Food Chemistry 131 (2012) 280–285

Contents lists available at SciVerse ScienceDirect

Food Chemistry

journal homepage: www.elsevier .com/locate / foodchem

Key meat flavour compounds formation mechanism in a glutathione–xyloseMaillard reaction

Ran Wang, Chao Yang, Huanlu Song ⇑Beijing Key Laboratory of Flavor Chemistry, Beijing Technology and Business University, China

a r t i c l e i n f o

Article history:Received 16 May 2011Received in revised form 14 June 2011Accepted 26 August 2011Available online 1 September 2011

Keywords:Formation mechanismMeat flavour compoundsGlutathione[13C5] XyloseGC-O-MSLC-TOF-MS

0308-8146/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.foodchem.2011.08.079

⇑ Corresponding author. Tel./fax: +86 10 68984025E-mail address: songhuanlu@yahoo.com.cn (H. Son

a b s t r a c t

The formation mechanism of meat flavours formed from a glutathione–xylose Maillard reaction wasstudied using a group of model reactions with [13C5] xylose/xylose (1:1), heated at 132 �C for 90 min. Vol-atiles, especially the aroma-active compounds, and non-volatiles were analysed respectively with GC-O-MS and LC-TOF-MS. Analysis of the mass spectra showed that furfural, 2-furfurylthiol and thiophenewere 13C5-labelled and hence stem from xylose, whereas 2-methyl-3-furanthiol, 2-methyl-thiopheneand 2-pentyl-thiophene, which showed similar formation patterns with only unlabelled compounds,may be from thiamine and/or cysteine carbons. In the process of Maillard reactions, GSH can be capableof cleaving in the position of glutamyl and cysteinyl, and then form 5-oxoproline or pyroglutamic acid(PCA), cysteine, and Cys-Gly dipeptide, which can form cyclic (Cys-Gly).

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Sulphur-containing volatile compounds are a major aroma classfound in vegetables, cooked meat, and other food (Block, 1992;Farmer & Mottram, 1990; Gasser & Grosch, 1988). Glutathione(c-L-glutamyl-L-cysteinylglycine, GSH) plays an important role inthe formation of sulphur-containing volatile compounds viahydrogen sulphide, which is released from the cysteine residuein GSH during the heating processing (Zheng, Brown, Walter,Mussinan, & Ho, 1997). It has been prevalently accepted that thesulphur-containing amino acids, cysteine and cystine are indis-pensable components for generation of a meat-like flavour throughthermal processing. They participated in the Maillard reaction andStrecker degradation to form those sulphur-containing com-pounds. In addition, both GSH and its Maillard reaction products(MRPs) were recently shown to have a taste-enhancing (‘‘kokumi’’)property (Dunkel, Koster, & Hofmann, 2007).

A wide range of peptides have been reported to be present infood systems and recognized as important flavour compounds. Inmany food systems such as meat (Mabrouk, 1976), sea food (Nogu-chi, Arai, Yamashita, Kato, & Fujimaki, 1975), and hydrolysed veg-etable protein (Aaslyng et al., 1998), peptides play significant roles,but the chemistry and mechanisms of production of aroma com-pounds, by peptides, have not been investigated to an appreciableextent.

ll rights reserved.

.g).

Cyclic dipeptides, known as diketopiperazines (DKPs), can beformed from tripeptides through degradation (Rizzi, 1989). Thereactivity of GSH may be related to its long peptide chain lengthand/or formation of DKP (Lu, 2006). Ueda, Yonemitsu, Tsubuku,Sakaguchi, and Miyajima, 1997 investigated the thermal degrada-tion of GSH, and found that GSH was degraded to half amountand formed pyroglutamic acid (PCA) and cyclic (Cys-Gly) (3-mer-captomethyl-2,5-diketopiperazine). The Cys-Gly dipeptide maybe formed from glutathione through 5-oxoproline (Kosower,1989), or PCA, and then formed cyclic (Cys-Gly) during the reac-tion; and the remaining straight Cys-Gly dipeptide may furtherparticipate in the Maillard reaction (Lu, 2006).

Isotopically labelled compounds are very useful for clarifyingthe Maillard reaction pathways and accurately quantifying the ar-oma compounds. In many approaches used in previous studies, thecarbon module labelling (CAMOLA) technique was developed(Schieberle, Bareth, Fischer, & Hofmann, 2002). 13C labelled precur-sors were always used to gain information on the fragmentation ofthese precursors during aroma formation (Cerny, 2007; Cerny &Briffod, 2007; Cerny & Davidek, 2003; Lee, Jo, & Kim, 2010).

The objective of this study was to elucidate the formationmechanism of meat flavours formed from xylose and GSH duringthe Maillard reaction, and detective DKP of the cyclic (Cys-Gly).[13C5] Xylose was used to trace back the origin of the carbons inthe productions. The intermediate and volatile compounds wereanalysed to elucidate mechanism of GSH participating in the Mail-lard reaction, then providing a foundation for future research ofoligopeptides.

Fig. 1. DKP of Cys-Gly dipeptide.

Table 1Model reactions.

Amount (mg)

A B C D E

Xyl 50 50 50 50 50[13C5] Xyl 50 50 50 50 50Thiamine 10 10 10 10 10HClGSH 57Glu 27.3 27.3Cys 29.2 36.8Gly 14.3 18.4Cys-Gly 29.7 57

Table 2Volatiles of the model reaction.

No. Compoundsa RIb Odour des

Sulphur-containing compounds1 Dimethyldisulphide 751 Sulphury2 2-Methyl-3-furanthiol 865 Meaty, vita3 2-Furfurylthiol 908 Roasted, co4 Thiophene 660 Scallion5 2-Me thy1-thiophene 774 Onion6 Dimethyl trisulphide 973 Mint7 2-Pentyl-thiophene 1168 Fatty

Aldehydes, Ketones8 2-Methyl-3-pentanone 712 –9 6-Hydroxy-2-hexanone 760 –10 Hexanal 799 Green, gra11 Furfural 831 Almond, p12 Heptanal 890 Cheesy, fat13 3-Octanone 982 Rusty14 3-Octanol 990 Earthy15 Octanal 1002 Fruity, sou16 Nonanal 1078 Green, stal

Heterocyclic compounds17 3-Methyl-2,5-piperazinedione 953 –18 3,6-Dimethylpiperazine-2,5-dione 1612 –19 3-Ethyl-lH-pyrrole 811 –20 2-Pentylfuran 987 Fruity21 D-Limonene 994 Sweet

22 4-Methyl-5-thiazoleethanol 1250 Roasted

Others23 1,2-Ethanediol monoformate <600 –24 1,5-Pentanediol dinitrate <600 –25 3-Methyl-1-butanol acetate 878 Solvent26 Butylbenzene 1062 –27 1-Methyl-2-(1-methylethyl)-benzene 1031 –28 1-Methyl-4-(1-methylethenyl)-benzene 1096 –29 Pentylbenzene 1163 –30 2-Methylbutylbenzene 1163 –

a Volatile compounds were identified by comparing the mass spectra, retention indicb RIs were determined using n-alkanes C7–C22 as external references.c Odour description was carried out at the Sniffer 9000 olfactometry.d Data are means of at least three assays; maximum SD + 10%.

R. Wang et al. / Food Chemistry 131 (2012) 280–285 281

2. Materials and methods

2.1. Chemicals

Cys-Gly (90% enrichment) and its DKP (Fig. 1) were fromApeptides (Shanghai, China). [13C5] Xylose (98% enrichment) andn-alkanes (C7–C22) (chromatographic reagent) were from Sigma–Aldrich (Santa clara, USA). HPLC-grade methanol, acetonitrile andtrifluoroacetic acid (TFA) were from Thermo Fisher Scientific (Wal-tham, USA), and HPLC-grade H2O was purified in the laboratory.The other chemicals were from Huihai Scientific Instruments Co.Ltd. (Beijing, China).

2.2. Model reactions

Based on a model system experiment reported by Lv & Song,(2010), the reactants listed in Table 1 were dissolved in distilledwater (25 ml, pH 4.8). The ratio of reactants was similar to the ratioin meat. To form more meat-like flavour, the reaction parametershad been optimised, and the solutions were optimally treaded for90 min in a High-pressure Stainless Reactor (Parr instrument Co.,Moline, USA) at 132 �C. Then they were cooled down rapidly usingrunning cool tap water, and stored at 4 �C for 24 h to afford the afterreactions.

criptionc Relative concentration (lg ll)d

A B C D E

0.279 0.157 0.242 0.515 0.227min 0.021 0.082 0.118 0.179 0.034ffee 0.049 0.066 0.170 – –

– 0.312 0.339 – –– – 0.111 – –– – 0.052 0.392 0.052– 0.005 0.006 – –

0.094 0.103 – – 0.1210.161 0.114 – – –

ss 0.089 0.063 0.051 0.112 0.073ungent 0.152 0.066 0.089 0.575 0.169ty 0.015 – – – 0.022

0.025 – – – –0.094 – – – –

r – – – – 0.011e – – – – 0.006

0.019 – – – –0.661 – – – –– 0.075 0.054 – –– 0.021 0.020 – –– 0.245 0.188 0.277 –

– – – – 0.025

0.102 – 0.104 0.135 –0.606 – – – 1.0380.046 0.099 – – –0.030 0.022 0.023 – 0.012– 0.070 0.040 0.060 0.008– 0.140 0.070 – 0.009– 0.003 0.003 0.023 –– – – – 0.002

es and odour with those of authentic reference compounds.

Table 3Proportion of isotopomers formed from xylose/[13C5] xylose (1:1) model reaction.

No. Compounds m/z (M+) m/z (analysed) Unlabeled carbon atoms (%)a Labeled carbon atoms (%)a Number of labelled carbon atoms

1 2-Methyl-3-furanthiol 114 114; 119 >99 <1 02 2-Furfurylthiol 114 114; 119 80 20 53 Thiophene 84 84; 88 88 12 54 2-Methyl-thiophene 98 98; 103 >99 <1 05 2-Pentyl-thiophene 154 154; 159 >99 <1 06 Furfural 96 96; 101 52 48 5

a Values represent the proportion between labelled and the indicated unlabelled isotopomers and are based on the abundance of the respective analysed ion signals.

282 R. Wang et al. / Food Chemistry 131 (2012) 280–285

2.3. Volatiles isolation

The capture of volatile components was undertaken in a StratumPurge & Trap Concentrator (Tekmar, Ohio, USA). 10 ml Maillardreaction productions with 1 ll internal standard solution (2-methyl-3-heptanone 1.68 lg ll�1 in methanol) was taken in thefritted sparge tube, and heated at 50 �C. Ultra-high purity nitrogenwas utilized as a purge, flowing at 40 ml/min. The other analyticalconditions were:

Trap temperature: purge 50 �C; desorption 250 �C; bake 260 �C.Time: purge 10 min; desorption 2 min; trap bake 10 min.The purge and trap concentrator was interfaced with a system

containing a gas chromatograph coupled with a mass spectrometerand an olfactometry.

Fig. 2. Possible formation pathway for 2-furfurylthiol, t

2.4. Gas chromatograph-olfactometry-mass spectrometer (GC-O-MS)analysis

The analysis of volatiles was performed on a GC 7890A cou-pled to a Triple Quad 7000B (both Agilent, Palo Alto, CA), andequipped with a Sniffer 9000 Olfactometer (Gerstel, KG, Ger-many). Separations in GC were performed on DB-5MS UI(30 m � 0.250 mm � 0.25 lm, J & W Scientific, Folsom, CA). Thecarrier gas used was ultra-high purity helium and the columnhad a flow rate of 1.2 ml/min. The oven temperature was pro-grammed from 40 �C for 3 min, then increases at 5 �C/min to200 �C, and then increasing at 15 �C/min to 260 �C, whereby itwas held for 5 min. The temperatures of the injector and theGC/MS interface were 250 �C and 280 �C. Electron-impact mass

hiophene and furfural from xylose and glutathione.

R. Wang et al. / Food Chemistry 131 (2012) 280–285 283

spectra were generated at 70 eV, with m/z scan range from 35 to550 amu, with the ion source temperature of 230 �C. Compoundswere identified according to NIST 2.0 mass spectra libraries in-stalled in the GC–MS equipment. GC-O was performed by threeexperienced panelists.

2.5. Compounds identification

The identification was based on the comparison of the massspectrum and retention index with reference compounds. TheirRI values and odour descriptions on DB-5MS column, with thoseof linear retention indices (RIs), having the same/similar odourquality and RI, previously reported in www.odour.org.uk. n-Al-kanes (C7–C22) were analysed under the same conditions to calcu-late LRIs:

LRI ¼ 100N þ 100nðtRa � tRNÞ=ðtRðNþnÞ � tRNÞwhich was described by Dool and Kratz (1963).

2.6. Quantification of DKP [Cyclice-(Cys-Gly)]

The amount of DKP in heated model solutions was determined bya HPLC 1200 system (Agilent, Palo Alto, CA). HPLC separation wasperformed using a ZORBAX SB-C18 (Agilent), 4.6 � 250 mm, 5 lm,at room temperature. The reaction components were eluted usinga liner acetonitrile (0.1% TFA)/H2O (0.1% TFA) gradient, 1–30% aceto-nitrile over 20 min. Chromatograms were monitored at 220 and254 nm and initial data were collected from 190 to 600 nm. Theinjection volume was 10 ll. DKP standard substances were preparedin aqueous solution (0.5–10.0 mg/l); the injection volume of all stan-dard substances was also 10 ll.

Fig. 3. Proposed formation of 3-methy

2.7. LC-TOF-MS detection of non-volatile components

The analysis of non-volatile components was performed usingan Agilent 6210 TOF-MS coupled to an Agilent 1200 Series HPLC.The separation of components was also carried out using an HPLCsystem (consisting of vacuum degasser, autosampler with thermo-stat, binary pump, and DAD system) equipped with a reversed-phase C18 column (ZORBAX Eclipse XDB-C18 4.6 � 50 mm,1.8 lm). The reaction components were eluted using a mobilephase of 15% methanol and 85% H2O. The flow rate was 0.2 ml/min. The TOF-MS was equipped with a positive electrospray ionisa-tion source. Ultra-high purity nitrogen (>99.7%) was used for thesheath gas and auxiliary gas at a pressure of 20 psi and 7 units.The temperature of the heated capillary was maintained at350 �C then the capillary voltage was set at 4000 V, and the appliedcollision offset energy was set at 175 V. The instrument wasscanned from m/z 100–1000. This mass range enabled the inclu-sion of three reference mass compounds, which produced ions atm/z 121.0508, 149.0233 and 922.0097. The injected sample vol-ume was 2 ll. Data obtained were processed with Analyst-QS soft-ware with accurate mass application. Compounds were identifiedaccording to NIST 2.0 mass spectra libraries.

3. Results and discussion

By comparing the mass spectra, RI values and aroma properties,with those of authentic standards, volatiles were identified quali-tatively and quantitatively. The results of volatiles of the modelreaction were listed in Table 2. Meat flavour compounds, such as2-methyl-3-furanthiol and 2-furfurylthiol, and many of other sul-phur-containing compounds, for example, dimethyldisulphide,

l-2,5-piperazine-dione from GSH.

284 R. Wang et al. / Food Chemistry 131 (2012) 280–285

thiophene, 2-methylthiophene, dimethyltrisulphide, 2-pentylthi-ophene were found in the Maillard reaction products. Table 3shows the isotope ratios of several aroma-active compounds fromthe model reaction, based on the molecular ions as generated byelectron impact. As indicated in Table 3, furfural had the isotopo-mers of [M+] (m/z 96) and [M++5] (m/z 101). Unlabelled and the5-fold-labelled molecules were present at approximately 1:1. Thatwas, the carbon skeleton of xylose chain remained intact for theformation of furfural. In the case of 2-furfurylthiol and thiophene,unlabelled and the 5-fold-labelled molecules did not appear at anapproximate ratio of 1:1, but 5-fold-labelled molecules was mea-sured in a certain percentage at 20% and 12%. So 2-furfurylthioland thiophene were referred to partly from xylose (Fig. 2), andunlabelled molecules may be from thiamine and/or cysteine car-bons. 2-Methyl-3-furanthiol, 2-methyl-thiophene and 2-pentyl-thiophene showed similar formation patterns with only unlabelledcompounds. They may be generated from thiamine and/or cys-teine carbons. Supposedly the carbons of the unlabelled 2-methyl-3-furanthiol are from thiamine, this way is well knownin scientific literature, on the other hand, the generation from cys-teine without involvement of reducing sugars seems unlikely(Cerny, 2007).

Table 2 showed the formula of 5 Maillard reaction systems. Ingeneral, the content of sulphur-containing compounds, including2-methyl-3-furanthiol, 2-furfurylthiol, thiophene etc. in the systemof amino acids and xylose/[13C5] xylose, was higher than that ofother systems. This may be related to the release of hydrogen sul-phide. According to Zhang, Chien, and Ho (1988) the release ofhydrogen sulphide from cysteine was fast and produced four timesas many volatiles as GSH under the same conditions. It was interest-ing to note that 3-methyl-2,5-piperazine-dione and 3,6-dimethyl-piperazine-2,5-dione were only found in GSH system. Both of themmay be formed from Cys-Gly dipeptide through the degradation ofGSH (Fig. 3). However, in Cys-Gly dipeptide system, Cys-Gly dipep-tide may be easier to form DKP of cyclic (Cys-Gly), and this can beproved by HPLC. DKP of cyclic (Cys-Gly) formed from the modelreaction was measured by HPLC. Compared to the retention timeof the standard substance, quantification of DKP was measured(Table 4). The generation of DKP from Cys-Gly dipeptide systemhad the highest yield than the other systems. This may be becauseDKP was easy transferred to 3-methyl-2,5-piperazine-dione and3,6-dimethylpiperazine-2,5-dione in GSH system.

The identification of non-volatile components was performedwith LC-TOF-MS, and nine major peak clusters were detected.[M+H]+ and/or [M+H+n]+ (n presented the number of 13C isotope)selected ions such as 122.0689, 130.0475, 140.0792 and144.0458 were analysed to find the compounds which these sig-nals distribute to. Based on reactant participating in Maillard reac-tion, the [M+H]+ peak of 122.0689 existed in all systems, and wasdistributed to cysteine. The [M+H]+ peak of 130.0475 was found inA, B, and D systems, which contained glutamyl or glutamate, so itwas distributed to PCA. Then it could be referred that GSH could becapable of splitting in the position of glutamyl and cysteinyl, andthen form 5-oxoproline or PCA, cysteine, and Cys-Gly dipeptide,which formed cyclic (Cys-Gly). A interesting intermediate was

Table 4The amount of DKP producing in the modelreaction.

Model reactions amount of DKP(mg/l)

A 0.420B 0.231C 0.379D 11.322E 17.960

found in the model reaction, as [M+H]+ peaks of 140.0792 and144.0458 was distributed to 1-(2-thienyl)-1-propanone, unla-belled and 4-times-labelled, and it may be from thiophene andthe fragment of xylose. LC-TOF-MS used to analyse the non-vola-tiles formed in the Maillard reaction may be a new attempt inthe study of the Maillard reaction.

4. Conclusion

In this study, key meat flavour compounds produced from theglutathione-specific Maillard reaction were successfully analysedby using carbon module labelling (CAMOLA) technique. The useof a combination of 13C5-labelled and unlabelled xylose in a 1:1 ra-tio explained the extent of fragmentation of the sugar skeleton.This study was very helpful for investigating the role of peptidesas precursors in the generation of meat flavour compounds.

Acknowledgements

This work was supported by Project of the National Natural Sci-ence Funds (31071510) and Academic Innovation Personnel Pro-gram of Universities Affiliated to Beijing Municipal Government(PHR200906110).

References

Aaslyng, M. D., Martens, M., Poll, L., Nielsen, P. M., Flyge, H., & Larsen, L. M. (1998).Chemical and sensory characterization of hydrolyzed vegetable protein, asavory flavoring. Journal of Agricultural and Food Chemistry, 46, 481–489.

Block, E. (1992). The organosulfur chemistry of the genus Allium-implications forthe organic chemistry of sulfur. Angewandte Chemie International Edition, 31,1135–1178.

Cerny, C. (2007). Origin of carbons in sulfur-containing aroma compounds from theMaillard reaction of xylose, cysteine, and thiamin. Food Science and Technology,40, 1309–1315.

Cerny, C., & Briffod, M. (2007). Effect of pH on the Maillard Reaction of [13C5] xylose,cysteine, and thiamin. Journal of Agricultural and Food Chemistry, 55, 1552–1556.

Cerny, C., & Davidek, T. (2003). Formation of aroma compounds from ribose andcysteine during the Maillard reaction. Journal of Agricultural and Food Chemistry,51, 2714–2721.

Dool, H. V. D., & Kratz, P. D. (1963). A generalization of the retention index systemincluding linear temperature programmed gas–liquid partitionchromatography. Journal of Chromatography, 2, 463–470.

Dunkel, A., Koster, J., & Hofmann, T. (2007). Molecular and sensory characterizationof c-glutamyl peptides as key contributors to the kokumi taste of edible beans(Phaseolus vulgaris L. Journal of Agricultural and Food Chemistry, 55, 6712–6719.

Farmer, L. J., & Mottram, D. S. (1990). Recent studies on the formation of meat-likearoma compounds. In Bessiere, Y., & Thomas, A. F (Eds.), Flavor Science andTechnology (pp. 113–116). Chichester, UK.

Gasser, U., & Grosch, W. (1988). Identification of volatile flavour compounds withhigh aroma values from cooked beef. Z. Lebensm Unters. Forsch, 186, 489–494.

Kosower E. M. (1989). Structure and reaction of thiols with special emphasis onglutathione. In D. Dopphin, R. Poulson, O. Avramovic, (Eds.), GlutathioneChemical, Biochemical, and Medical aspects. Part A (pp.103–111). New York:Wiley-Interscience Publication Press.

Lee, M. S., Jo, J. Y., & Kim, S. Y. (2010). Investigation of the aroma-active compoundsformed in the Maillard reaction between glutathione and reducing sugars.Journal of Agricultural and Food Chemistry, 58, 3116–3124.

Lu, Chih-Ying. (2006). Peptides as flavor precursors in Maillard reaction [D]. Rutgers:The state university of New Jersey, pp. 104.

Lv & Song, H. L. (2010). Comparison of the compounds with meat-like aroma in thedifferent model systems. Food and Fermentation Industries (Chinese), 36(1),160–162.

Mabrouk, A. F. (1976). In G. Charalambous, & I. Katz, (Eds.), Phenolic, Sulfur, andNitrogen Compounds in Food Flavors (pp. 146). Washington, DC: Am. Chem. Soc.

Noguchi, M., Arai, S., Yamashita, M., Kato, H., & Fujimaki, M. (1975). Isolation andidentification of acidic oligopeptides occurring in a flavor potentiating fractionfrom a fish protein hydrolysate. Journal of Agricultural and Food Chemistry, 23,49–53.

Rizzi, G. P. (1989). Heat-induced flavor from peptides. In T. H. Parliament, R. J.McGorrin, & C. T. Ho, (Eds.), Thermal Generation of Aromas (pp. 285–301). ACSSymposium Series 409; American Chemical Society: Washington, DC.

Schieberle, P., Bareth, A., Fischer, R., & Hofmann, T. (2002). The carbohydrate modullabeling-technique-A useful tool to clarify formation pathways of key aromaand taste compounds formed in Maillard-type reactions. Book of Abstracts, 10thWeurman Flavour Research Symposium, Beaune, France, June 24–28; INRA:Dijion, France, Abstract O25.

R. Wang et al. / Food Chemistry 131 (2012) 280–285 285

Ueda, Y., Yonemitsu, M., Tsubuku, T., Sakaguchi, M., & Miyajima, R. (1997). Flavorcharacteristics of glutathione in raw and cooked foodstuffs. BioscienceBiotechnology and Biochemistry, 61, 1977–1980.

Zhang, Y., Chien, M., & Ho, C. T. (1988). Agricultural and Biological Chemistry, 36,992–996.

Zheng, Y., Brown, S., Walter, O. L., Mussinan, C., & Ho, C. T. (1997). Formation ofsulfur-containing compounds from reactions of furanol and cysteine,glutathione, hydrogensulfide, and alanine/hydrogensulfide. Journal ofAgricultural and Food Chemistry, 45, 894–896.