Microbial oil rich in docosahexaenoic acid

Abstract

The present invention relates to a microbial oil rich in docosahexaenoic acid (DHA, C22:6n3), containing more than 60% DHA relative to the total lipid mass, and the use of said microbial oil for human or animal food, in particular food for newborns, children, or pregnant or lactating women.

Description

[Technical Field]

[0001] The present invention relates to a microbial oil enriched in docosahexaenoic acid (DHA, C22:6n3), which contains more than 60% DHA relative to the total mass of lipids and at least 80% triglycerides relative to the total mass of lipids. [Background Art]

[0002] Oils containing DHA are obtained from various sources, the best known of which are microorganisms such as fish, krill and microalgae. Many strains of microorganisms are known to produce PUFAs, in particular docosahexaenoic acid (DHA), arachidonic acid (ARA) or eicosapentaenoic acid (EPA), which are also represented by the ω3 and ω6 designations. These PUFAs are widely used in industry, particularly in food for humans or animals, or in cosmetics, and their industrial production has been continuously improved over many years (WO1997 / 037032, WO2001 / 054510, WO2013 / 136028, WO2015 / 004402, US2017 / 016036, US2017 / 335356). Selection criteria for strains suitable for industrial production are high biomass productivity, significant accumulation of triglycerides (TG) in lipids, and high PUFA content. Today, many known industrial strains meet these three criteria, and have a PUFA content of around 35% in lipids, and 50% in the best case.

[0003] However, there is a demand for concentrated oils with high PUFA content, i.e., a demand for the supply of concentrated products such as concentrated oil capsules that allow for a reduction in the number of unit doses required for an equivalent amount of PUFA. To obtain oils with high PUFA content (e.g., over 55% DHA), oils can be enriched by adding PUFAs (US2014 / 323569), and / or oils can be concentrated by converting triglycerides to ethyl esters with the use of solvents such as ethanol. Ethyl esters are artificial chemical forms and do not exist in nature. The bioavailability of fatty acids in the ethyl ester form is much lower than that of fatty acids in the triglyceride form (Ghasemifard et al., 2014). Furthermore, the above methods remove vitamins and antioxidants contained in crude oils. As a result, concentrated oils become more susceptible to oxidation.

[0004] To improve bioavailability, it is also possible to convert ethyl esters back to triglycerides ("reformed" triglycerides). Antioxidants can also be added to improve the oil's stability over time. However, this concentrated oil differs rather from the natural oil, namely, it undergoes several denaturation processes that alter its composition: fatty acid profile, vitamins, pigments, and other antioxidant molecules, resulting in the loss of natural protection from PUFAs. However, PUFAs are sensitive molecules that can change from cis-bonded to trans-bonded molecules, and are particularly sensitive to temperature (Tsuzuki W, 2012). It should be noted that the reformation of triglycerides is incomplete, and the oil processed in this way still contains varying proportions of ethyl esters, thus differing from the unprocessed oil. Ethanol released during transesterification (converting ethyl esters to triglycerides) is generally removed by evaporation. Nevertheless, trace amounts of ethanol remain in the concentrated oil.

[0005] Another reason why minimizing the oil processing process is desirable is the formation of impurities such as monochloropropanediols (2-MCPD, 3-MCPD) and glycidol and their derivatives (2-MCPD, 3-MCPD, fatty acid esters of glycidol). The presence of these impurities has been detected particularly after the refining and deodorization processes of fish oil (Miyazaki and Koyama, 2017). At present, there is little available information regarding the effect of the concentration process on the formation of impurities. However, the reformation of triglycerides from ethyl esters can lead to an increase in diglycerides, which are precursor compounds of impurities. The levels of glycidol (and glycidol esters) are subject to the (EU) 2018 / 290 / EC regulation, which limits their content in food: the concentration in edible oils shall not exceed 1000 μg / kg, except for edible oils intended for the preparation of infant and baby food, where the limit concentration is 500 μg / kg. For formulations for infants and toddlers, the maximum levels are even lower: 75 μg / kg in powders and 10 μg / kg in liquids. These levels are expected to be further reduced in 2019 (50 μg / kg in dispersed formulations and 6 μg / kg in liquids). Assessment of maximum MCPD concentrations is currently underway for oils and infant foods. Currently, regulations only apply to hydrolyzed vegetable proteins and soy sauce (limit: 20 μg / kg).

[0006] Therefore, it is important to obtain an oil that is naturally rich in PUFAs, whose composition is as close as possible to the lipid-soluble substances of the producing microorganisms, and which produces minimal impurities during processing. This makes the oil particularly suitable for incorporating DHA into food products. Its very low 3-MCPD and glycidol content, combined with its high DHA content, makes it ideal for preparing foods intended for infants and young children.

[0007] Furthermore, these concentrated oils are generally obtained through expensive and environmentally harmful processing methods.

[0008] Other known solutions involve creating genetically modified microorganisms to enhance the metabolic pathway for PUFA production (Hamilton et al., 2016) or mutants that are thought to produce more DHA (WO2017 / 09804). However, the technical solutions are limited, particularly in human food products, depending on the intended use of the resulting oil (Fedorova-Dahms I. et al., 2011).

[0009] There is a demand for oils in which PUFAs are naturally concentrated without requiring any processing other than extraction, i.e., oils produced by microorganisms where PUFAs are basically in the form of triglycerides. More specifically, there is a demand for oils with high PUFA content and low saturated fatty acid content. In addition to questions about oil quality, the interest in low saturated fatty acid content is directed towards oils that are lower viscosity, easier to use at an industrial level, and especially require less energy to handle.

[0010] The present invention satisfies the above requirements with a high-DHA oil containing at least 60% DHA relative to the total mass of lipids. This oil contains neither ethyl ester nor trace amounts of solvent (ethanol or methanol), and contains reduced amounts of 3-MCPD and glycidol (compared to oils containing more than 60% DHA currently on the market). [Overview of the project]

[0011] The present invention relates to a microbial oil containing docosahexaenoic acid (DHA), characterized in that the microbial oil contains at least 80% triglycerides and more than 60% DHA relative to the total mass of lipids, and the saturated fatty acid content is less than 25% relative to the total mass of lipids.

[0012] The present invention also relates to diluted microbial oils, which include microbial oils according to the present invention, rich in triglycerides and DHA, and mixed with other oils.

[0013] Another subject of the present invention is microbial biomass containing the oil according to the present invention, which is rich in triglycerides and DHA.

[0014] The present invention also relates to the use of the oil according to the present invention, or biomass containing the oil, which may be diluted, and which is rich in triglycerides and DHA, for food for humans or animals, in particular for food for neonates, children, or pregnant or lactating women.

[0015] Another area of ​​interest of the present invention is a food product characterized by containing the oil according to the present invention, which is rich in triglycerides and DHA, and may be diluted. [Modes for carrying out the invention]

[0016] The oil according to the present invention is a microbial oil containing more than 60% DHA, preferably at least 62% DHA, more preferably at least 65% DHA, more preferably more than 67% DHA, more preferably at least 70% DHA, and even more preferably 75% DHA, relative to the total mass of lipids.

[0017] The above-mentioned characteristics of the oil according to the present invention relate to both oil present in microbial biomass and oil extracted from said biomass, regardless of whether it is crude oil or refined oil.

[0018] The present invention also relates to a diluted oil comprising the oil according to the present invention in a mixture with other oils.

[0019] The present invention also relates to pharmaceutical, cosmetic, or food compositions comprising the oil according to the present invention, whether in the form of crude oil, refined oil, or diluted oil.

[0020] The present invention also relates to the use of the oil according to the present invention, or biomass containing this oil, whether in the form of crude oil, refined oil, or diluted oil, for human or animal food, particularly for food for neonates, children, or pregnant or lactating women.

[0021] The oil according to the present invention is a microbial-derived oil, obtained from the biomass of microbial cells that proliferate under conditions that enable cell proliferation (to produce biomass) and the production of oil with a high DHA content.

[0022] The oil according to the present invention is a microbial oil containing more than 60% DHA, preferably at least 62% DHA, more preferably at least 65% DHA, more preferably more than 67% DHA, more preferably at least 70% DHA, and even more preferably at least 75% DHA, relative to the total mass of lipids.

[0023] Preferably, the oil according to the present invention contains a high content of unsaturated fatty acids relative to saturated fatty acids. The unsaturated fatty acids contained in the oil according to the present invention are substantially DHA and DPA (docosapentaenoic acid, C22:5n6). The content of ARA (arachidonic acid, C20:4n6) is generally less than 0.5%, more preferably less than 0.3%, and favorably less than 0.1%. The content of EPA (eicosapentaenoic acid, C20:5n3) is generally less than 1.5%, favorably less than 1%, and more favorably less than 0.5%. The percentages of ARA and EPA are given relative to the total mass of lipids.

[0024] Advantageously, the combined content of DHA and DPA is at least 70%, advantageously at least 75%, more advantageously at least 80%, even more advantageously at least 85% relative to the total mass of lipids. In specific cases, the total amount of DHA+DPA accounts for up to 90% of the total mass of lipids. In the oil with the highest DHA content, which is at least 70%, the combined content of DHA and DPA is at least 80%, preferably 85%.

[0025] In the DHA-enriched oil according to the present invention, the DHA / DPA ratio is preferably at least 3, more preferably at least 4, and ranges from 4 to 9. In the oil with the highest DHA content, which is at least 70%, the DHA / DPA ratio is advantageously from 4 to 7.

[0026] The saturated fatty acid content is less than 25%, further less than 20%, more preferably less than 15%, even more preferably less than 10% relative to the total mass of lipids.

[0027] The saturated fatty acids are substantially palmitic acid (C16:0). Other saturated fatty acids are present in a content of less than 2%, further less than 1%, and are in particular pentadecanoic acid (C15:0), myristic acid (C14:0) or stearic acid (C18:0). Advantageously, C10 to C22 saturated fatty acids other than palmitic acid, in particular C10, C11, C12, C17, C20, C21 and C22 saturated fatty acids, are each independently present in trace amounts in a content of less than 0.1% each, or are absent (including 0% when considering the uncertainty of the analytical method). Percentages are given relative to the total mass of lipids.

[0028] The content of palmitic acid is preferably less than 20%, more preferably less than 15%, even more preferably less than 10% of the total mass of lipids.

[0029] In the oil with the highest DHA content, which is at least 70%, the content of saturated fatty acids C10 to C22 is preferably less than 15%, and more preferably less than 10%.

[0030] One method for measuring the high DHA content and low saturated fatty acid (SFA) content of the oil according to the present invention is to determine the DHA / SFA ratio.

[0031] The DHA / SFA ratio is favorably at least 2.5, preferredly at least 3, more favorably at least 5, and even more favorably at least 6. In some cases, the DHA / SFA ratio may be up to at least 8, and even more favorably at least 9. In the oil with the highest DHA content, which is at least 70%, the DHA / SFA ratio is at least 4, preferredly at least 6, more favorably at least 8, and up to approximately 9.

[0032] The high content of polyunsaturated fatty acids relative to saturated fatty acids (SFA) can also be measured by the (DHA + DPA) / SFA ratio.

[0033] The (DHA+DPA) / SFA ratio is favorably at least 2.5, preferredly at least 3, more favorably at least 4, and even more favorably at least 5. In some cases, the (DHA+DPA) / SFA ratio may be up to at least 8, and even more favorably at least 9. In the oil with the highest DHA content, which is at least 70%, the (DHA+DPA) / SFA ratio is at least 5, preferredly at least 7, more favorably at least 10, and up to approximately 11 or higher.

[0034] The oil according to the present invention is substantially in the form of triglycerides. Triglycerides account for at least 80%, preferably at least 90%, and more preferably at least 93% of the total mass of lipids. The triglyceride content is analyzed, for example, by thin-layer chromatography (Jouet et al., 2003).

[0035] The above-mentioned characteristics of the oil according to the present invention relate to both oil present in microbial biomass and oil extracted from said biomass, regardless of whether it is crude oil or refined oil.

[0036] In certain cases, depending on the process used, the extraction of oil from biomass may lead to a slight increase in the DHA and DPA content, which is advantageous for extracting these PUFAs compared to low molecular weight saturated fatty acids. However, this concentration does not substantially alter the intrinsic properties of the oil contained in the biomass, particularly the triglyceride content. In any case, the oil according to the present invention is an oil that has not undergone substantial alteration of its fatty acid content by, for example, the addition of PUFAs in the form of esters, concentration, and / or the removal of saturated fatty acids such as palmitic acid.

[0037] The oil according to certain embodiments of the present invention contains more than 10 mg, more than 30 mg / kg, preferably more than 40 mg / kg, more preferably more than 60 mg / kg, and at least 65 mg / kg of natural carotenoids per kg of oil. The carotenoids contained are mainly astaxanthin and beta-carotene. The oil contains more than 20 mg / kg, more than 30 mg / kg, and more preferably more than 40 mg / kg of astaxanthin. Canthaxanthin is also contained, but in small amounts. Other carotenoids such as lutein and zeaxanthin may be contained, but only to the detection limits of the method used. The term "natural carotenoids" means carotenoids that have not been added, i.e., those obtained from the same biomass from which the oil is obtained, and extracted from this biomass at the same time as the oil extraction. Natural carotenoids are produced from strains under heterotrophic fermentation conditions without any particular stimulation. Natural carotenoids are therefore present throughout the process and protect fatty acids, especially DHA, from oxidation. Because the refining process can remove pigments, refined oils may contain fewer carotenoids, if any.

[0038] The color of the oil is typically evaluated by measuring the Gardner index using a spectrophotometer, according to the method described in the AOCS Cc-13j-97 standard (revised 2017). The measurement scale includes 18 grades, from clear (1) to dark red / brown (18). Several carotenoids, including astaxanthin and beta-carotene, exhibit darker or lighter colors depending on their concentration. Therefore, the presence of carotenoids is reflected in a high Gardner index. Oils according to certain embodiments of the present invention have a Gardner index greater than 8, and even greater than 10, preferably between 12 and 17.

[0039] The oxidation of polyunsaturated fatty acids (PUFAs) in oils results in yellowing (relative to clear oils), and therefore the Gardner scale can be used to evaluate oil degradation because this can lead to a high Gardner value. However, oxidation of PUFA-rich oils can be more accurately measured by the anisidine index and peroxide index. The oil according to the present invention has a low anisidine index and a low peroxide index, which guarantee a low-oxidation product, and a high Gardner index due to the presence of carotenoids. The oil according to the present invention has an anisidine index of less than 5, more preferably less than 2, and more preferably less than 1.5, and a peroxide index of less than 5, more preferably 1 or less, and more preferably 0.5 or less.

[0040] The oil according to the present invention has a fairly low melting temperature, which decreases in correlation with increasing DHA content. The melting temperature is measured according to the ISO 6321 standard. In fact, oils containing more than 600 mg of DHA (or more than about 62% DHA) per gram of fatty acid have a melting temperature of less than 20°C, and even less than 5°C. Therefore, the above oil is liquid at room temperature. Oils containing more than 700 mg of DHA (or more than about 73% DHA) per gram of fatty acid have a melting temperature of less than -5°C. The low melting temperature makes storage and handling (especially pumping) easier, as it allows the oil to be stored in liquid form while being refrigerated or frozen to suppress deterioration. Oils that freeze during storage must be warmed for sampling and for addition to mixtures. However, temperature is a factor that promotes oxidation.

[0041] This characteristic is also reflected in the viscosity value measured by a viscometer (Viscoman, Gilson) at 22°C. The oil according to the present invention has a viscosity value of 50 Pa·s or less at room temperature, more preferably less than 40, and more preferably less than 30.

[0042] The oil according to the present invention is obtained by culturing microorganisms that produce DHA-rich oil. The microbial strains that enable the acquisition of such oil are industrial strains, that is, according to the present invention, the strains have a lipid content of at least 45%, preferably at least about 50%, of the dry product, and have the ability to grow at a cell density of at least 50 g / L, preferably at least 70 g / L, and more preferably at least 100 g / L.

[0043] Those skilled in the art are primarily familiar with industrial strains of PUFA-producing microorganisms found in thraustochytrids, dinoflagellates, diatoms, and eustigmatophytes, in particular industrial strains of DHA-producing microorganisms belonging to the genera Crypthecodinium, Schizochytrium, Thraustochytrium, or Aurantiochytrium.

[0044] The analysis of PUFA content in lipids is carried out according to standard methods for those skilled in the art, in particular, according to the method described in the following document: Gas Chromatographic Quantification of Fatty Acid Methyl Esters: Flame Ionization Detection vs. Electron Impact Mass Spectrometry, Dodds et al., Lipids, Vol. 40, no. 4 (2005).

[0045] More specifically, examples include Aurantiochytrium mangrovei CCAP4062 / 7 and CCAP4062 / 8 strains, and Schizochytrium sp. CCAP4087 / 7 strain, which produce oil containing more than 60% DHA relative to the total mass of lipids. The present invention also relates to the above strains capable of producing oil containing more than 60% DHA.

[0046] The industrial culture processes for producing fermentation mast used in oil production are autotrophic, heterotrophic, or mixed trophic, all of which are well known to those skilled in the art. Industrial culture in heterotrophic or mixed trophic form allows for cell densities of at least 50 g / L, preferably at least 70 g / L, and more preferably at least 100 g / L.

[0047] According to the present invention, "industrial culture" means culturing a strain in a medium suitable for strain propagation and PUFA production, and in a volume suitable for producing a sufficient amount to meet market demands.

[0048] These industrial cultivations are carried out by discontinuous "batch" fermentation, semi-continuous "half-batch" fermentation, or continuous fermentation. Fermentation tanks range from 1000L to 200m³. 3 It has a volume in the range of the supervolt.

[0049] A suitable culture medium is preferably a chemically defined medium containing a carbon source, a nitrogen source, a phosphorus source, and salts. "Chemically defined medium" means a medium in which the composition of each element is known. Advantageously, the medium does not contain large amounts or complex organic substances. Large amounts or complex organic substances refer to unrefined organic substances in the form of mixtures in which the exact composition and concentrations of the various components in the mixture are unknown and uncontrolled, and may exhibit significant batch-to-batch variability. Examples of large amounts or complex organic substances include yeast extracts, or peptones which are products of protein hydrolysis reactions, or abundant minerals such as marine mineral salts, or other complex growth agents in which the concentrations of each component are not determined.

[0050] Generally, industrial culture processes include a growth step to promote biomass production and a subsequent accumulation step to promote the production of lipids and PUFAs in particular. This process is most notably described in WO2001 / 054510. More recently, processes using culture conditions that simultaneously promote biomass production and PUFA production have been described, particularly the culture methods described in WO2012 / 035262, WO2015 / 004402, and WO2015 / 004403. Of course, those skilled in the art will be able to adapt the culture conditions, in particular the composition of the culture medium, the conditions for adding nutrients during cultivation, the temperature, the oxygen supply cycle, and the lighting conditions, to promote biomass production.

[0051] The temperature for industrial cultivation is advantageously above 17°C.

[0052] According to the present invention, “biomass” advantageously means an aggregate of microbial cells produced by culturing microbial cells, particularly by the culture method described above, wherein the cells may or may not maintain physical integrity. Therefore, it is understood that the biomass may contain degraded microbial cells in amounts from 0% to 100%. “Degraded” means that the physical integrity of the microbial cells may be altered, for example, as in lysed microorganisms resulting from a process of homogenization or enzymatic lysis. Once produced, the biomass may be left as is, separated from the culture medium, dried or not dried, degraded or not degraded.

[0053] The biomass may have a moisture content ranging from 1% to 90%, depending on whether it has been dried whole or partially.

[0054] Therefore, the present invention also relates to microbial biomass containing the oil already described.

[0055] According to the first embodiment, the biomass has a moisture content of 70% to 90%, preferably 80% to 85%. This is particularly true when the biomass essentially consists of cultured optimized industrial microorganisms after filtering the fermentation mast to separate the cultured microorganisms from the culture medium before drying.

[0056] According to another embodiment of the present invention, the biomass is whole or partially dried and has a moisture content of 1% to 10%, preferably 2% to 7%.

[0057] Biomass may be packaged for preservation or for use in food products, such as food supplements or food for human or animal consumption.

[0058] Methods for isolating the oil according to the present invention from biomass produced by the cultivation of microorganisms are well known to those skilled in the art. In particular, solid-liquid extraction based on the use of a solvent (liquid phase) to extract the oil contained in dry biomass (solid phase) by sprinkling or maceration; and liquid-liquid extraction based on separating the aqueous phase from the oil by decantation or centrifugation after pre-lysing the cells. Preferably, the extraction is carried out without the use of organic solvents. In particular, WO01 / 53512, WO02 / 10423, WO2014 / 122092, WO2015 / 092546 and WO2015 / 095694 can be cited.

[0059] Furthermore, a suitable method for improving the lipid extraction yield from microorganisms for PUFA-rich oils can be cited. This method consists of first lysing cells at a first temperature, then continuing to lysing cells at a second temperature lower than the first temperature, and then mechanically separating the oil from the lysed biomass (filtration, decantation).

[0060] Cell lysis is carried out by enzymatic lysis or mechanical lysis (grinding). The temperature of the first part of the lysis is preferably at least 50°C and kept below the temperature at which the oil composition deteriorates while promoting cell lysis, i.e., below 80°C, preferably at most 70°C.

[0061] The enzymes that may be used are known, particularly those described in WO2015 / 095688, WO2011 / 153246, US6750048 and WO2015 / 095694, and especially proteases or cellulases such as those commercially available by Novozyme under the names Alcalase 2.5L, Alcalase 2.4L, Novozym 37071, Flavourzyme 1000L, Novozym FM 2.4 L, Protamex, and Viscozyme. The conditions of use are those recommended by the supplier, and the temperature is the temperature recommended for optimal enzyme activity, at least 50°C and up to 70°C, preferably about 65°C. Advantageously, enzymatic lysis is carried out in an oxygen-poor atmosphere.

[0062] Mechanical dissolution methods are also known, particularly those using ball mills, mixers-dispersers, high-pressure homogenizers, pin mills or impact mills, ultrasound, and pulsed electric fields. Specifically, examples of ball mills include Netzsch / Discus-1000 and WAB / ECM-AP60; examples of high-pressure homogenizers include GEA / Ariete; examples of mixers-dispersers include Silverson / 700-X; examples of pin mills include Hosakawa / Contraplex; and examples of impact mills include Netzsch / Condux.

[0063] The first part of lysis is carried out under the usual conditions recommended by conventional cell lysis techniques, particularly with respect to the duration of the enzymatic lysis or grinding cycle.

[0064] The dissolution continuation process completes the dissolution by changing the operating conditions without prior extraction of the dissolved biomass. The dissolution temperature in the second part is at least 10°C lower than the dissolution temperature in the first part. Preferably, the temperature of the second part of dissolution is 40°C or lower, and advantageously within the range of 5°C to 40°C. The second part of dissolution, or the end of dissolution, carried out at a lower temperature, is advantageously performed for at least 30 minutes and advantageously up to 30 hours.

[0065] Mechanical separation of oil from dissolved biomass is also well known to those skilled in the art as gravity separation, and centrifugation as described in WO01 / 53512 is particularly well known. Continuous separation, in particular using centrifugal plate separators, is also possible. Such separators are known to continuously extract oil from a complex medium containing solid residue and water, as described in WO2010 / 096002, and are commercially available, in particular from Alfa Laval, Flottweg, or SPX Flow Technology Santorso. This continuous separation process is preferred in the process for obtaining oil according to the present invention.

[0066] The resulting oil is commonly called crude oil and can be used as is, or it can be refined, in particular, to facilitate preservation by suppressing unpleasant odors, or to alter its color to make it more consumer-friendly. Refining processes, especially degumming, purification, and deodorization, are well known to those skilled in the art. These processes (whole or partially) remove phospholipids, pigments, volatile substances, and free fatty acids. In practice, these methods do not substantially alter the relative content of saturated or unsaturated fatty acids or triglycerides in the resulting refined oil compared to crude oil.

[0067] The present invention also includes a container of a suitable volume for containing crude or refined oil rich in DHA, in quantities greater than 1 L, preferably greater than 10 L, more specifically in the 220 L range, and even more specifically in the 20 m 3 Regarding oil packaged in a single unit.

[0068] A container capable of holding a certain volume of oil or biomass and protecting it for storage and transport may be used by those skilled in the art. The volume of the container will, advantageously, be equal to or substantially greater than the volume of the oil or biomass being packaged in such a way that it limits the presence of air in the container and thus limits oxidation. The container will, advantageously, be opaque to prevent degradation of the product by light, particularly UV light. Advantageously, the container will be airtight so that any volume not filled with oil or biomass can be filled with an inert gas.

[0069] The oil according to the present invention can be mixed with other oils for its final use. This dilution alters the total content of DHA and other unsaturated fatty acids in the composition of the diluted oil. However, by considering the fatty acid profile of the oil used for dilution, it is still possible to determine the relative percentage of fatty acids derived from the DHA-rich oil according to the present invention and fatty acids derived from the dilution oil in the final oil.

[0070] Accordingly, the present invention also relates to diluted oils, including the oil according to the present invention mixed with other oils. The oil used to dilute the DHA-rich oil according to the present invention is generally and preferably a vegetable oil suitable for food consumption for humans and animals. In particular, examples include sunflower oil, rapeseed oil, soybean oil, walnut oil, sesame oil, hemp oil, hazelnut oil, argan oil, olive oil, flaxseed oil, or other oils suitable for food use. The added oil may be an oil containing other PUFAs, particularly ARA and / or EPA, particularly other oils of microbial origin or fish oil.

[0071] The present invention also relates to compositions comprising crude oil, refined oil, or diluted oil according to the present invention, or compositions comprising biomass according to the present invention.

[0072] The compositions according to the present invention may contain one or more excipients. An excipient is a mixture of one or more components used in the present invention to impart desired properties for the preservation and use of the composition, including food, pharmaceutical, cosmetic, and industrial compositions. An excipient is described as a "pharmaceutically acceptable" excipient when it is used in contact with human and animal tissues without causing excessive toxicity, irritation, allergic reactions, or other complications, and is added to a pharmaceutical composition whose properties are known by pharmacopoeia. Various excipients may be used, such as organic or inorganic bases, organic or inorganic acids, pH buffers, stabilizers, antioxidants, adhesives, release agents, coatings, external phase components, controlled release components, surfactants, humectants, fillers, softeners, or combinations thereof.

[0073] Depending on the purpose, the compositions according to the present invention are particularly pharmaceutical compositions, cosmetic compositions, nutritional supplement compositions, or food products.

[0074] Foods are intended for both human and animal use and include solid compositions, paste-like compositions, and liquid compositions. In particular, examples include general foods, milk, beverages, liquid products including therapeutic and nutritional beverages, functional foods, supplements, nutritional supplements, neonatal formula including formula for premature infants, foods for pregnant or lactating women, foods for adults, foods for the elderly, and animal feed.

[0075] The crude or refined DHA-rich oil or biomass containing the oil according to the present invention may be used directly as is, or may be added as an additive to oils, spreads, other lipid components, beverages, soy-based sauces, dairy products (milk, yogurt, cheese, ice cream), bakery products, nutritional products in the form of nutritional supplements (e.g., capsules or tablets), vitamin supplements, food supplements, powders diluted for beverages such as energy drinks, or milk powder for infant formulas, or powdered foods in the form of finished or semi-finished products, according to uses known to those skilled in the art.

[0076] Animal feed is also known to those skilled in the art. Animal feed is intended in particular for livestock such as cattle, pigs, chickens, sheep, and goats, or for fish farming, including for fish and shellfish.

[0077] Pharmaceutical compositions containing DHA-rich oils are also known to those skilled in the art, and the oils are used alone or in combination with other medical products.

[0078] The crude oil or refined oil according to the present invention, or biomass containing such oil, may be formulated in the form of a single-dose composition, particularly in the form of tablets, capsules, powders, or granules suitable for oral administration.

[0079] The advantage of the DHA-rich crude oil or refined oil, or biomass containing such oil, according to the present invention is that these mixtures and compositions can be used in smaller quantities.

[0080] The present invention also relates to the use of crude oil, refined oil, or diluted oil, or biomass containing the same, for human or animal food, in particular for food for neonates, children, or pregnant or lactating women.

[0081] Such uses are well known to those skilled in the art, and are described in particular in WO2010 / 107415 and on the DSM website (https: / / www.dsm.com / markets / foodandbeverages / en_US / products / nutritional-lipids / life-dha.html). [Examples]

[0082] Example 1: Fatty acid profile of high-DHA biomass containing thraustochytrids Strains of the thraustochytrids (Aurantiochytrium mangrovei-FCCB1897, FCCB1800, CCAP4062 / 8) were grown in ATCC790 (modified) medium in Erlenmeyer flasks. Similar results were obtained for strains of Schizochytrium sp. (particularly strain CCAP4087 / 7).

[0083] Once the culture reached a quiescent phase, the biomass was recovered by centrifugation, and then freeze-dried before analyzing the fatty acid composition of the biomass using GC-FID (a method conforming to ISO 12966-2).

[0084] Composition of the improved ATCC790 medium: Yeast extract 5.0g / L Peptone 5.0g / L D+ glucose 30.0 g / L Sea salt 20g / L

[0085] Table 1 shows the fatty acid composition contained in biomass. The results are expressed as a percentage of the total fatty acid content. SFA refers to saturated fatty acids.

[0086] [Table 1]

[0087] Example 2: Fermentation culture of high-DHA-containing strains The cultures were grown in 1-5 L bioreactors equipped with a dedicated automated system and computer station for monitoring. Two strains of Aurantiochytrium mangrovei were used, and two different culture protocols were employed. The system was adjusted to pH 5 by adding a base (NH4OH for Examples b1 and b2, and NaOH for Example a), and the pH was maintained throughout the culture period while supplying nitrogen (for Examples b1 and b2). The culture temperature was 30°C, then 22°C, and finally 18°C ​​at the end of the culture period.

[0088] CCAP4062 / 7 strain was used in Examples a and b1, and FCCB1897 strain was used in Example b2.

[0089] The composition of the culture medium is shown in Table 2.

[0090] [Table 2]

[0091] Glucose was added in the form of a fortifying solution with a carbon:nitrogen:phosphorus (CNP) molar ratio of 533:11:1 (Example a), or in a solution containing glucose alone (Examples b1 and b2).

[0092] Culture monitoring: Total biomass concentration was monitored by measuring the dry mass (filtered through a Whatman GF / F filter before measurement, then oven-dried at 105°C for a minimum of 24 hours). Fatty acid analysis was performed for the biomass according to the method in accordance with ISO 12966-2, and for the oil according to European Pharmacopoeia 9.0 (2.4.29).

[0093] Table 3 shows the fatty acid profiles of biomass obtained under conditions a, b1, and b2. The results are expressed as a percentage of the total fatty acid content.

[0094] [Table 3]

[0095] Example 3: Cultivation under industrial conditions 10m 3 or 180m 3 When grown in industrial-sized fermentation tanks such as the above tanks, by adding medium b and glucose in the form of a fortified solution with a carbon:nitrogen:phosphorus (CNP) molar ratio of 533:0.4:1, under the same conditions as in Example 2, the high-DHA-containing strain produces biomass with a similar fatty acid composition.

[0096] 10m 3 or 180m 3 Table 4 shows the fatty acid profile of the CCAP4062 / 7 strain biomass after cultivation in the tank. The results are expressed as a percentage of the total fatty acid content.

[0097] [Table 4]

[0098] Example 4: Oil extraction from biomass of high-DHA-containing strains Example 3 (180m) was prepared according to the method described in WO2015 / 095694 (Example 9). 3 Oil was extracted from biomass (in the tank). The fatty acid composition of this oil is similar to that of the biomass shown in Table 4.

[0099] Example 5: Oil extraction from biomass of high-DHA-containing strains The biomass produced under the same conditions as in Example 3 was extracted according to the following procedure: (a) Cell lysis by enzymatic means (e.g., Alcalase 2.5L or Alcalase 2.4L or Novozym 37071 from Novozymes) at a temperature of 65°C for 4 hours. (b) Continue dissolving for 30 minutes to 30 hours while gradually lowering the temperature from 5 to 40°C. (c) Mechanical separation of oil using a centrifugal plate separator.

[0100] The extraction yield is 60% of the lipids extracted from the biomass.

[0101] Table 6 shows the lipid profiles of oils extracted from biomass. The results are expressed as a percentage of the total fatty acid content.

[0102] [Table 5]

[0103] Example 6: Oil Quality: Antioxidants and Impurities Several heterotrophic fermentations were carried out according to the conditions of Example 3. Oil was extracted from the fermentation mast according to the conditions of Example 5. Carotenoids in the extracted oil were measured by LC / DAD according to the following methods: astaxanthin (including ester forms), reference method: DSM 1.5, 2009; saponified beta-carotene (total of cis and trans forms), reference method: EN 12823-2:2000; canthaxanthin, reference method: Roche Index No. 2264; lutein and zeaxanthin, reference method: Roche Index No. 2264.

[0104] [Table 6]

[0105] Impurities such as glycidol and 2- and 3-MCPD were also assayed in the same batch.

[0106] [Table 7]

[0107] Example 7: Viscosity The viscosity of the oils produced and extracted under the same conditions as in Example 5 was measured at different temperatures using a viscometer (Viscoman, Gilson). The melting temperature was evaluated according to ISO 6321 standard.

[0108] [Table 8]

[0109] 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 lipides 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 / j.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., Marechal E., Bligny R., Joyard J., Block M. A. (2003). Transient increase of phosphatidylcholine in plant cells in response to phosphate deprivation. FEBS Lett. 544 63-68.

Claims

1. A microbial oil containing docosahexaenoic acid (DHA), wherein the microbial oil contains at least 80% triglycerides and more than 60% DHA relative to the total mass of lipids, and the saturated fatty acid content is less than 25% relative to the total mass of lipids, and the oil has not undergone substantial alteration of its fatty acid content by the addition, concentration, and / or removal of saturated fatty acids of PUFAs, and the oil is either a crude oil extracted from microbial biomass or a refined oil, wherein the microorganism is selected from Schizochytrium sp. strain or Aurantiochytrium mangrovei strain.

2. The microbial oil according to claim 1, characterized in that it contains at least 65% DHA relative to the total mass of lipids.

3. The microbial oil according to claim 1 or 2, characterized in that it contains docosapentaenoic acid (DPA), and the combined content of DHA and DPA is at least 70% of the total mass of lipids.

4. The microbial oil according to claim 1, characterized in that it contains at least 70% DHA relative to the total mass of lipids.

5. The microbial oil according to claim 4, characterized in that the combined content of DHA and DPA is at least 80% of the total mass of lipids.

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

7. The microbial oil according to any one of claims 1 to 6, characterized by containing less than 0.5% ARA (arachidonic acid) relative to the total mass of lipids.

8. The microbial oil according to any one of claims 1 to 7, characterized by containing less than 1.5% EPA (eicosapentaenoic acid) relative to the total mass of lipids.

9. The microbial oil according to any one of claims 1 to 8, characterized in that the saturated fatty acid content is less than 15% of the total mass of lipids.

10. The microbial oil according to any one of claims 1 to 9, wherein the palmitic acid content is less than 10% of the total mass of lipids.

11. The microbial oil according to any one of claims 1 to 10, comprising more than 10 mg of natural carotenoids per 1 kg of microbial oil.

12. A microbial oil according to any one of claims 1 to 11, characterized in that its viscosity at room temperature is 50 Pa·s or less.

13. The microbial oil according to claim 12, characterized in that its viscosity at room temperature is less than 30 Pa·s.

14. The microbial oil according to any one of claims 1 to 13, wherein the quantity is greater than 1 liter and packaged in a container having a volume equal to or greater than said quantity, in such a manner that the presence of air in the container is restricted and oxidation is restricted.

15. A diluted microbial oil, characterized by comprising the microbial oil described in any one of claims 1 to 14, mixed with other oils.

16. A microbial biomass characterized by comprising the oil described in any one of claims 1 to 14, wherein the microorganism is selected from Schizochytrium sp. strain or Aurantiochytrium mangrovei strain.

17. The microbial biomass according to claim 16, The biomass includes an oil containing DHA obtained by the industrial cultivation of the aforementioned microorganisms. The oil in question further - At least 80% triglycerides relative to the total mass of lipids, - Contains more than 60% DHA relative to the total mass of lipids. - Furthermore, the saturated fatty acid content is less than 25% of the total lipid mass. A microbial biomass characterized by the following features.

18. Use of the oil according to any one of claims 1 to 15 or the biomass according to claim 16 or 17 for food for human or animal use.

19. The use according to claim 18 for food for newborns, children, or pregnant or breastfeeding women.

20. A food product characterized by containing the oil described in any one of claims 1 to 14.

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