Background of the invention Several thiols are impact aroma compounds in roasted and cooked food flavours. Currently, most flavour compounds are produced by chemical synthesis or by extraction from natural sources. However, decreasing or variable availability of certain natural sources due to climatic instability and the lack of convincing, chemosynthetic alternative have stimulated investigations to produce flavours in controlled biotechnologically processes. Accordingly, EP 0963706 discloses a flavor building block containing precursors which can generate thiols during reheating of a ready-to-eat dish.

Natural flavours are defined as"biologically derived aromas generated by microbial fermentation and by the action of endogenous or processing enzyme".

Among these enzymes, lipases and esterases have received special attention because of their effectiveness in regio-and enantioselective esterifications and transesterifications of organic acids and alcohols (Dordick, J. S. Enzymatic catalysis in monophasic organic solvents. Enzyme. Microb. Technol. 1989,11,194-211).

The use of enzymes in different media such as organic solvents or supercritical fluids, has been the subject of an extensive amount of research. These organic solvents offer many advantages over aqueous systems such as increased solubility of nonpolar substrates, reduction of water-dependant side reactions, enhanced thermal stability of enzymes and ease of products recovery from low boiling solvents.

Applications of hydrolytic enzymes, in particular lipases, have been also considered in treatments of various dairy products like creams, milk and cheese.

Moreover, they are the main catalysts in the production of numerous flavours or flavour enhancers such esters, alcohols, aldehydes and organic acids and particulary for sweet flavour area.

The present invention aims to provide a novel method for generating thiols and their disulfides by using enzymes as biocatalysts, and their use as enhancers or intensifiers of flavour in food products.

Summary of the invention Accordingly, this invention provides a process for generating natural thiols in which, at least one thioacetate of the general formula Rl SCOR2 in which: Rl is linear or branched alkyl, phenyl, aryl, alkenyl, and their alcohol, ketone and aldehyde derivatives, heterocycle such furanes, thiazole, thiazoline, thiophenes, pyrroles, subsituted or not, and terpenes.

R2 is CH3, C2H5 or C3H7 is enzymatically hydrolysed in the presence of a food-grade solvent or solvent mixtures thereof, the obtained thiols having in all cases the general formula Rl SH.

These thiols may be transformed into their disulfide derivatives with formula RISS, by oxydative processes.

In a preferred embodiment, the enzyme may be hydrolase such as lipases, esterases, carbonic anhydrases, and all enzymes and microorganisms with hydrolytic activity.

The food-grade solvent may be any food-grade organic solvent or any aqueous buffer or a mixture thereof.

This method provides a convenient way to produce natural thiols and disulfides which could be used as flavour enhancers or intensifiers of flavour in food products (thiols), in particular for roasty notes or for the in-situ aroma generation (disulfides).

Another object of the invention relates to the use of thioesters, and particularly thioacetate prepared by addition of an thiocarboxylic acid as thioacetic acid or salt derivatives thereof, on a-, ß-, or y-unsaturated alcohols, carbonyl compounds and cyclic or acyclic alkenes or by nucleophilic substitution of the hydroxyl group or a halogen with S-acetyl group in food-grade organic solvents, as substrate for bioconversion into thiols according to the present invention.

In one embodiment, thioacetate is generated by addition of thioacetic acid on a-, 0-or 7-unsaturated alcohols, carbonyl compounds, cyclic or acyclic alkenes, and heterocycles in food-grade organic solvents, for example.

In another embodiment, it is also generated by nucleophilic substitution of the hydroxyl group of saturated or unsaturated alcohols and halogens with S-acetyl group, in food-grade organic solvents, for example.

In a preferred embodiment, the thioacetates generated may be directly hydrolysed into thiols in the same reaction by using immobilized enzymes.

Another object of the invention relates to sulfur containing compounds, and particularly mercaptoterpenes and their thioacetate derivatives that are obtainable by the process according to the present invention.

Detailed description of the invention

In the present invention, the term"alkyl"refers to substituted or non substituted linear or branched chain carbon groups cyclic or not and heterocycles of 1 to 20 carbon atoms, preferably 1 to 6 carbon atoms.

According to the first object of the present invention, the thioester is preferably a thioacetate.

The present process may be carried out in any food-grade organic solvent.

Typical solvents suitable for use in the present process may include hexane, propane, propylene glycol, polyethylene glygol, super critical carbon dioxide, super critical propane or triacetin, for example. It is possible to use aqueous solvents, in particular water or aqueous buffer such as phosphate, borate or citrate for example.

The enzymes used in the present process may be any enzyme or microorganism having the ability to hydrolyse thioesters of the general formula as described above. Various enzymes, such as lipases, proteases, esterases, carbonic anhydrases, and all enzymes and microorganisms with hydrolytic activity, may be used as biocatalyst. The enzyme may be an hydrolytic enzyme from any origin or whole microorganisms with hydrolytic activities.

In a preferred embodiment, commercially available lipase from Candida cylindracea (EC 3.1.1. 3) or Immobilized enzyme (EC. 3.1.1.3) from Candida rugosa are used.

To carry out the process of the present invention, the enzyme and thioacetate are added to the solvent. The enzyme may then be present in the reaction solution in concentration of at least 1 IU/ml of solvent and preferably from about 4 IU to about 50 IU/ml of solvent. The ratio enzyme: thioacetate may be of about 10-500 IU of enzyme for about 3x10-5 to 1 OOxl 0-3 mol of the thioacetate.

The pH of the reaction in aqueous solutions may be maintained between about 4 and 10, and it is preferably comprised between 5.5 to 7.5.

The reaction time can be appropriately varied depending upon the amount of enzyme used and its specific activity and upon the reaction conditions (temperature, pH,...). It may be of at least 5 minutes and up to about 48 hours and preferably between 15min and 6 hours The thiols may be isolated from the reaction mixture and purified by known methodologies. In a preferred embodiment, the thiols formed in the supernatant can be extracted and separated, for example by centrifugation or by filtration, 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 heating conditions, for example at a temperature of < 70°C for spray drying.

In one embodiment, a mixture of various thioesters may be used. This mixture can be constituted by any compound as described above and the number can be illimited. It is possible to use mixture of S-3- (2-methylfurfuryl) thioacetate and S- furfuryl thioacetate, for example. The concentration ratio between these 2 compounds may be for instance, when the hydrolysis of the mixture is performed, from about 1/1 to 1/10 or 1/1 to 10/1.

In another preferred embodiment, mercaptoterpenes may be prepared. Thus, the thioester can have the following formula: R, SCOR2 in which the Rl is a monoterpene, a sesquiterpene, a diterpene, a triterpene, a tetraterpene or a polyterpene, and aldehydes, alcohols, ketones and oxides analogues and R2 is CH3, C2H5 or C3H7.

The thiols according to the invention and their disulfide derivatives obtained by further oxydative processes, or mixture thereof, could be applied as flavour compounds or flavour boosters for aroma foodstuffs.

The invention also concerns the use of a product of the preceding reaction containing thiols and their derivatives 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.

The disulfides formed in the hydrolysis reaction of a single thioacetate derivative or mixture, thereof, could be applied for in-situ generation of aroma as described in EP 0963 706.

All these compounds contribute significantly to the generation of flavours of the meat and savoury products and also roasted coffee and sweet flavours (fruity, tropical juices,..).

According to another object, the invention relates to the use of thioacetate prepared by addition of an thiocarboxylic acid as thioacetic acid or salt derivatives thereof, on a-, ß-, or y-unsaturated alcohols, carbonyl compounds and cyclic or acyclic alkenes or by nucleophilic substitution of the hydroxyl group or a halogen with S-acetyl group in food-grade organic solvents, as substrate for bioconversion into thiols by enzymatic hydrolysis said thioacetate in the presence of a food-grade solvent.

The substrate may then be a-, (3-or y-unsaturated alcohols (primary, secondary and tertiary), aldehydes, hydroxyketones, primary, secondary or tertiary halogens (alkyle, alkenyl, cyclalkyl,...), carbonyl compounds and cyclic or acyclic alkenes and heterocycles such furanes, pyrroles, thiazoline, thiazoles or thiophenes.

The sulfur source for thioacetates generation could be: thioacetic acid, potassium thioacetate, sodium thioacetate,... and all thiocarboxylic acids and their salt derivatives thereof. Thioacetic acid may also be obtained in natural form according to the patent EP 778350, for example.

The food grade organic solvent may be the same as described above. The ratio substrate: thioacetic acid is preferably comprised between 1: 0.2 to about 1: 5 depending on which thioacetate is to be prepared.

The reaction may be conducted at a temperature of about 10°C to 80°C preferably at 25 °C and during about 5 minutes to 48 hours.

The obtained thioacetates can be used in a food, as it is, in a flavouring composition or as a flavour enhancers. Such flavours may be incorporated in foods intended for human or animal consumption.

They can also be used as substrate for bioconversion into thiols as described above. Accordingly, the thioacetates generated may be directly hydrolysed into thiols in the same reaction by using immobilized enzymes.

According another object, the invention provides novel thiols and thioacetate derivatives and particularly mercaptoterpene derivatives that can be obtained by the process according to the present invention.

These compounds have the following formula: 1 2,3,4,5,6 7, 8, 2, 10, I 1, 37, 38, __, 41, 43,44,45, and 46.

The aroma character of these volatile compounds is described as meaty, savoury, roasted, vegetable, sulfury, garlic and onion-like.

In a preferred embodiment, these new aroma compounds are prepared by the process as described above.

The following examples are given by way of illustration only and in no way should be construed as limiting the subject matter of the present application. All percentages are given on the basis of weight except where specifically stated otherwise.

Figures: Fig. 1 : Effect of pH on enzymatic reaction and products stability: FF- Thioacetate (0.064 mmol), CCL (77 units), buffer 10 ml, pH 8.0

Fig 2: Effect of pH on enzymatic reaction and products stability : FF-Thioacetate (0.064 mmol), CCL (77 units), buffer 10 ml, pH 7.0 Fig 3: Effect of pH on enzymatic reaction and products stability : FF-Thioacetate (0.064 mmol), CCL (77 units), buffer 10 ml, pH 6.0 Fig 4: Effect of the quantity of lipase on the reaction rate: FF-Thiacetate (0.064 mmol), CCL (13 units), H20 (10 ml), 23 °C, pH 5.8 Fig. 5: Effect of the quantity of lipase on the reaction rate: FF-Thioacetate (0.064 mmol), CCL (65.6 units), H20 (10 ml), 23 °C, pH 5.8 Fig 6: Effect of the quantity of lipase on the reaction rate: FF-Thioacetate (0.064 mmol), CCL (77 units), H20 (10 ml), 23 °C, pH 5.8 Fig 7: Effect of the quantity of lipase on the reaction rate: FF-Thioacetate (0.064 mmol), CCL (262 units), H20 (10 ml), 23°C, pH 5.8 Fig 8: Effect of the quantity of lipase on the reaction rate: FF-Thioacetate (0.64 mmol), CCL (65 units), H20 (10 ml), 23 °C, pH 5.8 Fig. 9: Effect of the temperature on the reaction rate and product stability, Reaction at 4°C Fig 10: Effect of the temperature on the reaction rate and product stability Reaction at 23 °C Fig 11: Effect of the temperature on the reaction rate and product stability Reaction at 37 °C Fig 12: Influence of the quantity of enzyme on the reaction rate: MF-Thioacetate (0.065 mmol), Enzyme (CCL, 65.5 units), distilled water, 23 °C, pH 5.8 Fig 13: Influence of the quantity of enzyme on the reaction rate: MF-Thioacetate (0.065 mmol), Enzyme (CCL, 26.2 units), distilled water, 23 °C, pH 5.8 Fig 14: Influence of the quantity of enzyme on the reaction rate: MF- Thioacetate (0.065 mmol), Enzyme (CCL, 6.5 units), distilled water, 23 °C, pH 5.8 In examples 1 to 9, the chemicals were of analytical grade and were purchased from Fluka and Aldrich. 2-Methyl-3-furanthioacetate was purchased from Oxford. All the solvents were of analytical grade and were purified by distillation using a Vigreux column (60cm x 3cm). Lipase from Candida cylindracea (EC 3.1.1.3) was purchased from Sigma (943 units/mg solid, 1,310 units/mg protein, one

enzyme unit hydrolysed 1.0 microequivalent of fatty acid from olive oil in one hour atpH7. 2at37°C).

Immobilized enzyme (EC. 3.1.1.3,30 units/g) from Candida rugosa, supported on Sol-Gel-Ak was purchased from Fluka.

Gas chromatography and gas chromatography-olfactometry were performed on a Carlo Erba gas chromatograph (Mega 2 series) equipped with an automatic cold on-column injector, a flame ionisation detector (FID), a flame photometer detector (FPD) and sniffing port. Fused silica capillary columns were used (DB-1701 and DB-FFAP), 30 m x 0.32 mm, film thickness 0.25 am. The carrier gas was helium (80 kPa), make-up gas for the FID was nitrogen (40 kPa). The injected volume was 0.5 p1. The oven was temperature programmed as follows: 35°C (2 min), 40°C/min to 50°C (2 min), 8°C/min to 180°C, 10°C/min to 240°C (10 min). Retention indexes were calculated by linear interpolation.

GC-MS analyses were performed with a Finnigan MAT-8430 mass spectrometer connected to an HP 5890 gas chromatograph using the same conditions as described above. The MS-EI spectra were generated at 70 eV and MS-CI at 150 eV with ammonia as reagent gas and the mass range was 20 to 500 Da.

Example 1: hydrolysis of S-furfuryl thioacetate (FF-thioacetate) To 10 ml of a solution of S-furfuryl thioacetate (FF-thioacetate) (0.064 mmol) in distilled water or phosphate-buffer (0.2 M), different quantities of enzyme were added and reactions were performed at different temperatures for 1 min to 72 h with gentle magnetic stirring.

Samples were withdrawn at various time intervals. 500 u. I benzyl mercaptan in diethyl ether (2000 ppm) was then added as internal standard and the mixture extracted by distilled diethyl ether. The ether extracts were dried over sodium sulphate and concentrated using a Vigreux column (30 cm x 1 cm) to a volume of 2 ml. The concentrated solution was then analyzed by various chromatographic techniques (GC, GC-MS, GC-O) and the respective concentrations of thioacetates, thiols and disulfides were determinated using gas chromatography. The influence of the following parameters were studied: pH Temperature Ratio lipase/substrate Solvent (°C) (units/mmol) 6.0 4 13/0.064 Water 7.0 23 65.5/0.064 Phosphate buffer 8.0 37 77/0.064 n-hexane 65/0.64n-pentane

The reaction can also be performed in an organic solvent. Accordingly, to 10 ml of a solution of thioacetate (0.064 mmol) in distilled n-hexane or n-pentane, different quantities of immobilized lipase from Candida rugosa were added and reactions performed at room temperature with magnetic stirring. Samples were withdrawn at various time intervals, filtered and five u. l benzyl mercaptan in n- hexane (2000 ppm) was then added as internal standard. The samples were analyzed by gas chromatography.

Influence of the pH on the enzymatic activity and products stability To a solution of FF-thioacetate (0.064 mmol) in 10 ml of potassium phosphate buffer (0.2 M), lipase from Candida cylindracea (77 units) was added.

Reactions were performed at different pHs (6.0,7.0, and 8.0) and at room temperature (23 °C). A kinetic study was performed for each reaction and the influence of the pH on the reaction rate, the yield of FFT and the formation of disulfide were studied.

As shown in scheme 1, enzymatic hydrolysis of FF-thioacetate allowed to generate FFT which could be transformed into dimer by chemical oxidation. The yields of FFT and Di-FFT were determined after quantification by gas chromatography using an internal standard (benzyl mercaptan). Reaction controls were carried out in the same conditions for each pH and without enzyme. Results are summarized in the figures 1 to 3.

The maximum yield of FFT was obtained at pH 6.0 (73 %) whereas at pH 8.0 this maximum was only 54 %. However, the generation of Di-FFT was marginal in all cases. We conclude that the optimum pH for this reaction is from 5.5 to 7.0.

Influence of the quantity of the lipase on the reaction To study the influence of the quantity of lipase on the reaction rate and the overall yield, reactions were performed in water at pH 5.8 and at room temperature.

Different concentrations of enzyme were used proportionally to the substrate. Results are summarized in the figures 4 to 8.

As we can observed in figures 4 to 8, the concentration of enzyme influence the reaction rate but had no effect on the yield. In all cases, the maximum yield obtained was from 70% to 80% but this maximum could be obtained after lh (fig 7) when 65 to 262 units of enzyme were used to transform 0.064 mmol of substrate or after 24h (fig 8) were only 65 units were used to transform 0.64 mmol of substrate.

Influence of the temperature on the reaction rate and product stability To a solution of FF-thioacetate (0.064 mmol) in 10 ml of water at pH 5.8,100 mg of lipase from Candida cylindracea (77 units) were added. Reactions were performed at different temperatures (4,23, and 35 °C) and results are shown in figures 9-11.

As shown in these figures, the temperature had an influence on the reaction rate and on the stability of the product (FFT). In fact, at 4 °C the reaction is slower than at 23 and 37 °C, and the maximum of FFT yield was obtained only after 24h reaction time. However, at 4 °C the stability of FFT is higher than at 23 and 37 °C.

Moreover, no big difference was observed between reactions performed at 23 °C and 37 °C.

Example 2 : Hydrolysis of S-3- (2-Methylfuryl) thioacetate (MF-Thioacetate).

To a solution of MF-thioacetate (0.064 mmol) in 10 ml of distilled water, different amounts of lipase from Candida cylindracea (65.5,26.2 and 6.5 units) were added. The reactions were performed at room temperature (23 °C) and at pH 5.8. A kinetic study was performed for each reaction and the influence of the quantity of enzyme was studied. Results are summarized in the figures 4 to 8.

As shown in scheme 2, the enzymatic hydrolysis of MF-Thioacetate allowed to generate 2-methyl-3-furanthiol (MFT) which then could be transformed into Di- MFT by oxydative process. The yields of the generated products were determined by quantification on gas chromatograph using an internal standard (benzyl mercaptan).

Reaction controls were carried out in the same conditions but without enzyme and results shown no hydrolysis of MF-thioacetate.

As shown in these figures, the amount of enzyme influence not only the reaction rate but also the yield of MFT produced and its degradation into the corresponding disulfide (disulfide, bis (2-methyl-3-furyl)). The highest yield of MFT (88 %) was obtained after 15 min when high quantity of enzyme was used (Fig 12).

Example 3: Hydrolysis of mixture of S-3- (2-methylfuryl) thioacetate (MF- Thioacetate) and S-furfuryl thioacetate (FF-Thioacetate) Mixtures of FF-Thioactetate and MF-Thioacetate, at different concentrations, were incubated with different lipases from different origins (e. g. Candida cylindracea, Candida antarctica) in water and at room temperature. After a few minutes (15min-30min) all the substrates were transformed and a mixture of several compounds, principally disulfides, was obtained (Scheme 3).

A number of these disulfides have been identified amongst the volatile products of the Maillard reaction between sulphur-containing amino-acids and reducing sugars (Farmer and Mottram, 1990). Their odor has been described as meaty, roasted coffee and roasted meat. These disulfides could be used for in-situ aroma generation as described in the Patent Application EP 0963706 of 07. 05.1998.

Example 4: Reaction between thioacetic acid and prenyl alcohol : Synthesis of thioacetate derivatives of 3-methyl-2-buten-1-ol A solution of 1 mmol of 3-methyl-2-buten-1-ol and 1 mmol of thioacetic acid in 10 ml of freshly distilled n-hexane, was stirred at room temperature, 35 °C or 50 °C. Samples were withdrawn at different reaction times (Imin to 24h) and analyzed by GC. As soon as all the substrates are consumed, the reaction is stopped and the mixture analyzed by GC-MS to identify the reaction products. The obtained thioacetate derivatives were then enzymatically hydrolyzed in water or phosphate buffer as described in example 1 or in n-hexane or n-pentane as described in example 2.

Prenyl alcohol and thioacetic acid were reacted at the concentration ratio of 1 mmol and 0.5 mmol respectively, and at 40 °C. In these conditions, compound 1 was the predominant volatile generated in the reaction. However, when the two substrates were reacted at concentration ratio of 1 mmol to 2 mmol and at 40 °C, compound 2 was the predominant volatile in the mixture.

These two compounds 1 and 2 have never been reported. They were detected by GC-FID, GC-FPD and GC-O and were identified on the basis of their mass spectroscopy data analysis. By GC-O, these compounds were described as roasty, meaty, sulfury, onion and garlic-like.

Example 5: Synthesis and in-situ enzymatic hydrolysis of thioacetate derivatives of 3-methyl-2-buten-1-ol.

To a solution of 1 mmol of 3-methyl-2-buten-1-ol and 2 mmole of thioacetic acid in 10 ml of n-hexane, different amounts of immobilized lipase from Candida rugosa were added. The reactions were performed at 35 °C and under magnetic stirring. Samples were withdrawn at various time intervals, filtered, and five hundred 1 benzyl mercaptan in n-hexane (2000 ppm) were added. The mixture was then analyzed by GC and GC-MS.

To the obtained compounds 1 and 2 (see example 4), immobilized enzymes from different origins were added in distilled n-hexane or distilled water, and reactions were performed at 37 °C. Kinetic studies were carried-out and samples were analyzed by GC and GC-MS.

Enzymatic hydrolysis of compound 1 allowed to form compound 15 which was identified by comparison of its mass spectra, retention indices (RI) and odour quality with those of literature data (Holscher 1992).

However, the enzymatic hydrolysis of compound 2 by different hydrolytic enzymes in n-hexane, allowed to specifically form compound 3 which has never been reported. The aroma character of this volatile is meaty, sulfury, garlic and onion-like.

It should be pointed out that the enzymatic hydrolysis of compound 2 in n- hexane was regioselective because just one thioacetate (compound 2 contains two thioacetates) was hydrolyzed into thiol group.

The condensation between prenyl alcohol and thioacetic acid, and the enzymatic hydrolysis reactions were performed in a single step using the same conditions described above.

Example 6: Chemo-emzymatic synthesis of sulphury prenyl alcohol derivatives via prenyl formate This example shows the synthesis of thioacetates of 3-methyl-2-buten-1-ol via prenyl formate.

To a solution of 3-methyl-2-buten-1-ol (1 mmol) in 10 ml of distilled n- hexane, 1.1 mmol of formic acid was addded and the mixture stirred at room temperature overnight. The reaction was then stopped and the mixture analyzed by GC and GC-MS. To the generated prenyl formate, 1 mmol of thioacetic acid was

added in 10 ml of distilled n-hexane. After 4h, prenyl formate was transformed, the reaction was then stopped and mixture analyzed by GC and GC-MS.

The resulting thioacetate derivatives were then enzymatically hydrolyzed in water or in phosphate buffer or in n-hexane as described in example 1.

Prenyl formate 16 was obtained by incubation of prenyl alcohol and formic acid in hexane and at room temperature. This compound was then reacted with thioacetic acid to obtain compound 17 which was hydrolyzed by lipases as described above to obtain compound 18. The natural thiol 18 is of great importance for the aroma of roasted coffee, for example.

Example 7: synthesis of 3-mercapto-3-methylbutyl formate Compound 15 could be transformed into 3-mercapto-3-methylbutyl formate 21 by reaction with formic acid in n-hexane as described in example 6.

By analogy, compound 3 should be hydrolyzed by enzymes in water or buffer media to give compounds 19 and 20. Compounds 20 and 21 have been identified in roasted coffee earlier (Silwar 1982).

Example 8: Mercaptopropanone and thioacetate derivatives To a solution of hydroxypropanone 22 (1 mmol) in 10 ml of distilled n- hexane, formic acid (1.5 mmol) was added at 35 °C and under magnetic stirring.

After the transformation of all hydroxypropanone, the mixture was analyzed by GC and GC-MS. The main volatile identified in the mixture was compound 23. This molecule was then incubated with thioacetic acid in n-hexane to produce thioacetate derivative 24. The enzymatic hydrolysis of this latter, using the conditions described above, allowed to mercaptopropanone 25.

Mercaptopropanone has been detected in cooked beef liver, canned pork and sesame seed. This molecule is also an interesting precursor for Maillard reaction.

By analogy, mercaptopropanone 25 could also be obtained by interaction between hydroxy propanone and potassium thioacetate in polyethylene glycol or Triacetin followed by enzymatic hydrolysis.

Example 9: Preparation of mercapto-limonene and thioacetate derivatives A solution of 1 mmol of d-limonene 39 and 1 to 3 mmol of thioacetic acid in 10 ml of distilled n-hexane, was stirred at 35 °C. Samples were withdrawn at different reaction times (5 min to 24h) and analyzed by GC and GC-MS.

As soon as all the limonene was transformed, the reaction is stopped and the mixture analyzed by GC-MS to identify the reaction products. The obtained thioacetate derivatives (7, 8, 9, 42,43) were then enzymatically hydrolyzed in organic solvent or phosphate buffer as described in example 1 and 2.

The enzymatic hydrolysis allowed to form a mixture of thiols 26, 27,28 and 29.

Example 10: a-Terpineol derivatives 1. Experimental Equipement: Magnetic stirrer, IKA Labortechnic, model RCT basic; Thermomixer Eppendorf; Rotative evaporator, Buchi EL-131; pH-stat system, Metrohm models 691 pH meter + 665 Dosimat + 614 Impulsomat; Magnetic stirrer, Heidolph, model MR3003, equipped with an oil.

Reagents: All reagents were of analytical grade and were purchased from Fluka (thioacetic acid, ethanol, potassium thioacetate) and from Merck (a-terpineol, n- hexane, polyethylenglycol).

Enzymes: Lipase from Candida cylindracea (CCL) immobilized on Sol-Gel-AK (Fluka 62278); Lipase from Candida antarctica (CAL) immobilized on Sol-Gel-AK (Fluka 62217); Lipase from Candida cylindracea, type VII (Sigma L-1754); Novozyme 871 L (Novo Nordisk); Palatase 20000L (Novo Nordisk); Lipopane 50 BG (Novo Nordisk); Lipase AYS (Amano); Lipase AS (Amano); Pig Liver Esterase (PLE) (Sigma E-3019); Immobilized Pig Liver Esterase (Fluka 46064).

Generation of thioacetates: Ten mmoles of terpene (a-terpineol) were solubilized in 5 ml of ethanol and added to a solution of 20 mmol thioacetic acid in 94 ml of distilled n-hexane. The mixture was kept under magnetic stirring and at room temperature. Samples were withdrawn at different reaction times and analyzed by gaz chromatography. After five hours the terpene was completely transformed, the reaction was stopped and the mixture was analyzed by various chromatographic techniques. The obtained terpene thioacetate derivatives were then characterized based on their mass spectra.

Enzymatic hydrolysis of terpene : In phosphate buffer: To 2.3 ml of a solution of thioacetate (460 mg) in ethanol, different quantities of lipases or esterases in phosphate-buffer (43.7 ml, 100 mM), were added and reactions were performed at 25 °C for 1 min to 48 h with gentle magnetic stirring. Samples were withdrawn at various time intervals, acidified to pH 4.0 with hydrochloric acid and after addition of sodium chloride (saturation), the mixture was extracted with distilled dichloromethane. The organic phases were then dried over sodium sulphate and analyzed by various chromatographic techniques (GC, GC-MS, GC-O). A Kinetic study was performed using different enzyme/subtrat ratios.

In organic solvent: To 2.3 ml of a solution of thioacetate (460 mg) in ethanol, 43.7 ml of distilled n-hexane and different quantities of immobilized lipases or esterases were added. Reactions were performed at room temperature with magnetic stirring. Samples were withdrawn at various time intervals, filtered and analyzed by different chromatography techniques (GC, GC-MS, GC-O).

Chromatography analyses (GC, GC-O, GC-MS): Gas chromatography was performed on a Agilent gas chromatograph (6890 series) equipped with a flame ionisation detector (FID) and a flame photometer detector (FPD). Fused silica capillary column was used (DB-Wax), 30 m x 0.25 mm, film thickness 0.25 um. The carrier gas was helium (80 kPa), make-up gas for the FID was nitrogen (40 kPa). The injected volume was 0.2 ul. The oven was temperature programmed as follows: 20°C (1 min), 70°C/min to 60°C (2 min), 4°C/min to 220°C (20 min).

Gas chromatography-olfactometry was performed on a HP 5890 Series II gas chromatograph equipped with sniffing port. Retention indexes were calculated by linear interpolation.

GC-MS analyses were performed with a Finnigan MAT-8430 mass spectrometer connected to an HP 5890 gas chromatograph using the same conditions as described above. The MS-EI spectra were generated at 70 eV and MS-CI at 150 eV with ammonia as reagent gas and the mass range was 20 to 500 Da. a-Terpineol derivatives Characterization of thioacetate derivatives by GC-MS p-menthan-8-ol-2-acetvlthio 32: MS-EI, m/z (relative intensity): 230 (2) [M] +, 215 (12) [M-CH3] +, 212 (3) [M-H20] +, 172 (30) [M-C3H60C] +, 136 (100) [M-C2H602S] +, 121 (60) [M-C3H9O2S] + p-menthan-8-ol-2-acetvlthio 33: MS-EI, m/z (relative intensity): 230 (2) [M] +, 215 (6) [M-CH3] +, 212 (3) [M-H2O] +, 172 (28) [M-C3H601+, 13 6 (100) [M-C2H602S] +, 121 (55) [M-C3H902S] +

Characterization of thiol derivatives by GC-MS 2-mercapto-8-p-menthanol 37 or 38: MS-EI, m/z (relative intensity): 188 (2) [M] +, 170 (100) CM-H201+, 155 (10) [M-CH50] +, 136 (67) [M-H30S] + 2-mercapto-8-p-menthanol 37 or 38: MS-EI, m/z (relative intensity): 188 (6) [M] +, 170 (100) [M-H2O] +, 155 (15) [M-CH501+, 136 (70) [M-H30S] + Purification of mercapto-terpineol derivatives Silica gel column: Mercapto-p-menthanols were obtained as described above. The mixture was then purified on silica gel column using pentane-ether (1: 1 to 1: 9; v/v) as eluent.

Thin Laver Chromatography (TLC): The obtained fractions from column chromatography were analyzed by thin layer chromatography (TLC); mobile phase: pentane-ether (1: 9; v/v); detection reagents: 1) 6g vanillin dissolved in 197ml ethanol and 3ml sulfuric acid; 2) solution of sulfuric acid (10%) in ethanol; detection: 180 °C, 2 min.

High Performance Liquid Chromatography (HPLC) HPLC system: HP series 1100, equipped with a photodiode array detector. Column.

Nucleosil 110-7-OH. Detection: 254 nm, Mobile phase: pentane-ether 3: 7 (v/v), Mode: socratic, Injection: 50 il.

2. Results 2.1 Generation of thioacetate derivatives Thioesters are commonly used in the food industry as fruit flavours and are added to soups, meat sauces, dairy and baked goods and cheese (12). In this study, thioacetate derivatives were produced by reaction of a-terpineol 30 with thioacetic acid 31 in distilled n-hexane and at room temperature as shown in scheme 4.

Reactions between substrates 30 and 31 were performed at different concentration ratios (lmole/lmole, 2moles/lmole, lmole/2moles). Best results were obtained when 2 moles of thioacetic acid were reacted with 1 mole of a-terpineol. In fact, after 5 h reaction time, a-terpineol was completely transformed and the reaction mixture was analyzed by gas chromatography. The results are summarized in table 1.

The predominant volatiles generated in the reaction were compounds 32 and 33 and the sample was described as grapefruit, sweet, green and sulphury.

Table 1: Gas chromatography analyses of a-terpineol thioacetate derivatives Retention Index (DB-WAX) Odour description by GC-O FID FPD Compound 32 2420 2426 sulphury, roasted, meaty, green, exotic Compound 33 2435 2440 sulphury, intensive, green, fruity, sweet The aroma volatiles 32 and 33 detected by GC-FID, GC-FPD and GC-O were tentatively identified on the basis of their mass spectroscopy data analyses. The proposed structures for compounds 32 and 33, were then confirmed by nuclear magnetic resonance (NMR) of the corresponding thiols obtained by enzymatic hydrolysis of thioacetates 32 and 33 (see above). Moreover, according to the NMR data analyses, it seems that compounds 32 and 33 are probably two diastereoisomeric forms (and) of p-menthane-8-ol-2-acetyl thio. This indirect characterisation of compounds 33 and 34 is due to the difficulty to purify the two diastereoisomers 34 and 35 by low pressure liquid chromatography and HPLC.

Several sulphur-containing terpenes have been described as powerful flavor impact constituents of buchu leaf oil or of grapefruit juice. However, compounds 32 and 33 have never been reported in the literature. As shown in table 1, the aroma character of these two stereoisomers, was described as sulphury, fruity, roasted, green and sweet.

First application trials using a-terpineol thioacetate derivatives as top-notes were found very interesting for lighter cooked notes and in tropical fruits.

For practical and stability reasons, flavouring powders were also prepared. In fact, the thioacetates solution was incorporated into maltodextrine and the mixture was lyophilized. To evaluate the volatile losses, the powder and liquid phases were extracted and analyzed by gas chromatography and losses were evaluated to about 2%.

To study the relation between the stereochemistry and the aroma activity, it is necessary to determine the absolute configuration of the chiral aroma compounds 34 and 35. As shown in table 1, the two stereoisomeric forms exhibited different odour quality. This phenomenon has been observed and reported for other similar aroma compounds. In fact, it was showed that the four stereoisomers of 3-oxo-p-menthane- 8-acetyl thio 36, compound found in buchu leaf oil, exhibited different aroma character and odour quality as summarized in table 2.

Table 2: Aroma character of the four stereoisomers of 3-oxo-p-menthane-8-acetyl thio Absolute Sensory evaluation Configuration (0.1 % in water) 1 R, 4R musty sulphury note, intensive 1 S, 4S green, black currant, exotic, intensive 1R, 4S delicate, fruity, I 1 S, 4R stron, sweet, slightly pungent 2.2 Generation of thiol derivatives To do so, the mixture of a-terpineol thioacetate derivatives 32 and 33 obtained as described above, was reacted with hydrolytic enzymes (lipases, esterases) to obtain the corresponding thiols as shown in scheme 5.

2.3 Screening of enzymes

Several lipases, as listed above, have been used to perform the enzymatic hydrolysis of thioacetates into thiols. These enzymatic reactions were carried-out at different conditions such solvent (water, phosphate buffer or organic solvent when using immobilized enzymes), temperature (25°C, 30°C and 38°C) and pH (6.0,7.0 and 8.0). However, no hydrolytic activity was observed after 72 h reaction time. The hydrolysis of thioacetates was observed only when pig liver esterase (PLE) was used as biocatalyst.

2.4 Enzymatic hydrolysis of thioacetate derivatives with pig liver esterase (PLE) Because compounds 32 and 33, are slightly or not soluble in water, the hydrolysis was performed in 0.1 M phosphate buffer containing 5% of ethanol.

Reactions were carried-out at room temperature and at pH 8.0.

A kinetic study was performed, and after 48h all thioacetates were transformed. This result means that the hydrolysis reaction was not stereospecific with the crude pig liver esterase used in this study. Moreover, the same reaction was performed using purified PLE using the same conditions as described above. No stereoselectivity was observed because all thioacetates were transformed after 48h reaction time. Trials were also carried-out using immobilized PLE in organic solvent (n-hexane containing 5% ethanol) and at room temperature but no enzymatic hydrolysis was observed after 78 h reaction time.

2.5 Identification of thiol derivatives by chromatography analyses After 48 h enzymatic reaction with PLE, the mixture was extracted with dichloromethane or with distilled diethyl ether and the extract was analyzed by various chromatographic techniques (GC-FID, GC-FPD, GC-O, GC-MS). Results of these spectroscopy analyses are summarised in table 3. For the first time compounds 37 and 38 were tentatively identified on the basis of their mass spectroscopy characteristics. The global flavour character of the mixture after enzymatic hydrolysis was described as soup, bouillon, meat-like, sulfury, lemon-like, green and exotic. As shown in table 3, the aroma character of thiols on GC-O was described as sulphury, green, sweet, roasted and fresh grapefruit juice.

Table 3: Chromatography analyses of mercapto-terpineol derivatives

Retention Indice (DB-WAX) Odour description FID FPD by GC-O Compound 37 or 38 * 2012 2016 roasted, green, exotic, sweet Compound 37 or 38 * 2024 2029 sulphury, green, grapefruit, sweet * because the absolute configurations of thiols were not determined, we can't attribute retention indices and aroma character to compounds 37 and 38 To confirm the proposed structures by nuclear magnetic resonance (NMR), we purified thiol derivatives by liquid chromatography on silica gel followed by high performance liquid chromatography. This is the first time that these aroma compounds have been described and characterized.

Example 11: d-Limonene derivatives 1. Experimental as in example 10, replacing a-terpineol by limonene (Merck) 2. Limonene derivatives Limonen thioacetate derivatives were obtained as described in example 10. The enzymatic hydrolysis of these thioacetates was performed as described in example 10.

Characterization of thioacetates derivatives by GC-MS Thioacetate 40: MS-EI, m/z (relative intensity): 212 (8) [M] +, 169 (100) [M- COCH3] +, 136 (14) [M-HSCOCH3] +, 121 (25) [M-C3H70S] +

Thioacetate 41 : MS-EI, m/z (relative intensity): 212 (12) [M] +, 169 (100) [M- COCH3] +, 136 (16) [M-C2H40S] +, 121 (25) [M-C3H70S] + Thioacetate 9 : MS-EI, m/z (relative intensity): 212 (5) [M] +, 197 (8) [M-CH3] +, 169 (2) [M-COCH3] +, 136 (100) [M-HSCOCH3] +, 121 (48) [M-C3H70S] + Thioacetate 42, 43: MS-EI, m/z (relative intensity): 288 (11) [M] +, 247 (7) [M- COCH3] +, 213 (16) [M-SCOCH3] +, 169 (100) [M-C4H702S] +, 136 (77) [M- C4HgO2S2] . Characterization of thiol derivatives by GC-MS Mercapto-limonene 44 or 45: MS-EI, m/z (relative intensity): 170 (70) [M] +, 136 (100) [M-H2S] +, 121 (52) [M-H2S, CH zu 107 (70) [M-C2H7S] +, 95 (98) [M-C3H7S] + Mercapto-limonene 44 or 45: MS-EI, m/z (relative intensity): 170 (33) [M] +, 136 (90) [M-H2S] +, 121 (28) [M-H2S, CH3] +, 107 (42) [M-C2H7S] +, 95 (100) [M-C3H7S] + Mercapto-limonene 29: MS-EI, m/z (relative intensity): 170 (5) [M] +, 155 (3) [M- CH3] +, 136 (76) [M-H2S] +, 121 (100) [M-H2S, CH3] +, 107 (32) [M-C2H7S] +, 93 (85) M_CsH9SJ+ Mercapto-limonene 46: MS-EI, m/z (relative intensity): 204 (20) M+, 170 (100) [M-H2S] +, 155 (12) [M-H2S-CH3] +, 136 (84) [M-2H2S] + 2.1 Generation of thioacetate derivatives The well known sulfur-containing limonene derivative is 1-p-menthene-8- thiol 29. This aroma volatile is one of the most powerful flavor compound ever found in nature. It was described as key aroma component of the peel and juice oils from Citrus spp which are both important flavoring ingredients for the food and beverage industries. When adequately diluted, this powerful flavor-impact compound displays a guenine, unmistakable aroma of fresh grapefruit juice, in which it naturally occurs at or below the ppb level. The taste detection threshold of synthetic, racemic 29 is lower than 1.104 ppb in water. For (+)- (R)- and (-)- (S)- 29, the respective values found were 2.10-5 ppb and 8.10-5 ppb. These detection threshold are one of the lowest ever recorded for a naturally occuring flavour compound.

To produce this aroma compound and related isomers by biochemical process, reactions were performed using d-limonene 39 and thioacetic acid 31 as substrates to generate thioacetate derivatives. These reactions were carried-out as described in the experimental part above. As shown in scheme 6, several odour- active compounds were detected in the reaction mixture. The odour quality of the reaction mixture was described as tropical, sweet, fruity, sulfury and green.

All these thioacetate derivatives were detected by GC-FID and GC-FPD and were tentatively identified on the basis of their mass spectroscopy data analysis. In fact, by GC-MS three compounds with the same molecular weight (212 Da) were detected. Two of these molecules had identical mass-spectra while the third one had a different mass-spectra regarding the fragment intensities. Moreover, by analogy with results obtained for a-terpineol derivatives, compound 40 and 41 are probably two stereoisomers, while compound 9 is an positional isomer of 40 and 41 as shown in scheme 6. The proposed structure of compound 9 was confirmed by the generation and charaterization of the corresponding thiol 29 obtained by enzymatic hydrolysis as described below. As shown in scheme 6, two other molecules 42 and 43 with the same molecular weight (288 Da) were identified among the main compounds of the reaction mixture.

To confirm the proposed structures, it is required to purify all the aroma compounds and to analyse them by different nuclear magnetic resonance spectroscopy techniques (IH, 13C, Cosy, Noe, Hetcor....).

2.2 Generation of thiol derivatives To produce thiol derivatives, the mixture of thioacetates prepared as described above, was incubated with several lipases at room temperature. Reactions were performed in water or in phosphate buffer as described in the experimental part.

After 5 h all thioacetates were transformed, the reaction was stopped and the diethyl ether extract was analysed by GC-FID, GC-FPD and GC-MS. As shown in scheme 7, the main compounds detected in the reaction mixture were thiols 29, 44, 45

The global aroma character of the mixture after enzymatic hydrolysis was described as fruity, blackcurrant, exotic, intense, sulphury and fresh grapefruit-like.

Other sulphur-containing compound 46, was also identified in the reaction mixture by gas chromatography analyses.

Example 10: bouillon, soup (cooked meat type note) A culinary base mass was prepared by adding molten beef fat (6.00 g) to a mixture comprising the following ingredients: 10 g Mono Sodium Glutamate, 38 g table salt, 32 g maltodextrine, 5 g corn starch, 2 g yeast extract powder, 5 g red wine powder and sodium inosinate, piment, pepper and tartaric acid.

The mixture was homogenised and sieved, thus yielding a culinary base mass.

A bouillon (reference sample) was prepared by adding 250 ml of boiling water to 5.00 g of the culinary base mass and 0.10 g table salt. The resulting product showed a good basic bouillon character with week meat note.

250 ml boiling water was added to a mixture containing 5.0 g culinary base mass, 0. 3 g freeze-dried product (thioacetates or thiols preparations as described above), and 0.10 g table salt. The resulting bouillon had a very intense and pleasant aroma, which was preferred to the reference sample.

The flavour body of the bouillon containing the flavouring ingredient (thioacetates or thiols preparations as described above) was more intense and it had a pronounced beefy, roasted and vegetable-like character, which was absent in the reference bouillon.

Example 11: meat

We prepare sauce based on roasted chicken juice and we added sulphur- containing terpenes prepared according to this invention in liquid or powder form.

The flavouring mixtures of thioacetate or thiol derivatives were added in concentrations from 0.1 to 0.5 % by weight to the chicken juice. This addition resulted in an intensification of the meaty aroma character and freshness of the chicken juice.

Example 12: fruit juices The flavouring mixtures of thiols or thioacetates, or the pure aroma compounds prepared according to the invention, were added to different fruit juices such as obtained from the following fruits: Citrus fruits orange, grapefruit Tropical fruits passion fruit, mango Drupes peach, apricot Berries blackcurrant, raspberry The aroma mixtures or pure compounds were added to these juices in concentration of 0.05 to 0.1 % by weight. This addition of aroma compounds resulted in an intensification of aroma character and freshness of the fruit juices.

Scheme 1: Chemoenzymatic generation of FFT and corresponding Di-FFT E (enzyme), [O] (oxydation) Scheme 2: Chemoenzymatic transformation of MF-thioacetate E (enzyme: lipase), [O] (oxydation) Scheme 3: Disulfides generated by hydrolysis of mixture of FF-thioacetate and MF- thioacetate.

11 Bis (2-furfuryl) disulfide; 12 Bis (2-methyl)-3-furyl disulfide; 13 2-furfuryl- (2- methyl)-3-furyl disulfide n-hexane + "+ SH 0 O 30 31 32 33 Scheme 4: Generation of a-terpineol thioacetate derivatives su,,, su . f. Enzyme,-f- OH OH OH OH 34 35 37 38 Scheme 5: Enzymatic hydrolysis of a-terpineol thioacetate derivatives 39p O vs 39 31 40 41 9 II +, S II O O t5t QX 42 43 Scheme 6: Generation of limonene thioacetate derivatives w II o + vs 41 40 9 Enzyme *SHSH H SH 44 45 29 Scheme 7: Enzymatic hydrolysis of limonene thioacetate derivatives into thiols Formulas 1 to 46