Lipase-assisted generation of 2-methyl-3-furanthiol and 2-furfurylthiol from thioacetates

Article abstract of DOI:10.1021/jf0202335

Enzymatic hydrolysis of S-3-(2-methylfuryl) thioacetate and S-2-furfuryl thioacetate using lipase from Candida rugosa produced 2-methyl-3-furanthiol and 2-furfurylthiol, respectively. When reactions were carried out at room temperature and pH 5.8, 2-methy

Full text of DOI:10.1021/jf0202335

J. Agric. Food Chem. 2002, 50, 4087−4090 4087
Lipase-Assisted Generation of 2-Methyl-3-furanthiol and
-Furfurylthiol from Thioacetates
2
RACHID BEL RHLID,* WALTER MATTHEY-DORET, IMRE BLANK,
LAURENT B. FAY, AND MARCEL A. JUILLERAT
Nestl e´ Research Center, Nestec Ltd., Vers-chez-les-Blanc, P.O. Box 44,
1000 Lausanne 26, Switzerland
Enzymatic hydrolysis of S-3-(2-methylfuryl) thioacetate and S-2-furfuryl thioacetate using lipase from
Candida rugosa produced 2-methyl-3-furanthiol and 2-furfurylthiol, respectively. When reactions were
carried out at room temperature and pH 5.8, 2-methyl-3-furanthiol was produced in a optimal yield of
88% after 15 min of reaction, whereas 2-furfurylthiol was obtained in a yield of 80% after 1 h of
reaction time. Enzymatic hydrolysis was also performed in n-hexane, n-pentane, and water/propylene
glycol mixture. The reaction rates in these media were slower as compared to those in aqueous
medium; however, the reaction yields were quite similar. As expected, the stability of the generated
2-methyl-3-furanthiol and 2-furfurylthiol was better in n-hexane, n-pentane, and the water/propylene
glycol mixture as compared to that in water or phosphate buffer.
KEYWORDS: Thiols; thioacetates; 2-methyl-3-furanthiol; 2-furfurylthiol; lipase; hydrolysis; flavor
INTRODUCTION
odorants (14, 15). They especially seem to impart the charac-
teristic cooked and roasted notes to meats (16) and to roasted
coffee (17). However, thiols are very unstable molecules and
easily oxidize to the corresponding disulfides upon storage, even
at low temperature (18, 19). To overcome this drawback,
enzyme-assisted formation of thiols from thioacetates may be
an interesting approach if the reaction rate and yield are high.
In fact, thiols could be stored in their more stable thioacetate
form, and enzymatic hydrolysis could be achieved just before
their use.
In this study, thiols were generated by enzymatic hydrolysis
of thioacetates in aqueous and organic media using lipase from
Candida rugosa as biocatalyst and S-3-(2-methylfuryl) thio-
acetate and S-2-furfuryl thioacetate as model substrates.
Natural flavors are defined as biologically derived aroma
chemicals generated by microbial fermentation and/or by the
action of endogenous or technical enzymes (1). Among these
biocatalysts, lipases and esterases have received special attention
because of their effectiveness in regio- and enantioselective
esterification and transesterification of organic acids and alcohols
(2). Moreover, the use of lipases and esterases in different media,
such as organic solvents and supercritical fluids, has been
extensively studied (3-5). From an industrial point of view,
the organic media exhibit many advantages as compared to
aqueous systems: a decrease in microbial contamination, the
possibility of dissolving apolar solutes, a reduction of water-
dependent side reactions, the enhancement of the thermal
stability of enzymes, and finally the easy recovery of products
from low-boiling solvents. Because of their stability, effective-
ness, and ease to use, lipases and esterases have been considered
in many industrial applications such as dairy products (6).
MATERIALS AND METHODS
Materials. All chemicals were of analytical grade purchased from
Aldrich (Buchs, Switzerland; S-2-furfuryl thioacetate), from Oxford
Chemicals [Hartlepool, U.K.; 2-methyl-3-furanthiol and S-3-(2-methyl-
furyl) thioacetate], or from Merck (Darmstadt, Germany; sodium
chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate,
and anhydrous sodium sulfate). Lipase from C. rugosa (EC 3.1.1.4,
type VII) was purchased from Sigma (Buchs, Switzerland). Immobilized
lipase from C. rugosa (30 units/g of solid) on Sol-Gel-AK support was
purchased from Fluka (Buchs, Switzerland). Organic solvents were of
analytical grade purified by distillation using a Vigreux column (1 m
These enzymes are also the main biocatalysts used in the
production of numerous flavoring compounds, particularly esters
that are impact aroma compounds in fruit flavors (7-9).
However, only a few studies deal with thioesters, which are
used as aroma molecules in savory, baked, and dairy goods (10-
1
2). Among these thioester derivatives, thioacetates have
received little attention, and very few studies deal with the
generation and enzymatic hydrolysis of thioacetates to produce
thiols (13). These thiols are important constituents of food
aroma, and in many cases they have been proven to be impact
×
1 cm).
Enzymatic Hydrolysis of Thioacetates. Different amounts of
enzyme were added to 10 mL of a solution of thioacetate (0.064 mmol)
in distilled water or phosphate buffer (0.2 M). Enzymatic reactions
were performed at different temperatures (4, 23, and 37 °C) with gentle
magnetic stirring. Samples were withdrawn at various time intervals
(from 1 min to 72 h). A solution of benzyl mercaptan in diethyl ether
*
Author to whom correspondence should be addressed [telephone (+41)
2
1 785 8634; fax (+41) 21 785 8549; e-mail rachid.bel-rhlid@rdls.nestle.com].
1
0.1021/jf0202335 CCC: $22.00 © 2002 American Chemical Society
Published on Web 06/01/2002

4088 J. Agric. Food Chem., Vol. 50, No. 14, 2002
Bel Rhlid et al.
(500 µL, 2000 ppm) was then added as internal standard for quantifica-
tion. The mixture was extracted with diethyl ether, and the ether extracts
were dried over sodium sulfate and concentrated to a volume of 2 mL
using a Vigreux column (30 × 1 cm). The concentrated solution was
then analyzed by various chromatographic techniques. The respective
concentrations of thioacetates, thiols, and disulfides were determined
by gas chromatography. The influence of the following parameters was
studied: pH (6.0, 7.0, and 8.0), temperature (4, 23, and 37 °C), and
the ratio of enzyme (units) to substrate (millimoles) (13/0.064, 65.5/
0
.064, 77/0.064, and 65/0.64).
Hydrolysis of S-3-(2-Methylfuryl) Thioacetate in n-Hexane and
Figure 1. Generation of 2-furfurylthiol (2), 2-methyl-3-furanthiol (5), and
related disulfides.
n-Pentane. Different amounts of immobilized lipase from C. rugosa
were added to 10 mL of a solution of S-3-(2-methylfuryl) thioacetate
(0.064 mmol) in n-hexane or n-pentane. Reactions were performed at
room temperature with magnetic stirring. Samples were withdrawn at
various time intervals and then filtered, and a solution of benzyl
mercaptan in n-hexane (500 µL, 2000 ppm) was added as internal
standard. Samples were then analyzed by different chromatographic
techniques.
Hydrolysis of S-3-(2-Methylfuryl) Thioacetate in Water/Pro-
pylene Glycol Media. To a solution of thioacetate (10 mL, 0.064 mmol)
containing different ratios of water and propylene glycol (90/10, 70/
3
0, 50/50) was added lipase from C. rugosa (26 units). Reactions were
performed at room temperature under magnetic stirring. Samples were
withdrawn at various time intervals, and a solution of benzyl mercaptan
(
500 µL, 2000 ppm) in diethyl ether was added as internal standard.
Samples were then analyzed by different chromatography techniques.
Figure 2. Influence of pH on enzymatic hydrolysis of S-2-furfuryl
thioacetate at pH 5.8 (9), pH 6.0 (O), pH 7.0 (2), and pH 8.0 (4):
substrate, 0.064 mmol; temperature, 23 °C; enzyme, 65 units; solvent,
phosphate buffer.
Gas Chromatography. Gas chromatography-olfactometry (GC-
O) analyses were performed using a Carlo Erba gas chromatograph
(
Mega 2, Fisons Instruments, via Brechb u¨ hler, Schlieren, Switzerland)
equipped with an automatic cold on-column injector, a flame ionization
detector (FID), a flame photometric detector (FPD), and a sniffing port
the same conditions but without enzyme. No hydrolysis of
thioacetates was observed in any of the control samples.
Enzymatic Hydrolysis of S-2-Furfuryl Thioacetate. Influ-
ence of pH. The study of the influence of the pH on the
enzymatic hydrolysis and on the product stability was performed
in phosphate buffer and in water. Figure 2 shows the generation
of 2-furfurylthiol at the pH values of 5.8, 6.0, 7.0, and 8.0. The
substrate was completely transformed after 1 h of incubation.
The reaction rates were similar at all pH values studied.
However, the yields were different. A maximum yield of 74%
was obtained at pH 5.8, whereas the maximum yield at pH 8.0
was only 50%. Part of the 2-furfurylthiol was chemically
oxidized to the dimer: up to ∼10% of compound 3 was obtained
at all pH values studied after 24 h of reaction time.
(
20). Fused silica capillary columns (DB-1701 and DB-FFAP) were
used, both 30 m × 0.32 mm i.d., with a film thickness of 0.25 µm
J&W Scientific, Folsom, CA). The temperature program for the DB-
701 was as follows: 35 °C (2 min), 40 °C/min to 50 °C (1 min), 6
C/min to 240 °C (10 min). The temperature program for the FFAP
was as follows: 50 °C (2 min), 6 °C/min to 180 °C, 10 °C/min to 240
C (10 min). Linear retention indices (RI) were calculated according
(
1
°
°
to the method of van den Dool and Kratz (21).
Mass Spectrometry. Gas chromatography-mass spectrometry (GC-
MS) analyses were carried out using a MAT-8430 mass spectrometer
(Finnigan, Bremen, Germany) using the same GC conditions as
described above. The MS-EI spectra were generated at 70 eV and MS-
CI at 150 eV with ammonia as the reagent gas.
Bis(2-furfuryl) disulfide (3): MS-EI, m/z (relative intensity) 226 (20),
61 (10), 113 (5), 85 (10), 82 (20), 81 (100), 53 (75), 51 (10), 45 (20).
1
Influence of Enzyme Amount. Only a few examples of
enzymatic hydrolysis using thioesters as substrates have been
reported (22). The ability of common hydrolytic enzymes to
transform thioesters into thiols is not well documented. There-
fore, we studied the effect of the concentration of enzyme on
the reaction rate and on the overall yield. Trials were performed
in water, at pH 5.8 and room temperature. The range of enzyme
quantity varied from 13 to 262 units for the same substrate
concentration (0.064 mmol). As shown in Figure 3, the quantity
of enzyme influenced the reaction rate but had no significant
effect on the final yield of 2-furfurylthiol generated. For all trials,
a maximum yield of 70-80% of aroma compound 2 was
obtained.
The degradation of this volatile molecule was much higher
when the amount of biocatalyst was increased. However, this
degradation was insignificant when the concentration of the
substrate was increased 10-fold for the same quantity of enzyme
(enzyme, 13 units; substrate, 0.64 mmol; data not shown). This
phenomenon could be explained by the presence of side
activities due to the presence of impurities in the commercial
crude enzyme preparation.
Bis(2-methyl-3-furyl) disulfide (6): MS-EI, m/z (relative intensity)
26 (100), 183 (5), 146 (5), 114 (40), 113 (95), 86 (5), 85 (5), 73 (10),
2 (15), 61 (15), 51 (25), 45 (20).
2
7
RESULTS AND DISCUSSION
Thiols are potent odorants that play a key role in many food
flavors. We investigated the feasibility of enzymatic hydrolysis
of thioacetates to produce thiols as an approach to overcome
the problem of chemical instability, which limits their applica-
tion. In this study, S-2-furfuryl thioacetate (1) and S-3-(2-
methylfuryl) thioacetate (4) were chosen as model substrates.
Enzymatic reactions were performed in water, in phosphate
buffer, or in nonconventional media such as n-hexane and
n-pentane. As shown in Figure 1, enzymatic hydrolysis of
compounds 1 and 4 using lipase from C. rugosa as biocatalyst
generated 2-furfurylthiol (2) and 2-methyl-3-furanthiol (5),
respectively. By chemical oxidation, parts of these thiols were
transformed into the corresponding dimers: bis(2-furfuryl)
disulfide (3) and bis(2-methyl-3-furyl) disulfide (6). The yield
of thiols and related disulfides were determined after quantifica-
tion using benzyl mercaptan as an internal standard. For all of
the parameters studied, reaction controls were performed under
Influence of Temperature. To study the influence of the
temperature on the reaction rate and on the stability of the

Lipase-Assisted Generation of Thiols
J. Agric. Food Chem., Vol. 50, No. 14, 2002 4089
Table 1. Aroma Qualities and Thresholds of the Odorants 2-Furfurylthiol, 2-Methyl-3-furanthiol, S-3-(2-Methylfuryl) Thioacetate, and S-2-Furfuryl
Thioacetate
odor threshold in
odorant
-furfurylthiol
-methyl-3-furanthiol
bis(2-furfuryl) disulfide
bis(2-methyl-3-furyl) disulfide
aroma quality
air (ng/L)
water (µg/L)
2
2
sulfury, coffee-like, roasty, fresh note
sulfury, meat-like, sweet, boiled note
sulfury, roasty
0.01−0.02 (17)a
0.005−0.01 (23)
0.00015−0.0006 (25)
0.0006−0.0024 (25)
0.01 (24)
0.007 (24)
sulfury, meat-like, boiled note
0.00002 (26)
a
Literature given in parentheses.
Figure 3. Effect of enzyme amount on hydrolysis of S-2-furfuryl thioacetate
using 262 units (4), 77 units (2), 65 units (O), and 13 units (9): substrate,
Figure 5. Influence of quantity of enzyme on hydrolysis of S-3-(2-
methylfuryl) thioacetate using 65 units (9), 26 units (O), and 6.5 units
0.064 mmol; temperature, 23 °C; pH, 5.8; solvent, phosphate buffer.
(2): substrate, 0.064 mmol; temperature, 23 °C; pH, 5.8; solvent,
phosphate buffer.
yield of 88% was obtained after 15 min of reaction time when
6
5 units of enzyme was used for the hydrolysis, whereas the
yields were only 50 and 10% when 26 and 6.5 units of enzyme
were used, respectively. However, the degradation rate of aroma
compound 5 was proportional to the quantity of enzyme. This
phenomenon was similar to that discussed previously for
2
-furfurylthiol.
The data also show that hydrolysis of S-3-(2-methylfuryl)
thioacetate (4) is much more rapid as compared to that of S-2-
furfuryl thioacetate (1). For example, with 65 units of lipase
about 15% of 2 and 90% of 5 were released after 15 min of
hydrolytic reaction. Furthermore, these results show that odorant
Figure 4. Effect of temperature on formation of 2-furfurylthiol at 37 °C
2), 23 °C (O), and 4 °C (9): substrate, 0.064 mmol; enzyme, 65 units;
pH, 5.8; solvent, phosphate buffer.
(
5
is easily consumed as only 50% was left after an incubation
period of 1 h. This is well in line with the higher oxidative
instability of 5 as compared to 2, mainly leading to the dimer
product, trials were performed at 4, 23, and 37 °C using the
same enzyme-to-substrate ratio (65 units/0.064 mmol). Figure
6
(18). The loss of odorants 2 and 5 may affect the overall aroma
4
shows the generation of 2-furfurylthiol and its stability as
of the sample as the dimers formed show different aroma
qualities and thresholds levels (Table 1).
functions of the temperature. As expected, no significant
differences were observed when the reaction took place at 23
or 37 °C, whereas at 4 °C the reaction rate was much slower.
A maximum yield of ∼70% of 2-furfurylthiol was obtained
after 24 h at 4 °C and after 1 h and 45 min at 23 and 37 °C,
respectively. Moreover, 2-furfurylthiol was much more stable
at 4 °C as compared to 23 and 37 °C, leading mainly to the
dimer 3. Reaction controls were performed without enzyme at
all temperatures and pH values studied, and no hydrolysis of
thioacetate was observed.
Enzymatic Hydrolysis of S-3-(2-Methylfuryl) Thioacetate.
Effect of the Quantity of Enzyme. The study of the influence of
the enzyme amount on the hydrolysis of S-3-(2-methylfuryl)
thioacetate was performed at room temperature in a phosphate
buffer (pH 5.8) containing 0.064 mmol of substrate. The
concentration range of the lipase from C. rugosa varied from 0
to 65 units. Figure 5 shows the formation and degradation of
Enzymatic Hydrolysis in n-Hexane and n-Pentane. As
reported above, enzymatic hydrolysis of S-3-(2-methylfuryl)
thioacetate in water resulted in 2-methyl-3-furanthiol in good
yields (∼90%) and with high reaction rate. However, thiol 5
was completely transformed into the corresponding disulfide 6
after 2 h of reaction time. It has been reported that 2-methyl-
3-furanthiol is more stable in n-pentane and in dichloromethane
than in diethyl ether or in water (18). Thus, enzymatic hydrolysis
was performed in n-hexane and in n-pentane using immobilized
lipase from C. rugosa. The reaction rates in n-hexane and
n-pentane were slightly slower in comparison to those in water
or phosphate buffer; however, the yields of 2-methyl-3-furanthiol
generated were similar (85%). As expected, the stability of
aroma compound 5 was better in the organic solvents as
compared to water.
2
-methyl-3-furanthiol as a function of the enzyme concentration.
Enzymatic Hydrolysis in Water/Propylene Glycol Mixture.
Propylene glycol is a food grade solvent used particularly in
the flavor industry as most of the aroma compounds are stable
The quantity of enzyme had an influence not only on the reaction
rate but also on the yield of the target compound 5. Indeed, a

4090 J. Agric. Food Chem., Vol. 50, No. 14, 2002
Bel Rhlid et al.
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Figure 6. Enzymatic hydrolysis of S-3-(2-methylfuryl) thioacetate in water/
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on the stability of 2-methyl-3-furanthiol. Propylene glycol
concentrations of e10% in water as reaction medium did not
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odorant 5 (data not shown). However, the increase of propylene
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In conclusion, enzymatic hydrolysis of S-3-(2-methylfuryl)
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and in organic solvents, yielding the corresponding thiols. This
enzymatic hydrolysis was achieved in good yield and with high
reaction rate. The optimum pH was ∼5.5-6.5. The reaction
performed at room temperature was found to be a good
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ACKNOWLEDGMENT
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(
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JF0202335