Agric. Biol. Chem., 54 (7), 1631-1638, 1990 1631 Volatile Products Formed from L-Cysteine and Dihydroxy- acetone Thermally Treated in Different Solvents Joji Okumura, Tetsuya Yanai, Izumi Yajima and Kazuo Hayashi Kawasaki Research Center, T. Hasegawa Co., Ltd., 335 Kariyado, Nakahara-ku, Kawasaki 211, Japan Received November 21, 1989 Equimolecular amounts of L-cysteine and dihydroxyacetone were heated at 110°C for 3hr in different solvent systems such as deionized water, glycerine, or triglyceride. The resulting mixtures were vacuum steam-distilled and each distillate was extracted with ethyl ether. The volatiles in the ether extracts were analyzed by gas chromatographyand gas chromatograph\-massspectrometry. Differences in the quality and quantity of volatiles formed in the systems were observed. Pyrazines, thiazoles, thiophenes, and someother sulfur-containing compounds wereidentified in the volatiles. Dimethylpyrazines were formed as major volatiles in the glycerine and triglyceride systems but were minor in the water system. 2-Acetylthiazole in the triglyceride system and 2-acetylthiophene in the glycerine system were secondary abundant products. In the water system, l-mercapto-2-propanone was found as a major volatile compound, and thiophenes as the next dominants. The Maillard reaction is significant in the sugar alcohols, oils and fats, or their mixtures formation of flavors from various heated were used for various applications. It has been foods. In the flavor industry, the Maillard observed in some cases that the quality of reaction is used to produce simulated flavors flavor formed has been changed by the differ- of thermally treated foods, or 'processed fla- ence of solvent system. The water content of vors.' Numerousstudies have been attempted heating systems effects the Maillard reac- to identify the precursors of flavors produced tion.1^ Hartman et ai14) investigated the ef- by thermal treatment offoods such as meat,1'2* fects of water activity on the major volatiles peanuts,3'4* and cacao5); the flavor precursors produced from a meat flavor model system were basically composedof aminoacids and containing monosodium L-glutamate, l- sugars. To form cooked meat flavors, it has ascorbic acid, thiamine hydrochloride, and been shown that sulfur-containing amino acids cystine, and reported qualitative and quanti- are important precursors.6'7* Studies on the tative differences in volatiles produced. model reaction systems between sulfur- Arnoldi et al.15) reported that the rate of the containing amino acids and sugar have in- Strecker reaction of leucine and valine with dicated that manyinteresting compounds, es- fructose was higher in cocoa butter-water than pecially sulfur- and nitrogen-containing het- in water. Manypatents concerning the pro- erocyclics, found in the volatiles of heat- duction of synthetic meat flavor claim cysteine processed food, can be identified as the re- as one of the most important ingredients.2) action products in these systems.8~12) How- Dihydroxyacetone is a fission product of sugar ever, these model experiments have been and has high reactivity in the browning re- done in a single solvent such as an aqueous action.16) Dihydroxyacetone has appeared in a solution, rather low boiling organic solvent, patent to produce roasted meat flavor.17) Thus, triglyceride, or in the dry state, under various it is the purpose of this study to compare the heating conditions. In making 'processed fla- volatile compoundsproduced by the reaction vors,' many kinds of solvent such as water, of L-cysteine with dihydroxyacetone in dif- 1632 J. Okumura et al. ferent solvent systems; water, glycerine, or Table I. Flavor Qualities of Reaction Mixtures triglyceride. Glycerine was selected for its ther- from l-Cysteine-Dihydroxyacetone Systems mostability and the negligible reactivity of System Flavor description sugar alcohols in the Maillard reaction.18'19) As triglyceride, a saturated mediumchain tri- Roasted rice, sulfury glyceride was used for its high stability to heat Roasted, burnt rice, somewhat roasted meat and oxidation. Roasted rice (weak), sweet sulfury roasted, somewhatroasted chicken Materials and Methods Preparation of reaction mixtures. One-tenth mol of L- is presented in Table I. The characters of cysteine and 0.1 mol of dihydroxyacetone were dispersed flavors of the three reaction mixtures some- in 500g of deionized water, glycerine, or triglyceride. As what differed. The sensory results indicated triglyceride, O.D.O. was used. O.D.O. is a medium chain the triglyceride and glycerine systems gave triglyceride (fatty acid composition : octanoic acid (75%) and decanoic acid (25%)) manufactured by Nisshin Oil moreroasted notes and the water systemgave Mills Ltd., Tokyo, Japan. These model systems were a more sulfury note. Therefore, it would be heated at 1 10°C for 3 hr in a glass autoclave with stirring. expected that there would be somedifferences Isolation ofvolatiles. Five hundred grams of the reaction in reaction products. The GCprofiles of the product was mixed with 2000g of deionized water and volatiles in the reaction mixtures are shownin then steam-distilled under reduced pressure (30 mmHg/ Fig. 1. The peaks identified are numbered and 40°C). The distillate was condensed with a cold water the peaks not identified are no numbered. (3°C) and two cold traps (dry ice-acetone and liquid Table II lists the volatile components identified nitrogen). The distillate (about 1000ml) from each re- along with the peak identification numbers action product was extracted three times with 100ml of ethyl ether after saturation with sodium chloride. The and quantitative data. The volatile com- ether extracts were dried over anhydrous sodium sulfate ponents were identified by the comparison of and concentrated to about 0.1 g under atmospheric pres- GC retention indices and mass spectra of sure. Theconcentrate was analyzed and measuredby gas unknowns with those of authentic samples. chromatography (GC) and gas chromatography-mass The components corresponding to peaks 56 spectrometry (GC-MS). and 64 identified in the triglyceride system GCand GC-MS. Gas chromatographic analyses were were octanoic and decanoic acids, respectively. done on a Shimadzu GC-9A with a flame ionization These two components which could be con- detector (FID), a flame thermionic detector (FTD), sidered as the.degradation products of O.D.O. and a flame photometric detector (FPD), fitted with a 0.25mm i.d x 60m fused silica capillary column coated were excluded from calculating the peak area with PEG 20M. The column oven temperature was pro- ratio. Upon examination of Table II, quali- grammed from 70 to 220°C at 3°C/min. The nitrogen tative and quantitative differences in volatile carrier gas flow was 1 ml/min. Peak areas were integrated products identified existed amongthe reaction using a Shimadzu Model Chromatopac 1AXintegrator systems. connected to the gas chromatograph. A Hitachi Model M- Ten pyrazines were detected in the volatiles 80Bmass spectrometer wasused for mass spectral identifi- cation of the gas chromatographic componentsunder the from the reaction mixtures. These pyrazines following conditions: column, fused silica capillary col- have been identified in many model systems of sugar and amino-compounds.20) Pyrazines umn (0.25mm x 60m) coated with PEG 20M; oven tem- perature, programmed from 60 to 210°C at 2°C/min; were detected as major volatile products in the carrier gas, helium; ionizing voltage, 70eV; accelerating triglyceride and glycerine systems (total area: voltage, 3000 V; ion source temperature, 190°C. 60% in the triglyceride system; 70% in the glycerine system). 2,6-Dimethylpyrazine was the largest constituent of the total pyrazines, Results and Discussion and the next largest one was 2,5-dimethyl- Sensory evaluation of the reaction mixtures pyrazine. The distributions of pyrazines iden- Volatiles from L-Cysteine and Dihydroxyacetone 1633 Fig. 1. Gas Chromatograms of Volatiles Formed in L-Cysteine-Dihydroxyacetone Model Systems. tified in the triglyceride and glycerine systems der. However, this might be due to differences were very close to each other. A marked of nitrogen sources and reaction conditions. difference was observed in the total percent- The presence of larger alkyl- and cyclopenta- ages of pyrazines formed in the water system, pyrazines and 2-methylquinoxaline in the tri- where only 2,5- and 2,6-dimethylpyrazines glyceride and glycerine systems suggests that were detected as minor components (total the aldol condensation between dihydroxy- area: 5%). Shibamoto and Bernhard19) re- acetone and its dehydration products and ported that the largest amount of pyrazine further addition of alkyl group to pyrazine formed in dihydroxyacetone-ammonia system skeleton intermediates occur. was 2-methylpyrazine and 2,6-dimethylpyr- Thiazole compounds were abundant prod- azine was the next largest, and 2,5-dimethyl-, ucts in the triglyceride and glycerine systems, 2,3,5-trimethyl-, unsubstituted-, and 2,3- but were more varied in the glycerine system. dimethyl-pyrazine followed in decreasing or- In the water system, those were not abundant 1634 J. Okumura et al. Table II. Volatile Components Identified from l-Cysteine-Dihydroxyacetone Model Systems Triglyceride system Glycerine system Water system Compound PeakNo. Area % PeakNo. Area % PeakNo. Area % Pyrazines 6 6.9 ll 10.4 2,5-Dimethylpyrazine 7 2,6-Dimethylpyrazine 48.5 12 54.8 2,3, 5-Trimethylpyrazine 1.3 15 2.1 2-Ethyl-3,5-dimethylpyrazine 0.7 17 0.7 2-Ethyl-3,6-dimethylpyrazine