Decoding the Combinatorial Aroma Code of a Commercial Cognac by

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Decoding the Combinatorial Aroma Code of a Commercial Cognac by Application of the Sensomics Concept and First Insights into Differences to a German Brandy Verena Uselmann, and Peter Schieberle J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf506307x • Publication Date (Web): 31 Jan 2015 Downloaded from http://pubs.acs.org on February 6, 2015

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Journal of Agricultural and Food Chemistry

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Decoding the Combinatorial Aroma Code of a Commercial Cognac by Application of the Sensomics Concept and First Insights into Differences from a German Brandy

Verena Uselmann and Peter Schieberle#

Deutsche Forschungsanstalt für Lebensmittelchemie, Lise-Meitner-Straße 34, D-85354 Freising, Germany

#

Corresponding author:

Tel.: +49 8161 71 2932 Fax: +49 8161 71 2970 e-mail: [email protected]

ACS Paragon Plus Environment


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ABSTRACT. In the volatile fraction isolated from a commercial Cognac by means of

2

extraction/SAFE distillation, 39 odor-active areas were detected among which (E)-β-

3

damascenone showed the highest Flavor-Dilution (FD) factor of 2048 followed by 2-

4

and

5

methylpropanoate and ethyl (S)-2-methylbutanoate, as well as 4-hydroxy-3-methoxy-

6

benzaldehyde (vanilla-like) and 2-phenylethanol. The quantitation of 37 odorants by

7

stable isotope dilution assays, and a calculation of odor activity values (OAV; ratio of

8

concentration to odor threshold) resulted in 34 odorants with OAVs > 1. Among them

9

(E)-ß-damascenone, methylpropanal, ethyl (S)-2-methylbutanoate, ethyl methyl-

10

propanoate and ethyl 3-methylbutanoate together with ethanol were established as

11

key contributors to the Cognac aroma. Finally, the overall aroma of the Cognac could

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be mimicked by an aroma recombinate consisting of these 34 key odorants on the

13

basis of their natural concentrations in the Cognac using an odorless matrix to

14

simulate the influence of the non-volatile constituents. A comparison of the FD

15

factors of the key odorants identified in a German brandy to those in the Cognac

16

suggested the pair (E)-ß-damascenone and ethyl pentanoate as indicators to

17

differentiate various Cognacs from German, French and Spanish brandies. This was

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confirmed by calculating a ratio of the concentrations of (E)-ß-damascenone to ethyl

19

pentanoate for 12 Cognacs and 7 brandies from Germany, and 2 from France and

20

Spain, respectively.

3-methylbutanol,

(S)-2-methylbutanol,

1,1-diethoxyethane,

ethyl

21 22

KEYWORDS. Cognac; aroma extract dilution analysis; stable isotope dilution

23

analysis, (E)-ß-damascenone; ethyl pentanoate

24 25


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INTRODUCTION

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Flavor is an important quality parameter of brandies commonly manufactured by

28

distilling a wine prepared from white grapes followed by aging the distillate in oak

29

barrels. Cognac is a specialty brandy, and within the EU, this name can only be used

30

for brandies from a defined region in France. According to the Bureau National

31

Interprofessionell du Cognac, the Cognac Delimited Region covers a large part of the

32

Charente department, all of the Charente-Maritime, and several districts of the

33

Dordogne and Deux-Sèvres. Furthermore, Cognac must be produced according to

34

the regulations of the AOC (appellation d´origine controlé).

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For more than 40 years, investigations on the volatile compounds of Cognac have

36

been performed. Reinhard1 already identified and quantitated 9 volatile compounds,

37

among them methylpropanol, 3-methylbutanol and 2-methylbutanol with the highest

38

concentrations. Later, Postel and Adam2 determined 53 volatile compounds in

39

Cognac, and reported that esters with 24 representatives were the largest group

40

among the entire set of volatiles followed by alcohols with 17 constituents. Schreier

41

et al.3 identified 139 volatile compounds in Cognacs and brandies, and ethyl phenol,

42

nerolidol, trans- and cis-linalool and γ-nonalactone were reported for the first time as

43

brandy constituents. Ledauphin et al.4 quantitated ca. 150 compounds in aged

44

Cognac using gas chromatography/mass spectrometry. By comparing Cognac with

45

other brandies produced in France they found the highest contents of furfural, 5-

46

methylfurfural, furfuryl ethyl ether and 2-acetylfuran in Cognac. According to a

47

previous review by de Rijke and Heide,5 a total of 546 volatile compounds were

48

known as Cognac constituents.

49

By applying the molecular sensory science approach on food aromas,6,7 it has

50

been shown for a considerable number of foods that not the entire set of volatiles

51

present in a food is able to interact with the human olfactory receptors, but only a ACS Paragon Plus Environment


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smaller number of the so called key odorants is selectively detected by the receptors

53

and is, thus, able to generate aroma perception in the brain.7 To separate such key

54

odorants from the bulk of odorless volatile compounds, GC/Olfactometry (GC/O),

55

aroma extract dilution analysis (AEDA), and a calculation of odor activity values in

56

the so-called Sensomics approach are the methods of choice.6 However, there are

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only a few studies available aimed at identifying the compounds responsible for the

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odor of Cognac8 or brandies from different origins, respectively.9,10 Ferrari et al.8

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detected 19 odor-active components in a distillate from Cognac by means of

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GC/Olfactometry. Odorants showing the highest nasal impact frequency, a method

61

similar to AEDA, were 2- and 3-methylbutanoic acid, 2- and 3-methylbutanol and

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ethyl hexanoate.

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To the best of our knowledge, there is no publication available successfully

64

identifying the key odorants of Cognac by a systematic approach using the

65

Sensomics concept6 and in particular by using exact quantitative data to perform a

66

final simulation of the overall aroma by means of an aroma recombinate. Therefore,

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the aim of the present study was to locate the potent odorants in an aroma extract

68

from a commercial Cognac by application of the aroma extract dilution analysis and

69

to identify the most odor active compounds. Aroma compounds with the highest

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Flavor Dilution (FD) factors should be quantitated using stable isotope dilution

71

assays, their odor activity values (ratio of concentration to odor threshold) should be

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calculated on the basis of their odor thresholds in water/ethanol, and the results

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should finally be confirmed by means of an aroma recombinate. In addition, an

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aroma extract dilution analysis was applied on a German brandy to find out

75

differences in the key odorants, which may be useful to distinguish Cognac from

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brandies.

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MATERIAL AND METHODS

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Cognac. According to the label, the commercial Cognac investigated (Hennessy)

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was labeled V.S. and had been stored in Limousin oak barrels for two years. This

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selection was not done for advertising purpose nor does it imply any research

82

contract with the manufacturer, but was done on the basis of an hedonic preference

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test performed by a group of 16 trained panelists (average age: 30 years; 60%

84

women). The grape variety used for the production of the Cognac was Ugni Blanc,

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and the product was manufactured according to the regulation of the AOC Cognac.

86

Several batches of the same production of the Cognac year were bought in a

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German supermarket. The German brandy (Mariacron) was selected because it

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showed the clearest difference in the overall aroma when compared to the Cognac.

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Reference odorants. These were obtained from the following commercial

90

sources:

4-allyl-2-methoxyphenol,

91

acetate, ethyl hexanoate, 4-ethyl-2-methoxyphenol, ethyl 3-methylbutanoate, ethyl

92

octanoate,

93

furanone,

94

methylbutanol,

95

phenylacetic acid, (3S,4S)-cis- and (3S,4R)-trans-whiskylactone, ethyl butanoate,

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ethyl methylpropanoate, ethyl pentanoate, ethyl propanoate, methylpropanol, (E)-2-

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nonanal, ethyl (S)-2-methylbutanoate, ethyl decanoate, octanoic acid, acetic acid, 2-

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methylbutanoic acid, 3-methylbutanoic acid, 2,3-butandione, (Z)-3-hexenol, ethyl 2-

99

hydroxy-3-methylbutanoate and 2-phenylethanol were from Sigma-Aldrich Chemie

100

(Taufkirchen, Germany). Acetaldehyde, ethanol, 4-hydroxy-3-methoxybenzaldehyde

101

and 2-methoxyphenol were from Merck (Darmstadt, Germany); 4-hydroxy-2,5-

102

dimethyl-3(2H)-furanone from SAFC (Hamburg, Germany). Cyclohexanoyl acetate

103

was supplied by Lancaster (Griesheim, Germany); 1-hexanol (Fluka, Buchs,

4-ethylphenol, ethyl

α-damascenone,

ethyl

2-phenylacetate, 3-hydroxy-4,5-dimethyl-2(5H)-

2-isopropyl-3-methoxypyrazine, 3-methylbutyl

1,1-diethoxyethane,

acetate,

methylpropanal,

(E)-2-nonenal,


3-methylbutanal,

3-

phenylacetaldehyde,

2-


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Germany); 4-vinyl-2-methoxyphenol was from Alfa Aesar, Karlsruhe, Germany,

105

hexanoic acid from Acros Organics, Geel, Belgium, and (E)-β-damascenone was a

106

gift from Symrise (Holzminden, Germany).

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Isotopically labeled internal standards. Most of the isotopically labeled internal

108

standards were synthesized as listed in a previous study:11 In addition, [2H2]-

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methylpropanol,12

110

pentanoate,15 [2H3]-hexanoic acid,16 [13C2]-3-hydroxy-4,5-dimethyl-2(5H)-furanone,17

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[2H2]-3-methylbutanoic acid,18 [2H2]-octanoic acid,19 and [13C2]-phenylacetaldehyde20

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were synthesized as previously reported. [13C2]-Phenylacetic acid was bought from

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Merck (Darmstadt; Germany).

[2H2]-(Z)-3-hexenol,13

[2H2]-butanoic

acid,14

[2H5]-ethyl

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Isolation of the volatiles. For volatile isolation, the spirit (25 mL) was diluted with

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brine, extracted with diethyl ether (3 x 50 mL) and the combined extracts were

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washed three times with brine (25 mL). Non-volatile compounds were separated by

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high vacuum distillation using the SAFE distillation,21 the distillate was washed with

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brine (3 x 25 mL) and concentrated to ~ 100 µL using a Vigreux column (60 cm x 1

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cm i.d.) followed by microdistillation.22 The distillate was separated into a fraction

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containing the neutral/basic volatiles (NBF) and the acidic volatiles (AF) by treatment

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with an aqueous sodium bicarbonate solution as previously described.22

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Fractionation of the neutral/basic volatiles by column chromatography. The

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neutral/basic fraction was concentrated to ~ 1 mL and was applied onto a water-

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cooled glass column (30 cm x 1 cm) filled with silica 60 (30 g) in n-pentane. Using six

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n-pentane/diethyl ether mixtures (v/v) of increasing polarity (100 mL each; fraction A:

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100:0; fraction B: 95:5; fraction C: 90:10; fraction D: 80:20; fraction E: 50:50; fraction

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F: diethyl ether) the compounds were separated and each fraction was concentrated

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to ~ 1 mL as described above. These fractions were used to re-locate the odor-active

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compounds for identification by GC/MS. ACS Paragon Plus Environment

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Gas

Chromatography/Flame

Ionization

Detector

(GC/FID)

and

Gas

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Chromatography/Olfactometry (GC/O). The analyses were performed by means of

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a gas chromatograph type 8000 (Fisons Instruments, Mainz, Germany) equipped

133

with the following capillaries: DB-5 (30 m x 0.25 mm i.d.; 0.25 µm film thickness)

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(Varian, Palo Alto, USA) and DB-FFAP (30 m x 0.32 mm i.d.; 0.25 µm film thickness)

135

(J&W Scientific, Folsom, USA). For GC/O, the end of the capillary was connected to

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a deactivated Y-shaped glass splitter dividing the effluent into two equal parts, which

137

were transferred via two deactivated but uncoated fused silica capillaries (50 cm x

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0.25 mm) to a sniffing port and an FID, respectively. The sniffing port was held at

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180 °C, while the temperature of the FID was at 240 °C; nitrogen was used as the

140

make up gas. Sample injection (0.5 µL) was done at 40 °C, and after 2 min, the

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temperature were raised at 6 °C per min to 230 °C and held for 10 min. All aroma

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active areas annotated by 3 panelists were marked in the chromatogram, and the

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odor qualities were described. For the original extracts, sniffing time was ~ 15 min;

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i.e. the entire chromatogram were sniffed 0-15 min, 15 to 30 min and 30 to 45 min.

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Linear retention indices (RI) of the compounds were calculated from the retention

146

times of a series of n-alkanes C-8 to C-24.

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Aroma Extract Dilution Analysis (AEDA). The distillate was stepwise diluted

148

with diethyl ether to obtain the following dilutions 1:1, 1:2, 1:4, 1:8, 1:16, etc., and

149

finally 1:2048 of the original extract.22 Each odorant was, thus, assigned an FD factor

150

representing the final dilution in which the odorant could be detected. Sniffing was

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continued until no odorant was detectable by GC/O. Odor qualities were assigned on

152

the basis of a flavor language previously developed in our group.23 The AEDA was

153

performed in parallel by at least three panelists, and the results were averaged.

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Gas-chromatography/Mass

Spectrometry

(GC/MS).

Mass

spectra

were

recorded by means of a gas chromatograph 5890 series II (Hewlett-Packard, ACS Paragon Plus Environment


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Waldbronn, Germany) connected to an MAT 95 S sector field mass spectrometer

157

(Finnigan, Bremen, Germany). Mass spectra in the electron ionization mode (MS-EI)

158

were recorded at 70 eV, and mass spectra in the chemical ionization mode (MS-CI)

159

were measured at 115 eV with isobutane as the reactant gas.

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Quantitation by Stable Isotope Dilution Assays in Combination with Two-

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Dimensional High Resolution Gas Chromatography (TDGC/MS). The labeled

162

internal standards (2 to 10 µg; depending on the amounts of the respective analyte

163

estimated in a preliminary trial) dissolved in diethyl ether (0.5 mL) were added to

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aliquots of the spirit. After addition of brine (50 mL) and diethyl ether (50 mL), the

165

solution was stirred for 1 h. The combined extracts were dried over anhydrous

166

sodium sulfate and concentrated to ~ 100 mL at 37 °C using a Vigreux column (50

167

cm x 1 cm ID). The non-volatile material was removed by SAFE distillation at 40 °C,

168

and the distillate was concentrated to 100 µL by means of microdistillation.22

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For TDGC/MS a gas chromatograph Trace 2000 (ThermoQuest, Mainz, Germany)

170

coupled via a moving column stream switching system (ThermoQuest, Mainz,

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Germany) to a gas chromatograph CP 3800 (Varian, Darmstadt, Germany) was

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used. In the first dimension, the separation of the distillate was achieved on the FFAP

173

column. First, the retention time of the analyte under investigation was determined by

174

means of reference substances. Then, at the respective elution time, the effluent was

175

quantitatively transferred into a cold trap (-80 °C) and, after the cooling was turned

176

off, the trapped material was transferred onto the DB-5 column in the second oven.

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The effluent was finally monitored using an ITD Saturn 2000 ion trap mass

178

spectrometer (Varian, Darmstadt, Germany). Mass spectra in the chemical ionization

179

mode (MS-CI) were generated at 70 eV using methanol as reagent gas. The peak

180

areas of the selected ions of the labeled standard and the analyte were separately

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determined in the mass chromatogram by either using the molecular ions or selected ACS Paragon Plus Environment

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fragments given in Table 1. Concentrations were calculated and corrected using

183

response factors obtained by measuring defined mixtures of the respective labeled

184

and the unlabeled compound.20

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Quantitation of Ethanol. Ethanol was determined on the basis of density by weighing exactly 20 mL of a steam distillate of Cognac.

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Separation of (S)-2-, (R)-2- and 3-Methylbutanol. Due to co-elution on the DB-

188

FFAP column, the separation of the three methylbutanols was achieved on a chiral

189

BGB 174 E column (30 m x 0.25 mm ID, 0.25 µm film thickness) (BGB Analytic,

190

Anwill, Switzerland) by means of TD-HRGC/MS with the DB-FFAP column in the first

191

and the BGB 174 E column in the second dimension.

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Determination of Odor Thresholds. First, the purity of the reference odorants

193

was checked by GC/O. For the determination of the odor thresholds, a defined

194

amount of the purified aroma compound in ethanol (10 µL) was then pipetted into a

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Teflon vessel containing 25 mL of ethanol/water (6:4, v/v). After stepwise dilution

196

(1:1, v/v), the samples were judged by 16 trained assessors. Triangular tests were

197

performed using 25 mL of water/ethanol (6:4, v/v) as the control, and the odorants

198

were presented in Teflon vessels (45 mL) with lid in decreasing concentrations. The

199

assessors were recruited from the German Research Center for Food Chemistry,

200

and sensory evaluations were performed in a sensory panel room at 21 ± 1 °C. Odor

201

thresholds were calculated according to the method of § 64 LFGB, methods 00.90-7

202

and 00.90-9.24

203

Descriptive Profile Tests. The panelists were asked to rate the odor intensity of

204

the aroma attributes of Cognac from 0 (not perceivable), 1 (weak), 2 (significant) and

205

3 (strong) using a seven point scale of 0, 0.5, 1, 1.5 up to 3.0. Eight supra-threshold

206

aqueous solutions (25 mL in Teflon vessels) of 3-methylbutanol (malty), (E)-β-

207

damascenone (cooked apple-like), ethyl 3-methylbutanoate (fruity), 2-phenylethanol ACS Paragon Plus Environment


10 208

(flowery), 4-hydroxy-3-methoxybenzaldehyde (vanilla-like), phenylacetic acid (honey-

209

like), 2-methoxy-4-vinylphenol (clove-like) and (3S,4S)-cis-whiskylactone (coconut-

210

like) were used for the weekly training of the sensory panel. For the evaluation of the

211

Cognac aroma, the panelists had to evaluate the intensities of the eight odor qualities

212

represented by solutions of the reference aroma compounds on a seven point

213

intensity scale. The results of the aroma profile analysis were displayed in a spider

214

web diagram.

215

For the aroma recombinate, a mixture consisting of 34 reference key odorants in

216

the concentrations determined in the Cognac and showing OAVs ≥ 1 was dissolved

217

in water/ethanol (6:4, v/v) and the pH was adjusted to 3.9 with aqueous H2SO4 (1

218

mol/L) (recombinate A). For the second aroma recombinate (recombinate B) aliquots

219

of the same 34 compounds were added to an odorless Cognac matrix. To obtain this

220

matrix, a Cognac sample (100 mL) was sequentially extracted with diethylether and

221

distilled using the SAFE apparatus. The residue obtained was treated with water

222

followed by n-pentane and finally freeze-dried until odorless. An aliquot of the

223

residue, representing 100 mL of Cognac, was dissolved in water/ethanol (6:4, v/v)

224

and was added to 100 mL of the recombinate. An overall aroma profile of both

225

recombinates was determined in the same way as described above for Cognac.

226

Separately, the similarity of the overall aromas of the Cognac and the Cognac

227

recombinates were compared. The similarity was ranked using a seven point scale

228

from 0 (no similarity) to 3 (100% identical).

229 230

RESULTS AND DISCUSSION

231

In a first experiment, a sensory panel was asked to perform an aroma profile

232

analysis of the Cognac. On the basis of eight odor qualities agreed in a preliminary

233

session, the panel evaluated fruity, malty and baked-apple like attributes as the ACS Paragon Plus Environment

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important odor qualities, followed by clove- and honey-like impressions (Figure 1). To

235

assess the odorants responsible for these aroma qualities, first, the most appropriate

236

solvent for volatile extraction was selected out of pentane/diethyl ether (1:1, v/v),

237

dichloromethane and diethyl ether. The diethyl ether extract exhibited the most

238

similar aroma compared to the original Cognac, when a drop of the extract was

239

evaluated on a strip of filter paper, and thus this solvent was used for further

240

analyses.

241

Identification of Odor-Active Compounds. A distillate of 25 mL of Cognac

242

prepared by extraction/SAFE distillation was subjected to GC/O after concentration.

243

A total of 39 odor-active areas could be detected in the FD factor range of 8 to 2048.

244

Among them, the cooked-apple-like odor, which was smelled at position 26 (Figure 2)

245

showed the highest FD-factor followed by a fruity (5) and a malty (12) odor. Further

246

compounds with fruity notes (2, 8 and 18) showed somewhat lower FD factors. In

247

addition a flowery smelling odorant (31), the malty smelling (10) and the vanilla-like

248

smelling odorant (39) showed quite high FD factors of 256.

249

To identify the compounds responsible for these odors, first, the retention indices

250

of the odor-active areas were compared to an in-house database, suggesting

251

potential structures for each odorant. To confirm these, the compounds present in the

252

neutral/basic volatiles fraction were separated on silica gel. This procedure was

253

necessary to avoid co-elution of trace odorants with volatiles present in high amounts

254

in the extract. The odor-active areas were then re-located in the respective fractions

255

by GC/O, and mass spectra in the electron impact mode (MS-EI) and the chemical

256

ionization mode (MS-CI) were recorded.

257

Compound 26 with the highest FD-factor was, thus, identified as (E)-β-

258

damascenone (Figure 3). Compound 12 with the second highest FD factor consisted

259

of two compounds, (S)-2-methylbutanol (ee: > 99%) and 3-methylbutanol, which both ACS Paragon Plus Environment


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could be separated on a chiral BGB E 174 column. The ratio was determined to be

261

84% 3-methylbutanol and 16% (S)-2-methylbutanol. The fruity odors (2, 5 and 8)

262

were evoked by diethoxy ethane, ethyl methylpropanoate and ethyl (S)-2-

263

methylbutanoate. The flowery smell (31) was caused by 2-phenylethanol and the

264

vanilla-like odor was attributed to vanillin (39). The results of the further identification

265

experiments are summarized in Table 2. Besides phenylacetic acid (38) with an FD

266

factor of 128, other odor-active acids were detected, hexanoic acid (28), acetic acid

267

(19), butanoic acid (23) and 2-and 3-methylbutanoic acid (24), all with an FD factor of

268

16.

269

Compounds with clove-like and phenolic aroma constituents were 4-allyl-2-

270

methoxyphenol (35) with an FD factor of 64, 2-methoxyphenol (29), 4-ethyl-2-

271

methoxyphenol (33) and 2-methoxy-4-vinylphenol (36) all with an FD factor of 16

272

(Table 2). Compounds 30 and 32 with a coconut-like odor were identified as (3S,4S)-

273

cis- and (3S,4R)-trans-whiskylactone.

274

α-Damascone (25) was another norisoprenoid eliciting a cooked apple like smell.

275

This odorant, and also cyclohexanoyl acetate (16), 2-isopropyl-3-methoxypyrazine

276

(17), 2-isobutyl-3-methoxypyrazine (20), (E)-2-nonenal (21), 2-methoxy-4-vinylphenol

277

(36), 3-hydroxy-4,5-dimethyl-2-(5H)-furanone (37) and phenylacetic acid (38) were

278

detected for the first time in a Cognac or brandy, respectively.

279

Quantitation by Stable Isotope Dilution Assays

280

The AEDA is an appropriate method to find out odorants potentially contributing to

281

the overall aroma of foods. However, this method neither permits conclusions on the

282

influence of the food matrix, e.g., on odorant binding, nor on the interactions of

283

odorants at the receptor level when matching the overall odor impression of a given

284

food. For this reason the odor activity values were calculated as a next step.7 For this

285

purpose, the 37 aroma compounds, which had shown the highest FD factors (Table ACS Paragon Plus Environment

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2) were quantitated using stable isotope dilution assays. In addition, ethyl pentanoate

287

was quantitated, because it was found to be an important marker to differentiate

288

brandies from Cognac samples.

289

The results of the quantitative measurements (Table 3) revealed ethanol (316 g/L)

290

and 3-methylbutanol (1044 mg/L) as compounds with the highest concentrations,

291

followed by methylpropanol, acetic acid (126 mg/L), 2-methylbutanol (107 mg/L),

292

acetaldehyde (52 mg/L), hexanol (41 mg/L), 1,1-diethoxyethane (16.7 mg/L) and

293

hexanoic acid (12.4 mg/L). Most odorants were, however, present in low amounts

294

between 170 µg/L and 0.5 µg/L (Table 3).

295

Aroma compounds showing the lowest concentrations were (E)-2-nonenal (9.0

296

µg/L), 3-hydroxy-4,5-dimethyl-2(5H)-furanone (5.7 µg/L), cyclohexanoyl acetate (6.0

297

µg/L), ethyl pentanoate (3 µg/L) and 2-isobutyl-3-methoxypyrazine (0.5 µg/L).

298

Odor activity values (ratio of concentration to odor threshold) are a valuable tool to

299

correlate quantitative data with the volatility of a compound from the respective

300

matrix,6 but, it is necessary that the thresholds of single components are determined

301

in a matrix as close as possible to the food itself. Thus, for Cognac the odor

302

thresholds were determined in water/ethanol (6:4, v/v) as previously reported in a

303

study on whisky aroma.11 To indicate the importance of the matrix, Table 4 shows a

304

comparison of odor thresholds for selected odorants in water and in water/ethanol

305

(6:4, v/v). With the exception of phenylacetic acid, all odorants showed higher odor

306

thresholds in water, with (E)-ß-damascenone, 3-methylbutanol and methylpropanol

307

showing the largest difference between both media.

308

Using the odor thresholds in water/ethanol, the odor activity values for 34 Cognac

309

odorants were calculated (Table 5). The highest OAV was obtained for ethanol

310

(12690) and confirmed the dominating aroma contribution of ethanol in spirits as

311

recently found also for whisky11 or pear brandy.25 The concentrations of all 34 aroma ACS Paragon Plus Environment


14 312

compounds clearly exceeded their thresholds in water/ethanol, and among them (E)-

313

β-damascenone, due to its low odor threshold of 0.14 µg/L, had the second highest

314

OAV. Quite high odor activity values were also calculated for the fruity smelling

315

odorants ethyl (S)-2-methylbutanoate (195), ethyl methylpropanoate (120), ethyl 3-

316

methylbutanoate (100), ethyl hexanoate (97), ethyl butanoate (69) and ethyl

317

octanoate (61). Ethyl butanoate and ethyl hexanoate have also previously been

318

suggested by Webb et al.26 as important aroma compounds in Cognac.

319

To confirm the contribution of the 34 key aroma compounds to the overall aroma

320

of the Cognac, an aroma recombinate was prepared containing reference chemicals

321

of all odorants which had shown OAVs ≥ 1 in water/ethanol (6:4, v/v) in the

322

concentrations measured in Cognac (Table 3). First, a recombinate in water/ethanol

323

(6:4, v/v) was evaluated by the sensory panel. As shown in Figure 4A, in particular

324

the flowery and fruity quality was somewhat weaker than determined for Cognac

325

(Figure 4B), and the overall similarly was only rated 2.2 on scale from 0 to 3.

326

If all aroma compounds were identified and quantitated, it can be assumed that

327

non-volatile compounds may interact with the odorants in Cognac, thus, influencing

328

the overall aroma perception. This was also recently proven for red wine.27 Thus, to

329

evaluate the influence of the non-volatile fraction from Cognac, deodorized, non-

330

volatile material was isolated from Cognac, then added to the recombinate, and

331

again a descriptive profile analysis against the original Cognac was performed. Now,

332

the aroma profile of the recombinate (Figure 4C) was nearly identical with the aroma

333

profile of the Cognac (Figure 4B), and in the similarity test this recombinate received

334

2.6 out of 3.0 points.

335

Nordström28 suggested the formation of ethyl esters from lower fatty acids during

336

fermentation with Saccharomyces cerevisiae, and ethyl esters have been described

337

as the largest group among the volatile compounds in Cognac.2 Our study ACS Paragon Plus Environment

Page 14 of 36

Page 15 of 36


15 338

corroborated that ethyl esters make an important contribution to the aroma of

339

Cognac, since high odor activity values were calculated for ethyl (S)-2-methyl-

340

butanoate, ethyl methylpropanoate, ethyl 3-methylbutanoate and ethyl hexanoate.

341

The concentrations measured in the Cognac for ethyl 2-methylbutanoate, ethyl 3-

342

methylbutanoate and ethyl hexanoate were in good agreement with data reported

343

earlier by Schreier et al.3

344

Janacova et al.9 suggested methylpropanol, 2-methylbutanol and 3-methylbutanol

345

to be important in the aroma of Slovac brandies, and Webb et al.26 also quantitated

346

3-methylbutanol, 2-methylbutanol and methylpropanol in Cognac with quite similar

347

values as found here (3-methylbutanol: 1650 to 2190 mg/L, 2-methylbutanol: 330 to

348

430 mg/L and 2-methylpropanol: 910 to 1100 mg/L). These alcohols are long-known

349

as amino acid metabolites of yeast by the Ehrlich mechanism and have been

350

detected in many fermented foods. They were also recently shown by us to be key

351

odorants in whisky,11 Williams Christ brandy25 or wheat beer, respectively.29

352

Guichard et al.30 quantitated whisky lactones in different brandies. From the four

353

possible stereoisomers they only found the (3S,4S)-cis-whiskylactone and the

354

(3S,4R)-trans-whiskylactone, which is also in agreement with the result found here.

355

Mosandl and Kustermann31 reported that both enantiomers were mainly present in

356

American white oak (Quercus alba). Maga32 and Guymon and Crowell33 investigated

357

brandies aged in American and French oak barrels and found both whiskylactone

358

isomers in quite high concentrations in brandies aged in American oak, but in lower

359

concentrations in French oak aged brandies. The Cognac used in this study was

360

stored in French oak barrels, and, thus, the concentrations of these whiskylactones

361

were rather low, thus supporting the literature results.

362

In the present study a total of eight aroma compounds were detected for the first

363

time in the volatile fraction of Cognac, namely 2-methoxy-4-vinylphenol, phenylacetic ACS Paragon Plus Environment


Page 16 of 36

16 364

acid, (E)-2-nonenal, 3-hydroxy-4,5,-dimethyl-2(5H)-furanone, ethyl cyclohexanoyl

365

acetate,

366

damascone (Table 2). Among them, in particular 2-methoxy-4-vinylphenol,

367

phenylacetic acid and (E)-2-nonenal showed quite high odor activity values.

2-isopropyl-3-methoxypyrazine,

2-isobutyl-3-methoxypyrazine

and

α-

368

Compared to wine34 and other spirits25, the concentration of (E)-ß-damascenone

369

was quite high in the Cognac. Therefore, the odorant was quantitated in further

370

cognacs from different manufacturers, and interestingly the concentrations were quite

371

high in all spirits ranging from 108 µg/L to 330 µg/L (Table 6). By contrast, the

372

norisoprenoid was much lower in 7 German brandies (3 to 31 µg/L) as well as in

373

Spanish (8-12 µg/L) and two French brandies (30-40 µg/L).

374

Odorants in a German Brandy. For a first comparison of the key odorants in a

375

German brandy, the aroma extract dilution analysis was applied on a distillate from

376

the German brandy Mariacron. The data revealed distinct differences, but in

377

particular ethyl pentanoate was higher in the German brandy (Table 7). The

378

quantitation of this ester in the 12 cognacs and the 7 German brandies revealed clear

379

differences in their concentrations (Table 6). The same was true for two Spanish and

380

two French brandies. While only 3 to 13 µg/L of the ester were present in the 12

381

Cognacs analyzed, 68 to 320 µg/L of the odorant were present in the 7 German

382

brandies (Table 6).

383

By calculating the ratio of the ß-damascenone concentration to that of ethyl

384

pentanoate, the German and Spanish brandies could easily be differentiated from the

385

Cognacs. Even two French brandies (Dujardin and Napoleon) could easily be

386

differentiated

387

damascenone/ethyl pentanoate as a possible indicator for the differentiation of

388

Cognac from other brandies.

on

the

basis

of

these

data

suggesting


the

pair

(E)-ß-

Page 17 of 36


17 389

Compared to data reported on the key odorants in other spirits, the results

390

showed that the differences in the overall aromas of most distilled spirits aged in oak

391

barrels are obviously caused by differences in the concentrations of odor-active

392

constituents rather than by specific compounds present in the overall set of key

393

odorants. This is, however not a general rule, because for some spirits, such as pear

394

brandy,25 so called specialists7 among the key aroma compounds, like ethyl

395

decadienoate, can be the cause for overall differences in the aroma profile. Further

396

studies will be undertaken to examine the influence of single processing steps as

397

source of the key aroma compounds in brandies by means of systematic studies on

398

intermediates of the manufacturing process.

399



18 400

REFERENCES

401

1. Reinhard, C. Gas-chromatographic study of wine, distilled wine and brandy,

402

Wein-Wiss., 1968, 23, 475-486.

403

2. Postel, W.; Adam, L. Gas-chromatographic characterisation of brandy, Cognac

404

and Armagnac II. Concentrations of volatile contents. Branntweinwirts. 1980, 2,

405

154-164.

406 407

3. Schreier, P.; Drawert, F.; Schmid, M. Composition of neutral volatile constituents in grape brandies J. Agric. Food Chem. 1979, 27, 365-372.

408

4. Ledauphin, J.; Milbeau, C.; Barillier, D.; Hennequin, D. Differences in the volatile

409

compositions of French labeled brandies (Armagnac, Calvados, Cognac and

410

Mirabelle) using GC-MS and PLS-DA. J. Agric. Food Chem. 2004, 52, 5124-

411

5134.

412

5. De Rijke, D.; Heide, R., Flavor compounds in rum, cognac and whisky. In: Flavor

413

of distilled beverages: origin and development (Piggott, J.R.; ed.), Ellis Horwood,

414

Chichester, 1983, pp. 192-202.

415

6. Schieberle, P.; Hofmann, T. Mapping the combinatorial code of food flavors by

416

means of molecular sensory science approach. In: Chemical and Functional

417

Properties of Food Components Series. Food Flavors. Chemical, Sensory and

418

Technological Properties (Jelen, H.; ed.) CRC Press, Boca Raton, FL, USA,

419

ISBN 978-1-4398-1491-8, 2012, pp. 413-438.

420

7. Dunkel, A.; Steinhaus, M.; Kotthoff, M.; Nowak, B.; Krautwurst, D.; Schieberle, P.;

421

Hofmann, T. Nature's chemical signatures in human olfaction: a foodborne

422

perspective for future biotechnology. Angew. Chemie Int. Ed., 2014, 7124-7143.

423

8. Ferrari, G.; Lablanquie, O.; Cantagrel, R.; Ledauphin, J.; Payot, T.; Fournier, N.;

424

Guichard, E. Determination of key odorant compounds in freshly distilled Cognac


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using GC/O, GC/MS and sensory evaluation. J. Agric. Food Chem. 2004, 52,

426

5670-5676.

427

9. Janacova, A.; Sadecka, J.; Kohajdova, Z.; Spanik, J. The identification of aroma-

428

active compounds in Slovak brandies using GC-sniffing, GC/MS and sensory

429

evaluation. Chromatographia 2008, 67, 113-121.

430

10. Zhao, Y.; Xu, J.; Fan, W.; Jiang, W. Characterization of aroma compounds of four

431

brandies by aroma extract dilution analysis. Am. J. Enol. Viticult. 2009, 60, 269-

432

276.

433

11. Poisson, L.; Schieberle, P. Characterization of the key aroma compounds in an

434

American Bourbon whisky by quantitative measurements, aroma recombination

435

and omission studies. J. Agric. Food Chem. 2008, 56, 5820-5826

436

12. Schieberle, P. Primary odorants of pale lager beer. Differences to other beers

437

and changes during storage. Z. Lebensm. Unters. Forsch. 1991,193, 403-431.

438

13. Guth, H.; Grosch, W. Determination of soya-bean oil: Quantification of primary

439

flavor compounds using a stable isotope dilution assay. Lebensm. Wiss. Technol.

440

1990, 23, 513-522.

441

14. Kirchhoff, E.; Schieberle, P. Quantitation of odor-active compounds in rye flour

442

and rye sourdough using stable isotope dilution assays. J. Agric. Food Chem.

443

2002, 50, 5378-5385.

444 445

15. Kormarek, D. Key odorants in beer – Influence of storage on the flavor stability. PhD Thesis, 2001.

446

16. Jagella, T.; Grosch, W. Flavour and off-flavour compounds of black and white

447

pepper (Piper nigrum L.). III. Desirable and undesirable odorants of white pepper.

448

Eur. Food Res. Technol. 1999, 209, 27-31.



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17. Blank, I.; Schieberle, P. Analysis of the seasoning-like flavour substances of a

450

commercial lovage extract (Levisticum officinale Koch.). Flavour Fragr. J. 1993,

451

8, 191-195.

452

18. Guth, H.; Grosch, W. Identification of the character impact odorants of stewed

453

beef juice by instrumental analysis and sensory studies. J. Agric. Food Chem.

454

1994, 42, 2862-2866.

455

19. Czerny, M.; Schieberle, P. Influence of the polyethylene packaging on the

456

absorption of odor-active compounds from UHT-milk. Eur. Food Res. Technol.

457

2007, 225, 215-223.

458

20. Schuh, C.; Schieberle, P. Characterization of the key aroma compounds in the

459

beverage preared from Darjeeling black tea: quantitative differences between tea

460

leaves. J. Agric. Food Chem. 2006, 54, 916-924.

461

21. Engel, W.; Bahr, W.; Schieberle, P. Solvent assisted flavour evaporation – a new

462

and versatile technique for the careful and direct isolation of aroma compounds

463

from complex food matrices. Eur. Food Res. Technol. 1999, 209, 237-241.

464

22. Hinterholzer, A.; Schieberle, P. Identification of the most odor-active volatiles in

465

fresh, hand-extracted juice of Valencia late oranges by odor dilution techniques.

466

Flavor Fragr. J. 1998, 13, 49-55.

467

23. Czerny, M.; Christlbauer, M.; Christlbauer, M.; Fischer, A.; Granvogel, M.;

468

Hammer, M.; Hartl, C.; Hernandez, N.M.; Schieberle, P. Re-investigation on odor

469

thresholds of key food aroma compounds and development of an aroma

470

language based on odor qualities of defined aqueous odorant solutions. Eur.

471

Food Res. Technol. 2008, 228, 265-273.

472

24. Bundesamt für Verbraucherschutz und Lebensmittelsicherheit BLV. Sensorische

473

Prüfverfahren:

Dreiecksprüfung

(00.90-7).

Amtliche

474

Untersuchungsverfahren nach § 64 LFGB. 2005, Beuth Verlag Berlin. ACS Paragon Plus Environment

Sammlung

von

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21 475

25. Willner, B.; Granvogl, M.; Schieberle, P. Characterization of the key aroma

476

compounds in Bartlett pear brandies by means of the sensomics concept. J.

477

Agric. Food Chem. 2013, 61, 9583–9593.

478 479

26. Webb A.; Kepner, R. E.; Ikeda, R. M. Composition of a typical grape brandy fusel oil. Anal. Chem. 1952, 24, 1944-1949.

480

27. Frank S.; Wollmann N.; Schieberle P.; Hofmann T. Reconstitution of the flavor

481

signature of Dornfelder red wine on the basis of the natural concentrations of its

482

key aroma and taste compounds. J. Agric. Food Chem. 2011, 59, 8866-8874.

483

28. Nordström, K. Formation of esters from acids by brewer´s yeast, II. Formation

484

from lower fatty acids, J. Inst. Brew. 1963, 70, 42-55.

485

29. Langos, D.; Granvogl, M.; Schieberle, P. Characterization of the key aroma

486

compounds in two Bavarian wheat beers by means of the Sensomics approach.

487

J. Agric. Food Chem. 2013, 61, 11303-11311.

488

30. Guichard, E.; Fournier, N.; Masson, G.; Puech, J.-L. Stereoisomers of β-methyl-

489

γ-octalactone. I. Quantitation in brandies as a function of wood origin and

490

treatment of the barrels. Am. J. Enol. Vitcult. 1995, 46, 419-423.

491

31. Mosandl, A.; Kustermann, A. Stereoisomeric flavor compounds. XXX. HRGC

492

analysis of chiral γ-lactones from beverages and fruit preparations. Z. Lebensm.

493

Unters. Forsch. 1989, 189, 212-215.

494

32. Maga, J. Formation and extraction of cis- and trans-β-methyl-γ-octalactone from

495

Quercus alba in distilled beverage flavor: Recent developments, Piggott J.R. and

496

Paterson A. Eds; Ellis Horwood, Chichester, UK, 1989; pp. 171-176.

497 498

33. Guymon, J. F.; Crowell, E. A. GC-separated brandy components derived from French and American oaks. Am. J. Enol. Vitcult. 1977, 23, 113-120.



22 499

34. Kotseridis, Y.; Baumes, R.; Skouroumounis, G. Quantitative determination of free

500

and hydrolytically liberated β-damascenon in red grapes and wines using a

501

stable isotope dilution assay. J. Chromatogr. A 1999, 849, 245-254.

502 503


Page 22 of 36

Page 23 of 36


23 504

FIGURE CAPTIONS

505 506

Figure 1. Aroma profile analysis of the Cognac Hennessy.

507

Figure 2. Flavor Dilution (FD) chromatogram obtained by application of the aroma

508

extract dilution analysis on a distillate of the volatiles isolated from the Cognac.

509

Figure 3. Structures of the most odor active compounds identified in Cognac. The

510

alcohol isomers (12) could not be separated on the GC stationary phase used in

511

GC/Olfactometry.

512

Figure 4. Aroma profile analysis of: A. the aroma recombinate in water/ethanol (6:4,

513

v/v); B. the Cognac and C. the aroma recombinate with a non-volatile matrix

514

prepared from Cognac added.

515 516



Page 24 of 36

24 Table 1. Selected Ions (m/z) (M++1; MS-CI) of Analytes and Isotopically Labeled Standards (ILS) Used in the Stable Isotope Dilution Assays Analyte Isotope (m/z) label

compound

ILS (m/z)

MS response factor

C2

75

0.94

H3 H3

64 105

1.03 1.01

73

13

acetic acid ethyl propanoate

61 103

2

ethyl butanoate

117

2

H3

120

0.97

131

2

H5

136

0.86

145

2

H3

148

0.99

173

2

H3

176

0.90

ethyl decanoate

201

2

H5

206

1.02

ethyl methylpropanoate

117

2

H3

120

1.00

131

2

H5

136

1.00

131

2

H3

134

0.95

147

2

H5

152

0.93

157

2

H3

160

1.03

103

2

H4

107

0.89

83

2

H5

88

0.99

H4-6

195-197

1.01

1,1-diethoxyethane

ethyl pentanoate ethyl hexanoate ethyl octanoate

ethyl (S)-2-methylbutanoate ethyl 3-methylbutanoate (S)-ethyl 2-hydroxy-3-methylbutanoate cyclohexanoyl acetate hexanol (Z)-3-hexen-1-ol

191

(E)-ß-damascenone

2

2

71

2

H2

73

1.00

71

2

H2

73

1.00

57

2

H7

64

0.99

methylpropanal

73

2

H7

80

0.98

3-methylbutanal

87

2

H2

89

1.02

87

13

C4

91

0.89

141

2

H2

143

0.98

2-phenylethanol

105

13

C2

107

1.05

3-methylbutyl acetate

148

2

H2

150

0.98

157

2

H2

159

1.00

153

2

H3

156

0.94

165

2

H3

168

0.92

125

2

H2

127

0.99

153 129

2

H2 H2

155 131

1.05 0.98

3-methylbutanol (S)-2-methylbutanol methylpropanol

2,3-butandione (E)-2-nonenal

(3S,4S)-cis-whiskylactone 4-hydroxy-3-methoxybenzaldehyde 4-allyl-2-methoxyphenol 2-methoxyphenol 4-ethyl-2-methoxyphenol 3-hydroxy-4,5-dimethyl-2-furanone

2


Page 25 of 36


25 Table 2. Most Aroma-active Compounds (FD ≥ 8) Detected in a Distillate of Volatiles Prepared from Hennessy Cognac a

b

c

no.

odorant

odor quality

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

methylpropanalg 1,1-diethoxyethane ethyl acetate 3-methylbutanal ethyl 2-methypropanoate 2,3-butanedione ethyl butanoate ethyl 2-methylbutanoate ethyl 3-methylbutanoate 2-methylpropanol 3-methylbutyl acetate 2- and 3-methylbutanol ethyl hexanoate hexanol (Z)-3-hexen-1-ol cyclohexyl acetate 2-isopropyl-3-methoxypyrazine ethyl octanoate acetic acid 2-isobutyl-3-methoxypyrazine (E)-2-nonenal ethyl decanoate butanoic acid 2-and 3-methylbutanoic acid α-damascone (E)-ß-damascenone

malty fruity fruity malty fruity butter-like fruity fruity fruity malty fruity malty fruity green green aromatic, fruity earthy fruity vinegar earthy green flowery sweaty sweaty cooked apple-like cooked apple-like

RIe on

d

fract. C B B C B C B B B D B D B D D C C B AF D C B AF AF C C

FFAP

DB-5

880 900 920 930 980 983 1036 1045 1064 1100 1113 1213 1230 1352 1376 1396 1413 1424 1444 1495 1505 1618 1620 1657 1733 1789

Decoding the Combinatorial Aroma Code of a Commercial Cognac by

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