Generation of thiols by biotransformation of cysteine-aldehyde conjugates with baker's yeast

Article abstract of DOI:10.1021/jf026198j

Baker's yeast was shown to catalyze the transformation of cysteine-furfural conjugate into 2-furfurylthiol. The biotransformation's yield and kinetics were influenced by the reaction parameters such as pH, incubation mode (aerobic and anaerobic), and substrate concentration. 2-Furfurylthiol was obtained in an optimal 37% yield when cysteine-furfural conjugate at a 20 mM concentration was anaerobically incubated with whole cell baker's yeast at pH 8.0 and 30 °C. Similarly to 2-furfurylthiol, 5-methyl-2-furfurylthiol (11%), benzylthiol (8%), 2-thiophenemethanethiol (22%), 3-methyl-2-thiophenemethanethiol (3%), and 2-pyrrolemethanethiol (6%) were obtained from the corresponding cysteine-aldehyde conjugates by incubation with baker's yeast. This work indicates the versatile bioconversion capacity of baker's yeast for the generation of thiols from cysteine-aldehyde conjugates. Thanks to its food-grade character, baker's yeast provides a biochemical tool to produce thiols, which can be used as flavorings in foods and beverages.

Full text of DOI:10.1021/jf026198j

J. Agric. Food Chem. 2003, 51, 3629−3635 3629
Generation of Thiols by Biotransformation of
Cysteine−Aldehyde Conjugates with Baker’s Yeast
TUONG HUYNH-BA, WALTER MATTHEY-DORET, LAURENT B. FAY, AND
RACHID BEL RHLID*
Nestec Ltd., Nestle´ Research Center, Vers-chez-les-Blanc, P.O. Box 44,
1000 Lausanne 26, Switzerland
Baker’s yeast was shown to catalyze the transformation of cysteine-furfural conjugate into
2-furfurylthiol. The biotransformation’s yield and kinetics were influenced by the reaction parameters
such as pH, incubation mode (aerobic and anaerobic), and substrate concentration. 2-Furfurylthiol
was obtained in an optimal 37% yield when cysteine-furfural conjugate at a 20 mM concentration
was anaerobically incubated with whole cell baker’s yeast at pH 8.0 and 30 °C. Similarly to
2-furfurylthiol, 5-methyl-2-furfurylthiol (11%), benzylthiol (8%), 2-thiophenemethanethiol (22%), 3-meth-
yl-2-thiophenemethanethiol (3%), and 2-pyrrolemethanethiol (6%) were obtained from the corre-
sponding cysteine-aldehyde conjugates by incubation with baker’s yeast. This work indicates the
versatile bioconversion capacity of baker’s yeast for the generation of thiols from cysteine-aldehyde
conjugates. Thanks to its food-grade character, baker’s yeast provides a biochemical tool to produce
thiols, which can be used as flavorings in foods and beverages.
KEYWORDS: Aroma; baker’s yeast; cysteine conjugates; 2-furfurylthiol; benzylthiol; 5-methyl-2-
furfurylthiol; 2-thiophenemethanethiol; 3-methyl-2-thiophenemethanethiol; 2-pyrrolemethanethiol
INTRODUCTION
Furans such as 2-furfurylthiol and 5-methyl-2-furfurylthiol
are well-known aroma compounds, which have been identified
among the potent volatile flavors of roasted coffee (19).
Hofmann et al. reported 5-methyl-2-furfurylthiol as the most
potent odorant of thermally treated rhamnose/cysteine solution
(20). This compound was also identified among volatiles isolated
from yeast extracts (21).
High consumer demand for natural flavors has stimulated a
number of research activities aimed at developing novel
biocatalytically processed products with the use of microbial
cells or isolated enzymes. Recently, a number of review articles
have been published about the possibilities of using microorgan-
isms to perform biochemical conversions (1-7). These studies
showed that the microbial processes seem to be the most
promising ways to produce natural flavor compounds or
complex mixtures thereof (8-10). Many microorganisms are
able to produce flavor compounds by fermentation starting from
simple nutrients such as sugars, amino acids, and fatty acids.
These readily available natural precursors can be converted to
valuable flavors such as carboxylic acid esters or products of
lipid oxidation (e.g., alcohols, aldehydes, and ketones), which
contribute to the aroma of food (11-13). Little data is, however,
available on the biogeneration of sulfur-containing compounds,
e.g., thiols, heterocyclic thiols, and disulfides (14, 15). The
occurrence of such biotransformations requires more specific
precursors and follows specific biochemical pathways (11).
Many thiols have been isolated from cooked foods, including
meat and chocolate. Some of them are believed to be particularly
important in roasted coffee aroma (16, 17). For savory flavors,
thiols are among the most important aroma compounds due to
their odor characters and their low odor thresholds (18).
Thiophenes have been identified in a wide range of food
systems in which they significantly contribute to the sensory
properties. They are responsible for the mild sulfurous odor of
cooked meat (22) and chicken (23). The formation of thiophenes
in various foods and in model systems has been described (24-
27). The application of thiophene derivatives in foodstuffs to
enhance their flavor has also been reported (28).
Pyrrole and derivatives are a group of nitrogen-containing
heterocyclic compounds found among the Maillard reaction
products in processed foods. The formation of pyrroles is
thought to proceed via the Strecker degradation involving proline
or hydroxyproline and dicarbonyl compounds (29). Thiol-
containing pyrroles have been chemically synthesized (30).
However, their application as a flavoring material has not
received much attention (31, 32).
The majority of the thiols mentioned above was produced
by organic synthesis or by process flavors (Maillard reaction).
2-Furfurylthiol is the only heterocyclic-containing aroma thiol,
which has been generated via a biochemical process. This aroma
compound was produced by the biotransformation of cysteine-
furfural conjugate with various bacteria possessing â-(C-S)
* To whom correspondence should be addressed. Tel: (+41)21 785 8634.
Fax: (+41) 21 785 8549. E-mail: rachid.bel-rhlid@rdls.nestle.com.
10.1021/jf026198j CCC: $25.00 © 2003 American Chemical Society
Published on Web 05/02/2003

3630 J. Agric. Food Chem., Vol. 51, No. 12, 2003
Huynh-Ba et al.
Figure 1. Chemical structure of cysteine−aldehyde conjugates used for
Figure 2. Chemical structures of biogenerated aroma compounds.
incubation with baker’s yeast.
for 15 min with a nitrogen flow and the whole apparatus was kept
under nitrogen during the incubation period. The suspension was
adjusted to the desired pH, which was automatically maintained
throughout the reaction with 2 M sodium hydroxide solution using a
Metrohm pH-stat device model 691 (Metrohm, Herisau, Switzerland).
The cysteine-aldehyde conjugate (6 mmol, 20 mM) was then added
all at once.
lyase enzymatic activity (33). However, these bacteria are not
food-grade.
Not many food-grade microorganisms are known to possess
â-(C-S) lyase activity (14, 32). Only in Saccharomyces
cereVisiae is â-cystathionase known to occur (35). It cleaves
the C-S bond of the cysteine moiety in cystathionine. Therefore,
the capability of baker’s yeast was investigated for the genera-
tion of thiols from related cysteine-aldehyde conjugates. The
aim was to find a biochemical tool to produce thiols with the
use of a food-grade microorganism. Results of this study are
reported here focusing on the production of thiols possessing
furan, benzyl, thiophene, and pyrrole backbones.
Five milliliters of the reaction mixture was sampled at different
incubation times. The sample was adjusted to pH 4.0 with 2 M
hydrochloric acid. Five hundred microliters of a solution of benzylthiol
in diethyl ether (2000 ppm) was then added as an internal standard. In
the case of the biogeneration of benzylthiol, a solution of 2-furfurylthiol
in diethyl ether (2000 ppm) was used as the internal standard. After
sodium chloride (3 g) was added, the sample was extracted with diethyl
ether (3 × 15 mL). The diethyl ether extracts were separated from the
mixture by centrifugation (15 min, 5000 rpm), combined, dried over
sodium sulfate, and concentrated to a volume of 2 mL using a Vigreux
column (30 cm × 1 cm). The concentrated solution was then analyzed
by various gas chromatographic techniques. The yields of all thiols
are given in mol % relative to original corresponding cysteine conjugates
(20 mM). The concentrations of targeted thiols were calculated on the
basis of their peak area (gas chromatography-flame ionization detection
(GC-FID) and GC-FPD) relative to that of the internal standard
assuming that their response factors were equal to that of the internal
standard.
MATERIALS AND METHODS
Materials. All chemicals were of analytical grade and commercially
available. Furfural, 5-methyl-2-furfural, 2-thiophenecarboxaldehyde,
3-methyl-2-thiophenecarboxaldehyde, 2-pyrrolecarboxaldehyde, and
benzaldehyde were purchased from Fluka (Buchs, Switzerland), and
L-cysteine was purchased from Aldrich (Buchs, Switzerland). Fresh
yeast cream was from Hefe Schweiz AG (Stettfurt, Switzerland).
Organic solvents were purified by distillation using a Vigreux column
(60 cm × 3 cm).
GC Analyses (GC, GC-O, and GC-MS). GC and GC-olfactometry
(GC-O) analyses were performed using a Carlo Erba gas chromatograph
(Mega 2, GC 8000, Fisons Instruments, via Brechbu¨hler, Schlieren,
Switzerland) equipped with a cold on-column injector, FID and FPD
detectors, and a sniffing port (37). Fused silica capillary columns (DB-
1701, DB-Wax, and DB-FFAP) were used, all of 30 m × 0.32 mm
i.d. and film thickness 0.25 µm (J&W Scientific, Folsom, CA). The
temperature program for the DB-1701 column was 35 °C (2 min), 40
°C/min to 50 °C (1 min), 6 °C/min to 240 °C (10 min) and for the
DB-Wax and DB-FFAP columns, 35 °C (2 min), 6 °C/min to 180 °C,
10 °C/min to 240 °C (10 min). The injected volume for GC and GC-O
was 0.2 µL. Linear retention indices (RI) were calculated by linear
interpolation according to van den Dool and Kratz (38).
GC-MS analyses were performed on a MAT-8430 mass spectrometer
(Finnigan, Bremen, Germany) combined with an HP 5890 gas chro-
matograph using the above GC conditions. The mass spectrometer was
operated in both electron mode at 70 eV and positive chemical
ionization mode at 150 eV with ammonia as the reagent. The interface
temperature was 220 °C, and the source temperature was set at 180
°C. All data were processed with MassLib (MSP Friedli, Koeniz,
Switzerland). The known compounds were identified by comparison
of their RI and MS with literature data. All new compounds were
tentatively identified on the basis of their MS data analysis.
Preparation of Cysteine-Aldehyde Conjugates. Cysteine-alde-
hyde conjugates were prepared following a previously reported method
(36) with some modifications. Aldehyde (11 mmol, neat or dissolved
in ethanol) was added dropwise to a solution of cysteine (10 mmol) in
distilled water (30 mL). After it was stirred for 1 h at room temperature,
the resulting precipitate was filtered, washed with water and ethanol
(or a mixture thereof), and then dried under vacuum. The products were
analyzed by thin-layer chromatography (TLC) using n-butanol/acetic
acid/water (2:1:1) as eluant. The following cysteine-aldehyde conju-
gates (Figure 1) were prepared, and pure products (as shown by TLC)
were tentatively characterized by mass spectrometry.
Cysteine-Furfural (1A). Yield, 86%. MS-EI m/z (relative inten-
sity): 199 (48), 153 (14), 127 (15), 107 (100), 94 (56), 80 (46), 43
(76).
Cysteine-Benzaldehyde (1B). Yield, 97%. MS-EI m/z (relative
intensity): 209 (33), 176 (11), 164 (31), 137 (100), 130 (46), 117 (69),
104 (100), 77 (42).
Cysteine-5-Methyl-2-furfural (1C). Yield, 65%. MS-EI m/z (relative
intensity): 213 (100), 181 (11), 167 (28), 141 (14), 125 (33), 121 (60),
109 (75), 94 (35).
Cysteine-2-Thiophenecarboxyaldehyde (1D). Yield, 88%. MS-EI
m/z (relative intensity): 215 (26), 170 (20), 143 (90), 84 (20).
Cysteine-3-Methyl-2-thiophenecarboxyaldehyde (1E). Yield, 40%.
MS-EI m/z (relative intensity): 229 (94), 183 (35), 157 (55), 97 (38).
Cysteine-2-Pyrrolecarboxyaldehyde (1F). Yield, 75%. MS-EI m/z
(relative intensity): 198 (16), 154 (10), 132 (12), 120 (40), 111 (98).
General Bioconversion Procedure. Five liters of commercial fresh
yeast cream (20% dry weight) was centrifuged, and the supernatant
was discarded. The yeast cells were resuspended in 1 L of 0.1 M
phosphate buffer. The yeast suspension (300 mL) was then placed in
a 500 mL flask equipped with an electrode and a magnetic stirrer (500
rpm). The flask was immersed in an oil bath heated at 30 °C. When
the anaerobic assays were performed, the yeast suspension was flushed
RESULTS AND DISCUSSION
Biogeneration of 2-Furfurylthiol. To evaluate the capacity
of baker’s yeast to generate thiols, the cysteine-furfural
conjugate 1A was used as a model substrate targeting the
biogeneration of 2-furfurylthiol 2 (Figure 2) because of its
occurrence in food and reaction flavors and its typical sulfury,
roasted, and coffeelike odors. 2-Furfurylthiol 2 was generated
from cysteine conjugate 1A in a bioconversion yield of about

Biogeneration of Thiols
J. Agric. Food Chem., Vol. 51, No. 12, 2003 3631
Table 1. Volatile Components Identified in the Diethyl Ether Extract
from the Biotransformation of the Cysteine−Furfural Conjugate under
Aerobic Conditionsa
Table 2. Volatile Components Identified in the Diethyl Ether Extract
from the Biotransformation of the Cysteine−Furfural Conjugate under
Anaerobic Conditionsa
linear retention
linear retention
aroma qualityc
(GC-O)
aroma qualityc
(GC-O)
index
index
compoundb
FFAP
DB-1701
compoundb
FFAP
DB-1701
S-methylthioacetate
S-ethylthioacetate
2-methyl-1-propanol
thioacetic acid
1046
1080
1084
1124
758
cheese, eggy
sulfury, garlic, onion
2-methyl-1-propanol
3-methyl-1-butanol
acetic acid
2-furfurylthiol
2-furfural
2-furfurylmethyl sulfide
1-mercapto-2-propanol
2-mercapto-1-ethanol
2-furfuryl alcohol
3-methylbutanoic acid
S-furfuryl thioacetate
2-furanoic acid
1084
1204
1425
1435
1457
1485
840
844
785
998
908
840
711
981
844
951
785
998
908
1089
sulfury, onion, garlic
fruity, pungent, sulfury
burnt, roasted, onion
garlic, eggy
diethyl disulfide
3-methyl-1-butanol
2-methyl-1-butanol
acetic acid
2-furfurylthiol
2-furfural
2-furfurylmethyl sulfide
2-mercapto-1-ethanol
butanoic acid
2-furfuryl alcohol
3-methylbutanoic acid
S-furfuryl thioacetate
2-furanoic acid
1204
-
1089
1425
1435
1457
1485
1498
1618
1665
1669
1769
2433 d
2600
1498
1665
1669
1769
2433 d
2600
burnt, roasted, onion
almond, caramel-like
garlic, eggy
1012
1037
1275
burnt, roasted, savory
fried onion, burnt coffee
980
1012
1037
1275
bis(2-furfuryl) disulfide
1862
cooked sugar, ethereal
burnt, roasted, savory
fried onion, burnt coffee
a The reaction was carried out anaerobically at pH 6.9 and 30 °C. The substrate
concentration was 20 mM. b Identification was based on comparison of RI and
MS with literature data using the MassLib database. c GC-O was performed only
for the main aroma compounds. d DB-wax capillary column was used to determine
the RI.
bis(2-furfuryl) disulfide
1862
a The reaction was carried out aerobically at pH 6.9 and 30 °C. The substrate
concentration was 20 mM. b Identification was based on comparison of RI and
MS with literature data using the MassLib database. c GC-O has been performed
only for the main aroma compounds. d DB-wax capillary column was used to
determine the RI.
Figure 3. Hypothetical pathways leading to alcohols and acids from
cysteine−aldehyde conjugates.
Figure 4. Influence of incubation mode on the biogeneration of
2-furfurylthiol: aerobic (9) and anaerobic (2).
25% after 24 h of incubation with baker’s yeast (conditions:
20 mM substrate, pH 6.9, aerobic mode). Besides 2-furfurylthiol,
several other sulfur-containing compounds were detected in the
reaction mixture (Table 1). However, their concentrations were
quite low (<1%) except for thioacetic acid (10%) and bis(2-
furfuryl) disulfide, the dimer of 2-furfurylthiol (10%). Besides
the common metabolites from baker’s yeast itself, furfural and
in particular furfuryl alcohol were identified in the reaction
mixture by GC-MS. Furfural, possibly originating from hy-
drolysis of cysteine-furfural conjugate 1A, was enzymatically
reduced into 2-furfuryl alcohol (Figure 3).
To better understand the biotransformation of cysteine-
furfural conjugate 1A into 2-furfurylthiol 2, the influence of
the incubation conditions (e.g., incubation mode (aerobic,
anaerobic), pH, and substrate concentration) on the biotrans-
formation kinetic and yield were investigated.
Influence of Aerobic and Anaerobic Incubation Mode.
Biotransformation of the cysteine-furfural conjugate 1A (20
mM) was performed with baker’s yeast at pH 6.9 under aerobic
and anaerobic conditions. As shown in Tables 1 and 2, many
compounds, which are common metabolites from baker’s yeast,
were mostly generated under aerobic rather than anaerobic
conditions. Acetic acid and furfuryl alcohol were the major
compounds identified in both assays. As shown in Figure 4,
2-furfurylthiol 2 was generated with an optimal yield of 28%
after 48 h of reaction time and under nitrogen, while the yield
was only about 25% under aerobic conditions and after 24 h of
incubation. Moreover, the degradation of 2-furfurylthiol was
Figure 5. Influence of the pH on the biogeneration of 2-furfurylthiol at pH
6.0 (×), pH 6.9 (9), pH 8.0 (2), and pH 9.0 (b).
much more pronounced under aerobic than anaerobic mode. This
difference can be explained by the chemical oxidative dimer-
ization of 2-furfurylthiol 2 into bis(2-furfuryl) disulfide (10%,
data not shown), which is favored under oxidizing conditions.
Influence of pH. To determine the optimal pH for the
biotransformation of cysteine-furfural conjugate 1A (20 mM)
with baker’s yeast, anaerobic incubation trials were performed
at different pH values (e.g., 6.0, 6.9, 8.0, and 9.0). As shown in
Figure 5, the yield of 2-furfurylthiol 2 was less than 1% at pH
6.0 and pH 9.0, while at pH 6.9 and pH 8.0 it was 15 and 37%,
respectively, after 24 h of incubation. However, the amounts
of 2, which were optimally obtained after 24 h of incubation at
pH 8.0, decreased more dramatically with longer incubation

3632 J. Agric. Food Chem., Vol. 51, No. 12, 2003
Huynh-Ba et al.
Table 3. Volatile Components Identified in the Diethyl Ether Extract
from the Biotransformation of the Cysteine−Benzaldehyde Conjugatea
linear retention
aroma qualityc
(GC-O)
index
compoundb
FFAP
DB-1701
S-methylthioacetate
thioacetic acid
acetic acid
1-mercapto-2-propanol
2-mercapto-1-ethanol
benzaldehyde
2-methylthio-1-ethanol
propanoic acid
1046
1124
1425
1492
1498
758
711
785
cheese, eggy
sulfury, onion, garlic
acid, pungent
1240
almond, sweet aromatic
green, sulfury
fruity, sour
Figure 6. Effect of substrate concentration on the biogeneration of
2-furfurylthiol at 40 (9) and 20 (2) mM.
1525
1626
1664
899
1181
benzylthiol
cabbage, roasted
benzylmethyl sulfide
2-methylthio acetic acid
benzyl acetate
benzyl alcohol
phenylethanol
S-benzylthioacetate
3-methylthiopropanoic acid
4-methylthiobutanoic acid
benzoic acid
1256
1205
green, fresh, fruity
balsamic, fruity
fruity, sweet, floral
1875
1909
1986
2298
2384
2428
1450
1345
1345
a The reaction was carried out anaerobically at pH 6.9 and 30 °C. The substrate
concentration was 20 mM. b Identification was based on comparison of RI and
MS with literature data using the MassLib database. c GC-O was performed only
for the main aroma compounds.
mixture from the cysteine-benzaldehyde conjugate 1B incu-
bated with baker’s yeast. This aroma volatile was generated in
a yield of 8% after 72 h of incubation. Among other sulfur-
containing volatiles (Table 3), benzylmethyl sulfide and S-
benzyl thioacetate, which are derivatives of benzylthiol, were
also identified in the reaction mixture. As shown in Table 3,
the volatile components containing no sulfur, which were
identified in the diethyl ether extract of the reaction mixture,
can be divided into two groups with respect to their origin.
Compounds such as propanoic acid and phenyl ethanol are
common metabolites from baker’s yeast, while other compounds
were generated from the cysteine conjugate 1B. Benzyl alcohol
for instance was probably generated by enzymatic reduction of
benzaldehyde, which was itself released by hydrolysis of the
conjugate 1B (Figure 3). Other benzaldehyde derivatives were
also detected in the reaction mixture, i.e., benzyl acetate and
benzoic acid.
Biogeneration of 5-Methyl-2-furfurylthiol. Biotransformation
of cysteine-5-methyl-2-furfural conjugate 1C with whole cell
baker’s yeast (pH 8.0, 30 °C and at 20 mM substrate) generated
5-methyl-2-furfurylthiol 4 in a yield of 11%. Thiol 4 was the
most intense odorant among the important odor active volatiles
identified in the reaction mixture by different chromatographic
techniques (GC, GC-MS, GC-O) as listed in Table 4. Thiol 4
elicits an attractive coffeelike and roasted note. The odor quality
as well as the chromatographic and spectroscopic properties (RI,
MS) of this thiol were identical to those reported in the literature
(20, 21). The odor threshold value of this aroma compound 4
is very low (0.006 ng/L of air), which is comparable to that of
2-furfurylthiol 2 and 2-methyl-3-furanthiol known as very
intense odorants eliciting coffeelike and cooked meat flavor
notes (17, 18).
Figure 7. Hypothetical pathways leading to thiols from cysteine−aldehyde
conjugates using baker’s yeast as the biocatalyst.
times as compared to pH 6.9 (Figure 5). This decrease could
be partially attributed to the increase of dimerization of 2 into
bis(2-furfuryl) disulfide, which was favored at pH 8.0 as
compared to pH 6.9 (data not shown).
Influence of Substrate Concentration. The effect of substrate
concentration on the biogeneration of 2-furfuylthiol 2 by baker’s
yeast was studied under anaerobic mode at pH 6.9 and 30 °C.
Two substrate concentrations, 20 and 40 mM, were used. As
shown in Figure 6, compound 2 was generated in a yield of
28% after 48 h of reaction when the substrate concentration
was 20 mM. This yield dropped to 6% when the concentration
was doubled (i.e., 40 mM). Moreover, the yield of 6% was
reached after 3 days of incubation time showing slower kinetics
in the biogeneration of 2 at higher substrate concentrations.
These results indicate that the baker’s yeast enzymatic activities
involved in the transformation of cysteine-furfural conjugate
into 2-furfurylthiol are strongly influenced by substrate con-
centration.
Results from incubation trials with cysteine-furfural conju-
gate 1A clearly indicate the high capacity of baker’s yeast in
the biogeneration of 2-furfurylthiol 2. The biochemical pathways
could involve â-(C-S) lyase and dehydrogenase of baker’s yeast
(Figure 7). The optimal incubation conditions were pH 8.0,
substrate concentration 20 mM, and anaerobic incubation mode.
Biogeneration of Other Thiols. Positive results obtained with
2-furfurylthiol prompted us to investigate the capacity of baker’s
yeast to generate other thiols of various chemical structures.
Emphasis was given to heterocyclic-containing aroma thiols
occurring in food and food systems and useful as flavorings.
Biogeneration of Benzylthiol. As shown by GC-FPD analysis,
benzylthiol 3 was the major sulfur compound of the reaction
The major component identified in the reaction mixture was,
however, 5-methyl-2-furfuryl alcohol 5. It was obtained in a
yield of 80% relative to the substrate cysteine conjugate 1C.
This alcohol likely originated from the enzymatic reduction of
the corresponding aldehyde, itself released by hydrolysis of the
cysteine conjugate 1C (Figure 3). These results indicate a much

Biogeneration of Thiols
J. Agric. Food Chem., Vol. 51, No. 12, 2003 3633
Table 4. Most Odor Active Volatiles Identified in the Diethyl Ether Extract from the Biotransformation of the Cysteine−5-Methyl-2-furfural Conjugatea
linear retention
index
aroma quality
(GC-O)
compoundb
FFAP
DB-1701
MS-EI data m/z (relative intensity)
5-methyl-2-furfurylthiol
1497
1723
1840
1086
1103
1366
2026
coffee, roasted
128 (18), 113 (2), 95 (100), 80 (5)
112 (100), 97 (35), 95 (85), 43 (57)
170 (10), 127 (2), 95 (100), 43 (12)
254 (5), 189 (3), 127 (10), 95 (100)
5-methyl-2-furfuryl alcohol
S-(5-methyl-2-furfuryl)thioacetatec
bis(5-methyl-2-furfuryl) disulfide
burnt, caramel
sulfury, burnt
a The reaction was carried out anaerobically at pH 8.0 and 30 °C. The substrate concentration was 20 mM. b Identification was based on comparison of RI and MS with
literature data using the MassLib database. c Tentatively identified on the basis of MS data analysis.
Table 5. Most Odor Active Volatiles Identified in the Diethyl Ether Extract from the Biotransformation of the Cysteine−2-Thiophenecarboxaldehyde
Conjugatea
linear retention
index
aroma quality
(GC-O)
compoundb
FFAP
DB-1701
MS-EI data m/z (relative intensity)
2-thiophenemethanethiol
2-thiophenemethanol
2-thiophenecarboxylic acid
bis(2-thiophenemethyl) disulfide
1702
1950
2519
2641
1197
1216
1484
2232
meaty, roasted, grilled
savory, sulfury
sulfury, meaty, animal
roasted, burnt
130 (35), 97 (100)
114 (100), 97 (45), 85 (50)
128 (100), 111 (42), 83 (20), 45 (20)
258 (38), 194 (100), 129 (8), 97 (60)
a The reaction was carried out anaerobically at pH 8.0 and 30 °C. The substrate concentration was 20 mM. b Identification was based on comparison of RI and MS with
literature data using the MassLib database.
Table 6. Most Odor Active Volatiles Identified in the Diethyl Ether Extract from the Biotransformation of the
Cysteine−3-Methyl-2-thiophenecarboxaldehyde Conjugatea
linear retention
index
aroma quality
(GC-O)
compoundb
FFAP
DB-1701
MS-EI data m/z (relative intensity)
3-methyl-2-thiophenemethanethiol
3-methyl-2-thiophenemethanol
3-methyl-2-thiophenecarboxylic acid
1820
2034
2501
1294
1316
1590
coffee, roasted
fruity, pungent
animal, spicy
144 (23), 129(2), 111 (100), 67 (6)
128 (100), 113 (75), 111 (82), 99 (30)
142 (100), 125 (55), 97 (62), 45 (12)
a The reaction was carried out anaerobically at pH 8.0 and 30 °C. The substrate concentration was 20 mM. b Compounds tentatively identified on the basis of their MS
data analysis.
Table 7. Most Odor Active Volatiles Identified in the Ether Extract from the Biotransformation of the Cysteine−2-Pyrrolecarboxaldehyde Conjugatea
linear retention
index
aroma quality
(GC-O)
compound b
FFAP
DB-1701
MS-EI data m/z (relative intensity)
2-pyrrolemethanethiol
2-pyrrolemethyl thioacetate
2094
2293
1309
1492
earthy, sulfury
roasted, savory
113 (50), 80 (100), 86 (12), 53 (38)
155 (85), 112 (10), 80 (100), 68 (35), 43 (40)
a The reaction was carried out anaerobically at pH 8.0 and 30 °C. The substrate concentration was 20 mM. b Compounds tentatively identified on the basis of their MS
data analysis.
faster hydrolysis/reduction of the substrate as compared to its
biotransformation into 5-methyl-2-furfurylthiol 4.
carboxylic acid 8. In a similar way as with other cysteine-
aldehyde substrates, the hydrolysis of cysteine-2-thiophenecar-
boxaldehyde 1D followed by enzymatic reduction and oxidation
of the released aldehyde (Figure 2) might account for the
generation of compounds 7 and 8. 2-Thiophenemethanethiol 6
could be useful as a flavoring since it was described to impart
a coffeelike odor when evaluated in a base of either syrup or
soluble coffee (28).
Biogeneration of 3-Methyl-2-thiophenemethanethiol. The
biotransformation of cysteine-3-methyl-2-thiophenecarboxal-
dehyde conjugate 1E (20 mM) with baker’s yeast at pH 8.0
produced 3-methyl-2-thiophenemethanethiol 9 in 3% yield after
40 h of incubation. Its odor character was found to be roasted
and coffeelike by GC-O analysis (Table 6). The other com-
pounds identified in the reaction mixture were 3-methyl-2-
thiophenemethanol 10 and 3-methyl-2-thiophenecarboxylic acid
11. Alcohol 10 was by far the most abundant volatile component
Biogeneration of 2-Thiophenemethanethiol. Bioconversion of
cysteine-2-thiophene carboxaldehyde conjugate 1D was per-
formed at pH 8.0 and 30 °C. 2-Thiophenemethanethiol 6 was
obtained in a yield of 22% after 40 h of incubation. It was
characterized based on chromatographic properties and mass
spectra in comparison with literature data (39, 40). Thiol 6 was
the most intense odorant with roasted, grilled, and meatlike odors
among the volatiles identified in the diethyl ether extract as listed
in Table 5. Other sulfur-containing volatiles were identified in
the reaction mixture such as 2-thiophenemethanol 7, 2-thiophen-
ecarboxylic acid 8, and the dimer of the thiol 6. The alcohol 7
has been identified in many foods such as cooked beef (41),
cooked asparagus (42), and corn (43). As found by GC-O (Table
5), the alcohol 7 possesses a savory and sulfury odor character,
while sulfury, meaty, and animal odors were attributed to the

3634 J. Agric. Food Chem., Vol. 51, No. 12, 2003
Huynh-Ba et al.
(9) Imhof, R.; Bosset, J. O. Relationships between microorganisms
and formation of aroma compounds in fermented dairy products.
Z. Lebensm. Unters. Forsch. 1994, 198, 267-276.
(10) Schreirer, P. Enzymes and flavour biotechnology. AdV. Biochem.
Eng. Biotechnol. 1997, 55, 51-72.
and was obtained in a yield of 45%. The odor quality of this
volatile was pungent and fruity (GC-O data in Table 6). The
generation of compounds 10 and 11 again suggests hydrolysis
of the cysteine-aldehyde conjugate 1E and the subsequent
enzymatic reduction/oxidation of the released aldehyde.
Biogeneration of 2-Pyrrolemethanethiol. Biotransformation
of cysteine-2-pyrrole carboxyaldehyde conjugate 1F resulted
in two main odor active volatiles: 2-pyrrole methanethiol 12
and 2-pyrrolemethyl thioacetate 13 as tentatively identified by
GC-MS. Thiol 12 was obtained in a yield of 6% after 24 h of
incubation. The aroma character of thiol 12 was earthy,
fermented, and sulfury, while roasted and savory characters were
attributed to thioacetate 13, as analyzed by GC-O (Table 7).
Thiol 12 and its acetate 13 newly generated with the use of
baker’s yeast could, therefore, be of use as flavorings in foods.
(11) Berger, R. G. Aroma Biotechnology; Springer-Verlag: Berlin,
Heidelberg, New York, 1995.
(12) Vandamme, E. J. The search for novel microbial fine chemicals,
agrochemicals and biopharmaceuticals. J. Biotechnol. 1994, 37,
89-108.
(13) Patterson, R. L. S.; Charlwood, B. V.; MacLeod, G.; Williams,
A. A. Bioformation of flavours. The Royal Society of Chemistry;
Thomas Graham House: Cambridge, 1992.
(14) Tominaga, T.; Peyrot des Gachons, C.; Dubourdieu, D. A new
type of flavor precursors in Vitis Vinifera L. cv. Sauvignon
blanc: S-cysteine conjugates. J. Agric. Food Chem. 1998, 46,
5215-5219.
(15) Wakabayashi, H.; Wakabayashi, M.; Eisenreich, W.; Engel, K.
H. Stereoselectivity of the â-lyase-catalyzed cleavage of S-
cysteine conjugate of pulegone. Eur. Food Res. Technol. 2002,
215, 287-292
(16) Holscher, W.; Vitzthum, O. G.; Steinhart, H. Identification and
sensorial evaluation of aroma impact compounds in roasted
Colombian coffee. Cafe´ Cacao The´. 1990, 3, 205-212.
(17) Blank, I.; Grosch, W. Potent odorants of the roasted powder and
brew of Arabica coffee. Z. Lebensm. Unters. Forsch. 1992, 195,
239-245.
(18) Kerscher, R.; Grosch, W. Quantification of 2-methyl-3-furanthiol,
2-furfurylthiol, 3-mercapto-2-pentanone and 2-mercapto-3-pen-
tanone in heated meat. J. Agric. Food Chem. 1998, 46, 1954-
1958.
(19) Tressl, R.; Silwar, R. Investigation of sulphur-containing com-
ponents in roasted coffee. J. Agric. Food Chem. 1981, 29, 1078-
1082.
(20) Hofmann, T.; Schieberle, P. Identification of potent aroma
compounds in thermally treated mixtures of glucose/cysteine and
rhamnose/cysteine using aroma extract dilution techniques. J.
Agric. Food Chem. 1997, 45, 898-906.
(21) Werkhoff, P.; Bretschneider, W.; Emberger, R.; Gu¨ntert, M.;
Hopp, R.; Kopsel, M. Recent developments in the sulphur flavour
chemistry of yeast extracts. Chem. Mikrobiol. Technol. Lebensm.
1991, 13, 30-57.
CONCLUSION
The results obtained in this work indicate that baker’s yeast
possesses the enzymatic capacity to generate thiols from
cysteine-aldehyde conjugates. Thiols of various chemical
structures were obtained, indicating that the baker’s yeast
enzymes involved in the biochemical transformation, in par-
ticular the â-(C-S) lyase, are not substrate specific. Beside
thiols, the generation of alcohols and carboxylic acids of similar
structure as the starting aldehydes suggested the involvement
of baker’s yeast dehydrogenases and oxidases to transform the
aldehyde, which was likely released from cysteine-aldehyde
conjugates by chemical hydrolysis (Figure 3). The biogeneration
yield of thiols was high, in particular for 2-furfurylthiol 2 (37%).
Further yield improvement could be expected by continuous
removal of thiols upon generation, as demonstrated in previous
work on microbial production of 2-furfurylthiol (33). The
capacity of baker’s yeast, as demonstrated by this work, provides
a biochemical tool to produce thiols. These aroma compounds
thus obtained with a food-grade microorganism (baker’s yeast)
could be of great potential use as flavorings in foods and
beverages. These findings were included in a patent application
(44).
(22) Shibamoto, T. Heterocyclic compounds found in cooked meats.
ACKNOWLEDGMENT
J. Agric. Food Chem. 1980, 28, 237-243.
(23) Nonaka, M.; Black, D. R.; Pippen, E. L. Gas chromatographic
and mass spectral analyses of cooked chicken meat volatiles. J.
Agric. Food Chem. 1967, 15, 713-717.
We are grateful to S. Metairon for her help and skillful technical
assistance.
(24) Whitefield, F. B.; Mottram, D. S. Investigation of the reaction
between 4-hydroxy-5-methyl-3(2H)-furanone and cysteine or
hydrogen sulfide at pH 4.5. J. Agric. Food Chem. 1999, 47 (3),
1626-1634.
(25) Elmore, J. S.; Mottram, D. S. Formation of 2-alkyl-(2H)-
thiapyrans and 2-alkylthiophenes in cooked beef and lamb. J.
Agric. Food Chem. 2000, 48 (6), 2420-2424.
(26) Hofmann, T.; Schieberle, P. Identification of key aroma com-
pounds generated from cysteine and carbohydrates under roasting
conditions. Z. Lebensm. Unters. Forsch. 1998, 207 (3), 229-
236.
(27) Golovnya, R. V.; Misharina, T. A.; Garbuzov, V. G.; Medvedyev,
F. A. Volatile sulphur containing compounds in simulated meat
flavour and their comparison with the constituents of natural
aroma. Nahrung 1983, 27 (3), 237-249.
(28) Winter, M.; Gautschi, F.; Flament, I.; Stoll, M.; Goldman, I.
M.; Firmenich & Cie. Flavoring agent. United States Patent
3,976,802, 1976.
(29) Tressl, R.; Helak, B.; Martin, N. Formation of flavour compo-
nents from L-proline. Topics in FlaVor Research; Berger, R. G.,
Nitz, S., Schreirer, P., Eds.; H. Eichlon: Marzling-Hargenham,
1985.
LITERATURE CITED
(1) Krings, U.; Berger, R. G. Biotechnological production of flavours
and fragrances. Appl. Microbiol. Biotechnol. 1998, 49, 1-8.
(2) Bigelis, R. Flavor Metabolites and enzymes from filamentous
fungi. Food Technol. 1992, 46, 151-161.
(3) Cogan, T. M. Flavour production by dairy starter cultures. J.
Appl. Bacteriol. 1995, 79, 49-64.
(4) Feron, G.; Bonnarme, P.; Durand, A. Prospects for the microbial
production of food flavours. Trends Food Sci. Technol. 1996,
7, 285-293.
(5) Gatfield, I. L. Enzymatic and microbial generation of flavours.
World Ingredients 1996, 40, 31-35.
(6) Hagedorn, S.; Kaphammer, B. Bread. Microbial biocatalysis in
the generation of flavour and fragrance chemicals. Annu. ReV.
Microbiol. 1994, 48, 773-800.
(7) Cheetham, P. S. J. Combining the technical push and the business
pull for natural flavours. AdV. Biochem. Eng. Biotechnol. 1997,
55, 1-49.
(8) Janssens, L.; De Pooter, H. L.; Schamp, N. M.; Vandamme, E.
J. Production of flavours by microorganisms. Proc. Biochem.
1992, 27, 195-215.

Biogeneration of Thiols
J. Agric. Food Chem., Vol. 51, No. 12, 2003 3635
(30) Nakamura, T.; Matsumoto, M. Mercaptomethylation of aromat-
ics. Synth. Commun. 1999, 29 (2), 201-210.
liquid partition chromatography. J. Chromatogr. 1963, 11, 463-
471.
(31) Dubs, P.; Kuentzel, H. Aroma and flavouring materials. Ger.
Offen. DE 2644201, 1977.
(32) Winter, M.; Gautschi, F.; Flament, I.; Stoll, M.; Goldman, I.
M.; Firmenich & Cie. Flavoring agent from pyrrole sulfur
compounds. United States Patent 3,985,906, 1976.
(39) Vitzthum, O. G.; Werkhoff, W. Steam volatile constituents of
roasted coffee. Z. Lebensm. Unters. Forsch. 1976, 160, 277-
291.
(40) Hofmann, T.; Schieberle, P. Evaluation of the key odorants in a
thermally treated solution of ribose and cysteine by aroma extract
dilution techniques. J. Agric. Food Chem. 1995, 43, 2187-2194.
(41) Wilson, R. A.; Mussinan, C. J.; Katz, I.; Sanderson, A. Isolation
and identification of some sulphur chemicals present in pressure-
cooked beef. J. Agric. Food Chem. 1973, 5, 873-876.
(42) Tressl, R.; Holzer, M.; Apetz, M. Formation of flavour compo-
nents in asparagus. J. Agric. Food Chem. 1973, 25, 455-459.
(43) Buttery, R. G.; Stern, D. J.; Ling, L. C. Studies on flavour
volatiles of some sweet corn products. J. Agric. Food Chem.
1994, 42, 791-795
(33) van der Schaft, P.; van Geel, I.; de Jong, G.; ter Burg, N.
Microbial production of natural furfuryl thiol. In Trends in
FlaVour Research; Proceedings of the 7th Weurman Flavour
Research Symposium, The Netherlands, 15-18 June 1993;
Maarse, H., van der Heij, D. G., Eds.; Elsevier: Amsterdam,
1994; pp 437-448.
(34) Kerkenaar, A.; Schmedding, D. J.; Berg, J. Method for preparing
thiol compounds. European Patent Application EP 277688, 1993.
(35) Strathern, J. N.; Jones, E. W.; Broach, J. R. The molecular
biology of the yeast Saccharomyces. Metabolism and Gene
Expression; Cold Spring Harbor Laboratory: New York, 1982.
(36) Schubert, M. P. Compounds of thiol acids with aldehydes. J.
Biol. Chem. 1936, 114, 341-350.
(44) Huynh-Ba, T.; Jaeger, D.; Matthey-Doret, W. Production of
aroma-enhancing thiols. European Patent Application EP 770686,
1997.
(37) Blank, I.; Lin, J.; Arce Vera, F.; Welti, H. D.; Fay, L. B.
Identification of potent odorants formed by autoxidation of
arachidonic acid: Structure elucidation and synthesis of (E,Z,Z)-
2,4,7-tridecatrienal. J. Agric. Food Chem. 2001, 49, 2959-2965.
(38) van den Dool, H.; Kratz, P. A. Generalization of the retention
index system including linear temperature programmed gas-
Received for review December 6, 2002. Revised manuscript received
April 4, 2003. Accepted April 6, 2003.
JF026198J