Method for preparing a compound of formula (i) by fermentation

Abstract

The present invention relates to a method for preparing a compound of formula (I) in which n is between 0 and 5, preferably between 1 and 2; R, which can be identical or different, is chosen from the group made up of OH, OR1, formyl, CO2H, linear or branched alkyl chains, which are optionally substituted, comprising between 1 and 6 carbon atoms, alkenyls, which may be linear or branched and optionally substituted, comprising between 1 and 6 carbon atoms; R1 is chosen from the group made up of methyl, ethyl, propyl and butyl, comprising a step (a) in which a substrate is transformed into a compound of formula (I) by bioconversion in the presence of a microorganism, which method is characterised in that step (a) is performed under non-aseptic, preferably non-sterile, conditions.

Description

[0001] DESCRIPTION

[0002] Process for the preparation of a compound of formula (I) by fermentation

[0003] Field of invention

[0004] The present invention relates to a process for preparing a compound of formula (I) comprising a step (a) in which a substrate is transformed into a compound of formula (I) by bioconversion in the presence of a microorganism, characterized in that step (a) is carried out under non-aseptic conditions, preferably under non-sterile conditions.

[0005] Prior art

[0006] Vanillin can be obtained by various methods known to those skilled in the art, and in particular by the following two routes:

[0007] - A so-called natural route based on a biotechnological process comprising in particular the culture of a microorganism capable of allowing the biotransformation of a fermentation substrate into vanillin. Such a process is known in particular from application EP0885968 in which the fermentation substrate is ferulic acid. US patent 5017388 describes a process in which the fermentation substrate is eugenol and / or isoeugenol. These processes result in the preparation of a vanillin known as natural vanillin.

[0008] - A so-called synthetic route comprising classic chemical reactions from guaiacol not involving microorganisms. This process results in the preparation of a vanillin called synthetic vanillin.

[0009] Finally, vanillin can also be prepared according to a route described as "bio-based" in which the vanillin is derived from lignin, we can cite in particular documents US 2745796, DE1132113 and the article entitled "Preparation of lignin from wood dust as vanillin source and comparison of different extraction method" by Azadbakht et al in International Journal of Biology and Biotechnology, 2004, vol 1, No 4, pp 535-537, or prepared from materials of natural origin, we can cite in particular document WO 2019 / 020773. Frambinone can also be obtained by different methods and in particular by a bioconversion process as described in document CN 112391418.

[0010] The processes for producing so-called natural vanillin or frambinone are carried out in fermenters under sterile conditions. Sterilization is generally achieved by injecting steam into the devices used for a period generally exceeding 20 minutes. The reagents are also generally sterile, particularly by passing them through a filter membrane. These processes are therefore tedious and expensive to implement and require specific equipment that can be sterilized. The energy impact of these processes is also very significant.

[0011] The present invention aims to provide a process for preparing a compound of formula (I) which is simple to implement and whose ecological and / or energy footprint is improved.

[0012] Brief description

[0013] A first subject of the present invention relates to a process for preparing a compound of formula (I) comprising a step (a) in which a substrate is transformed into a compound of formula (I) by bioconversion in the presence of a microorganism, characterized in that step (a) is carried out under non-sterile conditions.

[0014] Detailed description

[0015] For the purposes of the present invention, and unless otherwise indicated, the expression "between ... and..." includes the limits.

[0016] For the purposes of the present invention, and unless otherwise indicated, the growth of a microorganism refers to a process in which said microorganism multiplies. The growth results in the production of biomass.

[0017] In the context of the present invention, and unless otherwise indicated, the term "bioconversion" refers to a biotechnological process in which a microorganism allows the transformation of a substrate into a bioconversion product. The microorganism may be a wild strain, genetically modified obtained by molecular biology or mutated, in particular by random or directed mutagenesis. In the context of the present invention, and unless otherwise indicated, the term "process carried out under aseptic conditions" refers to a process carried out under conditions free from any germ and / or microorganism.

[0018] In the context of the present invention, and unless otherwise indicated, the term "process carried out under sterile conditions" refers to a process carried out under conditions free from any germ and / or microorganism likely to be harmful to the microorganism capable of transforming a substrate into a compound of formula (I) used in the context of the process of the present invention. A harmful effect on the microorganism capable of transforming a substrate into a compound of formula (I) can be measured by the conversion rate of said substrate into a compound of formula (I).

[0019] In the context of the present invention, and unless otherwise indicated, asepsis or sterilization may be obtained by any method, in particular thermal, chemical, mechanical or by radiation, allowing the elimination of germs and / or microorganisms. Asepsis or sterilization may be carried out on the fluids, raw materials, and / or devices used in the context of the process according to the present invention. By way of illustration, the following methods may be mentioned in particular:

[0020] Sterilization by dry or moist heat, carried out by raising the temperature to a sufficient temperature, followed by maintaining this temperature for a sufficient period of time to eliminate germs and / or microorganisms, for example the temperature may be 121°C, then maintaining this temperature of 121°C for a sufficient period of time to eliminate the germs,

[0021] Membrane filtration or several successive membrane filtrations, the smallest cut-off diameter of which allows the elimination of any germ and / or microorganism

[0022] For the purposes of the present invention, and unless otherwise indicated, the term "reactor" refers to a container suitable for carrying out chemical reactions. For the purposes of the present invention, the reactor may be used after being sanitized or sterilized or without sanitization or sterilization.

[0023] For the purposes of the present invention, and unless otherwise indicated, the term "fermentation" refers to any biological reaction involving a microorganism such as bacterial growth, bioconversion, biosynthesis or biocatalysis. For the purposes of the present invention, and unless otherwise indicated, the term "fermenter" refers to a reactor capable of carrying out fermentations, for example a bioreactor. For the purposes of the present invention, the fermenter may be used after being sanitized or sterilized or without sanitization or sterilization.

[0024] In the context of the present invention and unless otherwise indicated, a compound of formula (I) obtained by a bioconversion process may be described as a natural compound of formula (I). In the context of the present invention and unless otherwise indicated, a frambinone obtained by a bioconversion process may be described as a natural frambinone.

[0025] In the context of the present invention and unless otherwise indicated, a vanillin obtained by a bioconversion process can be described as natural vanillin.

[0026] A first aspect of the present invention refers to a process for preparing a compound of formula (I) comprising a step (a) in which a substrate is transformed into a compound of formula (I) by bioconversion in the presence of a microorganism characterized in that step (a) is carried out under non-sterile conditions.

[0027] In the context of the present invention, a compound of formula (I) has the following formula:

[0028] Formula (I) in which n is between 0 and 5, preferably between 1 and 2,

[0029] R, identical or different, is chosen from the group consisting of OH, ORi, formyl, CO2H, linear or branched alkyl chains, optionally substituted, comprising between 1 and 6 carbon atoms, alkenyls, linear or branched, optionally substituted, comprising between 1 and 6 carbon atoms.

[0030] According to a particular embodiment, RI is selected from the group consisting of methyl, ethyl, propyl and butyl. According to a particular embodiment, R is selected from the group consisting of formyl -CHO, OH, OMe, OEt, OPr, OBu, methyl, ethyl, -(CH2)2-C(O)-CH3, -CH=CH-C(O)-CH3, -CH=CH- CO2H, -CO2H, -CH2-OH.

[0031] According to a particular embodiment, the compound of formula (I) is chosen from vanillin and frambinone.

[0032] In the context of the present invention, the microorganism may be any microorganism capable of forming the compound of formula (I) by bioconversion of a suitable substrate. The microorganism, in particular a bacterium or fimgus, may be a wild microorganism, genetically modified by molecular biology, mutated, by random or directed mutagenesis.

[0033] Preferably the microorganism is chosen from bacteria belonging to the order Actinomycetales, Caulobacterales or Pseudomonadales, preferably belonging to the family Streptomycetacae, Pseudonocardiacae, Micrococcaceae, Caulobacteraceae, or Pseudomonadaceae, preferably Streptomyces setonii, Amycolatopsis sp. Streptomyces psammoticus, Amycolatopsis thermoflava, Micrococcus sp, Caulobacter segnis, Pseudomonasfluorescens. or their mutants, even more preferably Streptomyces setonii, Amycolatopsis sp. Streptomyces psammoticus. Preferably, the bioconversion reaction is carried out in the presence of a strain available under the number ATCC39116, DSMZ 9991, DSMZ 9992, CCTCC 2015329, CCTCC 2011265, IMI 390106 or Zyl 926 or their mutants, preferentially ATCC39116, DSMZ 9991, DSMZ 9992, CCTCC 2015329, IMI 390106 or Zyl 926 or their mutants and very preferentially ATCC39116, DSMZ 9991, DSMZ 9992, CCTCC 2015329, IMI 390106 or Zyl 926.According to another embodiment, the microorganism may be chosen from genetically modified microorganisms belonging to the family Saccharomyces cerevisiae, Schizosaccharomyces pombe, E.coli, Corynebacterium glutamicul.

[0034] The microorganism used in the context of the present invention may also be a strain as described in WO 2014 / 102368, WO 2016 / 001203 or EP 2721148.

[0035] According to another embodiment, the microorganism is chosen from fungi belonging to the genus Aspergillus, Pycnoporus, Penicillium, preferably belonging to the family Aspergillus luchuensis, Aspergillus niger, Picnoporus cinnabarinus, Penicillium camamberti. In the context of the present invention, the substrate can be transformed by bioconversion into a compound of formula (I).

[0036] In the context of the present invention, the substrate can be transformed by bioconversion into frambinone. The substrate is generally selected from the group consisting of p-coumaric acid, p-hydroxybenzalacetone.

[0037] In the context of the present invention, the substrate can be transformed by bioconversion into vanillin. The substrate is generally selected from the group consisting of glucose, ferulic acid, eugenol, isoeugenol, coumaric acid, caffeic acid, L-tyrosine, vanillic acid, 4-vinyl guaiacol, preferably selected from glucose, ferulic acid and eugenol.

[0038] In the context of the present invention, step (a) of the method is carried out under non-aseptic, preferably non-sterile, conditions. In the context of the present invention, the expression "step (a) is carried out under non-aseptic, preferably non-sterile, conditions" indicates that the reactor or fermenter in which step (a) is carried out, and optionally the various feed or outlet pipes of the reactor or fermenter have not been sanitized, preferably have not been sterilized prior to their use in step (a). In the context of the present invention, the expression "step (a) is carried out under non-aseptic, preferably non-sterile, conditions" indicates that the fermentation substrate and / or the solvent used during step (a) have not been sanitized, preferably have not been sterilized prior to their use in step (a).In the context of the present invention, the expression "step (a) is carried out under non-aseptic, preferably non-sterile, conditions" indicates that the transfer of the biomass used for the fermentation has not been carried out aseptically, preferably has not been carried out sterilely. In the context of the present invention, the expression "step (a) is carried out under non-aseptic, preferably non-sterile, conditions" indicates that the gas(es), in particular air or oxygen, used during step (a) are not sanitized, preferably have not been sterilized prior to their use in step (a).

[0039] The absence of sanitization, preferably sterilization, of the equipment, reagents and air or oxygen used during bioconversion step (a) is particularly advantageous, in particular in that it allows the use of simpler equipment. The energy cost of the bioconversion process is also improved, in fact the absence of sanitization, preferably sterilization, reduces steam consumption and reduces the carbon footprint of the process.

[0040] In the context of the present invention, step (a) can be carried out in batch or fed-batch. According to another embodiment, step (a) can be carried out continuously. Step (a) can also be carried out in a cascade of perfectly stirred reactors in series.

[0041] According to a particular embodiment, the fermentation substrate is introduced into the reactor or fermenter. A biomass is then introduced into the reactor.

[0042] According to another particular embodiment, the fermentation substrate is introduced into the reactor containing the biomass.

[0043] Advantageously, the total concentration of fermentation substrate and compound of formula (I) is between 0.1 g / L and 50 g / L. According to a particular embodiment, the total concentration of fermentation substrate and compound of formula (I) is between 0.1 g / L and 25 g / L, preferably between 0.2 g / L and 20 g / L. According to another particular embodiment, the total concentration of fermentation substrate and compound of formula (I) is between 10 g / L and 50 g / L, preferably between 12 g / L and 40 g / L, very preferably between 15 g / L and 35 g / L.

[0044] The pH during step (a) is controlled and between 7 and 9. The pH can optionally be regulated by adding base or acid.

[0045] Step (a) is generally carried out at a temperature between 25 and 50°C, preferably between 28°C and 45°C. Step (a) is generally carried out with stirring. Step (a) is generally carried out under air or oxygen. In the context of the present invention, the pO2 during step (a) is controlled and optionally regulated. A person skilled in the art will be able to adapt the pO2 during step (a) to the needs of the microorganism capable of transforming a substrate into a compound of formula (I), preferably into vanillin or frambinone, very preferably into vanillin used in the context of the present invention. By way of illustration, the pO2 may be between 1% and 99%, preferably of the order of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%. pO2 corresponds to the dissolved oxygen concentration in the bioconversion medium relative to the dissolved oxygen concentration at saturation, without biomass.In particular, pO2 can be measured with a Hamilton OXYFERM FDA VP 225 O2 Sensor.

[0046] Advantageously, the process of the invention is not only more sustainable, due to the reduction in its energy consumption, while making it possible to obtain a yield of compound of formula (I) at least equivalent to those of the processes requiring asepsis or sterilization.

[0047] In general, bioconversion step (a) is preceded by a step (aO) during which a microorganism is cultured so as to obtain a sufficient quantity of biomass. This biomass can then be used in a bioconversion process according to the present invention.

[0048] The culture of the bacteria is generally carried out in an aqueous medium, in the presence of nutrients. In general, the culture medium comprises a carbon source, an organic or inorganic nitrogen source, inorganic salts and growth factors. A person skilled in the art will be able to adapt the composition of the culture medium to the needs of the microorganism capable of transforming a substrate into a compound of formula (I) used in the context of the method of the present invention.

[0049] The carbon source concentration is generally between 5 and 50 gL 4 , preferably between 20 and 34 gL 4 The nitrogen source, such as yeast extract, and growth factors are generally added at a concentration between 2 and 20 gL, preferably between 5 and 10 gL. 4 . In addition, magnesium ions, such as magnesium sulfate, can be added at a concentration between 0.1 and 5 gL 4, preferably between 0.5 and 1 gL 4 . In general, the pH of the culture is between 3 and 9, in particular between 4 and 8, and very preferably between 6 and 8. In general, the temperature of the culture is between 10 and 55°C, particularly preferably in the range of 30 to 50°C, and very preferably between 35°C and 45°C.

[0050] The culture period generally lasts between 15 minutes and 80 hours, preferably between 1 hour and 50 hours and very preferably between 5 hours and 40 hours. The duration of the culture period is variable and can be adapted according to the microorganism capable of transforming a substrate into a compound of formula (I) used in the context of the present invention. By way of illustration, the culture period can last until the carbon source, generally glucose, is almost completely consumed, preferably such that the concentration of carbon source is less than or equal to 15 gL 4 , preferably less than or equal to 5 gL 4 , very preferably less than or equal to 3 gL 4. According to another embodiment, the culture period can last until 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% of the carbon source introduced at the start of step (aO) has been consumed. Step (aO) is generally carried out in a fermenter, under aseptic conditions, preferably sterile.

[0051] In the context of the present invention, step (a) may be followed by a step (b) of purification of the compound of formula (I), obtained. In general, the purification comprises a first step in which the fermentation must is separated from the biomass.

[0052] The purification of the compound of formula (I) obtained at the end of step (a) can in particular be carried out by liquid / liquid extraction, by distillation, by crystallization, by evaporation in a scraped film evaporator, a falling film evaporator, in a partition evaporator, and / or by stripping. Nanofiltration and ultrafiltration steps can also be carried out as part of the purification step (b).

[0053] The purification of the vanillin obtained at the end of step (a) can in particular be carried out by liquid / liquid extraction, by distillation, by crystallization, by evaporation in a scraped film evaporator, a falling film evaporator, in a partition evaporator, and / or by stripping. Nanofiltration and ultrafiltration steps can also be carried out as part of the purification step (b). By way of illustration, the purification process can be carried out according to the processes described in WO2013 / 087795, WO 2014 / 114590, WO2018 / 146210, WO2021 / 019005.

[0054] According to another embodiment, vanillin can be continuously extracted from the fermenter using a vanillin separation device. For illustration purposes, the purification method can be carried out according to the method described in WO2017 / 025339. There is no particular limitation as to the choice of the vanillin separation device. Preferably, the vanillin separation device is a membrane filtration device. This type of device makes it possible to recover from the fermentation must a retentate containing the microorganism and a microorganism-free filtrate. The vanillin separation device can also allow the selective extraction of vanillin from the extracted fermentation must.

[0055] The present invention also relates to a compound of formula (I), preferably a vanillin or a frambinone, capable of being obtained according to the process of the present invention. Finally, the present invention refers to the use of the compound of formula (I), preferably vanillin or frambinone obtained according to the present invention as a flavoring in the field of human and animal food, pharmacy, or as a perfume in the cosmetics, perfumery and detergent industry. Examples

[0056] Example 1: cultivation

[0057] The strain Streptomyces setonii ATCC 39116 is cultivated under the conditions described in document WO 2017 / 025339.

[0058] Examples 2 and 3: Bioconversion process according to the invention and comparison

[0059] Growth medium containing biomass, prepared according to Example 1, is used. The pH of the growth medium is adjusted to 8.4 by adding 30 mol% sodium hydroxide and kept constant, with stirring and aeration.

[0060] A solution containing ferulic acid, 30 mol% sodium hydroxide, and water is prepared. This solution is then added to the fermenter or reactor containing the biomass. Conditions

[0061] Table 1

[0062] The following bioconversion results were obtained:

[0063] Table 2

[0064] Examples 4 and 5: Bioconversion process according to the invention and comparison

[0065] Growth medium containing biomass, prepared according to Example 1, is used. The pH of the medium is adjusted to 8.4 and a coumaric acid solution is introduced to obtain a final concentration of between 5 and 50 g / L. The reaction medium is maintained at 37°C and 170 rpm for 24 h. After 24 h of bioconversion, the biomass is removed by centrifugation, the supernatant is filtered and analyzed by HPLC. 4-hydroxybenzaldehyde is obtained in the same proportions according to Examples 4 and 5.

[0066] Terms

[0067] Table 3

[0068] Examples 6 and 7: Bioconversion process according to the invention and comparison

[0069] Growth medium containing biomass, prepared according to Example 1 is used.

[0070] 4-Hydroxybenzalacetone is mixed into the medium obtained after the growth period as described in Example 1. The mixture is incubated at 30°C with stirring (200 rpm) for 72 hours. After 72 hours of bioconversion, the biomass is removed by centrifugation, the supernatant is filtered and analyzed by HPLC. Frambinone is obtained in the same proportions according to Examples 6 and 7. Conditions

[0071] Table 4

[0072] Thus, the process according to the present invention makes it possible to obtain results at least as good as the process according to comparative example 2 in terms of vanillin concentration or comparative examples 4 and 6 for the preparation of 4-hydroxybenzaldehyde and frambinone. The results in Table 2 demonstrate that the process according to the present invention is particularly advantageous in that it makes it possible, in particular, to reduce the steam consumption of the process and simplifies handling by not requiring any sterilization of incoming materials involved in the process. These results are obtained while maintaining the yields of the bioconversion step (a).

Claims

DEMANDS 1. Process for preparing a compound of formula (I) Formula (I) in which n is between 0 and 5, preferably between 1 and 2, R, identical or different, is chosen from the group consisting of OH, ORi, formyl, CO2H, linear or branched alkyl chains, optionally substituted, comprising between 1 and 6 carbon atoms, alkenyls, linear or branched, optionally substituted, comprising between 1 and 6 carbon atoms, RI is chosen from the group consisting of methyl, ethyl, propyl and butyl, comprising a step (a) in which a substrate is transformed into a compound of formula (I) by bioconversion in the presence of a microorganism characterized in that step (a) is carried out under non-aseptic conditions.

2. Process for preparing a compound of formula (I) in which step (a) is carried out under non-sterile conditions.

3. A process for preparing a compound of formula (I) according to any one of claims 1 to 2 characterized in that the reactor or fermenter in which step (a) is carried out, optionally the various feed or outlet pipes of the reactor or fermenter have not been aseptic, preferably have not been sterilized prior to their use in step (a).

4. A process for preparing a compound of formula (I) according to any one of claims 1 to 3, characterized in that the fermentation substrate and / or solvent used in during step (a) were not aseptic, preferably were not sterilized prior to their use in step (a).

5. A process for preparing a compound of formula (I) according to any one of the preceding claims characterized in that the transfer of the biomass used for fermentation is not carried out under aseptic conditions, preferably not carried out under sterile conditions.

6. A process for preparing a compound of formula (I) according to any one of the preceding claims characterized in that the total concentration of fermentation substrate and of compound of formula (I) is between 0.1 g / L and 50 g / L.

7. A process for preparing a compound of formula (I) according to any one of the preceding claims characterized in that the gas or gases, preferably air or oxygen, used during step (a) are not aseptic, preferably not sterilized prior to their use in step (a).

8. A process for preparing a compound of formula (I) according to any one of the preceding claims, characterized in that the fermentation substrate is introduced into the reactor or fermenter, and the biomass is then introduced into the reactor.

9. A process for preparing a compound of formula (I) according to any one of claims 1 to 7 characterized in that the fermentation substrate is introduced into the reactor containing the biomass.

10. A process for preparing a compound of formula (I) according to any one of the preceding claims, further comprising a step (aO) of culturing a microorganism so as to obtain a sufficient quantity of biomass prior to step (a).

11. A method for preparing a compound of formula (I) according to any one of the preceding claims, further comprising a step (b) of purifying the compound of formula (I) obtained at the end of step (a).

12. A method for preparing a compound of formula (I) according to any one of the preceding claims, wherein the compound of formula (I) is selected from the group consisting of vanillin and frambinone.

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