Formation of Pyrazines in Aqueous Maltose/Glucose/Fructose-Glutamine Model Systems Upon Heating at Below 100'C

Food Sci. Tecl7no!. Res,. 10 (2), 1 99-2O4, 2004

Formation of Pyrazines in Aqueous Maltose/Glucose/Fructose-Glutamine Model Systems upon Heating at below 100'C

Kenji ITo* and Masataka MORl

Toba('co Scienc'e Resea/rJ7 Center Japa/7 Tohac'co l/7c., 6-2 Unlegaoka, Aoho-ku, Yok017anla 227-85J2, ,lupan

Received October 2 1 , 2003; Accepted December 1 6, 20O_3

Pyrazines generated from aqueous sugar-glutamine model systems heated at 90'C were investigated quantita- tively. To determine trace levels of pyrazines in aqueous matrices, an efficient method using solid-phase extraction (SPE) and gas chromatography/mass spectrometry (GCMS) was developed. A series of alkylpyrazines as well as trace levels of acetylpyrazines and bis-(2-furyl)pyrazines were detected in the model systems by this method. Remarkable difference in the formation of pyrazines between monosaccharide and disaccharide was observed. The yield of acetylpyrazines and bis-(2-furyl)pyrazines from maltose was larger than those from fructose and glucose, while that of alkylpyrazines was less. The pH dependency on the generation of pyrazines in maltose-glutamine model systems was also examined.

Keywords: sugar, glutamine, non-enzymatic browning reaction, pyrazine, pH dependency, solid-phase extraction

The volatiles generated from non-enzymatic browning of acetate in methanol at 80'C for I h (isomer rati0=2.8 : I on the sugars and amino acids (Maillard reaction) have been exten- GC analysis). The spectral data of the minor isomer was con- sively studied from the viewpoint of their chemical and sensory sistent with those of 2,5-bis-(2-furyl)pyrazine described In the properties in foods and beverages (Ledl & Schleicher, 1 990). literature (Takehara et a!., 1997); IH NMR (CDCl~) ~ 6.-58 (2H, Among them, pyrazine derivatives have received much atten- dd, ./=3.7, I .7 Hz), 7. 1 3_ (2H, dd, J=3.7, 0.6 Hz), 7.60 (2H, dd, tion because of their characteristic flavors and low odor thresh- ./= 1.7, 0.6 Hz), 8.91 (2H, s). The major isomer was tentatively olds (Wagner et a!., 1999). assigned as 2,6-bis-(2-furyl)pyrazine; IH NMR (CDC13) ~ 6.58 A Iarge number of studies have been carried out on pyrazine (2H, dd, ./=3.6, 1.7 Hz), 7.22 (2H, dd, ./=3_ .6, 0.6 Hz), 7.60 fonnation in sugar-amino acid systems mainly under roasting, (2H, dd, J= 1.7, 0.6 Hz), 8.81 (2H, s). frying and pyrolysls conditions at 120-200'C (Weenen et a/., The retention times on GCMS analysis and the fra_~:menta- 1992; Yaylayan et a/., 2000; Bemis-Yong et a/., 1993; Ames et tion pattern of these synthesized pyrazines are listed in Table I . a/., 200 1 ). However, some pyrazines were found in foods pro- Standu/r/ solutions The standard stock solutions con- cessed at below 100'C, Iike soy sauce (Yokotsuka, 1975) or the taining O. I to 2 mg/ml of pyrazine and pyrrole derivatives listed broth of cooked clams (Sekiwa et a/., 1 997). To our knowledge, in Table 2, as well as furans and reductones (Mori & Ito, there is very little literature referring to pyrazine f~ormation at 2004b), were prepared in acetonitrile. The standard stock solu- below 100'C (Shibamoto & Bernhard, 1976). tion of 2-vinylpyrazlne was prepared in dlchloromethane due to The aim of this study was to investigate the formation of its poor solubility in acetonitrile. Butyrated hydroxytoluene pyrazines at 90'C. An efficient method using solid-phase (BHT) was added to prevent oxidative degradation of the ana- extraction and GCMS was initially developed to detennine lytes. The solutions were stocked at _5'C in a refrigerator until trace levels of pyrazines, and was applied to aqueous sugar- use. The internal standards (1.S.) were 4,6-dimethylpyrimidine glutamine model systems. The effect of a sugar species (mal- (DMP) for pyrazine analysis, and p-bromophenethylalcohol tose, glucose, and fructose) and pH on the pyrazine foamation (BPA) for furan, reductone and pyrrole analysis. was also examined. Mode! reactions Equimolar amounts of D-glucose, D- fructose or D-maltose with L-glutamine were dissolved in water Materials and Methods in a glass vessel. This solution was heated at 90i2'C with stir- Chemicals Most of the chemicals were obtained com- ring in an oil bath. The initial pH was adjusted to 7 with NaO- mercially at the highest purity available (Sigma-Aldrich, TCI, Haq. In the pH-stat experiments, the pH of the solution was ini- Wako, ACROS or AVOCADO). Samples of 2-acetyl-5-meth- tially adjusted to 3_ , 4, 5, 6, 7 or 8 with HClaq or NaOHaq. Ex- ylpyrazine and 2-acetyl-6-methylpyrazine were prepared by the periments were perfumed at the constant pH by using a pH- reported method (Schwaiger et a/., 1984). stat device (Mettler-Toledo DL_55 titrator v.2.4) h~lled wlth A mixture of two isomers of bis-(2-furyl)pyrazine was syn- NaOHaq (2 or O. I mol//) and equipped with an electrode (Met- thesized by treatment of 2-hydroxyacetylfuran with ammonium tler HA405-DPA-SC-S81120) permitting measurements at above 90'C. Aliquots of the reaction mixture were taken at reg- *TO Whom correspondence should be addressed. E-mail: [email protected] ular time intervals and analyzed. 2 oo K. ITO and M. MORl

Table l. Retention times and mass spcctra of acetyl- and bis-(2-furyl)pyrazines. Compound Kl'i) MW MS, nl/z (relative intensity) 2-acetyl-6-methylpyrazine l ,694 136 136(lOO), 94(78), 93(55), 108(41 ), 66(38), 43(37), 3_ 9(26), 67(24), 53(13) 2-acetyl-_5-methylpyrazine l ,704 136 136( 100), 94(68), 93(63_ ), 43 (_38), 108(3 1 ), 67(28), 39(26), 66(23), 53(15) 2,6-bis-(2-furyl)pyrazine 2,749 212 212(lOO), 92(23_ ), 213_ (14), 63_ (13_ ), 21 l(lO), 183(lO), 64(lO), 1_5_5(8), 184(7) 2,5-bls-(2-furyl)pyrazine 2,780 212 212(lOO), 92(19), 213 (1_5), 63 (15), 183_ (14), 1_55(14), 64(9), 156(6), 106(_5) ") Kovat Index, calculated according to the retention time of n-alkanes on a HP-INNOWAX column.

Table 2. The SIM parameters and recoveries (c7c) of pyrazine and pyrrole derivatives after SPE treatments. H.O matrix!') Model matrix' ) Compound Kl") Target ion Qualifier ion R ( V.) RSD ("/.) R ('/.) RSD ("/.) Pyrroles 2-acetylpyrrole l ,986 94 09 oo 04 l 02 06 2-formylpyrrole 2,039 95 66 oo 04 99.3 06 I.S. p-bromophenethyla]cohol 2,546 l 69 171 Pyrazines pyrazine 1 222 80 53 47 O 18 40 7 24 2-methylpyrazine l 278 94 67 OO l 97 2 04 2,5-dimethylpyrazine l 3 34 108 42 02 l 98 8 02 2,6-dimethylpyrazine l 340 108 42 O1 1 98 3 06 2-ethylpyrazinc l 347 l 07 08 02 1 97 4 O 3 2,3-dimethylpyrazine l _358 l 08 67 02 l 99 1 03 2,3,_5-trimethylpyrazine l 417 42 22 02 1 99 l O1 2-ethyl-3_ -methylpyrazlne 1 417 121 22 02 l 97 8 02 2-vinylpyrazine 1 449 I 06 79 02 l Ol 2 2,_5-dimethyl-3-ethylpyrazine 1 4_56 135 36 02 97 8 03 2,6-dimethyl-3-ethylpyrazine 1 472 135 36 02 l 97 8 03 tetramethylpyrazine 1 486 l 36 ,54 Ol l 97 7 O1 2,3-diethyl-_5-methylpyrazine 1 50_5 150 35 02 I 96 ~3 03 2-acetylpyrazine 1 638 l 22 80 02 I 96 5 Ol 2-acetyl-3-methylpyrazine 1 635 1_36 94 04 99 9 lO 2-acetyl-3-ethylpyrazine 1 684 150 36 03 l 98 9 09 2-acetyl-6-methylpyrazine l 694 13_6 94 O] O 99 7 lO 2-acetyl-5-methylpyrazine l 704 136 94 O_3 Ol 1 2,6-bis-(2-furyl)pyrazine 2 749 212 92 99 4 lO 90 7 13 2,5-bis-(2-furyl)pyrazine 2 780 212 92 97 2 12 91 l lO I.S. 4,6-dimethylpyrimidine 1 ,38 1 l 08 81 "'Kovat Index, calculated according to the retention time of n-alkanes on a HP-FFAP column for pyrroles and on a HP-INNOWAX column for pyrazines. !') Standard solution (50 hLl) were spiked to 20 ml of H20. ') Standard solution (50 u1) were spiked to 20 ml of model matrix containing 2 ml of 2 M maltose-2 M glutamine reaction mixture (pH 5, 90'C for 4 h).

SPE procedure The SPE procedure described in our pre- under the same conditions. vious paper (Mori & Ito, 2004a) was used for pyrazine deriva- Pyrazines were chromatographed on an INNOWAX 30 mX tives with some modification. Two ml of the sample solution 0.25 mm i.d. (film thickness: 0.5 um, J&W Scientific). The and 50 u1 of I.S.(DMP) were placed in a 50-ml glass vial and oven temperature was programmed as follows: 40'C for 2 min, diluted to 20 ml with water . The pH of the mixture was 40-100'C at lO'C/min, 100-160'C at 3'C/min, 16C~260'C at adjusted to 9 by addition of NaOHaq. Then, the solution was 5'C/min, and then isothermally held at 260'C for 10 min. The loaded onto an SPE cartridge (OASISR HLB 0.5 g/6 cc LP, carrier gas was helium at a flow rate of 40 cm/s and the injector Waters) directly and passed through. The cartridge was washed split ratio was I : I O. The inlet and MSD interface temperature with I O ml of water and flushed with a nitrogen stream. The was 230'C and 250'C, respectively. Identification of pyrazines nitrogen flush was continued until no water was purged. After a was achieved by comparing their mass spectra and retention SepPak DryR (Waters) was attached to the tip of the cartridge, time with those of authentic specimens. this was eluted with 10 ml of diethyl ether. The quantitation was performed in selected ion mode (SIM) The same SPE procedure except for the pH adjustment of by comparing the peak area ratio of the target ions of analytes the sample solution (pH 4.5) and I.S. (BPA) was employed for wlth that of the internal standard using a linear standard curve. furans, reductones and pyrroles. GCMS analysis The qualitative and quantitative analysis Results and Discussion was accomplished by an Agilent 6890 gas chromatograph Pyrazines are usually present in very low concentration in equipped with a split/splitless injector and a 5973 mass selec- foods and beverages, particularly those processed under low tive spectrometer. temperature conditions. In order to determine amounts of these GC condition for furans and reductones was described in our compounds, a highly efficient analytical method is required. previous paper (Mori & Ito, 2004b). Pyrroles were analyzed Whereas application of an internal standard (Jusino et a/., Formation of Pyrazlnes 20 1

Table 3. The volatiles identified in the reaction of maltose, glucose and fructose with glutamine at inltial pH 7, 90'C for 2, 8 and 24 h. Mal-Gln") (umol/mol-Sugar) Glc-Glnh) (umol/mol-Sugar) Fru-Gln' ) (hLmollmol Sugar) Compound 2h 8h 24 h 2h 8h 24 h 2h 8h 24 h Pyrazincs 2-methylpyrazine l .O l 5.~59 -5,96 9._3 l 20,3 24.2 lOl 153 153 2,5-dimethylpyrazine '!) 0.23 0.93 l .8_5 l_3.8 15.5 l_5._5 2,6-dimethylpyrazine O. 1 5 O._37 0,83 O.9_3 5,19 9.3 1 l 90 2 24 220 2-ethylpyrazine O.32 0.37 0,2_3 0.6_5 0,93 0.32 l.16 l .90 2,3-dimethylpyrazine O. 1 2 l .62 1 .85 0.28 O.56 0.83 l .02 l .48 l .76 2,3,_5-trimcthylpyrazine 0.20 O. 1 6 0.3_ 7 0.4_5 O.70 0.94 2-ethyl-3-methylpyrazine O. 16 2-vinylpyrazine O, 1 2 0.19 O, 14 2.88 2.45 0.85 0.94 3.73 2.83 2,5-dimethyl-3-ethylpyrazine 2,6-dimethyl-3_ -ethylpyrazine tetramethylpyrazine 2,3-diethyl-5-methylpyrazine Totals (alkylpyrazines) 1 40 8.09 9.3-5 13.9 30.2 3_ S.3 3 08 400 396 2-acetylpyrazine O 16 0.78 0.86 l j6 2..38 2.54 0.25 I .02 1 .80 2-acetyl-6-methylpyrazine O. 1 1 O.26 0.29 0.70 l .84 2.46 2-acetyl-_5-methylpyrazine O. 1 3 0.23 0.40 1 .58 l .9 l O.8-5 2.3 9 3.3-5 Tota]s (acetylpyrazines) O. 16 0.9 l 1 .09 2.07 4.22 4.74 I .80 5,25 7.61 2,6-bis-(2-furyl)pyrazine O.28 5.45 O, 14 2.lO 0.02 O.2 l 2,5-bis-(2-furyl)pyrazine l.18 O, 1 2 0.83 O, 17 l .30 Totals (furylpyrazines) o 0.28 6.6~3 o O.26 2.93 o O. 1 9 1 ._5 1 Pyrroles 2-acetylpyrrole 32 2 8C 1 88 7 47 26 8 o 69 2.66 2-formylpyrrole 0.47 63 7 52 O 84 4 84 28 9 o.58 4 o_5 22.2 Furans 2-furfural 0.36 l .67 4 1 ._3 0.52 2 1 .6 346 0.57 25.9 311 2-acetylfuran 0.50 9.9 1 l .OO l 3 .O 2.9 1 24.2 5-methylfurfural 0.05 0.9_5 1 .0_5 19.0 l .05 1 1.0 2-f uranmethanol 8,27 98.4 69.5 2,40 32.2 198 2.50 49,4 24 I 2-hydroxyacetylfuran O._36 2. 1 8 21 ,2 O. 1 6 l ._5 l 7.34 0.99 72.2 268 _5-hydroxymethyl-2-furfural 4,76 24.0 l ,570 23. 1 1 , I OO 1 6, I OO 14.7 9~35 l 0,400 reductones cyclotene O. 1 8 O.58 0.09 0.49 2.86 0.45 2.54 maltol l.3 9 90.2 1,170 3 ,lO 3.25 4.92 2.06 2.94 2.46 f uraneol O._59 _5.90 5,82 l 8.3 16.7 _5.16 34.7 33_.l Totals (furans and reductones) l_5,l 218 _3,5 1 O 3~ 5.2 I , 1 80 l 6,700 26.0 1 , 1 20 l I ,300 pH of rpaction mi~ture 6,6 5.3 4,4 6,2 4.6 4.0 -5.7 4.~5 3*9 ") I M maltosc-1 M glutamine; !" I M glucose-1 M glutamine; ') I M fructose-1 M glutamine; heatcd at 90'C. 'h Not detected.

l 997) or a stable isotope dilution technique (Schieberle, 1995) unsubstituted pyrazine during the procedure may be due to its is effective for the quantitation of the selected pyrazines, the lit- weak affinity toward the SPE adsorbent. The recoveries of two erature is limited in this field. Recently, Herent & Collin ( 1998) isomers of bis-(2-furyl)pyrazine in the model matrix were optimized a method using vacuum distillation followed by suc- slightly decreased (9 1 %), while most analytes were quantita- cessive liquid~iquid extraction and gas chromatography. Even tively recovered (96-l02%). These results indicated that the though this technique is highly efficient, there still remain some effects of sugar, glutamine and other co-existing compounds on problems. The procedures are time-consuming, and the recov- the recovery and reproducibility were subtle in these model ery of each pyrazine, which has different polarity and volatility, experiments . varies from 40% to 90%. The qff;ects of suga/' species on py,'azine fol'mation To Quantitative analysis oj'pyr'azine and pyrrole de/~ivatives investigate the effects of a sugar species on the reaction prod- by SPE-GCMS In our preceding paper, we demonstrated the ucts in aqueous sugar-glutamine model systems, each I M conc. efficiency of SPE-GCMS for the quantitation of trace levels of of maltose, glucose and fructose with equimolar glutamine, in furan, reductone and organic acids in aqueous solution (Mori & which the initial pH was adjusted to 7, was heated at 90'C for Ito, 2004a). This technique was modified for pyrazines. On the 24 h. These sugars have widely been used as sweeteners in basis of their basic character, pH 9 was selected for the SPE, food and beverage industries, and we focused on the difference and DMP was applied as I.S. for the GCMS analysis. The SIM of pyrazine formation between monosaccharide and disaccha- parameters and the recoveries with relative standard deviation ride. Glutamine has up to now been employed as the nitrogen (RSD) are summarized in Table 2. Analyses were perforrned in source for several model studies on pyrazine formations triplicate. The recovery study was carried out in both water and (Hwang et a/., 1995; Chen & Ho, 1999), and has potential to aqueous solution (20 ml) containing 2 ml of the model matrix release ammonia under relatively low temperature ( 1 10'C) con- (2 M maltose-glutamine reaction product, pH 5-stat at 90'C for dition (Sohn & Ho, 1995). Samples were collected after 2, 8 4 h). The standard solution ( 10 u1) was spiked and treated by and 24 h heating, and the pH and amounts of volatiles were the usual SPE procedure. All analytes in the water matrix measured (Table 3). showed good recovery (97-l04%) and reproducibility Although the total yield of pyrazines was less than that of (RSD<2%) except for unsubstituted pyrazine. The loss of furans and reductones, a series of pyrazines were detected from 2O2 K. ITO and M. MORI these model systems. Whereas high temperature is advanta- Table 4. The volatiles identified in the reaction of maltose, glucose and fructose with glutamine at constant pH 8. _~:eous t~or pyrazine formation (Koehler & Odell, 1970; Shibam- hLmol/mol-Sugar oto & Bemhard, 1976), our results indicated that pyrazines Compound could be formed at below 100'C. Mal-Gln") Glc-Gln!') Fru-Gln' ) Marked din:'erences were observed in the yields of alkylpyra- Pyruzines 2-methylpyrazlnc l_51 l .490 4,000 zines. The order of the magnitude of their formation was fruc- 2,5-dimethylpyrazinc 3.43 48.8 95.5 tose>glucose>maltose. The total yield of alkylpyrazines 2,6-dimethylpyrazine 23.7 320 882 formed after 24 h from fructose was 40 times larger than that 2-ethylpyra7.inc 2.X l 7.3~5 6.80 2.3_ -dimcthylpyrazine 36.-5 4O.O 79.0 from maltose. Major products from fructose and glucose were 2,3_ ,5-trimcthylpyrazine 4.8 l 14.6 22.6 2-methylpyrazine and 2,6-dimethylpyrazine, and those from 2-ethyl-3-methylpyrazlne O.3 3 l.18 l .63 4.67 6.02 5.58 maltose were 2-methylpyrazine and 2,_3-dimethylpyrazine. 2-vinylpyrazlne 2.5-dimethyl-3-ethylpyrazine

Fig. l. The pH dependency of vola- a H- 2-methylpyrazine 600 b -X- 2-furanmethanol tile formation, a: alkyl- and acetylpyrazines, b: maltol and 2-furanmethanol, c: -~T 2,3-dimethylpyrazine HF maltol bis-(2-furyl)pyrazines, d: 2-hydroxy- 75 ~- 2-acetylpyrazine acetylfuran. The reactions were carried *':l out at 90'C for 4 h on 2 M maltose with o, -e- 2-acetyl-5-methylpyrazine 2 M glutamine under constant pH condi- uP= 400 tions. :ol: "O

o E ::t 200 25

O o

I~ 2,6-bis-(2-furyl)pyrazine 20 1 .O c d H:H 2-hydroxyacetylfuran -~- 2,5-bis-(2-furyl)pyrazine

*c9 o, :, uP 10 505E ~5 E ::~

0.0 o 3 4 5 6 7 3 4 5 6 7 pH pH

Table 5. Bis-(2-furyl)pyrazine formation from glucosamine, 2,5-fructosazine temperature on the formation of volatile compounds in cysteine/reduc- and 2-hydroxyacetylfuran with glutamine. ing sugar/starch mixtures during extrusion cooking. ./. A,~"'ic. Food umol/mol-Substrate Chen7., 49, 188_5-1894. Compound Bemis-Young, G.L.. Huang, J. and Bemhard, R.A. (1993_ ). Et~fect of pH GAu) 2,5 -FS!') 2_ HAF + Gln' ) on pyrazine formation in glucose-glycine model systems. Food Chem., Pyrazine 178 46, ~383-3~ 87. 2-methylpyrazine 58.5 Chen, J. and Ho, C.-T. ( 1998). Volatile compounds formed from thermal 2.6-dimethylpyrazine 32.4 degradation of glucosamine in a dry system. ./. Agj'!c. Food Chen7., 46, 2,6-bis-(2-furyl)pyrazine 83 1 l 97 1-1 974. 2,_5-bls-(2-furyl)pyrazine 818 Chen, J. and Ho. C.-T. (1999). Comparison of volatile generation in The reactions were carried out under constant pH 5 condition at 90'C for 4 h on serine/threonine/glutamlne-ribose/glucose/fructose model systems. ./. ") 20 mM glucosamine; !'} 20 mM 2,_5-fructosazine; ' ) 20 mM 2-hydroxyacetylfuran with 2 M _~lutamine. '!) Not detected. Ag,'ic. Food Chem., 47, 643-647. Herent, M-F. and Collin. S. ( 1998). Pyrazine and thiazole structural properties and their influence on the recovery of such derivatives in aroma extraction procedures. ./. Ag,'ic. Food Chem., 46, 1 975-1980. Hwang, H.-1., Hartman, T.G. and Ho, C.-T. (1995). Relative reactivities of amino acids in pyrazine formation. ./. Ag,~ic. Food Cl7enl., 43, 1 79- condition and generated no volatiles. On the basis of these l 84. results, it is indicated that the two isomers of bis-(2-furyl)pyra- Jusino, M.G., Ho, C.-T. and Tong, C.H. (1997). Quantitative analysis of zine could be foamed from the reaction of 2-HAF with pyrazines in a hydrophilic solid model system. ./. Food P/r)cess. P,'e- glutamine at below 100'C. sen'., 21, 409~24. The present study demonstrated that pyrazine derivatives Koehler, P.E. and Odell. V.O. ( 1970). Factors aff'ecting the formation of pyrazine compounds in sugar-amine reactions. J. Ag,'ic. Food Cl7enl., could be generated from aqueous sugar-glutamine model sys- 18, 89_5-898. tems even at below 100'C along with other volatiles formed by Ledl, F. and Schleicher, E. ( 1 99O). New aspects of the Maillard reaction the Maillard-type reaction. Since disaccharide like maltose has in foods and in the human body. Angcvt'. Chem, lnt. Ed. Eng., 29, -56-5- potential to generate flavor compounds with dift'erent aromatic ~594. characters from that of monosaccharides, it must contribute to Mori, M. and Ito, K. (2004a). Determinatlon of reductones, t~urans and organic acids in aqueous model systems using solid-phase extraction sensory attributes in actual food systems. and gas chromatography/mass spectrometry. Food Sci. Tecl7no/. Res., lO, 5 1-55. Acknov,'ledgment We thank Ms. Emiko Yasuda of JT Creative Service Mori, M. and Ito, K. (2O04b). Effect of pH on the fomaation of volatile for her technical assistance. products in non-enzymatic brownlng of maltose. Food Sci. Tecl7nol. Res., lO, 60~4. References Pittet. A.O. and Hruza, D.E. ( 1 974). Comparative study of flavor proper- Ames, J.M., Guy, R.C.E. and Kipping, G.J. (2001). Effect of pH and ties of thiazole derivatives. ./. Agric. Food Chen7., 22, 261~269. 2 04 K. ITO and M. MORI

Reese, G. and Baltes, R. ( 1992). Model reactions on roast aroma forma- Takehara, K., Isomura, K., Yamada. K., Ide, S. and Haraguchi, T. (1997). tion. Z. Lebensm. Unte/~s. Forsch., 194, 417~2 1 . Syntheses of 2,5-diarylpyrazine compounds and their fluorescence Schieberle, P. ( 1 99_5). Quantitation of important roast-smelling odorants properties (in Japanese). Kitakyushu Ko,~'yo Koto Senmon Gakko Ken- In popcom by stable Isotope dilution assays and model studies on fla- kyu Hokoku, 30, 107-1 12. vor formation during popping. ./. A,~""ic. Food C/7em., 43, 2442-2448. Wagner, R., Czemy, M., Bielohradsky, J. and Grosch, W. (1999). Struc- Schwaiger, W., Cornelissen, J.M. and Ward, J.P. (1984). A convenient ture-odor activity relationships of alkylpyrazines. Z. Lebensm. Unters. synthesis of alkyl- and arylpyrazinyl ketones. Food Chen7., 13, 225- Forsh. A: Food Res. Technol., 208, 3 08-_~ 1 6. 234 Weenen, H., Tjan, S.B., Valois, P.J., Bouter, N., Pos, A. and Vonk, H. Sekiwa, Y., Kubota, K. and Kobayashi, A. (1997). Characteristic flavor ( 1 992). Mechanism of pyrazine formation. In "ACS Symposium Series components in the brew of cooked clam (Me,'et,'ix !uso,'ia) and the 543 Thermally Generated Flavors," ed. by T.H. Parliament, M.J. Mor- efl~ect of storage on flavor formation. ./. Ag,'ic'. Food Chem., 45, 826- rello, and R.J. McGorrin. Am. Chem. Soc., Washington. D.C., pp. 830. l 42- I _5 7 . Shibamoto, T. and Bemhard, R.A. ( 1 976). Effect of time, temperature, Yaylayan. V.A., Keyhani, A, and Wnorowski, A. (2000). Formation of and reactant ratio on pyrazine formation in model systems. J. Agl'ic. sugar-specific reactive intennediates from 13C-labeled L-serines. ./. Food C/7em., 24, 847-852. Agric. Food C/7em., 48, 636-641 . Sohn, M. and Ho, C-T. ( 1 99_5). Ammonia generation during thennal deg- Yokotsuka, T. (1975). The flavor of shoyu (in Japanese). Ko,~yo, I12, 57- radation of amino acids. ./. A,~'/'i('. Food Chem., 43, 3_ OO1-3_ 003. 71.