CH654567A5 - Flavourings with a hydroxy-beta-damascone structure - Google Patents

Abstract

Compounds of the formula <IMAGE> in which one symbol R represents a hydroxy group and the other represents hydrogen, and also 2-hydroxy- beta -damascone and 4-hydroxy- beta -damascone are obtained by microbiological hydroxylation of beta -damascone. 4-Hydroxy- beta -damascone can also be obtained by chemical hydroxylation of beta -damascone. The novel compounds I represent flavourings which are particularly suitable for the aromatisation of smoking articles.

Description

** WARNING ** Beginning of DESC field could overlap end of CLMS **.

PATENT CLAIMS 1. Compounds of the formula

wherein one symbol R stands for a hydroxyl group and the other for hydrogen.

2. 2-hydroxy-ss-damascone as a compound according to claim 1.

3. 4-hydroxy-3-damascone as a compound according to claim 1.

4. Flavor compositions, characterized in that they contain a compound of the formula

wherein one symbol R stands for a hydroxyl group and the other for hydrogen.

5. Process for the preparation of compounds of the formula

wherein one symbol R stands for a hydroxyl group and the other for hydrogen, characterized in that damascone is microbiologically hydroxylated.

6. The method according to claim 5, characterized in that B-damascone is fermented with a fungus of the genus Botryodiplodia, Botryosphaeria, Lasiodiplodia, Rhizopus, Penicillium or Aspergillus.

7. The method according to claim 6, characterized in that the fungus Botryosphaeria rhodina CBS-175.26, Botryosphaeria rhodina CBS-3 () 6.58. Lasiodiplodia theobromae ATCC- 1 (19.36. Lasiodiplodia theobromae ATCC-285 .7 (1. Rhizopus arrhizus ATCC-l 11.45. Penicillium rubrum NRRL-11 () 55 or Aspergillus niger IFO-8541 used.

8. Process for the preparation of 4-hydroxy-i3-damascons characterized. that ss-damascone is chemically hydroxylated.

9. Use of compounds of the formula

wherein the one symbol R stands for a hydroxyl group and the other for hydrogen, as flavorings for the production of flavor compositions.

The invention relates to new flavorings. These are the compounds of the formula

wherein one symbol R stands for a hydroxyl group and the other for hydrogen.

The formula should accordingly be the 2-hydroxy-ss-damascone Ia and the 4-hydroxy-B-damascone Ib

namely the individual optical isomers as well as the racemic mixtures.

The invention also relates to a process for the preparation of the compounds of the formula I.

This process is characterized by the fact that Damascons, i.e. the connection of the Fnrmel

microbiologically hydroxylated.

The microbiological hydroxylation of II is preferably carried out using strains of the genera Botryodiplodia, Botryosphaeria, Lasiodiplodia, Aspergillus, Rhizopus and Penicillium.

Suitable strains are, for example: Aspergillus niger ATCC 16888, ATCC 9142 Aspergillus awamori IFO 4313 Aspergillus sojae IFO 5241 Aspergillus phoenicis IFO 8874 Botryosphaeria rhodina CBS 356.69, CBS 230.30, CBS 374.54, CBS 124.13 Lasiodiplodia theobromae ATCC 9055, ATCC 16391 Rhizopus arrhizus CBS 285.55, CBS 346.49 Rhizopus stolonifer CBS 382.52.

Preferred strains are: 1. Botryosphaeria rhodina CBS-175.26 2. Botryosphaeria rhodina CBS-306.58 3. Lasiodiplodia theobromae ATCC-109.36 4. Lasiodiplodia theobromae ATCC-285.7 () 5. Rhizopus arrhizus ATCC-111.45 6. Penicillium rubrum NR. Aspergillus niger IFO-8541 Strains Nos. 1 through 5 and 7 were available to the public prior to the filing date of the patent as disclosed in the catalogs of CBS List of Cultures 1972, pages 7, 9, 12 and 49; ATCC Catalog of Strains I, Thirteenth Edition 1978, pages vi, 292, 264 and 350; and IFO List of Cultures Fifth Edition 1972, pages ii, iii and 13 follows. Strain No. 6 is identical to Penicillium rubrum FERM P 3796, deposited with the Northern Regional Research Laboratory of the US-Department of Agriculture, 1915, North University Street , Peroria, Illinois 61604, USA, before March 1, 1978 and to the public on March 13, 1978.

These strains are characterized in particular by an excellent hydroxylation capacity (bioconversion) of the ring structure of the ss-damascone, the 4-position of the ring being preferably hydroxylated in all cases and optically active compounds being obtained. In addition, the 2-position is hydroxylated to a lesser extent.

The microorganisms can be used as a culture (suspension) (which means the mycelium and the corresponding nutrient solution), in the form of the mycelium or in processed form, e.g. B. can be used in the form of an enzyme extract prepared in a manner known per se from the culture suspension or the mycelium. The culture suspension can be prepared by inoculating a suitable medium with the microorganism.

Suitable media are those containing carbon sources, nitrogen sources, inorganic salts, etc. Contain nutrients suitable for the growth of the microorganism. Examples of carbon sources are glucose, fructose, sucrose, molasses and dextrin. Maltose, mannose, starch, e.g. B. corn starch, lactose and glycerin into consideration; Nitrogen sources are, for example, organic substances containing nitrogen such as peptone, meat extract. Yeast extract, soy flour, corn steep liquor (Cornsteep liquor) and casein, urea. Amino acids, or nitrogenous. inorganic compounds such as nitrates and inorganic ammonium salts. Inorganic salts that should be mentioned are phosphates or sodium, potassium, magnesium, calcium, manganese, iron and copper salts.

The cultivation of the microorganism can be carried out as a submerged culture, as a shaking culture or as a stationary culture (e.g. in fermenters); preferably the microorganism is cultivated under aerobic conditions.

It is expedient to work in a pH range of 3-8, in particular between 4-7.5. If necessary, inorganic or organic acids such as hydrochloric acid, acetic acid, oxalic acid, or bases such as. B. NaOH, or buffer systems such as phosphate, phthalate, tris (hydroxymethyl) aminomethane systems, or sugars, such as. B. glucose can be used.

The reaction temperature is generally between 15 and 35 "C, in particular between 2S32 C.

The process according to the invention is expediently carried out in such a way that the compound of the general formula II is added as substrate to the cultured, growing culture solution. The concentration of the substrate is expediently 0.5 g per liter to 20 g per liter. The fermentation according to the invention can be carried out by continuing the cultivation of the microorganism under the above-mentioned conditions. The reaction time can depend on the species and strain of the microorganism used, on the composition of the culture medium. vary depending on the substrate used and its concentration. A fermentation time of 18-216 hours is generally sufficient.

especially 18144 hours. The reaction temperature is generally between 15 and 35 "C, in particular between 2S32 C.

The fermentation according to the invention can also be carried out with the mycelium of the microorganism isolated from the culture solution or with an enzyme extract prepared in a manner known per se from the culture solution or the mycelium. In this case, the fermentation can expediently in aqueous solution, e.g. B. a buffer solution, in physiological salt solution, in fresh nutrient solution or in water.

The substrate II can be used as such or as a solution in a hydrophilic solvent such as acetone, dimethyl sulfoxide.

Methanol, ethanol, ethylene glycol, propylene glycol or dioxane can be added. It is also possible to add a surface-active agent or a dispersant to an aqueous suspension of the substrate, or to emulsify the substrate by treatment with ultrasound.

Conventional anti-foam agents can be used to combat foam, e.g. B. Silo oils (e.g.

UCON), polyalkylene glycol derivatives, or corn or soybean oil.

The substrate can be added to the culture of the microorganism during the cultivation or, as mentioned above, after the cultivation has ended.

The time at which the compound II is added to the pre-cultivated microorganism can be important. On the one hand, it is desirable to achieve as dense growth of the microorganisms as possible (larger biomass), on the other hand, overaging of the culture (which can be expressed, for example, in sporulation and color changes) if the cultivation time is too long, can have a negative influence on the transformation of linen. In the case of many microorganisms, the pH value initially drops by half to one unit (due to the production of acid). When the pH value begins to rise again (lysis!) And the biomass of the culture increases weakly or hardly at all, this is usually the best time. to add compound II. This is the time at which the exponential growth of the biomass is completed (determination: centrifugation and weighing). The optimal time for adding the compound II can also - if in The presence of glucose can be determined using a glucose analyzer (consumption of the added glucose). However, changes in the pH value during the preculture can also occur depending on the microorganism and medium used.

The course of the transformation of II can be followed with the aid of known gas chromatographic or thin-layer chromatographic methods. If, as a result, a strong decrease or even complete disappearance of II is found, the starting material is expediently added again in order to achieve the greatest possible utilization of the transformation capacity of the microorganism.

The fermentation product can be isolated from the reaction mixture (the fermentation broth) in a manner known per se, for example by extraction (which method is preferred) with an organic solvent that is immiscible with water, such as chloroform, methylene chloride (preferred) or methyl acetate, carbon tetrachloride , Butanol, diethyl ether. Ethyl acetate (preferred). Emulsions can occur during extraction with an organic solvent.

These can be broken up using methods familiar to those skilled in the art, e.g. B. by using filter aids. like cellite, centrifugation, etc.

The organic extractant, optionally after drying the extract with sodium sulfate, can be removed by distillation: the transformation product then remains as a yellowish to brownish oil.

Treatment with activated charcoal can now be carried out to decolorize. The reaction mixture can also undergo a gentle distillation under high vacuum, e.g. B. at pressures from 0.01 to 0.5 torr. A separation of the transformation product for the purpose of obtaining the individual compounds l can be carried out by chromatography (e.g.

Thick layer, such as column chromatography) on supports such as aluminum oxide. Silica gel or cellulose.

It's clear. that the mutants of the genera mentioned and the specifically mentioned microorganisms also produce the same transformation of II. Such mutants can easily by exposure to UV rays in a manner known per se. X-rays or common mutagenic substances such as B. acridine organs, can be obtained on the corresponding spores or mycelium suspensions.

Those obtainable by the process according to the invention.

isolated mixtures represent yellowish to brownish colored liquids, are insoluble in water, but soluble in organic solvents, such as. B. alcohols, ethers, ketones, esters. Hydrocarbons and halogenated hydrocarbons.

The compounds 1 can be isolated from the mixtures. As can be seen from the examples below, however, isolation is not even necessary, since the purified mixtures can already be used as flavorings.

In the latter case, the compounds 1 can be detected analytically (for example by gas chromatography coupled with mass spectroscopy) in the mixtures.

The identification can be done in such a way that the fermentation product from the fermentation broth, as described above, by extraction with an organic solvent, e.g. B.

Ethyl acetate (3x, volume = 1:: 1) is isolated. The extract can then be dried over Na2SO4 and the solvent distilled off. This can be done in as little as possible, e.g. B. 10 ml of ether: hexane = 1: 1 a solution can be created, and the solution by means of column chromatography on silica gel, for example 30 times the amount. z. B. Silica gel Merck. be separated into fractions using ether: hexane mixtures with an increasing proportion of ether, starting with 30: 3 (hexane: ether). and is stopped with ether alone. The eluates can be examined using conventional analytical methods (IR, NMR, MS). The compound la is eluted first, followed by compound lb.

A preparative production of Ia and Ib is also possible in this way.

Identification of I. For the spectroscopic characterization, samples of odorant mixtures were used, which were separated by chromatography on 30 times the amount of silica gel (Merck, silica gel 60; particle size 0.063-0.2 mm, 70-230 mesh).

The separation by means of a (Merck) ready-made column used further below in Example 3 can also be used for this purpose.

Data for compounds 4-hydroxy- (3-damascone: IR (film): 3450, 1640 cm 'NMR (CDCl3): 1.04 (6H), s. 1.64 (3H) s. 1.92 (3H ) Quartet J = 7, J = 1.5 Hz. 4 (1H) triplet J = 5 J = 6 Hz. 6.15 (in), multiplet J = 14, J = 1.5 Hz.

6.76 (pH), multiplet J = 7, J = 14 Hz.

MS: 208 (27), 193 (30), 175 (5), 165 (4), 152 (9), 139 (60), 123 (10), 109 (18), 95 (13), 77 (12 ), 69 (100), 55 (8), 43 (28).

2-Hydroxy-ss-damascone: IR (film): 3500, 1650 com ~ 'NMR (CDC13): 1.04 (3H), s. 1.06 (3H), s. 1.52 (3H) s. 1.92 (3H) quartet J = 7, J = 1.6 Hz.

3.57 (1H) quartet J = 3rd J = 8 Hz.

6.15 (1H) multiplet J = 15, J = 1.5 Hz.

6.76 (i) multiplet J = 7, J = 15 Hz.

MS: 208 (22), 193 (12), 175 (24), 165 (37), 149 (46), 139 (19), 121 (63), 107 (20), 93 (23), 79 (22 ), 69 (100), 55 (22), 41 (60).

As I said before. the purified mixtures or the compounds of the formula I have particular organoleptic properties which make them particularly suitable as flavorings.

The invention accordingly also relates to the use of compounds of the formulas as flavorings for the production of flavoring compositions.

The compounds of the formula I used according to the invention as flavorings or the mixtures primarily obtained by fermentation can in particular serve to flavor smokers' articles, such as cigarettes (tobacco), cigars (tobacco), snuff, etc., the compounds preferably not alone but can be used in the form of compositions with other flavors. Such flavor compositions containing the compounds of the formula according to the invention also form the subject of the invention. They are produced in a manner known per se: adding the mixtures containing the compounds I to known flavor compositions or mixing them with natural or synthetic compounds or mixtures suitable as constituents of flavor compositions.

In general, it can be said that the mixtures obtained by fermentation contain approx. 5990% of compounds of the formula, the ratio of Ia to Ib usually approx.

0.01: 1 to 0.5: 1.

As mentioned above, the compounds of the formula can be used as flavorings for the production or improvement, enhancement, enhancement or modification of tobacco.

The pronounced taste qualities of the compounds of the formula allow their use in low concentrations. A suitable dosage includes, for example, the range of 1 ppm - 1 (100 ppm, preferably 5 ppm - 50 () ppm in the finished product, i.e. the flavored tobacco.

The mixtures containing the compounds of the formulas can be mixed in a customary manner with the constituents used for tobacco flavor compositions or added to such aromas. The aromas used according to the invention are understood to mean flavor compositions which can be diluted in a manner known per se and distributed in the smokers' articles. They can be converted into the customary use forms, such as solutions, pastes or powders, by methods known per se. The products can be spray dried, vacuum dried or lyophilized.

The following carrier materials, thickeners, for example, are used for the production of such common forms of use. Flavor improvers, spices and auxiliary ingredients, etc. in question: Gum arabic, tragacanth, salts or brewer's yeast, alginates, carrageenan or similar absorbents; Maltol, spice oleoresins, smoke flavorings; Cloves, sodium citrate; Monosodium glutamate, disodium inosine 5'-monophosphate (IMP), disodium guanosine 5-phosphate (GMP); or special flavorings, water, ethanol, propylene glycol, glycerine.

The compositions appropriately contain about 0.1 to 10% by weight of compounds I.

The amount of the composition applied is preferably between 0.005 and about 0.05% by weight. The composition can be applied to the smokers' articles in any suitable manner, but preferably in the form of a solution or suspension, the application being carried out by spraying, dipping or in some other known manner. I can be added at any stage of the treatment of the tobacco, but preferably added after aging, ripening and shredding and before the tobacco is processed into cigarettes, etc.

Finally, the invention also relates to a chemical process for the preparation of the compound Ib from B-damascone.

This process is characterized in that ss- damascone is chemically hydroxylated.

The oxidation is expediently carried out using oxygen in the presence of an oxidation catalyst and a (metal) halide, such as ammonium bromide. Heavy metal salts are expediently used as the oxidation catalyst. Particularly suitable heavy metal salts are: cobalt-II-chloride, cobalt-II-acetate-bromide, cobalt-II-acetate, etc.

It is expedient to work in a solvent such as glacial acetic acid, cyclohexane, etc.

One works expediently in a temperature range from 20 to 150 "C, preferably at 50 to 9 () C. See also A.S. Hat wand H.S. Blanchard, Can. J. Chem. 43, 1306 (1965), A.

Onopchenko, J.G.D. Schulz and R. Seekircher. J. Org. 37, 1414 (1972).

Examples Table 1 A. Composition of suitable growth media Medium 1 Glucose 50.0 g Cornsteep liquor 10.0 g (corn steep liquor) KC1 0.5g KH2PO4 1.0g NaNO3 I. () G distilled water ad 1000 ml.

pH = 6.3 before sterilization (121 in an autoclave) medium 2 glucose 20.0 g Yeast extract 3.0 t NaNO3 1.0 g Glycine 1.0g KH2PO4 1.0g MgSO4.7H2O 0.5g KCI (1.5 g FeSO4'7H30 0.01 g Distilled water to 1000 ml.

pll = 6.3 before sterilization Medium 3 NUN03 2 * () g KCl 0.5g K2S04 O.35g FeSG47H2G 0.01g sucrose 30.0g yeast extract 1.0g magnesium glycerophosphate 0.5 g Tap water ad 1000 ml, pH = 6.8 after sterilization Medium 4 Malt extract 20.0 g soy flour 15.0 g Casein hydrolyzate 1.0 g yeast extract 1.0 g NaCl 1.0 g distilled water ad 1000 ml, pH = 6.3 before sterilization Table II B. Microorganisms (species) and growth media used Fungus cultures collection No. Bio-transformation: optimal growth media quantities II used in g / l (see Table I) Aspergillus niger IFO 8541 0.5 - 3 2, 3, 4 Aspergillus niger ATCC 9142 0.2 - 3 1, 3, 4 Aspergillus niger ATCC 16888 0.1-2 1, 2.3 Aspergillus awamori IFO 4314 0.1 - 0.4 2, 3 Aspergillus sojae IFO 5241 0.1 - 0 , 4 2, 3 Aspergillus phoenicis IFO 8874 0.1 - 0.5 2. 3 Botryosphaeria rhodina CBS 175.26 1-4 1, 2, 4 Botryosphaeria rhodina CBS 306.58 1 - 4 1, 2. 4 Botryosphaeria rhodina CBS 356.59 1 - 3 1, 2. 4 Botryosphaeria rhodina CBS 23 (1.30 0.5 - 2 1. 2. 4 Table II (continued) Botryosphaeria rhodina CBS 374.54 1-2 1,2.4 Botryosphaeria rhodina CBS 124.13 0.2 - 1 1, 2, 4 Lasiodiplodia theobromae ATCC 109.36 1 - 4 Lasiodiplodia theobromae ATCC 285.70 1 - 4 1, 2, 4 Lasiodiplodia theobromae ATCC 9055 0.1 - 2 Lasiodiplodia theobromae ATCC 16391 0.2 - 2.5 Rhizopus arrhizus ATCC 111.45 0.5-2 1,2,3,4 Rhizopus arrhizus CBS 285.55 0.5 - 1 1, 2, 3, 4 Rhizopus arrhizus CBS 346.49 0.1 - 1 1, 2, 4 Rhizopus stolonifer CBS 382.52 0.1 - 1 2, 3, 4 Botryodiaplodia theobromae IFO 6469 0.2 - 2 1, 2, 4 example 1 Cultivation of the microorganism The microorganisms in Table II are grown on agar plates (prepared with medium 2). For this purpose, spores or hyphae of the respective microorganism are spread onto the surface of the agar plates with the aid of a platinum tube and incubated at 28 ° C. for 8-10 days. The inoculum for the precultures is produced by making spores of these Plates suspended in 9 ml of a physiological NaCl solution and 1 ml of the spore suspension to 100 ml of the corresponding Growth medium in a 500 ml shake flask (Erlenmeyer flask with baffles). The flask is incubated at 28 C48 hours on a rotary shaker (15 (1-20 (1 rpm)).

8 ml of the resulting mycelium suspension are used to Inoculate precultures of 40 (1 ml in 11 shake flasks.

After an incubation time of 24-48 hours under the above conditions, a dense mycelium culture results (10O-200 gil wet mycelium, when centrifuged at 800 () g).

Hydroxylation of p-damascone with Botryosphaeria rhodina CBS 175.26.

A 7 () 1 fermenter (Bicicngeneering. Rüti). containing 451 des .Nlediullls 1 and 1 () ml of antifoam agent (polypropylene glycol 2 () 00) is inoculated with 1.61 of a 24-hour preculture of the mold (also grown on medium 1). The cultivation conditions are as follows: Stirring system: Circulation stirrer (900 rpm), temp. 28 "C.

Ventilation: 20 1 air / min.

After 24 hours of incubation, a biomass = 25 g dry matter / 1 is achieved: the glucose is practically completely converted and the pH of the culture broth drops to approx.

At this point in time, the addition of SS Damascone is started by continuously adding a P-Damascone / Ethanol = 1: 1 mixture via a metering pump within 4 days in such a way that approx. 10 C / o SS Damascone is obtained on the total ss-Damascone metabolites (the transformation products, the product mixture) is present (determination by means of gas chromatography). At the same time the pH of the culture is adjusted to 6.5. A total of 110 ml = 103 g ss-Damascone are fed into the fermenter within 4 days. After the addition is complete, stirring is continued for 8 hours, after which time practically all B-Damascone is consumed. The fermenter contents are then extracted twice using an Alva-Laval extractor (Girotester) using CH2Cl2 as the extraction agent. After drying over Na2SO4 and removing CH2Cl2 in vacuo, 111 g of a brownish oil with a fruity odor remain. The oil consists essentially of 4-hydroxy-ss-damascone with a small proportion of 2-hydroxy-ss-damascone (ratio approx. 80: 1). The compounds Ia and Ib can, as indicated above, be separated off by means of chromatography on silica gel.

The resulting compound 4-hydroxy-P-damascone has a rotation of a 22 = -30.8 ". The IR, MS and NMR data agree with the values given above.

Incorporated into cigarette tobacco (10-20 ppm), 4-hydroxy-ss-damascone shows extremely positive effects when smoking cigarettes. Compared to a cigarette with untreated tobacco, the salivating effect is strongly promoted. The side stream of smoke is felt to be much milder. But the tobacco aroma is also more noticeable in the main stream of smoke. The mouthfeel is much better.

The same organoleptic effects are achieved if the extracted brownish oil is worked into tobacco instead of 4-hydroxy-ss-damascone and the cigarettes are then smoked.

Example 2 Hydroxylation of ss-Damascone with Lasiodiplodia theobromae ATCC 28570.

The fermentation is carried out in analogy to Example 1, the cultivation of Lasiodiplodia theobromae ATCC 28570 being carried out on the same medium and within the same period of time as indicated in Example 1.

110 ml (103 g) ss-damascone are continuously added to the fermenter culture within 5 days. After working up the reaction mixture according to Example 1, 94.5 g of a brownish oil with a fruity odor are obtained. The oil consists mainly of 4-hydroxy-t3-damascone with a small amount of 2-hydroxy-damascone. The optical rotation of the 4 hydroxy-ss-damascon is: z22 = 42.2 ".

The IR, MS and NMR data agree with the values given above.

The compound of the formula Ib is incorporated into tobacco and produces essentially the same organoleptic effects as described in Example 1. The same also applies if the mixture obtained is used instead of the compound Ib.

Example 3 Hydroxylation of ss-Damascone with Aspergillus niger IFO 8541 A 101 fermenter (Biologische Verfahrenstechnik, Basel) containing 81% of medium 3 and 1 ml of antifoam agent (polypropylene glycol 2000) is inoculated with 400 ml of a 40-hour pre-culture of Aspergillus niger IFO 8541 (grown on medium 3). Fermentation conditions: Circulating stirrer: (400 rpm). Temp. 28 C ventilation: 5 1 / min.

After 41 hours of incubation, a biomass = 10 g dry weight / l is achieved, the pH value drops to 4.5. This pH is now adjusted to 6.5 again (1N NaOH) and an amount of 22 ml (20 g) as a ss-Damascone / ethanol = 1: 1 mixture is added continuously over the course of 4 days.

Again, care is taken to ensure that approx. 10% free ss-damascone is always present during the fermentation. The fermentation conditions are otherwise those of Examples 1 to 2, with the exception that the stirring is carried out at 600 rpm. After 4 days, the contents of the fermenter are extracted twice with ethyl acetate. The emulsions that form are filtered through Celite. After drying the combined ethyl acetate extracts over sodium sulfate and removing the solvent in vacuo, 16 g of a yellow oil with a pleasant fruity odor remain.

The oil consists essentially of 4-hydroxy-ss-damascone with a2r2 + 24.3 and 2-hydroxy-Q-damascone, the ratio of the two metabolites being 4: 1.

2-Hydroxy-ss-damascone can be converted extremely efficiently from 4-hydroxy-ss-damascone using a ready-made column, e.g. B.

a Merck chromatography ready-to-use column, such as Merck Lobar, size B, silica gel 60: 1.5 g metabolite mixture (oil), dissolved in the minimum amount (2 ml) of CH2C12, are added to the with CH2Cl3 (containing 5% ethyl acetate) applied column. It is eluted with CH2Cl2, which contains increasing amounts of ethyl acetate, up to a ratio of 6 to 4 (chromatography is carried out at a slightly increased pressure, at 0.2 bar).

The 2-hydroxy-ss-damascone is eluted first, followed by 4-hydroxy-B-damascone.

The 2-hydroxy-ss-damascone has an opt. Rotation of a2r2) = -2.22 ".

The data for IR, MS and NMR correspond to those given above.

The mixture and the isolated compounds I show organoleptic properties indicated in Examples 1 and 2.

Example 4 Hydroxylation of B-Damascone with Penicillium rubrum NRRL 11055 A 11 Erlenmeyer flask with 4 baffles, containing 300 ml of medium 1, is inoculated with 3 ml of a preculture of Penicillium rubrum NRRL 11055 and shaken at 28 ° C. on a rotary shaker (150 rpm) for 48 hours. After this time an extremely dense growth of the deep red becomes Mycels achieved: the biomass corresponds to 15 g of dry matter per 1. Within 4 days, 0.08 ml (0.75 g) B-Damascone in 0.3 ml of ethanol was added. Then it is extracted twice with 150 ml CH2Cl2 each time and the combined extracts are poured over Na2SO4 dried. After removing the extractant in In the vacuum, 0.26 g of a reddish, viscous oil with a fruity odor remains. The oil consists mainly of 4 hydroxy-ss-damascones with admixtures of 2-hydroxy-da mascon.

Incorporation of the oil into tobacco (10 ppm) leads to the im Example 1 given organoleptic properties.

Example 5 Hydroxylation of B-damascone with Rhizopus arrhizus ATCC 11145 The fermentation is carried out in analogy to Example 4.

A 60 hour old culture of Rhizopus arrhizus ATCC 11145 (grown in 300 ml of medium 4) is mixed with a total of 0.4 ml (0.37 g) p-damascone within 4 days: the procedure is such that 0.1 ml (0.093 g) B-damascone per day , dissolved in 0.3 ml of ethanol is added to the culture and shaken at 28 ° C. on a rotary shaker.

After the fermentation has ended, the reaction mixture is extracted twice with 300 ml of methylene chloride each time, the combined extracts are dried over Na2SO4 and the CH2Cl2 is removed in vacuo. There remains 0.35 g of a yellowish oil, the smell is fruity. The oil consists mainly of 4-hydroxy-B-damascone.

Incorporated into tobacco (15 ppm), a surprisingly mild smoke is achieved when smoking the cigarette. The tobacco aroma itself is enhanced.

Example 6 Hydroxylation of B-Damascone with Lasiodiplodia theobromae ATCC 109.36 The fermentation is carried out analogously to Example 4. A 24 hour old culture of Lasiodiplodia theobromae ATCC 109.36 (grown on 300 ml medium 2) is mixed with a total of 0.6 ml (0.56 g) ss-damascone within 4 days. The procedure is such that 0.15 ml of Damascone dissolved in 0.3 ml of ethanol is added per day and the mixture is shaken at 28 ° C. on a rotary shaker (150 rpm). The reaction mixture is then extracted twice with 300 ml of CH2Cl2 each time, the combined extracts are dried over Na2SO4 and the CH, Cl, is removed in vacuo. This leaves 0.48 g of a yellow oil with a fruity odor. The oil consists mainly of 4-hydroxy-B-damascone with (x = 41.6. By incorporating the oil into tobacco (10 ppm) the Smoking the cigarettes significantly enhances the tobacco aroma, while the cigarette smoke is much milder.

Example 7 Preparation of 4-hydroxy-ss-damascone In a sulphonation flask equipped with a KPG stirrer, gas inlet tube, thermometer and reflux condenser, 3.9 g of cobalt acetate tetrahydrate and 1.5 g of office ammonium bromide are mixed with 90 ml of glacial acetic acid and 90 ml of cyclohexane. The mixture is heated to 50 in an oil bath while stirring and passing in air. Then 15 g of SS Damascone are added.

Now air is passed through for 14 hours with stirring at 50. The reaction mixture is extracted with ether, washed with water and with sodium pyrosulfite solution until free of peroxide, then washed with 2N NaOH and water, dried over sodium sulfate and evaporated.

The crude product (14.6 g) is chromatographed over 440 g of silica gel. Elution with 20% ether in hexane gives 3 g of 1 (2,66-trimethyl-3-oxo-1-cyclohexen-1-yl) -2-buten-1-one; elution with ether gives 1.5 g of 1 (2,66-trimethyl-3-hydroxy-1-cyclohexen-1-yl) -2-buten-1-one (4-hydroxy-ss-damascone).

The IR, NMR and MS data of the compound IB agree with the values given above.

Example 8 Apricot flavor for tobacco Parts by weight Linalyl acetate (10% in ethanoi) A: 0.3 B: 0.3 Cinnamaldehyde (10 in ethanol) (1.4 (h4 geraniol (1.5 () .5 angelica root oil 0.5 () .5 butyric acid amyl ester 1. () L () valeric acid amyl ester 1.0 1.0 vanillin 2P 2. () C 18 aldehyde 2s ( ) 2. () Petitgrain oil Paraguay 2P 2.0 Benzaldehyde 2.5 2.5 Orange oil (1 () - concentrated) 5.0 5.0 C 14-Aldehyde 15.0 15.0 Ethanol 967.'R 957.8 Fermentation mixture of example 2 2 10.0 100 (). 0 1000.0 By adding the fermentation mixture (Comp. B) to the above composition, the existing fruity note is significantly enhanced. An additional note appears. reminiscent of fully ripe apricots.

After incorporating the above aroma into tobacco in a concentration of 100 glkg tobacco, the full fruity note of the apricots can be clearly identified. the existing tobacco note is also advantageously reinforced and rounded off. The above flavor is suitable as a so-called top flavor for flavoring tobacco. (10 ppm based on the fermentation mixture of Example 2).

Claims (9)

PATENT CLAIMS 1. Compounds of the formula wherein one symbol R stands for a hydroxyl group and the other for hydrogen. 2. 2-hydroxy-ss-damascone as a compound according to claim 1. 3. 4-hydroxy-3-damascone as a compound according to claim 1. 4. Flavor compositions, characterized in that they contain a compound of the formula wherein one symbol R stands for a hydroxyl group and the other for hydrogen. 5. Process for the preparation of compounds of the formula wherein one symbol R stands for a hydroxyl group and the other for hydrogen, characterized in that damascone is microbiologically hydroxylated. 6. The method according to claim 5, characterized in that B-damascone is fermented with a fungus of the genus Botryodiplodia, Botryosphaeria, Lasiodiplodia, Rhizopus, Penicillium or Aspergillus. 7. The method according to claim 6, characterized in that the fungus Botryosphaeria rhodina CBS-175.26, Botryosphaeria rhodina CBS-3 () 6.58. Lasiodiplodia theobromae ATCC- 1 (19.36. Lasiodiplodia theobromae ATCC-285 .7 (1. Rhizopus arrhizus ATCC-l 11.45. Penicillium rubrum NRRL-11 () 55 or Aspergillus niger IFO-8541 used. 8. Process for the preparation of 4-hydroxy-i3-damascons characterized. that ss-damascone is chemically hydroxylated. 9. Use of compounds of the formula wherein the one symbol R stands for a hydroxyl group and the other for hydrogen, as flavorings for the production of flavor compositions. The invention relates to new flavorings. These are the compounds of the formula wherein one symbol R stands for a hydroxyl group and the other for hydrogen. The formula should accordingly be the 2-hydroxy-ss-damascone Ia and the 4-hydroxy-B-damascone Ib namely the individual optical isomers as well as the racemic mixtures. The invention also relates to a process for the preparation of the compounds of the formula I. This process is characterized by the fact that Damascons, i.e. the connection of the Fnrmel ** WARNING ** End of CLMS field could overlap beginning of DESC **.