Article pubs.acs.org/JAFC
Comparison of Aroma-Active Volatiles in Oolong Tea Infusions Using GC−Olfactometry, GC−FPD, and GC−MS JianCai Zhu,† Feng Chen,†,‡ LingYing Wang,§ YunWei Niu,† Dan Yu,† Chang Shu,† HeXing Chen,† HongLin Wang,† and ZuoBing Xiao*,† †
Department of Perfume and Aroma Technology, Shanghai Institute of Technology, Shanghai, 201418, China Department of Food, Nutrition, and Packaging Sciences, Clemson University, Clemson, South Carolina 29634, United States § Shanghai Cosmax (China) Cosmetics Co., LTD, Shanghai, China ‡
S Supporting Information *
ABSTRACT: The aroma profile of oolong tea infusions (Dongdingwulong, DDWL; Tieguanyin, TGY; Dahongpao, DHP) were investigated in this study. Gas chromatography−olfactometry (GC−O) with the method of aroma intensity (AI) was employed to investigate the aroma-active compounds in tea infusions. The results presented forty-three, forty-five, and forty-eight aromaactive compounds in the TGY, DHP, and DDWL infusions, including six, seven, and five sulfur compounds, respectively. In addition, the concentration of volatile compounds in the tea infusions was further quantitated by solid phase microextraction−gas chromatography (SPME)−GC−MS and SPME−GC−flame photometric detection (FPD). Totally, seventy-six and thirteen volatile and sulfur compounds were detected in three types of tea infusions, respectively. Quantitative results showed that fortyseven aroma compounds were at concentrations higher than their corresponding odor thresholds. On the basis of the odor activity values (OAVs), 2-methylpropanal (OAV: 230−455), 3-methylbutanal (1−353), 2-methylbutanal (34−68), nerolidol (108−184), (E)-2-heptenal (148−294), hexanal (134−230), octanal (28−131), β-damascenone (29−59), indole (96−138), 6methyl-5-hepten-2-one (34−67), (R)-(−)-linalool (63−87), and dimethyl sulfide (7−1320) presented relatively higher OAVs than those of other compounds, indicating the importance of these compounds in the overall aroma of tea infusions. KEYWORDS: oolong tea infusions, sulfur compound, GC−FPD, aroma-active volatiles, GC−O
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INTRODUCTION
that those aroma compounds tend to be thermally unstable and are prone to oxidative reactions.12 Therefore, the aims of this research were (i) to identify volatile sulfurs in oolong tea infusions using flame photometric detection (FPD), (ii) to identify the key aroma compounds in oolong tea infusions by GC−O and OAV, and (iii) to characterize the aroma profile of oolong tea infusions by sensory evaluation.
Oolong tea is a well-known beverage, and its consumption is widely distributed throughout China. It is well-known for its flavor and other characteristics, by which it could increase digestion and fat transformation.1,2 In the preparation of oolong tea, young green shoots are freshly harvested and allowed to undergo semifermentation. The concept of fermentation, in tea, refers to reactions between oxidative enzymes and amino acids, tea polyphenols, which occurs in the cells of tea leaves.3,4 The degree of fermentation of oolong tea ranges from 20% to 80%. Volatile composition in oolong tea infusions is complex: however, only a few of these compounds are considered to be the key contributors to overall aroma.5 Thus, the identification of key aroma compounds from the complex mixture of oolong tea compounds is critical for flavor analysis. Recently, there have been several reports on oolong tea and its catechin content, volatile compounds, antioxidant activity, and gene expression.6−8 However, an investigation of the key aroma compounds in oolong tea is yet to be reported. Volatile sulfur compounds (VSC) are an important class of key aroma-inducing compounds, due to their low thresholds, strong odor impact, and wide distribution in food products.9,10 However, VSCs are often neglected in an aroma analysis. The reasons for this might be that those compounds are present at extreme trace levels in foods and conventional instruments are often not sensitive enough to identify these compounds.11 Another challenge in the isolation and identification of VSCs is © 2015 American Chemical Society
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MATERIALS AND METHODS
Tea Samples. A total of 30 samples of tea, including ten lightly fermented samples of tea (Dongdingwulong, DDWL) from 4 different brands, eleven moderately fermented samples of tea (Tieguanyin, TGY) from 5 different brands, and nine heavily fermented samples of tea (Dahongpao, DHP) from 4 different brands, were analyzed in the present study. The tea samples were purchased from the local markets in Shanghai, China. All samples were kept in their own packages with aluminum foil material and stored in a refrigerator (4 °C) until analyzed. Chemicals. Authentic standards were obtained from the following sources: α-terpineol, β-damascenone, β-ionone, 1-octanol, 1-octen-3ol, 1-pentanol, 1-penten-3-one, 1-penten-3-ol, 2,3-butanedione, 2,3octanedione, 2,3-pentanedione, 2,5-dimethylpyrazine, 2-acetylfuran, 2acetylpyrrole, 2-ethyl-1-hexanol, 2-ethylfuran, 2-heptanone, 2-methylReceived: Revised: Accepted: Published: 7499
May 12, 2015 July 31, 2015 August 9, 2015 August 10, 2015 DOI: 10.1021/acs.jafc.5b02358 J. Agric. Food Chem. 2015, 63, 7499−7510
Article
Journal of Agricultural and Food Chemistry butanal, 2-methylfuran, 2-methylpropanal, 2-nonanone, 2-pentylfuran, 2,3,5-trimethylpyrazine, 3-mercaptohexanol, 3-methylbutanal, 3-methylbutyl acetate, 3-methylbutanoate, 5-methyl-2-furfural, 6-methyl-5hepten-2-one, acetaldehyde, acetic acid, benzaldehyde, benzothiazole, benzyl alcohol, butanal, butyl acetate, camphor, cis-jasmone, cislinaloloxide, dihydroactinidiolide, dimethyl disulfide, dimethyl sulfide, dimethyl sulfoxide, dimethyl trisulfide, (E)-2-heptenal, (E)-2-hexenal, (E)-2-pentenal, (E,E)-2,4-heptadienal, (E,E)-2,4-hexadienal, ethyl acetate, (E)-2-octenal, ethanethiol, furan, furfural, geraniol, heptanal, heptanoic acid, heptanol, hotrienol, hexanal, hexanoic acid, hexanol, indole, methanethiol, methyl benzoate, methyl hexanoate, methyl jasmonate, methyl salicylate, nerolidol, nonanal, octanal, octanoic acid, pentanal, propanal, propanethiol, phenyl acetaldehyde, phenylethyl alcohol, (R)-(−)-linalool, S-methyl thioacetate, trans-linalooloxide, (Z)-3-hexen-1-ol, and (Z)-4-heptenal were purchased from Alfa Aesar Corporation (Tianjin, China). Furfurylthiol, methional, methionol, and C6−C30 n-alkane standard were purchased from Sigma-Aldrich Corporation (Shanghai, China). All of the chemical standards used above were of GC quality. Sample Preparation. Twenty grams of oolong tea was weighed and placed in a prewarmed tea pot, and 1000 mL of 80 °C distilled water was added. The tea was brewed for 5 min and swirled 10 times clockwise while brewing. Then, the tea was poured through a porcelain strainer into a prewarmed porcelain bowl. These samples were stored in a refrigerator (4 °C) after the tea infusions had cooled down to room temperature. Solid Phase Microextraction (SPME) Absorption of Aroma Compounds. The manual SPME holder, together with 20 mL vials, Teflon covers, and one 75 μm divinylbenzene-carboxyl-polydimethylsiloxane (DVB-CAR-PDMS) fiber, were purchased from Supelco, Inc. (Bellefonte, PA, USA). Before chemical absorption, the fiber was preconditioned for 30 min on an Agilent 6890 gas chromatograph (Agilent Technologies, USA) with the injector temperature of 250 °C. The main parameters that were known to influence the methodology were investigated, such as fiber, extraction time, extraction temperature, and sample volume. According to the results obtained (data not shown), optimized SPME experimental conditions were established, i.e., 30 min of extraction time, and a sample volume of 5 g. It is worth pointing out that the volatile compounds in tea infusions were very likely artifacts formed during the heat impact. Therefore, the extraction temperature was set at 25 °C. Then 5 g portions of the treated samples were transferred to the vials and sealed with a Teflon cover. The vials were kept at 25 °C on a water bath for 30 min with shaking at regular intervals. After the SPME fiber had been exposed in the upper space of the bottle for 30 min, it was then withdrawn and directly introduced to the GC injector for desorption and analysis. Calibration of Standard Curves. To obtain a matrix similar to that of tea infusion, model solution was prepared containing 25 mg/g caffeine, 5 mg/g (+)-catechin (C), 5 mg/g (−)-epicatechin (EC), 10 mg/g (−)-epicatechin gallate (ECG), 20 mg/g (−)-epigallocatechin (EGC), 10 mg/g (−)-gallocatechin gallate, 40 mg/g (−)-epigallocatechin gallate (EGCG), 4 mg/g theanine, 0.8 mg/g Glu, 0.5 mg/g Pro, 0.5 mg/g Val, and 0.5 mg/g Ser in Milli-Q deionized water.6,13,14 A standard stock solution was prepared containing 50 mg/kg methanethiol, 5 mg/kg ethanethiol, 2 mg/kg propanethiol, 1500 mg/ kg dimethyl sulfide, 10 mg/kg dimethyl trisulfide, 150 mg/kg dimethyl sulfoxide, 10 mg/kg dimethyl disulfide, 0.1 mg/kg 3-mercaptohexanol, 1 mg/kg S-methyl thioacetate, 2 mg/kg benzothiazole, 1 mg/kg methionol, 2 mg/kg methional, and 0.1 mg/kg furfurylthiol in Milli-Q deionized water. The standard stock solution was then diluted with water to 1:2, 1:10, 1:20, 1:50, and 1:200 strengths. In order to construct the calibration curves, 0.01 g of each of those diluted solutions of sulfur compounds and 0.01 g of the internal standard solution containing 2 mg/kg of dipropyl disulfide were introduced to the 5 g of model solution in a 20 mL vial, which was extracted by SPME, as was performed for the tea infusions. The standard curves were shown in the research, where y represented the peak area ratio (peak area of sulfur volatile standard/peak area of internal standard) and x represented the concentration ratio (concentration of sulfur volatile standard/
concentration of internal standard). The calibration curves were obtained from Chemstation software (Agilent Technologies Inc.) and used for calculation of volatiles in tea infusions. Quantitation of other volatile compounds was carried out by standard curves obtained by each compound from six different concentrations in water with the internal standard of 2-octanol (0.01 g of 5 mg/kg). GC−MS Identification of Aroma Compounds. The analyses were performed using a Hewlett-Packard 7890 GC with a 5975 mass selective detector (MSD) (Agilent Technologies) instrument operating under electron ionization (EI) mode (70 eV, ion source temperature 230 °C) with the quadrupole in a scanning mode (scan range was m/z 30−450 at a scan rate of 1 scan/s). Separation of compounds was achieved on Innowax-Wax (60 m × 0.25 mm i.d. × 0.25 μm film thickness, Agilent Technologies) and DB-5 (60 m × 0.25 mm i.d. × 0.25 μm film thickness, Agilent Technologies). Helium (purity = 99.999%) was used as a carrier with a constant flow velocity of 1 mL/min. The quadrupole mass filter was at 150 °C. The oven temperature was held at 40 °C for 6 min, then ramped to 100 °C at the rate of 2 °C/min, and ramped at the rate of 5 °C/min to 230 °C for the last 10 min. The volatile compounds were identified by comparing retention indices and authentic standards, or with mass spectra in the Wiley and NIST11 libraryies. The RIs were determined via sample injection with a homologous series of alkanes (C5−C30) (Sigma-Aldrich). Gas Chromatography−FPD. Gas chromatography equipped with a 5380 FPD detector from Agilent (Santa Clara, CA) was used in the sulfur mode. Separation of compounds was achieved on Innowax-Wax (60 m × 0.25 mm i.d. × 0.25 μm film thickness, Agilent Technologies, USA) and DB-5 (60 m × 0.25 mm i.d. × 0.25 μm film thickness, Agilent Technologies, USA). The oven temperature was held at 40 °C for 6 min, then ramped to 100 °C at the rate of 2 °C/min, and ramped at the rate of 5 °C/min to 230 °C for the last 10 min. GC was operated in a constant flow mode (1 mL/min) with helium as the carrier gas. The FPD detector was set at 250 °C, and the photomultiplier tube (PMT) voltage was set at 500 V. The sulfurcontaining compounds were confirmed by comparison with authentic standards on both columns, RI value matching, and aroma descriptor. The quantification method was identical to the GC analysis as described in the above section. The samples were run in triplicate. GC−Olfactometry Analysis. GC−olfactometry analysis was carried out on an Agilent 6890N GC equipped with flame ionization detection (FID) system and an olfactometer (Gerstel, Inc., Baltimore, MD). The Innowax (60 m × 0.25 mm i.d. × 0.25 μm film thickness, Agilent Technologies, USA) and DB-5 (60 m × 0.25 mm i.d. × 0.25 μm film thickness, Agilent Technologies, USA) were used. The oven temperature was held at 40 °C for 6 min, then ramped to 100 °C at the rate of 2 °C/min, and ramped at the rate of 5 °C/min to 230 °C for the last 10 min. The column effluent was split 1:1 into the FID and the heated sniffing port (250 °C) using two deactivated and uncoated fused silica capillaries. The column carrier gas was nitrogen at a constant flow rate of 2 mL/min. A panel of ten trained panelists (5 males and 5 females) evaluated the effluents enriched with purified, humidified air (120 mL/min). Each of the panelists was trained before sensory analysis in order to get familiar with an odor description by using solutions of artificial odorants. The aroma-active compounds perceived by ten panelists were recorded as the time for onset and end while sniffing the effluent from the sniffing mask. The panelists were also required to note the perceived odor characteristic and intensity. With the same time, the computer automatically recorded the retention time and sniffing time of aroma-active compound individually. The odor intensities were evaluated using a 6-point intensity scale from 0 to 5; “0” was none, “3” was moderate, and “5” was extreme. The experiment was replicated in triplicate by each panelist. Finally, the aroma intensity was the average from ten panelists. Odor Activity Values (OAV). The contribution of each odor to the overall fruit wine aroma was evaluated by the odor activity value, which was measured as the ratio of the concentration of each compound to its detection threshold. The threshold values were taken from information available in the references. 7500
DOI: 10.1021/acs.jafc.5b02358 J. Agric. Food Chem. 2015, 63, 7499−7510
Article
Journal of Agricultural and Food Chemistry Table 1. GC−O Identified Aroma-Active Compounds in Tea Infusions with the Method of Aroma Intensity RIb no.a 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 59
name methanethiol acetaldehyde dimethyl sulfide propanal 2-methylpropanal butanal 3-methylbutanal 2-methylbutanal pentanal 2,3-butanedione 1-penten-3-one 2,3-pentanedione dimethyl disulfide hexanal (E)-2-pentenal 1-penten-3-ol unknown1 heptanal (E)-2-hexenal (Z)-4-heptenal 1-pentanol octanal 2,5-dimethylpyrazine (E)-2-heptenal 2,3-octanedione 6-methyl-5-hepten-2-one unknown2 hexanol (Z)-3-hexen-1-ol nonanal 2,3,5-trimethylpyrazine (E,E)-2,4-hexadienal (E)-2-octenal cis-linaloloxide 1-octen-3-ol furfurylthiol unknown3 furfural methional trans-linalooloxide (E,E)-2,4-heptadienal 2-acetylfuran benzaldehyde (R)-(−)-linalool dimethyl sulfoxide 5-methyl-2-furfural unknown4 hotrienol phenyl acetaldehyde methionol methyl salicylate unknown5 β-damascenone geraniol 3-mercaptohexanol phenylethyl alcohol β-ionone benzothiazole cis-jasmone
Innowax 696 714 716 750 821 832 910 912 935 970 973 1054 1071 1084 1131 1157 1165 1174 1192 1230 1255 1280 1302 1315 1320 1336 1345 1360 1378 1385 1395 1405 1411 1420 1426 1432 1443 1455 1458 1465 1470 1490 1495 1537 1576 1598 1611 1623 1625 1723 1745 1776 1813 1847 1875 1925 1932 1950 1959
DB-5 502
