Determination of Volatile Sulfur Compounds Formed by the Maillard

Downloaded by UNIV OF DELAWARE MORRIS LIB on June 8, 2012 | http://pubs.acs.org Publication Date (Web): August 24, 2011 | doi: 10.1021/bk-2011-1068.ch011

Chapter 11

Determination of Volatile Sulfur Compounds Formed by the Maillard Reaction of Glutathione with Glucose Sang Mi Lee and Young-Suk Kim* Department of Food Science and Engineering, Ewha Womans University, Seoul 120-750, South Korea *E-mail: [email protected].

The volatile compounds formed from the thermal reaction of glutathione with glucose were analyzed by gas chromatographymass spectrometry (GC-MS). Sulfur-containing compounds dominated the volatiles in glutathione-Maillard reaction products (GSH-MRPs) and included 1 thiazole, 12 thiophenes, 2 polysulfides, 1 sulfur-substituted furan, and 1 miscellaneous. The carbohydrate module labeling (CAMOLA) experiment was employed to evaluate the relative importance of precursors to the formation pathways and elucidate the origin of the carbon skeleton for sulfur-containing compounds in GSH-MRPs. The isotopomeric distribution patterns showed that 2-ethylthiophene, 2,5-dimethylthiophene, 1-thiophen-2-ylethanone, 5-methylthiophene-2-carbaldehyde, and 1-thiophen-3-ylethanone can be formed from the intact carbon skeleton of a C-6 glucose chain, whereas 3methylthiophene-2-carbaldehyde occurs via the recombination of fragments that may originate from both GSH and glucose.

Volatile sulfur-containing compounds have been found in diverse foods such as vegetables, roasted coffee, roasted seeds, wheat bread, cooked meats, and many thermally processed foods (1–4). These sulfur-containing compounds are known to play an important role in contributing meaty flavor, in particular, to roasted and cooked meats. Sulfur-containing amino acids, such as cysteine, cystine,

© 2011 American Chemical Society In Volatile Sulfur Compounds in Food; Qian, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2011.


and methionine, are major precursors for the formation of the sulfur-containing compounds. During the thermal processing, reactive intermediates such as hydrogen sulfide are liberated from the sulfur-containing amino acids and participate in the Maillard reaction and Strecker degradation to form volatile sulfur-containing compounds (4, 5). Glutathione (γ-L-glutamyl-L-cysteinylglycine, GSH), a tripeptide, can form diverse volatile sulfur-containing compounds through the Maillard reaction during the heating processing. In earlier publications, Zheng et al. (6) reported that hydrogen sulfide is released from the cysteine residue in GSH and involved in the generation of volatile sulfur-containing compounds during thermal reactions. It can therefore lead to diverse sulfur-containing components, such as thiols, thiophenes, thiazoles, and polysulfides, which are related to savory and meaty-type flavor notes, through the Maillard reaction and Strecker degradation (1, 2, 6). Volatile compounds generated from the thermal decomposition of glutathione were compared to those of cysteine when heated under same conditions. In that study, Zhang et al. (7) reacted glutathione in an aqueous solution at 180°C and identified 17 compounds, including isomers of 3,5-dimethyl-1,2,4-trithiolane as the major compounds. Also, Ho et al. (8) determined the reactivity of Maillard volatile compounds generated from the thermal reaction of four cysteine-containing peptides, GSH, γ-glu-cys, cys-gly, and gly-cys, with glucose. Volatiles found in those model systems were mainly thiophenes, thiazoles, and cyclic polysulfides whereas pyrazines were minor components. The results might be due to the fact that hydrogen sulfide is released more easily than ammonia, and hydrogen sulfide inhibits the Strecker degradation and pyrazine formation (8). The use of isotopically labeled compounds proposed as precursors or intermediates in the formation of certain target molecules is a powerful technique to elucidate complex reaction pathways (9). The carbohydrate module labeling (CAMOLA) technique was developed to evaluate relative importance of different pathways that lead to a certain target molecule. In particular, this technique employes a combination of 13C6-labeled and unlabeled carbohydrates such as glucose and fructose at equal ratio to explain the extent of fragmentation of the sugar skeletons and the formation of key transient intermediates involved in the formation of flavor molecules (9). If these transient intermediates combine, isotopomers of the respective product are formed, being ruled statistically, from these modules. This approach has been used to clarify formation pathways and gain insight into the fragmentation of precursors of Maillaed reaction products (MRPs) (9, 10). Using CAMOLA technique, Schieberle demonstrated that the reaction conditions influence the formation pathway of furaneol from the thermal reaction of glucose and proline (9). Cerny and Davidek also performed CAMOLA approach to show that the carbon skeleton remains intact in the formation of 2-methyl-3-furanthiol, 2-furfurylthiol, and 3-mercapto-2-pentanone during the reaction of ribose/[13C5]-ribose with cysteine (11). Some sulfur-containing compounds, such as 2-methyl-3-furanthiol, 3-mercapto-2-pentanone, furfurylthiol, and 4,5-dihydro-2-methyl-3-furanthiol, were reported to be key odorants from the reaction of cysteine and thiamine with xylose. When [13C5]-xylose was used in the Maillard reaction instead 232 In Volatile Sulfur Compounds in Food; Qian, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2011.


of xylose, the carbon atoms of furfurylthiol were found to be completely labeled by 13C. This could demonstrate that the whole carbon skeleton of furfurylthiol was originated from xylose as the carbon precursor. In contrast, 4,5-dihydro-2-methyl-3-furanthiol was virtually unlabeled, indicating thiamine as the carbon source (12). Although GSH plays an important role in the formation of diverse volatile sulfur-containing compounds during the heating processing, sulfur-containing compounds from Maillard reaction products (MRPs) of GSH and the relative importance of their formation pathways have not been systematically studied yet. Therefore, the objective of this study was to elucidate the formation of volatile sulfur-containing compounds in GSH-MRPs, which can be formed from the interaction of GSH and glucose during the thermal reaction. The CAMOLA approach was employed to evaluate the relative contribution of precursors to the formation pathways and elucidate the origin of the carbon skeleton for sulfur-containing compounds .

Experimental Chemicals L-glutathione (GSH), D-glucose ([12C6]-D-glucose), n-alkane standards (C8C22), sodium sulfate, and an internal standard compound (ethyl trans-2-octenoate) were purchased from Sigma-Aldrich (St. Louis, MO). Dichloromethane of HPLC grade was obtained from Fisher Scientific (Seoul, South Korea). All authentic standard compounds used in this study were obtained from Sigma-Aldrich. For the CAMOLA study, [U-13C6]-D-glucose was obtained from ISOTEC (Milwaukee, WI). Model Maillard Reaction Systems GSH (0.01 M) and D-glucose (0.01 M) were dissolved in 100 mL of HPLC grade water (Fisher Scientific). The reaction mixtures were adjusted to pH 7.5 and then sealed in a 200 mL stainless steel cylinder. The cylinder was heated in a 160 °C drying oven for 2 h. After the thermal reaction, the cylinder was cooled in cold water before the cap was opened. Extraction of Volatile Maillard Reaction Products After the reaction mixture was cooled, the volatile components were extracted using a simultaneous steam distillation and solvent extraction (SDE) method with a Likens-Nickerson (L-N) apparatus with 50 mL of dichloromethane. Before the SDE, an internal standard compound (50µL of 100 ppm ethyl trans-2-octenoate in dichloromethane, w/v) was added for quantification. After the sample started boiling, SDE was run continuously for 2 h. The extract was dehydrated using anhydrous sodium sulfate and filtered on Advantec 110 mm filter paper (Toyo Roshi Kaisha, Tokyo, Japan) before concentrated to a final volume of 0.1 mL using a gentle stream of nitrogen gas. 233 In Volatile Sulfur Compounds in Food; Qian, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2011.


Analysis by Gas Chromatography-Mass Spectrometry (GC-MS) The volatile extracts from Maillard reaction products (MRPs) were analyzed by GC-MS, using a gas chromatograph and mass selective detector (6890N and 5975, respectively; Agilent Technologies, Palo Alto, CA) equipped with a DB-5 ms column (30 m length × 0.25 mm i.d. × 0.25 mm film thickness; J&W Scientific, Folsom, CA). Helium was run as a carrier gas at a constant column flow rate of 0.8 mL/min. A 1 µL aliquot of the MRP extract was injected into the GC column using the splitless injection mode. The oven temperature was initially held at 40 °C for 4min, raised to 200 °C at a rate of 2 °C/min, and then held there for 10 min. The temperatures of the injector and detector transfer line were 200 °C and 250 °C, respectively. The mass detectorwas operated in electron impact mode with ionization energy of 70 eV, a scanning range of 33-550 amu, and a scan rate of 1.4 scans/s. Identification of Volatile Compounds Volatile compounds were positively identified by comparing their mass spectral data, and linear retention indices (RIs) with those of authentic compounds. The RI of each compound was calculated using n-alkanes C8-C22 as external reference (13). Otherwise, tentative identification was made based on mass spectra in on-line Wiley database. The semiquantitative analysis of volatile compounds was performed by comparing their peak areas to that of the internal standard compound (50µL of 100ppm ethyl trans-2-octenoate in dichloromethane, w/v) on the GC-MS total ion chromatograms. Carbohydrate Module Labeling (CAMOLA) Experiment Equimolar amounts of 0.01 M fully labeled [13C6]-D-glucose and 0.01 M unlabeled D-glucose ([12C6]-D-glucose) were reacted with 0.01 M GSH in a drying oven at 160 °C for 2 h. The reaction mixture was then extracted using the SDE method as described above. The extracts were dehydrated over anhydrous sodium sulfate, concentrated to 0.1mL of final volume under a gentle stream of nitrogen gas, and then subjected to GC-MS analysis. Calculation of Isotopomer Proportions The isotopomer ratios were calculated using the relative signal intensities of analyzed ions in the mass spectrum of the respective compound. The values of the calculated isotopomer proportions for sulfur-containing compounds were corrected by subtracting the naturally occurring percentages of 13C (1.1%), 33S (0.76%), and 34S (4.20%). The loss of hydrogen observed with the molecular ion signal was determined in the labeled molecular ions by the ratio [M+ - 1]/[M+]. Additional data processing was required for 5-methylthiophene-2-carbaldehyde and 3-methylthiophene-2-carbaldehyde; calculation of the isotopomer ratio was based on the [M+ - 1] ion signal instead of [M+] because the former was 234 In Volatile Sulfur Compounds in Food; Qian, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2011.

more intense than the molecular ion signal. After correction, any isotopomer percentages below 1% were taken to be 0%.

Results and Discussion


Volatile Compounds in Glutathione-Maillard Reaction Products Table I lists the volatile compounds identified in glutathione and glucose (GSH-GLU) Maillard reaction products (MRPs), considering their mass spectral data, relative peak areas, and RIs on the DB-5 column, respectively. A total of 29 volatile compounds, including 1 thiazole, 12 thiophenes, 7 furans and furanones, 2 polysulfides, 1 pyrazine, 5 other nitrogen-containing heterocyclics, and 1 miscellaneous, were identified in GSH-GLU MRPs. The volatile compounds in GSH-GLU MRPs were primarily composed of sulfur-containing compounds. The chemical structures of these sulfur-containing compounds are shown in Figure 1. The major volatile compounds formed in the GSH-GLU MRPs were thiophene and its derivatives, the abundance of which could be due to the hydrogen sulfide easily released from the cysteine residue in GSH. It was reported that the release of hydrogen sulfide from GSH is much faster than that of ammonia (8). Therefore, thiophene derivatives dominated the sulfur-containing compounds, whereas thiazole derivatives, which need additional nitrogen for their formation, were almost undetectable in GSH MRPs. When Ho et al. (8) investigated the Maillard volatile products generated from cysteine-containing peptides, GSH, γ-glu-cys, cys-gly, and gly-cys, with glucose, they found that GSH produced larger amounts of thiophenes compared to thiazoles. Our study identified thiophenes and polysulfides as major components in GSH-GLU MRPs. Among thiophene derivatives, 2-ethylthiophene, 2,5dimethylthiophene, thiolan-3-one, thiophene-2-thiol, 1-thiophen-2-ylethanone, 5-methylthiophene-2-carbaldehyde, 3-methylthiophene-2-carbaldehyde, and 1-thiophen-3-ylethanone were detected as major components. These thiophenes have been identified in a wide range of food systems in which they significantly contribute to the characteristic odor properties (14). In particular, 2,5-dimethylthiophene has been identified in cooked beef, chicken, and pork liver (15). On the other hand, 3-methylthiophene-2-carbaldehyde was identified in chicken, whereas 5-methylthiophene-2-carbaldehyde, which has a roasted odor note, was found in cooked beef (16). Elucidation of Volatile Sulfur-Containing Compounds Formation GSH and glucose (equimolar amounts) were reacted at pH 7.5 in aqueous system at 160 °C for 2 h. The thermal reaction was carried out using a mixture (1:1) of unlabeled and 13C6-labeled glucose. Diverse sulfur-containing compounds were found in GSH MRPs. The mass spectra of volatile sulfur-containing compounds identified from the Maillard reaction of [12C6]-glucose/[13C6]-glucose and GSH were analyzed on the basis of the mass-to-charge ratios (m/z) of the molecular ions of the isotopomers, which exhibited signals with mass differences of up to M++ 6 as compared to those obtained from GSH-unlabeled 235 In Volatile Sulfur Compounds in Food; Qian, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2011.


glucose MRPs. Table II lists the identified volatile sulfur-containing compounds and the proportions of their 13C-labeled isotope molecules. The isotopomers indicated that the molecules comprise either unlabeled carbons, fully 13C-labeled carbons, or a mixture of labeled and unlabeled carbon fragments. In the case of 2-methylthiophene, 2,5-dimethylthiophene, thiophen-2-ylmethanol, 5-methylthiophene-2-carbaldehyde, and 3-methylthiophene-2-carbaldehyde, the values for the labeled [M+-1] ions were corrected by the ratio [M+]/([M+ - 1] due to the significant loss of hydrogen.

Table I. Volatile compounds formed from the thermal reaction of glutathione with glucose No

RIa

Possible compounds

Relative peak areab

IDc

0.015±0.001

A

Thiazoles 1

Determination of Volatile Sulfur Compounds Formed by the Maillard

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