Identification of the Key Odorants in Tahitian Cured Vanilla Beans (Vanilla Tahitensis) by GC-MS and an Aroma Extract Dilution Analysis

Biosci. Biotechnol. Biochem., 77 (3), 601–605, 2013

Identification of the Key Odorants in Tahitian Cured Vanilla Beans (Vanilla tahitensis) by GC-MS and an Aroma Extract Dilution Analysis

y Makoto TAKAHASHI, Yoko INAI, Norio MIYAZAWA, Yoshiko KUROBAYASHI, and Akira FUJITA

Technical Research Institute, R&D Center, T. Hasegawa Co., Ltd., 29-7 Kariyado, Nakahara-ku, Kawasaki, Kanagawa 211-0022, Japan

Received October 30, 2012; Accepted November 28, 2012; Online Publication, March 7, 2013 [doi:10.1271/bbb.120840]

The key odorants of Tahitian vanilla beans (Vanilla chocolate, and confectionery by the flavor industry,3) tahitensis) were characterized by a sensory evaluation, and in luxury applications such as gastronomy and aroma extract dilution analysis (AEDA), quantification, perfumery.4) and aroma reconstitution. Vanillin and anisaldehyde The aroma composition of Tahitian vanilla beans were identified in the same highest flavor dilution (FD) (Vanilla tahitensis) has been extensively analyzed by factor as the most characteristic odor-active compounds gas chromatography (GC) and high-performance liquid in Tahitian vanilla beans, followed by anisyl alcohol and chromatography (HPLC), and more than 200 com- anisyl acetate. Vanillin and anisyl alcohol were by far pounds have been found to be responsible for the flavor the most abundant odorants present with the highest profile of Tahitian vanilla beans.4,5) Lepters-Andrzejew- concentration in the beans, followed by acetic acid, ski et al. have reported Vanilla tahitensis to be different anisaldehyde, and anisyl acetate. A sensory evaluation from Vanilla planiforia in its aroma composition, with a of Tahitian vanilla beans and its reconstitute aroma smaller contribution of vanillin to the overall flavor, and concentrate characterized both samples as similar. to contain more aroma compounds than Vanilla plani- These results indicated vanillin, anisaldehyde, anisyl foria, particularly with anisyl compounds.6) alcohol, and anisyl acetate to be the key odorants While identifying odor-active compounds of Tahitian in Tahitian vanilla beans. 3-Methylnonane-2,4-dione vanilla beans would be important to describe its were identified for the first time in vanilla beans. characteristic aroma profile, it seems surprising that -Damascenone and phenylacetic acid were identified only two studies, using gas chromatography-olfactom- for the first time in Tahitian vanilla beans. etry (GC-O), have been focused on the odor-active compounds in Vanilla planiforia7,8) and only one study Key words: Tahitian vanilla bean (Vanilla tahitensis); focused on the odor-active compounds in Vanilla aroma extract dilution analysis (AEDA); tahitensis. The study performed by Brunschwig et al. vanillin; anisyl compound; quantification on the odor-active compounds of Tahitian vanilla beans by applying combined hedonic of aromatic response Vanilla is one of the world’s most popular flavoring measurement (CHARM) and odor specific magnitude materials applied by the food, confectionery, beverage, estimation (OSME) analyses has shown anisaldehyde perfume, and pharmaceutical industries. Commercial and guaiacol to be the key aroma compounds of Tahitian vanilla beans are the cured, dried, and conditioned pods vanilla beans among 38 odor-active components; these of fully mature fruit of the orchid genus Vanilla were representative of the predominant ‘‘spicy-anise’’ indigenous to Mexico.1) Although 110 species of vanilla and ‘‘phenolic’’ odor families. However, simultaneous are known, only 3 of them are commercially traded distillation extraction (SDE) was performed by isolating today: i) Vanilla planiforia, primarily cultivated in the vanilla bean aroma in hot water at 100 C for 2 h, and Madagascar, where the most abundant cured vanilla the CHARM and OSME analyses were performed by beans are produced, Indonesia, Uganda, India, Comoro using only seven limited attributes.4) Moreover, there Islands and Mexico, and traded all over the world; ii) have been no studies reported on the odor-active Vanilla pompona cultivated and used in Central America compounds of Tahitian vanilla beans by quantification where only a small amount of cured vanilla beans are and aroma reconstitution. produced and traded; and iii) Vanilla tahitensis culti- In this present study, we first compared Tahitian vated mainly in Tahiti island of French Polynesia, where vanilla beans to Madagascar vanilla beans to clarify the only a small amount of cured vanilla beans is produced, aroma profile by a sensory evaluation. A Tahitian vanilla and in Papua New Guinea, and traded in the global bean aroma concentrate for GC-O was prepared by market.2) Vanilla tahitensis has a unique aroma and can direct solvent extraction and distilled under reduced therefore be used as an ingredient for ice cream, pressure to extract the natural and overall aroma

y To whom correspondence should be addressed. Tel: +81-44-411-0134; Fax: +81-44-411-8353; E-mail: makoto [email protected] Abbreviations: AEDA, aroma extract dilution analysis; ANOVA, analysis of variance; CF, correction factor; CHARM, combined hedonic of aromatic response measurement analysis; df, degree of freedom; EI, electron impact; FD, flavor dilution; FID, flame ionization detector; GC, gas chromatography; GC-MS, gas chromatography-mass spectrometry; GC-MS-O, gas chromatography-mass spectrometry-olfactometry; GC-O, gas chromatography-olfactometry; HPLC, high-performance liquid chromatography; IS, internal standard; LSD, least significant difference; MCT, medium chain triglyceride; MS, mass spectrum; ms, mean square; OSME, odor specific magnitude estimation; RI, retention index; SDE, simultaneous distillation extraction; SEM, standard error measurement; SS, sum of squares 602 M. TAKAHASHI et al. components without changing the original aroma com- Linear retention indices (RIs) of the compounds were calculated from ponents during extraction and distillation. The odor- the retention times of n-alkanes. active compounds in Tahitian vanilla beans were then identified, quantified and reconstituted by using gas Gas chromatography-mass spectrometry-olfactometry (GC-MS-O). chromatography-mass spectrometry (GC-MS) and an The GC-MS-O analysis was performed with an Agilent 6890 GC combined with a 5973 mass selective detector and a sniffing port aroma extract dilution analysis (AEDA) to identify the equipped with a TC-WAX capillary column (0.25 mm i.d. 60 m). overall odor-active compounds in the vanilla beans. The effluent of the column at the end of the capillary was divided into two branches and respectively routed by deactivated fused silica Materials and Methods capillaries to the mass detector and sniffing port. The other conditions were the same as those applied for the GC-MS method just described. Plant materials. 14–16-cm Tahitian cured vanilla beans (Vanilla The odor-active volatiles were each identified by the agreement of their tahitensis) with 45% moisture which had been cultivated on Tahiti RI, MS, and odor quality data with those of the reference odorant in island in French Polynesia, and 13–16-cm Madagascar cured vanilla our laboratory. The GC-MS-O analysis was performed twice by three beans ‘‘red whole’’ (Vanilla planiforia) with a reddish chocolate brown experienced assessors. color and 25% moisture were purchased from the Dammann commercial source in Oakland (NJ, USA). AEDA. Flavor dilution (FD) factors for the odor-active compounds were determined by the AEDA method.10) The aroma concentrate of Chemicals. Standard compounds of acetic acid, anisaldehyde, Tahitian vanilla beans was diluted stepwise with diethyl ether to obtain 1 9 guaiacol, 3-methylbutanoic acid, 2-methylbutanoic acid, p-cresol, dilutions in a ratio range of 1:5 to 1:5 . Each dilution was analyzed by ethyl (E)-cinnamate, methyl (E)-cinnamate, eugenol, anisyl alcohol, the GC-MS-O system equipped with a TC-WAX capillary column. phenylacetic acid, vanillin, 3-phenylpropanoic acid, isovanillin, anet- hole, 2-methylpentanoic acid, 1,2-dimethoxybenzene and (2E,4E)- Sensory evaluation. deca-2,4-dienal (Sigma-Aldrich Japan, Tokyo, Japan); -damascenone Vanilla beans. Tahitian cured vanilla beans, these being the same (Firmenich, Meyrin, Switzerland); 4-vinylguaiacol (Wako Pure Chem- sample as that used for GC-MS and AEDA, and Madagascar cured icals, Osaka, Japan); anisyl acetate and methyl 2-phenylethyl ether vanilla beans ‘‘red whole’’ were cut into 1-cm pieces, frozen with (Tokyo Chemical Industry, Tokyo, Japan); and the emasol O-120V liquid nitrogen, and then ground into a fine powder in an AM-10 emulsifying agent (Kao, Tokyo, Japan) were used. 3-Methylnonane- laboratory mill (Nihonseiki Kaisha). Ten grams of each ground powder 2,4-dione was synthesized in our laboratory by the method reported in was directly put into a closed 30-mL sensory vial. the literature.9) All reagents and solvents were of analytical grade. Reconstituted aroma concentrate. Tahitian vanilla beans and a reconstituted Tahitian vanilla bean aroma concentrate were used as the Isolation of the volatiles. Tahitian vanilla beans were cut into 1-cm test samples evaluated by 11 panelists. The reconstituted Tahitian vanilla bean aroma concentrate was prepared by mixing the following pieces, frozen with liquid nitrogen, and then ground into a fine powder in an AM-10 laboratory mill (Nihonseiki Kaisha, Tokyo, Japan). Thirty 13 reference compounds: vanillin (317 mg), anisyl alcohol (315 mg), grams of the ground powder was extracted with diethyl ether acetic acid (73.8 mg), anisaldehyde (13.2 mg), anisyl acetate (2.51 mg), E (3 60 mL) at room temperature for 1 min, then filtered, and the guaiacol (1.98 mg), isovanillin (577 mg), methyl ( )-cinnamate (247 mg), p-cresol (78.9 mg), phenyl acetic acid (20.3 mg), 3-phenyl- filtrate was dried over Na2SO4 and concentrated to about 10 mL by E distilling off the solvent in a Vigreux column at 43 C. The volatiles in propanoic acid (15.3 mg), eugenol (6.98 mg) and ethyl ( )-cinnamate the concentrate were distilled at under 500 Pa at room temperature (1.79 mg) at concentrations based on the quantified data for the odor- active compounds (FD 125) in Tahitian vanilla beans. To the which was raised to 80 C in 20 min and then maintained at 80 C for mixture was then added 25 g of water, 1 g of emasol O-120V, and 10 min. The distillate was dried over Na2SO4 and concentrated to approximately 50 mg. medium chain triglyceride (MCT) to a total weight volume of 101 g, before mixing thoroughly. Evaluation attributes. To determine suitable terms to describe the Quantification of the volatiles. To accurately determine the aroma before the sensory evaluation, the panelists were presented with concentration of the peaks and to confirm the reproducibility of the Tahitian and Madagascar vanilla beans, together with a list of 44 aroma concentrate, an internal standard solution (300 mg) prepared descriptors.11) They collaboratively discussed, generated, and modified from anethole (6240 ppm), 2-methylpentanoic acid (172 ppm), methyl the descriptive terms after evaluating the vanilla beans. Consensus was 2-phenylethyl ether (6.47 ppm) and 1,2-dimethoxybenzene (177 ppm) reached after several consultations on seven attributes to be used as in methylene chloride was added to the ground vanilla beans, before descriptors for evaluating the vanilla bean aroma. These attributes were solvent extraction. The volatiles were then isolated as just described. floral, dried fruit-like, resinous, hay-like, metallic, phenolic, and sweet. Vanillin, anisyl alcohol, anisaldehyde and guaiacol in the concentrate Evaluation conditions. The panel consisted of 9 male and 4 female were quantified before distillation, and the other compounds were employees of the Technical Research Institute of T. Hasegawa Co., quantified after distillation. Calibration curves were constructed by Ltd. (Kawasaki, Japan). They had been trained to recognize and using a mixture of authentic samples of the odor-active compounds and quantify aromas of about 100 aroma chemicals and raw materials. Each internal standards. The absolute concentrations of the odor-active panelist was presented with a set of test samples coded by a random components in vanilla beans were determined by the ratio of the three-digit number. They were instructed to sniff each sample and rate percentage peak area of each compound to the internal standard and the intensities of the seven attributes on a seven-point linear scale from from calibration curves of the authentic samples. Extractions was 1 (none) to 7 (very strong). The evaluation was conducted in a quiet performed in triplicate. room kept at 23 C. Two booths were set up in the room, and each panelist was asked to perform the assessment in a separate booth to GC-MS. The GC-MS analysis was performed with an Agilent 6890 eliminate any influence from the other. GC combined with a 5973 mass selective detector and a flame Statistical analysis. Differences between the average scores for i) ionization detector (FID; 250 C) equipped with a TC-WAX capillary Tahitian vanilla beans and Madagascar vanilla beans ‘‘red whole’’ and column (0.25 mm i.d. 60 m; GL Sciences Co., Tokyo, Japan). The ii) the reconstituted aroma concentrate of Tahitian vanilla beans and effluent from the column at the end of the capillary was divided into the Tahitian vanilla beans were compared by an analysis of variance two branches and respectively routed by deactivated fused silica (ANOVA) and Tukey’s multiple comparison test. For ANOVA, the capillaries to the mass detector and FID. Each 1-mL sample was correction factor (CF), sum of squares (SS), degree of freedom (df), injected in the split mode (50:1) at a constant temperature of 250 C. mean square (ms), and F-value were calculated for each attribute The oven temperature was kept at 40 C for the initial 3 min and then (p < 0:05, and 0.01). For Tukey’s multiple comparison test, the increased to 230 C at a rate of 3 C/min under a constant helium standard error measurement (SEM) and least significant difference carrier gas flow of 1.8 mL/min. The mass spectrum (MS) in the (LSD) were calculated for each attribute (p < 0:05). The results were electron impact (EI) mode was recorded at 70 eV ionization energy. averaged for each attribute and plotted on a spider web diagram. Identification of the Key Odorants in Tahitian Vanilla Beans 603 Table 1. Sensory Evaluation of Vanilla Beans Floral ** 7 Average scorea Attribute 6 Tahitianb Madagascarc Sweet 5 Dried fruit−like * 4 Floral 6.1 3.4 3 Dried fruit-like 4.5 5.7 2 Resinous 4.4 5.8 1 Hay-like 3.7 4.3 Metallic 3.8 3.5 Phenolic Resinous** Phenolic 4.2 4.0 Sweet 4.3 5.5

a n ¼ 13. Asterisks on the right side of scores denote significant differences Metallic Hay−like by ANOVA ( p < 0:05 and p < 0:01) and Tukey’s multiple-comparison p < : test ( 0 05). Values correspond to those shown in Fig. 1. Fig. 1. Aroma Profile of the Tahitian Cured Vanilla Beans Compared bTahitian cured vanilla beans with Madagascar Cured Vanilla Beans: ( ) Tahitian Cured Vanilla cMadagascar cured vanilla beans ‘‘red whole’’ Beans and ( ) Madagascar Cured Vanilla Beans. Asterisks indicate significant differences between the samples Results and Discussion ( p < 0:05, p < 0:01).

Aroma profile of Tahitian and Madagascar vanilla which had not previously been reported to be an odor- beans active compound,4) was identified as the most odor- The characteristic aroma of Tahitian vanilla beans active with the same highest FD factor of 1953125. was compared with that of Madagascar vanilla beans Guaiacol had previously been reported to be the most which is universally known for its vanilla aroma. We odor-active compound in Tahitian vanilla beans,4) first evaluated the seven sensory attributes (floral, dried although its FD factor was much lower than that of fruit-like, resinous, hay-like, metallic, phenolic, and anisyl alcohol or anisyl acetate. Although odor-active sweet). The average scores for each attribute are shown compounds using only seven limited attributes were in Table 1 and plotted on a spider web diagram in identified by GC-MS, GC-O and the CHARM analy- Fig. 1. Since there were no significant differences in the sis,4,5,8) using the AEDA method in this study enabled us hay-like, metallic, phenolic, and sweet notes between to confirm the overall key odorants. These compounds the two vanilla bean samples, we characterized these as constituted 90% of the total aroma concentrate. Part of the basic aroma of cured vanilla beans. The aroma of the aroma concentrates could not be fully recovered Tahitian vanilla beans, however, was significantly more during the distillation process, so we tried to quantify the floral (p < 0:01), less resinous (p < 0:01) and less dried amount by using the above-mentioned method. It is of fruit-like (p < 0:05) than that of Madagascar vanilla interest from the results of this study that -damasce- beans. We concluded from these results, that the aroma none, which is a well-known key odorant in rose,12) of Tahitian vanilla beans was predominantly floral, wine13) and black tea14) was identified for the first time whereas that of Madagascar vanilla beans was resinous in Tahitian vanilla beans. 3-Methylnonane-2,4-dione and dried fruit-like. was identified for the first time in vanilla beans, and phenylacetic acid was also identified for the first time in Odor-active compounds of Tahitian vanilla beans Tahitian vanilla beans. AEDA applied to the aroma concentrate extracted and distilled from Tahitian vanilla beans revealed 20 odor- Quantitative analysis of the odor-active compounds in active areas with an FD factor of more than 25. The vanilla beans odor-active volatiles identified with an FD factor range The results of AEDA enabled 13 compounds of more than 25 are summarized in Table 2, 19 of these (FD = 125) to be quantified by using four internal compounds being unambiguously identified by the standards (anethole, 2-methylpentanoic acid, methyl 2- agreement of their RI, MS, and odor quality data with phenylethyl ether, and 1,2-dimethoxybenzene) whose RI those of the standard odorants in our laboratory. Vanillin values were different. The results for the quantification (sweet) and anisaldehyde (anise-like) had the highest FD are summarized in Table 3. Vanillin with a concentra- factor (FD-1953125) and were considered to be the most tion of 3.17 g/kg (FD-1953125) and anisyl alcohol with characteristic odor-active compounds. The other odor- a concentration of 3.15 g/kg (FD-390625) were by far active compounds with an FD factor higher than 390625 the most abundant odorants among the odor-active and 15625 were respectively anisyl alcohol (floral) and compounds with the highest concentration of all volatile anisyl acetate (floral). Acetic acid (acidic), guaiacol compounds of Tahitian vanilla beans identified by GC- (phenolic), methyl (E)-cinnamate (fruity), p-cresol MS, being followed by acetic acid (FD-625), anisalde- (fecal), ethyl (E)-cinnamate (fruity), eugenol (clove- hyde (FD-1953125), anisyl acetate (FD-15625), and like), phenylacetic acid (honey-like), 3-phenylpropanoic guaiacol (FD-125) in respective concentrations of acid (buttery), and isovanillin (phenolic) were identified 738 mg/kg, 132 mg/kg, 25.1 mg/kg, and 19.8 mg/kg. with the FD factors between 125 and 625. Anisaldehyde Comparatively high concentrations were also found for has been found to be the most odor-active compound in isovanillin (5.77 mg/kg), methyl (E)-cinnamate (2.47 an AEDA study and agreed with the CHARM analysis.4) mg/kg), p-cresol (0.789 mg/kg), phenylacetic acid Anisyl alcohol and anisyl acetate, both reported as odor- (0.203 mg/kg), and 3-phenylpropanoic acid (0.153 active compounds of Tahitian vanilla beans,4) were also mg/kg). Such components as eugenol (69.8 mg/kg) and found to contribute to the aroma character. Vanillin, ethyl (E)-cinnamate (17.9 mg/kg) were present in low 604 M. TAKAHASHI et al. Table 2. Odor-Active (FD = 25) Volatiles in Tahitian Cured Vanilla Beans

Odoranta Odor qualityb RI FD factor Identification modec Acetic acid acidic, sour 1430 625 MS, RI, GC-O 2-Methylbutanoic acid buttery, cheese-like 1691 25 MS, RI, GC-O 3-Methylbutanoic acid buttery, cheese-like 1693 25 MS, RI, GC-O 3-Methylnonane-2,4-dioned floral, medicinal 1739 25 RI, GC-O (2E,4E)-deca-2,4-dienald oily 1816 25 RI, GC-O -Damascenone raisin-like, fruity 1826 25 MS, RI, GC-O Guaiacol phenolic, medicinal 1863 125 MS, RI, GC-O Anisaldehyde anise-like, raspberry-like 2052 1953125 MS, RI, GC-O Methyl (E)-cinnamate fruity, cinnamon-like 2083 125 MS, RI, GC-O p-Cresol fecal 2084 125 MS, RI, GC-O Anisyl acetate floral, raisin-like 2132 15625 MS, RI, GC-O Ethyl (E)-cinnamate cinnamon-like, fruity 2145 125 MS, RI, GC-O Unknown cooked, meaty 2167 25 GC-O Eugenol clove-like, spicy 2169 125 MS, RI, GC-O 4-Vinylguaiacol phenolic, spicy 2207 25 MS, RI, GC-O Anisyl alcohol floral, anise-like 2276 390625 MS, RI, GC-O Phenylacetic acid buttery, honey-like 2512 125 MS, RI, GC-O Vanillin sweet, vanilla-like 2604 1953125 MS, RI, GC-O 3-Phenylpropanoic acid metallic, buttery 2672 125 MS, RI, GC-O Isovanillin phenolic, medicinal 2718 125 MS, RI, GC-O

aEach odorant was identified by the agreement of its mass spectrum, retention index, and odor quality with the reference odorant. bOdor quality perceived at the sniffing port. cMS, reference mass spectrum; RI, reference retention index; GC-O, gas chromatography coupled with olfactometry dTentatively identified odorant

Table 3. Concentration of Odor-Active (FD = 125) Volatiles in Table 4. Sensory Evaluation of Tahitian Cured Vanilla Beans and Tahitian Cured Vanilla Beans Reconstituted Aroma Concentrate

Mean Average scorea SDa Odorant concentration (mg/kg) Attribute Reconstituted aroma (mg/kg) Beansb concentratec Vanillinb 3170000 304000 Floral 4.9 4.3 Anisyl alcoholb 3150000 133000 Dried fruit-like 4.8 4.2 Acetic acidc 738000 90900 Resinous 4.2 3.7 Anisaldehydeb 132000 18700 Hay-like 4.6 3.6 Anisyl acetatee 25100 877 Metallic 3.5 3.6 Guaiacolb 19800 693 Phenolic 3.7 3.4 Isovanilline 5770 1270 Sweet 3.9 4.1 Methyl (E)-cinnamatee 2470 224 p-Cresole 789 33.3 an ¼ 11. Both samples were not significantly different in any attributes. Phenylacetic acidd 203 23.8 bTahitian cured vanilla beans 3-Phenylpropanoic acidd 153 24.9 cReconstituted aroma concentrate of Tahitian cured vanilla beans Eugenold 69.8 6.38 Ethyl (E)-cinnamated 17.9 1.83 beans contained moisture. The reconstituted aroma aStandard deviation (SD) calculated from quantitative data obtained from concentrate and the vanilla beans themselves were then three different samples. compared by using seven aroma attributes and evaluated bAnethole was used as the internal standard. c2-Methylpentanoic acid was used as the internal standard. for the overall similarity. The results show strong dMethyl 2-phenylethyl ether was used as the internal standard. similarity in all attributes between the samples (Table 4). e1,2-Dimethoxybenzene was used as the internal standard. The results for the FD factors, quantification, and sensory evaluation of the reconstituted aroma concentrate show amounts. The concentrations of the five odor-active that the Tahitian vanilla bean aroma could be recon- compounds with the highest FD factor range of 625- stituted by using the quantified data for 13 odor-active 1953125, vanillin, anisaldehyde, anisyl alcohol, anisyl volatiles identified by AEDA in the beans. acetate, and acetic acid, were also higher than the other The foregoing findings show that vanillin, anisalde- eight odor-active compounds with FD-125. hyde, anisyl alcohol, and anisyl acetate, which were the odor-active compounds within the highest FD factor Reconstituted aroma concentrate of Tahitian vanilla range of 15625-1953125, were the key odorants in beans Tahitian vanilla beans. To investigate the effect of the odor-active compounds in Tahitian vanilla beans, we prepared a reconstituted References aroma concentrate of Tahitian vanilla beans composed of 13 odor-active volatiles identified by AEDA. We mixed 1) Rain P and Lubinsky P, ‘‘Vanilla,’’ eds. Odoux E and Grisoni M, CRC Press, Boca Raton, pp. 251–259 (2011). the compounds at concentrations based on the quantified 2) Ranadive AS, ‘‘Handbook of Vanilla Science and Technology,’’ data for the Tahitian vanilla beans. Such solvents as eds. Havkin-Frenkel D and Belanger FC, Wiley-Blackwell, MCT, water and emasol O-120V were used, since the Chichester, pp. 141–161 (2011). Identification of the Key Odorants in Tahitian Vanilla Beans 605 3) Ehlers D, Pfister M, and Bartholomae S, Z. Lebensm. Unters. 8) Dignum MJW, Van der Heiden R, Kerler J, Winkel C, and Forsch., 199, 38–42 (1994). Verpoorte R, Food Chem., 85, 199–205 (2004). 4) Brunschwig C, Senger-Emonnot P, Aubanel ML, Pierrat A, 9) Naef R, Jaquir A, Velluz A, and Maurer B, J. Agric. Food George G, Rochard S, and Raharivelomanana P, Food Res. Int., Chem., 54, 9201–9205 (2006). 46, 148–157 (2012). 10) Schieberle P, ‘‘Characterization of Food: Emerging Methods,’’ 5) Da Costa NC and Pantini M, ‘‘Flavour Science Recent ed. Gaonkar AG, Elsevier, Amsterdam, pp. 403–431 (1995). Advances and Trend,’’ eds. Bredie WLP and Petersen MA, 11) Shimoda M, Sasaki H, Doi Y, Kameda W, and Osajima Y, Elsevier, Amsterdam, pp. 161–164 (2006). Nippon shokuhin kagaku kougaku kaishi (in Japanese), 36, 7–25 6) Lepters-Andrzejewski S, Brunschwing C, Collard F, and Dron (1989). M, ‘‘Vanilla,’’ eds. Odoux E and Grisoni M, CRC Press, Boca 12) Kovats ES, J. Chromatogr., 406, 185–222 (1987). Raton, pp. 205–228 (2011). 13) Pineau B, Barbe JC, Leeuwen CV, and Dubourdieu D, J. Agric. 7) Perez-Silva A, Odoux E, Brat P, Ribeyre F, Rodrigues-Jimenes Food Chem., 55, 4103–4108 (2007). G, Robles-Olvera V, Garcia-Alvarado MA, and Gunata Z, Food 14) Schuh C and Shieberle P, J. Agric. Food Chem., 54, 916–924 Chem., 99, 728–735 (2006). (2006).