Indian Patents. 264277:GAMMA-UNDECENOLACTONES AND PROCESS OF MAKING THE SAME

Title of Invention	GAMMA-UNDECENOLACTONES AND PROCESS OF MAKING THE SAME
Abstract	A gamma-undecenolactone corresponding to formula (I): in which the lactone ring can bear an unsaturation between carbon No. 2 and carbon No. 3, and in which R1 is a C7 alkenyl group bearing a single unsaturation located at C10-C11, or a C7 alkynyl group comprising several unsaturations, including one C10 alkenic unsaturation and at least one other alkenic unsaturation on a carbon other than C7.

Full Text	The present invention relates to a new lactone, gamma- undecenolactone; to the synthesis thereof via the biological process, in particular the stereoselective synthesis of each of its (R) or (S) isomers and to the uses thereof, in particular in the food and perfumery sector. "Natural" products are increasingly appreciated by the general public and, as a result, industries that use aromatic or odorant compounds concentrate their efforts on the development of "natural" aromatizing substances and preparations. Only substances that have been identified in nature can aspire to this label; they are therefore currently produced either from plants or from microorganisms; the latter are increasingly used, biotechnological processes now making it possible to synthesize natural molecules at reasonable expense. This is the case of gamma-lactones. Gamma-lactones are aromatic molecules that constitute the aroma and the flavor of many natural products. For example, gamma-heptalactone is known for its hazelnut or caramel aroma and taste, gamma-nonalactone has a fatty, creamy, or coconut aroma; gamma-decalactone and gamma-undecalactone have a peach or apricot aroma and taste. Gamma-lactones exist naturally, in their two enantiomeric forms (R) and (S) , the (R) enantiomer being, however, predominant. Gamma-lactones can be produced synthetically, or by biosynthesis by means of microorganisms. Thus, EP 371 568 describes a process for producing gamma-lactones by means of microorganisms that are acceptable for preparing food products, such as, in particular, Saccharomyces cerevisiae, Debaromyces hansenii or Candida boidinii. US 5,112,803 indicates that gamma-octalactone, and in particular its (R) and (S) optical isomers, can be used to form butter aromas and flavors, and describes a process for increasing the aroma or the flavor of materials that can be consumed, by adding significant amounts of optically active gamma-octalactones, and a mixture of various compounds which are by-products of the biological process for obtaining gamma- octalactones, described in said patent. The process described in US 5,112,803 indicates that, using caprylic acid, it is possible to obtain the two (R) and (S) isomers of gamma-octalactone by biosynthesis using strains of the genus Syncephalastratum sp. or Mortierella sp.; however, this process is not enantioselective. Gamma-lactones are of great value in the food flavoring industry and in the perfumery industry, and real industrial high stakes are involved in the production of products that have different organoleptic nuances. The object of the present invention is to propose a new gamma-lactone, gamma-undecenolactone, which has aromatic and gustative properties comparable to those of known gamma-lactones, but different from the latter, in particular pineapple and passion fruit aromas and flavors. It is known that the chirality of volatile molecules can induce differences in terms of olfactory percep- tion, and that the optical isomers of gamma-lactones do not all have the same organoleptic notes: there is therefore a considerable advantage in producing a specific optical isomer of a gamma-lactone, in parti- cular if this production is carried out according to a process that is at least as efficient, or even more efficient, than in the prior art and at a competitive cost. Another object of the invention is therefore to propose a process for the biological synthesis of gamma- undecenolactone that is efficient and economical, and in particular a process for the stereoselective synthesis of each of the (R) and (S) optical isomers of gamma-undecenolactone. The gamma-undecenolactone in accordance with the inven- tion corresponds to formula (I): in which the lactone ring can bear an unsaturation between carbon No. 2 and carbon No. 3, and in which R1 is a C7 alkenyl or alkynyl group bearing at least one unsaturation, including one C10-C11 alkenic unsatura- tion, said R1 group being optionally substituted. According to a preferred embodiment of the invention, Rl comprises a single unsaturation. Very preferably, this unsaturation is an alkenic unsaturation. According to another embodiment of the invention, R1 comprises several unsaturations, including one C10 alkenic unsaturation and at least one other alkenic unsaturation on a carbon other than C7. According to a specific embodiment of the invention, R1 is an optionally substituted alkene group having 7 carbon atoms, bearing a single unsaturation located at C10-C11. According to another preferred embodiment of the invention, R1 is not substituted with a halogen atom, in particular bromine or chlorine. According to a preferred embodiment of the invention, Rl is a C7H13 group, preferably the group CH2=CH-CH2-CH2- CH2-CH2-CH2-, and the lactone ring is saturated. According to another embodiment of the invention, R1 comprises a single alkynic unsaturation, preferably on carbon 10. The term "substituted alkenyl or alkynyl" is intended to mean an alkenyl or alkynyl in which at least one carbon bears at least one substituent group. The term "substituent group" is intended to mean in particular a hydroxyl group, a keto group, a thiol group, an alkyl group or an alkenyl group. The gamma-undecenolactone according to the invention contains an asymmetric carbon in position 4 and can be in the (R) or (S) configuration. The invention relates to both the (R)-gamma- undecenolactone and the (S)-gamma-undecenolactone, and the mixture, in any proportions whatsoever, of these two optical isomers, in particular the racemic mixture. The invention also relates to the biological prepara- tion of the gamma-undecenolactone of formula (I) , and in particular its biosynthesis via the microbial pathway, from at least one substrate, in particular undecylenic acid or one of its esters, preferably methyl or ethyl ester, using a microbial culture of a strain chosen from those that allow hydroxylation of the substrate, in particular at C4. This preparation comprises the following steps: a) selecting an appropriate strain, b) culturing said strain in an appropriate culture medium, said culturing being optionally preceded by a step consisting in preculturing the strain, c) adding a substrate that can be converted into gamma-undecenolactone of formula (1), d) bioconverting the substrate to gamma- undecenolactone of formula (I), e) recovering the gamma-undecenolactone of formula (I) thus produced. The appropriate microbial strains, targeted in step a) for the biosynthesis of the gamma-undecenolactone according to the invention, are those that allow specific hydroxylation of the substrate, in particular at C4. When the product resulting from the biosynthesis is for use in the food industry, the food-grade strains are of course preferred. Among the strains that allow stereo- selective hydroxylation, mention may in particular be made of the strains of the genus Aspergillus sp., Penicillium sp., Mucor sp., Mortierella sp. . Since the strains all belong to microorganism class 1, and since some are food strains, their use does not pose any specific problem either for the industrial production of lactone or for its possible use in food products. According to a specific embodiment of the invention, the strain used is of the genus Aspergillus sp., preferably Aspergillus oryzae, of which mention may be made of the following collections of strains: Aspergillus oryzae DSMZ 18 61, Aspergillus oryzae DSMZ 18 64, Aspergillus oryzae DSMZ 1147, Aspergillus oryzae DSMZ 63303, Aspergillus oryzae CBS 570.65, Aspergillus oryzae CBS 819.72, Aspergillus oryzae CBS 110.27, Aspergillus oryzae VMF 8 8 093. Among them, Aspergillus oryzae DSMZ 18 61 and Aspergillus oryzae CBS 110.27 are preferred. According to another specific embodiment, the strain used is of the genus Mortierella sp., of which mention may be made of the following collections of species: 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 them, the Mortierella isabellina strains will be preferred, in particular Mortierella isabellina DSMZ 1414, Mortierella isabellina CBS 100559 and Mortierella isabellina CBS 221.29. In fact, the inventors have noted that, surprisingly and unexpectedly, the use of a strain of the genus Aspergillus sp. results in the selective production of (R)-gamma-undecenolactone, and that the use of a strain of the genus Mortierella sp. results in the selective production of (S)-gamma-undecenolactone. According to one embodiment of the invention, Yarrowia lipolytica strains are excluded from the invention since they are not capable of bringing about hydroxyla- tion at C4. Advantageously, all strains which are not capable of specifically and stereoselectively producing hydroxylation at C4 are excluded from the present invention. Without wishing to be bound by any theory, it can be envisioned that the conditions for culturing the strains could be of importance in the stereoselectivity observed, and also in the quantitative aspect of the bioconversion. The culture targeted in step b) of the process according to the invention comprises the preparation of a culture, preferably a semi-concentrated culture, of the strains, for example by cell amplification, in an appropriate culture medium. This culture may be preceded by a preculture of the strains in a first culture medium more suitable for the first steps of multiplication of the strain. The culture conditions used in the stereoselective process of the invention should be such that they result in the production of a mycelium which exhibits swellings filled with inclusions (with peroxysomes in particular) . According to the preferred embodiment of the invention, the cell culture prepared has a "compot" mycelium composed of compartmentalized filaments with no conidiospores and exhibiting bulging structures, filled with these inclusions (peroxysomes). The culture conditions should in fact be particularly appropriate for preventing sporulation of the mycelium. Moreover, the inventors have been able to note that the physiological state of the mycelium, obtained in particular due to the use of the culture conditions described in the present application (compartmentalized mycelium comprising swellings and bulges filled with inclusions, in particular with peroxysomes) could have a considerable influence on the reaction yield and would make it possible to obtain yields greater than those of the prior art. The physiological state of the mycelium could also have an influence on the stereoselectivity of the reaction. Thus, according to a preferred embodiment of the invention, step b) of the process of the invention is a step consisting in culturing the strain in an appro- priate culture medium for obtaining a compartmentalized mycelium comprising swellings and bulges filled with inclusions, in particular with peroxysomes. Advanta- geously, the culture medium used according to the invention does not contain peptone. Preferably, the culture medium used according to the invention comprises malt and/or yeast extract. According to a preferred embodiment, the mycelium used for step c) is concentrated. Preferably, the concentration of the mycelium used for step c) is between 5 and 15 g/1, preferably 6 to 12 g/1, very preferably 7 to 10 g/1. It has been particularly noted that the production of (S)-gamma-lactone by the Mortierella strain is particu- larly promoted, in terms of stereoselectivity and in terms of yield, by the use of a swollen mycelium filled with inclusions as described above; in fact, the use of such a mycelium would make it possible to obtain a reaction product which has an optical rotation greater, in absolute value, than those of the prior art; furthermore, the yield obtained by means of the process according to the invention, and in particular by the use of a swollen mycelium filled with inclusions as described above, makes it possible to obtain yields greater than those of the prior art. Step c) of the process consists in adding the substrate to the cell culture. According to the invention, the biological synthesis of gamma-undecenolactone involves any appropriate substrate, and preferably undecylenic acid or one of its esters. Among the other preferred substrates, mention may be made of all substituted derivatives of undecylenic acid or of its esters. Undecylenic acid and the methyl ester and ethyl ester of undecylenic acid are particularly preferred sub- strates. It goes without saying that the substrate can be any appropriate substrate, or a mixture of various appropriate substrates, in particular a mixture of undecylenic acid and of one or more of its esters. According to an advantageous embodiment of the invention, the substrate is added to the mycelium according to a batchwise or fed-batch process. Accord- ing to a preferred embodiment, the undecylenic acid is added as a mixture with an auxiliary product, for example an oil, in particular any conventional food oil such as soybean, maize, sunflower, or the like, or synthetic short-chain fatty acid triglycerides such as miglyol, preferably sunflower oil which is hydrogenated or rich in oleic acid, prior to it being brought into contact with the mycelium. The presence of the auxiliary product makes it possible in particular to greatly decrease the corrosive or toxic effect of the substrate, in particular of undecylenic acid. According to one embodiment of the invention, the synthesis according to the invention using the Mortierella isabellina strain is carried out in a medium free of mineral oil. Advantageously, the substrate is added in concentra- tions of from 0.3 to 2.5 g/l/h. Advantageously, the amount of oil, preferably of plant oil, mixed with the substrate is from 100 to 500 g/1, preferably 150 to 300 g/1. A source of sugar, preferably of glucose, is also added to the medium, at the same time as the substrate, so as to ensure that the energy needs of the cells are covered. Advantageously, the concentration of glucose added is from 0.3 to 0.4 g/l/h. The pH can be adjusted, as needed, during the addition of the substrate and throughout the duration of the bioconversion that will follow, by means of the addition of any appropriate base. The pH should be between 4.5 and 8.5, preferably between 5.5 and 8, and preferably between 6 and 7.5. The temperature is preferably maintained between 27 and 30°C, during the bioconversion. The duration of the bioconversion may be from 30 to 120 hours, preferably from 48 to 72 hours. The bioconversion of the substrate to gamma-undeceno- lactone, covered in step d) of the process of the invention, is a step consisting of lactonization preceded by a reaction consisting of hydroxylation of the substrate at C4, carried out by the strain. A source of oxygen is required in order for it to be possible for this hydroxylation to be carried out. This source of oxygen is preferably a gas containing oxygen, very preferably air or oxygen. The gas is dissolved in a relatively large amount in the reaction medium. According to a preferred embodiment, and as is known in the prior art, antifoams, in particular silicone oils or polymers of polyethylene glycol esterified with fatty acids, are used to control the foam that may form during the bioconversion. Once the bioconversion, i.e. the specific and stereo- selective hydroxylation at C4, followed by the lactoni- zation, has been carried out, step e) of the process consists in recovering the gamma-undecenolactone by extraction, the extraction of the gamma-undecenolactone being carried out by any appropriate means. Advanta- geously, the extraction of the gamma-undecenolactone is carried out by hydrodistillation, optionally followed by an esterification intended to subsequently eliminate the substrate which has not reacted. Alternatively, the extraction of the gamma-undeceno- lactone is carried out by solvent extraction (cyclohexane, ethyl acetate) , after acidification of the medium. According to a variant of the process according to the invention, step e) of the process is not carried out, and instead, a step e') is carried out, which step consists in continuing the process at the end of step d) by means of an in situ reduction of the gamma- undecenolactone obtained, before extraction, so as to obtain, for example, the corresponding gamma-undeca- lactone. According to a specific embodiment, the reduction can be stopped so as to obtain a gamma- undecenolactone that has fewer unsaturations than that derived from the bioconversion of step d). According to this other embodiment, the process according to the invention is continued at the end of step d) by stopping the pH regulation of the fermenter, and adding an active dry yeast, which may be a baker's yeast, a wine-maker's yeast or a brewer's yeast, and a source of sugar, in particular of glucose, to the reactor. When the pH reaches the value of 5.5, it is regulated at 5.5 with an appropriate base, for example sodium hydroxide. The mixture is left to incubate in order for the reduction to take place, preferably for a period of 12 to 24 hours, and then the gamma-undecalactone is extracted under the conditions described in step e) . According to another variant, the gamma-undecenolactone can be reduced to gamma-undecalactone by means of a fresh culture of a reducing microorganism or a microorganism that is at least placed under reducing conditions, for example Saccharomyces cerevisiae or Pichia etchelsii, Pichia pastoris, Hansenula polymorpha, Bacillus subtilis or Lactobacillus brevis. According to a specific embodiment of the invention, the reduction in step e') results in the production of gamma-undecalactone and this gamma-undecalactone corresponds to the formula: in which R1 is an optionally substituted C7 alkyl group. The term "substituted alkyl" is intended to mean an alkyl, at least one carbon of which bears at least one substituent group. The term "substituent group" is intended to mean in particular a hydroxyl group, a keto group, a thiol group, an alkyl group or an alkenyl group. According to a preferred embodiment of the invention, Rl is the group C7H13, very preferably CH2=CH-CH2-CH2- CH2-CH2-CH2-. The gamma-undecalactone obtained according to this specific embodiment contains an asymmetrical carbon in position 4 which has the same configuration as that of the gamma-undecenolactone molecule from which it derives, since the reduction reaction does not modify the stereoisomerism of the molecule. Step e') can also be carried out and makes it possible to obtain a more saturated (R)- or (S)-undecenolactone or an (R)- or (S)-gamma-undecalactone, depending on the stereochemistry of the gamma-undecenolactone obtained in step d). These saturated lactones, the (R)-gamma-undecalactone and the (S)-gamma-undecalactone, can be used in the same applications as their unsaturated homologs: these lactones have odorant and gustative properties such that they can be used in all perfumery and food flavoring applications, in particular for the produc- tion of perfumes, of odorant substances, or of cosmetic or food compositions, or as a food additive. For the purpose of the present invention, the term "perfumery" denotes not only perfumery in the usual sense of the term, but also the other fields in which the odor of products is important. This may involve perfumery compositions in the usual sense of the term, such as fragrancing bases and concentrates, eaux de Cologne, eaux de toilette, perfumes and similar products; topical compositions - in particular cosmetic compositions - such as face and body creams, talcum powders, hair oils, shampoos, hair lotions, bath salts and oils, bath and shower gels, toilet soaps, body antiperspirants and deodorants, shaving lotions and creams, soaps, creams, toothpastes, mouthwashes, ointments, and similar products; and maintenance products, such as softeners, detergents, washing powders, air fresheners, and similar products. The term "odorant" is used to denote a compound which gives off an odor. The term "food flavoring" is intended to mean any use of the compounds of the invention for the flavoring of any human or animal, liquid or solid food product, in particular drinks, dairy products, ice creams. The gamma-undecenolactone, (R) or (S), or a mixture of (R) and (S) , and also the gamma-undecalactone, (R) or (S) , or a mixture of (R) and (S) , can be used as perfuming compositions in order to contribute to providing exotic, floral or fruity notes, which has resulted in the applicant registering the trademark "Tropicalone®" given to the gamma-undecenolactone. According to the applications, the (S) enantiomer or the (R) enantiomer, or else a mixture of the 2 enantiomers in proportions determined by a person skilled in the art, will be used. Preferably, the products according to the invention are used in amounts of between 0.0025% and 10% by weight relative to the total weight of the composition in which they are present. They may go to make up the composition of solids or of liquids, and in particular the composition of gels, creams, ointments and/or sprays. The products according to the invention can also be used in a composition that is itself odorant, or in a composition in which the odorant agent is used to mask or neutralize certain odors. Other characteristics and advantages of the present invention will emerge clearly upon reading the examples given hereinafter, which illustrate the invention without, however, limiting it. Example 1, step a: - Selection of strains All the strains of the collection are first inoculated onto MGY agar medium and incubated for 72 h at 27 °C; these strains are subsequently inoculated into one- liter Erlenmeyer flasks containing 100 ml of 1x malt medium and incubated for 24 h at 27°C. The substrate, undecylenic acid, is then added to the culture medium (5 g/1 in 10 doses) and the culture is maintained for a further 48 h to 120 h at 27°C. After olfaction and analyses of the gamma- undecenolactone concentration in the media, the most advantageous strains are selected; this was the case for the Mortierella isabellina CBS 100559, Mortierella isabellina CBS 221.29, Aspergillus oryzae DSMZ 1861 and Aspergillus oryzae CBS 110.27 strains, which were subsequently used for the fermenter optimization trials. Example 1, step b: - Preparation of cell cultures The Mortierella isabellina CBS 100559, Mortierella isabellina CBS 221.29, Aspergillus oryzae DSMZ 1861 or Aspergillus oryzae CBS 110.27 strain (origin = tube frozen at -80°C) is inoculated onto MGY agar and incubated at 27°C for 30 minutes. The above preculture is inoculated into 5 1 of 1x malt medium in a 6 1 fermenter: Mortierella isabellina Incubation is carried out at 27°C, 500 rpm, 3.5 1/1/h of air, open pH, for 30 hours. Aspergillus oryzae Incubation is carried out at 20°C, 500 rpm, 0.05 vvm of air, open pH, for 30 h and then at 25°C, 500 rpm, 0.05 vvm of air, open pH, for 24 hours. In both cases, a mycelium containing many large bulges full of inclusions (including peroxysomes) should be obtained. 125 1 of 1.5x malt medium are then prepared in a 300 1 fermenter: The medium is sterilized for 40 minutes at 121°C. The fermenter and its parts are sterile and pressurized. The temperature is stable and regulated at 27°C. The pressure is flushed and an air flow rate of 3.5 1/1/h, i.e. approximately 0.6 m3/h, is maintained. The base (10 N NaOH), the acid (85% H3PO4), the antifoam and the 6 1 fermenter which serves as inoculum are sterily brought together. The agitation speed is adjusted to 325 rpm, the antifoam is initiated, and then the inoculum (5 1) is inoculated, open pH. The agitation speed is maintained at 325 rpm and the aeration is increased to 2.2 m3/h (0.3 vvm) . Growth is allowed to continue for 24 hours, so as to have approximately 10 g/1 of mycelium on a dry weight basis: this mycelium should be "compot" and should consist of filaments comprising numerous bulges and swellings, without spores. Example 2, steps c and d: Conversion of the undecylenic acid by the Mortlerella sp. strains Once the amount and the quality of mycelium have been achieved, the undecylenic acid is dispensed at the flow rate of 0.3 g/l/h for 6 h, and then at the flow rate of 0.53 g/l/h for 72 h: i.e. a total of 40 g/1. This undecylenic acid is dispensed as a mixture with hydrogenated sunflower oil (1/4 acid-3/4 oil); this oil is therefore dispensed at the flow rates of 0.9 g/l/h and then 1.53 g/l/h. Glucose is continuously dispensed, in parallel, at the flow rate of 0.36 g/l/h for 72 h. The pH is regulated at 7.5 throughout the duration of the fermentation, with 5 N NaOH. The speed is increased to 505 rpm and aeration is carried out at the flow rate of 1 vvm, i.e. 12 m3/h. The conversion is pursued for 72 hours. A production of 6.5 g/1 of gamma-undecenolactone, the stereoisomerism of which is (S), is obtained. Example 3, steps c and d: Conversion of the undecylenic acid by the Aspergillus sp. strains Once the amount and the quality of the mycelium have been attained, the undecylenic acid is dispensed at the flow rate of 0.3 g/l/h for 6 h, and then of 0.53 g/l/h for 72 h: i.e. a total of 40 g/1. This undecylenic acid is dispensed as a mixture with hydrogenated sunflower oil (1/4 acid-3/4 oil). Glucose is continuously dispensed, in parallel, at the flow rate of 0.36 g/l/h for 72 h. The pH is regulated at 6.5 throughout the duration of the fermentation, with 5 N NaOH. Aeration is carried out at the flow rate of 0.5 vvm, i.e. 6 m3/h. The conversion is pursued for 80 hours. A production of 0.5 g/1 of gamma-undecenolactone, the stereoisomerism of which is (R), is obtained. Example 4, step e: Extraction-purification Acidification at pH 1.5 is carried out with 3 1 of 85% phosphoric acid. Heating is carried out at more than 100 °C for 30 minutes in order for the lactone to be essentially in its cyclized form and not in its open hydroxy acid form. The lactone is quantitatively determined, extraction solvent is added (preferably cyclohexane), and the mixture is stirred at ambient temperature for 1 hour. Centrifugation is carried out and the organic phase is recovered. The lactone is quantitatively determined. The solvent is concentrated and an oily "crude" is thus obtained. Vacuum distilla- tion is carried out. The "deresined" lactone and an exhausted oil are obtained. Purification is subsequently carried out by fractionating the lactone under vacuum. A product that is > 99% pure is obtained, which product is either gamma-undecenolactone (> 99% S) if a strain of Mortierella sp. was used, or gamma- undecenolactone (> 99% R) if a strain of Aspergillus sp. was used. Example 5, step e': Reduction of gamma-undecenolactone to gamma-undecalactone Instead of stopping the reaction, after the bioconversion of example 3, the process is continued as follows: The fermenter pH regulation is stopped. 200 g/1 of active dry yeast from commercial baker's yeast (i.e. 30 kg of yeast) and 100 g/1 of glucose (i.e. 15 kg of dry glucose or 30 kg of glucose syrup at 50%) are added to the medium. As soon as the pH reaches the value of 5.5, the pH is regulated at pH 5.5, with a 5 N NaOH base. Incubation is carried out for 12 to 24 h at 30°C, 325 rpm, air at 0.5 vvm. (S)-gamma-undecalactone or (R)-gamma-undecalactone are obtained, as appropriate. The extraction and the purification are then carried out in accordance with example 4 above. Example 6: Evaluation of the (S)-gamma-undecenolactone in perfumery The 99%-pure (S)-gamma-undecenolactone was tested on a sponge and in solution (of 5% in ethanol) : the head gives the impression of lactonic aldehyde of gamma- undecalactone intreleven aldehyde type (IFF), and very powerful. The background is very natural, lactonic and very powerful pineapple flesh. It was also tested in a formulation where it gives an interesting marine fruity note, for example in the following formula: Example 7: Evaluation of the (S)-gamma-undecenolactone in terms of a food flavoring The 99%-pure (S)-gamma-undecenolactone was tested at 10 ppm in mineral water: it has a woody and fruity note very different from that given by any other lactone used up until now in formulation; by comparison with (R)-gamma-decalactone, with (R) delta-decalactone and with (R) delta-dodecalactone, at the same dosage (10 ppm) , it has more of a coco flavor and is more woody with pineapple and passion fruit notes. In sugary water (10 ppm), it also has notes with a milk-like, very fatty, sugary, sweet characteristic. Its potency at 10 ppm gives it an advantage in formulation due to its unique milk characteristic, concentrated milk tendency. Also tested at 0.5 ppm and 1 ppm on a cream flavoring: it stands out better in the mouth at 0.5 ppm, giving a roundness to the cream and giving it a unique condensed milk-type characteristic. Example 8: Evaluation of the (S)-gamma-undecalactone in terms of food flavoring When compared at 99%-pure on a sponge with (R)-gamma- undecalactone, (S)-gamma-undecalactone gives less fatty, more natural and more "peachy" notes. When evaluated at 10 ppm in mineral water, it gives a characteristic peach and apricot taste. Example 9: Evaluation of the (S)-gamma-undecalactone in a food flavoring formulation The addition of gamma-undecenolactone to an exotic fruit flavoring increases the "exotic" appreciation, for example in the following formula: WE CLAIM: 1. A gamma-undecenolactone corresponding to formula (I): in which the lactone ring can bear an unsaturation between carbon No. 2 and carbon No. 3, and in which R1 is a C7 alkenyl group bearing a single unsaturation located at C10-C11, or a C7 alkynyl group comprising several unsaturations, including one C10 alkenic unsaturation and at least one other alkenic unsaturation on a carbon other than C7. 2. The gamma-undecenolactone as claimed in claim 1, in which the asymmetric carbon in position 4 is in the ( R) configuration. 3. The gamma-undecenolactone as claimed in claim 1, in which the asymmetric carbon in position 4 is in the (S) configuration. 4. The gamma-undecenolactone as claimed in any one of claims 1 to 3, in which the lactone ring does not contain an unsaturation and R1 is a C7H13 group preferably CH2=CH-CH2-CH2-CH2-CH2-CH2-. 5. A process for preparing a gamma-undecenolactone as described in any one of claims 1 to 4, wherein biosynthesis of said gamma-lactone is carried out via the microbial pathway, in a culture medium free of peptones, from at least one substrate, in particular undecylenic acid or one of its esters, preferably methyl or ethyl ester, using a microbial culture of a strain chosen from those that allow hydroxylation of the substrate at C4. 6. The process as claimed in claim 5, wherein it comprises the following steps: a) selecting an appropriate strain, chosen from those that allow hydroxylation of the substrate at C4, b) culturing said strain in an appropriate culture medium, said culturing being optionally preceded by a step consisting in preculturing the strain, c) adding a substrate that can be converted into gamma- undecenolactone of formula (I) as claimed in claim 1, d) bioconverting the substrate to gamma-undecenolactone of formula (I). e) recovering the gamma-undecenolactone of formula (I) produced. 7. The process as claimed in either one of claims 5 and 6, wherein said substrate is added in step c) as a mixture with at least one production auxiliary product, preferably chosen from oils, in particular sunflower oil that is hydrogenated or rich in oleic acid, mygliol or glucose, or a mixture of these ingredients. 8. The process as claimed in any one of claims 5 to 7, wherein step e) is an extraction of the gamma-undecenolactone by hydrodistillation, optionally followed by esterification and elimination of the substrate which has not reacted. 9. The process as claimed in any one of claims 5 to 7, wherein step e) is a solvent extraction of the gamma-undecenolactone obtained at the end of step d). 10. The process as claimed in any one of claims 5 to 7, wherein step e) is preceded by a step e') consisting of the in situ reduction of the undecenolactone obtained at the end of step d). 11 .The process as claimed in claim 10, wherein the product derived from said step e') is an undecalactone. 12. A stereoselective process for preparing (R) -gamma-undecenolactone as claimed in any one of claims 5 to 10, wherein the microbial strain targeted in step a) is chosen from strains of the genus Aspergillus sp., preferably Aspergillus oryzae, or from strains of the genus Mortierella sp., preferably Mortierella isabellina. 13.A perfumery composition, wherein it comprises an undecenolactone as claimed in any one of claims 1 to 4 in an amount comprised between 0.0025% and 10% by weight relative to the total weight of the composition. 14. The gamma-undecenolactone, as claimed in claims 1-3, is used in food composition. 15. The gamma-undecenolactone as claimed in claim 1-3, is used in a food additive. ABSTRACT Title: Gamma-undecenolactones and process of making the same. A gamma-undecenolactone corresponding to formula (I): in which the lactone ring can bear an unsaturation between carbon No. 2 and carbon No. 3, and in which R1 is a C7 alkenyl group bearing a single unsaturation located at C10-C11, or a C7 alkynyl group comprising several unsaturations, including one C10 alkenic unsaturation and at least one other alkenic unsaturation on a carbon other than C7.

Full Text

The present invention relates to a new lactone, gamma-
undecenolactone; to the synthesis thereof via the
biological process, in particular the stereoselective
synthesis of each of its (R) or (S) isomers and to the
uses thereof, in particular in the food and perfumery
sector.
"Natural" products are increasingly appreciated by the
general public and, as a result, industries that use
aromatic or odorant compounds concentrate their efforts
on the development of "natural" aromatizing substances
and preparations. Only substances that have been
identified in nature can aspire to this label; they are
therefore currently produced either from plants or from
microorganisms; the latter are increasingly used,
biotechnological processes now making it possible to
synthesize natural molecules at reasonable expense.
This is the case of gamma-lactones.
Gamma-lactones are aromatic molecules that constitute
the aroma and the flavor of many natural products. For
example, gamma-heptalactone is known for its hazelnut
or caramel aroma and taste, gamma-nonalactone has a
fatty, creamy, or coconut aroma; gamma-decalactone and
gamma-undecalactone have a peach or apricot aroma and
taste.
Gamma-lactones exist naturally, in their two
enantiomeric forms (R) and (S) , the (R) enantiomer
being, however, predominant.
Gamma-lactones can be produced synthetically, or by
biosynthesis by means of microorganisms. Thus, EP 371
568 describes a process for producing gamma-lactones by

means of microorganisms that are acceptable for
preparing food products, such as, in particular,
Saccharomyces cerevisiae, Debaromyces hansenii or
Candida boidinii.
US 5,112,803 indicates that gamma-octalactone, and in
particular its (R) and (S) optical isomers, can be used
to form butter aromas and flavors, and describes a
process for increasing the aroma or the flavor of
materials that can be consumed, by adding significant
amounts of optically active gamma-octalactones, and a
mixture of various compounds which are by-products of
the biological process for obtaining gamma-
octalactones, described in said patent. The process
described in US 5,112,803 indicates that, using
caprylic acid, it is possible to obtain the two (R) and
(S) isomers of gamma-octalactone by biosynthesis using
strains of the genus Syncephalastratum sp. or
Mortierella sp.; however, this process is not
enantioselective.
Gamma-lactones are of great value in the food flavoring
industry and in the perfumery industry, and real
industrial high stakes are involved in the production
of products that have different organoleptic nuances.
The object of the present invention is to propose a new
gamma-lactone, gamma-undecenolactone, which has
aromatic and gustative properties comparable to those
of known gamma-lactones, but different from the latter,
in particular pineapple and passion fruit aromas and
flavors.
It is known that the chirality of volatile molecules
can induce differences in terms of olfactory percep-
tion, and that the optical isomers of gamma-lactones do
not all have the same organoleptic notes: there is
therefore a considerable advantage in producing a

specific optical isomer of a gamma-lactone, in parti-
cular if this production is carried out according to a
process that is at least as efficient, or even more
efficient, than in the prior art and at a competitive
cost.
Another object of the invention is therefore to propose
a process for the biological synthesis of gamma-
undecenolactone that is efficient and economical, and
in particular a process for the stereoselective
synthesis of each of the (R) and (S) optical isomers of
gamma-undecenolactone.
The gamma-undecenolactone in accordance with the inven-
tion corresponds to formula (I):

in which the lactone ring can bear an unsaturation
between carbon No. 2 and carbon No. 3, and in which R1
is a C7 alkenyl or alkynyl group bearing at least one
unsaturation, including one C10-C11 alkenic unsatura-
tion, said R1 group being optionally substituted.
According to a preferred embodiment of the invention,
Rl comprises a single unsaturation. Very preferably,
this unsaturation is an alkenic unsaturation.
According to another embodiment of the invention, R1
comprises several unsaturations, including one C10
alkenic unsaturation and at least one other alkenic
unsaturation on a carbon other than C7.
According to a specific embodiment of the invention, R1
is an optionally substituted alkene group having 7

carbon atoms, bearing a single unsaturation located at
C10-C11.
According to another preferred embodiment of the
invention, R1 is not substituted with a halogen atom,
in particular bromine or chlorine.
According to a preferred embodiment of the invention,
Rl is a C7H13 group, preferably the group CH2=CH-CH2-CH2-
CH2-CH2-CH2-, and the lactone ring is saturated.
According to another embodiment of the invention, R1
comprises a single alkynic unsaturation, preferably on
carbon 10.
The term "substituted alkenyl or alkynyl" is intended
to mean an alkenyl or alkynyl in which at least one
carbon bears at least one substituent group. The term
"substituent group" is intended to mean in particular a
hydroxyl group, a keto group, a thiol group, an alkyl
group or an alkenyl group.
The gamma-undecenolactone according to the invention
contains an asymmetric carbon in position 4 and can be
in the (R) or (S) configuration.
The invention relates to both the (R)-gamma-
undecenolactone and the (S)-gamma-undecenolactone, and
the mixture, in any proportions whatsoever, of these
two optical isomers, in particular the racemic mixture.
The invention also relates to the biological prepara-
tion of the gamma-undecenolactone of formula (I) , and
in particular its biosynthesis via the microbial
pathway, from at least one substrate, in particular
undecylenic acid or one of its esters, preferably
methyl or ethyl ester, using a microbial culture of a
strain chosen from those that allow hydroxylation of

the substrate, in particular at C4.
This preparation comprises the following steps:
a) selecting an appropriate strain,
b) culturing said strain in an appropriate culture
medium, said culturing being optionally
preceded by a step consisting in preculturing
the strain,
c) adding a substrate that can be converted into
gamma-undecenolactone of formula (1),
d) bioconverting the substrate to gamma-
undecenolactone of formula (I),
e) recovering the gamma-undecenolactone of formula
(I) thus produced.
The appropriate microbial strains, targeted in step a)
for the biosynthesis of the gamma-undecenolactone
according to the invention, are those that allow
specific hydroxylation of the substrate, in particular
at C4.
When the product resulting from the biosynthesis is for
use in the food industry, the food-grade strains are of
course preferred. Among the strains that allow stereo-
selective hydroxylation, mention may in particular be
made of the strains of the genus Aspergillus sp.,
Penicillium sp., Mucor sp., Mortierella sp. . Since the
strains all belong to microorganism class 1, and since
some are food strains, their use does not pose any
specific problem either for the industrial production
of lactone or for its possible use in food products.
According to a specific embodiment of the invention,
the strain used is of the genus Aspergillus sp.,
preferably Aspergillus oryzae, of which mention may be
made of the following collections of strains:
Aspergillus oryzae DSMZ 18 61, Aspergillus oryzae DSMZ
18 64, Aspergillus oryzae DSMZ 1147, Aspergillus oryzae

DSMZ 63303, Aspergillus oryzae CBS 570.65, Aspergillus
oryzae CBS 819.72, Aspergillus oryzae CBS 110.27,
Aspergillus oryzae VMF 8 8 093.
Among them, Aspergillus oryzae DSMZ 18 61 and
Aspergillus oryzae CBS 110.27 are preferred.
According to another specific embodiment, the strain
used is of the genus Mortierella sp., of which mention
may be made of the following collections of species:
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 them, the Mortierella isabellina strains will be
preferred, in particular Mortierella isabellina DSMZ
1414, Mortierella isabellina CBS 100559 and Mortierella
isabellina CBS 221.29.
In fact, the inventors have noted that, surprisingly
and unexpectedly, the use of a strain of the genus
Aspergillus sp. results in the selective production of
(R)-gamma-undecenolactone, and that the use of a strain
of the genus Mortierella sp. results in the selective
production of (S)-gamma-undecenolactone.
According to one embodiment of the invention, Yarrowia
lipolytica strains are excluded from the invention

since they are not capable of bringing about hydroxyla-
tion at C4. Advantageously, all strains which are not
capable of specifically and stereoselectively producing
hydroxylation at C4 are excluded from the present
invention.
Without wishing to be bound by any theory, it can be
envisioned that the conditions for culturing the
strains could be of importance in the stereoselectivity
observed, and also in the quantitative aspect of the
bioconversion.
The culture targeted in step b) of the process
according to the invention comprises the preparation of
a culture, preferably a semi-concentrated culture, of
the strains, for example by cell amplification, in an
appropriate culture medium. This culture may be
preceded by a preculture of the strains in a first
culture medium more suitable for the first steps of
multiplication of the strain.
The culture conditions used in the stereoselective
process of the invention should be such that they
result in the production of a mycelium which exhibits
swellings filled with inclusions (with peroxysomes in
particular) . According to the preferred embodiment of
the invention, the cell culture prepared has a "compot"
mycelium composed of compartmentalized filaments with
no conidiospores and exhibiting bulging structures,
filled with these inclusions (peroxysomes). The culture
conditions should in fact be particularly appropriate
for preventing sporulation of the mycelium.
Moreover, the inventors have been able to note that the
physiological state of the mycelium, obtained in
particular due to the use of the culture conditions
described in the present application (compartmentalized
mycelium comprising swellings and bulges filled with

inclusions, in particular with peroxysomes) could have
a considerable influence on the reaction yield and
would make it possible to obtain yields greater than
those of the prior art. The physiological state of the
mycelium could also have an influence on the
stereoselectivity of the reaction.
Thus, according to a preferred embodiment of the
invention, step b) of the process of the invention is a
step consisting in culturing the strain in an appro-
priate culture medium for obtaining a compartmentalized
mycelium comprising swellings and bulges filled with
inclusions, in particular with peroxysomes. Advanta-
geously, the culture medium used according to the
invention does not contain peptone. Preferably, the
culture medium used according to the invention
comprises malt and/or yeast extract. According to a
preferred embodiment, the mycelium used for step c) is
concentrated. Preferably, the concentration of the
mycelium used for step c) is between 5 and 15 g/1,
preferably 6 to 12 g/1, very preferably 7 to 10 g/1.
It has been particularly noted that the production of
(S)-gamma-lactone by the Mortierella strain is particu-
larly promoted, in terms of stereoselectivity and in
terms of yield, by the use of a swollen mycelium filled
with inclusions as described above; in fact, the use of
such a mycelium would make it possible to obtain a
reaction product which has an optical rotation greater,
in absolute value, than those of the prior art;
furthermore, the yield obtained by means of the process
according to the invention, and in particular by the
use of a swollen mycelium filled with inclusions as
described above, makes it possible to obtain yields
greater than those of the prior art.
Step c) of the process consists in adding the substrate
to the cell culture. According to the invention, the

biological synthesis of gamma-undecenolactone involves
any appropriate substrate, and preferably undecylenic
acid or one of its esters. Among the other preferred
substrates, mention may be made of all substituted
derivatives of undecylenic acid or of its esters.
Undecylenic acid and the methyl ester and ethyl ester
of undecylenic acid are particularly preferred sub-
strates. It goes without saying that the substrate can
be any appropriate substrate, or a mixture of various
appropriate substrates, in particular a mixture of
undecylenic acid and of one or more of its esters.
According to an advantageous embodiment of the
invention, the substrate is added to the mycelium
according to a batchwise or fed-batch process. Accord-
ing to a preferred embodiment, the undecylenic acid is
added as a mixture with an auxiliary product, for
example an oil, in particular any conventional food oil
such as soybean, maize, sunflower, or the like, or
synthetic short-chain fatty acid triglycerides such as
miglyol, preferably sunflower oil which is hydrogenated
or rich in oleic acid, prior to it being brought into
contact with the mycelium. The presence of the
auxiliary product makes it possible in particular to
greatly decrease the corrosive or toxic effect of the
substrate, in particular of undecylenic acid. According
to one embodiment of the invention, the synthesis
according to the invention using the Mortierella
isabellina strain is carried out in a medium free of
mineral oil.
Advantageously, the substrate is added in concentra-
tions of from 0.3 to 2.5 g/l/h. Advantageously, the
amount of oil, preferably of plant oil, mixed with the
substrate is from 100 to 500 g/1, preferably 150 to
300 g/1.
A source of sugar, preferably of glucose, is also added

to the medium, at the same time as the substrate, so as
to ensure that the energy needs of the cells are
covered. Advantageously, the concentration of glucose
added is from 0.3 to 0.4 g/l/h.
The pH can be adjusted, as needed, during the addition
of the substrate and throughout the duration of the
bioconversion that will follow, by means of the
addition of any appropriate base. The pH should be
between 4.5 and 8.5, preferably between 5.5 and 8, and
preferably between 6 and 7.5.
The temperature is preferably maintained between 27 and
30°C, during the bioconversion. The duration of the
bioconversion may be from 30 to 120 hours, preferably
from 48 to 72 hours.
The bioconversion of the substrate to gamma-undeceno-
lactone, covered in step d) of the process of the
invention, is a step consisting of lactonization
preceded by a reaction consisting of hydroxylation of
the substrate at C4, carried out by the strain. A
source of oxygen is required in order for it to be
possible for this hydroxylation to be carried out. This
source of oxygen is preferably a gas containing oxygen,
very preferably air or oxygen. The gas is dissolved in
a relatively large amount in the reaction medium.
According to a preferred embodiment, and as is known in
the prior art, antifoams, in particular silicone oils
or polymers of polyethylene glycol esterified with
fatty acids, are used to control the foam that may form
during the bioconversion.
Once the bioconversion, i.e. the specific and stereo-
selective hydroxylation at C4, followed by the lactoni-
zation, has been carried out, step e) of the process
consists in recovering the gamma-undecenolactone by

extraction, the extraction of the gamma-undecenolactone
being carried out by any appropriate means. Advanta-
geously, the extraction of the gamma-undecenolactone is
carried out by hydrodistillation, optionally followed
by an esterification intended to subsequently eliminate
the substrate which has not reacted.
Alternatively, the extraction of the gamma-undeceno-
lactone is carried out by solvent extraction
(cyclohexane, ethyl acetate) , after acidification of
the medium.
According to a variant of the process according to the
invention, step e) of the process is not carried out,
and instead, a step e') is carried out, which step
consists in continuing the process at the end of step
d) by means of an in situ reduction of the gamma-
undecenolactone obtained, before extraction, so as to
obtain, for example, the corresponding gamma-undeca-
lactone. According to a specific embodiment, the
reduction can be stopped so as to obtain a gamma-
undecenolactone that has fewer unsaturations than that
derived from the bioconversion of step d). According to
this other embodiment, the process according to the
invention is continued at the end of step d) by
stopping the pH regulation of the fermenter, and adding
an active dry yeast, which may be a baker's yeast, a
wine-maker's yeast or a brewer's yeast, and a source of
sugar, in particular of glucose, to the reactor. When
the pH reaches the value of 5.5, it is regulated at 5.5
with an appropriate base, for example sodium hydroxide.
The mixture is left to incubate in order for the
reduction to take place, preferably for a period of 12
to 24 hours, and then the gamma-undecalactone is
extracted under the conditions described in step e) .
According to another variant, the gamma-undecenolactone
can be reduced to gamma-undecalactone by means of a
fresh culture of a reducing microorganism or a

microorganism that is at least placed under reducing
conditions, for example Saccharomyces cerevisiae or
Pichia etchelsii, Pichia pastoris, Hansenula
polymorpha, Bacillus subtilis or Lactobacillus brevis.
According to a specific embodiment of the invention,
the reduction in step e') results in the production of
gamma-undecalactone and this gamma-undecalactone
corresponds to the formula:

in which R1 is an optionally substituted C7 alkyl
group. The term "substituted alkyl" is intended to mean
an alkyl, at least one carbon of which bears at least
one substituent group. The term "substituent group" is
intended to mean in particular a hydroxyl group, a keto
group, a thiol group, an alkyl group or an alkenyl
group.
According to a preferred embodiment of the invention,
Rl is the group C7H13, very preferably CH2=CH-CH2-CH2-
CH2-CH2-CH2-.
The gamma-undecalactone obtained according to this
specific embodiment contains an asymmetrical carbon in
position 4 which has the same configuration as that of
the gamma-undecenolactone molecule from which it
derives, since the reduction reaction does not modify
the stereoisomerism of the molecule.
Step e') can also be carried out and makes it possible
to obtain a more saturated (R)- or (S)-undecenolactone
or an (R)- or (S)-gamma-undecalactone, depending on the
stereochemistry of the gamma-undecenolactone obtained

in step d).
These saturated lactones, the (R)-gamma-undecalactone
and the (S)-gamma-undecalactone, can be used in the
same applications as their unsaturated homologs: these
lactones have odorant and gustative properties such
that they can be used in all perfumery and food
flavoring applications, in particular for the produc-
tion of perfumes, of odorant substances, or of cosmetic
or food compositions, or as a food additive.
For the purpose of the present invention, the term
"perfumery" denotes not only perfumery in the usual
sense of the term, but also the other fields in which
the odor of products is important. This may involve
perfumery compositions in the usual sense of the term,
such as fragrancing bases and concentrates, eaux de
Cologne, eaux de toilette, perfumes and similar
products; topical compositions - in particular cosmetic
compositions - such as face and body creams, talcum
powders, hair oils, shampoos, hair lotions, bath salts
and oils, bath and shower gels, toilet soaps, body
antiperspirants and deodorants, shaving lotions and
creams, soaps, creams, toothpastes, mouthwashes,
ointments, and similar products; and maintenance
products, such as softeners, detergents, washing
powders, air fresheners, and similar products.
The term "odorant" is used to denote a compound which
gives off an odor.
The term "food flavoring" is intended to mean any use
of the compounds of the invention for the flavoring of
any human or animal, liquid or solid food product, in
particular drinks, dairy products, ice creams.
The gamma-undecenolactone, (R) or (S), or a mixture of
(R) and (S) , and also the gamma-undecalactone, (R) or

(S) , or a mixture of (R) and (S) , can be used as
perfuming compositions in order to contribute to
providing exotic, floral or fruity notes, which has
resulted in the applicant registering the trademark
"Tropicalone®" given to the gamma-undecenolactone.
According to the applications, the (S) enantiomer or
the (R) enantiomer, or else a mixture of the 2
enantiomers in proportions determined by a person
skilled in the art, will be used.
Preferably, the products according to the invention are
used in amounts of between 0.0025% and 10% by weight
relative to the total weight of the composition in
which they are present. They may go to make up the
composition of solids or of liquids, and in particular
the composition of gels, creams, ointments and/or
sprays.
The products according to the invention can also be
used in a composition that is itself odorant, or in a
composition in which the odorant agent is used to mask
or neutralize certain odors.
Other characteristics and advantages of the present
invention will emerge clearly upon reading the examples
given hereinafter, which illustrate the invention
without, however, limiting it.
Example 1, step a: - Selection of strains
All the strains of the collection are first inoculated
onto MGY agar medium and incubated for 72 h at 27 °C;
these strains are subsequently inoculated into one-
liter Erlenmeyer flasks containing 100 ml of 1x malt
medium and incubated for 24 h at 27°C. The substrate,
undecylenic acid, is then added to the culture medium
(5 g/1 in 10 doses) and the culture is maintained for a
further 48 h to 120 h at 27°C.

After olfaction and analyses of the gamma-
undecenolactone concentration in the media, the most
advantageous strains are selected; this was the case
for the Mortierella isabellina CBS 100559, Mortierella
isabellina CBS 221.29, Aspergillus oryzae DSMZ 1861 and
Aspergillus oryzae CBS 110.27 strains, which were
subsequently used for the fermenter optimization
trials.
Example 1, step b: - Preparation of cell cultures
The Mortierella isabellina CBS 100559, Mortierella
isabellina CBS 221.29, Aspergillus oryzae DSMZ 1861 or
Aspergillus oryzae CBS 110.27 strain (origin = tube
frozen at -80°C) is inoculated onto MGY agar and
incubated at 27°C for 30 minutes.
The above preculture is inoculated into 5 1 of 1x malt
medium in a 6 1 fermenter:

Mortierella isabellina
Incubation is carried out at 27°C, 500 rpm, 3.5 1/1/h
of air, open pH, for 30 hours.
Aspergillus oryzae
Incubation is carried out at 20°C, 500 rpm, 0.05 vvm of
air, open pH, for 30 h and then at 25°C, 500 rpm, 0.05
vvm of air, open pH, for 24 hours. In both cases, a
mycelium containing many large bulges full of
inclusions (including peroxysomes) should be obtained.
125 1 of 1.5x malt medium are then prepared in a 300 1
fermenter:

The medium is sterilized for 40 minutes at 121°C. The
fermenter and its parts are sterile and pressurized.
The temperature is stable and regulated at 27°C. The
pressure is flushed and an air flow rate of 3.5 1/1/h,
i.e. approximately 0.6 m3/h, is maintained. The base
(10 N NaOH), the acid (85% H3PO4), the antifoam and the
6 1 fermenter which serves as inoculum are sterily
brought together. The agitation speed is adjusted to
325 rpm, the antifoam is initiated, and then the
inoculum (5 1) is inoculated, open pH. The agitation
speed is maintained at 325 rpm and the aeration is
increased to 2.2 m3/h (0.3 vvm) . Growth is allowed to
continue for 24 hours, so as to have approximately
10 g/1 of mycelium on a dry weight basis: this mycelium
should be "compot" and should consist of filaments
comprising numerous bulges and swellings, without
spores.
Example 2, steps c and d: Conversion of the undecylenic
acid by the Mortlerella sp. strains
Once the amount and the quality of mycelium have been
achieved, the undecylenic acid is dispensed at the flow
rate of 0.3 g/l/h for 6 h, and then at the flow rate of
0.53 g/l/h for 72 h: i.e. a total of 40 g/1. This
undecylenic acid is dispensed as a mixture with
hydrogenated sunflower oil (1/4 acid-3/4 oil); this oil
is therefore dispensed at the flow rates of 0.9 g/l/h
and then 1.53 g/l/h. Glucose is continuously dispensed,
in parallel, at the flow rate of 0.36 g/l/h for 72 h.
The pH is regulated at 7.5 throughout the duration of
the fermentation, with 5 N NaOH. The speed is increased
to 505 rpm and aeration is carried out at the flow rate
of 1 vvm, i.e. 12 m3/h. The conversion is pursued for
72 hours.
A production of 6.5 g/1 of gamma-undecenolactone, the

stereoisomerism of which is (S), is obtained.
Example 3, steps c and d: Conversion of the undecylenic
acid by the Aspergillus sp. strains
Once the amount and the quality of the mycelium have
been attained, the undecylenic acid is dispensed at the
flow rate of 0.3 g/l/h for 6 h, and then of 0.53 g/l/h
for 72 h: i.e. a total of 40 g/1. This undecylenic acid
is dispensed as a mixture with hydrogenated sunflower
oil (1/4 acid-3/4 oil). Glucose is continuously
dispensed, in parallel, at the flow rate of 0.36 g/l/h
for 72 h. The pH is regulated at 6.5 throughout the
duration of the fermentation, with 5 N NaOH. Aeration
is carried out at the flow rate of 0.5 vvm, i.e.
6 m3/h. The conversion is pursued for 80 hours. A
production of 0.5 g/1 of gamma-undecenolactone, the
stereoisomerism of which is (R), is obtained.
Example 4, step e: Extraction-purification
Acidification at pH 1.5 is carried out with 3 1 of 85%
phosphoric acid. Heating is carried out at more than
100 °C for 30 minutes in order for the lactone to be
essentially in its cyclized form and not in its open
hydroxy acid form. The lactone is quantitatively
determined, extraction solvent is added (preferably
cyclohexane), and the mixture is stirred at ambient
temperature for 1 hour. Centrifugation is carried out
and the organic phase is recovered. The lactone is
quantitatively determined. The solvent is concentrated
and an oily "crude" is thus obtained. Vacuum distilla-
tion is carried out. The "deresined" lactone and an
exhausted oil are obtained. Purification is
subsequently carried out by fractionating the lactone
under vacuum. A product that is > 99% pure is obtained,
which product is either gamma-undecenolactone (> 99% S)
if a strain of Mortierella sp. was used, or gamma-
undecenolactone (> 99% R) if a strain of Aspergillus
sp. was used.

Example 5, step e': Reduction of gamma-undecenolactone
to gamma-undecalactone
Instead of stopping the reaction, after the
bioconversion of example 3, the process is continued as
follows:
The fermenter pH regulation is stopped. 200 g/1 of
active dry yeast from commercial baker's yeast (i.e.
30 kg of yeast) and 100 g/1 of glucose (i.e. 15 kg of
dry glucose or 30 kg of glucose syrup at 50%) are added
to the medium. As soon as the pH reaches the value of
5.5, the pH is regulated at pH 5.5, with a 5 N NaOH
base. Incubation is carried out for 12 to 24 h at 30°C,
325 rpm, air at 0.5 vvm. (S)-gamma-undecalactone or
(R)-gamma-undecalactone are obtained, as appropriate.
The extraction and the purification are then carried
out in accordance with example 4 above.
Example 6: Evaluation of the (S)-gamma-undecenolactone
in perfumery
The 99%-pure (S)-gamma-undecenolactone was tested on a
sponge and in solution (of 5% in ethanol) : the head
gives the impression of lactonic aldehyde of gamma-
undecalactone intreleven aldehyde type (IFF), and very
powerful. The background is very natural, lactonic and
very powerful pineapple flesh.
It was also tested in a formulation where it gives an
interesting marine fruity note, for example in the
following formula:

Example 7: Evaluation of the (S)-gamma-undecenolactone
in terms of a food flavoring
The 99%-pure (S)-gamma-undecenolactone was tested at
10 ppm in mineral water: it has a woody and fruity note
very different from that given by any other lactone
used up until now in formulation; by comparison with
(R)-gamma-decalactone, with (R) delta-decalactone and
with (R) delta-dodecalactone, at the same dosage (10
ppm) , it has more of a coco flavor and is more woody
with pineapple and passion fruit notes.
In sugary water (10 ppm), it also has notes with a
milk-like, very fatty, sugary, sweet characteristic.
Its potency at 10 ppm gives it an advantage in
formulation due to its unique milk characteristic,
concentrated milk tendency.
Also tested at 0.5 ppm and 1 ppm on a cream flavoring:
it stands out better in the mouth at 0.5 ppm, giving a
roundness to the cream and giving it a unique condensed
milk-type characteristic.
Example 8: Evaluation of the (S)-gamma-undecalactone in
terms of food flavoring
When compared at 99%-pure on a sponge with (R)-gamma-
undecalactone, (S)-gamma-undecalactone gives less
fatty, more natural and more "peachy" notes.
When evaluated at 10 ppm in mineral water, it gives a
characteristic peach and apricot taste.

Example 9: Evaluation of the (S)-gamma-undecalactone in
a food flavoring formulation
The addition of gamma-undecenolactone to an exotic
fruit flavoring increases the "exotic" appreciation,
for example in the following formula:

WE CLAIM:
1. A gamma-undecenolactone corresponding to formula (I):

in which the lactone ring can bear an unsaturation between carbon No. 2 and
carbon No. 3, and in which R1 is a C7 alkenyl group bearing a single
unsaturation located at C10-C11, or a C7 alkynyl group comprising several
unsaturations, including one C10 alkenic unsaturation and at least one other
alkenic unsaturation on a carbon other than C7.
2. The gamma-undecenolactone as claimed in claim 1, in which the
asymmetric carbon in position 4 is in the ( R) configuration.
3. The gamma-undecenolactone as claimed in claim 1, in which the
asymmetric carbon in position 4 is in the (S) configuration.

4. The gamma-undecenolactone as claimed in any one of claims 1 to 3, in
which the lactone ring does not contain an unsaturation and R1 is a C7H13
group preferably CH2=CH-CH2-CH2-CH2-CH2-CH2-.
5. A process for preparing a gamma-undecenolactone as described in any
one of claims 1 to 4, wherein biosynthesis of said gamma-lactone is
carried out via the microbial pathway, in a culture medium free of
peptones, from at least one substrate, in particular undecylenic acid or
one of its esters, preferably methyl or ethyl ester, using a microbial culture
of a strain chosen from those that allow hydroxylation of the substrate at
C4.
6. The process as claimed in claim 5, wherein it comprises the following
steps:

a) selecting an appropriate strain, chosen from those that allow
hydroxylation of the substrate at C4,
b) culturing said strain in an appropriate culture medium, said
culturing being optionally preceded by a step consisting in
preculturing the strain,
c) adding a substrate that can be converted into gamma-
undecenolactone of formula (I) as claimed in claim 1,

d) bioconverting the substrate to gamma-undecenolactone of formula
(I).
e) recovering the gamma-undecenolactone of formula (I) produced.
7. The process as claimed in either one of claims 5 and 6, wherein said
substrate is added in step c) as a mixture with at least one production
auxiliary product, preferably chosen from oils, in particular sunflower oil
that is hydrogenated or rich in oleic acid, mygliol or glucose, or a mixture
of these ingredients.
8. The process as claimed in any one of claims 5 to 7, wherein step e) is an
extraction of the gamma-undecenolactone by hydrodistillation, optionally
followed by esterification and elimination of the substrate which has not
reacted.
9. The process as claimed in any one of claims 5 to 7, wherein step e) is a
solvent extraction of the gamma-undecenolactone obtained at the end of
step d).

10. The process as claimed in any one of claims 5 to 7, wherein step e) is
preceded by a step e') consisting of the in situ reduction of the
undecenolactone obtained at the end of step d).
11 .The process as claimed in claim 10, wherein the product derived from said
step e') is an undecalactone.
12. A stereoselective process for preparing (R) -gamma-undecenolactone as
claimed in any one of claims 5 to 10, wherein the microbial strain targeted
in step a) is chosen from strains of the genus Aspergillus sp., preferably
Aspergillus oryzae, or from strains of the genus Mortierella sp., preferably
Mortierella isabellina.
13.A perfumery composition, wherein it comprises an undecenolactone as
claimed in any one of claims 1 to 4 in an amount comprised between
0.0025% and 10% by weight relative to the total weight of the composition.
14. The gamma-undecenolactone, as claimed in claims 1-3, is used in food
composition.

15. The gamma-undecenolactone as claimed in claim 1-3, is used in a food
additive.

ABSTRACT

Title: Gamma-undecenolactones and process of making the same.
A gamma-undecenolactone corresponding to formula (I):

in which the lactone ring can bear an unsaturation between carbon No. 2 and
carbon No. 3, and in which R1 is a C7 alkenyl group bearing a single unsaturation
located at C10-C11, or a C7 alkynyl group comprising several unsaturations,
including one C10 alkenic unsaturation and at least one other alkenic
unsaturation on a carbon other than C7.

GAMMA-UNDECENOLACTONES AND PROCESS OF MAKING THE SAME

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