Influence of Melanoidins on the Aroma Staling of Coffee Beverage

Chapter 15

Downloaded by PENNSYLVANIA STATE UNIV on July 26, 2012 | http://pubs.acs.org Publication Date: December 1, 2003 | doi: 10.1021/bk-2004-0871.ch015

Influence of Melanoidins on the Aroma Staling of Coffee Beverage T. Hofmann and P . Schieberle Deutsche Forschungsanstalt für Lebensmittelchemie, Lichtenbergstrasse 4, D-85748 Garching, Germany

Sensory analysis on aqueous biomimetic coffee aroma recombinates in the absence or presence of coffee melanoidins revealed that, in particular, the intensity of the roasty-sulfury aroma quality was reduced when melanoidins were present. Comparative aroma dilution analysis on the headspaces of aqueous solutions containing the total coffee volatiles, alone, or in mixture with melanoidins revealed that the losses of the odor-active thiols 2-furfurylthiol (FFT), 3-methyl-2-butenthiol, 3-mercapto-3methylbutyl formate, 2-methyl-3-furanthiol, and methane thiol are responsible for the aroma change. Quantification by means of stable isotope dilution assays confirmed the rapid loss of these thiols during warm-keeping of the coffee brew. Using synthetic [ H ]-FFT as an example, H NMR and LC/MS experiments gave strong evidence that thiols are covalently bound to the coffee melanoidins via Maillard-derived pyrazinium compounds formed by oxidation of 1,4-bis-(5-amino-5-carboxy-1-pentyl) pyrazinium radical cations (CROSSPY), which were recently identified as key intermediates in roasting-induced coffee melanoidin genesis. Using synthetic 1,4-diethyl diquaternary pyrazinium ions or N acetyl-L-lysine/glycolaldehyde and FFT, it was shown that 2-(2furyl)methylthio-1,4-dihydro-pyrazines, bis[2-(2-furyl)methylthio]-1,4-dihydro-pyrazines and 2-(2-furyl)methylthio-hydroxy1,4-dihydro-pyrazines were formed as the primary reaction products. On the basis of these results it can be concluded that the CROSSPY-derived pyrazinium intermediates are involved in the rapid covalent binding of thiols to melanoidins, and, consequently, contribute to the decrease in the sulfury-roasty odor note observed after preparation of the coffee brew. 2

2

2

α

200

© 2004 American Chemical Society In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.


201 The stimulatory, pleasant overall aroma is one of the most important attributes determining the consumer acceptance of a freshly prepared coffee brew. Unfortunately, this desirable aroma is not stable and rapidly changes shortly after preparation of the coffee brew. While numerous studies have addressed the aroma changes during storage of ground coffee powder (e.g. 1,2\ the chemistry responsible for this rapid aroma staling of coffee beverages is as of yet not understood. Recent investigations, combining instrumental analysis with human olfactory perception (e.g. GC/olfactometry), have revealed a rapid decrease in the concentrations of some odor-active thiols when coffee brews were manufactured at elevated temperatures. This is particulary the case in the manufacturing of instant coffee (3) as well as during heat sterilization of canned coffee drinks (4). Based on model experiments with odor-active disulfides and egg albumin, the decrease of thiols and disulfides in foods was recently suggested to be the result of an interchange with sulfhydryl and disulfide groups of proteins (5). It is, however, as yet not clear whether the aroma changes in coffee brews are due to similar reactions involving cysteine residues of the macromolecular coffee pigments, the so-called melanoidins, which are present in concentrations of more than 200 mg per cup of beverage. The purpose of this investigation was, therefore, (i) to characterize the key coffee odorants affected by melanoidins, and (ii) to elucidate the chemical mechanisms involved in the aroma change occurring during warm-keeping of coffee beverages.

Experimental Materials A biomimetic recombinate of coffee brew aroma was prepared using 25 key odorants in their "natural" concentrations ( 3000 Da) after freeze-drying, or separated by gel permeation chromatography on Sephadex G-25 fine (75 χ 5 cm i.d.; Pharmacia, Uppsala, Sweden) using water (4 mL/min) as the eluent (9). In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

202 Static headspace gas chromatography/olfactometry The aqueous aroma destillate (10 mL) alone, or mixed with coffee melanoidins (125 mg), respectively, was equilibrated in a septum-sealed vessel (240 mL) for 30 min at 40°C. Stepwise decreased headspace volumes (25 to 0.2 mL) were then analysed by headspace-HRGC/oIfactometry as recently reported (9)


Determination of headspace concentrations of thiols A solution of 3-methyl-2-buten-l -thiol (400 μg), 2-furfurylthiol (600 μg) and 3-mercapto-3-methylbutylformate (500 μg) dissolved in phosphate buffer (10 mL; 0.1 mmol/L; pH 6.0) alone, or mixed with melanoidins (125 mg), respectively, was equilibrated in a closed vessel (240 mL) at 30°C. The amounts of thiols in the headspace before and after melanoidin addition were determined 09. Quantification of thiols and disulfides by stable isotope dilution analysis 2-Furfurylthiol, 3-mercato-3-methylbutyl formate and bis(2-furfuryi) disulfide were quantified by means of stable isotope dilution analysis in coffee brews either freshly prepared, or kept warm in a thermo flask, and in binary mixtures containing thiols and melanoidins, respectively (6). Spectroscopic measurements GC/MS was performed using a CP 9001 gas chromatograph (Chrompack, Frankfurt, Germany) equipped with a fused silica capillary CP-WAX 52 CB (25m χ 0.32 mm, 1.2 μπι film thickness, Chrompack) and coupled with the mass spectrometer Incos X L (Finnigan, Bremen, Germany). LC/MS was performed with a LCQ-MS (Finnigan MAT GmbH, Bremen, Germany) using electrospray ionization (ESI). Electron paramagnetic resonance (EPR) and H NMR spectra were recorded on an ESP 300 and an AMX 500 spectrometer (Bruker, Rheinstetten, Germany). 2

Table I. Influence of warm-keeping on aroma quality of a coffee brew 0

Aroma quality sweet/caramel earthy sulfury/roasty smoky

Omin 1.6 1.9 2.3 2.0

Intensity after 60 min 2.1 1.8 1.2 2.3

210 min 2.5 1.9 0.4 2.3

a

The intensities of the given odour qualities were scored on a scalefrom0 (not detectable) to 3 (strong).

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

203


Results and Discussion In order to investigate the aroma change of a coffee beverage during warmkeeping, sensory analyses were performed on coffee brews freshly prepared or stored in thermosflasksfor 60 or 210 min, respectively (Table I). A drastic decrease of the intensity of the sulfury-roasty odor quality was observed with increasing storage time, e.g. on a scale from 0 (not detectable) to 3 (strong) the intensity dropped from 2.3 to 0.4 during storing of the brew for 210 min (Table I). In contrast, the intensity of the sweet/caramel-like and the smoky note only increased to some extent, and the earthy aroma quality did not change significantly. To study whether interactions between the coffee odorants and the melanoidins might be responsible for the aroma change observed, an aqueous aroma recombinate mimicking the overall aroma of an authentic fresh coffee brew was prepared using 25 coffee odorants in their "natural" concentrations (9). One aliquot of this recombinate was sensorially evaluated in comparison to an original coffee brew without any further additions (Table II), and the second aliquot was mixed with "natural" amounts of melanoidins (MW>3000 Da), which had been isolatedfromcoffee brew by means of ultrafiltration. In comparison to the aroma profile of afreshcoffee brew and the aroma recombinate, addition of melanoidins reduced, in particular, the intensity of the sulfury-roasty odor quality after an equilibration time of 30 min at 40°C (Table II). These data clearly demonstrate that the melanoidins are somehow involved in degradation or binding of the sulfury/roasty smelling key coffee odorants (9). Table II. Influence of coffee melanoidins (CM) on the overall aroma of a biomimetic coffee aroma recombinate (CAR) Aroma quality sweet/caramel earthy sulfury/roasty smoky

coffee brew 1.6 1.9 2.3 2.0

Intensity in CAW 2.1 1.7 2.1 1.4

CAR+CAf 1.9 1.9 1.2 1.6

a

The aroma profile of an biomimetic coffee aroma recombinate (CAR; 10 mL) was analysed in the absence or presence of coffee melanoidins (CM; 125 mg; MW>3000 Da) after storing for 30 min at 40°C. To systematically elucidate the roasty-sulfury odorants affected by the coffee melanoidins, the total volatiles and the melanoidins (MW>3000 Da), respectively, were separately isolated from a fresh coffee brew prior to the analytical experiments, and then recombined. In a first experiment, by application of the comparative aroma dilution analysis, the odor-active compounds in a stored model solution (A in Table III) only containing the coffee brew volatiles were compared to those of a second model (B in Table III) containing both, the volatile fraction and the melanoidins in their "natural" concentrations (10). The results revealed 16 odorants in the headspace of model A after incubation for 30 min with relative flavor dilution (rFD) factors of 2 to 256 (A in Table III). After



204 incubation with melanoidins (B in Table III), only 15 odorants were detectable, and, in addition, the rFD factors of all thiols were drastically decreased when melanoidins were present (10). The most pronounced effects were measured for 2-furfurylthiol (FFT), 3-methyl-2-buten-l-thiol (MBT), and 3-mercapto-3methylbutyl formate (MMBF), the rFD factors of which were decreased by factors of 16, 8 or 4, respectively (Table III). Also 2-methyl-3-furanthiol (MFT) was significantly decreased, and methane thiol could not be detected at all. In contrast, the aroma impacts of odor-active 2,3-diones, phenols or pyrazines, were not significantly affected upon addition of melanoidins (Table III). To further confirm this decrease in thiol concentration, the amounts of FFT and MMBF were quantified in coffee brews kept warm in a thermos flask for 0, 30, 60, 90 and 210 min (Figure 1). As given in Figure 1, the freshly prepared coffee beverage contained about 16.0 or 8.2 μg of FFT or MMBF, respectively. Warmkeeping of the brew then led to a drastic decrease in the concentrations of both thiols (JO). After 60 min the FFT concentration decreased by a factor of more than four compared to the fresh coffee brew. Extending the storage time to 210 min finally resulted in a complete loss of FFT, and only small amounts of MMBF were still detectable (Figure 1). These data, being well in line with the results of the comparative aroma dilution analysis (cf. Table III), clearly demonstrate that the decrease of the sulfury-roasty odor quality observed during storage of coffee brew is mainly due to the loss of odor-active thiols. Table HI. Comparative aroma dilution analysis of the headspace of aroma distillates incubated in the absence (A) or presence (b) of melanoidins 11

Odorant

rFDfactor

Aroma quality A

Β

128 256 Buttery butane-2,3-dione 128 128 Buttery pentane-2,3-dione 64 64 Malty 3-methylbutanal 32 64 2-methylbutanal Malty 32 32 Acetaldehyde Fruity 32 16 potato-like Methional 32 2 roasty, sulfury 2-furfurylthiol 32 32 Earthy 2-ethyl-3,5-dimethylpyrazine 32 32 2,3-diethyl-5-methylpyrazine Earthy 32 Phenolic Guaiacol 16 32 16 cabbage-like dimethyl trisulfide 16 16 green, earthy 2-isobutyl-3-methoxypyrazine 8 1 foxy, skunky 3-methyl-2-butenthiol 8 2 3-mercapto-3-methylbutyl formate Catty 4 2 meat-like 2-methyl-3-furanthiol 3 kDa) were stored for 30 min at 40°C. a


205


20

time (min) Figure 1. Influence of storage time on the concentrations of 2-furfurylthiol (FFT) and 3-mercapto-3-methylbutylformate (MMBF) in a coffee brew maintained at 80°C in a thermos flask. In order to study the mechanisms of the thiol/melanoidin interaction, aqueous solutions of FFT, MMBF, and MBT were incubated either in the absence, or in the presence of coffee melanoidins for 30 min at 30°C (9). The decrease in thiol concentration was determined by means of headspace/HRGC by comparing the control (without melanoidins) to the sample with added melanoidins (Figure 2).

Figure 2. Influence of storage time on the headspace concentrations of thiols in aqueous solutions of 2-furfurylthiol (FFT), 3-mercapto-3-methylbutyl formate (MMBF), 3-methyl-2-buten-l-thiol (MBT), and coffee melanoidins (MW>3000 Da) maintained at 30°C. In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

206 The results revealed that the amounts of each of the three thiols were strongly reduced in the presence of coffee melanoidins and showed that the decrease in concentration proceeds very rapidly (Figure 2), e.g. 50 % of FFT was lost after 20 min. After 30 min, the FFT was nearly absent in the headspace (P). In order to reveal whether these thiols are chemically degraded, or covalently bound to melanoidins, coffee melanoidins were incubated with [ H ]-2furfurylthiol ([ H ]-FFT) for 90 min at 30°C, then freed again from lowmolecular weight compounds by ultrafiltration, and,finally,analysed by H-NMR spectroscopy (10). As controls, aqueous solutions of coffee melanoidins and [ H ]-FFT, respectively, were analysed. As displayed in the H-NMR spectrum in Figure 3A, a solution of [ H ]-FFT in H 0 showed two resonance signals, one at 3.67 ppm corresponding to the deuterated methylene group in the odorant, and another at 4.70 ppm corresponding to the natural H-abundance in tap water. H NMR of the melanoidins isolated from coffee (Figure 3B) did not show any signals besides the natural H-abundance of the solvent (10). Coffee melanoidins, however, which had been pre-incubated with [ H ]-FFT, showed additional resonance at 3.0-4.2 ppm with a strong line broadening (Figure 3C) as typically found for compounds covalently linked to macromolecules (10). These data clearly confirmed the idea that the odor-active thiols are bound to the coffee melanoidins. 2

2

2

2

2

2

2

2


2

2

2

2

2

2

2

2

-JO (A)

8 £ (Ω

melanoidin (Β)

(Q

S

9 8 7 6 5 4 3 2 1 (ppm)

9 8 7 6 5 4 3 2 1 (ppm)

2

9 8 7 6 5 4 3 2 1 (ppm) 2

Figure 3. H-NMR spectra (500 MHz, H 0) of (A) [ HJ-FFT (1 mg/mL), (B) coffee melanoidins (100 mg/mL), (C) coffee melanoidins (100 mg/mL) after pre incubation (90 min, 30°C) with fH ]-FFT(2 mg) and purification. 2

2

Because recent studies had clearly shown that the addition of the reducing agent dithioerythrytol was not able to regenerate major amounts of the "free" thiol from coffee melanoidins, which had been preincubated with FFT (P), it can be speculated that the thiols do not bind via disulfide bonds of, e.g. cysteinyl residues, to the coffee melanoidins. Obviously, the thiols preferably react with other reactive sites present in the macromolecules. Model studies on potential binding sites To investigate the role of chlorogenic acid moieties present in coffee melanoidins, an aqueous solution of FFT was incubated for 30 min at 30°C either in the presence of free chlorogenic acid, or in the presence of chlorogenic acid, which had been thermally pretreated for 5 min at 230°C to simulate roasting conditions. After incubation with FFT the amounts of the thiol left were analyzed.



207 Neither untreated (A in Figure 4), nor pre-heated chlorogenic acid (B in Figure 4) showed strong binding activity, since the losses in both models were below 20 per cent (10). To study the role of Maillard-derived reaction products in thiol binding, a dry-heated protein/glucose mixture was stored together with FFT. The results showed that the thiol concentration was decreased by a factor of nearly two (C in Figure 4), thus demonstrating that Maillard-derived reaction products might play a role in thiol binding (10). Another experiment using a thermally processed mixture of protein and glycolaldehyde, a carbohydrate cleavage product present in roasted coffee in high amounts, led to a more pronounced effect, because the FFT concentration was reduced to below 30 % (D; Figure 4). Because the εamino groups of protein-bound lysine are known as primary targets in Maillard reactions during coffee roasting, the protein was substituted by N -acetyl-Llysine. The dark brown material formed upon roasting was most effective in thiol binding, because only 17 % of the "free" FFT were left in the headspace of the model reaction mixture (E; Figure 4). a

w/o

A

B

C

D

Ε

Figure 4. Relative amounts ofFFTpresent in the headspaces of aqueous FFT solutions stored in the presence of (w/o) no additives, (A) chlorogenic acid (20 S)f ( ) pre-heated chlorogenic acid (20 mg; 5 min at 230°C), (C) heated albumine/glucose (10 mg each; 5 min at 230°C), (D) glycolaldehyde/albumine (10 mg each; 5 min at 230°C), (E) glycolaldehyde/N -acetyl-L-lysine (10 mg each; 5 min at 230°C). m

B

a

It is well documented in the literature thatfreeradicals are present in roasted coffee (77-77), and that l,4-bis-(5-amino-5-carboxy-l-pentyl)pyrazinium radical cations (CROSSPY), which had been recently shown to be formed from proteinbound lysine side chains and glycolaldehyde, contribute to melanoidin genesis during coffee roasting (16,17). In order to gain more detailed insights into the role of radicals as potential thiol binding sites, coffee melanoidins were separated into fourfractionsby means of gel permeation chromatography (Figure 5, left), and the fractions obtained were investigated for their thiol binding as well as for their free radical activities (Figure 5, right). Incubation offractionsI to IV in the presence of FFT resulted in a complete loss of FFT after 30 min at 30°C (IV; Figure 5). Fraction I and II showed somewhat lower activities, whereas fraction III was least effective in FFT binding (10). Analysis of thefractionsby means of In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

208 EPR spectroscopy revealed that the radical activities run in parallel with the thiol binding activity, e.g. fraction IV showed the most pronounced effect in thiol binding and exhibited the highest radical activity, whereas fraction III had the lowest potential in thiol binding and the lowest radical activity (Figure 5, right). t [hi

H FFT degraded ED rel. radical activity

Sephadex G-25 fine

Percent of FFT degraded Downloaded by PENNSYLVANIA STATE UNIV on July 26, 2012 | http://pubs.acs.org Publication Date: December 1, 2003 | doi: 10.1021/bk-2004-0871.ch015

0

20

i

40 60

ι

80 100

ι

III

MM

0 absorbanceat λ=405ηπι

10

20 30

40 50

rel. radical activity

Figure 5. Separation of coffee melanoidins by gel permeation chromatography (GPC; left). Free radical activity in GPCfraction I to IV, and amount of FFT degraded in an aqueous solution ofFFT without (w/o), or in the presence of GPC fractions I to IV after 30 min at 30°C (right). The role of the CROSSPY radical in thiol binding 1,4-Bis-(5-amino-5-carboxy-1 -pentyl)pyrazinium radical cations, named CROSSPY (I in Figure 6), were recently identified in melanoidins isolated from coffee brew (16,17). These radical cations, found to participate in a redox cycle of reaction intermediates, are oxidized into diquaternary pyrazinium ions (II in Figure 6), which subsequently form 2-hydroxy-l,4-dihydropyrazines (III in Figure 6) upon hydratization, and regenerate the CROSSPY radicals upon a redox reaction with III (16,17). Due to their strong browning activity, the bis- and mono-hydroxylated 1,4-dihydropyrazines (IV and III in Figure 6) were recently proposed as penultimate monomers involved in melanoidin genesis (77), e.g. by oligomerization reactions via the dimer V (Figure 6). This redox cycle can be modelled with 1,4-diethyl pyrazinium diquaternary ions (Diquat) as a suitable template to mimic the reactions of lysine-bound pyrazinium derivatives (17). Characterization of the reaction products formed upon dissolving Diquat in water by LC/MS spectroscopy (Figure 7) revealed that all reaction intermediates proposed in Figure 6 are generated, namely the CROSSPY-type radical cation (m/z 138), the 2-hydroxy-1,4-diethyl-1,4dihydropyrazine (m/z = 155), the dihydroxy-l,4-di ethyl-1,4-dihydropyrazine (m/z = 171), and the bis-hydroxy dimer (m/z = 309). In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.


Figure 6. Proposed pathway of melanoidin genesis via CROSSPY (I) and diquaternary pyrazinium ions (II) as the key intermediates (R=protein-bound lysine side chain).

r r

( Ν" Y O H

138

100

155

s

5 0

109 HO

0

n^HO Γ

H.

(

/

309

171

α ϊ oo

150

200

250

300

350

400

450

500

m/z Figure 7. LC/MS spectrum of a solution of 1,4-diethylpyrazine diquaternary ions (Diquat) in water. In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

210 To study possible reactions between odor-active thiols and these CROSSPYassociated intermediates, an aqueous solution of Diquat was incubated in the presence of FFT at 30°C. Quantitation of the concentrations of FFT revealed a rapid decrease induced either by the addition of the Diquat solution, or the coffee melanoidins (Figure 8). Both, the Diquat-derived intermediates as well as the coffee melanoidins showed similar kinetics of FFT degradation. Although about 400 or 330 μg FFT, respectively, were bound to the coffee melanoidins or the Diquat-derived intermediates, respectively, less than 6 μg of the corresponding bis(2-furfuryl) disulfide (FFT-S ) were generated (10). These results clearly demonstrate that neither the Diquat solution, nor the coffee melanoidins have the potential to oxidize the thiol into the corresponding disulfide. Downloaded by PENNSYLVANIA STATE UNIV on July 26, 2012 | http://pubs.acs.org Publication Date: December 1, 2003 | doi: 10.1021/bk-2004-0871.ch015

2

time (min) Figure 8. Influence ofreaction time on the concentrations of2-furfurylthiol (FFT) and disulfide (FFT-S2) in the presence of melanoidins or Diquat-derived reaction intermediates, respectively. To elucidate the chemical mechanism of thiol binding, an aqueous solution of Diquat was incubated with equimolar amounts of FFT for 10 min at 30°C, and was analysed by LC/MS (Figure 9A). The mass spectrum exhibited a molecular ion at m/z = 251 (100%), which on the basis of its LC-MS spectrum (data not given) was proposed as the 2-(2-furyl)methylthio-l,4-dihydropyrazine (A in Figure 9). In addition, LC-MS gave evidence that the ions at m/z=363 and 267 correspond to bis[2-(2-furyl)methylthio]-l,4-dihydropyrazine and 2-(2-furyl) methylthio-hydroxy-l,4-dihydropyrazine, respectively (A in Figure 9). To further confirm these structures, the experiment was repeated with [ H ]-labelled FFT (B in Figure 9). Comparing the LC/MS spectra (B in Figure 9) with those measured in the non-labelled experiment (A in Figure 9) revealed an isotopic shift of the ions at m/z 251 to m/z 253, thus confirming the incorporation of two deuterium atomsfromthe methylene group of one molecule of FFT as given in the structure proposed for 2-(2-furyl)methylthio-l,4-dihydropyrazine (10). In addition, an isotopic shift of two and four units were observed for the ions at m/z 267 to 269 and m/z 363 to 367, respectively, thus verifying the structures, outlined in Figure 2

2

2

2


211

r


f •

(A)

•f

50-1

100

I 150

443 477 I

200

300

350

11

400

450

500

m/z

100 —r

(Β)

Ι

-

/ 100

150

250

200

I 300

350

400

450

500

m/z

Figure 9. LC/MS spectra of aqueous solutions of Diquat and (A) FFT, and (B) H2-FFT, respectively. 2

9, as 2-(2-furyl) methylthio-hydroxy-l,4-dihydropyrazine and bis[2-(2-furyl) methylthio]-1,4-dihydropyrazine (10). To get more closer to the food, in an additional experiment, the CROSSPY radical was generated in vitro from N -acetyl-L-lysine and glycolaldehyde prior to the addition of FFT. The aqueous solution containing CROSSPY and its corresponding pyrazinium-type redox partners (Figure 6) was incubated in the presence of FFT for 30 min at 30°C, and then analyzed by LC/MS spectroscopy a


212 (10). The mass spectrum obtained showed a quasi molecular ion at m/z 537 being well in line with the structure of 2-(2-furyl)methylthio-l,4-bis-(5-acetamino-5carboxy-1 -pentyl)-1,4-dihydropyrazine, the structure of which is given in Figure 10. Fragmentation of that ion gave the MS spectrum (ESI) displayed in Figure 10. Loss of 113 or 82, respectively, leads to the base ion at m/z 424 or 455, most likely corresponding to the cleavage of the 2-furfurylthio or the 2-furylmethyl group, respectively. The data clearly corroborated the results obtained for the Diquat model experiments and gave strong evidance that odor-active thiols are covalently linked to pyrazinium moieties in coffee melanodins. Downloaded by PENNSYLVANIA STATE UNIV on July 26, 2012 | http://pubs.acs.org Publication Date: December 1, 2003 | doi: 10.1021/bk-2004-0871.ch015

2

V

I ι

1

ι

1

1

ι

1

I i

1

519

i i

1

200 250 300 350 400 450 500 550 600 m/z Figure 10. LC/M§ spectrum obtainedfor m/z 537 in a thermally pre-treated N acetyl-L4ysine/glycolaldehyde mixture after incubation (30 min/30°C) with FFT. a

Taking all these data into account, the reaction pathways, displayed in Figure 11, were proposed for the binding of thiols to CROSSPY-associated reaction intermediates (10). Oxidation of CROSSPY (I) leads to diquaternary pyrazinium ions (II), which, in the absence of thiols, react with water to form the 2-hydroxy1,4-dihydropyrazine III (7,16,17), or, in the presence of thiols such as, e.g. the 2furfurylthiol, giveriseto 2-(2-furyl)methylthio-l,4-dihydropyrazine (IV in Figure 11). Involving the diquaternary ions these intermediates participate in Redox reactions, thus pumping additional molecules of 2-furfurylthiol into the melanoidin via 2-(2-furyl)methylthio-hydroxy-l,4-dihydropyrazine (V) and bis[2(2-furyl) methylthio]-l,4-dihydropyrazine (VI), respectively (10).


213 I protein!


I protein

I protein I

1 protein 1

protein I

HjQ

I protein"! Ill

Γι *1

Κ* n«

Λ

1 protein 1 II

1 protein 1

\U ν

Λ

1 protein 1 IV

Figure 11. Reaction pathways proposedfor the covalent binding of 2-furfurylthiol to CROSSPY-associated reaction intermediates in melanoidins. Thiol binding and radical activity in various food melanoidins In order to investigate whether odor-active thiols bind exclusively to coffee melanoidins, or to melanoidins in browned foods in general, aqueous solutions of FFT were incubated for 30 min at 30°C in the presence of melanoidins isolated from bread crust, dark beer, roasted coffee, and dark malt, and the headspace concentrations of the "free" thiol were determined. As given in Figure 12, dark malt showed nearly identical thiol binding activity as roasted coffee, e.g. less than 10% of FFT were recovered only. In comparison, the melanoidins isolated from bread crust as well as dark beer showed somewhat lower reactivity towards thiol compounds, because about 55 to 60% of "free" thiol were determined after incubation. Analysis of these food melanoidins by means of EPR spectroscopy revealed that the radical activities run in parallel with the thiol binding activity, e.g. malt and coffee melanoidins showed the most pronounced effect in thiol binding and exhibited by far the highest radical activity, whereas bread crust and dark beer had the lowest potential in thiol binding and the lowest radical activity. These data again indicate the tight relationship between thiol binding and free


214


radical activity of melanoidins, and clearly demonstrate that the binding of odorous thiols to melanoidins is not restricted to roasted coffee only, but is of general importance for the aroma of browned foods such as, e.g. cereals and beer.

w/o

bread crust dark beer

coffee

dark malt

Figure 12. Amount of "free " FFT in the headspace of aqueous solutions ofFFT incubatedfor 30 min at 30°C without (w/o), or in the presence of melanoidins isolatedfrom bread crust, dark beer, coffee and dark malt.

Conclusions On the basis of these results it might be concluded that CROSSPY-derived pyrazinium intermediates are involved in the rapid covalent binding of odorous thiols to coffee melanoidins, and are responsible for the decrease in the sulfuryroasty odor quality detected shortly after preparation of the coffee brew. Studies on how the activity of these binding sites might be influenced, e.g. by blocking the active binding sites, are ongoing and will help to elucidate novel possibilities how to stabilize the aroma quality and how to increase the aroma shelf-life of coffee-beverages.

References 1. 2. 3. 4. 5.

Holscher, W.; Steinhart, H. Z. Lebensm. Unters.-Forsch. 1992, 192, 33-38. Grosch, W.; Semmelroch, P.; Masanetz, C. In Proceedings of the 15 ASIC Colloquium, Montpellier, ASIC, Paris, France, 1994, pp 545-549. Semmelroch, P.; Grosch, W. Lebensm. Wiss. Technol. 1995, 28, 310-313. Kumazawa, K.; Masuda, H.; Nishimura, O.; Hiraishi, S. Nippon Shokuhin Kagaku Kaishi, 1998, 45, 108-113. Mottram, D.S.; Szauman-Szumski, C.; Dodson, A. J. Agric. Food Chem. 1996, 44, 2349-2351. th


215 6. 7. 8.


9. 10. 11. 12. 13. 14. 15. 16. 17.

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