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Characterization of the Key Aroma Compounds in Heat-processed Licorice (Succus Liquiritae) by Means of Molecular Sensory Science Juliane Wagner, Peter Schieberle, and Michael Granvogl J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04499 • Publication Date (Web): 18 Nov 2016 Downloaded from http://pubs.acs.org on November 18, 2016

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Journal of Agricultural and Food Chemistry

Characterization of the Key Aroma Compounds in Heat-processed Licorice (Succus Liquiritiae) by Means of Molecular Sensory Science

Juliane Wagner, Peter Schieberle, and Michael Granvogl*

Lehrstuhl für Lebensmittelchemie, Technische Universität München, Lise-Meitner-Straße 34, D-85354 Freising, Germany

*

Corresponding author Phone:

+49 8161 71 2987

Fax:

+49 8161 71 2970

E-Mail:

[email protected]

1

ACS Paragon Plus Environment


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ABSTRACT: Application of the sensomics concept elucidated the key odorants of

3

heat-processed licorice (Succus Liquiritiae). Forty-nine aroma-active compounds with

4

flavor dilution (FD) factors between 16 and 2048 were detected; 47 thereof were

5

identified, 23 for the first time in heated licorice. 4-Hydroxy-2,5-dimethylfuran-3(2H)-

6

one

7

methoxybenzaldehyde, 3-hydroxy-4,5-dimethylfuran-2(5H)-one, 3-hydroxy-2-methyl-

8

4H-pyran-4-one, and 2-methoxyphenol (all 1024). Forty-two substances were

9

quantitated by stable isotope dilution assays (SIDAs) and odor activity values (OAVs;

10

ratio of concentration to the respective odor threshold) were calculated revealing

11

OAVs ≥ 1 for 29 compounds. Thereby, 3-hydroxy-4,5-dimethylfuran-2(5H)-one,

12

2,3-butanedione, 2-methoxyphenol, and 1,8-cineole showed the highest OAVs in

13

Succus Liquiritiae. To validate the obtained data, a reconstitution model based on an

14

aqueous sucrose solution (50%) was prepared, containing all 29 odorants with an

15

OAV ≥ 1 in their naturally occurring concentrations. The recombinate elicited an

16

aroma profile matching very well with the profile of the original heat-processed

17

licorice, proving the correct identification and quantitation of all key aroma

18

compounds of Succus Liquiritiae.

revealed

the

highest

FD

factor

of

2048,

followed

by

4-hydroxy-3-

19 20

KEYWORDS: heat-processed licorice, Succus Liquiritiae, molecular sensory science

21

concept, aroma extract dilution analysis, stable isotope dilution assay, odor activity

22

value, aroma recombination

23


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3 24

INTRODUCTION

25

Licorice is a very popular confectionery in Europe and North America. The

26

characteristic ingredient of this candy is Succus Liquiritiae, a heat-processed

27

aqueous extract of raw licorice (Glycyrrhiza glabra L.). George Dunhill, an English

28

pharmacist, invented licorice confectionery by mixing heated licorice extract with

29

flour, sugar, and molasses.1 To date, the recipe has only been slightly modified with

30

gelatin and flavorings, in particular salmiac (ammonium chloride) and aniseed oil, as

31

additional ingredients.2 Succus Liquiritiae evokes a distinctively different aroma

32

compared to raw licorice, indicating thermally induced changes in the composition of

33

the odorants.

34

Up to now, only a few studies examined the volatile compounds of heated licorice.

35

In 1977, Frattini et al.3 investigated the volatile compounds of heated licorice

36

essential oil and identified 63 substances by gas chromatography-mass spectrometry

37

(GC-MS) and infrared spectroscopy (IR). The authors stated that only the mixture of

38

these aroma compounds is responsible for the characteristic aroma and not a single

39

substance. Tanaka et al.4 applied water distillation to Chinese licorice root

40

(Glycyrrhiza uralensis Fisch.) and subsequently identified 127 compounds in the

41

respective solvent extract, e.g., alcohols, aldehydes, ketones, acids, esters and

42

terpenoids. However, no studies were carried out to analyze the volatile compounds

43

in regard to their contribution to the overall aroma of heated licorice.

44

In accordance with our previous study, in which the key odorants of raw licorice

45

(Glycyrrhiza glabra L.) were characterized by the molecular sensory science

46

concept,5,6 the aim of the present study was to characterize the key aroma

47

compounds of a commercially heated licorice extract. This sensomics approach is

48

based on (i) the identification of the key odorants using aroma extract dilution

49

analysis (AEDA) in combination with gas chromatography-mass spectrometry, (ii) the ACS Paragon Plus Environment


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4 50

quantitation of the odorants by stable isotope dilution analysis (SIDA), (iii) the

51

calculation of odor activity values (OAVs; ratio of concentration to odor threshold) to

52

evaluate the contribution of each substance to the overall aroma, and, (iv) the

53

verification of the obtained results by recombination experiments.

54 55 56 57

MATERIALS AND METHODS Succus Liquiritiae. Succus Liquiritiae was purchased from Caesar & Loretz (Caelo) (Hilden, Germany).

58

Chemicals. The following reference compounds used for characterization of the

59

odorants were obtained from commercial suppliers: acetic acid, 2-acetyl-5-

60

methylfuran,

61

dichlorophenol, 2,6-dimethoxyphenol, dimethyl trisulfide, 2-ethyl-3,5(6)-dimethyl-

62

pyrazine,

63

dimethylfuran-2(5H)-one,

64

2-isopropyl-5-methylphenol, 5-isopropyl-2-methylphenol, linalool, 2-methoxyphenol,

65

2-methylbutanoic acid, 3-methylbutanoic acid, 4-methylphenol, 3-(methylthio)pro-

66

panal, (E,E)-2,4-nonadienal, (E,Z)-2,6-nonadienal, γ-nonalactone, octanoic acid, 2,3-

67

pentanedione, pentanoic acid, phenylacetaldehyde, and phenylacetic acid (Sigma-

68

Aldrich Chemie, Taufkirchen, Germany); 2-methylbutanal, 3-methylbutanal, and 1-

69

octen-3-one (Alfa Aesar, Karlsruhe, Germany); benzaldehyde, γ-dodecalactone,

70

hexanoic

71

propenyl)benzene (Fluka; Sigma-Aldrich Chemie); coumarin (Merck, Darmstadt,

72

Germany);

73

Germany); butanoic acid and 4-hydroxy-3-methoxybenzaldehyde (VWR, Darmstadt);

74

(E)-β-damascenone was kindly provided by Symrise (Holzminden, Germany).

acetylpyrazine,

anethole,

2-ethyl-5(6)-methylpyrazine,

acid,

indole,

2,3-butanedione,

γ-hexalactone, β-ionone,

hexanal,

(Lancaster

2,6-

3-hydroxy-4,5-

2-isobutyl-3-methoxypyrazine,

4-hydroxy-2,5-dimethylfuran-3(2H)-one,

(E,E)-2,4-decadienal

1,8-cineole,

Synthesis,


and

1-methoxy-4-(2-

Frankfurt-Griesheim,

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5 75 76

The following compounds were synthesized as previously reported: 2-acetyl-1pyrroline7 and trans-4,5-epoxy-(E)-2-decenal.8

77

Dichloromethane, diethyl ether (both VWR), and pentane (Merck) were freshly

78

distilled prior to use. Hydrochloric acid (37%), silica gel 60, and sodium carbonate

79

were purchased from Merck; liquid nitrogen was from Linde (Munich, Germany).

80

Stable

Isotopically

Labeled

Internal

Standards.

[2H3]-Acetic

acid,

81

[2H3]-hexanoic acid, [2H7]-4-methylphenol, and [13C2]-phenylacetic acid were obtained

82

from Sigma-Aldrich Chemie.

83

The following stable isotopically labeled internal standards were prepared as [2H2]-acetylpyrazine,9

[2H2-5]-2-acetyl-1-pyrroline,9

[2H5]-

84

described

85

benzaldehyde,10 [13C4]-2,3-butanedione,11 [2H2-4]-butanoic acid,12 [2H3]-1,8-cineole,13

86

[13C2]-coumarin,14 [2H4-7]-(E)-β-damascenone,15 [2H2-4]-(E,E)-2,4-decadienal,16 [2H5-8]-

87

2,6-dimethoxyphenol,17 [2H2]-γ-dodecalactone,18 [2H3]-2-ethyl-3,5-dimethylpyrazine,19

88

[2H4]-hexanal,20 [13C2]-3-hydroxy-4,5-dimethylfuran-2(5H)-one,21 [13C2]-4-hydroxy-2,5-

89

dimethylfuran-3(2H)-one,22

90

ionone,24 [2H7]-2-isopropyl-5-methylphenol,25 [2H2]-linalool,26 [2H3]-2-methoxphenol,19

91

[2H3]-1-methoxy-4-(2-propenyl)benzene,27

92

methylbutanoic acid,29 [2H3]-3-(methylthio)propanal,30 [2H2]-(E,E)-2,4-nonadienal,23

93

[2H2]-(E,Z)-2,6-nonadienal,16

94

octen-3-one,32

95

[13C2]-phenylacetaldehyde.28

96 97

previously:

[2H3]-4-hydroxy-3-methoxybenzaldehyde,23

[2H2]-3-methylbutanal,28

[2H3]-β-

[2H2]-3-

[2H2]-γ-nonalactone,18 [2H2]-octanoic acid,31 [2H3]-1-

[13C2]-2,3-pentanedione,33

[2H3]-pentanoic

acid,34

and

Concentrations of the isotopically labeled standards were determined as previously described.35

98

Isolation of the Volatiles. Succus Liquiritiae was cut into pieces, frozen in liquid

99

nitrogen, and finely ground in a commercial blender. An aliquot of the powder

100

obtained (25 g) was extracted with dichloromethane (2 x 125 mL) by stirring ACS Paragon Plus Environment


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6 101

vigorously for 2 x 1 h at room temperature. The combined organic extracts were

102

subjected to high vacuum distillation using the solvent assisted flavor evaporation

103

(SAFE)36 technique to separate the volatiles from the non-volatile material. The

104

distillate obtained was dried over anhydrous sodium sulfate, filtered, and

105

concentrated to ~0.5 mL by a Vigreux column (40 cm x 1 cm i.d.) and micro-

106

distillation.37

107

Fractionation of the Volatiles. For identification experiments, the volatiles of

108

Succus Liquiritiae (500 g) were isolated as described above and fractionation was

109

performed as recently described.6

110

Aroma Extract Dilution Analysis (AEDA) and Identification Experiments.

111

Flavor dilution (FD) factors of each aroma-active compound were determined by

112

diluting the distillate stepwise 1+1 (v+v) with dichloromethane and analyzing each

113

dilution by HRGC-O. The FD factor of an odorant is defined as the highest dilution, in

114

which its odor impression was perceived at the sniffing port for the last time. To avoid

115

an overlooking of odorants, HRGC-O of the concentrated distillate was performed by

116

three trained panelists. Aroma-active compounds with FD factors ≥ 16 were identified

117

on the basis of their retention indices determined on two capillary columns of different

118

polarities (DB-FFAP and DB-5), their odor qualities and intensities perceived at the

119

sniffing port, and their mass spectra obtained in electron ionization (EI) mode as well

120

as in chemical ionization (CI) mode in comparison with the data obtained from

121

reference compounds available in an in-house database containing >1000 aroma-

122

active reference volatiles.

123

High-Resolution Gas Chromatography-Olfactometry (HRGC-O). HRGC-O

124

was performed as recently described.6 For identification, linear retention indices of

125

each compound were determined.38


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7 126

High-Resolution Gas Chromatography-Mass Spectrometry (HRGC-MS) and

127

Two-Dimensional High-Resolution Gas Chromatography-Mass Spectrometry

128

(HRGC/HRGC-MS). Identification and quantitation of odorants present in higher

129

concentrations was performed by HRGC-MS as recently described.6 In cases where

130

major volatiles caused overlapping of an analyte, a two-dimensional setup

131

(HRGC/HRGC-MS) was applied.6

132

Quantitation by Stable Isotope Dilution Assays (SIDAs). To Succus Liquiritiae

133

powder (1 - 500 g, depending on the concentrations of the analyzed odorants),

134

dichloromethane (50 - 250 mL) and defined amounts of the respective internal

135

standards (0.5 - 5 µg; dissolved in diethyl ether; amounts depending on the

136

concentrations of the respective analytes determined in a preliminary experiment)

137

were added and the mixture was stirred for 1 h at room temperature. After decanting

138

of the solvent, another portion of dichloromethane was added and the mixture was

139

stirred again for 1 h. Both organic extracts were combined, filtered, and subjected to

140

SAFE distillation.36 Further workup was performed as described above for the

141

isolation of the volatiles.

142

To calculate the response factor (Rf) of each odorant, binary mixtures of defined

143

amounts of the analyte and the respective standard in five different mass ratios (5:1,

144

3:1, 1:1, 1:3, 1:5) were analyzed under the same conditions by (HRGC/)HRGC-MS

145

(Table 1).

146

Quantitation of 2- and 3-Methylbutanoic Acid. As both isomers could not be

147

separated by HRGC-MS in CI mode, a two-step approach was applied for

148

quantitation as previously described.39

149 150

Sensory Experiments. Orthonasal odor thresholds were determined in water as matrix using triangle tests.40



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8 151

For aroma profile analysis, water (15 mL) was added to Succus Liquiritiae powder

152

(10 g) and the resulting mixture was evaluated by the sensory panel in parallel with

153

the recombinate (15 mL), which was based on an aqueous sucrose (50%) model

154

solution containing all quantitated odorants with an OAV ≥ 1 in their naturally

155

occurring concentrations determined in Succus Liquiritiae. The intensities of the odor

156

attributes were rated from 0 (not perceivable) to 3 (strongly perceivable): seasoning-

157

like

158

methoxyphenol), fatty ((E,E)-2,4-nonadienal), eucalyptus-like (1,8-cineole), caramel-

159

like (4-hydroxy-2,5-dimethylfuran-3(2H)-one), aniseed-like (anethole), malty (3-

160

methylbutanal), and thyme-like (2-isopropyl-5-methylphenol).

(3-hydroxy-4,5-dimethylfuran-2(5H)-one),

gammon/like,

smoky

(2-

161 162

RESULTS AND DISCUSSION

163

Identification of Key Odorants in Succus Liquiritiae. After isolation of the

164

volatiles by solvent extraction and SAFE distillation,36 the extract elicited the typical

165

aroma of Succus Liquiritiae if put on a strip of filter paper. Using aroma extract

166

dilution analysis (AEDA), 49 odorants present in the flavor dilution (FD) factor range

167

between 16 and 2048 were located in the aroma extract (Figure 1). Compound 36

168

with a caramel-like odor showed the highest FD factor of 2048, followed by 30

169

(gammon-like, smoky), 33 (caramel-like), 42 (seasoning-like), and 49 (vanilla-like; all

170

FD factor of 1024).

171

The odor-active areas detected by HRGC-O were identified by comparison of

172

their retention indices on two capillary columns of different polarities, odor quality and

173

intensity perceived at the sniffing port as well as mass spectra generated in EI and CI

174

mode with data of the respective reference compounds. Following this procedure, 4-

175

hydroxy-2,5-dimethylfuran-3(2H)-one (36),

176

methyl-4H-pyran-4-one

(33),

2-methoxyphenol

(30), 3-hydroxy-2-

3-hydroxy-4,5-dimethylfuran-2(5H)-one


(42),

and

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9 177

4-hydroxy-3-methoxybenzaldehyde (49) were identified (Table 2 and Figure 2).

178

Altogether, 47 odorants were successfully identified, 23 thereof for the first time in

179

heat-processed licorice (Table 2).

180

Quantitation of Key Odorants in Succus Liquiritiae and Calculation of Odor

181

Activity Values (OAVs). Forty-two aroma-active compounds in Succus Liquiritiae

182

were quantitated by stable isotope dilution assays revealing acetic acid (1280 mg/kg)

183

and maltol (226 mg/kg) with the highest concentrations, followed by hexanoic acid

184

(11.0 mg/kg), pentanoic acid (9.06 mg/kg), 2,6-dimethoxyphenol (6.60 mg/kg), 4-

185

hydroxy-3-methoxybenzaldehyde (4.98 mg/kg), butanoic acid (3.75 mg/kg), 4-

186

hydroxy-2,5-dimethylfuran-3(2H)-one (3.63 mg/kg), octanoic acid (2.49 mg/kg), and

187

phenylacetic acid (1.15 mg/kg). Seven odorants occurred in a concentration range

188

between 100 and 1000 µg/kg, e.g., 3-hydroxy-4,5-dimethylfuran-2(5H)-one (610

189

µg/kg) eliciting a seasoning-like odor. Concentrations between 10 and 100 µg/kg

190

were analyzed for five compounds, among them 1-methoxy-4-(2-propenyl)benzene

191

(56.7 µg/kg) with an aniseed-like odor (Table 3).

192

To get knowledge about the importance of single odorants to the aroma of Succus

193

Liquiritiae, odor activity values (OAVs; ratio of concentration to respective odor

194

threshold) were calculated revealing 29 odorants with OAVs ≥ 1. Thereby, 3-hydroxy-

195

4,5-dimethylfuran-2(5H)-one showed the highest OAV of 1240, followed by 2,3-

196

butanedione (664), 2-methoxyphenol (485), 1,8-cineole (324), 2,6-dimethoxyphenol

197

(228), 4-hydroxy-3-methoxybenzaldehyde (94), and 4-hydroxy-2,5-dimethylfuran-

198

3(2H)-one (67). Thirteen odorants found with FD factors ≥ 16 during AEDA, not

199

considering matrix influences, resulted in an OAV < 1 (Table 3).

200

Aroma Recombination Studies. To verify the data obtained by identification and

201

quantitation, aroma recombination experiments were performed. Therefore, a

202

reconstitution model based on an aqueous sucrose (50%) solution was prepared by ACS Paragon Plus Environment


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mixing the aroma compounds with an OAV ≥ 1 in their naturally occurring

204

concentrations. Comparative aroma profile analysis of the recombinate and the

205

original Succus Liquiritiae showed a very good similarity (Figure 3).

206

Sources of Odorants and Comparison of Succus Liquiritiae and Raw

207

Licorice. 3-Hydroxy-4,5-dimethylfuran-2(5H)-one showed the highest OAV in Succus

208

Liquiritiae. Several pathways for the formation of this compound have already been

209

described,

210

hydroxyacetaldehyde leading to 2,3-dihydroxy-4-oxo-3-methylpentanal, which can

211

form sotolon after enolization and the release of water,42 or by thermal treatment of a

212

mixture of glucose and cysteine.43

e.g.,

by

aldol

reaction

of

diacetyl

(2,3-butanedione)

and

213

The butter-like smelling diketones 2,3-butanedione and 2,3-pentanedione can be

214

formed by various reactions of carbohydrate degradation products, e.g., by an aldol

215

reaction of acetaldehyde and hydroxyacetaldehyde (glycolaldehyde) leading to

216

diacetyl.44 The formation of the homologous 2,3-pentanedione can be suggested by

217

the similar reaction starting from propanal and hydroxyacetaldehyde.

218

The phenolic compounds 2-methoxyphenol, 4-hydroxy-3-methoxybenzaldehyde,

219

and 2,6-dimethoxyphenol were also key odorants of Succus Liquiritiae and are typical

220

odorants of heat-processed vegetable foods stemming from ferulic acid (2-

221

methoxyphenol,

222

dimethoxyphenol).45

4-hydroxy-3-methoxybenzaldehyde)

or

sinapic

acid

(2,6-

223

4-Hydroxy-2,5-dimethylfuran-3(2H)-one is also well-known to be generated upon

224

heat-processing. Several pathways have already been identified, e.g., from fructose-

225

1,6-bisphosphate, with acetylformoin as the key intermediate,46 or by aldol

226

condensation of carbohydrate degradation products, such as hydroxyacetone and 2-

227

oxopropanal, leading to 3,4-dihydroxy-2,5-dioxohexane, which can form 4-hydroxy-


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11 228

2,5-dimethylfuran-3(2H)-one after enolization, cyclization, and the release of

229

water.42,47

230

While Patton48 as well as Yaylayan and Mandeville49 reported on a possible heat-

231

induced generation of 3-hydroxy-2-methyl-4H-pyran-4-one (maltol) only from maltose

232

or lactose, Ito50 showed its formation by thermal degradation of sucrose in aqueous

233

solution at 120 °C. Thereby, maltol was identified by comparison of the results

234

obtained for the model solution by gas chromatography-mass spectrometry and

235

infrared spectroscopy with data from maltol as authentic reference compound.

236

Accordingly, amounts of > 10% of sucrose in licorice roots might explain the high

237

concentrations of maltol in Succus Liquiritiae.

238

In summary, licorice roots contain very high amounts of sugars (about 60% of

239

fructose, glucose, and sucrose), unequivocally leading, also in combination with

240

present amino compounds, e.g., amino acids, to many heat-induced odorants

241

shaping the overall aroma of Succus Liquiritiae. A comparison of the aroma-active

242

compounds in Succus Liquiritiae and raw licorice6 revealed a relatively similar

243

qualitative pattern. Thus, the different overall aroma impressions of the unheated and

244

heated material are mainly evoked by different concentrations of the odorants. In raw

245

licorice, aroma-active compounds formed by lipid peroxidation and monoterpenoids

246

had the greatest impact, whereas in Succus Liquiritiae Maillard reaction products,

247

sugar degradation products, and phenolic compounds showed the highest odor

248

activity values.

249



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13 (8) Schieberle, P.; Grosch, W. Potent odorants of the wheat bread crumb. Differences to the crust and effect of a longer dough fermentation. Z. Lebensm.Unters. Forsch. 1991, 192, 130-135. (9) Schieberle, P.; Grosch, W. Quantitative analysis of aroma compounds in wheat and rye bread crusts using a stable isotope dilution assay. J. Agric. Food Chem. 1987, 35, 252-257. (10) Ruisinger, B.; Schieberle, P. Characterization of the key aroma compounds in rape honey by means of the molecular sensory science concept. J. Agric. Food Chem. 2012, 60, 4186-4194. (11) Schieberle, P.; Hofmann, T. Evaluation of the character impact odorants in fresh strawberry juice by quantitative measurements and sensory studies on model mixtures. J. Agric. Food Chem. 1997, 45, 227-232. (12) Schieberle, P.; Gassenmeier, K.; Guth, H.; Sen, A.; Grosch, W. Character impact odour compounds of different kinds of butter. Lebensm.-Wiss. Technol. 1993, 26, 347-356. (13) Horst, K.; Rychlik, M. Quantification of 1,8-cineole and of its metabolites in humans using stable isotope dilution assays. Mol. Nutr. Food Res. 2010, 54, 15151529. (14) Rychlik, M. Quantification of free coumarin and its liberation from glucosylated precursors by stable isotope dilution assays based on liquid chromatography-tandem mass spectrometric detection. J. Agric. Food Chem. 2008, 56, 796-801. (15) Sen, A.; Laskawy, G.; Schieberle, P.; Grosch, W. Quantitative determination of β-damascenone in foods using a stable isotope dilution assay. J. Agric. Food Chem. 1991, 39, 757-759.



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14 (16) Guth, H.; Grosch, W. Deterioration of soybean oil: quantification of primary flavour compounds using a stable isotope dilution assay. Lebensm.-Wiss. Technol. 1990, 23, 513-522. (17) Willner, B.; Granvogl, M.; Schieberle, P. Characterization of the key aroma compounds in Bartlett pear brandies by means of the sensomics concept. J. Agric. Food Chem. 2013, 61, 9583-9593. (18) Poisson, L.; Schieberle, P. Characterization of the key aroma compounds in an American Bourbon whisky by quantitative measurements, aroma recombination, and omission studies. J. Agric. Food Chem. 2008, 56, 5820-5826. (19) Cerny, C.; Grosch, W. Quantification of character-impact odour compounds of roasted beef. Z. Lebensm.-Unters. Forsch. 1993, 196, 417-422. (20) Steinhaus, M.; Sinuco, D.; Polster, J.; Osorio, C.; Schieberle, P. Characterization of the key aroma compounds in pink guava (Psidium guajava L.) by means of aroma re-engineering experiments and omission tests. J. Agric. Food Chem. 2009, 57, 2882-2888. (21) Blank, I.; Schieberle, P.; Grosch, W. Quantification of the flavour compounds 3hydroxy-4,5-dimethyl-2(5H)-furanone and 5-ethyl-3-hydroxy-4-methyl-2(5H)-furanone by a stable isotope dilution assay. In Progress in Flavour Precursor Studies; Schreier, P., Winterhalter, P., Eds.; Allured Publishing: Carol Stream, IL, 1993, pp 103-109. (22) Blank, I.; Fay, L. B.; Lakner, F. J.; Schlosser, M. Determination of 4-hydroxy-2,5dimethyl-3(2H)-furanone and 2(or 5)-ethyl-4-hydroxy-5(or 2)-methyl-3(2H)-furanone in pentose sugar-based Maillard model systems by isotope dilution assays. J. Agric. Food Chem. 1997, 45, 2642-2648. (23) Guth, H.; Grosch, W. Odorants of extrusion products of oat meal - changes during storage (in German). Z. Lebensm.-Unters. Forsch. 1993, 196, 22-28.


Page 15 of 30


15 (24) Kotseridis, Y.; Baumes, R.; Skouroumounis, G. K. Synthesis of labelled [2H4]βdamascenone, [2H2]2-methoxy-3-isobutylpyrazine, [2H3]α-ionone, and [2H3]β-ionone, for quantification in grapes, juices and wines. J. Chromatogr. A 1998, 824, 71-78. (25) Fischer, A. Characterization of the odor-active compounds in peel oils of Jeruk Pontianak orange (Citrus nobilis Lour. var. microcarpa Hassk.) and Brazilian green mandarin (Citrus reticulata). Ph.D. thesis, Technical University of Munich, Munich, Germany, 2008. (26) Steinhaus, M.; Fritsch, H. T.; Schieberle, P. Quantitation of (R)- and (S)-linalool in beer using solid phase microextraction (SPME) in combination with a stable isotope dilution assay (SIDA). J. Agric. Food Chem. 2003, 51, 7100-7105. (27) Zeller, A.; Horst, K.; Rychlik, M. Study of the metabolism of estragole in humans consuming fennel tea. Chem. Res. Toxicol. 2009, 22, 1929-1937. (28) Granvogl, M.; Beksan, E.; Schieberle, P. New insights into the formation of aroma-active Strecker aldehydes from 3-oxazolines as transient intermediates. J. Agric. Food Chem. 2012, 60, 6312-6322. (29) Guth, H.; Grosch, W. Identification of the character impact odorants of stewed beef juice by instrumental analyses and sensory studies. J. Agric. Food Chem. 1994, 42, 2862-2866. (30) Sen, A.; Grosch, W. Synthesis of six deuterated sulfur containing odorants to be used as internal standards in quantification assays. Z. Lebensm.-Unters. Forsch. 1991, 192, 541-547. (31) Czerny M.; Schieberle P. Influence of the polyethylene packaging on the adsorption of odour-active compounds from UHT-milk. Eur. Food Res. Technol. 2007, 225, 215-223.



Page 16 of 30

16 (32) Lin, J.; Welti, D. H.; Vera, F. A.; Fay, L. B.; Blank, I. Synthesis of deuterated volatile lipid degradation products to be used as internal standards in isotope dilution assays. 2. Vinyl ketones. J. Agric. Food Chem. 1999, 47, 2822-2829. (33) Mayer, F.; Czerny, M.; Grosch, W. Influence of provenance and roast degree on the composition of potent odorants in Arabica coffees. Eur. Food Res. Technol. 1999, 209, 242-250. (34) Jagella, T.; Grosch, W. Flavour and off-flavour compounds of black and white pepper (Piper nigrum L.). III. Desirable and undesirable odorants of white pepper. Eur. Food Res. Technol. 1999, 209, 27-31. (35) Franitza, L.; Granvogl, M.; Schieberle, P. Influence of the production process on the key aroma compounds of rum: from molasses to the spirit. J. Agric. Food Chem. 2016, DOI: 10.1021/acs.jafc.6b04046. (36) Engel, W.; Bahr, W.; Schieberle, P. Solvent assisted flavour evaporation - a new and versatile technique for the careful and direct isolation of aroma compounds from complex food matrices. Eur. Food Res. Technol. 1999, 209, 237-241. (37) Bemelmans, J. M. H. Review of isolation and concentration techniques. In Progress in Flavour Research; Land, D. G., Nursten, H. E., Eds.; Applied Science: London, UK, 1979; pp 79-98. (38) van den Dool, H.; Kratz, P. D. A generalization of the retention index system including linear temperature programmed gas-liquid partition chromatography. J. Chromatogr. 1963, 11, 463-471. (39) Steinhaus, M.; Sinuco, D.; Polster, J.; Osorio, C.; Schieberle, P. Characterization of the aroma-active compounds in pink guava (Psidium guajava, L.) by application of the aroma extract dilution analysis. J. Agric. Food Chem. 2008, 56, 4120-4127.


Page 17 of 30


17 (40) Czerny, M.; Christlbauer, Ma.; Christlbauer, Mo.; Fischer, A.; Granvogl, M.; Hammer, M.; Hartl, C.; Moran Hernandez, N.; Schieberle, P. Re-investigation on odour thresholds of key food aroma compounds and development of an aroma language based on odour qualities of defined aqueous odorant solutions. Eur. Food Res. Technol. 2008, 228, 265-273. (41) Grosch, W.; Schieberle, P. Bread. In Volatile Compounds in Foods and Beverages; Maarse, H., Ed.; Marcel Dekker: New York, NY, 1991; pp 41-77. (42) Schieberle, P.; Hofmann, T. Flavor contribution and formation of heterocyclic oxygen-containing key aroma compounds in thermally processed foods. In Heteroatomic Aroma Compounds; ACS Symposium Series 826; Reineccius, G. A., Reineccius, T. A., Eds.; American Chemical Society: Washington, DC, 2002; pp 207226. (43) Hofmann, T.; Schieberle, P. Identification of potent aroma compounds in thermally treated mixtures of glucose/cysteine and rhamnose/cysteine using aroma extract dilution techniques. J. Agric. Food Chem. 1997, 45, 898-906. (44) Hofmann, T.; Schieberle, P. Flavor contribution and formation of the intense roast-smelling odorants 2-propionyl-1-pyrroline and 2-propionyltetrahydropyridine in Maillard-type reactions. J. Agric. Food Chem. 1998, 46, 2721-2726. (45) Tressl, R.; Kossa, T.; Renner, R.; Köppler, H. Gas chromatographic-mass spectrometric investigations on the formation of phenolic and aromatic hydrocarbons in food (in German). Z. Lebensm.-Unters. Forsch. 1976, 162, 123-130. (46) Schieberle, P. Formation of furaneol in heat-processed foods. In Flavor Precursors. Thermal and Enzymatic Conversions; ACS Symposium Series 490; Teranishi, R., Takeoka, G. R., Güntert, M., Eds.; American Chemical Society: Washington DC, 1992; pp 164-174.



Page 18 of 30

18 (47) Schieberle, P. The carbon module labeling (CAMOLA) technique. A useful tool for identifying transient intermediates in the formation of Maillard-type target molecules. Ann. N. Y. Acad. Sci. 2005, 1043, 236-248. (48) Patton, S. The formation of maltol in certain carbohydrate-glycine systems. J. Biol. Chem. 1950, 184, 131-134. (49) Yaylayan, V. A.; Mandeville, S. Stereochemical control of maltol formation in Maillard reaction. J. Agric. Food Chem. 1994, 42, 771-775. (50) Ito, H. The formation of maltol and isomaltol through degradation of sucrose. Agric. Biol. Chem. 1977, 41, 1307-1308.


Page 19 of 30


19 FIGURE CAPTIONS

Figure 1. Flavor dilution (FD) chromatogram on a DB-FFAP capillary column obtained by aroma extract dilution analysis (AEDA) of the volatile fraction of Succus Liquiritiae. Odorants with an FD factor ≥ 16 are illustrated. Numbering is identical to that in Table 2.

Figure 2. Structures of the most aroma-active compounds identified in Succus Liquiritiae (FD factors and odor impressions given in parentheses). Numbering is identical to that in Table 2.

Figure 3. Comparative aroma profile analysis of Succus Liquiritiae (solid line) and the respective recombinate (broken line).



Page 20 of 30

20 Table 1. Selected Ions (m/z) of Analytes and Stable Isotopically Labeled Standards as well as Response Factors (Rf) used in Stable Isotope Dilution Assays compound

isotope label

acetic acid

[2H3]

ion (m/z)a internal analyte standard 61 64

2

acetylpyrazine

[ H 2]

2-acetyl-1-pyrroline

2

123 c

[ H2-5] 2

125

Rfb 0.99 0.98

c

112

114-117

0.91

benzaldehyde

[ H 5]

107

112

0.86

2,3-butanedione

[13C4]

87

91

1.00

butanoic acid

[2H2-4]c

89

91-93c

0.96

1,8-cineole

[2H3]

137

140

0.85

coumarin

[13C2]

147

149

(E)-β-damascenone

2

c

2

c

2

c

[ H4-7]


[ H2-4]

2,6-dimethoxyphenol

191 153

0.99 c

0.83

c

0.96

c

195-198 155-157

[ H5-8]

155

160-163

0.97

γ-dodecalactone

[2H2]

199

201

0.76

2-ethyl-3,5-dimethylpyrazine

[2H3]

137

140

0.85

2-ethyl-3,6-dimethylpyrazined

-d

137

140d

0.85

[2H4]

83

87

0.90

hexanoic acid

2

[ H 3]

117

120

0.96

3-hydroxy-4,5-dimethylfuran-2(5H)-

13

[ C2]

129

131

1.00

[13C2]

129

131

0.95

4-hydroxy-3-methoxybenzaldehyde

[2H3]

153

156

0.97

3-hydroxy-2-methyl-4H-pyran-4-one

-e

127

131e

0.73

[2H3]

193

196

0.96

hexanal

one 4-hydroxy-2,5-dimethylfuran-3(2H)one

(maltol)e β-ionone

2

[ H 7]

151

158

0.98

f

-

151

f

158

0.98

linalool

[2H2]

137

139

0.94

2-methoxyphenol

[2H3]

125

128

0.96

2-isopropyl-5-methylphenol f

5-isopropyl-2-methylphenol


Page 21 of 30


21 Table 1. Continued. isotope label

compound 1-methoxy-4-(1-propenyl)benzeneg

-g

1-methoxy-4-(2-propenyl)benzene

Rfb 0.78

[2H3]

149

152

0.78

-h

87

89h

0.86

[ H 2]

87

89

2-methylbutanalh 2

3-methylbutanal

ion (m/z)a internal analyte standard 149 152g

i

-

3-methylbutanoic acid

0.86

103

i

105

0.78

[ H 2]

103

105

0.78

4-methylphenol

[2H7]

109

116

0.99

3-(methylthio)propanal

[2H3]

105

108

0.99

(E,E)-2,4-nonadienal

[2H2]

139

141

0.98

(E,Z)-2,6-nonadienal

[2H2]

139

141

0.98

157

159

0.74

145

147

i

2-methylbutanoic acid

2

2

[ H 2]

γ-nonalactone

2

octanoic acid

[ H 2]

1-octen-3-one

2

[ H2-3]

127

129-130

0.96

2,3-pentanedione

[13C2]

101

103

0.55

pentanoic acid

[2H3]

103

106

0.98

phenylacetaldehyde

[13C2]

121

123

0.99

phenylacetic acid

[13C2]

137

139

0.92

a

c

0.65 c

Ion used for quantitation in chemical ionization (CI) mode.

b

Response factor (Rf)

was determined by analyzing mixtures of known amounts of analyte and internal standard. of

c

Internal standard was used as a mixture of isotopologues.


was

dimethylpyrazine as internal standard.

performed e

by

d

Quantitation

2

[ H3]-2-ethyl-3,5-

using

Quantitation of 3-hydroxy-2-methyl-4H-

pyran-4-one was performed by using [13C2]-4-hydroxy-2,5-dimethylfuran-3(2H)-one as internal standard. f Quantitation of 5-isopropyl-2-methylphenol was performed by using [2H7]-2-isopropyl-5-methylphenol as internal standard.

g

Quantitation of 1-

methoxy-4-(1-propenyl)benzene (anethole) was performed by using [2H3]-1-methoxy4-(2-propenyl)benzene ([2H3]-estragole) as internal standard. 2

h

Quantitation of

2-methylbutanal was performed by using [ H2]-3-methylbutanal as internal standard.

i

Quantitation of 2-methylbutanoic acid was performed by using [2H2]-3-methylbutanoic acid as internal standard.



Page 22 of 30

22 Table 2. Important Aroma-Active Compounds (FD factor ≥ 16) Identified in Succus Liquiritiae no.a

odorantb

odor qualityc

retention indices on DB-FFAP

DB-5

FDd

lit.e

1/2

2-/3-methylbutanal

malty

916

657

64

-

3

2,3-butanedione

buttery

1011

619

256

4

4

2,3-pentanedione

buttery

1060

705

16

-

5

1,8-cineole

eucalyptus-like

1189

1028

32

-

6

1-octen-3-onef

mushroom-like

1304

969

32

4

7

2-acetyl-1-pyrroline

popcorn-like

1330

919

512

4

8

dimethyl trisulfide

cabbage-like, sulfury

1367

969

16

-

9/10

2-ethyl-5(6)-methylpyrazine

nutty, roasty

1384

983

16

3

11/12 2-ethyl-3,5(6)-dimethylpyrazine

earthy, roasty

1442

1080

32

4

13

vinegar-like

1447

612

128

4

cooked potato-like

1451

905

128

-

14

acetic acid f


f

15

2-isobutyl-3-methoxypyrazine

earthy, green bell pepper-like

1500

1171

16

4

16

benzaldehyde

bitter almond-like

1524

990

32

3

17


fatty, cucumber-like

1595

1159

16

4

18

2-acetyl-5-methylfuran

roasty

1610

1038

16

3

19

butanoic acid

sweaty

1620

829

64

-

20

acetylpyrazine

roasty, sweet

1626

1023

64

-


Page 23 of 30


23 Table 2. Continued. retention indices on no.a

odorantb

odor qualityc

DB-FFAP

DB-5

FDd

lit.e

21/22 2-/3-methylbutanoic acid

sweaty

1663

872

128

-

23


fatty, green

1688

1206

16

-

24

γ-hexalactone

coconut-like

1693

864

16

-

25

pentanoic acid

sweaty

1728

920

64

-

26


fatty, deep-fried

1774

1304

32

4

27

(E)-β-damascenonef

cooked apple-like

1822

1410

128

4

28

anethole

aniseed-like

1838

1284

128

4

29

hexanoic acid

sweaty

1843

1033

64

3

30

2-methoxyphenol

gammon-like, smoky

1865

1090

1024

3

31

unknown

foxy, phenolic

1884

1244

16

-

32

β-ionone

flowery, violet-like

1943

1497

32

-

33

3-hydroxy-2-methyl-4H-pyran-4-one (maltol) caramel-like

1977

1109

1024

3

34

trans-4,5-epoxy-(E)-2-decenalf

metallic

2018

1380

16

-

35

γ-nonalactone

coconut-like

2029

1368

256

3

36


caramel-like

2035

1080

2048

-

37

octanoic acid

carrot-like, mouldy

2041

1132

64

3

38

4-methylphenol

fecal, horse stable-like

2065

1080

64

-

39

2,6-dichlorophenol

phenolic, leather-like

2100

1216

256

-



Page 24 of 30

24 Table 2. Continued. retention indices on a

no.

b

c

odorant

odor quality

DB-FFAP

DB-5

FDd

lit.e

40


thyme-like

2144

1290

32

4

41


thyme-like

2144

1310

32

3

42

3-hydroxy-4,5-dimethylfuran-2(5H)-onef

seasoning-like

2194

1105

1024

-

43

2,6-dimethoxyphenol

clove-like

2275

1397

64

-

44

unknown

phenolic

2362

nd

16

-

45

γ-dodecalactonef

peach-like

2369

1678

32

-

46

indole

fecal, mothball-like

2433

1294

32

4

47

coumarin

woodruff-like

2473

1449

16

-

48

phenylacetic acid

honey-like, beeswax-like

2565

1276

512

-

49


vanilla-like

2570

1404

1024

4

a

Odorants were consecutively numbered according to their retention indices on capillary DB-FFAP. b Odorant identified by comparison

of its odor quality and intensity, retention indices on capillaries DB-FFAP and DB-5 as well as mass spectra (EI and CI mode) with data of reference compounds. e

c

Odor quality perceived at sniffing port. d Flavor dilution factor determined by AEDA on capillary DB-FFAP.

Compound was firstly reported as volatile compound in heated licorice in the given reference. f No unequivocal mass spectrum (EI

mode) was obtained; identification based on remaining criteria in footnote b. nd: not determined.


Page 25 of 30


25 Table 3. Concentrations, Orthonasal Odor Thresholds, and Odor Activity Values (OAVs) of Key Aroma Compounds (OAV ≥ 1) in Succus Liquiritiae concentrationa odor threshold

compound

(µg/kg)


610

in water (µg/L) 0.49c

OAVb 1240

c

2,3-butanedione

664

1.0

664

2-methoxyphenol

407

0.84c

485

1,8-cineole

356

1.1c

324

2,6-dimethoxyphenol

6600

29

228


4980

53c

94


3630

54

67

2,3-pentanedione

202

3-hydroxy-2-methyl-4H-pyran-4-one

226000

γ-nonalactone

318


339

52

3.9 5000

45

9.7c

33

15c

23


0.10

0.0045c

22


0.72

0.033

22

phenylacetic acid

1150

(E)-β-damascenone

0.19

octanoic acid

2490

acetic acid

1284000

17

68 c

0.013 d

190

15 13

c

99000

13 c

2-methylbutanal

15.3

1.5

10


56.7

6.0c

9


0.75

0.08

9

3-methylbutanal

3.57

0.5c

7


1.73

0.28

6

1-octen-3-one

0.05

0.013c

4

linalool

1.83

0.58

3


0.07e

0.027c

3

hexanoic acid

11000

4800

2

butanoic acid

3750

2400c

2


0.05

Table 3. Continued. ACS Paragon Plus Environment

0.046

1


Page 26 of 30

26 Table 3. Continued. compound

concentrationa

odor threshold

(µg/kg)

in water (µg/L)

OAVb

γ-dodecalactone

0.43

0.43c

1


0.38

0.43c

Characterization of the Key Aroma Compounds in ... - ACS Publications

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