Nutraceutical Beverages

Chapter 26

Downloaded by CORNELL UNIV on July 27, 2012 | http://pubs.acs.org Publication Date: December 1, 2003 | doi: 10.1021/bk-2004-0871.ch026

Identification of Maillard-Type Aroma Compounds in Winelike Model Systems of Cysteine-Carbonyls: Occurrence in Wine G. de Revel, S. Marchand, and A. Bertrand Faculté d'Œnologie, Université Victor Segalen Bordeaux 2, 351, Cours de la Libération, F-33405 Talence Cedex, France The role of carbonyl (acetoin and acetol) and dicarbonyl (glyoxal, methylglyoxal, diacetyl and pentan-2,3-dione) compounds associated with amino acids in the formation of Maillard-type odorous products under conditions close to those of wine, i.e. low pH, aqueous medium and low temperatures is described. Many carbonyl and α-dicarbonyl compounds were present in wine and their origins were studied in our laboratory and their contents specified. Among the amino acids present in wine, cysteine is most remarkable. In a first step, cysteine was quantified by using derivatizationHPLC-fluorimetry and the parameters of cysteine formation in wine were studied. Then, reaction products between cysteine and carbonyls, mainly heterocyclic compounds such as thiazole, 4-methylthiazole, 2,4-dimethylthiazole, 2acetylthiazole, 2-acetylthiazoline, trimethyloxazole, 2-methyl3-furanthiol, 2-furanmethanethiol and thiophene-2-thiol were identified. The descriptors are "very ripe fruit" for trimethyloxazole; "popcorn", "roasted", "peanuts" for thiazoles and "roasted coffee" or "burnt rubber" for furans and thiophenes. Some of these molecules were identified for the first time in wine. To determine the concentrations of all these compounds, quantitative assay methods were optimized using GC/MS for identification, GC/FPD for sulfur compounds, GC/NPD for nitrogenous compounds and derivatizationHPLC for carbonyl compounds. Also, attempt was made to detect these Maillard-type compounds in wines from various origins. © 2004 American Chemical Society In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

353


354 An important trust of our work is to define the role of lactic acid bacteria in wine aroma. In particular, we are interested (1) in the flavor of products synthesized by bacteria, and (2) in the formation of aroma by reactions between amino acids and carbonyl compounds later during the aging period of wine. Thus, an overview of the origin and evolution of carbonyl compounds in wine is presented. Moreover, we are interested in cysteine in wine, an amino acid present in small quantities but playing a well-known role in the reaction with carbonyl compounds. Thus, results of cysteine levels in wine are first presented, before describing a study of the products of reaction between amino acids and adicarbonyl compounds under mild conditions: ethanol/water 12%, a pH close to that of wine and temperature of 20 °C (a very low temperature in comparison to conditions in the Maillard reaction system). In this way, the heterocyclic compounds found in model solutions were quantified in wine.

Experimental Materials. Cysteine, diacetyl, pentan-2,3-dione, methylglyoxal, acetoin, acetol, butan-2,3-diol, propan-l,2-diol and compounds used for identification of final products (thiazole, 2-acetylthiazole and 2-acetyl-2-thiazoline) were purchased from Sigma Aldrich Chemical Co. (Fallavier, France) Inorganic reagents and solvents were all commercial products of analytical grade. Each cysteine / methylglyoxal mixture was prepared in stoichiometric conditions (20 mM, 10 mM, 5 mM, 0.4 mM, 0.2 mM and 0.1 mM) in an hydroalcoholic solution (12% vol.) and adjusted to pH 3.5 with IN HC1, or IN NaOH or KOH (/). The solutions were stored at 25 °C in the dark during various storage periods. Cysteine and heterocycle solutions (thiazole, 2-acetylthiazole, acetylthiazoline) were prepared at 20 mM and analyzed during a 3-week storage period. The compound 2-sulfanylethanal was synthesized by equimolar reaction (15 mM) between H S and glyoxal in ethyl acetate-like solvent. It was then derivatized by 0-(2,3,4,5,6-pentafluorobenzyle)hydroxylamine hydrochloride (PFBOA) (Aldrich) to form an oxime that was injected onto a chromatographic column coupled with different detectors (FID, NPD, FPD and MS). The carbonyl compounds were determined by direct injection or after derivatization (2). Hydroxyketones were quantified with a GC-FID system and a capillary Carbowax column (Biochrom, Vindelle, France). A l l dicarbonyl compounds were quantified in gas chromatography after derivatization. We used pentafluorobenzyl hydroxylamine hydrochloride derivatives and diaminobenzene derivatives. The latter method has proved more efficient (5). Good specificity was obtained with dicarbonyls and these could be determined without previous solvent extraction. Quinoxalines were detected at 313 nm. DAB derivatives could be injected after extraction with dichloromethane into a gas chromatograph and detected by mass spectrometry with selected ions. 2

In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

355


An HPLC method for the determination of free amino acids and especially that of cysteine in wine was reported by Pripis-Nicolau et al. (4). This technique involves the use of a pre-column derivatization with iodoacetic acid and ΟΡΑ. GC/FPD Analysis. Determination of the heterocycles (thiazole, 2acetylthyazole, 2-acetyl2-thiazoline) and N-(2-sulfanylethyl)-2-oxopropanamide (SOPA) was performed on a gas chromatograph (Agilent Tech. 6890, Massy, France) coupled with a flame photometric detector (FPD). Thiazol-2carboxaldehyde was used as an internal standard. The column was a HP 5 (30 m χ 0.53 mm χ 5 μιη) (Agilent Technologie, Massy, France). The oven temperature was kept at 50 °C for 1 min, programmed at a rate of 2 °C/min to 150 °C, then at a rate of 10 °C/min to 200 °C. Two millilieters of the headspace were injected. Light sulfurous compounds were determined by a second gas chromatograph (Agilent Tech. 5890) coupled with an FPD. Thiophene was used as an internal standard. An HP 5 column (30 m χ 0.53 mm χ 5 μπι) was used. The oven temperature was kept at 32 °C for 1 min, programmed at a rate of 10 °C/min to 100 °C, then at a rate of 20 °C/min to 180 °C, with the final step lasting 20 min. GC/NPD Analysis. After derivatization with PFBOA 2-Sulfanylethanal could be determined with an NPD under the same chromatographic conditions. GC/FID Analysis. Acetaldehyde analysis was performed by gas chromatography (Agilent Tech. 5890). The internal standard used was 4methylpentan-2-ol. The column was a capillary CP Wax 57 CB (50 m χ 0.25 mm χ 0.2 μιη) (Biochrom, Vindelle, France). The oven temperature was kept at 40°C for 5 min and programmed at a rate of 4 °C/min to 200°C; split injection at a flow of 60 ml/min. GC/MS Analysis. A first gas chromatograph (Agilent Tech. 5890) was coupled with a mass spectrometer (Agilent Tech. 5972A, electronic impact: 70 eV, eMV: 2.7 kV). A BP 21 polar column (50 m χ 0.25 mm χ 0.20 μιη) (SGE Sorl, Georges, France) was used to identify 2-sulfanylethanal. The oven temperature was programmed from 50 °C to 220 °C at a rate of 3°C/min, the initial step lasting 1 min and the final step lasting 40 min. The carrier gas was helium (1.6 mL/min). The injector was a splitless system: the splitless time was 20 s and split vent was 30 mL/min. For other heterocyclic compounds, a second gas chromatograph (Agilent Tech. 6890) was coupled with a mass spectrometer (Agilent Tech. 5972A, electronic impact: 70 eV, eMV : 2.7 kV). The column was a CP-sil 5CB (50 m χ 0.32 mm χ 0.12 μπι) (Varian Chrompack France, Les Ullis, France). The oven temperature was programmed from 50 °C to 220 °C, the initial step lasting 1 min, at a rate of 2 °C/min to 150 °C, then a rate of 5 °C/min to 220 °C and with a final step lasting 30 min. The carrier gas was


356 helium (1.7 ml/min). The injector was a splitless system. Quantitative determination of 2-acetylthiazole, 2-acetyl 2-thiazoline SOPA was done in SIM mode selecting ions of m/z =127 for 2-acetylthiazole, m/z = 129 for 2-acetyl 2thiazoline, m/z = 104 for SOPA and m/z = 113 for the internal standard (thiazole carboxaldehyde).


Results and Discussion The α-dicarbonyls in wine were glyoxal, methylglyoxal, diacetyl, and pentane-2,3-dione. Glyoxal was encountered in large quantities only in wine made from botrytized grapes (like Sauternes), while the other compounds were synthesized during alcoholic fermentation by yeast. Diacetyl and methylglyoxal were produced by bacteria in malolactic fermentation. These compounds could be reduced to hydroxyketones, i.e. acetoin, acetol and 3-hydroxypentan-2-one. In wine, hydroxyketones were then reduced to diols, i.e. butan-2,3-diol, propan1,2-diol and pentan-2,3-diol. In red wine, α-dicarbonyls were found at variable levels (concentrations in white wines were always lower). Diacetyl concentrations ranged from 1 to 4 mg/L. Methylglyoxal was between 0.04 and 0.5 mg/L after alcoholic fermentation and pentanedione between 1 and 2 mg/L during fermentation. However, infinishedwine the levels were less than 0.5 mg/L. During malolactic fermentation the levels of diacetyl and methylglyoxal increased and bacteria produced diacetyl between 3 and 8 mg/L. Diacetyl and other dicarbonyls decreased during fermentation to form hydroxyketones. Acetoin and acetol occurred at about 20 mg/L and hydroxypentanone at a maximum of about 3 mg/L. The evolution of α-dicarbonyls in wine was quite different between diacetyl and pentanedione due to the bacterial activity. Pentanedione was synthesized during alcoholic fermentation by yeast and later decreased. Its level in finished wine was low. On the other hand, diacetyl was synthesized by yeast and by bacteria and its level was higher. This is important for wine aroma whenever malolactic fermentation occurs. The dicarbonyl compounds in wine have a system of reduction. The reduction of vicinal dicarbonyls leads to a concomitant reduction in flavor intensity (Figure 1). The sensorial impact of α-dicarbonyl compounds is great. The odor of diacetyl is butter-like, while the aroma of methylglyoxal is lighter and fresher but is similar to diacetyl. Pentanedione is fatty but also has a strong toasted odor. Of all the compounds detected, only diacetyl was found in wine close to the odor threshold. The α-dicarbonyls have an incidence in wine technology, because they can combine with sulfur dioxide. In this case, they lose their aroma but the phenomenon is reversible when the S 0 disappears. After fermentation, dicarbonyls could be of interest in the aroma of aging wine so research was focused on the reactivity of these compounds with amino 2



357 acids. The first results showed that dicarbonyl could react with amino acids in the conditions of wine and could produce very interesting aromas, e.g. not only with valine, leucine, isoleucine and phenylalanine but also with methionine and cysteine. Because cysteine is an amino acid which has not been widely studied in wine, we have recently developed an HPLC method allowing the determination of all amino acids in wine (except proline) and their evolution during fermentations. Cysteine increased during alcoholic fermentation like ornithine, while other amino acids were consumed by yeast by about 90%. Cysteine levels in wine were low with a mean between 1 and 2 mg/L. For model solutions between dicarbonyls and cysteine or other amino acids, we prepared 20 mmoles of each one and analyzed the aroma by tasting or by GC/sniffing and volatile compounds, by GC/MS and also with various detectors (FID or specific FPD and NPD). Many odorous heterocycles like thiazole, acetylthiazole, alkyloxazole, thiophene and furane were detected (Figure 2). These compounds are well known products of the Maillard reaction between sugars and amino acids. This non-enzymatic reaction produces a number of aromatic compounds like HMF and furfural. It also produces not only small reactive molecules like hydrogen sulfide, ammonia and acetaldehyde by Strecker degradation of amino acids in the presence of dicarbonyl compounds, but also specific aldehydes and heterocyclic compounds. However, the reaction in wine is very different, since wine is not heated and the temperature is between 10 and 35 °C. Normally the wine is dry but we have seen wines which contain some a-dicarbonyls. Under the conditions of pH and temperature experienced in wine, many products, including specific aldehydes were found with amino acids such as valine, leucine, isoleucine, phenylalanine and methionine (7). With cysteine, no specific labile aldehydes were found, but many heterocycles were present. In addition, H S, methanethiol, carbon disulfide, alkylthiazole, alkylpyrazine, alkyloxazole; 2-acetyl-2-thiazoline and 2-acetylthiazole were detected. In these heterocyclic compounds, the influence of pH and temperature on the degradation of cysteine and the formation of 2-acetylthiazole was investigated. Cysteine degradation was observed with three carbonyl compounds, namely acetoin, diacetyl and methylglyoxal. The decomposition of cysteine was faster at pH 8.0 than at pH 3.5 and was particularly slow in the presence of acetoin. With regard to the reaction products, higher amounts of acetylthiazole and 2-acetyl-2-thiazoline were formed at the lower pH. At pH 3.5 the solution released popcorn, roasted hazelnuts, while at pH 8, we detected rotten egg, cabbage and roasted meat notes. Therefore, an acid pH close to that of wine is more favorable for the formation of acetylthiazole and acetylthiazoline. No large differences were observed in terms of olfactory nuance between 10 and 40 °C, nor in terms of the compounds identified in the solution. 2



Diacetyl

Acetoin


Butan-2,3diol


Methylglyoxal

Propan-1,2-diol

Figure 1. Redox system of the different dicarbonyl and related compounds in wine.

Acetol


so

in


Figure 2. Heterocyclic compounds identified in cysteine/dicarbonyl solutions.

Figure 3: Heterocyle evolution in cysteine / methylglyoxal solution (0.1 mM).


In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003. N

SH

Acetylthiazole

2-Furanmethanethiol

4-Methylthiazole

"

Thiophene-2-thiol

%"NH

Acetylthiazoline

Figure 3. Heterocyle evolution in cysteine / methylglyoxal solution (0.1 mM).

Trimethyloxazole

Thiazole

Q

//


On


1 33

2 43

0 0

0 0

Champagne (7 samples) Average (Mg/L) 5 Frequency (%) 86

Wines for Champagne (3 samples) Average (Mg/L) 3 Frequency (%) 67

Languedoc red wines (7 samples) Average (Mg/L) 4 Frequency (%) 100

Average (Mg/L) Frequency (%)

0 0

4-methiazole

(6 samples) 5 67

Médoc

St Emilion (6 samples) Average (Mg/L) 4 Frequency (%) 100

thiazole

Table I. Thiazoles in wine.

1 29

3 33

4 71

4 83

1 17

2,4-dimethiazole

0 29

1 33

1 71

2 100

1 67

2-acthiazole


1 29

6 67

6 57

5 100

3 50

2-acthiazoline


363 During the degradation of cysteine at 2 mmole and at 10 °C, cysteine was found to be degraded; at the same time, 2-acetyl-2-thiazoline increased with a maximum after 3 h, while 2-acetylthiazole increased regularly. Acetylthiazoline presented a characteristic curve of an intermediate reaction (/). This molecule may be oxidized in acetylthiazole. A mechanism was proposed by Griffith and Hammond (5), where 2-acetylthiazoline served the intermediate during the formation of acetylthiazole from cysteine and methylglyoxal. The reaction was conducted at low temperature but at pH 8. Moreover, thiazole and acetylthiazole were found in the solution of methylglyoxal and cysteine after 5 min, but no acetylthiazoline if cysteine and methylglyoxal were diluted to 0.4 mM ((5). In this case acetylthiazoline appeared only after 1414 h (Figure 3). In our lab, we have determined an unknown compound which is present during the formation of acetylthiazole from 2-acetylthiazoline, and this compound has also been determined in the solutions of cysteine and methylglyoxal which was termed N-(2-sulfanylethyl)-2-oxopropanamide or SOPA (7,5). It is now necessary to confirm that under mild conditions how thiazole, acetylthiazole and acetylthiazoline are synthesized and the role of the Schiff base or SOPA in these formations. The initial results on heterocycles found in wine are shown in Table I; thiazole, 4-methylthiazole, 2,4-dimethylthiazole, 2-acetylthiazole and 2-acetyl2-thiazoline have now been determined. All these heterocycles are found in wine, except for some wines in which 4-methylthiazole and 2-acetylthiazole are present. Champagne and Medoc wines have been found to have the highest concentrations. Only 2-acetyl-2-thiazoline has levels close to its aroma threshold.

Acknowledgment Students in the laboratory: Benoît Touquette, Jacques Hitier, Harald Keim, Laura Pripis-Nicolau, Jean Christophe Barbe. We thank the "Comité Interprofessionnel des Vins de Bordeaux" for theirfinancialsupport.

References 1.

Pripis-Nicolau, L.; de Revel, G.; Bertrand, A. J. Agric. Food Chem. 2000, 48, 3761-3766. 2. de Revel, G.; Bertrand, A. In Trends in Flavour research, Proc of the 7 Weurman Flavour Research Symposium, 15-18 June 1993 ; Maarse H., van der Heij D.G., Eds.; Elsevier : Zeist, The Netherlands, 1994; pp 353-361. 3. de Revel, G.; Pripis-Nicolau, L.; Barbe J.C.; Bertrand A . J. Sci. Food Agric 2000, 80, 102-108. th


364 4. 5. 6.

7.


8.

Pripis-Nicolau, L.; de Revel, G.; Marchand, S.; Anocibar-Beloqui, Α.; Bertrand, A J. Sci. Food Agric. 2001, 81, 731-738. Griffith, R.; Hammond, G. J. Dairy Sci. 1988, 72, 604-613. Hitier, J.; de Revel, G.; Marchand, S.; Bertrand, A . Formation of 2acetylthiazole from methylglyoxal and cysteine in mild conditions, in preparation. Marchand, S.; de Revel, G.; Bertrand, A . J. Agric. Food Chem. 2000, 48, 4890-4895. Merchand, S.; de Revel, G.; Vercauteren, J.; Bertrand, A. J. Agric. Food Chem. 2002, 50, 6160-6164.


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