Characterization of the Key Odorants in Commercial Cold-Pressed

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Characterization of the Key Odorants in Commercial Cold-Pressed Oils from Unpeeled and Peeled Rapeseeds by the Sensomics Approach Gwendola Pollner, and Peter Schieberle J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05321 • Publication Date (Web): 21 Dec 2015 Downloaded from http://pubs.acs.org on December 29, 2015

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Journal of Agricultural and Food Chemistry

Characterization of the Key Odorants in Commercial Cold-Pressed Oils from Unpeeled and Peeled Rapeseeds by the Sensomics Approach

Gwendola Pollner, Peter Schieberle*

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

* Corresponding author Phone +49 8161 712932 Fax +49 8161 712970 E-mail [email protected]

ACS Paragon Plus Environment


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1

ABSTRACT. By application of the aroma extract dilution analysis (AEDA) on the volatile

2

fraction isolated from commercial cold-pressed rapeseed oil prepared from unpeeled seeds,

3

35 odor-active constituents in the flavor dilution (FD) factor range of 8 – 8192 were

4

detected. The identification experiments showed that the earthy, pea-like smelling 2-

5

isopropyl-3-methoxypyrazine showed the highest FD-factor of 8192, followed by 1-octene-

6

3-one (FD 4096) and (E,Z)-2,6-nonadienal with an FD of 2048. After quantitation of the 16

7

key odorants showing FD-factors ≥ 32 by stable isotope dilution assays and a determination

8

of their odor thresholds in deodorized sunflower oil, odor activity values (OAV; ratio of

9

concentration to odor threshold) could be calculated. The results indicated 2-isopropyl-3-

10

methoxypyrazine,

11

(cucumber-like) with the highest OAVs. To confirm that the key aroma compounds were

12

correctly identified and quantitated, a recombination experiment was performed by mixing

13

the reference odorants in the same concentrations as they occurred in the rapeseed oil

14

using odorless sunflower oil as the matrix. The recombinate showed a very good

15

agreement with the overall aroma of the oil. In a commercial rapeseed oil prepared from

16

peeled seeds, the same odorants were identified, however, in particular the FD factor of

17

dimethylsulfide (DMS) was clearly higher. Quantitation of DMS in ten commercial rapeseed

18

oils from either peeled and unpeeled seeds revealed significant differences in DMS, but no

19

influence of the peeling process on the amounts of DMS was found. The data can serve as

20

a basis for the quality assessment of cold-pressed rapeseed oil.

(E,E)-2,4-nonadienal

(deep-fried,

fatty)

and

(E,Z)-2,6-nonadienal

21 22

KEYWORDS. aroma extract dilution analysis, stable isotope dilution assay, rapeseed oil,

23

dimethylsulfide, [13C4]-(E,E)-2,4-decadienal

24


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1 25

INTRODUCTION

26

Rapeseed oil is commonly produced from the unpeeled black seeds of Brassica napus,

27

a variety which has been developed from a hybridization between wild cabbage (Brassica

28

oleracea) and turnip rape (Brassica rapa). About 1.500 black seeds are present on one

29

plant; the oil content is approximately 45%. The residue consists of protein (27%), fiber

30

(12%), water (10%) and carbohydrates (5%).1 Until a few decades ago, rapeseed oil was

31

not suitable for human nutrition, because it contained up to 30% erucic acid acid eliciting a

32

negative effect on the metabolism of kidney, spleen and thyroid in animal experiments.2

33

Since the 1970s, new cultures were developed, which are practically free from erucic acid

34

(< 1%) and at the same time low in glucosinolates (< 18 mmol/kg).3 In the US and Canada

35

the oil produced from the new cultivars is assigned as canola oil. Today, because of its fatty

36

acid composition, rapeseed oil is considered to be very appropriate for human nutrition.

37

Special emphasize is put on the correlation between linoleic (ω-6) and linolenic (ω-3) acid,

38

and only a spoonful of this oil should cover the daily recommendation of ω-3-fatty acids by

39

90%.

40

Cold-pressed rapeseed oil is manufactured from either unpeeled or peeled rapeseeds.

41

Although it is believed by the industry that the aromas of these two types differ, studies on

42

the odor-active compounds among the abundant, mostly odorless volatile constituents of

43

cold-pressed oils produced from either peeled or unpeeled oils are scarce.

44

Most of the previous publications were focused on the oxidation stability of refined

45

rapeseed oil. First results on volatile compounds of refined rapeseed oil were published by

46

Snyder et al.4 They analyzed the headspace of fresh and stored oil, and found pentanal,

47

hexanal, (E)-2-heptenal, octanal and nonanal. Raghavan et al.5 also analyzed the

48

headspace of fresh and stored refined rapeseed oil by means of dynamic headspace gas

49

chromatography and reported 9 µg/kg 2-pentenal, and 113 µg/kg hexanal in the fresh oil.



2 50

Jeleń et al.6 analyzed the volatile compounds formed during peroxidation of different

51

plant oils, among them cold-pressed and refined rapeseed oil. The compounds were

52

isolated by headspace solid-phase micro-extraction (HS-SPME), then characterized by GC-

53

MS, and selected aldehydes were quantitated using GC with FID detection. Initially, they

54

found a total aldehyde concentration of 436 µg/L for cold-pressed rapeseed oil, with

55

hexanal as the most abundant compound. After 10 d of storage at 60 °C, 2-heptenal

56

appeared with the highest concentration of 7.4 mg/L, followed by hexanal and nonanal. The

57

oil samples, containing the lowest amounts of volatiles, were perceived as the most

58

desired, whereas stored oils with higher amounts of volatiles showed lower acceptance.

59

Later,7 the same group used HS-SPME-GC/MS to analyze volatiles in refined rapeseed oil,

60

which was stored up to 12 d at 60 °C. Thirty-seven volatile compounds were detected in the

61

12 d stored oil, 28 of which could be identified. Predominant ones were hexanal, 2,4-

62

heptadienal, 2-heptenal and 1-penten-3-ol, while only two compounds were detected in the

63

unstored refined rapeseed oil, namely hexanal and 6-methyl-5-hepten-2-one. Recently,

64

Tynek et al.8 compared the volatiles in cold-pressed rapeseed oil from six different rape

65

varieties. They measured the total volatile compounds and selected “oxidation markers”,

66

such as propanal, hexanal and nonanal. The total volatile content in the six oil samples

67

ranged from 20 – 600 µg/kg.

68

It is well accepted in the literature that not the entire set of volatiles in a food is involved

69

in aroma perception, but only those compounds present in concentrations above their odor

70

thresholds.9 But, up to now, the most odor-active compounds in cold-pressed rapeseed oil

71

have not been analyzed by application of the Sensomics (molecular sensory science)

72

concept.9 Sensomics is defined as a systematic study aimed at decoding the chemical

73

fingerprint of a given food needed to cause a characteristic aroma perception in the human

74

brain. Guth and Grosch10 were the first and only to differentiate between volatiles and


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3 75

aroma compounds of a refined rapeseed oil. By application of the aroma extract dilution

76

analysis (AEDA), 23 odor-active compounds were reported, eight of them with a Flavor

77

Dilution (FD) factor ≥ 16. Among them, 1-octene-3-one, (Z)-1,5-octadien-3-one, (Z)-2-

78

nonenal, (E)-2-nonenal, 3-methyl-2,4-nonandione and trans-4,5-epoxy-(E)-2-decenal were

79

found to have the highest FD factors.

80

The overview of the literature indicates that the key aroma compounds of cold-pressed

81

rapeseed oil were not yet elucidated. Furthermore, no comparison was done between cold-

82

pressed oils manufactured from either unpeeled or peeled seeds. Thus, the aim of this

83

study was (i) to characterize the odor-active compounds in a commercial cold-pressed oil

84

prepared from unpeeled seeds by application of an AEDA, (ii) to quantify the most

85

important odorants by means of stable isotope dilution assays, and, finally, (iii) to evaluate

86

their contribution to the overall aroma by recombination experiments. In addition, a

87

commercial rapeseed oil prepared from peeled seeds was analyzed for comparison.

88 89

EXPERIMENTAL PROCEDURES

90

Oil Sample. The cold-pressed oils from unpeeled and peeled seeds were obtained from

91

a German oil mill. The oils was stored at 6 °C in brown glass bottles prior to analysis. For

92

the identification and quantitation of the aroma compounds different batches were used.

93

Chemicals.

Hydrochloric

acid, sodium

hydroxide, sodium

thiosulfate,

sodium

94

hydrogencarbonate, ammonium chloride, sodium sulfate as well as silica gel for flash

95

chromatography (silica gel 60, 15-40 µm) were obtained from Merck (Darmstadt, Germany).

96

Dess-Martin

97

prepared according to a published procedure.11 All other chemicals were obtained from

98

Sigma-Aldrich (Steinheim, Germany) in the highest available grade of purity. Diethyl ether,

periodinane

(1,1,1-triacetoxy-1,1-dihydro-1,2-benziodoxol-3(1H)-one)


was


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4 99 100

dichloromethane, and pentane were freshly distilled prior to use. Argon and liquid nitrogen were obtained from Linde (Munich, Germany).

101

Reference Aroma Compounds. The following reference compounds were obtained

102

from the suppliers given in parentheses: acetic acid, 2-sec-butyl-3-methoxypyrazine,

103

dimethyl sulfide, dimethyl trisulfide, heptanal, hexanoic acid, 2-isobutyl-3-methoxypyrazine,

104

2-isopropyl-3-methoxypyrazine, (R)-limonene, 3-methylbutanal, 3-methylbutanol, methyl

105

butanoate, (S)-2-methylbutanoic acid, 3-methylbutanoic acid, (E,Z)-2,6-nonadienal, (E,E)-

106

2,4-nonadienal,

107

octalactone, octanal, (E)-2-octenal, pentanoic acid, phenylacetaldehyde, phenylacetic acid,

108

2-phenylethanol and α-pinene (Sigma Aldrich Chemie, Taufkirchen, Germany). 2,3-

109

Butanedione, (E)-2-decenal, linalool, methylpropanoic acid, hexanal and 1-hexanol (Fluka,

110

Sigma-Adrich

111

methoxybenzaldehyde (Merck, Darmstadt, Germany) and (E,E)-2,4-decadienal (Lancaster,

112

Mühlheim am Main, Germany). 2-Methylbutanal and 1-octene-3-one (Alfa Aesar, Karlsruhe,

113

Germany). (Z)-4-Heptenal was a gift from Symrise, Holzminden, Germany. The following

114

compounds were synthesized as reported in the literature: (E,E,Z)-2,4,6-nonatrienal12 and

115

trans-4,5-epoxy-(E)-2-decenal.13

(E,Z)-2,6-nonadienol,

Chemie,

Taufkirchen,

γ-nonalactone,

Germany).

nonanal,

Butanoic

acid

(E)-2-nonenal,

and

γ-

4-hydroxy-3-

116

Isotopically Labeled Internal Standards. [13C4]-(E,E)-2,4-decadienal. As detailed

117

below, the target compound was synthesized in a three-step sequence through a Horner-

118

Wadsworth-Emmons reaction of [13C2]-triethylphosphono acetate with [13C2]-(E)-2-octenal,14

119

followed by a reduction of the labeled ester into [13C4]-(E,E)-2,4-decadienol with

120

diisobutylaluminium hydride (DIBAL) and, finally, an oxidation with Dess-Martin-periodinane

121

to the corresponding α,β-unsaturated aldehyde (Figure 1).

122

[13C4]-Ethyl-(E,E)-2,4-decadienoate. Methylmagnesium bromide (3 mol/L, 0.2 mL) was

123

added dropwise to a stirred solution of [13C2]-triethylphosphono acetate (0.11 g, 0.5 mmol)


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5 124

in anhydrous tetrahydrofuran (10 mL) at RT under an argon atmosphere. After stirring for

125

15 min, [13C2]-(E)-2-octenal (70 mg, 0.5 mmol) was added, and the mixture was refluxed for

126

2.5 h. The reaction was stopped by addition of a saturated aqueous solution of NH4Cl (15

127

mL), and the pH was adjusted to ∼7.0 with hydrochloric acid (1 mol/L). The solution was

128

extracted three times with diethyl ether (total volume: 100 mL), the combined organic

129

phases were washed with a saturated aqueous solution of NaHCO3 (20 mL), then dried

130

over anhydrous Na2SO4 and, after filtration, evaporated to dryness under reduced pressure.

131

MS-EI, m/z (%): 56 (37), 69 (58), 70 (59), 85 (56), 101 (100), 129 (86), 155 (16), 200

132

(12).

133

[13C4]-(E,E)-2,4-Decadienol. The [13C4]-ethyl-(E,E)-2,4-decadienoate obtained was

134

dissolved in anhydrous n-hexane (10 mL) and DIBAL-H (1 mol/L, 1.5 mL) was added

135

dropwise at 0 °C with stirring. After 1 h at RT, the reaction was stopped by addition of a

136

saturated aqueous solution of NH4Cl (15 mL), and hydrochloric acid (5 mol/L) was added

137

dropwise to dissolve the precipitate formed. Extraction with diethyl ether (total volume: 100

138

mL) was followed by treatment of the combined organic phases with a saturated aqueous

139

solution of NaHCO3 (20 mL). The organic layer was dried over anhydrous Na2SO4 and, after

140

filtration, evaporated to dryness under reduced pressure.

141

MS-EI, m/z (%):58 (77), 70 (67), 83 (70), 87 (100), 95 (14), 112 (8), 140 (7), 158 (8).

142

[13C4]-(E,E)-2,4-Decadienal. The [13C4]-(E,E)-2,4-decadienol obtained was treated with a

143

suspension of Dess-Martin-periodinane (0.31 g, 0.74 mmol) in anhydrous dichloromethane

144

for 2 h at 20 °C. The crude product was purified by flash chromatography to yield the target

145

compound in an overall yield of 4%.

146

MS-EI m/z (%): 57 (23), 70 (15), 85 (100), 99 (7), 156 (4).

147

The following isotopically labeled standards were synthesized as previously reported:

148

[2H2]-butanoic acid,15 [2H4]-hexanal,16 2-isobutyl-3-[2H3]-methoxypyrazine,17 2-isopropyl-3-



6 149

[2H3]-methoxypyrazine and [2H3]-3-methylbutanal18 [2H2]-3-methylbutanoic acid,19 [2H2]-

150

(E,E)-2,4-nonadienal,20 [2H2]-(E,Z)-2,6-nonadienal and [2H2-4]-1-octene-3-one,21 [2H2]-γ-

151

octalactone,22 [2H2-4]-octanal,23 and [13C2]-phenylethanol.24

152 153

[2H6]-Dimethyl sulfide and [2H6]-dimethyl trisulfide were obtained from Sigma-Aldrich Chemie (Taufkirchen, Germany).

154

Isolation of the Volatiles. An aliquot of the oil (200 g) was diluted with 150 mL

155

dichloromethane and submitted to Solvent Assisted Flavor Evaporation (SAFE).25 To avoid

156

overlapping peaks during gas chromatography, the SAFE distillate was extracted with an

157

aqueous saturated solution of NaHCO3 (total volume: 150 mL) to remove the acidic

158

volatiles and to isolate the neutral/basic fraction (NBF). The combined aqueous layers

159

containing the acidic volatiles as salts (AF) were then adjusted to pH 2 with hydrochloric

160

acid and extracted with dichloromethane (total volume: 150 ml). Both fractions were

161

washed with brine, dried over anhydrous Na2SO4, filtered and concentrated to ~150 µL

162

using Vigreux columns of different sizes.

163

High-Resolution Gas Chromatography-Olfactometry (HRGC-O). The distillates were

164

analyzed by means of a Fisons Instruments gas chromatograph 8000 (Mainz, Germany)

165

using the following J&W Scientific fused silica capillaries: DB-FFAP and DB-5, each 30 m ×

166

0.25 mm i.d., 0.25 µm film thickness (Folsom, CA). The sample (injection volume: 1 µl) was

167

applied by the cold-on-column injection technique at 40 °C, and the temperature of the oven

168

was held isothermally for 2 min, then raised at 6 °C/min to 230 °C, and held isothermally for

169

5 min. The carrier gas helium was directed by a y-type glass splitter into two deactivated

170

fused silica capillaries (50 cm each, 0.2 mm, I.D.) one leading to a sniffing port operated at

171

200 °C, and the other to a flame-ionization detector (FID) held at 220 °C. A constant

172

pressure of 80 kPa resulting in a flow rate of 1.2 mL/min was applied. Retention indices


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7 173

were calculated by co-chromatography of the sample with a homologous series of n-

174

alkanes.

175

Aroma Extract Dilution Analysis (AEDA). Flavor dilution (FD) factors were determined

176

by AEDA of the volatiles present in the respective acidic and the neutral/basic fraction,

177

using the FFAP capillary column. The original extracts were diluted stepwise with solvent

178

(1+1 by volume), each dilution was analyzed by HRGC-O (injection volume: 1 µL), and the

179

odor impression perceived at the odor port was assigned. This process was repeated on all

180

dilutions until no odor could be perceived. Hence, each single aroma-active compound was

181

assigned a flavor dilution (FD) factor displaying the last dilution in which the odor was

182

perceivable. The results were drawn as an FD-chromatogram, with the x-axis

183

corresponding to the retention index, and the y-axis (exponential scale) to the FD factors.

184

Three experienced panelists performed the sensory analysis to avoid overlooking of odor-

185

active compounds, and the results obtained were averaged.

186

High-Resolution Gas-Chromatography/Mass Spectrometry (HRGC/MS). For the

187

identification of the volatile compounds, mass spectra were generated by means of a

188

Finnigan MAT 95 S mass spectrometer (Bremen, Germany) at 70 eV in the electron

189

ionization (MS-EI) mode and at 110 eV in the chemical ionization (MS-CI) mode with

190

isobutane as the reagent gas using the GC capillaries described above.

191

Static Headspace High-Resolution Gas-Chromatography/Mass Spectrometry (HS-

192

HRGC/MS); Aroma Dilution Analysis. For the identification and quantitation of highly

193

volatile compounds, static headspace HRGC/MS was used. The system consisted of a

194

Thermo Scientific Trace Ultra gas chromatograph (Dreieich, Germany) with a Chrompack

195

purge-and-trap-(PTI/TCT)-injection system 4001 (Frankfurt, Germany) coupled to an Varian

196

ion-trap mass spectrometer Saturn 2100 T (Darmstadt, Germany). Sampling was

197

performed automatically by a Varian Combi Pal autosampler (Darmstadt, Germany) with a



8 198

gas-tight syringe. After sampling, the volatile compounds were collected in a cryo trap

199

cooled with liquid nitrogen at - 150 °C. By rapidly heating the trap to 250 °C, the compounds

200

were transferred onto the capillary column. The detection of the compounds was carried out

201

by mass spectrometry, and the effluent was simultaneously sniffed using a Y-type glass

202

splitter and two uncoated fused silica capillaries (50 cm x 0.3 mm i.d.). Flavor dilution (FD)

203

factors of highly volatile compounds were determined by GC-O/headspace using the DB-5

204

capillary column. Decreasing headspace volumes (20 mL, 10 mL, 5 mL, and so on) taken

205

from different vessels were analyzed, until no compound was perceived at the sniffing-port.

206

By definition, detection of an aroma-active compound in the first headspace volume (20 mL)

207

corresponded to an FD factor of 1.

208

Quantitation of Odorants by Stable Isotope Dilution Assays (SIDA). Between 1 to

209

500 g of the rapeseed oil was used for the quantitation to obtain concentrations between

210

0.5 and 5 µg/ml of each compound in the concentrated distillate. The sample was spiked

211

with defined amounts of the labeled standards, and was stirred for 60 min for equilibration.

212

The volatiles were then isolated by SAFE as described above, and mass spectra were

213

recorded in the chemical ionization mode. The concentrations of 2-/3-methylbutanal,

214

hexanal, octanal, 2- and-3-methylbutanoic acid, butanoic acid, (E,E)-2,4-decadienal, 2-

215

phenylethanol and γ-octalactone were determined using a Varian gas chromatograph 431

216

coupled to an ion trap mass spectrometer 220-MS (Darmstadt, Germany). The oven was

217

equipped with an Agilent DB-FFAP capillary column (30 m, 0.25 mm I.D., 0.25 µm film

218

thickness, Waldbronn, Germany). The remaining compounds were quantitated by means of

219

two-dimensional gas chromatography/mass spectrometry. The system consisted of a

220

Thermo Quest Trace 2000 series gas chromatograph (Egelsbach, Germany) coupled to a

221

Varian CP 3800 gas chromatograph and a Varian Saturn 2000 ion trap mass spectrometer

222

(Darmstadt, Germany). Mass spectra were recorded in MS-CI using methanol as the


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9 223

reagent gas. Quantitation of dimethyl sulfide was performed by static headspace analysis

224

as described above for the aroma dilution analysis. The samples were weighed into a

225

headspace vessel, d6-dimethyl sulfide was added and the vessel was immediately sealed

226

with a gas-tight septum. The samples were equilibrated by continuous stirring for 30 min at

227

30 °C prior to analysis. Mixtures of the respective labeled and unlabeled compounds were

228

prepared in five different mass ratios (1+5, 1+3, 1+1, 3+1, 5+1) and analyzed by HRGC-MS

229

to calculate the response factor (RF) for each component from the peak areas of the

230

selected mass fragments (Table 1).

231

Sensory evaluation. For the aroma profile analysis, a sensory panel of 17 to 25

232

panelists was recruited. Their ability to assess and evaluate the smell and intensity of

233

different odor compounds was frequently trained. Sensory analysis was performed in a

234

sensory room with single booths. Solution of the following aroma compounds in water (50

235

fold above the odor threshold) were used as references for the aroma descriptors: dimethyl

236

sulfide (cabbage-like), 3-methylbutanal (malty), hexanal (green, grassy), 2-isopropyl-3-

237

methoxypyrazine (pea-like), (E,Z)-2,6-nonadienal (cucumber-like) and (E,E)-2,4-nonadienal

238

(fatty). The panelists were asked to rank the intensities of the six different aroma attributes

239

on a seven point scale (0; 0.5; 1.0; 1.5; 2.0; 2.5; 3.0) with 0 (not perceivable) and 3

240

(strongly perceivable). All samples were presented in white, non-transparent teflon vessels.

241

Aroma Recombination Experiments. Aroma models were prepared in deodorized

242

sunflower oil using the concentrations of the aroma compounds determined in the rapeseed

243

oil. The recombinate and the oil were each placed in closed teflon vessels (15 g each) and

244

presented to the panelists at room temperature. In a first session, the similarity of the

245

recombinate with the oil was evaluated on a seven point scale from 0 to 3, and in a second

246

session, the intensities of the single attributes were judged.

247



10 248

RESULTS AND DISCUSSION

249

Identification of Odor-Active Compounds. First, the cold-pressed oil prepared from

250

unpeeled seeds was evaluated by a sensory panel using six aroma descriptors defined in

251

preliminary sessions. In the aroma profile, pea-like and fatty qualities dominated, followed

252

by a cucumber-like and a green, grassy odor (Figure 2). To identify the odorants

253

responsible for the overall aroma, the volatile fraction was isolated by solvent extraction

254

followed by SAFE distillation. When an aliquot of the distillate was put on a stripe of filter

255

paper and checked by a sensory panel against the aroma of the oil, a very good similarity

256

was judged. Application of HRGC/O on the entire distillate then revealed 45 odor-active

257

areas among the oil volatiles, which were ranked by AEDA on the basis of their FD factors.

258

Among the odorants showing FD factors above 8, a compound with a pea-like odor (23,

259

Figure 3) reached the highest FD factor, followed by the following four odor-active

260

compounds: a mushroom-like note (16), a fatty-cucumber smelling compound (36), a

261

grassy-green odorant (10) and a compound with a citrus-like odor (15). Also an intense

262

perception was evoked by an earthy, bell-pepper (29) and a sweaty (40) smelling

263

compound.

264

For the identification of the compounds responsible for the perceived odors, the

265

analytical and sensory attributes of the odor-active areas (retention index on two capillary

266

columns of different polarity, the odor quality as well as odor intensity) were compared to

267

data available in an in-house database produced from ~ 1000 odor-active reference

268

volatiles previously identified in different foods. Thus, a structure was suggested which was

269

finally confirmed by mass spectrometry (MS-EI; MS-CI) using the respective reference

270

compounds for comparison. The most intense aroma-active compounds were identified as

271

2-isopropyl-3-methoxypyrazine (23; FD 8192; Figure 4) smelling like peas, 1-octene-3-one

272

(16; FD 4096) with a mushroom-like odor and the cucumber-like smelling (E,Z)-2,6-


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11 273

nonadienal (36; FD 2048). Somewhat lower FD-factors were determined for the grassy,

274

green smelling hexanal (10; FD 1024) and octanal with a citrus-like odor (15; FD 1024). In

275

addition, 3-isobutyl-2-methoxypyrazine (29) and 2- and 3-methylbutanoic acid (40)

276

appeared with high FD-factors (Figure 4). The headspace above the oil was additionally

277

analyzed by static headspace gas chromatography-olfactometry, because highly volatile

278

odorants might be lost during distillation and the concentration steps. Only one additional,

279

cabbage-like smelling compound could be detected which was identified as dimethyl sulfide

280

(1; Table 2). Altogether, 43 odorants with FD-factors between 2 and 8192 were identified in

281

the cold-pressed rapeseed oil (Table 2). In comparison with available literature data,

282

surprisingly 32 out of the 46 aroma-active compounds were identified for the first time even

283

as constituents of cold-pressed rapeseed oil in this study. In particular 2-isopropyl-3-

284

methoxypyrazine (23) and (E,Z)-2,6-nonadienal (36) are worth mentioning, because both

285

appeared with the highest FD factors.

286

Quantitation of Important Odorants and Calculation of Odor Activity Values

287

(OAVs). In order to confirm the contribution of the aroma-active compounds to the overall

288

aroma, accurate quantitative measurements are required. Thus, a total of 16 odorants

289

showing FD factors ≥ 32 were quantitated in the oil by means of stable isotope dilution

290

assays (SIDA) using isotopically labeled reference compounds as internal standards. The

291

highest concentration of 1.9 mg/kg oil was measured for hexanal, while concentrations

292

between 130 and 360 µg/kg oil were found for octanal and 2- and 3-methylbutanoic acid

293

(Table 3). Concentrations below 10 µg/kg were determined for both methoxypyrazines as

294

well as for dimethyl sulfide and dimethyl trisulfide.

295

To assess the contribution of the odorants to the overall rapeseed oil aroma, odor

296

activity values (OAV; ratio of concentration to odor threshold) were calculated. The

297

corresponding odor thresholds in odorless sunflower oil were taken from our previously



12 298

published data,26-31 except for 2-methylbutanoic acid which was newly determined. The

299

highest OAV among the rapeseed oil volatiles was determined for 2-isopropyl-3-

300

methoxypyrazine, despite its rather low concentration of 7.6 µg/kg (Table 4). Also a high

301

OAV was calculated for the fatty smelling (E,E)-2,4-nonadienal. OAVs below 10 were

302

determined for (E,Z)-2,6-nonadienal, 3-methylbutanoic acid, 2- and 3-methylbutanal,

303

hexanal, octanal, 1-octene-3-one and dimethyl sulfide. On the other hand, the

304

concentrations of 2-isobutyl-3-methoxypyrazine and 2-methylbutanoic acid scarcely

305

exceeded their odor thresholds. Butanoic acid and (E,E)-2,4-decadienal, which were

306

present in quite high concentrations in the rapeseed oil (Table 3), showed OAVs below 1

307

and, thus, should not contribute to the aroma of this plant oil. In general, the quite low OAVs

308

reflect the overall weak to moderate odor intensity of the rapeseed oil aroma.

309

Aroma Recombination Experiments. As a final step of the Sensomics concept,9 it is

310

important to validate the quantitative data on the basis of an aroma recombination. This

311

way, interactions of a mixture of key odorants at the human odorant receptor level can be

312

addressed. The aroma recombinate of the rapeseed oil was prepared in refined, odorless

313

sunflower oil, the same matrix as used for the odor thresholds, and contained all 16 aroma

314

compounds in the concentrations given in Table 3. The aroma profile of the recombinate

315

and the rapeseed oil were then compared by a trained sensory panel. The similarity

316

between the rapeseed oil and the recombinate in sunflower oil was ranked with 2.5 on a

317

scale from 0 to 3. In particular the grassy, green, the cabbage-like and the malty odor

318

attributes in the recombinate were well in agreement with those in the oil sample (Figure 5).

319

The fatty and cucumber-like notes were ranked a bit higher in the recombinate, whereas the

320

pea-like smelling odor was perceived a bit weaker. However, the results allow the

321

assumption that the most important aroma compounds were correctly characterized. Except

322

2-isopropyl-3-methoxypyrazine, which is proposed to be formed biosynthetically from the


Page 14 of 38

Page 15 of 38


13 323

amino acid valine, most of the important aroma compounds are undoubtedly secondary

324

degradation products of unsaturated fatty acids. For example, hexanal and (E,E)-2,4-

325

nonadienal are known to be generated from the 13- and 10-hydroperoxide of linoleic acid,

326

respectively, and (E,Z)-2,6-nonadienal from 9-hydroperoxy-10,12,15-octadecatrienoic acid.

327

Aroma Compounds in Cold-pressed Rapeseed Oil Manufactured from Peeled

328

Seeds. Peeling of rapeseeds before pressing of the oil is known to obtain oils with different

329

aroma compared to those from unpeeled seeds. In a commercial cold-pressed oil

330

manufactured from peeled seed, a distinct cabbage-like aroma quality was detected (Figure

331

6), an odor quality which had been ranked low in the oil from unpeeled seeds (Figure 2). On

332

the other hand, the pea-like and fatty odors appeared with a similar intensity (Figures 2 and

333

6). Isolation of the volatile fraction from this oil, and application of the AEDA followed by

334

identification experiments also revealed the earthy smelling compound 23 with the highest

335

FD factor (Figure 7), which was identified as 2-isopropyl-3-methoxypyrazine. This was in

336

agreement with the data for the oil from the unpeeled sample (Figure 2). However, in the oil

337

from peeled seeds, two further methoxypyrazines with earthy notes (27 and 29; Figure 7)

338

showed higher FD factors than in the oil from the unpeeled seeds. The identification

339

experiments revealed in addition hexanal (10), 1-octene-3-one (16) and 2- and 3-

340

methylbutanoic acid (40) as further key odorants differing, however, in the FD factor

341

compared to those in the oil from the peeled seeds.

342

One of the main differences with respect to the aroma compounds in both oils was the

343

clearly higher FD factor of the cabbage-like smelling dimethylsulfide (DMS) in the oil from

344

the peeled seeds (1; Table 5). This prompted us to measure the concentration of

345

dimethylsulfide in this oil. The results showed 424.0 µg/kg dimethylsulfide compared to only

346

4.1 µg/kg in the oil from the unpeeled seeds (Table 2). The thioether is known to be

347

generated from S-methyl methionine,32 a reaction which may occur either during a thermal



14 348

treatment or enzymatically after cell damage. To clarify whether the peeling process may

349

cause the formation of DMS, the odorant was quantitated in ten commercial rapeseed oils

350

either manufactured from peeled or unpeeled seeds. The results, however, indicated that

351

the peeling is obviously not the reason for the formation of DMS. In two oils from peeled (D

352

and E, Table 6) low concentrations of the odorant were measured, while three oils from

353

unpeeled seeds (F, G and H, Table 6) also contained high concentrations. Studies to

354

elucidate the processing conditions needed to mitigate DMS formation are underway.

355 356


Page 16 of 38

Page 17 of 38


15 357

REFERENCES

358

(1)

Matthäus, B. Influence of the manufacturing process on the sensory perception of

359

rapeseed oil (in German). Bundesforschungsanstalt für Ernährung und Lebensmittel,

360

Institut für Lipidforschung, 2005.

361

(2)

heart from rats fed rapeseed oil. Res.Vet. Sci. 1978, 25, 318-322.

362 363

(3)

(4)

Snyder, J. M.; Frankel, E. N.; Selke, E. Capillary gas chromatographic analyses of headspace volatiles from vegetable oils. J. Am. Oil Chem. Soc. 1985, 65, 1675-1679.

366 367

Frauen, M. Quality and yield in winter oilseed rape. 61. Tagung der Vereinigung der Pflanzenzüchter und Saatgutkaufleute Österreichs 2010, 109-113.

364 365

Umemura, T.; Slinger, S. J.; Bhatnagar, M. K.; Yamashiro, S. Histopathology of the

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Raghavan, S. K.; Connel, D. R.; Khayat, A. Canola oil flavor quality evaluation by

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dynamic headspace gas chromatography. In Lipids in Food Flavors (ACS Symposium

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Series); Ho, C.; Hartmann, T. G. (eds.); American Chemical Society: Washington, DC,

370

1994, pp 92-330.

371

(6)

Jeleń, H. H.; Obuchowska, M.; Zawirska-Wojtasiak, R.; Wąsowicz, E. Headspace

372

solid-phase microextraction use for the characterization of volatile compounds in

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vegetable oils of different sensory quality. J. Agric. Food Chem. 2000, 48, 2360-2367.

374

(7)

Jeleń, H. H.; Mildner-Szkudlarz, S.; Jasińska, L.; Wąsowicz, E. A headspace-SPME-

375

MS method for monitoring rapeseed oil autoxidation. J. Am. Oil Chem. Soc. 2007, 84,

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509-517.

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Tynek, M.; Pawlowicz, R.; Gromadzka, J.; Tylingo, R., Wardencki, W.; Karlovits, G.

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their characteristics, properties, and differences. Eur. J. Lipid Sci. Technol. 2012, 114,

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

382

Hofmann, T. Nature’s chemical signatures in human olfaction: A foodborne

383

perspective for future biotechnology. Angew. Chem. Int. Ed. 2014, 53, 7124-7143.

384

(10) Guth, H.; Grosch, W. Comparison of stored soya-bean and rapeseed oils by aroma

385

extract dilution analysis. Lebens. Wiss. Technol. 1990, 23, 59-65.

386

(11) Dess, D. B.; Martin, J. C. Readily accessible 12-I-5 oxidant for the conversion of

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primary and secondary alcohols to aldehydes and ketones. J. Org. Chem. 1983, 48,

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4155-4156.

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(12) Schuh, C.; Schieberle, P. Characterization of (E,E,Z)-2,4,6-nonatrienal as a character impact aroma compound of oat flakes. J. Agric. Food Chem. 2005, 53, 8699 - 8705. (13) Schieberle, P.; Grosch, W. Potent odorants of the wheat bread crumb. Z. Lebensm. Unters. Forsch. 1991, 192, 130-135. (14) Kiefl, J.; Pollner, G.; Schieberle, P. Sensomics analysis of key hazelnuts odorants

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(Corylus avellana L. ´Tonda Gentile´) using comprehensive two-dimensional gas

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chromatography in combination with time-of-flight mass spectrometry (GCxGC/TOF-

396

MS). J. Agric. Food Chem. 2013, 61, 5226-5235.

397 398

(15) Schieberle, P.; Gassenmeier, K.; Guth, H.; Sen, A.; Grosch, W. Character impact odor compounds of different kinds of butter. Lebensm. Wiss. Technol. 1993, 26, 347-356.

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(16) Steinhaus, M.; Wilhelm, W.; Schieberle, P. Comparison of the most odor-active

400

volatiles in different hop varieties by application of a comparative aroma extract

401

dilution analysis. Eur. Food Res. Technol. 2007, 226, 45-55.

402 403 404 405

(17) Semmelroch, P.; Laskawy, G.; Blank, I.; Grosch, W. Determination of potent odorants in roasted coffee by stable isotope dilution assays. Flav. Fragr. J. 1995, 10, 1-7. (18) Semmelroch, P.; Grosch, W. Studies on character impact odorants of coffee brews. J. Agric. Food Chem. 1996, 44, 537-543.


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(19) Guth, H.; Grosch, W. Identification of the character impact odorants of stewed beef

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juice by instrumental analyses and sensory studies. J. Agric. Food Chem. 1994, 42,

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2862-2866.

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(20) Guth, H.; Grosch, W. Quantitation of potent odorants of virgin olive oil by stable isotope dilution assay. J. Am. Oil Chem. Soc. 1993a, 70, 513-518.

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(21) Guth, H.; Grosch, W. Deterioration of soya-bean oil: quantification of primary flavor

412

compounds using stable isotope dilution assay. Lebensm. Wiss. Technol. 1990b, 23,

413

513-522.

414

(22) Fukuzawa, S.; Nakanishi, A.; Fujinami, T.; Sakai, S. Samarium(II) di-iodide induced

415

reductive coupling of α,β-unsaturated esters with carbonyl compounds leading to a

416

facile synthesis of γ-lactone. J. Chem. Soc. Perkin Trans. 1988, 1, 1669-1675.

417

(23) Blekas, G.; Guth, H. Evaluation and quantification of potent odorants of Greek virgin

418

olive oils. Dev. Food Sci. 1995, 37A, 419-427.

419

(24) Schuh, C.; Schieberle, P. Characterization of the key aroma compounds in the

420

beverage prepared from Darjeeling black tea: quantitative differences between tea

421

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

422

(25) Engel, W.; Bahr, W.; Schieberle, P. Solvent assisted flavour evaporation – a new and

423

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

424

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

425

(26) Burdack-Freitag, A.; Schieberle, P. Changes in the key odorants of italian hazelnuts

426

(Coryllus avellana L. Var. Tonda Romana) induced by roasting. J. Agric. Food Chem.

427

2010, 58, 6351-6359.

428

(27) Guth, H.; Grosch, W. Deterioration of soya-bean oil: quantification of primary flavor

429

compounds using stable isotope dilution assay. Lebensm. Wiss. Technol. 1990, 23,

430

513-522.



18 431 432 433 434 435 436

(28) Reiners, J.; Grosch, W. Odorants of virgin olive oils with different flavor profiles. J. Agric. Food Chem. 1998, 46, 2754-2763. (29) Kubíckovà, J.; Grosch, W. Quantification of potent odorants in Camembert cheese and calculation of their odour activity values. Int. Dairy J. 1998, 8, 17-23. (30) Wagner, R.; Grosch, W. Key odorants of french fries. J. Am. Oil Chem. Soc. 1998, 75, 1385-1392.

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(31) Czerny, M.; Christlbauer, Ma.; Christlbauer, Mo.; Fischer, A.; Granvogl, M.; Hammer,

438

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

439

of key food aroma compounds and development of an aroma language based on odor

440

qualities of defined aqueous odorant solutions. Eur. Food. Res. Technol. 2008, 228,

441

265-273.

442

(32) Scherb, J.; Kreissl, J.; Haupt, S.; Schieberle, P. Quantitation of S-methylmethionine in

443

raw vegetables and green malt by a stable isotope dilution assay using LC-MS/MS:

444

Comparison with dimethyl sulfide formation after heat treatment. J. Agric. Food Chem.

445

2009, 57, 9091-9096.

446 447


Page 20 of 38

Page 21 of 38


19 448

LEGENDS TO THE FIGURES

449

Figure 1. Synthetic route used in the preparation of [13C4]-(E,E)-2,4-decadienal.

450

Figure 2. Aroma profile of commercial cold-pressed rapeseed oil manufactured from

451

unpeeled rape seeds.

452

Figure 3. Flavor dilution chromatogram obtained by application of the AEDA on the entire

453

volatile fraction isolated from cold-pressed rapeseed oil manufactured from unpeeled

454

seeds. Compounds with FD ≥ 32 are given.

455

Figure 4. Structures of the most important odor-active compounds in a commercial cold-

456

pressed rapeseed oil from unpeeled seeds.

457

Figure 5. Comparison of the aroma profiles of cold-pressed rapeseed oil from unpeeled

458

seeds (A) and the aroma recombinate (B).

459

Figure 6. Aroma profile of cold-pressed rapeseed oil manufactured from peeled seeds.

460

Figure 7. Flavor dilution chromatogram obtained by application of the AEDA on the entire

461

set of volatiles isolated from cold-pressed rapeseed oil manufactured from peeled seeds.

462



Page 22 of 38

20 Table 1. Isotopically Labeled Standards, Selected Ions, and Response Factors Used in the Stable Isotope Dilution Assays ion (m/z) analyte internal standard RFa butanoic acid [2H2]-butanoic acid 89 91 0.88 13 (E,E)-2,4-decadienal [ C4]-(E,E)-2,4-decadienal 153 157 0.94 2 63 69 0.97 dimethyl sulfide [ H6]-dimethyl sulfide 2 dimethyl trisulfide [ H6]-dimethyl trisulfide 127 133 0.91 2 hexanal [ H4]-hexanal 83 87 1.02 2 167 170 0.94 2-isobutyl-3-methoxypyrazine [ H3]-2-isobutyl-3-methoxypyrazine 2 2-isopropyl-3-methoxypyrazine [ H3]-2-isopropyl-3-methoxypyrazine 153 156 0.87 2 87 89 0.99 2- and 3-methylbutanal [ H2]-3-methylbutanal 2 2- and 3-methylbutanoic acid [ H2]-3-methylbutanoic acid 103 105 0.97 2 (E,E)-2,4-nonadienal [ H2]-(E,E)-2,4-nonadienal 139 141 0.96 2 (E,Z)-2,6-nonadienal [ H2]-(E,Z)-2,6-nonadienal 139 141 0.89 2 γ-octalactone [ H2]-γ-octalactone 143 145 0.98 2 b octanal [ H2-4]-octanal 111 113-115 0.76 2 b 1-octene-3-one [ H2-4]-1-octene-3-one 127 129-131 0.79 2 2-phenylethanol [ H2]-2-phenylethanol 105 107 0.86 a b MS response factor determined by analyzing defined mixtures of the analyte and the internal standard by MS-CI. The internal standard was used as a mixture of isotopologues. odorant

labeled standard


Page 23 of 38


21 Table 2. Important Aroma Compounds (FD ≥ 2) Identified in Cold-pressed Rapeseed oil Manufactured from Unpeeled Seeds no.a aroma compoundb 1 3 5 6 7 8 10 11 12 13 14 15 16 19 20 21 22 23 24 26 27

dimethyl sulfide 2- and 3-methylbutanal 2,3-butanedione α-pinene ethyl butanoate ethyl 2-methylbutanoate hexanal unknown limonene 3-methyl-1-butanol (Z)-4-heptenal octanal 1-octene-3-one 1-hexanol dimethyl trisulfide nonanal (E)-2-octenal 2-isopropyl-3-methoxypyrazine acetic acid (E,E)-2,4-heptadienal 2-sec-butyl-3-methoxypyrazine

29 2-isobutyl-3-methoxypyrazine 30 (E)-2-nonenal 31 propanoic acid

odor qualityc cabbage-like malty buttery resinous fruity fruity grassy, green fruity citrus-like malty fishy citrus-like mushroom-like grassy cabbage-like citrus-like, soapy fatty, nutty pea-like vingar-like fatty, flowery earthy earthy, bell-pepperlike fatty, cardboard-like sweaty

retention index on FFAP DB-5 520  933 652 981 593 1009 934 1027 803 1045 847 1078 800 1153  1189 1033 1206 737 1236 901 1280 1003 1293 979 1350 872 1361 968 1389 1103 1422 1059 1427 1094 1443 612 1478 1010 1497 1174

FDd

previously identified as volatile in rapeseed oil

2 32 8 8 4 16 1024 2 2 4 8 1024 4096 2 32 16 2 8192 8 8 16

     

1516

1184

256

1527 1534

1160 836

16 8


4-8     4,6 9   4,7,8 7  7 5,7   7,9 


Page 24 of 38

22 Table 2. continued no.a aroma compoundb 32 33 35 36 37 38 40 41 42 43 45 47 48 49 50 52 53 54 57 58 59 60

linalool unknown 2-methylpropanoic acid (E,Z)-2,6-nonadienal butanoic acid (E)-2-decenal 2- and 3-methylbutanoic acid (E,E)-2,4-nonadienal 3-methyl-2,4-nonanedione pentanoic acid (E,Z)-2,6-nonadienol (E,E)-2,4-decadienal hexanoic acid (E,E,Z)-2,4,6-nonatrienale 2-phenylethanol γ-octalactone trans-4,5-epoxy-(E)-2-decenale γ-nonalactone 3-hydroxy-4,5-dimethyl-2(5H)-furanonee unknown phenylacetic acid 4-hydroxy-3-methoxybenzaldehyde

odor qualityc flowery fatty sweaty, cheese-like cucumber-like sweaty, cheese-like fatty, tallowy sweaty, cheese-like fatty strawy sweat, cheese-like cucumber-like fatty sweaty, pungent oat flakes honey-like coconut-like metallic coconut-like seasoning-like smoky honey-like vanilla-like

retention index on FFAP DB-5 1539 1102 1549  1558 789 1575 1153 1619 820 1633 1261 1660 872 1699 1216 1708 1251 1726 914 1757 1170 1801 1317 1835 1018 1877 1274 1905 1116 1920 1284 1997 1382 2029 1360 2195 1108 2457  2565 1261 2574 1406


FDd 4 8 8 2048 32 8 256 128 8 16 16 64 4 16 32 32 16 8 8 4 16 16

previously identified as volatile in rapeseed oil      7  6 9   5,7 6,7    9     

Page 25 of 38


23 Table 2. Footnotes a

Numbering refers to Figure 3. bThe compound was identified by comparison of its mass spectra and retention indices on two

different capillary columns (FFAP and DB-5) as well as the odor quality and intensity perceived at the GC odor port with data for the respective reference compound. cOdor quality perceived at the odor port. dFlavor dilution factor determined by AEDA on capillary FFAP. eNo unequivocal mass spectrum was obtained, identification is based on the remaining criteria given in footnote b.



Page 26 of 38

24 Table 3. Concentrations of 16 Important Aroma Compounds in Cold-pressed Rapeseed oil Manufactured from Unpeeled Seeds concn. (µg/kg)

range (µg/kg)

na

hexanal

1900

1890 – 1940

3

octanal

360

353 – 375

3

3-methylbutanoic acid

150

146 – 147

2

2-methylbutanoic acid

130

127 – 128

2

butanoic acid

81

73 – 89

4

(E,E)-2,4-decadienal

36

34.7 – 38.5

3

3-methylbutanal

36

33.8 – 39.1

2

(E,Z)-2,6-nonadienal

34

32.4 – 34.8

3

(E,E)-2,4-nonadienal

33

30.3 – 35.9

3

2-phenylethanol

26

23.6 – 27.6

2

γ-octalactone

13

11.8 – 14.2

4

1-octene-3-one

11

9.0 – 12.5

3

2-methylbutanal

10

9.2 – 11.0

2

aroma compound

2-isopropyl-3-methoxypyrazine

7.6

7.6 – 7.7

3

dimethyl sulfide

4.1

3.9 – 4.2

2

2-isobutyl-3-methoxypyrazine

1.1

0.9 – 1.2

3

dimethyl trisulfide a Number of analyses with different work-ups.

0.8

0.8 – 0.8

4


Page 27 of 38


25 Table 4. Odor Thresholds (OT) of Key Odorants Determined in Sunflower Oil and Odor Activity Values (OAVs) in Cold-pressed Rapeseed oil Manufactured from Unpeeled Seeds odorant 2-isopropyl-3-methoxypyrazine (E,E)-2,4-nonadienal (E,Z)-2,6-nonadienal 3-methylbutanoic acid 3-methylbutanal hexanal octanal 2-methylbutanal 1-octene-3-one dimethyl sulfide 2-isobutyl-3-methoxypyrazine (S)-2-methylbutanoic acid

OT (µg/kg)a 0.054 26 1.48 26 3.8 27 22 28 5.4 28 300 28 56 28 2.2 28 2.0 28 1.2 29 0.8 30 113c

OAVb 141 22 9 7 7 6 6 5 5 3 1 1

butanoic acid 135 14

Characterization of the Key Odorants in Commercial Cold-Pressed

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