EP3849344A1

EP3849344A1 - Oil of microorganisms rich in docosahexaenoic acid

Info

Publication number: EP3849344A1
Application number: EP19765744.8A
Authority: EP
Inventors: François GODART; Adeline LAPENDRIE
Original assignee: Fermentalg SA
Current assignee: Fermentalg SA
Priority date: 2018-09-14
Filing date: 2019-09-13
Publication date: 2021-07-21
Also published as: US20240141395A1; CN113766836A; WO2020053372A1; US20220042054A1; FR3085825B1; KR20210093850A; FR3085825A1; CA3112621A1; BR112021004896A2; AU2019340853A1; JP2022500048A

Abstract

The present invention relates to an oil of microorganisms rich in docosahexaenoic acid (DHA, C22:6n3), comprising more than 60 % of DHA relative to the total mass of fat and to the use thereof for human or animal feed, in particular for feeding infants, children, or pregnant or lactating women.

Description

OIL FROM MICROORGANISMS RICH IN DOCOSAHEXAENOIC ACID

FIELD OF THE INVENTION

The present invention relates to an oil of microorganisms rich in docosahexenoic acid (DHA, C22: 6n3), comprising more than 60% of DHA relative to the total mass of fat and at least 80% of triglycerides relative to the total mass of fat.

STATE OF THE ART

Oils containing DHA come from several sources, the best known of which are fish, krill and microorganisms such as microalgae. Many strains of PUFA-producing microorganisms are known, in particular docosahexaenoic acid (DHA), arachidonic acid (ARA) or eicosapentaenoic acid (EPA) also identified by the signs w3 and w6. These PUFAs are widely used in industry, in particular for human or animal food or in cosmetics, and their industrial production has been subject to constant improvement for many years (WO 1997/037032, WO 2001/054510, WO 2013/136028 , WO 2015/004402, US 2017/016036, US 2017/335356). The criteria for selecting strains suitable for industrial production are their high biomass productivity, a large accumulation of triglycerides (TG) and a high PUFA content in fat. Many industrial strains are known today which meet these first three criteria, with a PUFA content in fat of the order of 35%, or even 50% in the best of cases.

However, there is a demand for concentrated oils with high PUFA contents, either for the supply of concentrated products, such as concentrated oil capsules which make it possible to reduce the catches for an equivalent quantity of PUFA. To obtain oils with a high PUFA content (for example greater than 55% of DHA), the oils can be enriched by the addition of PUFA (US 2014/323569) and / or the oils are concentrated by a process which transforms the triglycerides to ethyl esters involving the use of solvents such as ethanol. Ethyl esters are an artificial chemical form, they are not present in nature. The bioavailability of fatty acids in the form of ethyl esters is much lower than in the form of triglycerides (Ghasemifard et al., 2014). In addition, the process removes the vitamins and antioxidants that are present in the crude oil. As a result, the concentrated oil is more vulnerable to oxidation.

It is possible to convert these ethyl esters again to triglycerides, these are “reformed” triglycerides, in order to improve the bioavailability. Antioxidants can also be added to increase the stability of the oil over time. However, this concentrated oil is quite different from natural oil, it has undergone many transformation processes which have changed its composition: profile in fatty acids, vitamins, pigments and other antioxidant molecules, depriving PUFAs of their natural protection. However, PUFAs are sensitive molecules, especially at the temperature which can convert cis bonds into trans bonds (Tsuzuki W, 2012). It should also be noted that the re-formation of the triglycerides is not complete, the oil thus treated still contains a variable proportion of ethyl esters, which distinguishes it from the untreated oil. The ethanol released during transesterification (conversion of ethyl esters to triglycerides) is generally eliminated by evaporation. However, traces of ethanol remain in the concentrated oil.

Another reason for wanting to minimize the oil treatment processes is the formation of contaminants such as monochloropropanediol (2-MCPDs, 3-MCPDs) and glycidol as well as their derivatives (esters of 2-MCPDs, of 3-MCPDs and fatty acid esters of glycidol). The presence of these contaminants has been detected in particular following the stages of purification and deodorization of fish oil (Miyazaki and Koyama, 2017). There is little data available at this time on the impact of concentration processes on the formation of contaminants. However, the re-formation of triglycerides from ethyl esters can cause an increase in diglycerides, precursor compounds of contaminants. The level of glycidol (and glycidol esters) is subject to regulation (EU) 2018/290 / EC in order to limit its content in food: the concentration must not exceed 1000 pg / kg in oils edible except for edible oils intended for the preparation of food for babies and infants where the limit is 500 pg / kg. In baby and infant formulas, the maximum content is even lower: 75 pg / kg in powders and 10 pg / kg in liquids. This content will be further reduced (50 and 6 pg / kg respectively) in 2019. The assessment of maximum MCPD concentrations is currently underway for oils and baby foods. For the moment, the regulations only concern hydrolyzed vegetable proteins and soy sauce (limit of 20 pg / kg).

It is therefore advantageous to obtain an oil naturally rich in PUFAs, the composition of which is as close as possible to the liposoluble substances of the producer microorganism, with a minimum of contaminants produced during the treatments. This makes it particularly suitable for the integration of DHA in food products. Its very low content of 3-MCPD and glycidol, combined with its high content of DHA makes it ideal for the preparation of food intended for babies and infants.

In addition, these concentrated oils are generally obtained by expensive treatment methods which are not respectful of the environment.

Another known solution consists in generating genetically modified microorganisms in order to seek to promote the metabolic pathways for the production of PUFA (Hamilton & al., 2016) or murtants supposed to produce more DHA (WO 2017/09804). However, the choice of technical solutions is limited by the use made of the oils obtained, especially in human food (Fedorova-Dahms I. & al., 201 1).

There is a need for oils that are naturally concentrated in PUFAs, which do not require treatments other than extraction methods, that is to say for which the PUFAs are essentially in the form of triglycerides such as produced by microorganisms. More particularly, there is a need for oils with a high PUFA content and a lower content of saturated fatty acids. In addition to the question of the quality of the oils, the interest of a low content of saturated fatty acids goes towards a less viscous oil, easier to use at industrial levels, in particular requiring less energy for its handling.

The invention responds to this request with an oil with a high DHA content, comprising at least 60% DHA relative to the total mass of fat. This oil contains no ethylesters, no traces of solvent (ethanol or methanol) and a reduced content of 3- MCPD and glycidol (compared to oils containing more than 60% of DHA currently on the market).

STATEMENT OF THE INVENTION

The present invention relates to a microbial oil which comprises docosahexenoic acid (DHA), characterized in that it comprises at least 80% of triglycerides relative to the total fat mass, more than 60% of DHA relative to the total mass of fat and the content of saturated fatty acids is less than 25% relative to the total mass of fat.

It also relates to a diluted microbial oil which comprises a microbial oil rich in triglycerides and in DHA according to the invention, mixed with another oil.

Another subject of the invention is a biomass of microorganisms which comprises an oil rich in triglycerides and DHA according to the invention.

The invention also relates to the use of an oil rich in triglyceride and in DHA according to the invention optionally diluted or of a biomass which contains this oil for human or animal consumption, in particular for the feeding of newborn babies. , children, or pregnant or breastfeeding women.

Another object of the invention is a food, characterized which comprises an oil rich in triglycerides and in DHA according to the invention, possibly diluted.

DETAILED DESCRIPTION OF THE INVENTION

The oil according to the invention is a microbial oil which comprises more than 60% of DHA relative to the total mass of fat, advantageously at least 62% of DHA, more advantageously at least 65% of DHA, preferably more than 67%, more preferably at least 70%, even more preferably 75% of DHA relative to the total mass of fat.

These characteristics of the oil according to the invention relate both to the oil as present in the biomass of microorganisms and to the oil extracted from this biomass, whether it is raw or purified.

The invention also relates to a diluted oil, comprising the oil according to the invention mixed with another oil.

The invention also relates to a pharmaceutical, cosmetic or food composition which comprises an oil according to the invention, whether it is crude, refined or diluted.

The invention also relates to the use of an oil according to the invention, crude, refined or diluted, or of a biomass containing the oil, for human or animal food, in particular for feeding newborns. , children, or pregnant or breastfeeding women.

DETAILED DESCRIPTION OF THE INVENTION

The oil according to the invention is an oil of microbial origin, obtained from a biomass of cells of microorganisms cultivated under conditions allowing both cell growth (to produce the biomass) and the production of an oil high in DHA.

The oil according to the invention is a microbial oil which comprises more than 60% of DHA relative to the total mass of fat, advantageously at least 62% of DHA, more advantageously at least 65% of DHA, preferably more than 67%, more preferably at least 70%, even more preferably at least 75% of DHA relative to the total mass of fat.

Preferably, the oil according to the invention has a high content of unsaturated fatty acids compared to saturated fatty acids. The unsaturated fatty acids in the oil according to the invention are essentially DHA and DPA (docosapentanoic acid, C22: 5n6). The ARA (arachidonic acid, C20: 4n6) content is generally less than 0.5%, or even less than 0.3%, advantageously less than 0.1%. The content of EPA (eicosapentaenoic acid, C20: 5n3) is generally less than 1.5%, advantageously less than 1%, more advantageously less than 0.5%. The percentages in ARA and EPA are given relative to the total mass of fat.

Advantageously, the cumulative DHA and DPA content is at least 70% relative to the total fat mass, advantageously at least 75%, more advantageously at least 80%, or even more than 85 % based on the total mass of fat. In some cases, the total DHA + DPA represents up to 90% of the total fat mass. For oils with the highest DHA content, at least 70%, the cumulative DHA and DPA content is at least 80%, preferably at least 85%.

For an oil rich in DHA according to the invention, the DHA / DPA ratio is preferably at least 3, more preferably at least 4, which can range from 4 to 9. For oils where the DHA content is the higher, at least 70%, the DHA / DPA ratio is advantageously from 4 to 7. The content of saturated fatty acids is less than 25% relative to the total mass of fat, or even less than 20%, more preferably less than 15%, even more preferably less than 10%.

Saturated fatty acids are mainly palmitic acid (C16: 0). The other saturated fatty acids are present at a content of less than 2%, or even less than 1%, in particular pendadecylic acid (C15: 0) or myristic acid (C14: 0) or even stearic acid (C18: 0). Advantageously, the saturated C10 to C22 fatty acids other than palmitic acid are, independently of each other, present in trace amounts, each in a content of less than 0.1%, or even absent (0% taking into account the uncertainties analytical methods), in particular for saturated fatty acids C10, C1 1, C12, C17, C20, C21 and C22. The percentages are given relative to the total mass of fat.

The palmitic acid content is preferably less than 20% of the total fat mass, more preferably less than 15%, even more preferably less than 10%.

For oils with the highest DHA content, at least 70%, the content of C10 to C22 saturated fatty acids is preferably less than 15%, more preferably less than 10%.

One way of measuring the high DHA content of the oil according to the invention and the low content of saturated fatty acids (AGS) is to establish a DHA / AGS ratio.

It is advantageously at least 2.5, preferably at least 3, more preferably at least 5, even more preferably at least 6. It can go up to at least 8 in certain cases, or even d '' at least 9. For oils where the DHA content is the highest, at least 70%, the DHA / AGS ratio is at least 4, preferably at least 6, more preferably at least 8, up to approximately 9.

We can also measure the high content of polyunsaturated fatty acids compared to saturated fatty acids (AGS) by making the ratio (DHA + DPA) / AGS.

It is advantageously at least 2.5, preferably at least 3, more preferably at least 4, even more preferably at least 5. It can go up to at least 8 in certain cases, or even d '' at least 9. For oils with the highest DHA content, at least 70%, the ratio (DHA + DPA) / AGS is at least 5, preferably at least 7, more preferably from at least 10, to about 1 1 or more.

The oils according to the invention are essentially in the form of triglycerides. The triglycerides represent at least 80% of the total mass of fat, advantageously at least 90%, more advantageously at least 93% of the total mass of fat. The triglyceride content is for example analyzed by thin layer chromatography (Jouet et al., 2003). These characteristics of the oil according to the invention relate both to the oil as present in the biomass of microorganisms as in the oil extracted from this biomass, whether it is crude or purified.

In some cases, depending on the process used, the extraction of oil from biomass can lead to a slight increase in the content of DHA and DPA, favoring the extraction of these PUFAs compared to saturated fatty acids of lower molecular weight. However, this concentration does not substantially modify the intrinsic properties of the oil contained in the biomass, in particular the triglyceride content. In all cases, the oil according to the invention is an oil which has not undergone substantial changes in its fatty acid content by the addition of PUFA, for example in the form of esters, by concentration and / or by eliminating saturated fatty acids like palmitic acid.

The oil according to a particular embodiment of the invention contains more than 10 mg of native carotenoids per kg of oil, or even more than 30 mg / kg, preferably more than 40 mg / kg, even more preferably more than 60 mg / kg, or even at least 65 mg / kg. The carotenoids present are mainly astaxanthin and beta-carotenes. The oil contains more than 20 mg / kg of astaxanthin, or even more than 30 mg / kg, more preferably more than 40 mg / kg. Canthaxanthin is also present, but in smaller quantities. Other carotenoids such as lutein and zeaxanthin may be present but they are at the limit of detection of the method used. The term "native carotenoids" means that the carotenoids have not been added, they come from the same biomass as the oil and are extracted from this biomass at the same time as the oil. They are produced by the strain under fermentation conditions in heterotrophy, without any particular stimulus. These native carotenoids are therefore present throughout the process, protecting fatty acids, in particular DHA, against oxidation. The refining process can remove pigments, so the refined oil may contain less, if not more, carotenoids.

The color of the oil is usually evaluated by measuring the Gardner index, according to the method described in the AOCS Ce 13d-97 standard (revised in 2017) with a spectrophotometer. The measurement scale includes 18 grades, ranging from transparent (1) to dark red / brown (18). Some carotenoids, including astaxanthin and beta-carotenes, show more or less intense coloring depending on their concentration. Their presence is therefore reflected in a higher Gardner index. The oil according to a particular embodiment of the invention has a Gardner index greater than 8, or even greater than 10, preferably between 12 and 17.

The Gardner scale is traditionally used to assess the aging of oils because the oxidation of oils rich in polyunsaturated fatty acids (PUFAs) can result in yellowing of the color (for a transparent oil), therefore a Gardner value higher. However, the oxidation of oils rich in PUFAs is more precisely measured by the anisidine index and the peroxidation index. The oils according to the invention have both low anisidine and peroxide indices, which indicate a poorly oxidized product, and a high Gardner index, due to the presence of carotenoids. The oils according to the invention have an anisidine index less than 5, see less than 2, preferably less than 1.5, and a peroxidation index less than 5, see less than or equal to 1, preferably less than or equal to 0.5.

The oils according to the invention have a fairly low melting temperature, which decreases in correlation with the increase in the level of DHA. The melting temperature is measured according to standard ISO 6321. Indeed, oils, with more than 600 mg of DHA / g of fatty acids (that is to say more than 62% of DHA) have a melting temperature below 20 ° C, see less than or equal to 5 ° C. They are therefore liquid at room temperature. Oils with more than 700 mg of DHA per g of fatty acids (i.e. approximately more than 73% of DHA) have a melting temperature below -5 ° C. A low melting temperature facilitates storage and handling (pumping in particular), since it is possible to keep the oil in liquid form while refrigerating it in order to limit aging. Oils that freeze during storage must be reheated for sampling and for their integration into mixtures. Temperature is an accelerating factor for oxidation.

This property is also reflected in the viscosity value, measured by a viscometer at 22 ° C (Viscoman, Gilson). The oils according to the invention have a viscosity value at room temperature less than or equal to 50 Pa.s, or even less than 40, preferably less than 30.

The oils according to the invention are obtained by culturing microorganisms producing oils rich in DHA. The strains of microorganisms which make it possible to obtain such oils are industrial strains, that is to say according to the invention, strains whose fat content represents at least 45% of the dry matter, preferably at least about 50 % of the dry matter, and which have a growth capacity at a cell density of at least 50 g / L, preferably at least 70 g / L, more preferably at least 100 g / L.

Those skilled in the art are well aware of the industrial strains of PUFA producing microorganisms mainly from traustochytrids, dinophyceae, diatoms, eustigmatophyceae, in particular microorganisms of the genera Crypthecodinium, Schizochytrium, Traustochytrium or Aurantiochytrium for the production of DHA.

The analysis of the PUFA content in the fat is carried out according to the usual methods of those skilled in the art, in particular described in the following article: Gas Chromatography Quantification of Fatty Acid Methyl Esters: Flame lonization Detection vs. Electron Impact Mass Spectrometry, Dodds et al., Lipids, Vol. 40, no. 4 (2005). More particularly, the strains Aurantiochytrium mangrovei CCAP4062 / 7 and CCAP4062 / 8 and Schizochytrium sp. CCAP4087 / 7 which produce oils comprising more than 60% of DHA compared to the total mass of fat. The invention also relates to these strains capable of producing oils comprising more than 60% of DHA.

The industrial culture methods of microorganisms for the production of a fermentation wort which will then be used for the production of oil are well known to those skilled in the art, whether in autotrophic, heterotrophic or mixotrophic mode. Industrial culture in heterotrophy or mixotrophy makes it possible to obtain cell densities of at least 50 g / L, preferably at least 70 g / L, more preferably at least 100 g / L.

By "industrial culture" is meant according to the invention a culture of the strains in a culture medium suitable for their growth and for the production of PUFA and in a volume suitable for the production of sufficient quantities to address a market.

These industrial cultures are produced by fermentation in batch mode called "batch", in semi-continuous mode called "fed batch" or in continuous mode. Fermenters have volumes that can range from 1000 L to more than 200 m ³ .

The suitable culture medium is preferably a chemically defined culture medium which comprises a source of carbon, a source of nitrogen, a source of phosphorus and salts. By "chemically defined culture medium" is meant a culture medium in which the content of each element is known. Advantageously, the medium does not contain rich or complex organic materials. By rich or complex organic materials is meant unpurified organic materials, in the form of mixtures for which the exact composition and the concentrations of the various components of the mixture are not known with exactitude, not controlled, and can present a significant variability. from one batch to another. As an example of rich or complex organic material, mention may be made of yeast extracts or peptones which are products of a protein hydrolysis reaction or also rich mineral materials such as for example marine mineral salts or other growth agents. complex, having no fixed concentration of each of their components.

Generally, industrial culture methods include a growth step to promote the production of biomass, then an accumulation step to promote the production of fat and PUFA in particular. This is particularly the case for the method described in patent application WO 2001/054510. More recently, methods have been described implementing culture conditions which concomitantly favor the production of biomass and that of PUFAs. Mention will in particular be made of the culture methods described in applications WO 2012/035262, WO 2015/004402 and WO 2015/004403. Of course, those skilled in the art can adapt the culture conditions, in particular the composition of the medium, the conditions for adding nutrients during the culture, temperature cycles, oxygenation and lighting conditions to promote the production of biomass.

The temperatures of industrial culture are advantageously higher than 17 ° C.

Advantageously, the term "biomass" according to the invention means a set of cells of microorganisms produced by their culture, in particular by the methods described above, cells which may or may not have retained their physical integrity. It is therefore understood that said biomass can comprise an amount of cells of degraded microorganisms ranging from 0% to 100%. By "degraded" is meant that the physical integrity of said cells of microorganisms could have been altered such as for example lysed microorganisms, resulting for example from a process of homogenization or enzymatic lysis. Once produced, this biomass can be raw, just separated from its culture medium, dried or not, degraded or not.

Biomass, depending on whether it is dried or not, wholly or in part, can include a humidity level of 1% to 90%.

The invention therefore also relates to a biomass of microorganisms comprising an oil as defined above.

According to a first embodiment, the biomass has a humidity rate of 70% to 90%, preferably 80% to 85%. This is particularly the case when it essentially consists of optimized industrial microorganisms cultivated after filtration of the fermentation must to separate the cultivated microorganisms from the culture medium, before drying.

According to another embodiment of the invention, the biomass is dried, wholly or in part, and has a humidity rate of 1% to 10%, preferably from 2% to 7%.

The biomass may be conditioned for its storage or for its use as such, for example as a food supplement or food for human or animal consumption.

The methods for isolating an oil according to the invention from a biomass produced by the culture of microorganisms are well known to those skilled in the art. Mention will be made in particular of the solid-liquid extraction which is based on the use of a solvent (liquid phase) for extracting the oil contained in the dried biomass (solid phase) by spraying or maceration; the liquid-liquid extraction which is based on the separation of the aqueous phase from the oil after the prior lysis of the cells then decantation or centrifugation. Preferably the extraction is done without organic solvents. The applications WO 01/53512, WO 02/10423, WO 2014/122092, WO 2015/092546 and WO 2015/095694 will be mentioned in particular.

Mention will also be made of a preferred method which makes it possible to improve the extraction yields of fat from microorganisms for oils rich in PUFA. This method consists in carrying out cell lysis at a first temperature, the latter being continued at a second temperature lower than the first, then a mechanical separation of the oil from the lysed biomass (filtration, decantation).

Cell lysis is done by enzymatic or mechanical lysis (grinding). The temperature of the first lysis part is preferably at least 50 ° C. while remaining below temperatures which would degrade the composition of the oils in addition to promoting cell lysis, that is to say temperatures below 80 ° C, preferably at most 70 ° C.

The enzymes capable of being used are known, in particular described in WO2015 / 095688, WO201 1/153246, US6750048 and WO2015 / 095694, in particular proteases or cellulases such as the enzymes sold by the company Novozyme under the names Alcalase 2, 5 L, Alcalase 2.4 L, Novozym 37071, Flavourzyme 1000 L, Novozym FM 2.4 L, Protamex, Viscozyme. The conditions of implementation are those recommended by the supplier, the temperature being that recommended for optimal activity of the enzymes, at least 50 ° C. and up to 70 ° C., preferably approximately 65 ° C. Advantageously, the enzymatic lysis is carried out under an atmosphere poor in oxygen.

Mechanical lysis methods are also well known, in particular by ball mill, mixer-disperser, high pressure homogenizer pin mill or impact mill, ultrasound, pulsed electric fields. Mention may in particular be made for the ball mill: Netzsch / Discus-1000; WAB / ECM-AP60; for the high pressure homogenizer: GEA / Ariete; for the mixer-disperser: Silverson / 700-X, for the spindle mill: Hosakawa / Contraplex; for the impact mill: Netzsch / Condux.

The first part of the lysis is carried out under the usual conditions recommended in the state of the art for cell lysis, in particular in terms of duration of the enzymatic lysis or the grinding cycles.

The lysis continuation stage makes it possible to complete the latter by modifying the conditions of implementation without having to extract the lysed biomass beforehand. The lysis temperature in this second part is at least 10 ° C lower than that in the first part. Preferably, the temperature of the second lysis part is less than or equal to 40 ° C, advantageously ranging from 5 ° C to 40 ° C. This second part of lysis at a lower temperature, or end of lysis, is advantageously carried out for at least 30 ’, which can range up to 30 h.

The mechanical separation of an oil from a lysed biomass is also well known to those skilled in the art, as a gravity separation, in particular by centrifugation as described in patent application WO 01/53512. One can also use a continuous separation, in particular by centrifugal separator with plates. Such separators are known to continuously extract oils from complex media comprising solid residues and water, as described in patent application WO 2010/096002, in particular sold by the companies Alfa Laval, Flottweg or SPX Flow Technology Santorso, in particular. This continuous separation step is preferred in the process used to obtain the oil according to the invention.

The oil obtained is generally an oil called crude oil, which can be used as it is or be subject to refining, in particular to facilitate its conservation, by preventing it from becoming rancid, or to modify its color so as to make it more acceptable to a consumer. These refining steps are well known to those skilled in the art, including degumming, clarification and deodorization. They make it possible to eliminate (all or in part) phospholipids, pigments, volatiles and free fatty acids. In fact, these methods do not substantially modify the relative content of fatty acids, saturated or unsaturated, nor the triglyceride content of the refined oil obtained compared to the crude oil.

The invention also relates to a packaged oil comprising a container of volume suitable for containing said oil, the oil being an oil rich in DHA as defined above, crude or refined, and packaged in an amount of oil greater than 1 L, advantageously in an amount of oil greater than 10 L, more particularly in an amount of oil of the order of 220 L, and more particularly in an amount of oil of the order of 20m ³ .

Any container capable of containing the volume of oil or biomass and protecting them for their conservation and transport may be used by a person skilled in the art. Advantageously, the volume of the container will be equal to or substantially greater than that of the oil or of the biomass conditioned so as to limit the presence of air in the container and limit oxidation. The container will advantageously be opaque in order to avoid deterioration of the product by light rays, in particular UV. Advantageously, the container will be airtight so that any volume not occupied with oil or biomass can be filled with an inert gas.

The oil according to the invention can be mixed with other oils for their end use. This dilution changes the overall content of DHA and other unsaturated fatty acids in the composition of the diluted oil. However, it is possible to identify in the final oil, in view of the fatty acid profile of the oil used for the dilution, the relative percentages of fatty acids which come from the oil rich in DHA according to the invention and from dilution oil.

The invention therefore also relates to a diluted oil, comprising the oil according to the invention mixed with another oil. The oils used to dilute the oil rich in DHA according to the invention are generally and preferably vegetable oils suitable for human or animal food consumption. Mention will in particular be made of sunflower, rapeseed, soybean, walnut, sesame, hemp, hazelnut, argan, olive, flax, or any other oil suitable for food use. The added oil can also be an oil comprising other PUFAs, in particular ARA and / or IΈRA, in particular other oils of microbial origin or also fish oils.

The invention also relates to a composition which comprises an oil according to the invention, whether it is crude, refined or diluted, or which which comprises the biomass according to the invention.

A composition according to the invention can comprise one or more excipients. An excipient is a component, or mixture of components, which is used in the present invention to impart desirable characteristics to the composition for its storage and use, including foods and pharmaceutical, cosmetic and industrial compositions. An excipient can be described as a "pharmaceutically acceptable" excipient when it is added to a pharmaceutical composition whose properties are known from the pharmacopoeia to be used in contact with human and animal tissues without excessive toxicity, irritation, allergic reaction or other complications . Different excipients can be used as an organic or mineral base, an organic or mineral acid, a pH buffer, a stabilizer, an antioxidant, an adhesion agent, a separation agent, a coating agent, an external phase component , a controlled release component, a surfactant, a humectant, a filler, an emollient, or combinations thereof.

Depending on their destination, the compositions according to the invention are in particular pharmaceutical, cosmetic, nutraceutical or food compositions.

The food is intended for both humans and animals and includes solid, pasty or liquid compositions. Mention will in particular be made of common foods, liquid products, including milks, drinks, therapeutic drinks and nutritional drinks, functional foods, supplements, nutraceuticals, infant formulas, including premature infant formulas, foods for pregnant or lactating women, adult foods, geriatric foods and animal foods.

The oil rich in DHA according to the invention, whether it is crude or refined or the biomass which contains it can be used directly as or added as an additive in an oil, a spread, another fatty ingredient, a drink, a soy-based or soy-based sauce, dairy products (milk, yogurt, cheese, ice cream), bakery products, nutritional products, for example in the form of a nutritional supplement (in the form of capsules or tablet), vitamin supplements, food supplements, powders for beverages, such as energy drinks or milk powders for infant formulations, finished or semi-finished powdered food products, etc. uses known to those skilled in the art.

Animal feed is also known to those skilled in the art. They are in especially intended for farm animals, such as cows, pigs, chickens, sheep, goats or in fish farming for crustaceans or farmed fish.

Pharmaceutical compositions comprising an oil rich in DHA are also known to those skilled in the art, the oil being used alone or in combination with other drugs.

The crude or refined oil according to the invention or the biomass which contains it, can be formulated in the form of single-dose compositions, in particular in the form of tablets, capsules, capsules, powders, granules, suitable for administration. per os.

The advantage of the oil rich in DHA according to the invention, whether it is crude or refined, or the biomass which contains it, is that it can be used in lesser amounts in these mixtures and compositions.

The invention also relates to the use of an oil according to the invention, crude, refined or diluted or else the biomass which contains it, for human or animal food, in particular for feeding newborns, children , or pregnant or breastfeeding women.

Such uses are well known to those skilled in the art, in particular described in patent application WO 2010/107415 and on the website of the company DSM (https://www.dsm.com/markets/foodandbeverages/en_US/ products / nutritional-lipids / life- dha.html)

EXAMPLES

EXAMPLE 1 Profile of Fatty Acids of the Biomass of Traustochytrides with a High DHA Content

Traustochytrid strains (Aurantiochytrium mangrovei - FCCB1897, FCCB1800, CCAP4062 / 8) are cultured in an Erlenmeyer flask in ATCC 790 culture medium (modified). Similar results are obtained with strains of Schizochytrium sp. ((in particular with the strain CCAP4087 / 7).

Once the cultures are in stationary phase, the biomass is recovered by centrifugation and then lyophilized before the analysis of the composition of the biomass in fatty acids by GC-FID (method adapted from standard ISO 12966-2).

Composition of the modified ATCC 790 medium:

Yeast extract 5.0 g / L

Peptone 5.0 g / L

D + -Glucose 30.0 g / L

Sea salts 20 g / L

Table 1 represents the composition of fatty acids contained in the biomass. The results are expressed as a percentage relative to the total fatty acid content. AGS are saturated fatty acids. Table 1

EXAMPLE 2 Cultures in a fermenter of strains with a high content of DHA

The cultures are carried out in fermenters (bioreactors) of 1 to 5 L useful with dedicated automated systems and supervision by computer station. They are carried out using two strains of Aurantiochytrium mangrovei and with two different culture protocols. The system is regulated at pH 5 via the addition of base (NH ₄ OH for example b1 and b2 and with NaOH for example a) with adjustment of the pH carried out over the entire duration of the culture, and providing a supply of nitrogen ( in the context of examples b1 and b2). The culture temperature was set at 30 ° C then 22 ° C and finally 18 ° C at the end of the culture. The strain CCAP4062 / 7 is used for example a and b1 while the strain FCCB1897 is used for example b2.

The composition of the culture media is given in Table 2.

Table 2

Additions of glucose in the form of an enrichment solution are made with a molar ratio of carbon: nitrogen: phosphorus (CNP) of 533: 11: 1 (example a) or with a solution composed entirely of glucose (examples b1 and b2).

Crop monitoring:

The total biomass concentration is monitored by measuring the dry mass (filtration through a GF / F filter, Whatman, then drying in an oven, at 105 ° C, for at least 24 h before weighing). The fatty acid analyzes are carried out according to a method adapted from ISO 12966-2 for biomass, and according to the European Pharmacopoeia 9.0 (2.4.29.) For oils.

The fatty acid profiles of the biomasses obtained with conditions a, b1 and b2 are given in Table 3. The results are expressed as a percentage relative to the total fatty acid content.

Table 3

EXAMPLE 3: Culture under industrial conditions

The strains with a high DHA content produce a biomass of similar fatty acid composition when they are cultivated in industrial-size fermenters, such as 10 m ³ or 180 m ³ tanks, under conditions similar to the example. 2, with culture medium b and additions of glucose in the form of an enrichment solution are made with a molar ratio of carbon: nitrogen: phosphorus (CNP) of 533: 0.4: 1.

The fatty acid profiles of the biomass of the strain CCAP4062 / 7 after culture in tanks of 10 m ³ and 180 m ³ are given in Table 4. The results are expressed in percentage with respect to the content of total fatty acids .

Table 4

EXAMPLE 4 Extraction of the Oil from the Biomass of Strains with a High DHA Content

The oil is extracted from the biomass of Example 3 (180 m ³ tank) according to a method described in WO2015 / 095694 (Example 9). The fatty acid composition of the oil is similar to that of the biomass, given in Table 4.

EXAMPLE 5 Extraction of the Oil from the Biomass of Strains with a High DHA Content

The extraction of the biomass produced under the same conditions as in Example 3 is carried out by following the sequence (a) cell lysis by enzymatic route (eg with 2.5 L Alcalase or 2.4 L Alcalase or Novozym 37071 from Novozymes) for 4 hours at a temperature of 65 ° C,

(b) continuation of the lysis by lowering the temperature between 5 and 40 ° C, for a period of between 30 minutes and 30 hours,

(c) mechanical oil separation by centrifugal plate separator.

The extraction yield is 60% of lipids extracted from the biomass.

The lipid profile of the oil extracted from the biomass is given in Table 6. The results are expressed as a percentage relative to the total fatty acid content. Table 6

EXAMPLE 6: oil quality: antioxidants and contaminants

Several heterotrophic fermentations are carried out according to the conditions of Example 3. The oil is extracted from the fermentation must according to the conditions of Example 5. The carotenoids are measured in the extracted oil, by LC / DAD, according to the following methods: Astaxanthin (ester forms included), Reference method: DSM Ver. 1.52009; Beta-carotene (sum of cis- & trans-), saponified, Reference method: EN 12823-2: 2000; Canthaxanthin, Reference method: Roche Index No. 2264; Lutein & Zeaxanthin, Reference method: Roche Index n ° 2264.

Table 7

Contaminants such as glycidol and 2- and 3-MCPDs are also dosed in the same batches.

Table 8

EXAMPLE 7 viscosity

The viscosity of the oil produced and extracted under conditions similar to Example 5 is measured by a viscometer (Viscoman, Gilson) at different temperatures. The melting temperature is evaluated according to ISO 6321 standard.

Table 9

REFERENCES

- EP 0 223 960; EP 1 001 034

- US 2014/323569, US 2017/016036, US 2017/335356

- WO 1994/008467; WO 1997/037032; WO 2001/054510; WO 03/049832; WO

2010/107415; WO 2012/035262; WO 2013/136025; WO 2013/136028; WO

2014/146098; WO 2015/004402; WO 2015/004403; WO 2015/150716; WO

2016/030631, WO 2017/094804

Fedorova-Dahms I. & al., Safety evaluation of DHA-rich algal oil from Schizochytrium sp, Food and Chemical Toxicology, 2011, 49, 3310-3318

Folch J, et al., A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem. 1957 May; 226 (1): 497-509

- Hamilton M. & al., Heterotrophic Production of Omega-3 Long-Chain Polyunsaturated Fatty Acids by Trophically Converted Marine Diatom Phaeodactylum tricornum, Marine Drugs, 2016, 14, 53

- Omega-3 long chain fatty acid "bioavailability": a review of evidence and methodological considerations.

- Ghasemifard S, Turchini GM, Sinclair AJ. Prog Lipid Res. 2014 Oct; 56: 92-108. doi: 10.1016 / d. plipres. 2014.09.001. Epub 2014 Sep 16. Review.

Wakako TSUZUKI, Study of the Formation of trans Fatty Acids in Model Oils (triacylglycerols) and Edible Oils during the Heating Process, JARQ 46 (3), 215 - 220 (2012)

Kinuko Miyazaki ^* and Kazuo Koyama, An Improved Enzymatic Indirect Method for Simultaneous Determinations of 3-MCPD Esters and Glycidyl Esters in Fish Oils, J. Oleo Sci. 66, (10) 1085-1093 (2017)

Jouhet J., Maréchal E., Bligny R., Joyard J., Block MA (2003). Transient increase of phosphatidylcholine in plant cells in response to phosphate deprivation. FEBS Lett. 544 63-68. Pct

(original in electronic form)

(This sheet is not part of the international application nor does it count as a sheet thereof)

Pct

(original in electronic form)

RESERVED AT THE RECEIVING OFFICE

RESERVED AT THE INTERNATIONAL BUREAU

Claims

1. Microbial oil which comprises docosahexenoic acid (DHA), characterized in that it comprises at least 80% of triglycerides relative to the total mass of fat, more than 60% of DHA relative to the total mass of matter fatty and the content of saturated fatty acids is lower than 25% compared to the total mass of fatty matter.

2. Microbial oil according to claim 1, characterized in that it comprises at least at least 65% of DHA.

3. Microbial oil according to one of claims 1 or 2, characterized in that it comprises docosapentanoic acid (DPA) and in that the cumulative content of DHA and DPA is at least 70% relative to to the total mass of fat.

4. Microbial oil according to claim 1, characterized in that it comprises at least 70% of DHA.

5. Microbial oil according to claim 4, characterized in that the cumulative content of DHA and DPA is at least 80% relative to the total mass of fat.

6. Microbial oil according to one of claims 1 to 5, characterized in that the DHA / DPA ratio is at least 4.

7. Microbial oil according to one of claims 1 to 6, characterized in that the content of saturated fatty acids less than 15% relative to the total mass of fat.

8. Microbial oil according to one of claims 1 to 7, characterized in that it has a viscosity at room temperature less than or equal to 50 Pa.s.

9. Microbial oil according to claim 7, characterized in that it has a viscosity of less than 30 Pa.s.

10. Diluted microbial oil, characterized in that it comprises a microbial oil according to one of claims 1 to 9, mixed with another oil.

1 1. Biomass of microorganisms, characterized in that it comprises an oil according to one of claims 1 to 9.

12. Use of an oil according to one of claims 1 to 10 or a biomass according to claim 1 1 for human or animal food, in particular for the feeding of newborns, children, or women pregnant or nursing.

13. Food, characterized in that it comprises an oil according to claims 1 to 10.