Preparation of thiols with food-acceptable micro-organisms

Flavorant compounds and compositions for food substances are prepared in a food-acceptable manner by bio-conversion of a cysteine S-complex with a food-acceptable micro-organism, particularly a yeast or filamentous fungus, which, upon incubation with the complex, splits the complex at a terminal carboxyl group in a beta position to yield a product including a thiol and at least one metabolite compound, and the product is isolated.

The invention relates to the preparation of thiols, in particular natural 
flavouring thiols, by bioconversion. 
Thiols are among the volatile sulphur-containing substances which are 
considered to be responsible for the meaty note of various meats, for 
example, chicken, beef and pork, or which are components of the flavour of 
coffee. 
Meat flavourings are generally produced by a thermal reaction between a 
reducing sugar and an amino acid in the presence of a sulphur-containing 
compound as the source of sulphur, for example cysteine, at a high 
temperature for a certain period of time, optionally in the presence of 
fat. Such a reaction provides a complex mixture of aromatic substances 
entering into the composition of the flavour of meat. It is known, for 
example from U.S. Pat. No. 5,182,194, that certain micro-organisms which 
cannot be used in food are able to provide a beta-C-S-lyase enzyme capable 
of producing thiols from cysteine S-complexes. 
SUMMARY OF THE INVENTION 
The object of the invention is to provide natural thiols which are obtained 
by bio-conversion and which are capable of being used as a flavoring 
ingredient for foods. 
The process according to the invention is characterized in that a cysteine 
S-complex with the formula: 
##STR1## 
in which: R.sub.1 is H, C.sub.1-7 linear or branched alkyl, phenyl, 
benzyl, 2-R.sub.2 -2-R.sub.3 -vinyl, 5-R.sub.2 -furan-2-yl, 5-R.sub.2 
-thiofuran-2-yl, 
R.sub.2 and R.sub.3 are similar or different and represent H or CH.sub.3, 
R.sub.4 is H, or forms with R.sub.1 a 2-R.sub.5 -5-R.sub.6 
-tetrahydrofuran-3-ylidene, or 2-R.sub.5 -5-R.sub.6 
-2,3-dihydrofuran-3-ylidene group, in which R.sub.5 and R.sub.6 are 
similar or different and represent H or CH.sub.3, 
or R.sub.4 is a CH(OH)R.sub.7 group and R.sub.7 represents H, C.sub.1-4 
-alkyl or phenyl, 
R.sub.8, R.sub.9 and R.sub.10 are similar or different and represent H or 
C.sub.1-4 -alkyl, 
R.sub.11 represents H, C.sub.1-4 -alkyl, phenyl, C.sub.1-4 -alkoxy, or 
C.sub.1-4 alkylamino and n is 0 or 1, 
is put into contact with a micro-organism which can be used in food, having 
an enzymatic activity of the beta-C-S-lyase type. 
Within the context of the invention, an R.sub.1 radical with C.sub.1-7 is 
preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, 
sec-butyl, n-hexyl or n-heptyl. 
An R.sub.8 to R.sub.10 radical is preferably H or methyl and R.sub.11 is 
preferably methyl, ethyl, phenyl, methoxy, ethoxy, methylamino or 
ethylamino. 
Included in the present invention are products of the preceding reaction 
which comprise thiols and metabolites and the use of the products in a 
food, as they are, in a flavouring composition, or as a flavour enhancer, 
and thus, the flavorant products may be incorporated in foods intended for 
human or animal consumption. 
DETAILED DESCRIPTION OF THE INVENTION 
In practice of the present invention, under the action of beta-C-S-lyase, 
the cysteyl radical of the complex compound is split in the beta position 
from the end carboxyl group leading to the corresponding thiol. 
When n is 0, the compound formed by the action of beta-C-S-lyase is then an 
alpha-mercapto aldehyde, an alpha-mercaptoacetone, an alpha-mercaptoester 
or an alpha-mercaptoamide. 
When n=1, the compound formed by the action of beta-C-S-lyase is then a 
beta-mercapto aldehyde, a beta-mercaptoacetone, a beta-mercaptoester or a 
beta-mercaptoamide. 
The preferred compounds liberated by beta-C-S-lyase are furfuryl thiol 
(FFT), benzyl thiol, 5-methylfurfuryl thiol, 2-methylfuran-3-thiol, 
2,5-dimethylfuran-3-thiol, 2-methyl-tetrahydrofuran-3-thiol and 
3-methyl-2-butene-1-thiol. 
These compounds contribute significantly to the generation of flavours of 
the meat, chicken, beef and pork type, and also of coffee as regards 
furanthiols and 3-methyl-2-butene-1-thiol. 
It has been found that the preceding preferred compounds can be partly 
metabolized into furfuryl methyl sulphide (Me-FFT), S-furfurylthio acetate 
(Ac-FFT) and bis-furfuryl disulphide (Di-FFT). These metabolites also 
possess a characteristic meaty note and may also contribute to the 
formation of flavour. 
According to the invention, the micro-organisms involved are those which 
can be used in food and are capable of possessing enzymatic activity of 
the beta-C-S-lyase type. In the present specification, a micro-organism 
which can be used in foods means any food-quality, or food-acceptable, 
micro-organism, whether this be for human or animal consumption. 
These are preferably yeasts and filamentous fungi or moulds. Among the 
yeasts, reference may particularly be made to baker's yeast, Saccharomyces 
cerevisae, but also, for example, brewer's yeast, and the yeasts Candida 
versatilis, Debaromyces hansenii, Saccharomyces bayanus and Saccharomyces 
rouxii. Among the filamentous fungi, reference may be made, for example, 
to Aspergillus oryzae, Penicillium roqueforti and Penicillium camemberti. 
Cysteine complexes able to act as a substrate in bioconversion according to 
the invention may be easily obtained by reacting an aldehyde or a ketone 
of the formula R.sub.1 --CHO or R.sub.1,R.sub.4 --CO or a ketone, ester or 
amide, alpha, beta-unsaturates of the formula CR.sub.8,R.sub.9 
=(CR.sub.10).sub.n COR.sub.11, in which R.sub.1 to R.sub.11 have the 
preceding meanings, with cysteine in a water-alcohol medium at room 
temperature. 
The incubation reaction with a micro-organism takes place under conditions 
for activating the C-S-lyase enzyme of the micro-organism. This may be 
under anaerobic or aerobic conditions, preferably anaerobic, for 5 to 72 
hours and preferably for 12 to 48 hours at pH 6-9, preferably at pH 7-8, 
with medium to strong stirring and at a temperature of 20.degree. to 
50.degree. C. and preferably around 30.degree. C. The concentration of the 
substrate, namely of the complex in the culture medium is from 1 to 100 
mmol and preferably 20 to 40 mmol. 
In particular, in the case of baker's yeast, it is preferred to operate 
under anaerobic conditions, for example under an inert gas such as 
nitrogen, so as to minimize the generation of metabolites of the baker's 
yeast itself and conversion products of such metabolites, for example by 
oxidation, and hence to direct fermentation to the production of thiols. 
Baker's yeast may be used in the form of a cream or extract. It is 
preferably fresh, in particular 0 to 18 days old, and advantageously 0 to 
8 days old, kept in a refrigerator. 
Incubation may be carried out by adding the substrate in one or more steps, 
preferably progressively, taking into account the kinetics of the 
enzymatic reaction, so that the latter does not inhibit the activity of 
the enzyme. With the same idea in mind, it could be possible to consider 
increasing the quantity of substrate and to isolate the volatile thiols as 
they are produced, so as to increase the yield and rate of the lysis 
reaction. For this purpose, volatile organic compounds can be attached to 
a support, for example a resin, and then eluted. They can also be 
extracted during incubation in a two-phase medium with the aid of a 
water-immiscible solvent, preferably of food quality, for example pentane 
or hexane. 
Following incubation, the thiols may be extracted with a solvent, 
preferably of food quality, for example pentane or hexane, and can be 
purified by conventional methods known to a person skilled in the art. 
As a variant, the thiols formed in the supernatant can be isolated, for 
example by centrifuging, and then optionally concentrated and dried, for 
example by spraying or lyophilizing in the presence of a solid support, 
for example maltodextrin. Drying takes place under moderate conditions, 
for example at a temperature of &lt;70.degree. C. under vacuum. 
The invention also concerns the use of a product of the preceding reaction 
containing thiols and their metabolites in a food, as it is, in a 
flavouring composition or as a flavour enhancer. 
Such flavours may be incorporated in foods intended for human or animal 
consumption. In the present specification a micro-organism which can be 
used in foods is understood to mean any food quality micro-organism, 
whether this be for human or animal consumption.

EXAMPLES 
The following examples illustrate the invention. In these examples, 
percentages and parts are by weight unless indicated to the contrary. 
Examples 1 to 4 
Preparation of cream of baker's yeast 
Commercial cream of baker's yeast, Saccharomyces cerevisae, containing 
22-28% dry matter, was clarified by centrifuging and the supernatant was 
discarded. The sediment was then mixed with a 0.1M aqueous solution of 
phosphate buffer at pH 7.5. 
Preparation of a cysteine-furfural complex or 
2-(2-furyl)-1,3-thiazolidine-4-carboxylic acid 
7.7 g (63.6 mmol) of cysteine were dissolved in 200 ml of a 40% aqueous 
solution of ethanol in a reactor. A solution of 7 g (72.9 mmol) of 
furfural in 20 ml of ethanol was then added drop-wise with stirring. The 
light yellow solution was left stirring and the appearance of a 
precipitate was observed at the end of 1 hour. After having placed the 
reactor in an ice bath for 1 hour, the precipitate was separated by 
filtration, washed with 2.times.50 ml of ethanol and dried under reduced 
pressure. 10.95 g of a whitish powder of cysteine-furfural complex were 
thus obtained with a yield of 86.5%. 
The structure of the compound, verified by electron impact mass 
spectrography (MS/EI) and by nuclear magnetic resonance of the proton in 
dimethyl sulphoxide (.sup.1 H-NMR) showed the presence of two cis/trans 
diastereoisomers in a ratio of 1:2. 
Incubation 
100 ml of the preceding cream of whole baker's yeast cells were then placed 
in a reactor fitted with a pH electrode, a condenser and a magnetic 
stirrer revolving at 600 rpm. The reactor was placed in an oil bath 
thermostatically controlled at 30.degree. C. The reactor was linked to a 
pH-stat keeping the pH at the chosen value (6.9 or 8) by addition of a 2N 
aqueous solution of sodium hydroxide. In the case of an anaerobic test, 
nitrogen was bubbled through the suspension for 15 min by means of a tube 
in the outlet from the condenser and the reactor was kept under nitrogen 
for the period of incubation. 0.4 g (2 mmol) and 0.8 g (4 mmol) 
respectively of 2-(2-furyl)-1,3-thiazolidine-4-carboxylic acid were added 
to this and 5 ml samples were taken at various incubation times. 
Analysis of bioconversion results 
After having adjusted the pH of the sample to 4 with a 2N aqueous solution 
of hydrochloric acid, 500 microlitres of a solution of benzyl thiol in 
pentane (2000 ppm) were added as an internal standard. After adding 3 g of 
sodium chloride, the sample was extracted with 3.times.15 ml of diethyl 
ether, the extracts were separated from the reaction medium by 
centrifuging (15 min., 5000 rpm), and these were combined, dried over 
sodium sulphate and concentrated in a Vigreux column to a volume of 2 ml, 
and the concentrated solution obtained was placed in a freezer at 
-20.degree. C. until analysed. 
Analysis by gas chromatography was carried out on a Carlo Erba Mega 2 
chromatograph fitted with a cold injector and a flame ionization detector 
(GC-FID). A flame photometric detector (GC-FPD) was used for the sulphur 
compounds. The capillary columns were DB-5, DB-1701, DB-Wax and DB-FFAP, 
30 m.times.0.32 mm, film thickness 0.25 micron, from J & W Scientific, 
Folsom, USA. The carrier gas was helium (65 kPa) with an addition of 
nitrogen (40 kPa) for the GC-FID. The retention indices (RI) were 
calculated by linear interpolation. 
The analyses were confirmed by establishing spectra produced by coupling a 
gas chromatograph with an electron impact mass spectrograph (GC/EI-MS, HP 
5890/HP 5971) under the same operating conditions as for GC-FID. 
The results for the principal sulphur compounds relating to aerobic 
incubation are shown below in table 1 and those concerning anaerobic 
incubation in table 2. 
TABLE 1 
______________________________________ 
RI (DB- RI (DB- RI RI RI (DB- 
Wax), Wax), (FFAP), 
(DB5), 1701), 
Compound 
FID FPD FPD FPD FPD 
______________________________________ 
S-methyl 
1037 1039 1048 -- -- 
thiocetate 
S-ethyl 1087 1088 1090 -- -- 
thioacetate 
Thioacetic 
-- 1148 -- -- 713 
acid 
Diethyl 1208 1192 -- -- -- 
disulphide 
2-thio-1- 
-- 1502 1500 -- -- 
ethanol 
S-furfuryl- 
2215 2217 -- -- -- 
thio 
thioacetate 
FFT 1434 1438 1437 908 1000 
Me-FFT 1486 1487 1487 998 1091 
Ac-FFT 1768 1769 1770 1159 1276 
Di-FFT 2570 2570 2602 1690 1864 
______________________________________ 
TABLE 2 
______________________________________ 
RI (DB- RI (DB- RI RI RI (DB- 
Wax), Wax), (FFAP), 
(DB5), 1701), 
Compound 
FID FPD FPD FPD FPD 
______________________________________ 
FFT 1435 1440 1438 911 1000 
Me-FFT 1487 1487 1487 998 1091 
Ac-FFT 1768 1768 1769 1159 1276 
Di-FFT -- 2570 2600 1689 1864 
______________________________________ 
--: not determined 
It was noted that aerobic incubation produced other sulphur compounds in 
addition to those sought (FFT, Me-FFT, Ac-FFT and Di-FFT). These other 
compounds probably resulted from the metabolism of cysteine. 
Anaerobic incubation mainly produced the compounds sought. 
Table 3 below gives the yield of FFT, calculated as a % of the starting 
complex, as a function of the incubation conditions. In examples 1-4, the 
yeast used was 4 days old. 
TABLE 3 
______________________________________ 
Example 1 2 3 4 
______________________________________ 
Aerobic + - - - 
Anaerobic - + + + 
pH 6.9 6.9 6.9 8 
Concentration 
20 20 40 20 
of substrate 
(mmol) 
Yield of FFT 
(%) after 
(days) 
1 25.4 14 2.2 36.9 
2 15 27.8 5 27.5 
3 11 26 6 16 
4 6 24 5 12 
7 2 8 6 8 
______________________________________ 
Example 5 
Incubation was carried out under the same conditions as for example 2, 
except for the 8 day yeast. Table 4 below gives the total yield of FFT and 
its metabolites as well as individual yields. 
TABLE 4 
______________________________________ 
Yield After After After After After 
(%) 1 day 2 days 3 days 4 days 
7 days 
______________________________________ 
Total 8.1 16.5 17.4 18.1 16.6 
FFT 6.2 15 14 12.5 13.2 
Me-FFT -- 0.5 0.5 1 0.5 
Ac-FFT 1.9 1 1.9 2.6 0.5 
Di-FFT -- -- 1 2 2.4 
______________________________________ 
--: not determined 
Example 6 
Incubation was carried out under the same conditions as for example 4, 
except for the 8 day yeast. Table 5 below gives the total yield of FFT and 
its metabolites as well as individual yields. 
TABLE 5 
______________________________________ 
Yield After After After After After 
(%) 1 day 2 days 3 days 4 days 
7 days 
______________________________________ 
Total 41 32 22 17 24 
FFT 37 28 17 13 8 
Ac-FFT 2 -- -- -- -- 
Di-EFT 2 4 5 4 16 
______________________________________ 
--: not determined 
Example 7 
Incubation was carried out under the same conditions as for example 2, 
except that fresh yeast (0 days) and 18 day yeast were used respectively. 
In order to carry out incubation with an 18 day yeast, the yeast was kept 
in a refrigerator at 4.degree. C. Just before use, the phosphate buffer 
solution was removed by centrifuging and was replaced by the same volume 
of fresh buffer. 
Table 6 below gives the yield of FFT. 
TABLE 6 
______________________________________ 
FFT 
Yield After After After After After 
(%) 1 day 2 days 3 days 4 days 
7 days 
______________________________________ 
Fresh 1 16 24 -- -- 
yeast 
18 day 13 22 22.5 20 9 
yeast 
______________________________________ 
--: not determined 
Examples 8 to 10 
Proceeding as in example 2, the following complexes were incubated: 
8. Cysteine-benzaldehyde and its bioconversion into benzyl thiol was 
obtained. 
9. 5-methyl-furfuryl-cysteine and its bioconversion into 5-methyl-furfuryl 
thiol was obtained, and 
10. 2-methyl-tetrahydrofuranyl-cysteine, which led to 
2-methyl-tetrahydrofuran-3-thiol. 
Examples 11 to 13 
Proceeding in a similar manner to that used in example 2, conversion of 
cysteine-furfural into FFT was obtained by incubation with the following 
micro-organisms: 
11. Candida versatilis 
12. Debaromyces hansenii 
13. Saccharomyces bayanus.