Aroma-Active Compounds in Bartlett Pears and Their Changes during

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Aroma-Active Compounds in Bartlett Pears and Their Changes During the Manufacturing Process of Bartlett Pear Brandy Bianca Zierer, Peter Schieberle, and Michael Granvogl J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04612 • Publication Date (Web): 16 Nov 2016 Downloaded from http://pubs.acs.org on December 1, 2016

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Journal of Agricultural and Food Chemistry

Aroma-Active Compounds in Bartlett Pears and Their Changes During the Manufacturing Process of Bartlett Pear Brandy

Bianca Zierer, 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]

ACS Paragon Plus Environment


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ABSTRACT: Application of aroma extract dilution analysis to Bartlett pears and the

2

fermented mash produced thereof revealed 24 and 34 aroma-active compounds in

3

the flavor dilution (FD) factor range between 8 and 8192. Twenty-eight compounds,

4

which have not been described before in Bartlett pears or in fermented pear mash,

5

were identified. While ethyl (E,Z)-2,4-decadienoate (pear-like, metallic odor

6

impression), hexyl acetate (green, fruity), and acetic acid (vinegar-like) showed the

7

highest concentrations in Bartlett pears, ethanol (ethanolic), acetic acid, 3-methyl-1-

8

butanol (malty), 1-hexanol (grassy, marzipan-like), (S)-2- and 3-methylbutanoic acid

9

(sweaty), and 2-phenylethanol (flowery, honey-like) were present at the highest

10

amounts in the fermented mash. The key aroma compounds were quantitated in

11

each pear brandy production step (pears, fermented mash, distillate, and aged

12

distillate) by stable isotope dilution analysis showing a clear influence of each step on

13

the overall aroma of the spirit and, consequently, revealing clearly changing

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concentrations (e.g., of ethyl (S)-2-methylbutanoate, (E)-β-damascenone, ethyl (E,Z)-

15

2,4-decadienoate, and ethyl (E,E)-2,4-decadienoate) and different aroma perceptions

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during the manufacturing process. In addition, the concentrations of the so-called

17

“pear esters” ethyl (E,Z)-2,4-decadienoate and ethyl (E,E)-2,4-decadienoate were

18

determined in 6 different pear varieties (Abate Fetel, Anjou, Bartlett, Forelle, Kaiser

19

Alexander, and Packham´s Triumph) clearly demonstrating the aroma potential of the

20

variety Bartlett, which is mostly used for brandy production due to the high amounts

21

of both esters eliciting a typical pear-like odor impression.

22 23

KEYWORDS: Bartlett pears, Bartlett pear brandy, production, fermentation,

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distillation, aging, aroma extract dilution analysis, stable isotope dilution analysis

25 26 ACS Paragon Plus Environment

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INTRODUCTION

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The identification of volatile compounds in Bartlett pears and its products, e.g.,

29

Bartlett pear brandy, has been of interest in several studies during the last 50 years

30

due to their pleasant and intensive aroma. Thereby, especially esters of (E,Z)-2,4-

31

decadienoic acid were postulated as character impact compounds.1 In the past, 85

32

volatiles in Bartlett pears and 106 volatile compounds in Bartlett pear brandy have

33

been identified, including several esters and alcohols.2 Suwanugal et al.3 analyzed

34

the volatile compounds in eight pear varieties, e.g., Bartlett, Packham´s Triumph,

35

Anjou, and Forelle, identifying methyl and ethyl esters of 2,4-decadienoic acid in

36

each

37

characterization of the most aroma-active compounds of two different Bartlett pear

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brandies with a clearly different aroma applying the molecular sensory science

39

concept4 was only performed once.5 Thereby, 44 aroma-active compounds were

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quantitated by stable isotope dilution assays resulting in clear differences in the

41

concentrations of several key aroma compounds and, consequently, in odor activity

42

values, e.g., for ethyl (E,Z)-2,4-decadienoate, ethyl (E,E)-2,4-decadienoate, and (E)-

43

β-damascenone.

variety,

but

with

clearly

different

amounts.

However,

a systematic

44

It is well-known that beside the raw material also the manufacturing process

45

influences the overall aroma of a final spirit. Bricout6 identified and compared the

46

volatiles of fresh Bartlett pears with those of Bartlett pear brandies. The formation of

47

13 volatiles during the fermentation step, such as isopentanol, 1-hexanol, 2-

48

methylbutanoic acid, butanoic acid, and 2-phenylethanol was shown. Later, Meinl7

49

analyzed the changes of several volatile compounds, such as 3-methylbutyl acetate

50

and 2-phenylethanol, during fruit brandy production. However, up to now, no data on

51

concentrations changes of important key aroma compounds during the single

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production steps of Bartlett pear brandy are available. ACS Paragon Plus Environment


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Therefore, the aim of the present study was to analyze the influence of the

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manufacturing process on the aroma of Bartlett pear brandy using the molecular

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sensory science concept4 including (i) the identification of the key odorants in all

56

production steps (pears, mash, distillate, and aged distillate) by aroma extract dilution

57

analysis (AEDA) in combination with gas chromatography-mass spectrometry and (ii)

58

the quantitation of the most potent odorants by stable isotope dilution assays (SIDAs)

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in pears, mash, distillate, and aged distillate. These data at hand will provide

60

producers the knowledge about possibilities to positively influence the overall aroma

61

of the final brandy.

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MATERIALS AND METHODS

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Bartlett Pears, Fermented Mash, Distillate, and Aged Distillate. The pear

64

varieties Abate Fetel, Anjou, Forelle, Kaiser Alexander, and Packham´s Triumph

65

were purchased from a local farmers market. Bartlett pears (originating from South

66

Tyrol, Italy), fermented mash, distillate, and aged distillate were obtained from a

67

small-sized premium distillery in Bavaria. For fermentation, water, sulfuric acid (to

68

avoid growth of undesired microorganisms), and a pure cultured yeast strain were

69

added to the crushed pears. Distillation was performed after a fermentation time of

70

two weeks. The distillate obtained containing the heart fraction was then stored in

71

special clay jugs for 2 years.

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Pears were pitted, cut into pieces, frozen in liquid nitrogen, vacuum-tight packed,

73

and stored at -25 °C prior to analysis. The fermented mash was diluted 1+1 (v+v)

74

with a saturated aqueous calcium chloride solution to prevent further enzymatic

75

reactions and was also stored at -25 °C. Distillate and aged distillate were stored at

76

room temperature in the dark.

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Chemicals. The following reference compounds were commercially obtained:

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acetic acid, allyl-2-methoxyphenol (eugenol), 2,3-butanedione, butyl acetate, (E,E)ACS Paragon Plus Environment

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2,4-decadienal, γ-decalactone, decanoic acid, (E)-2-decenal, 1,1-diethoxyethane,

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2,6-dimethoxyphenol (syringol), ethyl (E)-cinnamate, ethyl (E,Z)-2,4-decadienoate,

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ethyl hexanoate, 4-ethyl-2-methoxyphenol (4-ethylguiacol), ethyl 2-methylbutanoate,

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ethyl (S)-2-methylbutanoate, ethyl methylpropanoate, ethyl octanoate, ethyl 3-phenyl-

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propanoate, hexanoic acid, 1-hexanol, (Z)-3-hexenal, hexyl acetate, 3-hydroxy-4,5-

84

dimethylfuran-2(5H)-one, linalool, 2-methoxyphenol (guaiacol), 2-methylbutanal,

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3-methylbutanal, 2-methylbutanoic acid, (S)-2-methylbutanoic acid, 3-methylbutanoic

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acid, 2-methyl-1-butanol, 3-methyl-1-butanol, 3-methylbutyl acetate, 4-methylphenol

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(p-cresol), methylpropanol, 3-(methylthio)propanal, 3-(methylthio)propanol, (E,E)-2,4-

88

nonadienal, γ-nonalactone, (E)-2-nonenal, phenylacetaldehyde, phenylacetic acid, 2-

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phenylethanol, 2-phenylethyl acetate, 3-phenylpropanoic acid, and 4-propyl-2-

90

methoxyphenol (4-propylguaiacol) (Sigma-Aldrich Chemie, Taufkirchen, Germany);

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butanoic acid, ethanol, and 4-hydroxy-3-methoxybenzaldehyde (vanillin) (VWR

92

International, Darmstadt, Germany); 4-hydroxy-2,5-dimethylfuran-3(2H)-one (Fluka,

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Sigma-Aldrich), and 1-octen-3-one (Alfa Aesar, Karlsruhe, Germany). 1-(2,6,6-

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Trimethyl-1,3-cyclohexadien-1-yl)-2-buten-1-one ((E)-β-damascenone) was kindly

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provided by Symrise (Holzminden, Germany).

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The following reference compounds were synthesized as previously reported:

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ethyl (E,E)-2,4-decadienoate,5 2-methoxy-4-methylphenol (2-methoxy-p-cresol, 4-

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methylguaiacol),8 and (Z)-1,5-octadiene-3-one.9

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Calcium chloride and sodium sulfate were from VWR International. Liquid

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nitrogen was from Linde (Munich, Germany). Dichloromethane, diethyl ether, and

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pentane (Merck, Darmstadt) were freshly distilled prior to use.

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Stable Isotopically Labeled Standards. The following stable isotopically labeled

103

standards were synthesized according to the literature: [13C4]-2,3-butanedione,10

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[2H2]-butanoic acid,11 [2H4-7]-(E)-β-damascenone;12 [2H2-4]-(E,E)-2,4-decadienal,13 ACS Paragon Plus Environment


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[2H2]-γ-decalactone,14 [13C2]-1,1-diethoxyethane,15 [2H3]-ethyl butanoate,10 [2H5]-ethyl

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(E)-cinnamate,15 [2H2]-ethyl (E,Z)-2,4-decadienoate,5 [2H3]-ethyl hexanoate,15 [2H2-4]-

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4-ethyl-2-methoxyphenol,16 [2H3]-ethyl 2-methylbutanoate,17 [2H3]-ethyl 3-methylbuta-

108

noate,17

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hexanal,19 [2H4]-1-hexanol,19 [2H2]-(Z)-3-hexenal,13 [2H3]-hexyl acetate,20 [2H3]-4-

110

hydroxy-3-methoxybenzaldehyde,21

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nol,23

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acid,26 [2H2]-3-methyl-1-butanol,21 [2H2]-3-methyl butylacetate,27 [2H2]-(E,E)-2,4-nona-

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dienal,13 [2H2]-γ-nonalactone,14 [2H2]-(E)-2-nonenal,13 [2H2-3]-1-octen-3-one,28 [13C2]-

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phenylacetaldehyde,29 [13C2]-2-phenylethanol,29 [13C2]-2-phenylethyl acetate,15 and

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[2H2-4]-4-propyl-2-methoxyphenol.18

116 117

[2H5]-ethyl methylpropanoate,17 [2H5]-ethyl 3-phenylpropanoate,18 [2H4]-

[2H3]-2-methoxyphenol,24

[2H2]-linalool,22

[2H3]-2-methoxy-4-methylphe-

[2H2]-3-methylbutanal,25

[2H2]-3-methylbutanoic

Concentrations of the isotopically labeled standards were determined as previously described.30

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Isolation of the Volatiles. Bartlett Pears and Corresponding Fermented Mash.

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An aliquot of frozen Bartlett pear pieces (150 g) were ground by means of a

120

commercial blender. To inhibit enzymatic reactions, saturated calcium chloride

121

solution was mixed 1+1 by vol. with the obtained pear mash as well as with an aliquot

122

of the fermented pear mash obtained from the distillery (150 mL). To both samples,

123

dichloromethane (150 mL) was added and the mixtures were stirred for 1 h at room

124

temperature. For separation of the aqueous and the organic layer, each mixture was

125

centrifuged (4500 rpm, 15 min, 20 °C; centrifuge GR 412; Jouan, Unterhaching,

126

Germany). After separation of the organic phase, the extraction was repeated. Then,

127

the organic phases were combined, dried over anhydrous sodium sulfate, and the

128

volatiles were isolated by means of the solvent assisted flavor evaporation (SAFE)

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technique.31 The extract was concentrated to ~2 mL using a Vigreux column (50 cm x

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1 cm i.d.) and, finally, to ~500 µL by microdistillation.32 ACS Paragon Plus Environment

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Distillate and Aged Distillate. For isolation of the volatiles in the distillate and the

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aged distillate, water (25 mL) was added to an aliquot (25 mL) of the samples. The

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volatiles of the diluted samples were extracted with diethyl ether (3 x 50 mL), the

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combined organic phases were washed with distilled water (3 x 50 mL), and,

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afterward, the extracts were dried over anhydrous sodium sulfate. After solvent

136

assisted flavor evaporation (SAFE),31 the distillates obtained were concentrated as

137

described above.

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

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determination of linear retention indices (RIs)33 for each odorant was performed as

140

recently described.34

141

Aroma Extract Dilution Analysis (AEDA). For AEDA, pears, mash, distillate,

142

and aged distillate were extracted and the volatiles were isolated by SAFE

143

distillation31 as described above. The distillates obtained were concentrated to the

144

same final volume (500 µL) and diluted stepwise 1+1 (v+v) with dichloromethane.

145

The original distillate and each dilution were analyzed by HRGC-O to determine the

146

flavor dilution (FD) factors. The original distillate was analyzed by three experienced

147

panelists to avoid overlooking of odor-active compounds.

148 149

High-Resolution Gas Chromatography-Mass Spectrometry (HRGC-MS) for Identification. HRGC-MS for identification was performed as recently described.35

150

Quantitation by Stable Isotope Dilution Analysis (SIDA). To aliquots of the

151

pears and fermented pear mash (1 - 250 g) as well as of the distillate and the aged

152

distillate (0.5 - 90 mL), the stable isotopically labeled internal standards were added

153

(amounts depending on the concentration of the analytes determined in preliminary

154

experiments). After stirring for 30 min at room temperature for equilibration, the

155

workup procedure was performed as described above for the isolation of the volatiles



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prior to HRGC-MS or two-dimensional high-resolution gas chromatography-mass

157

spectrometry (HRGC/HRGC-MS).

158

For determination of the respective response factor, mixtures of known amounts

159

of the unlabeled analyte and the labeled internal standard in five different ratios (5:1,

160

3:1, 1:1, 1:3, 1:5) were analyzed in the same way.

161

High-Resolution Gas Chromatography-Mass Spectrometry for Quantitation.

162

Quantitation of butanoic acid, decanoic acid, (S)-2- and 3-methylbutanoic acid, 2-

163

and 3-methyl-1-butanol, and 2-phenylethanol was performed using a gas chromato-

164

graph type 431 (Varian, Darmstadt) equipped with a DB-FFAP column (30 m x 0.25

165

mm i.d., 0.25 µm, J&W Scientific; Agilent Technologies, Waldbronn, Germany)

166

coupled to a 220 ion trap mass spectrometer (Varian).35

167

Two-Dimensional High-Resolution Gas Chromatography-Mass Spectro-

168

metry (HRGC/HRGC-MS). In the case of a trace compound being overlapped by a

169

major volatile, HRGC/HRGC-MS was performed by a TRACE 2000 series GC

170

(ThermoQuest) equipped with a DB-FFAP column (30 m x 0.32 mm i.d., 0.25 µm film

171

thickness, J&W Scientific) coupled to a CP 3800 gas chromatograph (Varian)

172

equipped with an OV-1701 column (30 m x 0.25 mm i.d., 0.25 µm film thickness,

173

J&W Scientific) as recently described.35

174

Enzymatic Quantitation of Ethanol. Ethanol was enzymatically determined

175

using an enzyme kit (R-biopharm, Darmstadt). All samples were analyzed according

176

to the instructions of the manufacturer using an UV-VIS photometer (UV-2401PC UV-

177

VIS; Shimadzu, Duisburg, Germany).

178

Separation of Ethyl (R)-2- and Ethyl (S)-2-Methylbutanoate, (R)- and (S)-

179

Linalool as well as (R)-2- and (S)-2-Methylbutanoic Acid. Enantiomers of ethyl 2-

180

methylbutanoate,36 linalool,22 and 2-methylbutanoic acid36 were separated by

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HRGC/HRGC-MS using a DB-FFAP column (30 m x 0.32 mm i.d., 0.25 µm film ACS Paragon Plus Environment

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thickness; J&W Scientific) in the first dimension and a chiral BGB-176 column (30 m

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x 0.25 µm i.d., 0.25 µm film thickness; BGB Analytik, Böckten, Switzerland) in the

184

second dimension.

185

Aroma Profile Analysis (APA). For APA, a sensory panel rated the intensities of

186

selected odor attributes on a linear seven-point scale in steps of 0.5 from 0 (not

187

perceivable) to 3 (strongly perceivable). The following reference compounds were

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selected to define the descriptors: fruity (ethyl (S)-2-methylbutanoate), pear-

189

like/metallic

190

(hexanal), smoky/clove-like (2,6-dimethoxyphenol), sweaty (3-methylbutanoic acid),

191

malty (3-methyl-1-butanol), sour (acetic acid), flowery/honey-like (2-phenylethanol),

192

and baked apple-like ((E)-β-damascenone). The panel consisted of 20 experienced

193

assessors participating in weekly sensory sessions to train their abilities to recognize

194

and describe different aroma qualities. The samples (15 mL) were presented in

195

covered glass vessels (40 mm i.d., total volume = 45 mL). Sensory experiments were

196

performed in a sensory room at 21 ± 1 °C equipped with single booths.37

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RESULTS AND DISCUSSION

(ethyl

(E,Z)-2,4-decadienoate),

ethanolic

(ethanol),

green/grassy

198

To compare the overall aroma during the processing steps, aroma profile

199

analyses of pears, fermented mash, and distillate were performed (Figure 1). As

200

expected, the ethanolic odor quality showed no intensity in the pears, clearly

201

increased in the fermented mash, and showed the highest intensity in the distillate. A

202

clear difference was observed for the fruity, malty, green, sweaty, and sour attributes.

203

The pear-like odor quality was evaluated with the highest intensity in the fruits and in

204

the distillate, but at a lower level in the fermented mash.

205

Identification of Odorants in Bartlett Pears. Ethyl (E,Z)-2,4-decadienoate and

206

ethyl (E,E)-2,4-decadienoate have previously been described as character impact

207

compounds of Bartlett pears, which are well-known to have a pleasant and intense ACS Paragon Plus Environment


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aroma and, thus, are famous for pear brandy production.5 This fact was corroborated

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in the actual study by quantitation of both so-called “pear esters”, eliciting a typical

210

pear-like odor impression, in six different varieties. Bartlett pears showed the highest

211

amount of 17800 µg/kg for both isomers, whereas in Abate Fetel and Kaiser

212

Alexander only 21 µg/kg and 20 µg/kg, respectively, were present, clearly proving the

213

aroma potential of the variety Bartlett (Table 1).

214

Next, the sensomics approach4 was applied to all samples to systematically

215

identify the key odorants contributing to the overall aroma. First, the volatile fractions

216

of Bartlett pears, mash, distillate, and aged distillate were extracted with

217

dichloromethane, followed by SAFE distillation.31 The distillates obtained were

218

evaluated on a strip of filter paper and elicited the typical aroma qualities of the

219

original samples indicating a successful extraction and cleanup of the odorants. Next,

220

the most aroma-active compounds were located by aroma extract dilution analysis

221

(AEDA) using high-resolution gas chromatography-olfactometry (HRGC-O).

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Twenty-four aroma-active compounds were detected in the flavor dilution (FD)

223

factor range of 8 to 1024. Among them, the highest FD factor of 1024 was obtained

224

for 17 (cooked potato-like odor impression), followed by 21a, b (sweaty) and 24

225

(baked apple-like, grape juice-like; both FD factor of 512), 30 (metallic), 32 (coconut-

226

like; both FD factor of 256), 4 (fruity), and 14 (metallic, geranium-like; both FD factor

227

of 128) (Table 2).

228

For identification, odor quality and odor intensity at the sniffing port, retention

229

indices on two stationary GC phases of different polarities, and mass spectra

230

generated in EI and CI mode were compared with the data of our in-house database

231

containing ~1000 aroma-active reference compounds. Following this procedure,

232

compounds 17, 21a, b, and 24 were identified as 3-(methylthio)propanal, (S)-2- and

233

3-methylbutanoic acid, and (E)-β-damascenone. The enantiomeric ratio of 2ACS Paragon Plus Environment

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methylbutanoic acid was determined to be >99% of the (S)-enantiomer. Further

235

compounds also high in FD factors were ethyl 2-methylbutanoate (4) with an

236

enantiomeric ratio of 90/10 (S/R), (Z)-1,5-octadien-3-one (14), trans-4,5-epoxy-(E)-2-

237

decenal (30), and γ-nonalactone (32) (Table 2).

238

The compound eliciting a pear-like, metallic aroma was identified as ethyl (E,Z)-

239

2,4-decadienoate (25). Besides, further esters with a fruity odor impression were

240

determined, e.g., butyl acetate (5), 3-methylbutyl acetate (8), ethyl hexanoate (11),

241

and hexyl acetate (12).

242

Identification of Odorants in Fermented Pear Mash. The manufacturing

243

process was performed by a small-sized premium distillery in Bavaria. Therefore, 5.5

244

L of water and sulfuric acid were added to 20 kg of the analyzed pears and the

245

mixture was fermented for two weeks. At the end of the fermentation, the mash was

246

used for the characterization of the aroma-active compounds. AEDA in combination

247

with identification experiments revealed ethanol (2), ethyl 2-methylbutanoate (4), 2-

248

and 3-methyl-1-butanol (10a, b), acetic acid (16), (S)-2- and 3-methylbutanoic acid

249

(21a, b), 2-phenylethanol (28), and phenylacetic acid (41) as important aroma

250

compounds. Clear changes in FD factors gave first insights into the formation or

251

degradation of aroma-active compounds during fermentation (Table 2).

252

It is well-known, that besides ethanol further odorants, such as 2- and 3-methyl-1-

253

butanol, 2-phenylethanol, phenylacetaldehyde, and phenylacetic acid, are formed

254

during fermentation due to yeast activity, according to the Ehrlich pathway.38 In

255

addition, 1-hexanol,39 short-chain fatty acids, and esters40 arise. However, the FD

256

factors of some odorants, e.g., 3-(methylthio)propanal (17), decreased.

257

Quantitation of Important Aroma-Active Compounds in Bartlett Pears and

258

the Corresponding Fermented Mash. AEDA is a screening method to reduce the

259

huge number of volatiles to a limited set of key aroma-active compounds, which have ACS Paragon Plus Environment


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to be quantitated for the characterization of the overall aroma of a certain food. Thus,

261

a total of 24 aroma-active compounds, proven to be important for the aroma of the

262

final spirit,5 were quantitated in pears and fermented mash by means of stable

263

isotope dilution assays (SIDAs) and, in addition, ethanol via photometric detection

264

using an enzyme kit.

265

During fermentation, the concentrations of (S)-2- and 3-methylbutanoic acid

266

(Bartlett pears (BP): 235 µg/kg vs fermented mash (FM): 11500 µg/1.275 kg), acetic

267

acid (6410 µg/kg vs 244000 µg/1.275 kg) as well as the corresponding esters 3-

268

methylbutyl acetate (8.16 µg/kg vs 368 µg/1.275 kg) and ethyl 2-methylbutanoate

269

(14.4 µg/kg vs 40.9 µg/1.275 kg) clearly increased (Table 3), which was in

270

accordance to previous studies.30,39,40 Additionally, the concentrations of γ-

271

decalactone (2.19 µg/kg vs 10.9 µg/1.275 kg) and γ-nonalactone (2.87 µg/kg vs 4.65

272

µg/1.275 kg) increased. A formation of γ-lactones from long-chain unsaturated fatty

273

acids dependent on the used yeast variety was already discussed.41

274

Clearly lower concentrations were detected for ethyl (E,Z)-2,4-decadienoate

275

(14500 µg/kg vs 1810 µg/1.275 kg), ethyl (E,E)-2,4-decadienoate (3300 µg/kg vs 134

276

µg/1.275 kg), and hexyl acetate (10900 µg/kg vs 481 µg/1.275 kg). The

277

concentrations of 2-methylbutanal (11.3 µg/kg vs 5.97 µg/1.275 kg), 3-methylbutanal

278

(36.1 µg/kg vs 13.8 µg/1.275 kg), and (E,E)-2,4-decadienal (2.08 µg/kg vs 0.69

279

µg/1.275 kg) also decreased (Table 3). An unspecific reduction of (E,E)-2,4-

280

decadienal and 3-methylbutanal during a model fermentation of whisky mash was

281

previously reported.42 All in all, quantitation experiments showed a clear influence of

282

the fermentation on important odorants confirming the sensorial experiments (Figure

283

1).

284

Influence of the Distillation on Aroma-Active Compounds of Fermented Pear

285

Mash. Based on the comparison of the aroma profile analyses of the fermented ACS Paragon Plus Environment

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13 286

mash and the corresponding distillate, the influence of the distillation to the overall

287

aroma was also evident. While the odor impressions sour, malty, and sweaty were

288

dominant in the mash and nearly not perceivable in the distillate, the odor qualities

289

pear-like, ethanolic, fruity, and baked apple-like were the most intense in the distillate

290

(Figure 1).

291

During distillation, the concentrations of both isomers of ethyl 2,4-decadienoate

292

increased. However, a clearly higher increase was detected for ethyl (E,E)-2,4-

293

decadienoate (FM: 134 µg/1.275 kg vs distillate (D): 2080 µg/0.105 L) compared to

294

ethyl (E,Z)-2,4-decadienoate (1810 µg/1.275 kg vs 8860 µg/0.105 L) (Table 4). Thus,

295

during distillation a heat-induced isomerization occurred, which was already reported

296

by Gordienko et al.43 investigating the thermally induced conversion of various

297

unsaturated esters in (E,Z)-configuration into the more stable (E,E)-isomer. The

298

increase of both esters was also confirmed in the present by a lab-scale model

299

experiment simulating the distillation process. Therefore, water, diluted sulfuric acid,

300

and [2H2]-ethyl (E,Z)-2,4-decadienoate as internal standard were added to a Bartlett

301

pear mash, and the mixture was heat-processed for 1 h under reflux. Quantitation of

302

Bartlett pear esters before and after thermal influence confirmed the concentration

303

increase already seen during distillation in the real process, which might be explained

304

by a heat-induced release of ethyl (E,Z)-2,4-decadienoate and ethyl (E,E)-2,4-

305

decadienoate encased in the pear paring during distillation. Meinl7 already described

306

an inhomogeneous distribution of both isomers in Bartlett pears and higher

307

concentrations in the pear paring compared to the core.

308

The distillation process had also a decisive effect on the concentrations of other

309

key odorants leading to an increase of phenylacetaldehyde, 1,1-diethoxyethane, (E)-

310

β-damascenone,

311

methylbutanal (Table 4). To explain the formation of 1,1-diethoxyethane, Vocke42

(E,E)-2,4-decadienal,

linalool,

2-methylbutanal,


and

3-


Page 14 of 38

14 312

described a model distillation of an ethanolic solution containing acetic acid and

313

acetaldehyde. Although the requirement for an acetalization was given in this

314

experiment, no additional 1,1-diethoxyethane was found. Thus, it can be assumed

315

that 1,1-diethoxyethane might be formed from precursors or catalysts present in the

316

pear mash.

317

The clear increase of (E)-β-damascenone during distillation of the fermented pear

318

mash can be explained from glycosidically bound precursors in the fruit, which are

319

able to release the aroma compound under acid catalyzed and heat-induced

320

conditions44 and corroborates recently reported data during rum production.30

321

The increase of (E,E)-2,4-decadienal can be explained by autoxidation of linoleic

322

acid, possible under the conditions applied.45 Vocke42 also observed a clear increase

323

of (E,E)-2,4-decadienal after distillation of the mash during whisky production.

324

Due to the elevated temperature, the amounts of the Strecker aldehydes 2- and

325

3-methylbutanal as well as phenylacetaldehyde, formed by decarboxylation and

326

oxidative deamination of the respective amino acids, also increased. Münch and

327

Schieberle46 studied thermally treated yeast extracts and found high levels of these

328

aldehydes.

329

However, also lower concentrations of aroma-active compounds were analyzed,

330

e.g., for 2-phenylethanol, vanillin, and (S)-2- and 3-methylbutanoic acid (Table 4).

331

Odorants with a clearly higher or lower boiling point compared to ethanol might be

332

discriminated in the distillate and may cause a loss of aroma. By means of a model

333

distillation, it was shown that especially vanillin and 2-phenylethanol showed this

334

phenomenon.42

335

Influence of the Aging on Important Aroma-Active Compounds. After

336

distillation, the heart fraction was stored in special clay jugs and was analyzed after a

337

period of 2 years. Especially the amounts of ethyl esters, such as ethyl 2ACS Paragon Plus Environment

Page 15 of 38


15 338

methylbutanoate, ethyl 3-phenylpropanoate, ethyl hexanoate, and ethyl (E,E)-2,4-

339

decadienoate increased during aging (Table 5). Edwards et al.47 already described

340

esterification reactions of fatty acids with alcohols, especially ethanol, during the

341

aging of wine, leading to higher concentrations of ethyl esters. Surprisingly, a

342

sensory evaluation revealed no changes of the wine. In a comparable study on rum,

343

Franitza et al.30 also reported about an increase of several ethyl esters during aging

344

of three years. A continuous increase of ethyl esters in a model solution simulating

345

aging conditions was also shown for whisky.42

346

The concentrations of both so-called pear esters ethyl (E,E)-2,4-decadienoate

347

and ethyl (E,Z)-2,4-decadienoate increased during aging, however, ethyl (E,E)-2,4-

348

decadienoate showed a clearly higher increase. In addition to the already mentioned

349

thermally induced conversion into the more stable (E,E)-isomer,43 a conversion under

350

aging conditions was also previously described.48

351

1,1-Diethoxyethane showed an increase by a factor of 1.5. Brandes et al.49

352

described its formation during aging of plum and apricot brandies in dependency of

353

pH value and temperature.

354

In contrast, the concentrations of 2,3-butanedione, (E)-2-nonenal, and (E,E)-2,4-

355

nonadienal decreased during aging, which was already shown for the aging of

356

whisky.42

357

These results proved that the aging step also influenced the concentrations of key

358

odorants, although to a lesser extent compared to the fermentation and distillation

359

process. The final Bartlett pear brandy will be obtained by addition of water, adjusting

360

the desired alcohol content of 40% by vol.

361

In conclusion, a total of 28 aroma-active compounds were characterized in the

362

volatile fraction of Bartlett pears and fermented Bartlett pear mash for the first time.

363

The quantitative studies clearly demonstrated the importance and the influence of ACS Paragon Plus Environment


Page 16 of 38

16 364

every single production step on the overall aroma of the final spirit. These data

365

facilitate knowledge about the aroma changes occurring during each step and, thus,

366

manufacturers will have possibilities to optimize their product in regard to its final

367

aroma, which is an important quality criterion for consumers.


Page 17 of 38


17

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(2)

Nijssen L.; Maarse H.; Ingen-Visher C. A. VFC – Volatile compounds in foods. Zeist (The Netherlands): TNO Quality of Life, Version 16.1, 1963-2016 (database).

(3)

Suwanagul, A.; Richardson, D. G. Identification of headspace volatile compounds from different pear (Pyrus communis L.) varieties. VIIth International Symposium on Pear Growing. Acta Hortic. 1998, 475, 599-603.

(4)

Schieberle, P.; Hofmann, T. Mapping the combinatorial code of food flavors by means of molecular sensory science approach. In Chemical and Functional Properties of Food Components Series. Food Flavors. Chemical, Sensory and Technological Properties; Jelen, H., Ed.; CRC Press: Boca Raton, FL, 2012; pp 413-438.

(5)

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.

(6)

Bricout, J. The aromatic constituents in pear brandies (in French). Ind. Alim. Agric. 1977, 94, 277-281.

(7)

Meinl, J. Changes of Volatile Compounds during the Processing Steps of Fruit Brandy Production (in German). Ph.D. thesis, Technical University of Munich, Munich, Germany, 1995.

(8)

Arnarp, J.; Bielawski, J.; Dahlin, B. M.; Dahlman, O.; Enzell, C. R.; Petterson, T. Alkyl and alkenyl substituted guaiacols found in cigarette smoke condensate. Acta Chem. Scand. 1989, 43, 44-50.



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(10) 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. (11) Schieberle, P.; Gassenmeier, K.; Guth, H.; Sen, A.; Grosch, W. Character

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flavour compounds using a stable isotope dilution assay. Lebensm.-Wiss. Technol. 1990, 26, 513-522. (14) 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. (15) Guth, H. Quantification and sensory studies of character impact odorants of

different white wine varieties. J. Agric. Food Chem. 1997, 45, 3027-3032. (16) Semmelroch, P.; Laskawy, G.; Blank, I.; Grosch, W. Determination of potent

odorants in roasted coffee by stable isotope dilution assay. Flavor Fragrance J. 1995, 10, 1-7. (17) Guth, H.; Grosch, W. Quantitation of potent odorants of virgin olive oil by stable

isotope dilution assays. J. Am. Oil Chem. Soc. 1993, 70, 513-518.


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19 (18) Schmitt, R. On the Role of Ingredients as Sources of Key Aroma Compounds in

Crumb Chocolate. Ph.D. thesis, Technical University of Munich, Munich, Germany, 2005. (19) 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. (20) Fuhrmann, E.; Grosch, W. Character impact odorants of the apple cultivars

Elstar and Cox Orange. Nahrung/Food 2002, 46, 187-193. (21) Schieberle, P. Primary odorants of pale lager beer. Z. Lebensm.-Unters. Forsch.

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in beer using a solid-phase microextraction (SPME) in combination with stable isotope dilution assay (SIDA). J. Agric. Food Chem. 2003, 51, 7100-7105. (23) Frauendorfer, F.; Schieberle, P. Identification of the key aroma compounds in

cocoa powder based on molecular sensory correlations. J. Agric. Food Chem. 2006, 54, 5521-5529. (24) Czerny, M.; Grosch, W. Quantification of character-impact odour compounds of

roasted beef. Z. Lebensm.-Unters. Forsch. 1993, 196, 417-422. (25) 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. (26) Guth, H.; Grosch, W. Identification of the character impact odorants of stewed

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wheat bread (French-type) - influence of pre-ferments and studies on the formation of key odorants during dough processing. Z. Lebensm.-Unters. Forsch. 1995, 201, 241-248. (28) 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. (29) Schuh, C.; Schieberle, P. Characterization of the key aroma compounds in the

beverage prepared from Darjeeling black tea: quantitative differences between tea leaves and infusion. J. Agric. Food Chem. 2006, 54, 916-924. (30) 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. (31) 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. (32) 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. (33) Van den Dool, H.; Kratz, P. D. A generalization of the retention index system

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Kralj Cigić, I.; Zupančič-Kralj, L. Changes in odour of Bartlett pear brandy influenced by sunlight irradiation. Chemosphere 1999, 38, 1299-1303.

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Brandes, W.; Karner, M.; Eder, R. Rate-determining parameters and sensory impact of the formation of 1,1-diethoxyethane during storage of distillates (in German). Mitt. - Hoehere Bundeslehr- Versuchsanst. Wein- Obstbau, Klosterneuburg 2005, 55, 76-84.


Page 23 of 38


23

FIGURE CAPTIONS Figure 1. Aroma profile analysis of Bartlett pears (solid line), fermented Bartlett pear mash (broken line), and distillate (dotted line).



Page 24 of 38

24

Table 1. Concentrations of Ethyl (E,Z)-2,4-decadienoate (EZD) and Ethyl (E,E)-2,4decadienoate (EED) in Different Pear Cultivars concentrations (µg/kg)a pear cultivar EZD Bartlett Packham´s Triumph Anjou

a

14500

EED 3300

1570

84.2

644

32.2

Forelle

19.1

7.31

Abate Fetel

15.8

5.09

Kaiser Alexander

14.1

5.79

Mean values of triplicates, differing not more than 15%.


Page 25 of 38


25

Table 2. Most Aroma-Active Compounds (FD ≥ 8) in the Aroma Distillate of Pitted Bartlett Pears (BP) and Fermented Bartlett Pear Mash (FM) RId on a

no.

b

compound

FD factorse in

c

odor quality

DB-FFAP

DB-5

BP

FM

1

3-methylbutanal

malty

967

658

32

32

2

ethanol

ethanolic

984

Aroma-Active Compounds in Bartlett Pears and Their Changes during

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