Characterization of Key Aroma-Active Compounds in Raw and

Chapter 8

Downloaded by COLUMBIA UNIV on November 29, 2016 | http://pubs.acs.org Publication Date (Web): November 18, 2016 | doi: 10.1021/bk-2016-1237.ch008

Characterization of Key Aroma-Active Compounds in Raw and Roasted White Mustard Seeds (Sinapis alba L.) Eva Ortner, Peter Schieberle, and Michael Granvogl* Lehrstuhl für Lebensmittelchemie, Technische Universität München, Lise-Meitner-Straße 34, D-85354 Freising, Germany *E-mail: [email protected]

Thermal processing of food leads to the formation of different desired quality aspects like aroma, taste, and color. Changes in the overall aroma are based, for example, on the degradation of odorless precursors or on interactions of food ingredients forming numerous odorants. To get a deeper insight into these changes induced by roasting of white mustard seeds on a molecular basis, the Molecular Sensory Science Concept including comparative aroma extract dilution analysis (cAEDA), identification experiments, quantitation via stable isotope dilution analysis (SIDA), and calculation of odor activity values (OAVs, ratio of concentration to odor threshold) was used. All odorants increased significantly during roasting, e.g., by a factor of 240 for 2-furanmethanethiol (from 2.84 to 690 µg/kg), of 190 for 3-methylbutanal (from 42.2 to 8030 µg/kg), and of 150 for 2-acetyl-1-pyrroline (from 0.16 to 24.4 µg/kg).

Introduction White mustard (Sinapis alba L.) belongs to the Brassicaceae and is suitable for a wide range of applications in foods. The seeds are used for dry milling to obtain the flour, for wet milling to get mustard pastes, and in spice mixtures. In Indian kitchen, they are well-known for their unique and highly attractive odor generated by deep-frying, whereas in Europe they are mainly used untreated in pickling or boiling vegetables such as cabbage or sauerkraut. Roasting has © 2016 American Chemical Society Granvogl et al.; Browned Flavors: Analysis, Formation, and Physiology ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


gained significant importance in food industry as a multfunctional process for numerous foods to produce a characteristic flavor, e.g., green coffee beans, hazelnuts, and peanuts (1–3). It is the complexity of precursors and reaction pathways that delivers the characteristic flavor associated with each type of food. Although mustard seeds are a popular spice, highly appreciated for their delicious aroma before and after thermal treatment, only a few studies have already been carried out to investigate the volatiles of processed seed products and plant parts, focused on volatile glucosinolate degradation products responsible for the pungent aroma qualities (4). Thereby, the volatiles of mustard seeds and products thereof have mostly been identified by instrumental-analytical methods like GC-MS, showing isothiocyanates, thiocyanates, sulfides, and nitriles as major constituents (5–7). In the past, the efficiency of the formation of desirable aroma-active compounds during the roasting process was already shown for white mustard seeds, e.g., 2-furanmethanethiol, 3-(methylthio)propanal, and 4-hydroxy-2,5-dimethylfuran-3(2H)-one were proven as main odorants (8). But, up to now, no data on the contribution of single odorants to the overall aroma of roasted seeds as well as quantitative changes in the key aroma compounds during heat-processing are available. To get a deeper insight into the changes of odorants induced by roasting of white mustard seeds, the Molecular Sensory Science Concept was applied to raw and roasted seeds.

Experimental Part Materials and Methods Seeds White mustard seeds (Sinapis alba L.) were obtained from Ostmann Gewürze (Dissen a.T.W., Germany). For heat-processing, raw mustard seeds (200 g) were roasted in a convection oven (Binder, Tuttlingen, Germany) for 30 min at 140 °C.

Stable Isotopically Labeled Internal Standards [2H3]-Dimethyl sulfide (Sigma-Aldrich, Taufkirchen, Germany) was commercially obtained. The following internal standards were synthesized according to published methods: [2H3]-acetylpyrazine (9), [13C5]-2-acetyl-1pyrroline (10), [13C4]-2,3-butanedione (11), [2H3]-2,3-diethyl-5-methylpyrazine (12), [2H6]-dimethyl trisulfide (13), [2H3]-2-ethyl-3,5-dimethylpyrazine and [2H3]-2-ethyl-3,6-dimethylpyrazine (12), [2H2]-2-furanmethanethiol (14), [13C2]4-hydroxy-2,5-dimethylfuran-3(2H)-one (15), [2H3]-methanethiol (16), [2H2]2-methylbutanal and [2H2]-3-methylbutanal (17), [2H3]-3-(methylthio)propanal (14), [13C2]-2,3-pentanedione (18), [13C2]-phenylacetaldehyde (19), and [2H2-5]-2-propionyl-1-pyrroline (9). 104 Granvogl et al.; Browned Flavors: Analysis, Formation, and Physiology ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Isolation of the Volatiles


Raw or roasted seeds (200 g each) were frozen in liquid nitrogen and ground by a commercial blender (Privileg, Fürth, Germany). After the addition of diethyl ether (2 x 250 mL), the mixture was vigorously stirred for 2 x 1 h at room temperature. Afterwards, the volatiles were separated from the non-volatiles by means of the solvent assisted flavor evaporation (SAFE) technique (20). The distillate obtained was dried over anhydrous sodium sulfate, filtered, concentrated at 40 °C to ~ 3 mL using a Vigreux column (50 cm × 1 cm i.d.), and finally to ~ 100 μL by micro-distillation.

Aroma Extract Dilution Analysis (AEDA) The flavor dilution (FD) factors of the odorants were determined by diluting the concentrated extract stepwise 1+1 (v+v) with diethyl ether. Every dilution step was analyzed twice by high-resolution gas chromatography-olfactometry (HRGCO).

High-Resolution Gas Chromatography-Olfactometry (HRGC-O) HRGC-O was performed by means of a Trace GC Ultra gas chromatograph (Thermo Fisher Scientific, Dreieich, Germany) equipped with either a DB-FFAP or a DB-5 fused silica capillary (each 30 m × 0.25 mm i.d., 0.25 μm film thickness; J&W Scientific; Agilent, Waldbronn, Germany). The samples were manually injected by the cold on-column technique at 40 °C. After 2 min, the temperature was raised at 6 °C/min to 230 °C (DB-FFAP) or 240 °C (DB-5), respectively, and held for 5 min. The flow rate of the carrier gas helium was 1.2 mL/min. At the end of the column, the effluent was split 1:1 by a Y-shaped splitter (Chrompack, Frankfurt, Germany). Two deactivated fused silica capillaries of the same length (30 cm × 0.18 mm i.d.) led either to a flame ionization detector (FID) held at 250 °C or to a sniffing port (230 °C), respectively.

High-Resolution Gas Chromatography-Sector Field Mass Spectrometry For identification experiments, mass spectra were generated by a gas chromatograph 5890 series II (Hewlett-Packard, Waldbronn, Germany) coupled to a MAT 95 S sector field mass spectrometer (Finnigan MAT, Bremen, Germany). Mass spectra in the electron ionization (EI) mode were generated at 70 eV and in the chemical ionization (CI) mode at 115 eV using isobutane as reactant gas. Samples were manually injected by the cold on-column technique (40 °C). 105 Granvogl et al.; Browned Flavors: Analysis, Formation, and Physiology ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Quantitation of Odorants by Stable Isotope Dilution Assays (SIDAs) Mustard seeds were frozen with liquid nitrogen and ground to a fine powder by a commercial blender. Diethyl ether (100−400 mL) and aliquots of the internal standards (0.5−5 μg; dissolved in dichloromethane or diethyl ether; amounts depending on the concentrations of the respective analytes determined in a preliminary experiment) were added to the powder (1−400 g) and the suspension was stirred for 2 h at room temperature. Further work-up was performed as described above for the isolation of the volatiles and quantitation was performed via high-resolution gas chromatography-mass spectrometry (HRGC-MS) or two-dimensional high-resolution gas chromatography-mass spectrometry (HRGC/HRGC-MS). For quantitation of dimethyl sulfide, the ground seeds (1 g) were placed into headspace vials (volume 20 mL) and the labeled standard (0.2-1 µg) was added. After equilibration (28 °C, 30 min), aliquots of the headspace were withdrawn and analyzed by static headspace high-resolution gas chromatography-mass spectrometry (SH-HRGC-MS). For quantitation of methanethiol, the ground seeds (0.5-1 g) were filled into headspace vials (20 mL) and sealed with a gas-tight septum. A defined volume of the labeled methanethiol was injected into the headspace vials with a gas-tight syringe. After equilibration (28 °C, 30 min), aliquots of the headspace were withdrawn and analyzed by SH-HRGC-MS. For each odorant, a response factor was calculated by analyzing binary mixtures of defined amounts of the unlabeled and the labeled compound in at least five different mass ratios (5:1, 3:1, 1:1, 1:3, 1:5).

High-Resolution Gas Chromatography-Mass Spectrometry (HRGC-MS) For quantitation, HRGC-MS was performed by a Varian 431 gas chromatograph (Darmstadt, Germany) equipped with a DB-FFAP capillary column (30 m × 0.25 mm i.d., 0.25 μm film thickness) coupled to an ion trap mass spectrometer Saturn 2000 (Varian). Mass spectra were generated in CI mode at 70 eV with methanol as reactant gas. Samples were injected at 40 °C by means of a Combi PAL autosampler (CTC Analytics, Zwingen, Switzerland) using the cold on-column technique.

Two-Dimensional High-Resolution Gas Chromatography-Mass Spectrometry (HRGC/HRGC-MS) If overlapping peaks were observed, HRGC/HRGC-MS was performed using a Trace 2000 series gas chromatograph (ThermoQuest, Egelsbach, Germany) equipped with a DB-FFAP column (30 m × 0.25 mm i.d., 0.25 μm film thickness) in the first dimension coupled to a Varian CP-3800 equipped with an OV-1701 column in the second dimension (30 m × 0.25 mm i.d., 0.25 μm film thickness) (both J&W Scientific). The system was finally connected to an ion trap mass 106 Granvogl et al.; Browned Flavors: Analysis, Formation, and Physiology ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

spectrometer Saturn 2000. Heart-cuts were done by means of the moving column stream switching system (ThermoQuest). Mass spectra were generated in CI mode (70 eV) using methanol as reactant gas. Samples were injected at 40 °C by means of a Combi PAL autosampler using the cold on-column technique.


Static Headspace Aroma Dilution Analysis (SH-ADA) on the basis of High-Resolution Gas Chromatography-Olfactometry/Mass Spectrometry (SH-HRGC-O/MS) Decreasing headspace volumes (10−0.16 mL) were withdrawn with a gas-tight syringe (Innovative Labor Systeme, Stützerbach, Germany) by a Combi PAL autosampler. The volatiles were cryo-focused and transferred onto a DB-5 fused silica capillary column (30 m × 0.25 mm i.d., 0.5 μm film thickness; J&W Scientific) placed in a Trace GC Ultra. At the end of the column, the effluent was transferred either to a sniffing port (230 °C) or an ion trap mass spectrometer Saturn 2100 T (Varian). Mass spectra were generated in CI mode at 70 eV with methanol as reagent gas (21).

Aroma Profile Analysis A panel consisting of at least 15 experienced panelists evaluated the characteristic aroma descriptors, determined in preliminary sensory experiments. For each descriptor, a reference solution in water at a concentration 100-fold above the respective odor threshold was provided. The intensities of the respective aroma qualities were ranked on a linear seven-point scale (steps of 0.5) from 0 (non perceivable) to 3 (strongly perceivable). Analyses were performed in a sensory room equipped with single booths at 21 ± 1 °C. The samples (5 g) were presented in covered glass vessels (40 mm i.d., total volume = 45 mL) (22).

Orthonasal Odor Thresholds (OTs) For the calculation of odor activity values (OAVs), orthonasal odor thresholds were determined in sunflower oil as recently described (22).

Results and Discussion Changes Induced by the Roasting Process To characterize the sensory changes during heat-processing, first, a comparative aroma profile analysis describing the orthonasal aroma impressions of raw and roasted mustard seeds was performed. The overall aroma of raw seeds was dominated by pea-like and earthy attributes, whereas after the roasting process a pleasant aroma eliciting popcorn-like, coffee-like, caramel-like, and malty odor impressions was generated (Figure 1). 107 Granvogl et al.; Browned Flavors: Analysis, Formation, and Physiology ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Figure 1. Comparative aroma profile analysis of raw (dotted line) and roasted white mustard seeds (solid line)

To identify the compounds being responsible for the changes of the overall aroma impression, comparative aroma extract dilution analysis on the basis of HRGC-O and comparative aroma dilution analysis on the basis of static headspace HRGC-O was carried out. The aroma-active areas were related to chemical structures by identification experiments. For this purpose, the analytical data of reference compounds were compared with data of the corresponding analytes in the sample. Differences in FD factors determined for the same odorants in raw and roasted seeds allowed first insights into the influence of the thermal treatment on the odorants (Table 1). Roasty and/or earthy smelling compounds like 2-ethyl-3,6-dimethylpyrazine, 2-ethyl-3,5-dimethylpyrazine, and 2,3-diethyl-5-methyl-pyrazine were not detectable during GC-O of the raw seeds, but showed high FD factors in the roasted seeds. Furthermore, 2,3-pentanedione (butter-like), acetylpyrazine (popcorn-like, roasty), and 2-propionyl-1-pyrroline (popcorn-like, roasty) were only perceived after the roasting process. Some other odorants like 4-hydroxy-2,5-dimethylfuran-3(2H)-one (caramel-like), 3-(methylthio)-propanal (cooked potato-like), dimethyl trisulfide (cabbage-like), 2-acetyl-1-pyrroline (popcorn-like, roasty), and 2,3-butanedione (butter-like) showed significantly higher FD factors after thermal treatment (Table 1). During AEDA of the distillate, highly volatile compounds might not be detected due to a possible overlapping by the solvent or due to losses during the work-up procedure. To avoid overlooking of these compounds, static headspace HRGC-O using a series of decreasing gas volumes was applied to the raw and freshly roasted seeds. Thereby, four further compounds, namely dimethyl sulfide (asparagus-like), methanethiol (putrid, cabbage-like, sulfury) as well as 2- and 3-methylbutanal (both malty) were identified. Both methylbutanals were only perceived after the roasting process, whereas the FD factors of methanethiol and dimethyl sulfide significantly increased in the roasted seeds (Table 2). 108 Granvogl et al.; Browned Flavors: Analysis, Formation, and Physiology ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Table 1. Aroma-active Compounds Identified in White Mustard Seeds Showing Increased FD factors after Roasting


odoranta

odor

qualityb

RIc (DB-FFAP)

FD factord in mustard seeds raw

roasted

2-propionyl-1-pyrroline

popcorn-like, roasty

1382

Characterization of Key Aroma-Active Compounds in Raw and

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