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 thioesters, and their use as enhancers or intensifiers of flavour in food products.

Summary of the invention Accordingly, this invention provides a process for preparing natural thioacetates by addition of an thiocarboxylic acid as thioacetic acid or salt derivatives thereof, on a-, (3-, 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.

In one embodiment, thioacetate is generated by addition of thioacetic acid on a-,-or y-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.

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

In a last aspect, the invention relates to the use of said thioester as flavour compounds or flavour boosters for aroma foodstuffs.

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.

The substrate may be a-, P-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.

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 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 35 °C and during about 5 minutes to 24 hours.

The thioacetates according to the invention 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 thioacetate derivatives obtainable by the process according to the present invention.

These compounds have the formula: 1,2,3,4,5,6,7,8, and 9 The aroma character of these volatile compounds is described as meaty, savoury, roasted, vegetable, sulfury, garlic and onion-like. All these compounds contribute significantly to the generation of flavours of the meat and savoury products and also roasted coffee.

In a preferred embodiment, these new aroma compounds can be 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.

In examples 1 to 4, the chemicals were of analytical grade and were purchased from Fluka and Aldrich. All the solvents were of analytical grade and were purified by distillation using a Vigreux column (60cm x 3cm).

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, um. The carrier gas was helium (80 kPa), make-up gas for the FID was nitrogen (40 kPa). The injected volume was 0.5 fJ. l. 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: 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 thioacetate derivatives were then enzymatically hydrolyzed in water or phosphate buffer as described in example 1 or in n-hexane 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 2: 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.

Prenyl formate 10 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 11.

Example 3: synthesis of 3-mercapto-3-methylbutyl formate Compound 12 could be transformed into 3-mercapto-3-methylbutyl formate 13 by reaction with formic acid in n-hexane as described in example 2.

By analogy, compound 3 should be hydrolyzed by enzymes in water or buffer media to give compounds 14 and 15. Compounds 15 and 13 have been identified in roasted coffee earlier (Silwar 1982).

Example 4: Mercaptopropanone and thioacetate derivatives To a solution of hydroxypropanone 17 (1 mmol) in 10 ml of distilled n- hexane, formic acid (1.5 mmol) was added at 40 °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 18. This

molecule was then incubated with thioacetic acid in n-hexane to produce thioacetate derivative 19.

Example 5: 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, limonene, n-hexane, polyethylenglycol).

Generation of thioacetates : Ten mmoles of terpene (a-terpineol or limonene) 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.

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 pm. The carrier gas was helium (80 kPa), make-up gas for the FID was nitrogen (40 kPa). The injected volume was 0.2 p1. 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-acetylthio 20: 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-C3H902S] + p-menthan-8-ol-2-acetylthio 21 : MS-EI, m/z (relative intensity): 230 (2) [M] +, 215 (6) [M-CH3] +, 212 (3) [M-H20] 172 (28) [M-C3H60] , 136 (100) [M-C2H602S] +, 121 (55) [M-C3H902S] + 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 pl.

2. RESULTS 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 22 with thioacetic acid 23 in distilled n-hexane and at room temperature as shown in scheme 1 Reactions between substrates 22 and 23 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 20 and 21 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 20 2420 2426 sulphury, roasted, meaty, green, exotic Compound 21 2435 2440 sulphury, intensive, green, fruity, sweet The aroma volatiles 20 and 21 detected by GC-FID, GC-FPD, GC-O were tentatively identified on the basis of their mass spectroscopy data analyses. The proposed structures for compounds 20 and 21, were then confirmed by nuclear magnetic resonance (NMR) of the corresponding thiols obtained by enzymatic

hydrolysis of thioacetates 20 and 21 (see above). Moreover, according to the NMR data analyses, it seems that compounds 20 and 21 are probably two diastereoisomeric forms (and) of p-menthane-8-ol-2-acetyl thio. This indirect characterisation of compounds 20 and 21 is due to the difficulty to purify the to diastereoisomers 22 and 23 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 22, 23 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 necessary to determine the absolute configuration of the chiral aroma compounds 22 and 23. 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 24, 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) 1R, 4R musty sulphury note, intensive 1S, 4S green, black currant, exotic, intensive 1R, 4S delicate, fruity, sweet 1 S, 4R strong, sweet, slightly pungent

Example 11: d-Limonene derivatives 1. Experimental as in example 6 2. Limonene derivatives Limonene thioacetate derivatives were obtained as described in example 6.

Characterization of thioacetates derivatives by GC-MS Thioacetate 26: MS-EI, m/z (relative intensity): 212 (8) [M] +, 169 (100) [M- COCH3] +, 136 (14) [M-HSCOCH3] +, 121 (25) [M-C3H70S] + Thioacetate 27 : MS-EI, m/z (relative intensity): 212 (12) [M] +, 169 (100) [M- COCH3] +, 136 (16) [M-C2H40S] +, 121 (25) [M-C3H70S] + Thioacetate 28: 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 29,30: MS-EI, m/z (relative intensity): 288 (11) [M] +, 247 (7) [M- COCH3] +, 213 (16) [M-SCOCH3] +, 169 (100) [M-C4H702S] +, 136 (77) [M- C4H802S2] Generation of thioacetate derivatives Reactions were performed using d-limonene 25 and thioacetic acid 23 as substrates to generate thioacetate derivatives. These reactions were carried-out as described in the experimental part above. As shown in scheme 2, 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 26 and 27 are probably two stereoisomers, while compound 28 is an positional isomer of 26 and 27 as shown in scheme 2. As shown in scheme 2, two other molecules 29 and 30 with the same molecular weight (288 Da) were identified among the main compounds of the reaction mixture.

Example 8: 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: Mono Sodium Glutamate 10.05 g Table Salt 38.00 g Sodium inosinate 0.10 g Piment Saromex 1.00 g Pepper Saromex 0.15 g Red Wine powder FIS 5.00 g Tartaric acid 0.15 g Corn starch 5.10 g Yeast extract powder 2.25 g Maltodextrine 31.55 g 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 (powder preparation of thioacetates 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 (powder preparation of thioacetates 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 9: 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 10: 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.