J. Agric. Food Chem. 1998, 46, 2721−2726
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Flavor Contribution and Formation of the Intense Roast-Smelling Odorants 2-Propionyl-1-pyrroline and 2-Propionyltetrahydropyridine in Maillard-Type Reactions Thomas Hofmann and Peter Schieberle* Institut fu¨r Lebensmittelchemie and Deutsche Forschungsanstalt fu¨r Lebensmittelchemie, Lichtenbergstrasse 4, D-85748 Garching, Germany
Application of aroma extract dilution analysis on two different model mixtures of proline and glucose, reacted under aqueous or dry-heating conditions, revealed 2-propionyl-1-pyrroline (PP) and 2-propionyltetrahydropyridine (PTHP; occurring in two tautomers), besides 2-acetyl-1-pyrroline and 2-acetyltetrahydropyridine, as important roast-smelling odorants in both mixtures. A comparison of the isotope distribution in PP and PTHP formed from either 13C6-labeled or unlabeled glucose suggested a formation pathway for both odorants from the same intermediate, 1-pyrroline, when reacted with either 2-oxobutanal (yielding PP) or 1-hydroxy-2-butanone (yielding PTHP). 2-Oxobutanal, a possible precursor of 1-hydroxy-2-butanone, was shown to be formed in high yields (29 mol %) by reacting acetaldehyde and glycolaldehyde, two well-known degradation products of carbohydrates. Keywords: Maillard reaction; labeling experiments; 2-acetyl-1-pyrroline; 2-acetyltetrahydropyridine; 2-propionyl-1,4,5,6-tetrahydropyridine; 2-propionyl-3,4,5,6-tetrahydropyridine; 2-propionyl-1-pyrroline INTRODUCTION
The Maillard-type reaction between the amino acid proline and reducing carbohydrates is well-known to generate popcorn-like, roasty odors upon thermal treatment (Hunter et al., 1969; Lane and Nursten, 1983). 2-Acetyltetrahydropyridine [ATHP, occurring in an equilibrium of two tautomers; cf. Tressl et al. (1981) and Schieberle (1990a)] and 2-acetyl-1-pyrroline (AP) exhibit such odor notes at the very low odor thresholds of 0.2 and 0.02 ng/L (in air), respectively (Schieberle, 1995a). Both odorants have earlier been identified in the volatile fraction of several Maillard-type model reactions containing proline (Hunter et al., 1969; Tressl et al., 1981), and it had, therefore, been proposed that ATHP and AP mainly contribute to the overall roasty odors of such processed flavors. The application of gas chromatography olfactometry (GCO) techniques, such as Charm analysis, or aroma extract dilution analysis (AEDA) [cf. review by Schieberle (1995b)] allows the evaluation of the relative odor potencies of a single volatile in complex extracts of volatile compounds by means of the “extract dilution to odor threshold in air” approach. Using Charm analysis, Roberts and Acree (1994) recently proved the two tautomers of ATHP to be the key odorants (74% of Charm) in a thermally treated (200 °C; 1 min; aqueous conditions) equimolar mixture of proline and glucose, followed by AP (18% of Charm). 2,3Butanedione (4% of Charm) and 4-hydroxy-2,5-dimethyl-3(2H)-furanone (4% of Charm) were found to contribute less to the overall odor. Similar results for ATHP and AP had earlier been reported for a heated proline/2-oxopropanal mixture (Schieberle, 1990a). * Author to whom correspondence should be addressed (telephone +49-89-289 141 70; fax +49-89-289 141 83; e-mail
[email protected]).
Besides AP and ATHP, 2-propionyl-1-pyrroline (PP) has been identified as a further key odorant in freshly popped corn (Schieberle, 1991) and, very recently, in the roasted skin of fried chicken (Kerscher and Grosch, personal communication). 2-Propionyltetrahydropyridines had been tentatively identified in thermally treated proline/glucose systems (Tressl et al., 1981). However, to date, these compounds have not been characterized in foods or reaction flavors based on sensory and analytical data. To get insight into the precursors and formation of the propionyl homologues of AP and ATHP, the key odorants formed during thermal treatment of proline/ glucose mixtures were reinvestigated by AEDA and their formation was elucidated using 13C-labeled glucose. EXPERIMENTAL PROCEDURES Chemicals. 2,3-Butanedione, 1-hydroxy-2-butanone, acetaldehyde, glycolaldehyde, 1,2-diaminobenzene, and 4-hydroxy-2,5-dimethyl-3(2H)-furanone were from Aldrich (Steinheim, Germany). [13C6]Glucose was from Sigma (Munich, Germany). Syntheses: Preparation of Roast Odorants. AP and ATHP were prepared using the new synthetic approaches described recently (Hofmann and Schieberle, 1998a). PP was prepared from 1-pyrroline and 2-oxobutanal as previously reported (Schieberle, 1991). 1-Pyrroline was generated by oxidation of L-proline as described recently (Schieberle, 1991). [13C4]-2,3-Butanedione was synthesized as recently described (Schieberle and Hofmann, 1997a). 2-Propionyltetrahydropyridine (PTHP). On the basis of a reaction route verified recently for the homologous ATHP (Hofmann and Schieberle, 1998b), 1-pyrroline (0.1 mmol) and 1-hydroxy-2-butanone (0.1 mmol) were reacted at 100 °C in phosphate buffer (50 mL; 0.1 mol/L; pH 7.0) for 30 min. The volatiles formed were isolated by extraction with diethyl ether followed by sublimation in vacuo (Sen et al., 1991). 1H NMR
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Hofmann and Schieberle
Figure 1. Mass spectra (MS/EI) of 2-propionyl-3,4,5,6-tetrahydropyridine (I; A) and 2-propionyl-1,4,5,6-tetrahydropyridine (II; B).
Figure 2. Mass spectra of 2-acetyl-3,4,5,6-tetrahydropyridine (A) and 2-acetyl-1,4,5,6-tetrahydropyridine (B).
and 13C measurements in an aliquot of the distillate gave signals for 2-propionyl-1,4,5,6- and 2-propionyl-3,4,5,6-tetrahydropyridine, which were in agreement with those published recently by De Kimpe and Keppens (1996). Highresolution gas chromatography on an SE-54 column- showed two main products eluting at retention indices (relative to n-alkanes) of 1148 (I) and 1239 (II). Their mass spectra are displayed in Figure 1 [A (I) and B (II)]. By MS/CI, for both a molecular mass of 139 was established. To correlate the GC peaks with the structure of the tautomers, compound I was isolated by preparative gas chromatography using the method described recently (Schieberle, 1991). The signals obtained by 1H NMR were identical with the data for the synthetic 2-propionyl-3,4,5,6-tetrahydropyridine (De Kimpe and Keppens, 1996). On the basis of this result, compound II was assigned as 2-propionyl-1,4,5,6-tetrahydropyridine. This order of elution is further corroborated by the following results: It was recently shown (Hofmann and Schieberle, 1998b) that on an SE-54 column the imine tautomer of 2-acetyl-3-methyltetrahydropyridine is eluted before the enamine tautomer. Furthermore, the higher intensity of the fragment m/z 57 (CH3CH2CtO+) found for I (Figure 1A) is more reasonable for the imine tautomer (cf. parts A and B of Figure 1). Similar differences were also found in the mass spectra of the homologous 2-acetyltetrahydropyridines, also showing a more intense fragment of the acetyl group m/z 43 (CH3CtO+) in the imine tautomer (cf. parts A and B of Figure 2). Both propionyltetrahydropyridines showed the same low odor threshold of 0.2 ng/L in air. Model Reactions: Isolation of the Volatiles. Two different model systems were studied. In mixture I, a diluted aqueous mixture of L-proline (2 mmol) and D-glucose (1 mmol) was boiled in phosphate buffer (200 mL; pH 7.0; 0.1 mol/L) for 2 h and the volatiles formed were simultaneously steamdistilled and extracted using the apparatus described by Nickerson and Likens (1966). In mixture II, L-proline (2 mmol) and D-glucose (1 mmol) were mixed with silica gel (2.7 g containing 300 µL of the same phosphate buffer) and then
heated for 10 min at 160 °C in closed glass vials (1 cm i.d.; total volume ) 10 mL). Mixture II was extracted with diethyl ether (total volume ) 300 mL), and the volatiles were isolated by sublimation in vacuo (Sen et al., 1991). The distillates obtained from both mixtures were concentrated to 0.2 mL by distilling off the solvent at 35 °C using a Vigreux column (60 cm × 1 cm i.d.) followed by microdistillation (Schieberle, 1991). The most odor-active volatiles were then evaluated by AEDA as described previously (Schieberle, 1991). Labeling Experiments. In mixtures I and II, D-glucose was substituted by [13C6]-D-glucose and, after thermal treatment, the volatile fractions were isolated as described above. The carbon isotope ratios in the labeled odorants generated were determined by mass chromatography of the cluster of molecular ions generated in the electron impact mode. The relative concentration of each isotopomer was calculated by using the computer of the mass spectrometer. The data were compared with results obtained for the respective unlabeled aroma compounds. High-Resolution Gas Chromatography (HRGC)/Mass Spectrometry (MS). HRGC was performed with a type 5160 gas chromatograph (Fisons Instruments, Mainz, Germany) by using the following capillaries: FFAP (30 m × 0.32 mm fused silica capillary, free fatty acid phase, 0.25 µm; J&W Scientific, Fisons Instruments, Mainz, Germany) and SE-54 (30 m × 0.32 mm fused silica capillary DB-5; 0.25 µm; J&W Scientific, Fisons Instruments). The samples were applied by the oncolumn injection technique at 40 °C. After 2 min, the temperature of the oven was quickly raised at 40 °C/min to 50 °C (SE-54) or 60 °C (FFAP), respectively, held for 5 min isothermally, then raised at 6 °C/min to 230 °C, and held for 15 min. The flow of the helium carrier gas was 2.5 mL/min. At the end of the capillary, the eluate was split 1:1 (by volume) into an FID and a sniffing port using deactivated but uncoated fused silica capillaries (50 cm × 0.32 mm). The FID and the sniffing port were held at 180 °C. Linear retention indices (RI) of the compounds were calculated from the retention times
Formation of 2-Propionyl-1-pyrroline and 2-Propionyltetrahydropyridine
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Table 1. Key Odorants (FD g 16) Generated in Thermally Treated Proline/Glucose Reaction Mixturesa no. 1 2 3 4 5 6 7 8 9
odorantb
odor qualityc
RI on SE-54
2,3-butanedione 2-acetyl-1-pyrroline 2-propionyl-1-pyrroline 2-acetyl-3,4,5,6-tetrahydropyridine 4-hydroxy-2,5-dimethyl-3(2H)-furanone 2-acetyl-1,4,5,6-tetrahydropyridine 2-propionyl-3,4,5,6-tetrahydropyridine 2-propionyl-1,4,5,6-tetrahydropyridine unknown
buttery popcorn-like popcorn-like popcorn-like caramel-like popcorn-like popcorn-like popcorn-like roasty
592 922 1024 1049 1068 1145 1148 1239 1352
FD factord in mixture I mixture II 16 64 16 4096
