Heteroatomic Aroma Compounds - American Chemical Society

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Chapter 18 Volatile Compounds Formed in a Glucose-Selenomethionine Model System Guor-Jien Wei and Chi-Tang Ho Department of Food Science, Rutgers University, 65 Dudley Road, New Brunswick, NJ 08901-8520

Selenium (Se) is an essential component of enzyme glutathione peroxidase, which is important in the protection of red blood cell membranes and other tissues from damage by peroxides. Wheat and meat are the main source of Se in the diet, and selenomethionine is the primary form found in these foods. The Maillard reaction is a complex degradative reaction and occurrs extensively in processed foods as well as in vivo, where proteins, amino acids, nucleic acids, and amino phospholipids react nonenzymatically with reducing sugars. This study investigates the Maillard reaction of selenomethionine and glucose. The effects of pH, time, and added diallyl disulfide on the generation of volatile compounds were studied using a model system. The organoselenium and mixed organoselenium-sulfur compounds generated were identified by ΕΙ/MS and NH -CI/MS. 3

© 2002 American Chemical Society

Reineccius and Reineccius; Heteroatomic Aroma Compounds ACS Symposium Series; American Chemical Society: Washington, DC, 2002.

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Intoduction

Chemical Properties of Selenium


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Selenium, the 34 element on the periodic table, was first discovered by Berzelius in 1817. The name selenium is derived from the Greek word meaning "the moon". It be longs to Group V I A (oxygen, sulfur, selenium, tellurium, polonium) of the periodic system and possesses similar chemical properties with sulfur. Selenium is a semi-metal. Essentially, it is a nonmetal from a chemical viewpoint; however, it possesses some metallic characteristics. Selenium has six stable isotopes: 74 (0.87%); 76 (9.02%); 77 (7.58%); 78 (23.52%); 80 (49.82%); 82 (9.19%), and is one of the few nonmetal elements which has variable valence within the redox range of biological systems. The four natural oxidation states are as follows: (0), elemental selenium, selenodiglutathione (dipeptide); (-2), sodium selenide (Na Se), hydrogen selenide (H Se); (+4), sodium selenite (Na Se0 ), selenium dioxide (Se0 ), selenious acid (H Se0 ); and (+6), sodium selenate (Na^eC^), selenic acid (H Se0 ). Selenium (0) can be reduced to hydrogen selenide, oxidized to selenious acid or selenites, and further oxidized to selenic acid or selenates (/). Selenium can easily replace sulfur to form a large number of organic selenium compounds (dimethyl selenide, trimethylselenium). Selenium occurs primarily as selenides on the side chains of selenocysteine at physiological p H values. Selenomethionine in nature occurs almost exclusively as the /enantiomer. Inorganic selenate and selenite predominates in water. Organic selenium compounds (primarily selenomethionine and selenocysteine), which are incorporated into plant proteins, account for the major portion of selenium in vegetables and in cereals (2). Selenocystine is an oxidation product of selenocysteine, in which two - S e H groups become one Se-Se group. In the body, selenocystine is metabolized to selenocysteine. Selenocysteine is the form of selenium is present in all currently known enzymes (e.g., selenoprotein P, glutathione peroxidase), which contain selenium. 2

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Biological Functions of Selenium Selenium is an essential nutrient with a recommended dose of 50-200 μg/day considered being adequate and safe for adults (5). Observations in Japanese fishermen suggest that a selenium intake of 10 to 200 times above normal does not produce toxic effects. However, ingestion of 31 mg/day for 11 days produced toxicity, while individuals who ingested 312-617 mg/day


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chronically reported toxicity. More selenium is required i f diets are also deficient in vitamin E. Specifically, selenium is an essential component of glutathione peroxidase, which destroys hydrogen peroxide and hydroperoxides; protecting cell membranes from oxidative damage. Vitamin Ε is also implicated in this system in which the role is to prevent the formation of lipid hydroperoxides (4).

Bioavailability The bioavailability as well as the toxic potential for selenium and selenium compounds is related to chemical form, and most important, to solubility. Selenates are relatively soluble compounds, similar to sulfates, but selenites and elemental selenium are virtually insoluble. Elemental selenium is probably not absorbed from the gastrointestinal track. Absorption of selenite is from the duodenum. The bioavailability of selenium, including selenomethionine and selenocysteine, in plant-derived foods is high while the bioavailability of selenium from animal-derived food is low to moderate. Animal studies suggest that vitamins A and C promote absorption of selenite, although vitamin C could also be expected to reduce selenite to elemental Se, which is not absorbed (5).

Organoselenium Compounds in Garlic Genus Allium plants, especially garlic (Allium sativum), contain more selenium than other vegetables. Garlic is claimed to contain selenoproteins, and ingestion of garlic is well known to cause bad breath. Now it is clear that garlic breath odor is from the lungs but not from particles of garlic retained in the mouth (5). Several compounds have been identified as responsible for the garlic flavor; they are allyl methyl sulfide, allyl methyl disulfide, diallyl sulfide, diallyl disulfide, hydrogen sulfide, 2-propenethiol, and (+)-limonene (7). Compared to organosulfur compounds, selenium is present in a very low amount in garlic. The ratio of S:Se in fresh garlic is 1.2 χ 10 :1. During analysis, selenium compounds usually coelute with other compounds. Because of the low concentrations present in foods, selenium analysis had not drawn much attention from flavor chemists before the 1990's. In 1995 Cai et al. (8) reported the organoselenium compounds generated in human breath after ingestion of garlic. Several organoselenium and mixed organoselenium-sulfur compounds were identified. The major seleniumcontaining compounds found in garlic breath were dimethyl selenide (Me Se), allyl methyl selenide (MeSeAll), methanesulfenoselenoic acid methyl ester 4

2


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(MeSeSMe), 2-propenesulfenoselenoie dimethyl diselenide (MeSeSeMe).

acid methyl ester (MeSeSAll), and


Maillard Reaction The Maillard reaction is one of the most important reactions on flavor and color development in foods, especially during thermal processing of foods. It may create desirable aroma components like in baking and roasting processes. In some cases, Maillard reaction is undesirable such as in the formation of offflavors or dark colors during storage or in dehydrated foods. The Maillard reaction is a complex series of chemical reactions initiated by the interaction of a free amino group and a carbonyl group. The reaction is a degradative reaction and responsible for the generation of many volatiles. In 1998, Tsai et al. (Ρ) studied the selenomethionine-glucose model system, and five organoselenium compounds were identified. The main objectives of our study were to gain a better understanding of the formation of organoselenium in food systems. Our study investigated the effects of time of heating and pH on the volatile compounds formed in a selenomethionine-glucose model system. In addition, interactions between organoselenium and sulfur compounds formed by the Maillard reaction were monitored.

Experimental

Thermal Reaction and Isolation of Volatiles Selenomethionine (0.15 g) and glucose (0.2 g) were dissolved in either 25 mL of water (unbuffered solution) or 25 m L 0.05 M sodium phosphate buffer solution, and the p H of the solutions were adjusted to 3.0, 5.0, 7.0 and 9.0 using 85% phosphoric acid or 1 M N a O H solution. The solution was sealed in a 100 m L glass bottle. The solutions were heated at 160 °C for 40, 60 and 80 minutes. After the reaction, the solution was adjusted to p H 7, and extracted with 50 mL of CH C1 . The organic phase was concentrated to 2 m L by a KudernaDanish concentrator, then to 1 m L under N . 2

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Interactions between Organoselenium and Sulfur Compounds Selenomethionine (0.15 g), glucose (0.2 g) and diallyl disulfide (10 μ ι ) were dissolved in 25 mL 0.05 M sodium phosphate buffer solution, and the p H of the solutions were adjusted to 7.0. The solution was sealed in a 100 m L glass bottle. The reaction time was 60 minutes and the reaction temperature is 160°C. After reaction, the solution was adjusted to p H 7, and extracted with 50 m L of CH C1 . The organic phase was concentrated to 2 mL by a Kuderna-Danish concentrator, then to 1 m L under N .


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Gas Chromatographic Analysis The volatile compounds isolated from the thermal reaction systems were analyzed by a Siemens SiChromat 2-8 Gas Chromatograph with a AS-20 autoinjection system. The G C was equipped with a HP-5 fused silica capillary column (25 m χ 0.32 mm i.d.; 0.52 μπι film thickness) and a flame ionization detector. For each sample, 1 μΐ^ was injected with a split ratio of 15:1. The G C was run with injector and detector temperatures of 275°C. The column temperature was programmed from 40°C to 285°C at a rate of 5°C/min.

GC/Mass Spectrometry Analysis G C / M S analysis was performed by an HP 5989A coupled with an H P 5890 II G C . Mass spectra were obtained by EI at 70 eV or N H - C I and a mass scan from 40-550 amu. 3

Results and Discussion

Volatile Compounds from Selenomethionine-Glucose Model System The G C / M S profile and compounds identified are shown in Figure 1. The most abundant peak in this chromatogram is dimethyl diselenide, which possess strong garlic and somewhat metallic flavors. Selenium-containing compounds can easily be identified by ΕΙ/MS or N H - C I / M S because of the isotopic distribution. The ΕΙ/MS spectrum of dimethyl diselenide, with the monoisotopic molecular ion at m/z 190, is shown in Figure 2. 3


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The heated model systems, the solvent extracts and the concentrates all possessed an aroma which can be described as roasted, nutty and garlic-like. It is believed that pyrazines are responsible for the roasted aroma and dimethyl diselenide is responsible for the garlic flavor. Due to the flavor properties of the extracts, this study focuses on the nitrogen-containing compounds, ethyl selenoacetate and dimethyl diselenide, although several selenium-containing and sugar degradation products were also identified.

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Figurel. GC/MS profile of volatile aroma compounds formedfrom glucoseselenomethionine model system: 1= hydroxyacetone, 2 = 3-hydroxy-2-butanone, 3 = pyrazine, 4 = 1-hydroxy-2-butanone, 5 = methylpyrazine, 6 = 2furanmethanol, 7 = dimethyl diselenide, 8 = 2,6-dimethylpyrazine, 9 = ethylpyrazine, 10 = ethyl selenoacetate, 11 = trimethylpyrazine, 12 - 4hydroxy-5-methyl-3(2H)-furanone, 13 = 2-hydroxy-3-methyl-2-cyclopenten-lone, 14 = 2,5-dimethyl-4-hydroxy-3(2H)-furanone, 15 = 3-ethyl-5-methyl2 (5H) -furanone, 16= 5-methyl-lH-pyrrole-2-carboxaldehyde, 17 = selenotrithiolane, 18 = internal standard.

Effects of Reaction Time and p H The p H is known to play an important role in the Maillard reaction. The formation of colored compounds in the Maillard reaction increases with increasing pH, especially at pH values above 7. Color formation is due to the formation of melanoidin pigments from reaction of amino groups with Maillard intermediates, which are inhibited at a lower p H as a result of the protonation of amino group. Higher pH conditions tend to favor the formation of nitrogen-


287 containing volatiles, such as pyrazines, but some volatiles are favored under acid conditions (10), such as furanthiols and disulfides, which are believed to be important in meat aroma and favored by lower pHs (77).


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175 171 160

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Figure 2. ΕΙ/MS spectrum of dimethyldiselenide. In our model systems, after reaction all buffered solutions with pH3 were colorlessand those with pH7 and pH9 were brown. In terms of the effect of heating time, the longer the heating times the darker the resulting solutions. The yields of pyrazines, ethyl selenoacetate and dimethyldiselenide at different times and p H values are listed in Tables I-III. Unlike pyrazines and dimethyldiselenide, the formation of ethyl selenoacetate is favored in low pH, which has the maximum yield at pH3 and 80 min (Figure 3), and the yield decreases with increasing pH.

Table I. Yields of Major Volatile Compounds (mg/g of glucose) Generated in the Model System Heated for 40 Minutes (buffered solutions) Compounds Pyranize Methylpyrazine Dimethyl diselenide 2,6-Dimethylpyrazine Ethylpyrazine Ethyl selenoacetate Trimethylpyrazine

pH3 ND ND 0.061 ND ND 0.027 ND


pH7 0.021 0.022 0.743 0.039 0.019 0.022 0.006

ND: not identified


pH9 0.041 0.014 0.938 0.014 0.005 0.009 0.003

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Table II. Yields of Major Volatile Compounds (mg/g of glucose) Generated in the Model System Heated for 60 Minutes (buffered solutions) Compounds Pyranize Methylpyrazine Dimethyl diselenide 2,6-Dimethylpyrazine Ethylpyrazine Ethyl selenoacetate Trimethylpyrazine


pH5 ND ND 0.041 ND 0.002 0.044 ND

pH7 0.029 0.033 0.545 0.067 0.008 0.016 0.011

pH9 0.024 0.164 1.162 0.144 0.027 0.010 0.043

ND: not identified

Table III. Yields of Major Volatile Compounds (mg/g of glucose) Generated in Model System Heated for 80 Minutes (buffered solutions) Compounds Pyranize Methylpyrazine Dimethyl diselenide 2,6-Dimethylpyrazine Ethylpyrazine Ethyl selenoacetate Trimethylpyrazine


pH5 0.014 0.009 0.134 0.018 0.008 0.098 0.008

pH7 0.252 0.202 2.099 0.239 0.034 0.077 0.098

pH9 0.260 0.169 1.490 0.191 0.027 0.007 0.052

ND: not identified

It is well known that pyrazines are only produced at p H values above 5.5, and the yield of pyrazines formed in the Maillard model system increased with p H values in the range of 5 to 9 (72). However, in our model system, 4 out of 5 pyrazines and dimethyl diselenide exhibited a maximum yield at pH7, 80 min. The yields of dimethyl diselenide at different times and p H values is shown in Figure 4. The possible mechanism for the formation of dimethyl diselenide from the Maillard reaction of selenomethionine is shown in Figure 5. The products from the Maillard reaction are p H dependent due to the breakdown of the Amadori product during the intermediate stages. In many model studies on the effect of p H on the volatile products of the Maillard reported, large p H changes were usually used. However, it has also reported that small changes in pH over the range 4.5-6.5 may have significant effects on the types and concentrations of the volatile products in model systems such as those that contain cysteine and ribose (13).



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Figure 3. The yields of ethyl selenoacetate at 160 °C and different times and pHs.

Figure 4. The yields of dimethyl diselenide at 160°C and different times and pHs.


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Figure 5. The formation of dimethyl diselenide.

In terms of p H change, in unbuffered model systems a p H change of 3 or more units is not unusual during heating, and this can affect both the rate and the pathway on the formation of volatile and colored products (14). Thus, it is very important to maintain a constant pH during heating when model systems are used to study the influence of pH on the Maillard reaction. In our study, it is shown that the pH change of an unbuffered solution is significant after heating (Table IV), and this p H change dramatically affected the formation of pyrazines (Figure 6).

Table IV. pH Changes of Buffered and Unbuffered Systems Buffered Systems 7.01 8.99 0 7 8.92

Initial p H Final p H

Unbuffered Systems 6.98 9.01 4/78 5Λ2

Interactions Between Organoselenium and Sulfur Compounds In order to investigate the potential interaction of organoselenium and organosulfur compounds, the model reaction of selenomethionine, glucose and diallyl disulfide was performed. The G C / M S profile of the reaction products is shown in Figure 7. Several organosulfur, organoselenium and mixed organoselenium-sulfur compounds were identified in this model system. They are allyl methyl selenide (MeSeAll), diallylsulfide, CH -Se-S-CH , CH -Se-SC H - C H = C H (Figure 8), CH -Se-Se-S-CH , CH -Se-S-S-CH -CH=CH , C H Se-Se-S-CH -CH=CH , 2-acetyl-thiophene, and 5-methyl-2-thiophenecarboxaldehyde. As an example, Figure 8 shows the ΕΙ/MS spectrum of MeSeSAll. Some of them have previously been identified in garlic (8). 3

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Figure 6. The yields ofpyrazines in buffered and unbuffered solutions,

(I6O0C, 80 min).


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«

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GC/MS

Figure 7. profile of volatile compounds formedfrom glucoseselenomethionine model system with diallyl disulfide. 1 = hydroxyacetone; 2 = allyl methyl selenide; 3 = CHs-Se-S-CHs; 4 = diallylsulfide; = dimethyldiselenide;

57 = CH3-Se-S-S-CH2-CH=C 6 = CH3-Se-S~CH - CHs-Se-Se-S-C

thiophenecarboxaldehyde; 9 = 2-acetylthiophene; 10 11 = 4-hydroxy-5-oxohexanoic acid lactone, 12 =

CH3-Se-Se-S-CH2-CH=CH2;

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100 110 1 20 130 1 40 1 50 160 1 70 1 80

Figure 8. ΕΙ/MS spectrum of 2-propenesulfenoselenoic acid methyl ester (MeSeSAll).


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References 1.


2. 3. 4. 5. 6. 7. 8. 9. 10.

11.

12.

13. 14.

Bard, A . J. Encyclopedia of Electrochemistry of the Elements, Vol. IV. Marcel Dekker: New York, N Y . 1975. Wilber, C. G. Clinical Toxicology. 1980, 17, 171-230. National Academy of Sciences. Recommended Dietary Allowances 9 ed, 1980, National Academy of Sciences: Washington, D C . Holkstra, W. G . Fed. Proc., 1975, 34, 2083-2088. Robison, J. R.; Robison, M. F.; Levander, Ο. Α.; Thomson, C. D. Am. J. Clin. Nutr. 1985, 41, 1023-1031. Buchan, R. F. J. Am. Med. Assoc. 1974, 227, 559-560. Minami, T.; Boku, T.; Inada, K.; Morita, M.; Okazaki, Y . J. Food Sci. 1989, 54, 763-765. Cai, X. J.; Block, E.; Uden, P.C.; Quimby, B . D.; Sullivan, J. J. J. Agric. Food Chem. 1995, 43, 1751-1753. Tsai, J. H.; Hiserodt, R. D.; Ho, C.-T.; Hartman, T. G.; Rosen, R. T. J. Agric. Food Chem. 1998, 46, 2541-2545. Tressl, R.; Helak, B.; Martin, N.; Kersten, E. In Thermal Generation of Aromas; Parliament, T. H.; McGorrin, R. J.; Ho, C.-T., Eds.; American Chemical Society: Washington, D C , 1989; pp 156-171. Mottram, D. S.; Whitfield, F. B . In Thermally Generated Flavors; Parliament, T. H . ; Morello, M. J.; McGorrin, R. J. Eds.; American Chemical Society: Washington, DC, 1994; pp 180-191. Leahy, M . M.; Reineccius, G. A . In Thermal Generation of Aromas; Parliament, T. H . ; McGorrin, R. J.; Ho, C.-T., Eds.; American Chemical Society: Washington, D C , 1989; pp 196-208. Mottram, D. S.; Leseigneur, A. In Flavour Science and technology; Bessiere, Y . and Thomas, A . F., Eds.; Wiley: Chichester, 1990; pp 121-124 Whitfield, F. B . ; Mottram, D. S.; Brock, S.; Puckey, D. J.; Salter, L . J. J. Sci. Food Agric. 1988, 42, 261-272. th


Heteroatomic Aroma Compounds - American Chemical Society

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