215 1103 1199

ethyl acetate ethyl 2-methylpropanoate 1-propanol ethyl butanoate ethyl 2-methylbutanoate ethyl 3-methylbutanoate hexanal 2-methylpropanol 3-methylbutyl acetate ethyl pentanoate 1-butanol heptanal 3-methylbutanol ethyl hexanoate styrene 1-pentanol octanal 1,1,3-triethoxypropane 3-hydroxy-2-butanone ethyl heptanoate ethyl lactate 1-hexanol nonanal 2-nonanone ethyl octanoate (E)-2-octenal acetic acid methional 1-heptanol 3-methylbutyl hexanoate furfural 2-decanone ethyl nonanoate (E)-2-nonenal propanoic acid 1-octanol 2-methylpropanoic acid butanoic acid ethyl decanoate (E)-2-decenal phenylacetaldehyde 3-methylbutanoic acid ethyl benzoate diethyl butanedioate methionol pentanoic acid naphthalene (E)-2-undecenal ethyl 2-phenylacetate 2-phenylethyl acetate (E,E)-2,4-decadienal hexanoic acid benzyl alcohol ethyl 3-phenylpropanoate 2-phenylethanol γ-octalactone heptanoic acid 4-methylguaiacol

pineapple apple alcoholic pineapple berry apple grassy wine banana apple alcoholic grassy nail polish fruity plastic fruity fatty vegetal buttery fruity fruity floral soapy floral fruity fatty vinegar cooked potato alcoholic fruity sweet, almond fruity fruity fatty vinegar fruity cheesy cheesy fruity fatty floral cheesy floral fruity cooked potato cheesy mothball fatty rosy, honey floral fatty sweaty, cheesy floral floral rosy, honey coconut rancid, unpleasant smoky

3663

basis of IDa MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS,

aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma, aroma,

RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI

fractionb

CXFLc

CXBDc

NBF NBF NBF + AF NBF NBF NBF NBF NBF + AF NBF NBF NBF+AF NBF NBF+AF NBF NBF NBF+AF NBF NBF NBF NBF NBF+AF AF NBF NBF NBF NBF AF NBF+AF AF NBF NBF+AF NBF NBF NBF AF AF AF AF NBF NBF NBF AF NBF NBF NBF AF NBF NBF NBF NBF NBF AF NBF+AF NBF NBF+AF NBF AF AF

81 243 3 3 9 81 243 3 81 1

81 27 3 9 9 27 9 3 243 3 1 9 27 3

81 9 1 3 729 3 9 9 3 1 729 9 9 81 81 243 1 1 1 3 81 243 1 9 243 1 9 9 3 1 1 81 3 3 3 3 81 9 3 3 27 9 3

1 27 3 3 3 3 1 3 81 3 243 9 3 9 27 27 3 81 3 27 81 3 1 9 27 9 1 1 3 81 1 3 9 1 9 3 3 243 9 9 3

DOI: 10.1021/jf506238f J. Agric. Food Chem. 2015, 63, 3660−3668

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Journal of Agricultural and Food Chemistry Table 2. continued RI

FD factor

no.

FFAP column

DB-5 column

59 60 61 62 63 64

2007 2010 2018 2033 2060 2185

991 1297 1359 1281 1168 1181

aroma compd phenol 4-ethylguaiacol γ-nonalactone (E)-cinnamaldehyde octanoic acid 4-ethylphenol

descriptor phenol, medicinal clove coconut cinnamon cheesy smoky

basis of ID MS, MS, MS, MS, MS, MS,

aroma, aroma, aroma, aroma, aroma, aroma,

a

RI RI RI RI RI RI

fraction AF AF NBF NBF AF AF

b

CXFLc

243 1 9

CXBDc 3 81 81 3 27 3

a MS, compounds were identified by MS spectra; aroma, compounds were identified by comparison to reference standards by GC−O; RI, compounds were identified on FFAP and DB-5 by comparison to reference standards. bFraction(s) in which odorant was detected by GC−O after fractionation. AF, acidic/water-soluble fraction; NBF, neutral and basic fraction. cCXFL, chixiang aroma-type finished liquor; CXBD, chixiang aromatype base distillate.

The total concentration of five unsaturated aldehydes in CXFL liquor was the highest, up to 1040 μg/L, notably higher than any other aroma-type liquors. Besides, CXBD and DJ liquor had higher concentrations of 100−200 μg/L whereas total concentrations of these five unsaturated aldehydes in SSFL, SFL, LFL, CFL, LBGXFL, RSLFL, FXFL, and TXFL liquors were less than 100 μg/L, far lower than that in CXFL liquor (Table 4). Diethyl pimelate, diethyl suberate, and diethyl azelate were included in the quantification experiments because they were frequently described as important aroma compounds in the literature.7 The concentrations of diethyl pimelate, diethyl suberate, and diethyl azelate were 687, 228, and 2310 μg/L, respectively. Odor Activity Values. The thresholds were determined in 46% hydroalcoholic solution (by volume). As indicated in Table 5, odor thresholds of 24 aroma compounds were determined, and the odor thresholds in liquor ranged from 7.12 (methional) to 1 280 000 μg/L (diethyl azelate). So contribution to the liquor would depend not only on the concentration of an odorant but also its odor threshold. To evaluate the contribution of quantified odorants to the overall aroma of the liquor, odor activity values (ratio of concentration to its odor threshold) were calculated for each aroma compound listed in Table 5. A total of 34 odorants were calculated with OAVs ≥1. These odorants should be important contributors to the characteristic aroma of CXFL liquor. The highest OAV in finished liquor was ethyl 2-methylpropanoate (OAV = 336), followed by ethyl octanoate (OAV = 157) and hexanal (OAV = 69). (E)-2-Decenal, (E)-2-octenal, (E,E)-2,4decadienal, and (E)-2-nonenal, which give “fatty” flavor,2,21 were detected with OAVs 31, 28, 5, and 3, respectively. The CXFL liquor was much higher than any other liquors in the OAVs of (E)-2-decenal, (E)-2-octenal, (E,E)-2,4-decadienal, and (E)-2-nonenal (Table 4). Noticeably, (E)-2-nonenal was detected with OAV > 1 only in the CXFL liquor. Two odorants, 2-phenylethanol (rose- or honey-like-aroma) and methionol (cooked potato), might contribute to the overall aroma according to their OAVs (>1). However, diethyl pimelate, diethyl suberate, and diethyl azelate were calculated with OAVs ≤ 1; therefore, they could not contribute to the overall aroma. Aroma Recombination and Omission/Addition Experiments. To confirm that the aroma compounds were indeed the important odorants of chixiang aroma-type liquor, aroma recombination and omission/addition experiments were undertaken, based on the quantitative results of finished liquor. For this purpose, an aroma recombinate was prepared in a 38% hydroalcoholic solution (by volume), by addition of 34

sensory data were analyzed by one-way analysis of variance (ANOVA) by use of SPSS15.0 (SPSS Inc., Chicago, IL). Omission/Addition Tests. In triangular tests with forced choice, a recombinant model, in which one odorant or a class of odorants (Table 6) was omitted/added, was evaluated against two complete recombinants. The testing samples were arranged in a random four-digit code, and each test was repeated in triplicate. The assessors were asked to sniff the samples and estimate the differing one. The significance of the differences was evaluated by the method as reported.17

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RESULTS AND DISCUSSION Aroma Compounds by Gas Chromatography−Olfactometry in Chixiang Aroma-type Liquor. A total of 64 odorants were perceived by GC−O in chixiang finished liquor and its base distillate. In finished liquor (CXFL), 56 odorants could be detected by AEDA. Among them, octanal and nonanal obtained the highest FD factor (FD ≥729), which gave intense fatty and soapy aromas, respectively. A total of 61 aroma compounds were identified in base distillate (CXBD). 3Methylbutyl acetate, acetic acid, and 2-phenylethanol (FD ≥ 243) had the highest FD factor, which elicited intense banana, vinegar, and rose- or honey-like aromas, respectively. As shown in Table 2, (E)-2-octenal, (E)-2-nonenal, (E)-2-decenal, (E)-2undecenal, and (E,E)-2,4-decadienal were first detected in CXFL and CXBD liquors, which exhibited intense fatty aromas. Unsaturated aldehydes usually gave off-flavor in beer,18,19 and they have been proposed as main note of the roasted mini-pig pork meat aroma.20 Methionol was detected in both liquors (FD ≥ 1 in CXFL and CXBD), which was previously regarded as one of the characteristic aromas of chixiang aroma-type liquor.5,7 Diethyl pimelate, diethyl suberate, and diethyl azelate were not detected by GC−O in either liquor. Concentration of Aroma Compounds in Chixiang Aroma-type Liquor. To gain a deeper insight into the aroma of chixiang aroma-type liquor, a total of 59 compounds, 56 odorants detected by AEDA and three dibasic acid diethyl esters (diethyl pimelate, diethyl suberate, and diethyl azelate), were quantified in CXFL liquor (Table 3). As exhibited in Table 3, ethyl lactate had the highest concentration (585 mg/ L), followed by ethyl acetate (385 mg/L) and 3-methylbutanol (354 mg/L). In addition, the data in Table 4 demonstrate that the highest concentrations of (E)-2-octenal, (E)-2-nonenal, (E)-2-decenal, (E)-2-undecenal, and (E,E)-2,4-decadienal were found in CXFL liquor among 10 aroma-type Chinese liquors. (E)-2-Octenal was the most abundant unsaturated aldehyde in CXFL liquor with concentration exceeding 400 μg/L, followed by (E)-2-decenal (375 μg/L) and (E)-2-nonenal (156 μg/L). 3664

DOI: 10.1021/jf506238f J. Agric. Food Chem. 2015, 63, 3660−3668

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Journal of Agricultural and Food Chemistry Table 3. Standard Curve and Concentrations of 59 Aroma Compounds in Chixiang Aroma-type Finished Liquor CXFLa no.

aroma compd

21 1 13 27 8 55 3 57 2 38 52 63 37 35 53 44 22 4 46 20 45 9 67 25 43 7 14 19 12 42 33 61 23 17 65 36 29 62 26 40 18 6 54 24 66 32 39 34 50 56 49 10 15 48 51 30 5 28 47

d

ethyl lactate ethyl acetated 3-methylbutanold acetic acidf 2-methylpropanold 2-phenylethanold 1-propanold heptanoic acidf ethyl 2-methylpropanoate butanoic acidf hexanoic acidf octanoic acidf 2-methylpropanoic acidf propanoic acidf benzyl alcohol diethyl butanedioate 1-hexanol ethyl butanoate pentanoic acidf ethyl heptanoate methionol 3-methylbutyl acetate diethyl azelate ethyl octanoate ethyl benzoate hexanalg ethyl hexanoate 3-hydroxy-2-butanoneg heptanalg 3-methylbutanoic acidf ethyl nonanoate γ-nonalactone nonanalg octanalg diethyl pimelate 1-octanol 1-heptanol (E)-cinnamaldehyde (E)-2-octenalg (E)-2-decenalg 1,1,3-triethoxypropane ethyl 3-methylbutanoate ethyl 3-phenylpropanoate 2-nonanoneg diethyl suberate 2-decanoneg ethyl decanoate (E)-2-nonenalg 2-phenylethyl acetate γ-octalactone ethyl phenylacetate ethyl pentanoate styrene (E)-2-undecenalg (E,E)-2,4-decadienalg 3-methylbutyl hexanoate ethyl 2-methylbutanoate methionalg naphthalene

quantified ion e

1.04 1.47e 0.770e 43 0.720e 0.830e 0.830e 60 43 60 60 60 43 74 79 101 56 71 60 88 106 43 199 88 105 44 88 240 70 60 88 85 57 43 125 56 70 131 250 250 103 88 104 58 185 58 88 250 104 85 91 88 104 250 276 70 57 299 128

IS IS1 IS1 IS1 IS2 IS1 IS1 IS1 IS2 IS3 IS2 IS2 IS2 IS2 IS2 IS5 IS3 IS4 IS3 IS2 IS3 IS4 IS3 IS3 IS3 IS5 IS6 IS3 IS6 IS6 IS2 IS3 IS3 IS6 IS6 IS3 IS4 IS4 IS5 IS6 IS6 IS3 IS3 IS5 IS6 IS3 IS6 IS3 IS6 IS5 IS3 IS5 IS3 IS5 IS6 IS6 IS3 IS3 IS6 IS3

slope

52.2

6.12 21.6 4.02 2.71 9.81 3.18 9.06 43.0 7.95 18.9 8.97 2.72 0.390 4.53 1.71 2.04 0.130 0.210 9.37 0.440 28.7 4.60 1.21 0.120 29.9 1.17 0.750 5.68 1.07 4.19 3.55 2.46 4.04 3.39 2.96 0.290 1.39 0.160 1.78 0.0400 3.05 0.380 19.2 0.260 0.440 0.250 9.78 3.73 0.210 4.25 14.3 0.0200

intercept

n

R2

LOD (μg/L)

2.39

6

0.993

2800

1.38 −1.41 0.980 0.740 0.490 0.410 0.460 2.90 −3.97 8.33 6.89 0.150 5.51 0.160 4.91 2.66 1.63 0.0300 −1.51 0.0800 −0.180 0.210 0.0400 1.11 0.150 0.140 0.790 0.540 0.290 −0.320 0.680 1.25 7.18 0.310 0.640 0.660 0.100 0.410 0.0800 0.0700 2.29 0.0300 0.120 0.0300 0.0800 0.130 4.56 2.12 0.0200 −0.280 0.0400 0.0100

6 7 8 7 7 8 6 9 11 12 11 6 12 9 11 8 11 11 12 7 10 7 8 10 10 10 7 8 8 11 8 10 10 11 8 11 11 7 12 8 10 11 8 11 9 8 11 11 11 7 9 9

0.993 0.998 0.999 0.996 0.984 0.999 0.997 0.993 0.994 0.996 0.996 0.984 0.990 0.993 0.999 0.996 0.992 0.999 0.999 0.997 0.999 0.998 0.999 0.998 0.995 0.999 0.997 0.993 0.992 0.996 0.994 0.999 0.999 0.998 0.996 0.999 0.996 0.994 0.997 0.995 0.999 0.998 0.991 0.997 0.998 0.997 0.999 0.999 0.999 0.992 0.999 0.995

2930 3.65 2580 938 887 1960 214 56.9 27.4 2.64 14.0 194 16.2 316 50.6 86.6 11.4 2.33 32.7 2.92 11.4 70.8 362 13.4 20.3 1.25 10.7 25.7 12.6 8.12 14.5 1.61 1.06 38.5 4.04 14.2 8.50 10.8 3.33 0.710 0.660 0.800 2.89 0.570 28.9 2.16 0.650 0.440 3.39 0.790 3.47 0.130

3665

recovery (%)

90

86 95 88 95 80 84 92 108 109 99 99 92 85 102 110 98 111 99 87 91 95 99 90 110 105 104 98 106 100 89 97 102 101 96 101 109 104 101 94 99 106 96 90 94 85 107 98 89 115 93 106 91

avgb (μg/L)

RSDc (%)

585000 385000 354000 260000 233000 127000 88200 25600 19300 19200 17400 11300 9300 8560 7130 5100 3820 3690 3470 3260 3090 2510 2310 2030 1880 1760 1600 1390 1180 1090 950 866 854 759 687 612 574 471 422 375 350 344 314 252 228 183 161 156 147 132 125 108 102 47.3 35.6 24.5 10.3 6.97 3.41

7 6 8 8 6 7 7 7 3 6 6 8 7 9 8 3 5 3 8 4 6 2 2 3 2 3 4 6 4 7 5 4 3 2 2 4 2 3 9 5 3 3 5 4 2 6 4 4 5 2 2 3 3 4 5 4 2 4 2

DOI: 10.1021/jf506238f J. Agric. Food Chem. 2015, 63, 3660−3668

Article

Journal of Agricultural and Food Chemistry Table 3. continued

CXFL, chixiang aroma-type finished liquor. bAverage concentration of triplicates. cRelative standard deviation. dQuantified by GC-FID. Calibration factors of aroma compounds detected by GC-FID. fQuantified by LLME combined with GC−MS. gDetected by GC−MS after derivatization with PFBHA.

a e

Table 4. Concentrations and OAVs of Unsaturated Aldehydes in Different Aroma-type Chinese Liquorsa (E)-2-octenal liquor

avgb (μg/L)

SSFL SFL LFL CFL LBGXFL RSLFL HLFL FXFL TXFL CXBD CXFL

22.2 33.4 28.4 18.9 27.1 20.8 33.7 28.5 22.4 96.4 422

(E)-2-nonenal

OAV

RSD (%)

avg (μg/L)

1 2 2 1 2 1 2 2 1 6 28

9 7 8 10 6 7 9 10 9 6 8

13.2 17.0 20.5 10.1 14.4 14.4 25.7 14.0 14.0 16.2 156

(E)-2-decenal

OAV

RSD (%)

avg (μg/L)