Composition of oil from microorganisms enriched in DHA or EPA diglycerides

Abstract

Microorganism Oil Composition Enriched with DHA or EPA Diglycerides. The present invention relates to a microorganism oil composition enriched with eicosapentaenoic acid or docosahexaenoic acid predominantly in the form of diglycerides, as well as a process for obtaining it. To this end, the invention relates to a microorganism oil composition characterized in that it comprises: - an eicosapentaenoic acid or docosahexaenoic acid content greater than or equal to 500 mg / g of composition, and, relative to the total amount of glycerides: - a quantity of diglycerides greater than 45%, - a quantity of monoglycerides and diglycerides greater than 60%.

Description

Title of the invention: Composition of oil from microorganisms enriched with DHA or EPA diglycerides

[0001] The present invention relates to a composition of microorganism oil enriched with eicosapentaenoic acid or docosahexaenoic acid mainly in the form of diglycerides, as well as a process for obtaining it.

[0002] For several years now, it has been recommended to enrich one's diet with polyunsaturated fatty acids due to their proven beneficial role in many physiological reactions and pharmaceutical functions.

[0003] Among the polyunsaturated fatty acids of interest, we distinguish those belonging to the omega-3 family such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and those belonging to the omega-6 family such as arachidonic acid (ARA).

[0004] DHA and EPA have been the subject of numerous physiological studies and their essential role in infants, children and adults is now known. DHA is known for its essential role in the development of the brain and retina and in the preservation of cognitive functions. EPA, for its part, contributes to proper cardiac function and has anti-inflammatory properties.

[0005] One way to increase the intake of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in one's diet is to consume concentrates made from oils, particularly fish oils or microalgae. Microalgae belonging to the genus Thraustochytrium or Schizochytrium are, for example, rich sources of long-chain polyunsaturated omega-3 fatty acids. The microalga Schizochytrium sp can produce between 20% and 35% of docosahexaenoic acid, by total weight of fatty acids (Hadley et al., 2017) (Hammond et al., 2001). In concentrates, the DHA or EPA contents have been increased compared to the contents of natural oils.

[0006] The methods for concentrating, or enriching, oils in DHA or EPA involve, in a first step, the separation of fatty acids from glycerol by ethanolic transesterification using a chemical catalyst or by enzymatic ethanolic transesterification. In a second step, the enrichment in DHA or EPA is carried out by molecular distillation, allowing the separation of the compounds according to their vapor pressure, usually their molecular weight.

[0007] By way of illustration, mention may be made of international patent application WO 2013 / 178936 which describes a process for enriching with DHA in the form of ethyl esters of a microalgal oil belonging to the Thraustochytriales sp family.

[0008] In these concentrated oils of the state of the art, the polyunsaturated fatty acids are found in the form of triglycerides or in the form of ethyl esters.

[0009] Another important qualitative element of the oil, besides the content of polyunsaturated fatty acids that can be obtained, is its bioavailability.

[0010] Thus, several studies describe the interest of fatty acids in the form of diglycerides compared to the form of triglycerides. Diglycerides consist of a glycerol skeleton on which two fatty acids are esterified. They can be found in the form of three stereoisomers snl,2, sn-1,3 or sn-2,3.

[0011] The intake of fat in the form of diglycerides has been shown to be beneficial in the management of diseases such as obesity, cardiovascular disease and diabetes (Yee-Ying Lee et al.; Maki et al.; Saito et al.; Flickinger and Masuto; Prabhavathi Devi et al.) and more generally in the nutritional prevention of metabolic syndrome.

[0012] Specifically, diglycerides allow: - Reduction of blood cholesterol levels, - Stimulation of beta oxidation, - Reduction of fat accumulation and reduction of body mass, - Modulation of glucose metabolism, - Improving bone health by increasing mineral density bone and improvement of bone microstructure.

[0013] It is accepted that these effects are due to the very structure of diglycerides. By comparison, numerous clinical studies, such as Tomonori Nagao et al., demonstrate that triglycerides, which have a different structure, do not allow the effects mentioned above to be obtained. Indeed, due to their structure, diglycerides use a different metabolic pathway for digestion and absorption than other fats, particularly triglycerides. For example, diglycerides ingested in sn-1,3 form are transformed into l-(3) monoglycerides in the small intestine, which are subsequently re-esterified to a very small extent into triglycerides. By comparison, ingested triglycerides are transformed into 2-monoglycerides, which are subsequently converted into triglycerides.

[0014] It is therefore clear that diglycerides are of interest in the pharmaceutical, nutraceutical, and food fields. In the field of food products, they could be used as a substitute for other fats that are less beneficial, or even harmful, in terms of health.

[0015] It therefore appears necessary, and this is an aim of the invention, to be able to provide oils concentrated in polyunsaturated fatty acids DHA or EPA, which are essentially in the form of diglycerides, and which have a high diglyceride content.

[0016] Furthermore, the good bioavailability of long-chain polyunsaturated fatty acids in the form of monoglycerides has also been verified (Valenzuleaa et al.). This study shows that supplementation with DHA monoglycerides allows for a higher DHA content in plasma and red blood cells than that found when DHA is provided in the form of ethyl esters or in the form of triglycerides (Effect of Supplementation with Docosahexaenoic Acid Ethyl Ester and sn-2 Docosahexaenyl Monoacylglyceride on Plasma and Erythrocyte Fatty Acids in Rats, Ann Nutr Metab, 2005;49:49-53).

[0017] It therefore also appears interesting to be able to offer oils concentrated in polyunsaturated fatty acids DHA or EPA, which also have a high monoglyceride content.

[0018] The present invention thus relates to a composition of microorganism oil enriched in polyunsaturated fatty acids, which is characterized in that it comprises:

[0019] - a content of eicosapentaenoic acid and / or docosahexaenoic acid greater than or equal to 500 mg / g of composition,

[0020] and, relative to the total quantity of glycerides:

[0021] - a quantity of diglycerides greater than 45%,

[0022] - a quantity of monoglycerides and diglycerides greater than 60%.

[0023] The composition according to the invention therefore has an excellent fatty acid profile with a high content of polyunsaturated fatty acids DHA or EPA in the form of diglycerides.

[0024] For the purposes of the invention, the term "enriched in polyunsaturated fatty acids" means a composition comprising, after implementing suitable concentration processes, a higher quantity, for example increased by a factor of 1.5 or 2, of polyunsaturated fatty acids than that which the composition initially presented.

[0025] Also, within the meaning of the invention, the term “total quantity of glycerides” means the sum of the quantities of monoglycerides, diglycerides, triglycerides and ethyl esters of fatty acids.

[0026] The oil composition can be obtained from microalgae of the genus Thraustochytrium, Schizochytrium, Nannochloropsis, Isochrysis, Phaeodactylum, Nitzchia, Staurosira, Crypthecodinium or Ulkenia.

[0027] Microalgae of the genera Thraustochytrium and Schizochytrium will preferably make it possible to obtain an oil composition enriched in docosahexaenoic acid (DHA).

[0028] Microalgae of the genera Nannochloropsis, Isochrysis, Phaeodactylum or Nitzchia will make it possible to preferentially obtain an oil composition enriched in eicosapentaenoic acid (EPA).

[0029] Advantageously, the composition comprises, relative to the total quantity of glycerides:

[0030] - a quantity of triglycerides less than 30%,

[0031] - a quantity of diglycerides between 45 and 75%,

[0032] - a quantity of monoglycerides between 10 and 30%.

[0033] Preferably, the composition comprises, relative to the total quantity of glycerides: - a quantity of triglycerides between 10 and 30%, - a quantity of diglycerides between 50 and 70%, - a quantity of monoglycerides between 15 and 25%.

[0034] According to a first embodiment, the composition comprises a DHA content greater than or equal to 600 mg / g of composition, more preferably greater than 700 mg / g of composition.

[0035] According to a second embodiment, the composition comprises an EPA content greater than or equal to 600 mg / g of composition, preferably greater than 700 mg / g of composition.

[0036] Another object of the invention is to provide a process for preparing a composition of microorganism oil having the characteristics described above.

[0037] The invention therefore also relates to a process for preparing an oil composition enriched with omega-3 polyunsaturated fatty acids, characterized in that it comprises:

[0038] (a) A step of obtaining an oil of microorganisms comprising acid docosahexaenoic acid or eicosapentaenoic acid in the form of ethyl esters in an amount greater than or equal to 500 mg / g of composition,

[0039] (c) a step of structuring docosahexaenoic acid or acid eicosapentaenoic in the majority form of diglycerides by reaction between the oil and one or more enzymes in the presence of glycerol,

[0040] (d) a step of removing glycerol,

[0041] (e) a short path molecular distillation step of the oil under vacuum. Step a#:

[0042] A microorganism oil comprising docosahexaenoic acid or eicosapentaenoic acid in the form of ethyl esters in an amount greater than or equal to 500 mg / g of composition can for example be obtained commercially. For example, we can cite the oil marketed under the name Omegavie Algae oils, by the company Polaris, France.

[0043] Alternatively, an oil enriched with docosahexaenoic acid in the form of ethyl esters can be obtained by implementing the following steps:

[0044] (i) a reaction step between an oil of microorganisms comprising docosahexaenoic acid in the form of triglycerides and an alcohol in the presence of a chemical or enzymatic catalyst,

[0045] (ii) a step of concentration of docosahexaenoic acid by molecular distillation under high vacuum.

[0046] Step (i) is a transesterification step which will allow fatty acids to be obtained in the form of ethyl esters (EE), which are then concentrated in step (ii).

[0047] The starting oil in step (i) may be, for example, an oil from microalgae of the genera Thraustochytrium or Schizochytrium.

[0048] An oil enriched with eicosapentaenoic acid in the form of ethyl esters can, for its part, be obtained by implementing the following steps:

[0049] (i) a reaction step between an oil of microorganisms comprising acid eicosapentaenoic acid and an alcohol in the presence of a chemical catalyst,

[0050] (ii) a step of concentration and purification of fatty acid ethyl esters including eicosapentaenoic acid ethyl esters by short path molecular distillation under high vacuum.

[0051] (iii) a step of concentration of eicosapentaenoic acid by molecular distillation on a rectification column.

[0052] Here again, step (i) is a transesterification step which will allow fatty acids to be obtained in the form of ethyl esters (EE).

[0053] The starting oil in step (i) may be, for example, a microalgae oil of the genera Nannochloropsis, Isochrysis, Phaeodactylum or Nitzchia.

[0054] Alternatively, it is possible, by implementing the same process, to obtain on the one hand, an oil enriched in docosahexaenoic acid and, on the other hand, an oil enriched in eicosapentaenoic acid, in the form of ethyl esters, by implementing the following steps:

[0055] (i) a reaction step between an oil of microorganisms comprising at the both docosahexaenoic acid and eicosapentaenoic acid, in the form of triglycerides, and an alcohol in the presence of a chemical or enzymatic catalyst,

[0056] (ii) a first step of molecular distillation under high vacuum of the oil from step (i), in a scraped film evaporator coupled to a rectification column comprising at least seven theoretical plates, and recovery of a first residue and a first distillate,

[0057] (iii) a second step of molecular distillation under high vacuum of the residue recovered in step (ii), in said scraped film evaporator coupled to the rectification column comprising at least seven theoretical plates, and recovery of a second residue and a second distillate.

[0058] Indeed, certain varieties of microalgae, such as those of the genus Thraustochytrium and Schizochytrium, although containing mainly docosahexaenoic acid, can also contain significant quantities of eicosapentaenoic acid. The above process allows the separation and concentration of eicosapentaenoic acid and docosahexaenoic acid. The second distillate obtained contains EPA and the second residue contains DHA. Step c#:

[0059] Step (c) is carried out using the oils concentrated in DHA or EPA obtained in step (a).

[0060] Preferably, the structuring step (c) is carried out according to a molar ratio between docosahexaenoic acid or eicosapentaenoic acid and glycerol of 1.

[0061] Preferably again, in the process according to the invention, step (c) comprises:

[0062] - mixing, in a vacuum reactor, with stirring and at a temperature of 35 to 40°C., docosahexaenoic acid or eicosapentaenoic acid and the enzyme(s), and,

[0063] - additions of glycerol in several portions, the additions being spaced apart by a duration between 1h and 3h.

[0064] The agitation parameter may be between 300 and 400 RPM and the vacuum parameter may be less than 15mBar.

[0065] Very good results in terms of diglyceride content are obtained when glycerol is added gradually.

[0066] The total quantity of glycerol will therefore be divided into three or four portions of identical quantity. The first portion will be added when mixing the fatty acids and the enzyme(s), then the other portions will be added one after the other by successive additions spaced 1 to 3 hours apart, preferably 2 hours.

[0067] The reaction can thus last between 4 and 10 hours in total.

[0068] According to a first embodiment, the enzyme used during step (c) is a lipase B produced by Candida antarctica. This enzyme is, for example, commercially available under the name Lipozyme 435®.

[0069] According to a second embodiment, step (c) is carried out using a mixture of two enzymes, the mixture being composed of 70-80% of a lipase B produced by Candida antarctica and 30-20% of a lipase produced by Thermomyces lanuginosus.

[0070] Very good results are obtained in particular with a mixture composed of 75% lipase B produced by Candida antarctica and 25% lipase produced by Thermomyces lanuginosus.

[0071] A lipase produced by Thermomyces lanuginosus is, for example, commercially available under the name Lipozyme TL IM®.

[0072] This regio-selective enzyme is advantageous because it allows the diglyceride level to be increased, and in particular, Sn-1,n-3 diglycerides. Indeed, if EPA or DHA, molecules with a high spatial size, are fixed in the Sn-2 position of the glycerol during the reaction initiated by the Candida antarctica enzyme, for example in the form of Sn-2 monoglycerides, the grafting of a second fatty acid becomes more difficult, especially for forms highly concentrated in EPA or DHA. The addition of the specific Thermomyces lanuginosus Sn-1,n-3 enzyme, in addition to the lipase B of Candida antarctica, makes it easier to graft a second fatty acid onto the glycerol.

[0073] The total quantity of enzymes used during step © is of the order of 6 to 8%, preferably 7.5%, relative to the quantity of ethyl esters in the oil. Step d#:

[0074] Step (d) aims to remove the residual glycerol. It can be carried out by decantation. Step e#:

[0075] Step (e) is a short-path molecular distillation step of the oil under vacuum which allows the separation of the molecules according to their molecular weight and, thus, the concentration of the EPA or DHA mainly in the form of diglycerides.

[0076] Preferably, this step takes place in a short path distiller at a wall temperature above 150°C, for example between 160 and 220°C, and a vacuum below 0.02 mBar. Step b#:

[0077] According to a particular embodiment, the method comprises, between step (a) and step (c): - a step (b) of structuring docosahexaenoic acid or eicosapentaenoic acid in the form of free fatty acids.

[0078] This structuring step is a saponification reaction which results in the formation of free fatty acids which are then used during the subsequent step (c).

[0079] This embodiment advantageously makes it possible to obtain, at the end of the process according to the invention, higher diglyceride and monoglyceride contents than the embodiments in which step (b) is not implemented.

[0080] Advantageously, according to this particular embodiment, the process comprises, following step (e) of short-path molecular distillation: - a step (f) of recovering a fraction comprising docosahexaenoic acid or eicosapentaenoic acid in free form and in the form of monoglycerides and of reintroducing this fraction into step (c).

[0081] The invention also relates to an oil composition, characterized in that it comprises a mixture of an oil composition as defined above, or obtained by a process as described above, and at least one other oil.

[0082] An oil composition according to the invention may in fact be mixed with other oils such as vegetable oils or microorganisms, having other qualitative characteristics. For example, it will be possible to mix an oil enriched with EPA essentially in the form of glycerides and obtained according to the invention with an oil enriched with DHA in the majority form of triglycerides or ethyl esters. Other mixtures are naturally possible.

[0083] The invention also relates to a food supplement characterized in that it comprises an oil as defined above or obtained by a process as described above.

[0084] A dietary supplement can be in any form, such as a capsule, a tablet, a liquid, a gel.

[0085] The invention also relates to a food product characterized in that it comprises an oil composition as defined previously or obtained by a process as described previously.

[0086] A food product may be any product intended for food, in any form. This may be, in particular, a nutritional formulation such as an infant or dietary preparation.

[0087] The invention also relates to a pharmaceutical or nutraceutical composition characterized in that it comprises an oil composition as defined previously or obtained by a process as described previously.

[0088] Finally, the invention relates to an oil composition as defined above or obtained by a process as defined above, for its use in the prevention and / or treatment of obesity, overweight, bone diseases, in the reduction of cholesterol levels and the modulation of glucose metabolism.

[0089] The invention will be better understood by reading the following exemplary embodiments.

[0090] Example 1: Obtaining microalgae oils enriched with DHA or EPA mainly in the form of diglycerides

[0091] Step (a) Obtaining microalgae oils comprising DHA or EPA mainly in the form of ethyl esters.

[0092] 1) Obtaining micro-algae oil comprising DHA: H1 oil (DHA)

[0093] The starting oil is a crude oil produced by the microalgae strain Schizochytrium sp T18 marketed by the company Mara Renewables Corporation. It initially contains 329 mg / g of DHA. Step (i)#: transesterification

[0094] A transesterification reaction is carried out on a biomass of 1800 kg of microalgae oil using 450 kg of ethanol and 21.6 kg of sodium ethylate, in a suitable reactor.

[0095] The reaction temperature is 50°C and the reaction time is 1 h. At the end of the reaction, the excess ethanol is evaporated under vacuum, then the mixture is cooled to a temperature of approximately 30°C and then subjected to decantation for 1 h. The light phase is recovered and then the glycerol is drained. A second decantation is carried out for 30 sec. The residual glycerol and monoglycerides are drained.

[0096] An acid water wash is then carried out by adding 17% of demineralized water containing 2.5% of phosphoric acid (75%) while stirring for 20 sec. The mixture is decanted for 20 sec and the aqueous phase is drained. This is followed by vacuum drying (pressure < 90 mbar) at 60°C for a time greater than 2 hours.

[0097] At the end of this step, the oil contains 329 m / g of DHA in the form of ethyl esters.

[0098] Step (ii): DHA concentration by molecular distillation

[0099] The oil is then passed through a degasser and then through a scraped film evaporator. The vapors are then distilled through a rectification column which is coupled to the evaporator supplied by UIC GmbH. Refluxing the distillate, i.e. reintroducing the distillate into the column, can increase the separation efficiency of the latter. The column used contains approximately seven theoretical plates. The distillation residue is recovered and represents the DHA-enriched fraction.

[0100] The operating conditions are as follows: Evaporator temperature: 225°C; Rectification column vacuum: 0.1 mbar; Reflux rate 75%, Temperature (top of column): 195°C.

[0101] The amount of DHA in the form of ethyl esters is 758 mg / g.

[0102] 2) Obtaining micro-algae oil comprising EPA: oilH2(EPA)

[0103] The starting oil is a crude oil produced by the strain Nannochloropsis sp obtained after culturing the biomass for about eight days under photoautotrophic conditions. The extraction of the fat from the biomass is carried out by ethanolic maceration. After separation of the remaining solid phase, the ethanol is evaporated under vacuum.

[0104] EPA produced by photoautotrophy is found incorporated into membrane lipids, either in the form of Glycolipids, or in the form of Phospholipids, or in the form of free fatty acids. The triglyceride form is poorly represented. Step (i)#: transesterification

[0105] A transesterification reaction is carried out on a biomass of 100 kg of microalgae oil using approximately 200 kg of ethanol and 10 kg of sulfuric acid, in a suitable reactor (reflux 60°C > 100). The transesterification is carried out by acid means, with sulfuric acid.

[0106] Step (ii): EPA concentration by short path molecular distillation under vacuum

[0107] The conditions are an evaporator wall temperature between 175 and 180°C and a vacuum of less than 0.1 mbar.

[0108] Step (iii): EPA concentration by molecular distillation on a rectification column

[0109] The operating conditions are as follows: Evaporator temperature: 230°C; Rectification column vacuum: 0.07 mbar; Reflux rate 65%, Temperature (top of column): 107°C, Temperature (bottom of column): 147°C.

[0110] The amount of EPA in the form of ethyl esters is 735 mg / g.

[0111] 3) Obtaining micro-algae oil comprising DHA and micro-algae oil algae containing EPA from the same process: H3 oil (DHA) and H4 oil (EPA)

[0112] The starting oil is a crude oil produced by the microalgae strain Schizochytrium sp marketed by the company DSM under the brand name Life's DHA 60. It initially contains 360 mg / g of DHA and 184 mg / g of EPA. Step (i): transesterification

[0113] A transesterification reaction is carried out on a biomass of 19 kg of microalgae oil using 4.75 kg of ethanol and 222 g of sodium ethylate, in a suitable reactor.

[0114] The reaction temperature is 50°C and the reaction time is 1 h. At the end of the reaction, the excess ethanol is evaporated under vacuum, then the mixture is cooled to a temperature of approximately 30°C and then subjected to decantation for 1 h. The light phase is recovered and then the glycerol is drained. A second decantation is carried out for 30 sec. The residual glycerol and monoglycerides are drained.

[0115] An acid water wash is then carried out by adding 3.2 kg of demineralized water containing 76.4 g of phosphoric acid (75%) while stirring for 20 sec. The mixture is decanted for 20 sec. and the aqueous phase is drained. This is followed by vacuum drying (pressure < 90 mbar) at 60°C for a time greater than 2 hours.

[0116] At the end of this step, the oil contains EPA and DHA in the form of ethyl esters.

[0117] Step (ii): first step of molecular distillation

[0118] The oil is then passed through a degasser and then through a scraped film evaporator. The vapors are then distilled through a rectification column which is coupled to the evaporator supplied by UIC GmbH. The aim here is to remove the lightest fatty acids while retaining the DHA and EPA. The column used contains seven theoretical plates. The distillation residue is recovered and represents the fraction enriched in EPA and DHA.

[0119] The operating conditions are as follows: Evaporator temperature: 225°C; Rectification column vacuum: less than 0.1 mbar; Reflux rate 70%, Temperature (bottom of column): 190°C, Temperature (top of column): 135°C.

[0120] At the end of this step, a residue fraction and a distillate fraction are obtained. The residue fraction contains EPA and DHA.

[0121] Step (iii): second step of molecular distillation

[0122] The operation of step (II) is repeated on the residue. This is then returned to the degasser and then passes through the scraped film evaporator. The vapors are then distilled through the rectification column coupled to the evaporator as in the case of the previous step (ii). The aim here is to separate the DHA and the EPA.

[0123] The operating conditions are as follows: Evaporator temperature: 235°C; Rectification column vacuum: less than 0.05 mbar; Reflux rate 60%, Temperature (bottom of column): 202°C, Temperature (top of column): 160°C.

[0124] At the end of this step, a fraction of distillate containing 733 mg / g of EPA and a fraction of residue containing 706 mg / g of DHA are obtained.

[0125] The oils H1(DHA), H2(EPA), H3(DHA) and H4(EPA) thus all contain an EPA or DHA content greater than 700 mg / g of oil and are ready to be used in the following steps.

[0126] Step (c): structuring of docosahexaenoic acid or eicosapentaenoic acid in the majority form of diglycerides by reaction between the oil and one or more enzymes in the presence of glycerol.

[0127] Option 1: Reaction in the presence of a single enzyme

[0128] Raw materials and enzyme:

[0129] 180g of an oil obtained in step (a),

[0130] 4 portions each of 12.5 g of glycerol (100%), or 50 g of glycerol in total,

[0131] 13.5 g of lipase B from Candida antarctica (Lipozyme 435®),

[0132] Molar ratio of docosahexaenoic acid or eicosapentaenoic acid to glycerol of 1.

[0133] 180g of oil and 12.5g of glycerol were mixed in a reactor, with the enzyme. The mixture was heated to 37°C, stirred at 330 rpm, and placed under vacuum. The remaining glycerol was added in four portions, 12.5 g every two hours. The vacuum was moderate for the first eight hours of the reaction, i.e., about 15 mbar, then increased, i.e., below 5 mbar.

[0134] Option 2: Reaction in the presence of a mixture of enzymes

[0135] Step (c) proceeds in the same manner as described previously except that the following mixture of enzymes is used.

[0136] 10.1 g of lipase B from Candida antarctica (Lipozyme 435®),

[0137] 3.4 g of lipase from Thermomyces lanuginosus (Lipozyme TL IM®).

[0138] Step d) elimination of glycerol

[0139] The glycerol is removed by decantation.

[0140] Step (e) short path molecular distillation of the oil under vacuum

[0141] The conditions are an evaporator wall temperature between 160 and 220°C and a vacuum of less than 0.02 mbar. Results

[0142] At the end of the process, oils are obtained having more than 700 mg / g of DHA or EPA according to the following glyceridic profile, expressed as a percentage relative to the quantity of total glycerides (analysis carried out by gas chromatography) (Table 1):

[0143] [Tables 1] Starting oil at step a) Step (c) with lipase B from Candida antarctica Step (c) with lipase B from Candida antarctica and lipase e from Thermomyces lanugin osus Glyceride profile at the end of step (e) Glyceride profile at the end of step (e) Oil containing more than 700 mg / g of DHA in the form of ethyl esters H1(DHA) or H3(DHA) 55% DG 15% MG 30% TG Oil containing more than 700 mg / g of EPA in the form of ethyl esters H2(EPA) or H4(EPA) 50% DG 22% MG 25% TG 52% DG 22% MG 23% TG

[0144] As a percentage of the total amount of monoglycerides, diglycerides, triglycerides and fatty acid ethyl esters; MG: monoglycerides; DG: diglycerides; TG: triglycerides

[0145] In all cases, the EPA or DHA fatty acids are mainly in the form of diglycerides. It is noted that the use of the combination of enzymes makes it possible to increase the diglyceride content if we compare the results obtained with the starting oils H2(EPA) and H4(EPA) using in step c) an enzyme versus a mixture of two enzymes. In addition, the reaction was faster, which is advantageous.

[0146] The total contents of diglycerides and monoglycerides are high, with, for each oil, a quantity greater than 70%.

[0147] Example 2: another example of obtaining a microalgae oil enriched with DHA mainly in the form of diglycerides

[0148] This example proceeds in the same way as example 1, starting in step a) with an H1(DHA) or H3(DHA) oil, except that the process implements, between step (a) and step (c), a step (b) and a step (f) after step (e).

[0149] Step b) structuring of docosahexaenoic acid or eicosapentaenoic acid in the form of free fatty acids.

[0150] For each oil resulting from step a), a saponification reaction intended to obtain the fatty acids in free form is carried out.

[0151] Step c): takes place in an identical manner to that described in example 1.

[0152] At the end of step c) the oil contains 57% diglycerides, 24% monoglycerides, 8% triglycerides and 11% free fatty acids.

[0153] Step d): takes place in an identical manner to that described in example 1.

[0154] Step e): takes place in an identical manner to that described in example 1.

[0155] Step f): the implementation of step e) allows the recovery of a fraction containing DHA in monoglyceride form and in free form which is rerouted and reintroduced in step c). Results

[0156] At the end of the process, oils are obtained having more than 700 mg / g of DHA or EPA according to the following glyceridic profile, expressed as a percentage relative to the quantity of total glycerides (analysis carried out by gas chromatography) (Table 2):

[0157] [Tables2] Starting oil at step a) Step (c) with lipase B from Ca ndida antarctica Glyceride profile from step (e) Oil containing more than 700 mg / g of DHA in the form of ethyl esters Hl(DHA) or H3(DHA) 68% DG 22% MG 10%TG

[0158] As a percentage of the total amount of monoglycerides, diglycerides, triglycerides and fatty acid ethyl esters; MG: monoglycerides; DG: diglycerides; TG: triglycerides

[0159] DHA fatty acids are mainly in the form of diglycerides.

[0160] The total contents of diglycerides and monoglycerides are very high, with, for each oil, a quantity of 90%.

Claims

Claims

1. Microorganism oil composition enriched with polyunsaturated fatty acids, characterized in that it comprises: - a content of eicosapentaenoic acid or docosahexaenoic acid greater than or equal to 500 mg / g of composition, and, relative to the total quantity of glycerides: - a quantity of triglycerides of between 10 and 30%, - a quantity of diglycerides of between 50 and 70%, - a quantity of monoglycerides of between 15 and 25%. - a quantity of monoglycerides and diglycerides greater than 60%.

2. Composition according to one of the preceding claims, characterized in that it comprises a docosahexaenoic acid content greater than or equal to 600 mg / g of composition, more preferably greater than 700 mg / g of composition.

3. Composition according to one of claims 1 to 2, characterized in that it comprises an eicosapentaenoic acid content greater than or equal to 600 mg / g of composition, preferably greater than 700 mg / g of composition.

4. Process for preparing an oil composition enriched with omega-3 polyunsaturated fatty acids, characterized in that it comprises: (a) A step of obtaining an oil from microorganisms comprising docosahexaenoic acid or eicosapentaenoic acid in the form of ethyl esters in an amount greater than or equal to 500 mg / g of composition, (c) a step of structuring the docosahexaenoic acid or eicosapentaenoic acid predominantly in the form of diglycerides by reaction between the oil and one or more enzymes in the presence of glycerol, said step (c) comprising: - mixing, in a vacuum reactor, with stirring and at a temperature of 35 to 40°C., docosahexaenoic acid or eicosapentaenoic acid and the enzyme(s), and, - additions of glycerol in several times in identical portions, the additions being spaced apart by a duration of between 1h and 3h, (d) a step of elimination of the glycerol, (e) a step of short-path molecular distillation of the oil under vacuum.

5. A method according to claim 4, characterized in that step (c) is carried out in a molar ratio between docosahexaenoic acid or eicosapentaenoic acid and glycerol of 1.

6. Method according to one of claims 4 to 5, characterized in that step (c) is carried out using a lipase B produced by Candida antarctica.

7. Method according to one of claims 4 to 5, characterized in that step (c) is carried out using a mixture of two enzymes, the mixture being composed of 70 to 80% of a lipase B produced by Candida antarctica and 20 to 30% of a lipase produced by Thermomyces lanuginosus.

8. Method according to one of claims 4 to 7, characterized in that it comprises, between step (a) and step (c): (b) a step of structuring docosahexaenoic acid or eicosapentaenoic acid in the form of free fatty acids.

9. Method according to claim 8, characterized in that it comprises, following step (e): (f) A step of recovering a fraction comprising docosahexaenoic acid or eicosapentaenoic acid in free form and in the form of monoglycerides and of reintroducing this fraction in step (c).

10. Oil composition characterized in that it comprises a mixture of an oil composition as defined in one of claims 1 to 3 or obtained by a process according to one of claims 4 to 9 and at least one other oil.

11. Food supplement characterized in that it comprises an oil composition as defined in one of claims 1 to 3 or obtained by a process according to one of claims 4 to 9.

12. Food product characterized in that it comprises an oil composition as defined in one of claims 1 to 3 or obtained by a process according to one of claims 4 to 9.

13. Pharmaceutical or nutraceutical composition characterized in that it comprises an oil composition as defined in one of claims 1 to 3 or obtained by a process according to one of claims 4 to 9.

14. Oil composition as defined in one of claims 1 to 3 or obtained by a process according to one of claims 4 to 9, for its use in the prevention and / or treatment of obesity, overweight, bone diseases, in reducing cholesterol levels and modulating glucose metabolism.