Modified vegetable oil containing 13-hydroxy-9(z)-octadecenoic acid, and method for preparing same

Abstract

The invention relates to a modified vegetable oil comprising, in percentages by weight expressed relative to the total weight of the free fatty acids in the modified vegetable oil, at least 10% of 13-hydroxy-9(Z)-octadecenoic acid (13-HOD). The invention also relates to a method for preparing a modified vegetable oil, comprising the following steps: - hydrolysis by biocatalysis in the presence of a lipase of the triglycerides of the vegetable oil in order to obtain linoleic acid in free form; - hydration by biocatalysis in the presence of a linoleic acid hydratase in order to obtain the modified vegetable oil.

Description

DESCRIPTION TITLE: Modified vegetable oil containing 13-hydroxy-9(Z)-octadecenoic acid, and its preparation process

[0001] The present invention relates to a modified vegetable oil containing 13-hydroxy-9(Z)-octadecenoic acid (hereinafter abbreviated "13-HOD"), as well as its preparation process.

[0002] A vegetable oil generally comprises, in mass percentages expressed in relation to the mass of said vegetable oil, between about 95% and about 99% triglycerides, the other constituents of the vegetable oil being minor.

[0003] Triglycerides are glycerides in which the three hydroxyl groups of glycerol are esterified by fatty acids, which may be identical or different. Triglycerides are thus esters derived from one molecule of glycerol and three fatty acid molecules, or in other words, glycerol-fatty acid triesters.

[0004] Depending on the plant species from which it is derived and its extraction method, a vegetable oil is more or less rich in saturated, polyunsaturated, and monounsaturated fatty acids. The distribution of these different fatty acids gives the vegetable oil its specific physical properties (for example, its texture and fluidity).

[0005] Thus, a vegetable oil comprises a mixture of fatty acids, predominantly in the form of triglycerides. Depending on the extraction method, free fatty acids may be present, but in very small quantities. A vegetable oil can be obtained by mechanical extraction, particularly by pressing. It may be refined or unrefined. It can be used for food or cosmetic purposes.

[0006] 13-HOD, whose chemical structure (1) is as follows:

[0007] 13-HOD is a hydroxylated derivative of linoleic acid, which is a natural fatty acid. Therefore, 13-HOD is a hydroxylated fatty acid.

[0008] Linoleic acid has the following chemical structure (2):

[0009] Linoleic acid is present, in varying quantities, in many vegetable oils (for example grapeseed or sunflower oil), mainly in the form of triglycerides and therefore very little in the form of free fatty acids.

[0010] Unlike linoleic acid, vegetable oils do not naturally contain 13-HOD. In other words, there is no naturally occurring 13-HOD.

[0011] However, 13-HOD has interesting cosmetic properties.

[0012] In addition, 13-HOD is known for its use as an intermediate in the preparation of lactones, which are chemical compounds used in a wide variety of applications such as food, polymers, agriculture, and especially in cosmetics and perfumery.

[0013] Therefore, 13-HOD synthesis routes implemented during lactone preparation processes have already been disclosed.

[0014] In this regard, US patent 11,499,171 B2 describes a process for producing delta-decalactone which includes the following steps: i) a step of synthesizing 13-HOD from linoleic acid and in the presence of a modified microorganism producing a protein of a defined amino acid sequence and having 13-hydratase linoleate activity; followed ii) by a step of synthesizing said delta-decalactone from the 13-HOD thus formed in step i), this step ii) being carried out in the presence of a microorganism having beta-oxidation activity.

[0015] Step i) is a biocatalytic step. It involves an enzymatic transformation carried out in vitro, using an enzyme produced by a modified microorganism. In this regard, it is important to remember that the operating conditions of biocatalysis (such as substrate concentrations, buffer solution, temperature, pH, solvent, agitation, etc.) must be precisely defined, controlled, and managed to promote optimal enzyme activity and thus convert, in the case of US patent 11,499,171 B2, linoleic acid (in other words the substrate) into 13-HOD optimally, that is to say in a controlled, rapid, reproducible manner and with the highest possible conversion rate.

[0016] Thus, in US patent 11,499,171 B2, 13-HOD is used as an intermediate in the synthesis of delta-decalactone. Furthermore, it is obtained from a pure compound, linoleic acid.

[0017] However, as mentioned above, linoleic acid is present in certain vegetable oils (for example, grapeseed or sunflower oil).

[0018] That is why it is also known to synthesize 13-HOD from a vegetable oil containing linoleic acid.

[0019] In this regard, patent application IT 2019 / 00015713 Al describes a process for preparing lactones (in particular dodecalactone and decalactone) for use as perfumes and flavorings, from the transformation of a vegetable oil; said process comprises the following steps, all carried out in the same bioreactor: - the hydrolysis of triglycerides present in vegetable oil in order to obtain fatty acids (in particular oleic, linoleic and linolenic acid) in free form, by means of an enzyme (lipase); - the hydration of the double bonds of the fatty acids thus released in the previous step by means of bacterial fermentation with the use of probiotic microorganisms expressing hydratase enzymes; - the microbiological degradation of the fatty acids thus hydrated into lactones, by means of yeasts, preferably oleaginous yeasts.

[0020] In the process described in patent application IT 2019 / 00015713 A1, after the hydration step, the 13-HOD formed in the bioreactor is immediately converted into lactones. As in US patent 11,499,171 B2, 13-HOD is therefore solely an intermediate in the synthesis of lactones.

[0021] Furthermore, in the process described in patent application IT 2019 / 00015713 A1, the triglyceride hydrolysis step is carried out by biocatalysis, while the hydration step is performed by bioconversion. This lactone preparation process puts thus implementing in the same bioreactor two very different enzymatic reaction techniques.

[0022] Indeed, unlike biocatalysis, where the goal is to optimize enzyme activity, in bioconversion, the enzyme transforming the substrate is present in living biomass produced through a fermentation process, and the aim is to improve the microorganism's growth. To achieve this, bioconversion takes place in a culture medium containing the nutrients necessary for the microorganism's growth, and the operating conditions (such as temperature, pH, bioconversion time, osmotic conditions, etc.) must be compatible with the microorganism. Furthermore, the substrate to be converted during bioconversion is diluted in the fermenter containing the culture medium, which is primarily composed of water, and the biomass.

[0023] However, it is known that the operating conditions of bioconversion are not always compatible with the implementation of a process on an industrial scale and can complicate the purification steps of the desired final product. Furthermore, bioconversion has the drawback of requiring long reaction times, which are not always suitable for industrial-scale implementation.

[0024] In other words, the preparation of 13-HOD as described in the first 2 steps of the process of patent application IT 2019 / 00015713 Al is not fully satisfactory, due to the use of bioconversion for the hydration step and the inherent disadvantages of this technique which have been recalled above.

[0025] Finally, since the process described in patent application IT 2019 / 00015713 A1 has the ultimate goal of obtaining lactones, 13-HOD is only one of the intermediate products obtained after the hydration step, among other fatty acids that are also transformed, notably from the transformation of oleic acid and linolenic acid. Indeed, after the hydrolysis of triglycerides, the resulting mixture is a complex mixture of various fatty acids in free form. This process was not developed specifically to synthesize 13-HOD, and therefore the yield of 13-HOD obtained during this process is low.

[0026] Referring in particular to example 4 of this Italian patent application describing the obtaining of a mixture of lactones at a concentration of 1 g / L of culture medium from 12 mL of linseed oil (i.e. a mass of 11.1 g because of density of 0.926 g / mL and which contained 17% of linoleic acid relative to the total mass of fatty acids) in 2 L of said culture medium, we deduce a production yield of only 18% of said mixture of lactones.

[0027] However, since the concentration of 13-HOD synthesized during this production process cannot exceed the aforementioned concentration of 1 g / L, this process described in patent application IT 2019 / 00015713 Al has a low productivity in 13 HOD, and this in addition with the disadvantage of a high dilution of linseed oil (namely 5.5 g / L) in the culture medium.

[0028] Thus, in light of the prior art mentioned above, it is clear that the 13-HOD preparation processes known to date, and implemented to use 13-HOD as an intermediate in lactone synthesis, are not entirely satisfactory, particularly in terms of yields and industrial-scale feasibility. Indeed, the quantities of 13-HOD obtained with these state-of-the-art processes are small. Furthermore, the productivity of these processes is relatively low.

[0029] Furthermore, in the interest of sustainable development, the cosmetics industry is paying increasing attention to the naturalness of its ingredients. In this regard, vegetable oils are natural compounds that perfectly meet all these requirements. This is why they are so popular in current cosmetic formulations.

[0030] Ingredients of natural origin are therefore prized and, when ingredients cannot be sourced naturally, the preferred alternative is to synthesize them from natural products and / or with green chemistry processes (including bioconversion or biocatalysis).

[0031] As explained above, 13-HOD is a compound with interesting cosmetic properties but is not naturally present in vegetable oils.

[0032] Therefore, given these requirements for naturalness in cosmetic development, it would be highly beneficial to have a process 13-HOD preparation: - whose starting product would be a vegetable oil containing linoleic acid, namely a perfectly natural product, - whose stages would be in line with green chemistry, - perfectly industrializable, and which would allow the 13-HOD to be obtained with high productivity, i.e. with high yields of conversion of linoleic acid into 13-HOD and from a high concentration of starting product (i.e. vegetable oil) in order to improve the industrial performance of said process and to avoid the use of large quantities of solvents involving subsequent treatment of used solvents.

[0033] The inventors have succeeded in achieving all these objectives by developing a modified vegetable oil containing 13-HOD, as well as a process for preparing a modified vegetable oil containing 13-HOD.

[0034] The invention thus relates to a modified vegetable oil which is characterized in that it comprises, in mass percentages expressed in relation to the total mass of free fatty acids contained in said modified vegetable oil, at least 10%, preferably at least 20%, more preferably at least 30% and even more preferably at least 40%, of 13-HOD.

[0035] The vegetable oil modified according to the invention may comprise, in mass percentages expressed in relation to the total mass of free fatty acids contained in said modified vegetable oil, between 10% and 80%, preferably between 20% and 70%, more preferably between 30% and 60%, of 13-HOD.

[0036] For the purposes of this invention, "modified vegetable oil" means a vegetable oil that does not exist in a natural form. Indeed, as explained above, vegetable oils do not naturally contain 13-HOD.

[0037] Unlike so-called "natural" vegetable oils, which, as explained above, are characterized by the fact that almost all of their fatty acids are in the form of triglycerides, the vegetable oil modified according to the invention can be a vegetable oil whose specific characteristic is that it essentially comprises fatty acids in free form. The vegetable oil modified according to the invention can comprise between 80% and 100%, preferably between 90% and 99%, of fatty acids in free form, relative to the total mass of fatty acids it contains.

[0038] Given the cosmetic properties of 13-HOD, the vegetable oil modified according to the invention exhibits interesting cosmetic properties.

[0039] The modified vegetable oil according to the invention may contain a small amount of triglycerides. For example, the modified vegetable oil according to the invention may contain, as a mass percentage relative to the total mass of said modified vegetable oil, less than 20%, preferably less than 10%, and more preferably less than 5%, of triglycerides. The modified vegetable oil according to the invention may also contain, as a mass percentage relative to the total mass of said modified vegetable oil, between 0.5% and 20%, preferably between 1% and 15%, and more preferably between 3% and 10%, of triglycerides.

[0040] Preferably, the vegetable oil modified according to the invention does not contain glycerol or possibly traces of glycerol.

[0041] The mass percentage of glycerol expressed relative to the total mass of said vegetable oil may be at most 5%, preferably 2%. In one embodiment of the invention, said mass percentage of glycerol may be between 0.05% and 5%, preferably between 0.1% and 3%, more preferably between 0.2% and 2%.

[0042] Vegetable oil can be chosen from the group consisting of grape seed, safflower, borage, peanut, wheat germ, corn, sunflower, blackberry, nigella, evening primrose, pumpkin seed, hemp, cottonseed, walnut, soybean, argan and rapeseed oil, taken alone or in mixtures thereof.

[0043] The invention also relates to a process for preparing a modified vegetable oil comprising 13-HOD which is characterized in that it comprises at least the following steps: a) at least one vegetable oil is available, of which part or all of the triglycerides comprise linoleic acid; b) the triglycerides of at least one vegetable oil are hydrolyzed by biocatalysis, in the presence of a lipase, to obtain a reaction medium containing a hydrolyzed vegetable oil which comprises linoleic acid in free form; c) Free-form linoleic acid is hydrated by biocatalysis in the presence of a hydratase to obtain a reaction medium containing the modified vegetable oil comprising 13-HOD, said hydratase being selective and hydroxylating the double bond at position 13 of the linoleic acid; d) Optionally, the modified vegetable oil comprising 13-HOD is isolated from the reaction medium obtained at the end of step c).

[0044] In the context of the disclosure of the present invention, "reaction medium" means a medium in which an enzymatic reaction is carried out by biocatalysis (namely in steps b) and c)) or, where appropriate, an isolation step (namely step d)) during the implementation of the preparation process according to the invention.

[0045] At the end of step b), a hydrolyzed vegetable oil is obtained.

[0046] At the end of step b), all or part of the linoleic acid, which was initially present in the vegetable oil as triglycerides, was released. During step b), other fatty acids besides linoleic acid that were initially present in the vegetable oil were also released. The inventors observed that during step b), linoleic acid was released preferentially over the other fatty acids.

[0047] Compared to prior art using vegetable oil as a starting product (for example, the aforementioned IT application 2019 / 00015713 A1), the process for preparing a modified vegetable oil containing 13-HOD according to the invention is unique in that the two enzymatic reaction steps are carried out by biocatalysis. This gives it the advantages of being easily industrialized and having a productivity far superior to that which could be obtained if the enzymatic reactions were carried out by bioconversion. Furthermore, all the drawbacks associated with bioconversion, as mentioned above, are avoided.

[0048] In addition, quite surprisingly, although step c) of hydration is carried out on a hydrolyzed vegetable oil, in other words a complex mixture containing fatty acids in free form including linoleic acid, a very satisfactory productivity and rate of conversion of linoleic acid to 13-HOD are obtained.

[0049] However, given this complex mixture, it was far from certain that such a satisfactory conversion rate of linoleic acid to 13-HOD could be achieved. Indeed, as detailed in the experimental section below, comparative tests showed that the biocatalytic hydration of oleic acid, which is also released after the hydrolysis of vegetable oil, resulted in lower conversion rates of oleic acid to its hydroxylated form.

[0050] The vegetable oil available at step a) may have been obtained by mechanical extraction, in particular by pressing. It may be refined or unrefined. It may have a food or cosmetic use.

[0051] The vegetable oil available in step a) can be a mixture of at least two vegetable oils.

[0052] The vegetable oil available at step a) can be chosen from the group consisting of grapeseed, safflower, borage, peanut, wheat germ, corn, sunflower, blackberry, black cumin, evening primrose, pumpkin seed, hemp, cottonseed, walnut, soybean, argan, and rapeseed oils, either alone or in mixtures thereof. These vegetable oils naturally have high linoleic acid content.

[0053] Preferably, the vegetable oil is grapeseed oil.

[0054] Preferably, the mass percentage of linoleic acid contained in the vegetable oil available at step a), expressed in relation to the total mass of fatty acids contained in said vegetable oil, is at least 10%, more preferably at least 20%, even more preferably at least 50% and most preferably at least 65%.

[0055] The vegetable oil available at step a) may comprise, in mass percentages expressed in relation to the total mass of fatty acids included in said vegetable oil, between 10% and 90%, preferably between 25% and 85%, more preferably between 45% and 80%, of linoleic acid.

[0056] The linoleic acid in the vegetable oil from step a) is primarily in the form of triglycerides. As explained above, a vegetable oil (so-called "natural" oil) consists mainly of triglycerides (between 95% and 99% of the mass of the vegetable oil).

[0057] The vegetable oil available in step a) may contain fatty acids other than linoleic acid. These may be saturated, such as palmitic, stearic, or arachidic acid. They may also be unsaturated, such as oleic, linolenic, or erucic acid.

[0058] In step b) of the preparation process, lipase transforms triglycerides into a mixture of free fatty acids and glycerol. Free linoleic acid is among the free fatty acids obtained at the end of step b). Glycerol is thus a byproduct of the enzymatic reaction in step b).

[0059] Advantageously, at the end of step b), all of the linoleic acid present as triglycerides in the vegetable oil from step a) has been released into the reaction medium. In other words, advantageously, at the end of step b), all of the linoleic acid is in free form.

[0060] Advantageously, step b) of biocatalytic hydrolysis is carried out by incubating a lipase with said vegetable oil in a reactor.

[0061] Preferably, step b) is carried out in an aqueous medium with a volume percentage of vegetable oil expressed relative to the volume of the reaction medium, which is at least 20%, preferably at least 30%.

[0062] The vegetable oil is thus diluted in the aforementioned aqueous medium. In other words, the vegetable oil is added to an aqueous medium.

[0063] Preferably, in step b), the volume percentage of vegetable oil expressed relative to the volume of the reaction medium can be at least 20%. Below 20%, the productivity of the preparation process according to the invention may be reduced because the dilution of the vegetable oil may be too great.

[0064] Advantageously, the volume percentage of vegetable oil, expressed relative to the volume of the reaction medium, can be between 20% and 80%, more preferably between 30% and 70%. Above 80%, the conversion rate of linoleic acid to 13-HOD in step c) may be limited.

[0065] Preferably, in step b), the mass concentration of the vegetable oil expressed in relation to the volume of the reaction medium can be at least 185 g / L.

[0066] Advantageously, the mass concentration of the vegetable oil expressed in relation to the volume of the reaction medium can be between 185 g / L and 740 g / L, more preferably between 276 g / L and 650 g / L.

[0067] The volume of the reaction medium corresponds to the sum of the volume of the vegetable oil and the volume of the aqueous medium.

[0068] The pH of the aqueous medium is advantageously between 6 and 8. This constitutes optimal conditions for step b) of hydrolysis. The pH of the aqueous medium is advantageously fixed by a buffer solution that may be included in the aqueous medium.

[0069] The aqueous medium may, for example, include a sodium phosphate buffer solution which fixes the pH of said aqueous medium to 7.

[0070] Step b) of hydrolysis can be carried out in an aqueous medium with a pH between 6 and 8 and at a temperature between 25°C and 70°C, preferably between 40°C and 60°C, even more preferably between 45°C and 55°C.

[0071] Step b) of hydrolysis can be carried out over a period of between 1 and 48 hours, preferably between 5 and 36 hours, and more preferably between 10 and 30 hours. In an advantageous embodiment of the invention, the duration of step b) of hydrolysis is 24 hours.

[0072] Advantageously, the lipase and the amount of lipase are appropriately chosen so that the complete hydrolysis of the triglycerides of the vegetable oil is carried out during step b) in at most 48 hours, more preferably in at most 24 hours.

[0073] Advantageously, lipase belongs to class EC 3.1.1., in particular EC 3.1.1.3.

[0074] Preferably, lipase is of natural origin.

[0075] Lipase can be produced by Streptomyces sp. It can also be produced by Candida sp., particularly Candida antarctica.

[0076] Advantageously, lipase is expressed in recombinant form.

[0077] Advantageously, lipase has been obtained by culturing bacteria such as E. coli or yeasts such as Pichia pastoris, Saccharomyces cerevisiae, Yarrowia lipolytica or fungi such as Aspergillus sp.

[0078] In step b), the lipase can be immobilized, for example by adsorption, microencapsulation, inclusion, crosslinking or chemical fixation on an insoluble support.

[0079] In step b), lipase may also be in the form of a culture supernatant (e.g. a mixture of proteins expressed by the culture strain) or in the form of a biomass including whole cells expressing lipase and expressed lipase.

[0080] When lipase is used in the form of biomass, i.e. when it is present in whole cells, the amount of biomass used in step b) of hydrolysis can be appropriately chosen so that its concentration is between 5 g / L and 30 g / L of the reaction medium.

[0081] The quantity of biomass to be implemented in step b) can be chosen appropriately so that the mass percentage of said biomass expressed in relation to the mass of vegetable oil (i.e. the vegetable oil available in step a) is between 0.9% and 5.4%.

[0082] In an advantageous embodiment of the invention, at the end of step b) and prior to step c), the reaction medium thus obtained is decanted so as to isolate the organic phase which includes fatty acids in free form.

[0083] In this advantageous embodiment of the invention, step c) is then carried out with the organic phase thus isolated.

[0084] More specifically and advantageously, the organic phase thus isolated can be diluted in an aqueous medium before carrying out step c).

[0085] The pH of this aqueous medium is advantageously between 5 and 8 and can be fixed by a buffer solution included in said aqueous medium. These conditions will be optimal for step c) of hydration.

[0086] The aqueous medium may, for example, include a buffer solution containing a mixture of citrate and sodium hydroxide which fixes the pH of said aqueous medium to 6.

[0087] Of course, this step of isolating the organic phase is not mandatory. Therefore, step c) can also be perfectly carried out with the reaction medium obtained at the end of step b), without the need to modify its pH, inactivate the lipase, or, if applicable, remove the biomass containing the lipase.

[0088] The reaction medium in step c) may advantageously include a surfactant. The surfactant may be a polysorbate (for example, polysorbate 20, polysorbate 40, or polysorbate 80).

[0089] The volume percentage of surfactant (e.g., polysorbate) expressed relative to the volume of the reaction medium in step c) may be less than 5%, preferably less than 2%. For example, the volume percentage of surfactant is between 0.5 and 1.5%.

[0090] In one embodiment of the invention, the reaction medium of step c) may be devoid of surfactant.

[0091] During step c), the free linoleic acid contained in the hydrolyzed vegetable oil obtained at the end of step b) is totally or partially converted into 13-HOD.

[0092] More specifically, in step c), the hydratase is selective. Indeed, it hydroxylates the double bond at position 13 of linoleic acid.

[0093] Advantageously, the hydratase implemented in step c) belongs to class EC4.2. Preferably, the hydratase is a hydratase that belongs to class EC4.2.1. More preferably, it is a 13-linoleate hydratase.

[0094] Preferably, hydratase is of natural origin.

[0095] Hydratase can be derived from Lactobacillus sp., preferably Lactobacillus acidophilus.

[0096] Advantageously, hydratase is expressed in recombinant form.

[0097] Advantageously, hydratase has been obtained by culturing bacteria such as E. coli or yeasts such as Pichia pastoris, Saccharomyces cerevisiae, Yarrowia lipolytica or fungi such as Aspergillus sp.

[0098] Advantageously, step c) of hydration by biocatalysis is carried out by incubating a hydratase with the hydrolyzed vegetable oil obtained at the end of step b) in a reactor.

[0099] In step c), the hydratase can be immobilized, for example by adsorption, microencapsulation, inclusion, crosslinking or chemical fixation on an insoluble support.

[0100] In step c), the hydratase may also be in the form of a culture supernatant (e.g. a mixture of proteins expressed by the culture strain) or in the form of a biomass including whole cells expressing the hydratase and the expressed hydratase.

[0101] When hydratase is used in the form of biomass, the amount of biomass used in step c) of hydrolysis can be appropriately chosen so that its concentration is between 55 g / L and 555 g / L of the reaction medium.

[0102] The quantity of biomass to be implemented in step c) can be chosen appropriately so that the mass percentage of said biomass expressed in relation to the mass of hydrolyzed vegetable oil is between 10% and 100%.

[0103] When hydratase is used in the form of biomass, the ratio of the mass of hydrolyzed vegetable oil to the mass of said biomass may be between 1:1 and 10:1, preferably between 1:1 and 5:1, even more preferably between 1:1 and 3:1.

[0104] Step c) of hydration can be carried out at a temperature between 15°C and 50°C, preferably between 20°C and 45°C, even more preferably between 25°C and 40°C.

[0105] Hydration step c) can be carried out over a period of between 1 and 48 hours, preferably between 5 and 36 hours, and more preferably between 10 and 30 hours. In an advantageous embodiment of the invention, the duration of hydration step c) is 24 hours.

[0106] Preferably, steps b) and c) are carried out successively. Indeed, the optimum temperatures for carrying out step b) of hydrolysis (around 50°C) are different from those for carrying out step c) of hydration (around 30°C).

[0107] Hydratase is added to the reaction medium obtained at the end of step b), if necessary in a reaction medium containing an aqueous medium and the organic phase if the latter was isolated at the end of step b). Indeed, hydratase does not act on the triglycerides of the vegetable oil but selectively on the linoleic acid released at the end of step b).

[0108] The preparation process according to the invention has the remarkable advantage that step c), the hydration of linoleic acid to 13-HOD, can be carried out directly in the reaction medium obtained at the end of step b), i.e., without the need to purify the linoleic acid released at the end of step b) or to isolate the organic phase containing the released fatty acids, including linoleic acid. The inventors have, in fact, discovered quite surprisingly that the hydratase activity is not inhibited during step c), even in the absence of purification of the linoleic acid and / or isolation of the organic phase containing it.

[0109] In one embodiment of the invention, steps b) and c) can be carried out without isolating the hydrolyzed vegetable oil thus obtained at the end of step b) from the reaction medium obtained at the end of step b) and by transferring this reaction medium into another reactor in order to carry out step c). In other words, in this embodiment of the invention, the reactors of steps b) and c) are two different reactors.

[0110] In another embodiment of the invention, steps b) and c) can be carried out without isolating the hydrolyzed vegetable oil thus obtained at the end of step b) from the reaction medium obtained at the end of step b) and in a monotopic manner.

[0111] By "steps b) and c) can be implemented in a monotopic manner", we mean that steps b) and c) can be carried out in the same reactor.

[0112] Of course, the choice of the reactor or reactors (for example a container) in which steps b) and c) of the preparation process according to the invention are carried out is perfectly within the reach of a person skilled in the art.

[0113] Preferably, in step c), the volume percentage of hydrolyzed vegetable oil, expressed relative to the volume of the reaction medium, can be at least 20%. Below 20%, the productivity of the preparation process according to the invention may be reduced because the dilution of the hydrolyzed vegetable oil may be excessive.

[0114] Advantageously, the volume percentage of hydrolyzed vegetable oil, expressed relative to the volume of the reaction medium, can be between 20% and 80%, more preferably between 20% and 70%. Above 80%, the conversion rate of linoleic acid to 13-HOD in step c) may be limited.

[0115] The volume of the reaction medium corresponds to the sum of the volume of the hydrolyzed vegetable oil and the volume of the aqueous medium.

[0116] Preferably, in step c), the mass concentration of the hydrolyzed vegetable oil expressed relative to the volume of the reaction medium can be at least 185 g / L.

[0117] Advantageously, the mass concentration of the hydrolyzed vegetable oil expressed in relation to the volume of the reaction medium can be between 185 g / L and 740 g / L, more preferably between 276 g / L and 650 g / L.

[0118] Preferably, step c) is carried out in such a way that, at the end of this step c), the conversion rate of linoleic acid to 13-HOD is greater than or equal to 30%, more preferably greater than or equal to 50%. In an advantageous embodiment of the invention, the conversion rate of linoleic acid to 13-HOD at the end of step c) may be between 60% and 80%.

[0119] Optionally, at the end of step c) of the preparation process according to the invention, a step d) is carried out, said step d) consisting of isolating from the reaction medium obtained at the end of step c) the modified vegetable oil comprising 13-HOD.

[0120] The implementation of step d) is perfectly within the capabilities of a person skilled in the art. Possible implementation methods for this step d) are detailed below as examples.

[0121] If not all the free fatty acids present in the reaction medium are in acidic form during step d), the reaction medium obtained at the end of step c) may firstly, it must be acidified so that all the free fatty acids present in said reaction medium are in acidic form.

[0122] In one embodiment of the invention, the reaction medium obtained at the end of step c) can be acidified to a pH of 3, preferably to a pH of 2, more preferably to a pH of 1. To do this, an acidic aqueous solution (for example, a concentrated hydrochloric acid solution) is added to the reaction medium so as to obtain a reaction medium that is acidified.

[0123] This acidification step is entirely optional. Indeed, all the fatty acids present in the reaction medium obtained at the end of step c) can be in acidic form.

[0124] In other words, the optional acidification step can be implemented to hydrolyze the remaining acids and remove the remaining glycerol from the aqueous phase.

[0125] Once the free fatty acids are in their acidic form, they can be extracted from the reaction mixture using an organic solvent. The choice of organic solvent is readily available to anyone skilled in the art. Examples include ethyl acetate, 2-methyltetrahydrofuran, methyl tert-butyl ether, methoxycyclopentane, dichloromethane, isopropyl acetate, or isobutyl acetate. This extraction of free fatty acids can optionally be repeated several times. The resulting organic phases are then combined to obtain a single organic phase containing the free fatty acids in their acidic form.

[0126] The organic phase can be filtered, preferably with a membrane (e.g., a glass microfiber membrane). Finally, the organic solvent can be removed, for example by evaporation, to isolate the modified vegetable oil containing 13-HOD.

[0127] The 13-HOD-modified vegetable oil that has been isolated may not contain glycerol, or possibly only traces of glycerol. This is because, during step d) of isolating the modified vegetable oil, the glycerol remains in the aqueous phase. The mass percentage of glycerol, expressed relative to the total mass of the modified vegetable oil thus isolated, may be at most 5%, preferably at most 2%. in an embodiment of the invention, said mass percentage of glycerol may be between 0.05% and 5%.

[0128] The invention also relates to a modified vegetable oil containing 13-HOD directly obtained by the preparation process as described above.

[0129] The invention and its advantages are illustrated in the examples below.

[0130] Examples:

[0131] Material :

[0132] The following raw materials were used during the experiments: - a grape seed oil marketed by the company DKSH France SA; - linoleic acid marketed by the company Sigma Aldrich; - oleic acid marketed by the company TCI; - the enzymes E4375, E4026 and E4027 which are enzymes from commercial kits marketed under the trade name SEQ.ENZYM® by the company Seqens.

[0133] More specifically, the fatty acid distribution in grape seed oil, established according to European Pharmacopoeia standard 2.4.22, was as follows: - between 0.0% and 2.0% of trans fatty acids; - between 6% and 10% palmitic acid; - between 0.0% and 0.5% palmitoleic acid; - between 3% and 6% stearic acid; - between 13% and 40% oleic acid; - between 58% and 77% linoleic acid; - between 0.0% and 0.5% linolenic acid; - between 0.0% and 0.3% arachidic acid; - between 0.0% and 2.0% erucic acid.

[0134] Grape seed oil thus consisted mainly of linoleic acid, oleic acid, palmitic acid and stearic acid.

[0135] The enzyme E4375 was a lipase.

[0136] The enzyme E4026 was a hydratase that was appropriately chosen to hydroxylate linoleic acid at the double bond in position 13.

[0137] The enzyme E4027 was a hydratase that was appropriately chosen to hydroxylate oleic acid at the double bond in position 10.

[0138] The process for preparing a modified vegetable oil containing 13-HOD according to the invention was implemented as follows on grape seed oil:

[0139] Step b) of hydrolysis:

[0140] In an IL reactor, 300 mL of grape seed oil were diluted in 200 mL of a potassium phosphate buffer solution at a concentration of 100 mmol / L and pH 7.

[0141] A biomass containing the E4375 enzyme was added to the reactor. The mass of the biomass was chosen appropriately so that its mass corresponded to 9% of the mass of the grape seed oil.

[0142] The reaction mixture thus obtained was stirred in the reactor at 52°C for 24 hours. By monitoring by high-performance liquid chromatography (HPLC), the complete conversion of triglycerides from grape seed oil into free fatty acids and glycerol was observed.

[0143] Once the complete conversion of triglycerides had been achieved, the reaction medium was then acidified with an aqueous solution of hydrochloric acid at a concentration of 1 mol / L.

[0144] Next, ethyl acetate was added to the acidified reaction mixture. The reaction mixture was then filtered through cotton under vacuum. Finally, the organic phase was isolated and the ethyl acetate was evaporated.

[0145] At the end of this isolation step, 240 g of hydrolyzed grape seed oil were obtained.

[0146] Step c) Hydration:

[0147] 55.5 mg of hydrolyzed grapeseed oil (60 pL) was introduced into a 4 mL bottle. A potassium phosphate buffer solution at a concentration of 0.1 mol / L and pH 6.5 was added to the bottle to dilute the hydrolyzed grapeseed oil to a concentration of 30 g / L in the reaction medium. Additionally, Tween® 40 surfactant was added to obtain a medium The reaction mixture included hydrolyzed grapeseed oil, a buffer solution, and a surfactant. The amount of surfactant was carefully chosen to ensure a volume percentage of 0.25% in the reaction mixture. The reaction mixture was stirred at 40°C. Then, E4026 hydratase, contained in a biomass, was added.

[0148] After 24 hours of incubation at 40°C, it was observed that 68% (mass percentage) of the linoleic acid in the hydrolyzed grapeseed oil was converted into 13-HOD. A modified grapeseed oil containing 13-HOD was thus obtained using the preparation method according to the invention.

[0149] The modified grapeseed oil thus obtained was analyzed. It comprised, in mass percentages expressed relative to the total mass of free fatty acids in said modified grapeseed oil: - 20.5% linoleic acid, - 17% oleic acid, - 7% palmitic acid, - 3.5% stearic acid, - 1% trans-oleic fatty acid, - 51% of 13-HOD.

[0150] It is noted that 13-HOD is the predominant fatty acid among the free fatty acids present in the modified seed oil according to the invention.

[0151] Further experiment: hydration of pure linoleic acid:

[0152] In addition, another experiment was carried out by performing step c) described above in the same way, with the sole exception that the 60 pL of hydrolyzed grape seed oil were replaced by 60 pL of pure linoleic acid.

[0153] After 24 hours of incubation at 40°C, it was observed that 67% (mass percentage) of pure linoleic acid was transformed into 13-HOD.

[0154] It is thus observed that, with the preparation process according to the invention, the conversion rate of linoleic acid from grapeseed oil, which was initially predominantly in the form of triglycerides, is equivalent to that obtained from pure linoleic acid. The preparation process according to the invention makes it possible to obtain a modified vegetable oil containing 13-HOD with a high yield of 13-HOD, comparable to that which would be obtained with pure linoleic acid (68% versus 67%).

[0155] Comparative example:

[0156] Hydration step of oleic acid present in hydrolyzed seed oil:

[0157] 55.5 mg of hydrolyzed grapeseed oil (60 pL) was placed in a 4 mL bottle. A 0.1 mol / L potassium phosphate buffer solution at pH 6.5 was added to the bottle to dilute the hydrolyzed grapeseed oil to a concentration of 30 g / L in the reaction medium. Tween® 40 surfactant was also added, resulting in a reaction medium containing hydrolyzed grapeseed oil, the buffer solution, and the surfactant. The amount of surfactant was adjusted to achieve a volume percentage of 0.25% in the reaction medium. The reaction medium was stirred at 40°C. Then, E4027 hydratase, in biomass form, was added.

[0158] After 24 hours of incubation at 40°C, it was observed that 42% (mass percentage) of the oleic acid contained in the hydrolyzed grape seed oil was hydroxylated.

[0159] Further experiment: hydration of pure oleic acid:

[0160] Furthermore, another experiment was carried out by performing step c) described above in the same way, with the sole exception that the 60 pL of hydrolyzed grape seed oil were replaced by 60 pL of pure oleic acid.

[0161] After 24 hours of incubation at 40°C, it was observed that 95% (mass percentage) of the oleic acid had been hydroxylated.

[0162] We observe that with hydrolyzed seed oil, the conversion rate of oleic acid into its hydroxylated form is much lower than that obtained from the pure form of oleic acid (42% versus 95%).

[0163] This comparative example confirms that a hydrolyzed vegetable oil is a complex mixture, meaning that it contains fatty acids in free form, resulting from the hydrolysis of the vegetable oil's triglycerides, and that, consequently, it is not possible to predict, from such a complex mixture, which fatty acids will be transformed into their hydroxylated form by enzymatic reaction via biocatalysis with a conversion rate that is comparable to that which would be obtained from the corresponding fatty acids in their pure form.

[0164] Experimentation in which steps b) and c) of the preparation process according to the invention were carried out in a monotopic manner:

[0165] An experiment was carried out by performing step c) following step b) without isolating the hydrolyzed vegetable oil obtained at the end of step b).

[0166] Step b):

[0167] In an IL reactor, 300 mL of grape seed oil were diluted in 200 mL of a potassium phosphate buffer solution at a concentration of 100 mmol / L and pH 7.

[0168] A biomass containing the E4375 enzyme was added to the reactor. The mass of the biomass was chosen appropriately so that its mass corresponded to 9% of the mass of the grape seed oil.

[0169] The resulting reaction mixture was stirred in the reactor at 52°C for 24 hours. High-performance liquid chromatography (HPLC) analysis confirmed the complete conversion of grape seed oil triglycerides into free fatty acids and glycerol. The reaction mixture was then used directly in step c) without prior acidification or extraction with an organic solvent.

[0170] Step c):

[0171] 9.4 mL of the reaction medium obtained at the end of step b) above were used to carry out step c). In a 50 mL bottle, 1% by volume of Tween 20® and 2.12 g of hydratase E4026 were added to this volume of reaction medium (called "crude").

[0172] After 24 hours of incubation at 40°C, it was observed that 73% (mass percentage) of pure linoleic acid was transformed into 13-HOD.

[0173] This experiment thus demonstrates that it is possible to eliminate an extraction step after step b). Indeed, as explained above, the preparation process according to the invention has the significant advantage that step c), the hydration of linoleic acid to 13-HOD, can be carried out directly in the medium. reaction obtained at the end of step b), namely without the need to purify the linoleic acid released at the end of step b) or the need to isolate the organic phase containing the released fatty acids, including linoleic acid.

Claims

DEMANDS 1. Modified vegetable oil, characterized in that it comprises, in mass percentages expressed in relation to the total mass of free fatty acids contained in said modified vegetable oil, at least 10%, preferably at least 20%, more preferably at least 40%, of 13-hydroxy-9(Z)-octadecenoic acid (hereinafter abbreviated as "13-HOD").

2. Modified vegetable oil according to claim 1, characterized in that it comprises, in mass percentages expressed in relation to the total mass of said modified vegetable oil, less than 20%, preferably less than 10%, more preferably less than 5%, of triglycerides.

3. Modified vegetable oil according to claim 1 or 2, characterized in that said vegetable oil is selected from the group consisting of grape seed, safflower, borage, peanut, wheat germ, maize, sunflower, blackberry, nigella, evening primrose, pumpkin seed, hemp, cotton, walnut, soybean, argan and rapeseed oil, taken alone or in mixtures thereof.

4. A process for preparing a modified vegetable oil comprising 13-HOD, characterized in that it comprises at least the following steps: a) at least one vegetable oil is made available, of which some or all of the triglycerides comprise linoleic acid; b) the triglycerides of at least one vegetable oil are hydrolyzed by biocatalysis in the presence of a lipase to obtain a reaction medium containing a hydrolyzed vegetable oil comprising linoleic acid in free form; c) the linoleic acid in free form is hydrated by biocatalysis in the presence of a hydratase to obtain a reaction medium containing the modified vegetable oil comprising 13-HOD, said hydratase being selective and hydroxylating the double bond at position 13 of the linoleic acid; d) optionally, the modified vegetable oil comprising 13-HOD is isolated from the reaction medium obtained at the end of step c).

5. A process for preparing a modified vegetable oil according to claim 4, characterized in that the vegetable oil available in step a) is selected from the group consisting of grape seed oil, safflower oil, borage oil, peanut oil, wheat germ oil, corn oil, sunflower oil, blackberry oil, black cumin oil, evening primrose oil, pumpkin seed oil, hemp oil, cottonseed oil, walnut oil, soybean oil, argan oil and rapeseed oil, taken alone or in mixtures thereof.

6. A process for preparing a modified vegetable oil according to any one of claims 4 to 5, characterized in that step b) is carried out in an aqueous medium with a volume percentage of the vegetable oil expressed relative to the volume of the reaction medium, which is at least 20%, preferably at least 30%.

7. A process for preparing a modified vegetable oil according to any one of claims 4 to 6, characterized in that step b) of hydrolysis is carried out in an aqueous medium having a pH between 6 and 8 and at a temperature between 25°C and 70°C, preferably between 40°C and 60°C, even more preferably between 45°C and 55°C. 8 Process for preparing a modified vegetable oil according to any one of claims 4 to 7, characterized in that the hydratase implemented in step c) is a 13-linoleate hydratase.

9. A process for preparing a modified vegetable oil according to any one of claims 4 to 8, characterized in that steps b) and c) are carried out without isolating the hydrolyzed vegetable oil thus obtained at the end of step b) from the reaction medium obtained at the end of step b) and in a monotopic manner.

10. A process for preparing a modified vegetable oil according to any one of claims 4 to 9, characterized in that the conversion rate of linoleic acid to 13-HOD at the end of step c) is greater than or equal to 30%, preferably greater than or equal to 50%.

Images