Elucidation of Thermally Induced Changes in Key Odorants of White

Oct 3, 2016 - ... dilution analysis (SIDA), followed by the calculation of odor activity values (OAVs) using odor thresholds determined in refined sun...
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Elucidation of Thermal Induced Changes in Key Odorants of White Mustard Seeds (Sinapis alba L.) and Rapeseeds (Brassica napus L.) using Molecular Sensory Science Eva Ortner, Michael Granvogl, and Peter Schieberle J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03625 • Publication Date (Web): 03 Oct 2016 Downloaded from http://pubs.acs.org on October 12, 2016

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Journal of Agricultural and Food Chemistry

Elucidation of Thermal Induced Changes in Key Odorants of White Mustard Seeds (Sinapis alba L.) and Rapeseeds (Brassica napus L.) using Molecular Sensory Science Eva Ortner, Michael Granvogl,* and Peter Schieberle

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

*Corresponding author: Tel.: +49 8161 712987 Fax: +49 8161 712970 e-mail: [email protected]

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ABSTRACT: Heat-processing of Brassica seeds led to the formation of a

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characteristic pleasant popcorn-like and coffee-like aroma impression compared to

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the mainly pea-like aroma of the corresponding raw seeds. To analyze this

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phenomenon on a molecular basis, raw and roasted white mustard seeds and

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rapeseeds were analyzed using the Sensomics approach. Application of comparative

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aroma extract dilution analysis (cAEDA) and identification experiments to raw and

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roasted (140 °C, 30 min) mustard seeds revealed 36 odorants (all identified for the

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first time) and 47 odorants (41 newly identified), respectively. Twenty-seven odorants

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in raw and 43 odorants in roasted (140 °C, 60 min) rapeseeds were found, which

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were all described for the first time. Among the set of volatiles, 2-isopropyl-3-

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methoxypyrazine (earthy, pea-like) and 4-ethenyl-2-methoxyphenol (clove-like,

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smoky) showed high FD factors in both raw seeds. 4-Hydroxy-2,5-dimethylfuran-

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3(2H)-one (caramel-like), 2,3-diethyl-5-methylpyrazine (earthy), dimethyl trisulfide

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(cabbage-like), and 2-acetyl-1-pyrroline (popcorn-like) were present at high FD

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factors in both roasted Brassica seeds. Odorants, differing in cAEDA or showing high

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FD factors in at least one of the seeds, were quantitated by stable isotope dilution

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analysis (SIDA), followed by the calculation of odor activity values (OAVs) using odor

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thresholds determined in refined sunflower oil. Eighteen aroma compounds in raw

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and 28 in roasted mustard seeds as well as 14 in raw and 25 in roasted rapeseeds

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revealed OAVs ≥ 1. All four aroma recombinates, prepared by mixing the odorants

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showing OAVs ≥ 1 in their natural occurring concentrations, showed a very good

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similarity with the original seeds and, thus, proved the successful characterization of

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the respective key odorants.

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KEYWORDS: mustard seeds, rapeseeds, aroma extract dilution analysis, stable

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isotope dilution analysis, odor activity value, aroma recombinate ACS Paragon Plus Environment

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INTRODUCTION

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White mustard (Sinapis alba L.) and rape (Brassica napus L.) belong to the

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Brassicaceae. Mustard seeds are used for dry milling to obtain the flour, for wet

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milling to get mustard pastes, and in spice mixtures. In Indian kitchen, they are well-

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known for their unique and highly attractive odor generated by deep-frying, whereas

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in Europe they are mainly used untreated in pickling or boiling vegetables such as

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cabbage or sauerkraut. In the last decades, roasting has gained significant

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importance in food industry as a versatile process for numerous foods to produce

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characteristic aromas. Although mustard seeds are a popular spice, highly

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appreciated for their delicious flavor, only a few studies have already been performed

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to investigate the volatiles of processed products like the seed oil1 or plant parts.2 In

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1981, the identification of 9 carbonyls, 6 pyrazines, 6 sulfur compounds, 2 amines,

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and 1 nitrile was reported for roasted brown mustard seeds.3 Application of aroma

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extract dilution analysis (AEDA) to a distillate of roasted (200 °C, 10 min) white

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mustard seeds showed 2-furanmethanethiol, 3-(methylthio)propanal, and 4-hydroxy-

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2,5-dimethylfuran-3(2H)-one as important odorants. Furthermore, 3-methylbutanal,

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2,3-pentanedione, 3-mercapto-2-pentanone, 2-acetyl-2-thiazoline, and 3-hydroxy-

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4,5-dimethylfuran-2(5H)-one were identified in the roasted seeds eliciting a strong

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coffee-like overall aroma impression. In contrast, the panelists described the smell of

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moderately roasted (160 °C, 10 min) seeds as peanut-like, with chicken-like, sulfury,

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earthy, roasty, and popcorn-like by-notes.4

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Rapeseeds might probably be the best known member within the

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Brassicaceae, which are used for the production of edible oil. Sensory experiments

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showed that the oil prepared from toasted rapeseeds (variety Alto) was significantly

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more preferred by panelists compared to the oil from untoasted seeds.5 The first

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differentiation between volatiles and aroma-active compounds of refined rapeseed oil ACS Paragon Plus Environment

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was reported by Guth and Grosch.6 Further studies decoded the overall aroma

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impression by characterization of the key odorants in commercially cold-pressed oils

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from unpeeled and peeled rapeseeds using the Sensomics approach.7,8

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But, up to now, studies comparing the key aroma compounds in raw and

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roasted mustard seeds or rapeseeds on the basis of Molecular Sensory Science

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including AEDA, identification experiments, stable isotope dilution analysis (SIDA),

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calculation of odor activity values (OAVs), and aroma recombination, were not

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performed. Volatiles have mainly been identified by instrumental-analytical methods

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like gas chromatography-mass spectrometry and, in addition, no quantitative data on

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thermally induced changes of the key aroma compounds of mustard seeds and

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rapeseeds are currently available.

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Therefore, the aim of the present study was the application of the Molecular

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Sensory Science Concept to raw and roasted mustard seeds and rapeseeds i) to

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identify the key aroma compounds, ii) to quantitate the most important odorants, iii) to

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calculate the respective odor activity values, and iv) to simulate the overall aroma by

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recombination experiments to get deeper insights into the changes induced by

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roasting of the seeds.

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MATERIALS AND METHODS

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Seeds. White mustard seeds (Sinapis alba L.) were obtained from Ostmann

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Gewürze (Dissen a.T.W., Germany) and rapeseeds (Brassica napus L.) from Oil +

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more (Straßberg, Germany).

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Chemicals. The following reference odorants were commercially available: acetic

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acid, acetylpyrazine, 2-acetyl-2-thiazoline, 2-aminoacetophenone, 2-sec-butyl-3-

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methoxypyrazine, 1,8-cineole, 2,3-diethyl-5-methylpyrazine, dimethyl sulfide, 2-ethyl-

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3,5-dimethylpyrazine, 2-ethyl-3,6-dimethylpyrazine, 2-furanmethanethiol, γ-hexa-

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lactone,

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methanethiol,

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3-(methylthio)propanal,

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2,3-pentanedione, pentanoic acid, phenylacetic acid (Sigma-Aldrich Chemie,

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Taufkirchen, Germany); 2-ethyl-5-methylpyrazine, 2-ethyl-6-methylpyrazine (Pyrazine

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Specialities, Ellenwood, GA); butanoic acid, 4-hydroxy-3-methoxybenzaldehyde

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(Merck, Darmstadt, Germany); 2,3-butanedione, (E,E)-2,4-decadienal, (E)-2-decenal,

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dimethyl disulfide, ethyl phenylacetate, ethylpyrazine, 2-formylthiophene, hexanal,

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hexanoic

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propanoic acid, γ-octalactone, octanal, 2-phenylethanol (Fluka; Sigma-Aldrich);

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4-ethenyl-2-methoxyphenol,

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3-methylbutanal, 1-octen-3-one (Alfa Aesar, Karlsruhe, Germany); dimethyl trisulfide

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and phenylacetaldehyde (Acros Organics, Geel, Belgium).

indole,

2-isobutyl-3-methoxypyrazine,

2-methylbutanoic

acid,

acid,

2-isopropyl-3-methoxypyrazine,

3-methylbutanoic

(E,E)-2,4-nonadienal,

acid,

4-methylphenol,

γ-nonalactone,

(E)-2-nonenal,

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

3-methylindole,

4-mercapto-4-methyl-2-pentanone,

methyl-

2-methylbutanal,

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The following reference odorants were synthesized according to published

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methods: 2-acetyl-1-pyrroline,9 2-acetyl-3,4,5,6-tetrahydropyridine,10 2-ethenyl-3-

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ethyl-5-methylpyrazine,11

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decenal.12

2-propionyl-1-pyrroline,9

and

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

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Argon and liquid nitrogen were obtained from Linde (Munich, Germany). Diethyl

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ether and dichloromethane were freshly distilled prior to use (Merck). All chemicals

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were at least of analytical grade.

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Stable Isotopically Labeled Standards. The following standards were

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commercially obtained: [2H9]-2-methylbutanoic acid (EQ Laboratories, Augsburg,

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Germany); [2H3]-hexanoic acid (Cambridge Isotope Laboratories, Tewksbury, MA);

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[13C2]-acetic acid, [2H6]-dimethyl disulfide, [2H3]-dimethyl sulfide, [2H12]-hexanal,

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[2H8]-4-methylphenol, and [13C2]-phenylacetic acid (Sigma-Aldrich). ACS Paragon Plus Environment

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The following internal standards were synthesized according to published [2H3]-acetylpyrazine,13

[13C5]-2-acetyl-1-pyrroline,9

[2H3]-2-aminoaceto-

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methods:

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phenone,14

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methoxypyrazine,17

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methylpyrazine,20 [2H6]-dimethyl trisulfide,21 [2H3]-4-ethenyl-2-methoxyphenol,22 [2H3]-

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2-ethyl-3,5-dimethylpyrazine

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furanmethanethiol,23 [13C2]-4-hydroxy-2,5-dimethylfuran-3(2H)-one,24 [2H3]-4-hydroxy-

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3-methoxybenzaldehyde,25 [2H3]-2-isobutyl-3-methoxypyrazine,26 [2H3]-2-isopropyl-3-

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methoxypyrazine,17 [2H2]-2-methylbutanal,27 [2H2]-3-methylbutanal,28 [2H3]-3-(methyl-

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thio)propanal,23 [2H2]-γ-nonalactone,29 [2H2]-(E)-2-nonenal,19 [2H2]-γ-octalactone,30

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[2H2-4]-1-octen-3-one,19 [13C2]-2,3-pentanedione,31 [2H3]-pentanoic acid,32 [13C2]-

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phenylacetaldehyde,33 and [2H2-5]-2-propionyl-1-pyrroline.13

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[2H3]-Methanethiol was synthesized prior to use and its concentration was

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determined as previously described.34

[13C4]-2,3-butanedione,15

[2H2]-butanoic

[2H2]-1,8-cineole,18

and

acid,16

[2H3]-2-sec-butyl-3-

[2H2]-(E)-2-decenal,19

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

[2H3]-2-ethyl-3,6-dimethylpyrazine,20

[2H2]-2-

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Determination of the Concentrations of Stable Isotopically Labeled

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Compounds. Concentrations of the stable isotopically labeled standards were

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determined by means of a gas chromatograph TRACE GC 2000 (ThermoQuest,

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Egelsbach, Germany) equipped with a flame ionization detector (FID) and a DB-

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FFAP column (30 m x 0.32 mm i.d., 0.25 µm film thickness; J&W Scientific; Agilent

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Technologies, Waldbronn, Germany). First, the FID response factor was determined

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for each unlabeled reference compound using methyl octanoate as internal standard.

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Then, the concentration of the labeled standard was calculated via the peak areas of

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the labeled compound and methyl octanoate using the FID response factor

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determined for the unlabeled compound.

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Isolation of the Volatiles. Raw or roasted seeds (200 g each) were frozen in

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liquid nitrogen and subsequently ground in a commercial blender (Privileg, Fürth, ACS Paragon Plus Environment

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Germany). The powder obtained was extracted with diethyl ether (2 x 250 mL) by

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stirring vigorously for 2 x 1 h at room temperature. Afterwards, the volatiles were

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separated from the non-volatiles using the solvent assisted flavor evaporation

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(SAFE) technique.35 The distillate obtained was dried over anhydrous sodium sulfate,

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filtered, concentrated at 40 °C to ∼ 3 mL by a Vigreux column (50 cm × 1 cm i.d.) and

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finally to ∼ 100 µL by micro-distillation.36

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Comparative Aroma Extract Dilution Analysis (cAEDA). To enable a

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comparison between the raw and roasted seeds, the same amounts were extracted,

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subjected to SAFE distillation, concentrated to the same final volume, and, finally, the

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same volume was used for high-resolution gas chromatography-olfactometry

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(HRGC-O). To avoid a potential overlooking of odorants, the aroma-active areas of

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the original distillate were evaluated by three experienced persons. The flavor dilution

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(FD) factors of the odorants were determined by diluting stepwise the extract with

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diethyl ether (1+1, v+v). Every dilution was analyzed twice by HRGC-O, differing not

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more than one FD factor. For each odorant, the respective FD factor, correlating to

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the highest dilution, in which the compound was perceivable for the last time, was

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assigned.

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

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was performed by a TRACE GC Ultra (ThermoQuest) equipped with either a DB-

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FFAP or a DB-5 fused silica capillary column (both 30 m × 0.25 mm i.d., 0.25 µm film

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thickness; J&W Scientific). Aliquots (1 µL) of the samples were injected manually by

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the cold on-column technique (40 °C). After 2 min, the temperature was raised at

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6 °C/min to 230 °C (DB-FFAP) or 240 °C (DB-5), respectively, and held for 5 min.

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The flow rate of the carrier gas helium was 1.2 mL/min. At the end of the column, the

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effluent was split 1:1 by a Y-type quick-seal glass splitter (Chrompack, Frankfurt,

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Germany). Two deactivated fused silica capillaries of the same length (30 cm × ACS Paragon Plus Environment

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0.18 mm i.d.) led either to an FID (250 °C) or to a sniffing port (230 °C). Linear

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retention indices (RIs) of the compounds were calculated using a series of n-alkanes

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(C6−C26 for DB-FFAP and C6−C18 for DB-5, respectively) as described previously.37

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High-Resolution Gas Chromatography-Sector Field Mass Spectrometry. For

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identification experiments, mass spectra were generated by a gas chromatograph

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5890 series II (Hewlett-Packard, Waldbronn, Germany) coupled to a MAT 95 S

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sector field mass spectrometer (Finnigan MAT, Bremen, Germany). Mass spectra in

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the electron ionization (EI) mode were generated at 70 eV and in the chemical

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ionization (CI) mode at 115 eV using isobutane as reactant gas. Aliquots (1 µL) of the

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samples were manually injected by the cold on-column technique (40 °C). The same

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capillary columns and temperature programs were used as mentioned above for

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HRGC-O.

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Two-Dimensional High-Resolution Gas Chromatography-Olfactometry/Mass

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Spectrometry (HRGC/HRGC-O/MS) for Identification. The HRGC/HRGC-O/MS

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system consisted of a gas chromatograph Mega-2 (Fisons Instruments, Egelsbach,

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Germany) equipped with a DB-FFAP fused capillary in the first dimension coupled to

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a gas chromatograph CP-3800 (Varian, Darmstadt) equipped with a DB-5 column in

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the second dimension (both 30 m × 0.25 mm i.d., 0.25 µm film thickness, J&W

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Scientific). The end of the second column was connected to an ion trap mass

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spectrometer Saturn 2000 (Varian), and, in parallel, to a sniffing port via a Y-splitter,

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enabling a simultaneous generation of mass spectra (recorded in EI mode, 70 eV)

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and the perception of the corresponding odor qualities of the respective aroma-active

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compounds. The elution range containing the compounds of interest was transferred

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from the first GC column into a cold trap (cooled with liquid nitrogen to -100 °C) by a

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moving column stream switching (MCSS) system (ThermoQuest). Then, the trap was

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immediately heated to 230 °C and the aroma compounds were transferred onto the ACS Paragon Plus Environment

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second GC column. Aliquots (1-3 µL) of the samples were manually injected at 40 °C

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using the cold on-column technique.

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Quantitation of Odorants by Stable Isotope Dilution Assays (SIDAs).

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Different amounts of seeds (1−400 g; depending on the concentrations of the

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respective odorants determined in a preliminary experiment) were used for

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quantitation of the aroma-active compounds. After grinding, diethyl ether (100-

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400 mL) and the labeled internal standards (0.5-5 µg; dissolved in diethyl ether or

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dichloromethane; amounts depending on the concentrations of the analytes) were

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added and the suspension was stirred for 2 h at room temperature. The further work-

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up was done as described above for the isolation of the volatiles and quantitation

191

was performed via high-resolution gas chromatography-mass spectrometry (HRGC-

192

MS) or two-dimensional high-resolution gas chromatography-mass spectrometry

193

(HRGC/HRGC-MS).

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High-Resolution Gas Chromatography-Ion Trap Mass Spectrometry (HRGC-

195

MS) for Quantitation. HRGC-MS for quantitation was performed by a Varian GC 431

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equipped with a DB-FFAP fused silica capillary column (30 m × 0.25 mm i.d.,

197

0.25 µm film thickness; J&W Scientific) coupled to an ion trap mass spectrometer

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Varian 220-MS. Mass spectra were generated in CI mode (70 eV) using methanol as

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reactant gas. Aliquots (2 µL) of the samples were injected at 40 °C by means of a

200

Combi PAL autosampler (CTC Analytics, Zwingen, Switzerland) using the cold on-

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column technique. The peak areas of the analyte and labeled standard were

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determined separately by using the respective mass traces of the protonated

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molecular masses or selected fragments (Table 1).

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Determination of the Ratio of 2-Methylbutanoic Acid to 3-Methylbutanoic

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Acid. As both isomers could not be separated by HRGC-MS, first, the sum of both

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compounds was determined by SIDA using CI mode. To differentiate the two ACS Paragon Plus Environment

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odorants, the sample was re-analyzed by HRGC-MS in EI mode (70 eV) and the ratio

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of 2- and 3-methylbutanoic acid was determined using the intensities of the

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fragments m/z 60 (3-methylbutanoic acid) and m/z 74 (2-methylbutanoic acid). Five

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defined mixtures of 2- and 3-methylbutanoic acid (90:10; 70:30; 50:50; 30:70; 10:90)

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were analyzed and a calibration curve was drawn plotting the intensity ratio of m/z 60

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over the sum of m/z 60 + m/z 74 against the percentage of 3-methylbutanoic acid in

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the mixture, as recently described.38

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Two-Dimensional High-Resolution Gas Chromatography Mass Spectrometry

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(HRGC/HRGC-MS) for Quantitation. If an overlapping of peaks was observed,

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HRGC/HRGC-MS was performed by a TRACE GC 2000 equipped with a DB-FFAP

217

column in the first dimension coupled to a gas chromatograph CP-3800 (Varian)

218

equipped with an OV-1701 column in the second dimension (both 30 m × 0.25 mm

219

i.d., 0.25 µm film thickness; both J&W Scientific). The system was finally connected

220

to an ion trap mass spectrometer Saturn 2000 (Varian). Heart-cuts were done by

221

means of the MCSS system. Mass spectra were generated in CI mode (70 eV) using

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methanol as reactant gas. Aliquots (2 µL) of the samples were injected at 40 °C by

223

means of a Combi PAL autosampler using the cold on-column technique.

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Static Headspace Aroma Dilution Analysis on the basis of High-Resolution

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Gas Chromatography-Olfactometry/Mass Spectrometry (SH-HRGC-O/MS). For

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SH-HRGC-O, decreasing headspace volumes (10−0.16 mL) were withdrawn with a

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gas-tight syringe (Innovative Labor Systeme, Stützerbach, Germany) by a Combi

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PAL autosampler as previously described.39

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For quantitation of dimethyl sulfide, the ground seeds (1 g) were placed into

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headspace vials (volume 20 mL), the labeled standard (0.2-1 µg) was added, and the

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vial was immediately sealed with a gas-tight septum. After equilibration (28 °C,

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30 min), aliquots of the headspace were withdrawn and analyzed by static

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headspace high-resolution gas chromatography-mass spectrometry (SH-HRGC-MS).

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For quantitation of methanethiol, the ground seeds (0.5-1 g) were filled into

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headspace vials (20 mL) and sealed with a gas-tight septum. A defined volume of the

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labeled methanethiol was injected with a gas-tight syringe through the septum into

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the headspace vials. After equilibration (28 °C, 30 min), aliquots of the headspace

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(2 mL) were withdrawn and analyzed by SH-HRGC-MS. The volatiles were cryo-

239

focused and transferred onto a DB-5 fused silica capillary column (30 m × 0.25 mm

240

i.d., 0.5 µm film thickness; J&W Scientific) in a TRACE GC Ultra coupled to an ion

241

trap mass spectrometer 2100 T (Varian). Mass spectra were generated in CI mode

242

(70 eV) using methanol as reactant gas.

243

Determination of Response Factors. For each odorant, a response factor was

244

calculated by analyzing binary mixtures of defined amounts of the unlabeled analyte

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and the labeled standard in five different mass ratios (5:1, 3:1, 1:1, 1:3, 1:5; Table 1)

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under the same conditions used for the samples.

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Aroma Profile Analysis (APA). All samples were evaluated by a sensory panel

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consisting of at least 15 experienced panelists.40 Characteristic aroma descriptors,

249

determined in preliminary sensory trials, were used. For each descriptor, an aqueous

250

reference solution at a concentration 100-fold above the respective odor threshold of

251

the odorant was provided. The intensity of each aroma quality was ranked on a linear

252

seven-point scale (steps of 0.5) from 0 (not perceivable) to 3 (strongly perceivable).

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The following compounds given in parentheses were chosen for the respective odor

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attributes used for APA of raw seeds: earthy (2,3-diethyl-5-methylpyrazine),

255

green/grassy

256

putrid/cabbage-like/sulfury (methanethiol), fatty/green ((E)-2-nonenal), and cabbage-

257

like (dimethyl trisulfide). For APA of roasted seeds, the following descriptors were

(hexanal),

earthy/pea-like

(2-isopropyl-3-methoxypyrazine),

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used: earthy (2,3-diethyl-5-methylpyrazine), popcorn-like (2-acetyl-1-pyrroline), malty

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(3-methylbutanal), caramel-like (4-hydroxy-2,5-dimethylfuran-3-(2H)-one), coffee-like

260

(2-furanmethanethiol), roasty (acetylpyrazine), cabbage-like (dimethyl trisulfide), and

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fatty/green ((E)-2-nonenal). Analyses were performed in a sensory room equipped

262

with single booths at 21 ± 1 °C. Samples (5 g) were presented in covered glass

263

vessels (40 mm i.d., total volume = 45 mL).40

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Orthonasal Odor Thresholds (OTs). For the calculation of odor activity values

265

(OAVs), orthonasal odor thresholds were determined in refined sunflower oil as

266

recently described.40

267

Aroma Reconstitution Experiments. On the basis of the quantitative data

268

obtained for each sample (raw and roasted mustard seeds, raw and roasted

269

rapeseeds), recombination experiments were carried out. Therefore, an odorless

270

matrix was used, to which all aroma compounds with OAVs ≥ 1 were added in their

271

natural occurring concentrations. To closely simulate the original matrix, the seeds

272

were deodorized by solvent extraction using pentane, diethyl ether, and

273

dichloromethane (each 250 mL for 24 h); traces of solvent residues were gently

274

removed by lyophilization. Considering the natural fat content (35% for mustard

275

seeds and 43% for rapeseeds, respectively), the odorless powder was soaked with

276

refined sunflower oil containing the odorants. The original seeds and the

277

recombinates were evaluated as described above for APA.

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RESULTS AND DISCUSSION

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Identification of Aroma-active Compounds in Raw Mustard Seeds and

280

Rapeseeds. First, the volatiles were extracted with diethyl ether followed by high

281

vacuum distillation using SAFE technique.35 The distillates obtained exhibited the

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typical characteristic overall aroma of each kind of ground seeds when a drop of the

283

concentrated extract was put on a strip of filter paper, proving the successful ACS Paragon Plus Environment

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extraction of all key aroma compounds. Next, the extracts were subjected to AEDA

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as a screening method to differentiate between the aroma-active compounds and the

286

bulk of odorless volatiles. Application of AEDA revealed 39 aroma-active regions in

287

raw mustard seeds and 27 aroma-active regions in raw rapeseeds with an FD factor

288

range between 4 and 1024. In the distillate of raw mustard seeds, 19 (earthy, pea-

289

like), 51 (clove-like, smoky), and 55 (honey-like, beeswax-like) showed the highest

290

FD factor of 1024, followed by 52 (foxy) and 56 (vanilla-like) with an FD factor of 512

291

(Table 2; Figures 1 and 2). In the distillate of raw rapeseeds, 51 revealed the highest

292

FD factor of 1024, followed by 19, 27 (bell pepper-like), and 52 (all 256).

293

For identification of the aroma-active compounds, the respective odor quality and

294

intensity perceived at the sniffing port, the retention indices on two capillary columns

295

of different polarity, and mass spectra in EI and CI mode recorded by HRGC-MS or

296

HRGC/HRGC-MS (if trace odorants co-eluted with other volatiles present in higher

297

amounts) were compared with the data of an in-house database containing > 1000

298

aroma-active reference compounds.

299

Following this procedure, 2-isopropyl-3-methoxypyrazine (19), 4-ethenyl-2-

300

methoxyphenol (51), phenylacetic acid (55), 2-aminoacetophenone (52), and

301

4-hydroxy-3-methoxybenzaldehyde

302

compounds in raw white mustard seeds and were reported for the first time as aroma

303

constituents of these seeds (Figure 2).

(56)

were

characterized

as

aroma-active

304

The clove-like, smoky smelling 4-ethenyl-2-methoxyphenol (51) was also

305

identified as the odorant with the highest FD factor in raw rapeseeds, followed by

306

2-isopropyl-3-methoxypyrazine (19), 2-aminoacetophenone (52), and 2-isobutyl-3-

307

methoxypyrazine (27). Altogether, 36 odorants were successfully identified in raw

308

mustard seeds and 27 aroma-active compounds in raw rapeseeds.

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14 309

Identification of Aroma-active Compounds in Roasted Mustard Seeds and

310

Rapeseeds. To get a freshly roasted material, first, mustard seeds (140 °C, 30 min)

311

and rapeseeds (140 °C, 60 min) were heat-processed in a convection oven (Binder,

312

Tuttlingen, Germany), then frozen with liquid nitrogen, and finally ground. The

313

panelists evaluated both roasted seeds with a very pleasant popcorn-like and coffee-

314

like overall aroma impression. The aroma distillates obtained by solvent extraction of

315

the roasted materials followed by distillation fully represented the aroma of the

316

roasted seeds. Application of AEDA to the distillates revealed a total of 50 aroma-

317

active areas in the FD factor range up to 2048 for roasted mustard seeds and 44

318

areas for roasted rapeseeds. The odor-active compounds were subsequently

319

identified following the procedure described above.

320

For roasted mustard seeds, 4-hydroxy-2,5-dimethylfuran-3(2H)-one (49; caramel-

321

like), 4-ethenyl-2-methoxyphenol (51), and phenylacetic acid (55) showed the highest

322

FD factor of 2048, followed by 2,3-butanedione (1; butter-like), 2-acetyl-1-pyrroline

323

(13; popcorn-like), dimethyl trisulfide (15; cabbage-like), 2-ethyl-3,6-dimethylpyrazine

324

(21; roasty, earthy), 3-(methylthio)propanal (23; cooked potato-like), and 4-hydroxy-

325

3-methoxybenzaldehyde (56) (all 1024) as well as 2,3-pentanedione (2; butter-like)

326

and trans-4,5-epoxy-(E)-2-decenal (47; metallic) (both 512; Table 2 and Figure 3).

327

For roasted rapeseeds, 2,3-diethyl-5-methylpyrazine (25; earthy) and 4-hydroxy-

328

2,5-dimethylfuran-3(2H)-one (49) were characterized as the most aroma-active

329

compounds during AEDA (both FD factor of 2048), followed by dimethyl trisulfide

330

(15), 2-isopropyl-3-methoxypyrazine (19), and 4-ethenyl-2-methoxyphenol (51) (all

331

1024) (Table 2).

332

Due to the fact that the isolation procedure might discriminate highly volatile

333

compounds with low boiling points or there might be an overlapping with the solvent

334

during HRGC-O, static headspace analyses were applied. In addition to the results ACS Paragon Plus Environment

Page 15 of 56

Journal of Agricultural and Food Chemistry

15 335

obtained by AEDA, four further compounds, namely methanethiol (HS1; putrid,

336

cabbage-like, sulfury), dimethyl sulfide (HS2; asparagus-like), 3-methylbutanal (HS3;

337

malty), and 2-methylbutanal (HS4; malty), were identified in the ground seeds via

338

static headspace aroma dilution analysis in combination with static headspace high-

339

resolution gas chromatography-mass spectrometry. The Strecker aldehydes 2- and

340

3-methylbutanal were only detectable in the roasted seeds (Table 3).

341

Quantitation of the Key Odorants by Stable Isotope Dilution Assays (SIDAs).

342

Next, the odorants previously identified with high FD factors at least in one of the raw

343

or roasted samples were quantitated by means of SIDAs using the respective stable

344

isotopically labeled internal standards (Table 1). Quantitation of the aroma-active

345

compounds in raw mustard seeds revealed the highest concentration for acetic acid

346

(7620 µg/kg), followed by 1,8-cineole (455 µg/kg), dimethyl sulfide (392 µg/kg),

347

hexanoic acid (284 µg/kg), and phenylacetic acid (171 µg/kg). The popcorn-like

348

smelling 2-acetyl-1-pyrroline (0.16 µg/kg) was only detected in trace amounts

349

(Table 4).

350

In raw rapeseeds, acetic acid (7030 µg/kg) was also the most abundant odorant,

351

followed by pentanoic acid (4930 µg/kg), hexanoic acid (1140 µg/kg), dimethyl sulfide

352

(980 µg/kg), hexanal (164 µg/kg), and γ-nonalactone (164 µg/kg). Butanoic acid,

353

4-ethenyl-2-methoxyphenol as well as 2- and 3-methylbutanoic acid were present at

354

concentrations > 100 µg/kg. Many compounds were present only in trace amounts,

355

like

356

(1.07 µg/kg),

357

(0.29 µg/kg) (Table 4).

2-isobutyl-3-methoxypyrazine 1-octen-3-one

(1.98 µg/kg),

(0.47 µg/kg),

and

2-isopropyl-3-methoxypyrazine 2,3-diethyl-5-methylpyrazine

358

After thermal treatment, 39 aroma-active compounds in mustard seeds and 34

359

odorants in rapeseeds were quantitated. Again, acetic acid (11800 µg/kg) was

360

present at the highest concentration in roasted mustard seeds, but this time followed ACS Paragon Plus Environment

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16 361

by the malty smelling Strecker aldehydes 3-methylbutanal (8030 µg/kg) and

362

2-methylbutanal (2390 µg/kg) as well as the caramel-like 4-hydroxy-2,5-dimethyl-

363

furan-3(2H)-one (2010 µg/kg). The thermal treatment led to a significant increase of

364

2-furanmethanethiol (from 2.84 µg/kg to 690 µg/kg), the earthy smelling pyrazine 2-

365

ethyl-3,6-dimethylpyrazine (from 5.16 µg/kg to 279 µg/kg), and the butter-like

366

smelling ketones 2,3-butanedione (from 2.72 µg/kg to 98.0 µg/kg) and 2,3-

367

pentanedione (from < LoD to 88.8 µg/kg). Additionally, some popcorn-like and roasty

368

smelling odorants occurred after roasting, e.g., acetylpyrazine (2.08 µg/kg) and 2-

369

propionyl-1-pyrroline (1.21 µg/kg) (Table 4).

370

For roasted rapeseeds, the highest concentrations were determined for acetic

371

acid (12000 µg/kg) and 4-ethenyl-2-methoxyphenol (3590 µg/kg). After thermal

372

treatment, again 3-methybutanal (2400 µg/kg) and 2-methylbutanal (361 µg/kg) as

373

well as 4-hydroxy-2,5-dimethylfuran-3(2H)-one (1380 µg/kg) increased to remarkable

374

concentrations. In addition, the formation of some sulfur compounds like dimethyl

375

disulfide (from 0.24 µg/kg to 340 µg/kg) or dimethyl trisulfide (from < LoD to

376

400 µg/kg) was observed.

377

Calculation of Odor Activity Values (OAVs). To get information about the

378

contribution of a single odorant to the overall aroma of the seeds, OAVs (ratio of

379

concentration to respective odor threshold) were calculated for each odorant. Due to

380

the fat content of 35% in mustard seeds and 43% in rapeseeds (determined by acid

381

hydrolysis according to Weibull-Stoldt prior to Soxhlet method), orthonasal odor

382

thresholds were determined in refined sunflower oil as matrix.

383

For raw mustard seeds, 18 odorants showed an OAV ≥ 1. The highest OAV was

384

calculated for dimethyl sulfide (OAV=151), followed by 2-furanmethanethiol (149), 2-

385

isopropyl-3-methoxypyrazine (114), dimethyl trisulfide (44), 2-isobutyl-3-methoxy-

386

pyrazine (33), methanethiol (31), and 1,8-cineol (27) (Table 5). ACS Paragon Plus Environment

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Journal of Agricultural and Food Chemistry

17 387

After thermal treatment, the sulfur containing compounds 2-furanmethanethiol

388

(36300), methanethiol (617), dimethyl trisulfide (453), and dimethyl sulfide (298) were

389

proven to have a significant influence on the typical aroma of roasted white mustard

390

seeds. Moreover, 2-acetyl-1-pyrroline (460; popcorn-like), 3-methylbutanal (535;

391

malty), and 2,3-pentanedione (296; butter-like) reached high OAVs.

392

For raw rapeseeds, 13 of the quantitated compounds were present in

393

concentrations above their respective odor thresholds. The highest OAVs were

394

obtained for dimethyl sulfide (377; asparagus-like), 2-isopropyl-3-methoxypyrazine

395

(107; pea-like, earthy), 2-isobutyl-3-methoxypyrazine (50; bell pepper-like), and

396

methanethiol (44; putrid, cabbage-like, sulfury) (Table 6).

397

For roasted rapeseeds, 2-furanmethanethiol (14200), dimethyl trisulfide (13300),

398

methanethiol

(1160),

dimethyl

sulfide

(962),

2,3-pentanedione

(687),

and

399

3-methylbutanal (160) showed the highest OAVs, similar to roasted mustard seeds

400

(cf. Tables 5 and 6).

401

All odorants showing OAVs < 1 should not contribute to the overall aroma,

402

although they were detected during AEDA, not considering the influence of the matrix

403

on the aroma release. For example, high FD factors were obtained for the vanilla-like

404

smelling 4-hydroxy-3-methoxybenzaldehyde in raw and roasted mustard seeds,

405

whereas its concentrations did not exceed the odor threshold in oil.

406

Aroma Recombination and Aroma Profile Analysis (APA). To validate the

407

data obtained by identification and quantitation, aroma recombination experiments

408

were performed. For this purpose, all odorants showing an OAV ≥ 1 were dissolved

409

in ethanol and added to refined sunflower oil, not exceeding the threshold of ethanol

410

in oil (850 µg/kg). This oily aroma solution was added to the respective deodorized

411

seed powders obtained after solvent extraction and lyophilization resulting in a

412

mixture representing the naturally occurring odorant concentrations in the seeds. The ACS Paragon Plus Environment

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18 413

aroma recombinates and the original seed samples were evaluated using APA,

414

which was performed by a trained sensory panel rating each aroma quality on a

415

scale from 0 (no intensity) to 3 (high intensity) in steps of 0.5.

416

APA of the original raw mustard seeds and the recombinate showed a very good

417

similarity. The pea-like, earthy, and cabbage-like odor attributes were perceived with

418

the highest intensities (Figure 4A). The sensory panel also found a good agreement

419

of the overall aroma of the original raw rapeseeds compared to the respective

420

recombinate, only lacking in the green, grassy odor impression (Figure 4B). APA of

421

the freshly roasted seed samples and the respective recombinates revealed a perfect

422

similarity (Figures 4C and 4D). All in all, APA of the seeds and the recombinates

423

clearly demonstrated the change from the mainly pea-like aroma of the raw seeds to

424

the pleasant popcorn-like and coffee-like aroma of the roasted seeds. Finally, these

425

data confirmed a successful characterization of the key aroma compounds for raw

426

and roasted mustard seeds and rapeseeds.

427

Formation

of

Odor-Active

Compounds

during

Heat-Processing.

A

428

comparison of the amounts of key odorants found in raw mustard seeds and

429

rapeseeds and in the corresponding roasted seeds showed clear differences for

430

some

431

processing by a factor of 243 (from 2.84 µg/kg to 690 µg/kg) in mustard seeds and

432

from < LoD (0.27 µg/kg) to 271 µg/kg in rapeseeds, respectively. The formation

433

pathway of 2-furanmethanethiol was investigated via model systems by reacting

434

various monosaccharides with cysteine, glutathione, or thiamine as sulfur source,

435

indicating 2-furancarbaldehyde as the key intermediate.41,42

compounds.

2-Furanmethanethiol

increased

enormously

during

heat-

436

4-Hydroxy-2,5-dimethylfuran-3(2H)-one was found in high concentrations in both

437

roasted seeds, resulting in OAVs of 74 for mustard seeds and 51 for rapeseeds,

438

respectively. The concentration of the caramel-like smelling odorant increased by a ACS Paragon Plus Environment

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Journal of Agricultural and Food Chemistry

19 439

factor of 73 in roasted mustard seeds and reached a concentration of 1380 µg/kg in

440

roasted rapeseeds, whereas it was not detectable in raw rapeseeds. Furanones

441

emerge as typical carbohydrate degradation products during thermal treatment of

442

foods. Model studies showed that the furanone was formed by dehydration of

443

reducing monosaccharides, indicating acetylformoin as important intermediate.43

444

In addition, the concentrations of the malty smelling Strecker aldehydes 2- and

445

3-methylbutanal

considerably

increased

during

roasting

in

both

seeds.

446

2-Methylbutanal rose by a factor of 54 in mustard seeds and 26 in rapeseeds,

447

whereas 3-methylbutanal reached even higher factors of 190 (mustard seeds) und

448

282 (rapeseeds). Phenylacetaldehyde increased by a factor of 96 in mustard seeds

449

and of 4 in rapeseeds. In contrast, the amount of the Strecker aldehyde

450

3-(methylthio)propanal only moderately rose by a factor of 9 (mustard seeds) and 2

451

(rapeseeds). The formation of these aldehydes during roasting starts from their

452

parent amino acids isoleucine, leucine, phenylalanine, and methionine, reacting with

453

various α-dicarbonyl compounds formed by carbohydrate degradation, e.g.,

454

2-oxopropanal or deoxyosones.44

455

The concentrations of the popcorn-like smelling 2-acetyl-1-pyrroline and

456

2-propionyl-1-pyrroline also increased in the roasted seeds. 2-Acetyl-1-pyrroline

457

showed a strong rise in mustard seeds by a factor of 153. 2-Propionyl-1-pyrroline

458

was not detectable in unroasted mustard seeds, but reached a concentration of

459

1.21 µg/kg after thermal treatment. Both compounds were not detectable in raw

460

rapeseeds and were present at concentrations of 4.43 µg/kg (2-acetyl-1-pyrroline)

461

and 1.45 µg/kg (2-propionyl-1-pyrroline) after heat-processing. Both pyrrolines are

462

known as degradation products of proline in the presence of reducing carbohydrates,

463

proving 1-pyrroline as the key intermediate of the thermally induced formation.45,46

ACS Paragon Plus Environment

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20 464

Numerous thermally generated aroma-active pyrazines increased significantly in

465

roasted mustard seeds, e.g., 2-ethyl-3,6-dimethylpyrazine by a factor of 54 or 2,3-

466

diethyl-5-methylpyrazine by a factor of 8. The increase of all pyrazines was also

467

observed in rapeseeds, e.g., for 2-ethyl-3,6-dimethlypyrazine (from < LoD to

468

1380 µg/kg) or for 2-ethyl-3,5-dimethylpyrazine (< LoD to 23.3 µg/kg). The postulated

469

formation pathways of these pyrazines is based on a condensation of two

470

aminoketones, of two aminoaldehydes, or of an aminoketone and an aminoaldehyde,

471

followed by the addition of the Strecker aldehyde of alanine (acetaldehyde) to the

472

formed dihydropyrazines, which were suggested as intermediates.47,48 The proposed

473

source of the ethyl group could be confirmed using isotopically labeled alanine.49

474

Also higher concentrations of the butter-like smelling diketones 2,3-butanedione

475

(98.0 µg/kg) and 2,3-pentanedione (88.8 µg/kg) were observed in roasted mustard

476

seeds, while the raw seeds showed an amount of 2.72 µg/kg for 2,3-butanedione and

477

2,3-pentanedione was not detectable (< 0.63 µg/kg). In rapeseeds, the concen-

478

trations increased by a factor of 51 for 2,3-butanedione and 25 for 2,3-pentanedione.

479

A possible formation pathway of 2,3-butanedione was postulated by an aldol reaction

480

from acetaldehyde and hydroxyacetaldehyde (glycolaldehyde).45 The formation of the

481

homologous 2,3-pentanedione can be suggested by the similar reaction starting from

482

propanal and hydroxyacetaldehyde.

483

In summary, the generation of new aroma-active compounds as well as the

484

increase of the concentrations of numerous key odorants during roasting of white

485

mustard seeds and rapeseeds explains on a molecular basis the formation of the

486

characteristic popcorn-like and coffee-like aroma of the roasted seeds, completely

487

different to the mainly pea-like aroma of the raw seeds. Thus, raw Brassica seeds

488

show a huge potential to generate aroma-active compounds from a natural source,

ACS Paragon Plus Environment

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Journal of Agricultural and Food Chemistry

21 489

which is demanded by the consumers, enabling various future possibilities for the

490

use in food production.

491 492

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22

493

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494

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induced reductive coupling of α,β-unsaturated esters with carbonyl compounds

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beverage prepared from Darjeeling black tea: quantitative differences between tea

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

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

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

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

Engel, W.; Bahr, W.; Schieberle, P. Solvent assisted flavour evaporation - a

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36.

Bemelmans, J. M. H. Review of isolation and concentration techniques. In

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Progress in Flavour Research; Land, D. G., Nursten, H. E., Eds.; Applied Science:

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London, UK, 1979; pp 79-98.

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37.

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1141-1144.

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Characterization of the aroma-active compounds in pink guava (Psidium guajava, L.)

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by application of the aroma extract dilution analysis. J. Agric Food. Chem. 2008, 56,

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4120-4127.

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39.

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compounds in organically grown, raw West-African peanuts (Arachis hypogaea) and

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in ground, pan-roasted meal produced thereof. J. Agric. Food Chem. 2008, 56,

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10237-10243.

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40.

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

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thresholds of key food aroma compounds and development of an aroma language

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based on odour qualities of defined aqueous odorant solutions. Eur. Food Res.

610

Technol. 2008, 228, 265-273.

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41.

612

treated solution of ribose and cysteine by aroma extract dilution techniques. J. Agric.

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Food Chem. 1995, 43, 2187-2194.

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42.

615

mechanism in a glutathione–xylose Maillard reaction. Food Chem. 2012, 131, 280-

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Precursors, Thermal and Enzymtic Conversions, ACS Symposium Series, no. 490;

Schieberle, P. Primary odorants in popcorn. J. Agric. Food Chem. 1991, 39,

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Sinuco,

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Czerny, M.; Christlbauer, Ma.; Christlbauer, Mo.; Fischer, A.; Granvogl, M.;

Hofmann, T.; Schieberle, P. Evaluation of the key odorants in a thermally

Wang, R.; Yang, C.; Song, H. Key meat flavour compounds formation

Schieberle, P. Formation of furaneol in heat-processed foods. In Flavor

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27 619

Teranishi, R., Takeoka, G. R., Güntert, M., Eds.; American Chemical Society:

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Washington, DC, 1992, pp 164-174.

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44.

622

formation. Food Rev. Int. 2008, 24, 416-435.

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45.

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roast-smelling odorants 2-propionyl-1-pyrroline and 2-propionyltetrahydropyridine in

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Maillard-type reactions. J. Agric. Food Chem. 1998, 46, 2721-2726.

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46.

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pyrroline - important intermediates in the generation of the roast-smelling food flavor

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compounds 2-acetyl-1-pyrroline and 2-acetyltetrahydropyridine. J. Agric. Food Chem.

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1998, 46, 2270-2277.

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47.

631

diethyl-5-methylpyrazine formed in roasted beef. Z. Lebensm.-Unters. Forsch. 1994,

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198, 210-214.

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sugar-ammonia model systems. J. Agric. Food Chem. 1977, 25, 609-614.

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alkylpyrazines in the Maillard reaction. J. Agric. Food Chem. 1995, 43, 2818-2822.

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637 638

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28 639

FIGURE CAPTIONS

640 641

Figure 1. Flavor dilution (FD) chromatogram on a polar DB-FFAP capillary column

642

obtained by AEDA from a distillate of raw mustard seeds (FD ≥ 32) (numbering refers

643

to Table 2).

644 645

Figure 2. Structures of key aroma-active compounds identified in raw mustard seeds

646

(FD factors and odor descriptions in parentheses; numbering refers to Table 2).

647 648

Figure 3. Structures of key aroma-active compounds identified in roasted mustard

649

seeds (FD factors and odor descriptions in parentheses; numbering refers to

650

Table 2).

651 652

Figure 4. Aroma profile analyses of raw mustard seeds (solid line) and the respective

653

recombinate (dotted line) (A), aroma profile analyses of raw rapeseeds (solid line)

654

and the respective recombinate (dotted line) (B), aroma profile analyses of roasted

655

mustard seeds (solid line) and the respective recombinate (dotted line) (C), and

656

aroma profile analyses of roasted rapeseeds (solid line) and the respective

657

recombinate (dotted line) (D).

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29 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 ion (m/z)a

isotope

Rfb

odorant label analyte

standard

acetic acid

[13C2]

61

63

0.93

acetylpyrazine

[2H3]

123

126

0.99

2-acetyl-1-pyrroline

[13C5]

112

117

0.91

2-aminoacetophenone

[2H3]

136

139

0.85

2,3-butanedione

[13C4]

87

91

0.92

butanoic acid

[2H2]

89

91

0.99

2-sec-butyl-3-methoxypyrazine

[2H3]

167

170

0.86

1,8-cineole

[2H2]

155

157

0.98

(E)-2-decenal

[2H2]

155

157

0.87

2,3-diethyl-5-methylpyrazine

[2H3]

151

154

0.93

dimethyl disulfide

[2H6]

95

101

0.98

dimethyl sulfide

[2H3]

63

66

0.84

dimethyl trisulfide

[2H6]

127

133

0.99

4-ethenyl-2-methoxyphenol

[2H3]

151

154

1.00

2-ethyl-3,5-dimethylpyrazine

[2H3]

137

140

0.97

2-ethyl-3,6-dimethylpyrazine

[2H3]

137

140

0.87

2-furanmethanethiol

[2H2]

81

83

0.98

hexanal

[2H12]

101

113

0.97

hexanoic acid

[2H3]

117

120

1.00

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

[13C2]

129

131

0.94

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30 Table 1. continued ion (m/z)a

isotope

Rfb

odorant label analyte

standard

4-hydroxy-3-methoxybenzaldehyde

[2H3]

153

156

0.99

2-isobutyl-3-methoxypyrazine

[2H3]

167

170

0.86

2-isopropyl-3-methoxypyrazine

[2H3]

153

156

0.98

methanethiol

[2H3]

49

52

0.58

2-methylbutanal

[2H2]

87

89

0.77

3-methylbutanal

[2H2]

87

89

0.85

2-methylbutanoic acid

[2H9]

103

112

0.81

3-methylbutanoic acid

-c

103

112c

0.81

4-methylphenol

[2H8]

109

117

0.98

3-(methylthio)propanal

[2H3]

105

108

0.93

γ-nonalactone

[2H2]

157

159

0.79

(E)-2-nonenal

[2H2]

141

143

0.90

γ-octalactone

[2H2]

143

145

0.95

1-octen-3-one

[2H2-4]d

127

129-131d

0.71

2,3-pentanedione

[13C2]

101

103

0.81

pentanoic acid

[2H3]

103

106

0.88

phenylacetaldehyde

[13C2]

121

123

0.81

phenylacetic acid

[13C2]

137

139

0.74

2-propionyl-1-pyrroline

[2H2-5]d

126

128-131d

0.99

a

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.

c

Quantitation of 3-methylbutanoic acid was performed using [2H9]-2ACS Paragon Plus Environment

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Journal of Agricultural and Food Chemistry

31 methylbutanoic acid as internal standard. For further details see Materials and Methods.

d

Internal standard was used as a mixture of isotopologues.

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32

Table 2. Important Aroma-active Compounds (FD ≥ 4) Identified in Aroma Distillates of Raw and Roasted Mustard Seeds and Rapeseeds FD factorsd

retention indices on

no.a odorantb

raw

odor qualityc DB-FFAP

roasted

raw

roasted

DB-5 mustard seeds

rapeseeds

1

2,3-butanedione

butter-like

996

592

8

1024

8

64

2

2,3-pentanedione

butter-like

1064

697