DK2297328T3 - Fremgangsmåde til fremstilling af naturlig 9-decen-2-on ved biokonvertering af undecylensyre under anvendelse af en skimmelsvamp - Google Patents

Description

METHOD FOR PRODUCING NATURAL 9-DECEN-2-ONE BY THE BIOCONVERSION OF UNDECYLENIC ACID USING A MOULD AND THE USE THEREOF IN PERFUMERY AND FOOD FLAVOURING

The present invention relates to the bioconversion, using a mould, of undecylenic acid into 9-decen-2-one, the latter being used in perfumery and food flavouring applications.

The Applicant Company has shown that 9-decen-2-one (or dec-9-en-2-one or methyl octenyl ketone, RN CAS 35194-30-0) is a methylketone with olfactory and taste properties such that it may be used in all applications in the aromatics industry (flavourings and fragrances). Indeed, it has a green and fatty note as well as fruity notes, notably of pear, apple, pineapple, passion fruit and exotic fruit in general.

This ketone, just like other methyl ketones, is formed by a metabolic pathway which takes the first steps of β-oxidation after oxidation of the fatty acid. This metabolic pathway for the formation of methyl ketones is used by mycetes during the detoxification of fatty acids.

As can be seen by the representation of the metabolic pathway (Figure 1), the substrate used during the bioconversion is undecylenic acid, which is obtained by cracking (physical operation transforming the crude product into finer compounds) ricinoleic acid obtained from castor oil then purified by dry distillation. Finally, following a decarboxylation step, the methyl ketone obtained has one less carbon than the original fatty acid.

To the Applicant Company's knowledge, there is no other biotechnological method today that describes and/or enables the synthesis of 9-decen-2-one. Indeed, this molecule can only be obtained to date by chemical synthesis, and with mediocre yields. Furthermore, synthetic flavourings have the disadvantage of being less valued by consumers than natural flavourings. Consequently, the present invention aims to provide an alternative method for obtaining natural aromatic molecules, in particular those obtained from 9-decen-2-one, by biological processes, notably bioconversion, implementing microorganisms such as moulds.

For the purposes of the present invention, "bioconversion" means the biological transformation of a substrate, preferably from a natural source, in order to obtain natural flavouring substances, which may be formulated in flavourings or fragrances classified as being natural.

In a surprising and unexpected way, the Applicant Company discovered that it was possible to satisfy this objective using a production method of 9-decen-2-one, characterised by the bioconversion of undecylenic acid using a mould, said method comprising the following steps: a) culturing of said mould; b) addition of undecylenic acid as bioconversion substrate, in the presence of oil; c) bioconversion of the substrate into 9-decen-2-one; d) extraction and purification of the 9-decen-2-one.

Thus, the present invention relates to a method of producing 9-decen-2-one, characterised by the bioconversion of undecylenic acid using a mould belonging to the family Mortierellacaeu or to the family Mucoraceae, preferably the genus Mortierella, genus Mucor or genus Rhizopus, said method comprising the following steps: a) culturing of said mould; b) addition of the undecylenic acid substrate with a flow rate of 0.1 to 0.9 g/L/h in the presence of oil; c) bioconversion of the substrate into 9-decen-2-one, d) extraction and purification of the 9-decen-2-one.

For the purposes of the present invention, the term "mould" corresponds to unicellular or multicellular filamentous microscopic fungi. Among moulds, Phycomycetes and Septomycetes are distinguished. Within the Phycomycetes, phylum Oomycota and phylum Zygomycota are found. Within the Septomycetes, phylum Basidiomycota, phylum Ascomycota and phylum Deuteromycota are found.

In one embodiment of the disclosure, the mould according to the disclosure belongs to the phylum Deuteromycota, class of Hyphomycetes, order of Tuberculariales, family of Aspergillaceae. More preferably, said mould belongs to the genus Aspergillus or Penicillium. In a first preferred embodiment, said mould belongs to the species Aspergillus oryzae or to the species Aspergillus niger. In a second preferred embodiment, said mould belongs to the species Penicillium roquefortii or to the species Penicillium camembertii.

Among the Aspergillus oryzae moulds, mention may be made of the following collection strains: Aspergillus oryzae DSMZ1861, Aspergillus oryzae DSMZ1864, Aspergillus oryzae DSMZ1147, Aspergillus oryzae DSMZ 63303, Aspergillus oryzae CBS 570.65, Aspergillus oryzae CBS 819.72, Aspergillus oryzae CBS 110.27.

Among the Aspergillus niger moulds, mention may be made of the following collection strains: Aspergillus niger DSMZ 823, Aspergillus niger DSMZ 2466.

Among the Penicillium roquefortii moulds, mention may be made of the following collection strains: Penicillium roquefortii CBS 221-30, Penicillium roquefortii PRB 18, Penicillium roquefortii DSMZ 1079, Penicillium roquefortii DSMZ 1080.

Among the Penicillium camembertii moulds, mention may be made of the strain Penicillium camembertii DSMZ 1233.

The mould according to the invention belongs to the phylum of Zygomycota, class of Zygomycetes, order of Mucorales, family of Mortierellaceae or family of Mucoraceae.

More preferably, said mould belongs to the genus Mortierella or to the genus Mucor or to the genus Rhizopus, and even more preferably said mould belongs to the species Mortierella isabellina or Mortierella rammniana or to the species Mucor racemosus or Mucor miehei or to the species Rhizopus arrhizus or Rhizopus oryzae.

Among the moulds belonging to the genus Mortierella, mention may be made of the following collection strains: Mortierella isabellina DSMZ 1414, Mortierella isabellina CBS 100559, Mortierella isabellina CBS

221.29, Mortierella isabellina CBS 194.28, Mortierella isabellina CBS 208.32, Mortierella isabellina CBS

224.35, Mortierella isabellina CBS 560.63, Mortierella isabellina CBS 167.80, Mortierella isabellina CBS

493.83, Mortierella isabellina CBS 309.93, Mortierella isabellina CBS 250.95, Mortierella isabellina CBS 109075, Mortierella ramanniana CBS 112.08, Mortierella ramanniana CBS 219.47, Mortierella ramanniana CBS 243.58, Mortierella ramanniana CBS 478.63, Mortierella ramanniana CBS 852.72, Mortierella ramanniana CBS 366.95, Mortierella ramanniana CBS 101226.

Among the moulds belonging to the genus Mucor, mention may be made of the following collection strains: Mucor javanicus DSMZ 1222, Mucor racemosus DSMZ 62760, Mucor rouxii DSMZ 1191.

Among the moulds belonging to the genus Rhizopus, mention may be made of the following collection strains: Rhizopus arrhizus oryzae DSMZ 905, Rhizopus oryzae DSMZ 2199, Rhizopus delemar DSMZ 853, Rhizopus niveus DSMZ 2194, Rhizomucor miehei DSMZ 1330.

The culturing targeted in step a) of the method according to the invention, comprises the preparation of a culture, preferably semi-concentrated, for example by cellular amplification, in an appropriate culture medium. This culturing is preceded by pre-culturing of the strain in a first culture medium more suited to the initial multiplication steps.

In a preferred embodiment of the invention, the culture time of said mould varies between 20 and 30 hours and in fact depends on the status of the mycelium growth, the latter being observable through a microscope. It is indeed preferable that the minimum biomass of the mould is between 7 and 15 g/L and more preferably between 9 and 12 g/L, before proceeding to the bioconversion according to the invention.

Step b) of the method consists in adding the substrate to the cell culture. According to the invention, synthesis of 9-decen-2-one via biological means involves the most appropriate substrate, which is undecylenic acid. According to an alternative (not according to the invention), the substrate is one of the esters of undecylenic acid, the methyl ester or the ethyl ester of undecylenic acid. It goes without saying that the substrate may be a mixture of various appropriate substrates, in particular a mixture of undecylenic acid and one or more or its esters.

According to the invention, said undecylenic acid is added to the fermentation medium with a flow rate of 0.1 to 0.9 g/L/h, preferably 0.2 to 0.7 g/L/h and very preferably 0.3 to 0.55 g/L/h.

Step c) of the method consists in the bioconversion of undecylenic acid into 9-decen-2-one.

The duration of this bioconversion phase is generally from 36 to 96 hours.

Due to the high toxicity of undecylenic acid towards mycetes, an adaptation period may be necessary in order to prepare the mould to convert the substrate. Thus, according to a preferred embodiment, the bioconversion step of the method according to the invention comprises a first adaptation phase of the mould in which the said undecylenic acid is added at a flow rate of 0.1 to 0.5 g/L/h, then a second phase in which the undecylenic acid is added at a flow rate of 0.25 to 0.9 g/L/h. Advantageously, the duration of said adaptation phase is less than 20 hours, preferably less than 12 hours and more preferably less than 6 hours.

According to a preferred embodiment, undecylenic acid is added in the presence of oil. Said oil being selected from the group comprising standard food oils such as soybean, corn, sunflower oils, or other triglycerides containing short-chain fatty acids such as Miglyol, or even white oils such as paraffin oils or mineral oils composed of long-chain hydrocarbons. In a preferred manner, said oil is partially hydrogenated, or rich in oleic acid, sunflower oil.

Preferably, said oil is added in the following proportions relative to the substrate: undecylenic acid/oil = % undecylenic acid - % oil or 1/3 undecylenic acid - 2/3 oil or Zi undecylenic acid - % oil.

Advantageously, said oil is added to the fermentation medium at a flow rate of 0.4 to 4.0 g/L/h, preferably 0.5 to 3 g/L/h and very preferably from 0.75 to 2.5 g/L/h.

As with all types of microorganisms under forced bioconversion conditions, mycetes need a source of carbon enabling them to cover their energy needs, such as for example a carbohydrate source, preferably glucose or maltose. The provision of glucose throughout the bioconversion is preferred. Consequently, according to another preferred embodiment of the invention, some glucose or maltose is added simultaneously to the undecylenic acid and oil. Advantageously, said glucose or said maltose is added to the fermentation medium at a flow rate lower than 1 g/L/h, preferably lower than 0.75 g/L/h and very preferably lower than 0.5 g/L/h.

According to another preferred embodiment of the invention, the addition of said undecylenic acid, said oil and optionally said glucose or maltose into the fermentation medium is done continuously over 5 to 95 hours, preferably over 24 to 72 hours and more preferably over 24 to 48 hours.

According to another preferred embodiment, bioconversion according to the invention is carried out at a temperature of between 25°C and 35°C, preferably between 27°C and 30°C.

According to another preferred embodiment, the pH of the fermentation medium according to the invention is from 5 to 8, preferably from 5.5 to 7.5 and more preferably from 6 to 7. Within the scope of the invention, the pH is controlled by using sodium hydroxide (for example sterile 5N NaOH or another base conventionally used in fermentation (NH4OH, KOH, etc.)).

During bioconversion, it is important to maintain adequate stirring as well good aeration of the fermentation medium. In the fermenter, stirring can be created using several stirring blades. Advantageously, and depending on the size of the fermenter, for example for a 6-litre fermenter, adequate stirring necessary according to the invention is from 200 to 1200 rpm, and preferably from 300 to 900 rpm. As for aeration, this may be carried out by injecting air at the level of the stirring blades.

Advantageously, adequate aeration of the fermentation medium according to the invention is less than or equal to 1 vvm, preferably between 0.2 and 0.8 vvm and more preferably between 0.3 and 0.7 vvm.

In another preferred embodiment, the bioconversion according to the invention is stopped by the addition of an acid into the culture medium. Said acid may be selected from the group comprising phosphoric acid, sulphuric acid, hydrochloric acid and citric acid. Preferably, said acid is phosphoric acid or citric acid.

In step d), extraction of 9-decen-2-one may be carried out by hydrodistillation or by extraction using a solvent selected from the group comprising cyclohexane, methylcyclohexane and ethyl acetate. Preferably, the solvent used is cyclohexane or a cyclohexane - ethyl acetate mixture.

Purification of the 9-decen-2-one may then be carried out by deresination followed by fractional distillation.

This extraction/purification method thus enables a molecule that is more than 98% pure to be obtained with an extraction yield in the order of 75 to 85%.

The bioconversion yield into 9-decen-2-one relative to the substrate used, undecylenic acid, is around 25 to 35%.

The disclosure also relates to the 9-decen-2-one obtained by the method as described above. A subject of the disclosure is the use of 9-decen-2-one (CAS 35194-30-0) in all perfumery and food flavouring applications, particularly in the manufacture of perfumes, fragrant materials, cosmetic or food compositions, or as a food additive.

In one preferred embodiment, a subject of the disclosure is the use of 9-decen-2-one obtained by the method as described above in all perfumery and food flavouring applications, particularly for the manufacturing of perfumes, fragrant materials, cosmetic or food compositions or as a food additive.

For the purposes of the present disclosure, the term perfumery means not only perfumery in the usual meaning of the term, but also the other fields in which the smell of products is important. This may be, in a non-exhaustive way, perfume compositions in the usual sense of the term, such as perfume bases and concentrates, eaux de Cologne, eaux de toilette, perfumes and similar products; topical compositions - in particular cosmetics - such as facial creams and body creams, talcum powders, hair oils, shampoos, hair lotion, ointments, bath salts and oil, shower and bath gels, toilet soaps, antiperspirants and deodorants, shaving creams and lotions, soaps, creams, toothpastes, mouthwashes, pomades and similar products; and maintenance products such as softeners, detergents, washing powders, air fresheners, aerosols and similar products.

The term fragrant is used to designate a compound that gives off a fragrance.

By food flavouring, we mean any use of the compounds of the disclosure to flavour any liquid, solid, human or animal foodstuff notably drinks, dairy products, ice creams, sweets, chewing gums, etc., but also for the flavouring of tobacco. 9-decen-2-one may thus be used in perfume compositions or food flavouring compositions to contribute to giving exotic, floral or fruit notes, particularly pineapple, pear, passion fruit, exotic fruit. Depending on the applications, this 9-decen-2-one molecule will be used in proportions determined by a person skilled in the art.

Preferably, 9-decen-2-one will be used in quantities between 0.00025% and 30%, preferably between 0.0025 and 20%, more preferably between 0.025 and 10%, by weight relative to the total weight of the composition in which it is present. It could be in the composition of solids or liquids and notably in the composition of gels, creams, pomades and/or sprays. 9-decen-2-one could also be used in a composition which is itself fragrant, or in a composition in which the fragrant agent is used to mask or neutralise certain odours.

Figure 1 describes the bioconversion of undecylenic acid into 9-decen-2-one. EXAMPLES Example 1:

Strains Mortierella isabellina (A), Aspergillus oryzae (B) and Penicillium roquefortii (C) [origin = tube frozen at - 80°C] were seeded onto malt agar and incubated at 27°C (for A) or 30 °C (for B and C) for 72 hours.

The preceding preculture was seeded into 3.5 L of malt medium in a 6 L fermenter:

Malt extract: 50 g/L

Yeast extract: 7.5 g/L pH 6.5

Mortierella isabellina (A)

Said strain was incubated at 27°C for 24 hours under stirring at 500 rpm and aeration at 0.3 vvm. Aspergillus oryzae (B)

Said strain was incubated at 30°C for 24 hours under stirring at 600 rpm and aeration at 0.5 vvm. Penicillium roquefort» (C)

Said strain was incubated at 30°C for 24 hours under stirring at 600rpm and aeration at 0.3 vvm.

The dry weight of mycelium at the end of culturing for each strain was around 9 to 11 g/L.

The undecylenic acid was then dispensed with a flow rate of 0.15 g/L/h for 6 hours then at a flow rate of 0.33 g/L/h for 72 hours; i.e. a total of 25 g/L. This undecylenic acid was then dispensed as a mixture with hydrogenated sunflower oil (1/3 acid-2/3 oil); this oil was then dispensed at flow rates of 0.6 g/L/h then 1 g/L/h. In parallel, glucose was dispensed continuously at a flow rate of 0.36 g/L/h for 72 hours. The pH was regulated to 6.5 throughout the entire conversion with 5N NaOH. The speed was increased to 900 rpm with aeration at 1 vvm for Mortierella isabellina and 0.3 vvm for Aspergillus oryzae or Penicillium roquefortii. The conversion was continued for 72 hours.

Under non-optimised conditions, a production of between 5 and 7 g/L of 9-decen-2-one was obtained. Example 2: Conversion with Aspergillus oryzae (B)

Aspergillus oryzae (origin = tube frozen at -80°C) was seeded onto malt agar and incubated at 30°C for 72 hours.

The preceding preculture was seeded into 3.5L malt medium (same concentration as in Example 1).

This was incubated at 30°C, 600 rpm, 0.5 vvm air, free pH for 24 hours. A dry weight of 11 g/L was obtained.

The undecylenic acid was then dispensed at a flow rate of 0.5 g/L/h for 6 hours then at a flow rate of 0.9 g/L/h for 48 hours: i.e. a total of 46 g/L. This undecylenic acid was then dispensed as a mixture with hydrogenated sunflower oil (1/4 acid - % oil); this oil was then dispensed at flow rates of 1.5 g/L/h then 2.7 g/L/h. In parallel, glucose was continuously dispensed at a flow rate of 0.36g/L/h over 48 hours. The pH was regulated to 6 throughout the entire conversion duration with 5N NaOH. The speed was increased to 900 rpm and aeration flow rate of 0.3 vvm. The conversion was continued for 48 hours. A production of 16.5 g/L of 9-decen-2-one was obtained, i.e. a bioconversion yield of 35%.

Example 3: Conversion with Mortierella isabellina (A)

Mortierella isabellina (origin = tube frozen at -80°C) was seeded onto malt agar and incubated at 27°C for 72 hours.

The preceding preculture was seeded into 3.5 L malt medium (same concentration as in Example 1).

This was incubated at 27°C with stirring at 600 rpm and aeration at 0.5 vvm for 24 hours. A dry weight of 11.3 g/L was obtained.

The undecylenic acid was then dispensed at a flow rate of 0.27 g/L/h for 6 hours then at a flow rate of 0.477 g/L/h for 48 hours: i.e. a total of 46 g/L. This undecylenic acid was then dispensed as a mixture with hydrogenated sunflower oil (1/4 acid - % oil); this oil was then dispensed at flow rates of 0.8 g/L/h then 1.43 g/L/h. In parallel, glucose was continuously dispensed at a flow rate of 0.32 g/L/h for 48 hours. The pH was regulated to 7.5 throughout the entire conversion duration with 5N NaOH. The speed was increased to 900 rpm and aeration flow rate of 1 vvm. The conversion was continued for 48 hours. A production of 8 g/L of 9-decen-2-one was obtained, i.e. a bioconversion yield of 33%.

Example 4: Conversion with Penicillium roquefortii (C)

Penicillium roquefortii (origin = tube frozen at -80°C) was seeded onto malt agar and incubated at 30°C for 72 hours.

The preceding preculture was seeded into 3.5 L malt medium (same concentration as in Example 1).

This was incubated at 30°C with stirring at 600 rpm and aeration at 0.3 vvm for 22 hours. A dry weight of 10 g/L was obtained.

The undecylenic acid was then redispensed at a flow rate of 0.25 g/L/h for 6 hours then at a flow rate of 0.45 g/L/h for 48 hours, then at a flow rate of 0.25 g/L/h for 24 hours: i.e. a total of around 30 g/L. This undecylenic acid was then dispensed as a mixture with hydrogenated sunflower oil (1/3 acid - 2/3 oil); this oil was then dispensed at flow rates of 0.5 g/L/h then 0.9 g/L/h then 0.5 g/L/h. In parallel, glucose was continuously dispensed at a flow rate of 0.36 g/L/h for 72 hours. The pH was regulated to 6.5 throughout the entire conversion duration with 5N NaOH. The speed was increased to 900 rpm and aeration flow rate of 0.6 vvm. The conversion was continued for 48 hours. A production of 10.5 g/L of 9-decen-2-one was obtained, i.e. a bioconversion yield of 35%.

Example 5: extraction - purification

Acidification to pH 1.5 to 2 with phosphoric acid 85% was performed. The extraction solvent cyclohexane was added and stirring was performed for 1 hour at ambient temperature. The mixture was centrifuged and the organic phase was collected. This was dosed with methyl ketone. The solvent was concentrated and an oily "crude" product was thus obtained. Distillation under vacuum was performed. The "deresinated" lactone and a depleted oil were obtained. Purification of the molecule was then carried out by fractional distillation under vacuum. A product with a purity of over 99% was obtained.

Example 6: Evaluation of the 9-decen-2-one in perfumery

The 99% pure 9-decen-2-one has been tested on smelling paper and in solution (at 5% in ethanol): the top note is powerful and homogenous with rose, aldehydic, citronellyl acetate, coriander leaf and marine notes. The base has pear notes.

Example 7: Evaluation in food flavouring

The 99% pure 9-decen-2-one has been tested at 8 ppm in mineral water: it has fruity notes: pear, apple and pineapple and green and fatty notes.

It has been deemed of interest for adding sophistication to natural exotic fruit formulations, which is all the more interesting since there are few natural molecules available to achieve this aim. In particular, the "Pineapple" tonality of 9-decen-2-one constitutes an attractive alternative to replace allyl caproate, which is currently the most commonly used molecule in formulations to obtain a pineapple-type tonality.

Example 8: Use in food flavouring and perfumery formulations

In an exotic fruit flavouring, the addition of 9-decen-2-one enables a typical "pineapple" tonality to be obtained. In the following formula, the replacement weight for weight of the allyl caproate, usually used in the formulation, by 9-decen-2-one compensated very advantageously for the "pineapple" tonality given by the allyl caproate.

Natural ethyl caproate 50 g

Natural maitoi 20 g

Natural Isoamyi acetate 35 g

Natural ethyl Isovaierate 37 g

Natural menthol codex 8 g

Natural ethyl methyl-2-butyrate 75 g

Natural ethyl acetate 50 g

Natural isoamyl butyrate 12 g

Natural ethyl butyrate 35 g

Refined camphor 2 g

Natural 9-decen-2-one 200 g

Natural ethyl alcohol qs 1000 g

The use at 50 ppm of this mixture in a still drink with juice gives it very characteristic "pineapple'' notes; the same is true when this mixture is used at 200 ppm to give a "pineapple" character to a sorbet.

Still in natural food flavouring formulation, 9-decen-2 one enables "pear" notes of interest to be obtained, for example in the following formula:

Natural caproic acid 1 to 10 g

Natural Isoamyi alcohol 20 to 60 g

Natural hexyl alcohol 10 to 50 g

Natural ethyl caprate 20 to 80 g

Natural ethyl caprylate 50 to 150 g

Natural Isoamyi acetate 50 to 150 g

Natural propyl acetate 50 to 150 g

Natural hexyl acetate 50 to 150 g

Natural butyl acetate 50 to 150 g

Natural ethyl acetate 50 to 150 g

Natural 9-decen-2-one 20 to 50 g

Propylene glycol qs. 1000 g

The use at 100 ppm of this mixture in a drink slightly sweetened at 5% and slightly acidified (0.1% citric acid) gives it very characteristic "pear" notes, even if the dose of 9-decen-2-one is very low (2 to 5 ppm).

In perfumery formulation, 9-decen-2-one enables the "flowery, green, violet" side and the "aldehydic fruity" note of a shampoo to be enhanced, for example in the following formula in which 9-decert-2-one is used at 1%, which makes the perfume note of the shampoo more complex and more rounded:

Benzyl acetate 70 g

Phenylethyl alcohol 100 g y-Undecalactone 4g cis-isoambrettolide 10 g

Anisaldehyde 25 g

Bacdanol 80g

Citroneilol 85 g

Ethylvaniiiin 2 g

Geraniol 40 g

Methyl dihydrojasmonate 120 g

Oxacyclobexadecen-2-one 100 g

Heliotropin 20 g 2-acetyl-l,2,3,4,5,6,7,8-octa- hydro-2,3,8,8-tetra-methyinaphthalene 110 g

Isoeugenol 2 g 4-(1,l-dimethylethyl)-a-methylbenzene-propanal 110 g

Linalooi 60 g

Methyl ionone 30 g

Vanillin 10 g 2-octynoic add, methyl ester 12 g 9-decen-2-one 10 g

Total lOOGg

In the case of a different perfumery application, that of a softener, the presence of 9-decen-2-one dosed at 0.5% gives the power and enhances the "apple" and "pear" fruity side of the softener fragrance, as indicated in the following formula:

Benzyl acetate 7Qg

Phenoxyallyl acetate 30g

Phenylethyl alcohol 80g

Hexylcinnamaldehyde 40g C12 aldehyde 2g cisiscambrettolide 60g

Anisaldehyde 30g 2-cyclohexylidene-2-phenylacetonitrile 20g

Bacdanol 50g

Citronellol 50g

Coumarin 80g

Dihydromyrcenol IQOg

Acetic acid, 3aA5,6,7,7a-hexahydro-'4/7-methanO"lH"indenyl ester 65g 2-acetyl-l,2,3,4,5,6,7,8-octahydro-2,3,8,8-tetramethylnaphthalene 106g

Methylionantheme 40g

Patchouli essence 30g

Cyclohexyl salicylate lOOg

Dimethyl 3-cyclohexene-l-carboxaidehyde 8g

Ambroxan 5% DPG 6g

Galbanum essence 10% DPG lOg

Indole 10% DPG 8g 9-decen-l-oi 10% DPG lOg 9-decen-2-one 5g

Total lOOOg

Finally, in the case of a cream, 9-decen-2-one gives the cream accentuated flora! notes, for example in the following application:

Benzyl acetate 49g cis-3-hexeny! acetate 2g

Llnalyl acetate 85g

Phenylethyl alcohol 65g y-undecaiactone 6g

Aliy! amyl glycolate 20g

Methyl anthranilate 5g

Anisaldehyde lOg

Methylbenzoate 8g

Citronellol 45g

EthyivaniNin llg

Geranie! 35g

Methyl hydrojasmonate 130g

Heliotropin Sg cis-3-hexenoi 2g ct-ionone 23g β-ionone 4g 2-acetyl 1,2,3,4,5,6,7,8-oetahydro-2,3,8,8-tetramethylnaphthalene 75g 4-(1,l-dimethylethyl)-a-methyl benzenepropanal 70g

Linaloo! lOOg

Patchouli essence llg y-nonalactone 27g cis-3-hexenyi salicylate 35g

Hexyl salicylate 75g

Terpineol 48g

Dimethyl 3-cyclohexene-l-carboxaidehyde 4g

Vanillin 2g

Acetyl cedrene 12g

Sandaiore 12g

Dimethyl benzyl carbinol butyrate Ig

Methyl 2,4-dihydroxy-3.,6-dimethyi- benzoate 13g 9-decen-2-one lOg

In the presence of 9-decen-2-one, the cream has more top notes, with much more intense green and fresh notes.

Example 9 Toxicity Tests

The molecule has been tested on 3 rabbits according to the experimental protocol established by "the OECD guideline No. 404 dated 24 April 2002" and the test "method B.4of the Directive No. 2004/73/EC): Acute dermal irritation". it thus emerges that 9-decen-2-one is non-irritating and that no hazard symbol or risk phrases are required for its use (according to the "EEC Directives 67/548/59 and 99/45").

Similarly, the molecule has been tested on 3 rabbits according to the experimental protocol established by "the OECD guideline No. 405 dated 24 April 2.002" and the test "method B.5 of the Directive No. 2004/73/EC): Acute eye irritation".

It also thus emerges that 9-decen-2-one is non-irritating and that no hazard symbol or risk phrases are required for its use (according to the "EEC Directives 67/548/59 and 99/45").

Similarly, the molecule has been tested on 3 rabbits according to the experimental protocol established by "the OECD guideline No. 405 dated 24 April 2002" and the test "method B.5 of the Directive No. 2004/73/EC): Acute eye irritation".

It also thus emerges that 9-decen-2-one is non-irritating and that no hazard symbol or risk phrases are required for its use (according to the "EEC Directives 67/548/59 and 99/45").

Finally, the value obtained in the LD50 test (acute ora! toxicity (see the same Directive), i.e. 2500 mg/kg), has enabled the molecule to be classified as non-hazardous.

Due to its harmiessness, it thus appears that this molecule may be used in formulations and, due to its confirmed presence in mango and pineapple, it may be used in food flavouring and perfumery applications that may claim a "natural" label.

Claims (18)

1. Fremgangsmåde til fremstilling af 9-decen-2-on, kendetegnet ved biokonvertering af undecylensyre under anvendelse af en skimmelsvamp tilhørende familien Mortierellaceae eller familien Mucoraceae, hvilken fremgangsmåde omfatter følgende trin: a) dyrkning af skimmelsvampen; b) tilsætning af undecylensyresubstrat med en hastighed på fra 0,1 til 0,9 g/L/h under tilstedeværelse afolie; c) biokonvertering af substratet til 9-decen-2-on, d) ekstrahering og oprensning af 9-decen-2-on.

2. Fremgangsmåde ifølge krav 1, kendetegnet ved at skimmelsvampen hører til slægten Mortierella, til slægten Mucor eller til slægten Rhizopus.

3. Fremgangsmåde ifølge krav 1 eller 2, kendetegnet ved at biokonverteringstrinnet omfatter en første fase til tilpasning af skimmelsvampen, hvor undecylensyren tilsættes med en hastighed på fra 0,1 til 0,5 g/L/h, derefter en anden fase, hvor undecylensyre tilsættes med en hastighed på fra 0,25 til 0,9 g/i/h.

4. Fremgangsmåde ifølge krav 3, kendetegnet ved at varigheden af tilpasningsfasen er mindre end 20 timer.

5. Fremgangsmåde ifølge krav 3, kendetegnet ved at varigheden af tilpasningsfasen er mindre end 5 eller 12 timer.

6. Fremgangsmåde ifølge et hvilket som helst af kravene 1 til 5, kendetegnet ved at olien vælges fra gruppen omfattende konventionelle spiselige olier eller triglycerider dannet ud fra kortkædede fedtsyrer, eller hvide olier.

7. Fremgangsmåde ifølge et hvilket som helst af kravene 1 til 6, kendetegnet ved at olien tilsættes til fermenteringsmediet med en hastighed på mellem 0,4 og 4,0 g/L/h.

8. Fremgangsmåde ifølge et hvilket som helst af kravene 1 til 7, kendetegnet ved at glucose eller maltose tilsættes simultant til undecylensyren og til olien.

9. Fremgangsmåde ifølge krav 8, kendetegnet ved at glucosen eller maltosen tilsættes til fermenteringsmediet med en hastighed på mindre end 1 g/L/h.

10. Fremgangsmåde ifølge krav 8, kendetegnet ved at glucosen eller maltosen tilsættes til fermenteringsmediet med en hastighed på mindre end 0,75 g/L/h.

11. Fremgangsmåde ifølge krav 8, kendetegnet ved at glucosen eller maltosen tilsættes til fermenteringsmediet med en hastighed på mindre end 0,5 g/L/h.

12. Fremgangsmåde ifølge et hvilket som helst af kravene 1 til 11, kendetegnet ved at tilsætningen af undecylensyre, af olien til fermenteringsmediet udføres kontinuerligt i 5 til 96 timer.

13. Fremgangsmåde ifølge et hvilket som helst af kravene 8 til 11, kendetegnet ved at tilsætningen af undecylensyren, af olien og af glucosen eller maltosen til fermenteringsmediet udføres kontinuerligt i 5 til 96 timer.

14. Fremgangsmåde ifølge et hvilket som helst af kravene 1 til 13, kendetegnet ved at biokonverteringen udføres ved en temperatur på mellem 25 °C og 35 °C.

15. Fremgangsmåde ifølge et hvilket som helst af kravene 1 til 13, kendetegnet ved at biokonverteringen udføres ved en temperatur på mellem 27 til 30 °C.

16. Fremgangsmåde ifølge et hvilket som helst af kravene 1 til 15, kendetegnet ved at pH af fermenteringsmediet er mellem 5 og 8.

17. Fremgangsmåde ifølge et hvilket som helst af kravene 1 til 16, kendetegnet ved at beluftningen af fermenteringsmediet er mindre end eller lig med 1 vvm.

18. Fremgangsmåde ifølge et hvilket som helst af kravene 1 til 17, kendetegnet ved at biokonverteringen stoppes ved, at der til dyrkningsmediet tilsættes en syre valgt fra gruppen omfattende fosforsyre, saltsyre, svovlsyre eller citronsyre.