Method for extracting oil rich in polyunsaturated fatty acids (PUFA)

Abstract

The present invention relates to a method for the extraction of microbial oils rich in polyunsaturated fatty acids (PUFAs), in particular rich in docosahexaenoic acid (DHA, C22:6n3), especially oils containing more than 60% PUFAs relative to the total lipid mass.

Description

[Technical field]

[0001] The present invention relates to a method for the extraction of oils rich in polyunsaturated fatty acids (PUFAs), in particular microbial oils rich in docosahexaenoic acid (DHA, C22:6n3), especially oils containing more than 60% PUFAs relative to the total lipid mass. [Background technology]

[0002] Today, there is a demand for concentrated oils with high PUFA content, i.e. for the provision of concentrated products such as concentrated oil capsules that allow to reduce the number of unit doses required for an equivalent amount of PUFA. To obtain oils with high PUFA content (e.g. more than 55% DHA), the oils are concentrated by a method that involves the conversion of triglycerides to ethyl esters, involving the use of a solvent such as ethanol. Ethyl esters are an artificial chemical form and do not occur in nature. The bioavailability of fatty acids in the form of ethyl esters is much lower than that of fatty acids in the form of triglycerides (Ghasemifard et al., 2014). The above methods also remove vitamins and antioxidants contained in the crude oil. As a result, concentrated oils are more vulnerable to oxidation.

[0003] It is therefore advantageous to obtain oils that are naturally rich in PUFAs, whose composition is as close as possible to the lipid-soluble substances of the producing organism, with minimal impurities generated during processing. However, the extraction of PUFA-rich oils faces certain difficulties with a high PUFA content that limits the amount of fatty acids that can be extracted from the biomass.

[0004] Currently used extraction methods involve the addition of sodium (WO2011 / 153246), solvents (US2014 / 350222) and / or high temperatures for several hours (WO2015 / 095694), which can impair the quality of the resulting oil and its by-products (defatted biomass). Indeed, temperature is a factor inducing the formation of trans fatty acids from cis fatty acids. However, regulations state that the maximum content of trans fatty acids in edible oils is less than 1%. The formation of trans fatty acids reduces the quality of the oil, since the fatty acids of PUFA-producing microorganisms are naturally in the cis structure. Temperature can also cause the formation of the toxic compounds 2-MCPD, 3-MCPD and glycidol, the content of which is regulated (glycidol) or is in the process of being regulated (2- and 3-MCPD). It is important to optimize the quality of the oil and to minimize it as much as possible, especially for its use in infant foods.

[0005] Adding sodium during extraction in the form of sodium sulfate or sodium chloride is a known method (WO2011 / 153246). Unfortunately, sodium is found in defatted biomass. This reduces the benefits of biomass that is also rich in protein, especially for animal feed. Also, the efficiency of methods involving sodium addition and temperatures below 70°C is limited in terms of extraction yield from biomass that contains more than 60% PUFA.

[0006] The present invention meets the above-mentioned demands with a novel method for extracting high PUFA content lipids, improving oil extraction yields, particularly the extraction of PUFAs from oil-bearing biomass, while maintaining oil quality. Summary of the Invention

[0007] The present invention relates to a novel method for extracting oil rich in PUFAs from biomass of oil producing organisms containing oil, comprising: The method comprises: (a) lysing cells of a biomass of said oil producing organism; (b) mechanically separating the oil from the dissolved biomass to recover crude oil; Including, The step (a) of lysing the cells comprises the following two parts: (a1) a first part carried out at a first temperature, then (a2) a second part that continues to carry out the dissolution at a second temperature that is lower than the first temperature; The present invention relates to a method comprising the steps of:

[0008] The method according to the invention can be carried out with any type of organism that produces oil with a high PUFA content, whether it is an animal, in particular a marine organism, an oil seed plant, or a microorganism.

[0009] The method according to the invention is particularly suitable for extracting microbial oils with high PUFA content. The method according to the invention does not involve the addition of a solvent for the oil extraction.

[0010] The present invention also relates to a PUFA-rich oil obtained by the process according to the invention, whether it is a crude oil, a refined oil, or a diluted oil comprising the oil extracted by the process according to the invention in admixture with other oils.

[0011] The present invention also relates to a pharmaceutical, cosmetic or food composition comprising an oil obtained by the process according to the invention, whether it is a crude, refined or diluted oil.

[0012] The present invention also relates to the use of the crude, refined or diluted oil obtained by the method according to the invention for food products for human or animal consumption, in particular for consumption by newborns, children or pregnant or lactating women.

[0013] The present invention also relates to a composition, in particular a nutritional composition or a food product, comprising the crude, refined or diluted oil obtained by the method according to the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] PUFAs are well known to those skilled in the art, especially for their use as food additives or for preparing functional foods, especially baby milk, pregnant women or lactating women.In particular, mention can be made of docosahexaenoic acid (DHA, C22:6n3), DPA (docosapentaenoic acid, C22:5n6), arachidonic acid (ARA, C20:4n6) or eicosapentaenoic acid (EPA, C20:5n3).Depending on the production organism considered, the oil contains DHA, EPA or ARA, or a mixture of several PUFAs.

[0015] ARA-producing organisms are well known, in particular filamentous fungi of the genus Mortierella alpina. EPA-producing organisms are also well known, in particular Nannochloropsis gaditana. DHA-producing organisms are also well known, in particular protists of the genus Aurantiochytrium or Schizochytrium. Plants are also good producers of PUFAs, especially at the seed stage: linseed (Linum usitatissimum L.) oil can contain more than 50% alpha-linolenic acid (ALA); rapeseed (Brassica napus) is also a well-known producer of ALA. Plants do not produce long-chain fatty acids (greater than C18). However, recently, genetically modified Camelina sativa plants have been engineered to produce DHA (Mansour et al., 2014) or EPA (Ruiz-Lopez et al., 2014). Seeds of oilseed plants typically contain between 20% (soybean) and 70% (dehydrated coconut) lipids (Rosenthal et al., 1996).

[0016] The method according to the invention is particularly suitable for extracting oil rich in PUFAs from oil-producing microorganisms, these microorganisms being in particular chosen from filamentous fungi and protists.

[0017] The strains of microorganisms making it possible to obtain such an oil are industrial strains, i.e. according to the invention, the strains have a fat content of at least 45% of the dry matter, preferentially at least about 50%, and are capable of growing at a cell density of at least 50 g / L, preferentially at least 70 g / L and more preferentially at least 100 g / L.

[0018] Those skilled in the art are primarily familiar with industrial strains of PUFA-producing microorganisms from the Thraustochytrids, Dinophyceae, Diatoms, Eustigmatophyceae, and in particular DHA-producing industrial strains of the genera Crypthecodiniu, Schizochytrium, Thraustochytrium or Aurantiochytrium.

[0019] In particular, Crypthecodinium cohnii M64245, Crypthecodinium cohnii FJ821501, Crypthecodinium cohnii CCAP 1104 / 3 (WO2016030631), Crypthecodinium cohnii CCAP 1104 / 5 or Crypthecodinium cohnii CCAP 1104 / 4, Aurantiochytrium limacinum AB022107; Aurantiochytrium limacinum HM042909; Aurantiochytrium limacinum JN986842; Aurantiochytrium limacinum SL1101 JN986842; Aurantiochytrium mangrovei; Aurantiochytrium mangrovei DQ323157; Aurantiochytrium mangrovei DQ356659; Aurantiochytrium mangrovei DQ367049; Aurantiochytrium mangrove CCAP 4062 / 2; Aurantiochytrium mangrovei CCAP 4062 / 3; Aurantiochytrium mangrovei CCAP 4062 / 4; Aurantiochytrium mangrove CCAP 4062 / 5; Aurantiochytrium mangrove CCAP 4062 / 6; Aurantiochytrium mangrove CCAP 4062 / 1; Aurantiochytrium sp. AB052555; Aurantiochytrium sp. AB073308; Aurantiochytrium sp. ATCC PRA276 DQ836628; Aurantiochytrium sp. BL10 FJ821477; Aurantiochytrium sp. LY 2012 PKU Mn5 JX847361; Aurantiochytrium sp. LY2012 JX847370; Aurantiochytrium sp. N1-27; Aurantiochytrium sp.SD116; Aurantiochytrium sp. SEK209 AB290574; Aurantiochytrium sp. SEK217 AB290572; Aurantiochytrium sp. SEK 218 AB290573; Aurantiochytrium sp. 18W-13a; Botryochytrium radiatum; Botryochytrium radiatum Raghukumar 16; Botryochytrium radiatum SEK353; Botryochytrium sp.; Botryochytrium sp. BUTRBC 143; Botryochytrium sp. Raghukumar 29; Oblongichytrium minutum; Oblongichytrium multirudimentalis; Oblongichytrium sp. SEK347; Parieticytrium sarkarianum; Parieticytrium sarkarianum SEK351; Parieticytrium sarkarianum SEK364; Parieticytrium sp. F3-1; Parieticytrium sp. H1-14; Parieticytrium sp. NBRC102984; Phytophthora infestation; Schizochytrium aggregate DQ323159; Schizochytrium aggregate DQ356661; Schizochytrium aggregate; Schizochytrium limacinum; Schizochytrium limacinum OUC166 HM042907; Schizochytrium mangrove; Schizochytrium mangrove FB1; Schizochytrium mangrove FB3; Schizochytrium mangrove FB5; Schizochytrium minute; Schizochytrium sp. ATCC20888 DQ367050; Schizochytrium sp.KGS2 KC297137; Schizochytrium sp. SKA10 JQ248009; Schizochytrium sp. ATCC 20111; Schizochytrium sp. ATCC 20888 (WO1991007498); Schizochytrium sp. ATCC 20889; Schizochytrium sp. ATCC 26185; Schizochytrium sp. BR2.1.2; Schizochytrium sp. BUCAAA 032; Schizochytrium sp. BUCAAA 093; Schizochytrium sp. BUCACD 152; Schizochytrium sp. BUCARA 021; Schizochytrium sp. BUCHAO 113; Schizochytrium sp. BURABQ 13; Schizochytrium sp. BURARM 802; Schizochytrium sp. CCAP 4087 / 3 (WO2017012931); Schizochytrium sp. CCAP 4087 / 1; Schizochytrium sp. CCAP 4087 / 4; Schizochytrium sp. CCAP 4087 / 5; Schizochytrium sp. FJU-512; Schizochytrium sp. KH105; Schizochytrium sp. KK17-3; Schizochytrium sp. KR-5; Schizochytrium sp. PJ10.4; Schizochytrium sp. SEK 210; Schizochytrium sp. SEK 345; Schizochytrium sp. SEK 346; Schizochytrium sp. SR21; Schizochytrium sp. TIO01; Sicyoidochytrium minutum SEK354; Sicyoidochytrium minutum NBRC 102975, Sicyoidochytrium minutum NBRC 102979; Thraustochytriidae sp.BURABG162 DQ100295; Thraustochytriidae sp. CG9; Thraustochytriidae sp. LY2012 JX847378; Thraustochytriidae sp. MBIC11093 AB183664; Thraustochytriidae sp. NIOS1 AY705769; Thraustochytriidae sp. #32 DQ323161; Thraustochytriidae sp. #32 DQ356663; Thraustochytriidae sp. RT49 DQ323167; Thraustochytriidae sp. RT49 DQ356669; Thraustochytriidae sp. RT49; Thraustochytriidae sp. Thel2 DQ323162; Thraustochytriidae sp. Thel2; Thraustochytrium aggregate; Thraustochytrium aggregatum DQ356662; Thraustochytrium aureum; Thraustochytrium aureum DQ356666; Thraustochytrium gaertnerium; Thraustochytrium kinnei; Thraustochytrium kinnei DQ323165; Thraustochytrium motivum; Thraustochytrium multirudimentale; Thraustochytrium pachydermum; Thraustochytrium roseum; Thraustochytrium sp. 13A4.1; Thraustochytrium sp. ATCC 26185; Thraustochytrium sp. BL13; Thraustochytrium sp. BL14; Thraustochytrium sp. BL2; Thraustochytrium sp. BL3; Thraustochytrium sp. BL4; Thraustochytrium sp. BL5; Thraustochytrium sp.BL6; Thraustochytrium sp. BL7; Thraustochytrium sp. BL8; Thraustochytrium sp. BL9; Thraustochytrium sp. BP3.2.2; Thraustochytrium sp. BP3.3.3; Thraustochytrium sp. caudivorum; Thraustochytrium sp. CHN-1; Thraustochytrium sp. FJN-10; Thraustochytrium sp. HK1; Thraustochytrium sp. HK10; Thraustochytrium sp. HK5; Thraustochytrium sp. HK8; Thraustochytrium sp. HK8a; Thraustochytrium sp. KK17-3; Thraustochytrium sp. KL1; Thraustochytrium sp. KL2; Thraustochytrium sp. KL2a; Thraustochytrium sp. ONC-T18; Thraustochytrium sp. PJA10.2; Thraustochytrium sp. TR1.4; Thraustochytrium sp. TRR2; Thraustochytrium striatum; Thraustochytrium striatum ATCC24473 (WO2017131188); Thraustochytrium striatum DQ323163; Thraustochytrium striatum DQ356665; Thraustochytrium visurgense; Ulkenia amoeboidea SEK 214; Ulkenia profunda; Ulkenia profunda BUTRBG 111; Ulkenia sp.; Ulkenia sp.ATCC 28207 (WO1998003671); Ulkenia visurgensis; Ulkenia visurgensis BURAAA 141; Ulkenia visurgensis ATCC 28208; Thraustochytrids deposited with the ATCC under the accession numbers PTA-9695, PTA-9696, PTA-9697 and PTA-9698 (US2010-239533).

[0020] Phaeodactylum tricornutum (Pt1) Bohlin Strain 8.6 CCMP2561 (WO2015008160); Nitzschia brevirostris (CCAP 1052 / 21; WO2013136025); Nitzschia laevis (UTEX 2047; WO2008004900); Pythium irregular (US9074160); Nannochloropsis limnetica (WO2016059262); Nannochloropsis saline; Nannochloropsis avicula; Nannochloropsis acceptata; Nannochloropsis oculata CCAP849 / 1; Nannochloropsis pseudotenelloides; Nannochloropsis follows; Nannochloropsis sp. CCAP211 / 46; Nannochloropsis saprophyla; Chlorella protothecoids CCAP211 / 17 (WO201115041); Chlorella ellipsoidea; Chlorella minutessima; Chlorella zophienesis; Chlorella luteoviridis CCAP211 / 3; Chlorella kessleri; Chlorella strain CCAP211 / 8K; Chlorella fiusca var. vacuolate; Chlorella sp.; Chlorella emersonii; Monodus subterraneus.

[0021] For the production of oils rich in ARA, particular mention may be made of Mortierella elongata IFO8570, Mortierella exigua IF08571, Mortierella hygrophila IF05941, Mortierella alpina IF08568, ATCC16266, ATCC32221, ATCC42430, CBS219.35, CBS224.37, CBS250.53, CBS343.66, CBS527.72, CBS529.72, CBS608.70, and CBS754.68 (WO2015 / 095696).

[0022] The method according to the invention is particularly suitable for extracting PUFAs from organisms that contain oils rich in PUFAs, especially from organisms that contain oils with a PUFA content of more than 50% based on the total lipid mass.The method according to the invention is the best method for extracting PUFAs from microorganisms that produce oils that contain more than 60% PUFAs based on the total lipid mass, advantageously at least 62% PUFAs, more advantageously at least 65% PUFAs, preferably more than 67%, more preferentially at least 70%, even more preferentially 75% PUFAs.

[0023] However, the advantages seen in high PUFA content also apply when extracting PUFAs from microorganisms that produce low yields of PUFA-rich oils but still contain significant amounts of PUFAs, up to 40% to 50% of the total lipid mass.

[0024] According to a preferred embodiment of the invention, the PUFA extracted is DHA or a DHA+DPA mixture and the microorganism used is a microorganism producing an oil rich in DHA or DHA+DPA.

[0025] In particular, Aurantiochytrium mangrovei CCAP 4062 / 2; Aurantiochytrium mangrovei CCAP 4062 / 3; Aurantiochytrium mangrovei CCAP 4062 / 4; Aurantiochytrium mangrovei CCAP 4062 / 5; Aurantiochytrium mangrovei CCAP 4062 / 6; Aurantiochytrium mangrovei CCAP 4062 / 1; Schizochytrium sp. CCAP 4087 / 3; Schizochytrium sp. CCAP 4087 / 1; Schizochytrium sp. CCAP 4087 / 4; Schizochytrium sp. CCAP 4087 / 5; or Thraustochytrids deposited with the ATCC under accession numbers PTA-9695, PTA-9696, PTA-9697 and PTA-9698. The method according to the invention may be carried out with microorganisms producing oils especially rich in DHA, in particular Aurantiochytrium mangrovei CCAP4062 / 7 and CCAP4062 / 8 and Schizochytrium sp. CCAP4087 / 7.

[0026] The biomass of the producing organism can be subjected to a first treatment allowing cell lysis, for example for the seeds of oilseed plants, the biomass may be subjected to a series of known treatments to separate the kernels from the husks, firstly to grinding and suspending the grinding, before lysis is then carried out.

[0027] For microbial biomass derived from the culture of microorganisms, the biomass can be obtained directly from the fermentation must. The biomass may be washed to remove certain soluble components (in particular by filtering to remove the fermentation medium and washing with water) and resuspended for the dissolution step (a). The microbial biomass can also be a dried or freeze-dried biomass that is stored and resuspended before the dissolution step. The above-mentioned pretreatments of microbial oils to facilitate the implementation of the dissolution step (a) are well known to those skilled in the art.

[0028] The cell lysis step (a) is advantageously carried out on a biomass suspension, which preferably has a dry matter content of between 5 and 20 percent by weight, typically between 8 and 10 percent by weight.

[0029] The cell lysis is carried out by enzymatic or mechanical lysis. The temperature of the first part of the lysis is preferably at least 50°C and below the temperature that promotes cell lysis while degrading the composition of the oil, i.e. below 95°C, even below 90°C, preferably below 80°C and even more preferentially below 70°C.

[0030] Usable enzymes are known, in particular proteases or cellulases, such as those described in WO2015 / 095688, WO2011 / 153246, US6750048 and WO2015 / 095694, in particular those marketed by Novozyme under the names Alcalase 2.5L, Alcalase 2.4L, Alcalase 3.0T, Novozym 37071, Flavourzyme 1000L, Novozym FM 2.4L, Protamex, Viscozyme. The conditions of use are those recommended by the supplier, and the temperature is that recommended for optimal enzyme activity, which is at least 50°C and at most 70°C, preferably about 65°C. Advantageously, the enzymatic dissolution is carried out in an oxygen-poor atmosphere. In general, the oxygen concentration is less than 1% by weight.

[0031] Mechanical dissolution methods are also well known, in particular by ball mills, mixer-dispersers, high-pressure homogenizers, pin or impact mills, ultrasound, pulse electrolysis. Apparatus for carrying out these mechanical dissolution methods (name of manufacturer in parentheses) include, in particular, for ball mills: Discus-1000 (Netzsch); ECM-AP60 (WAB); for high-pressure homogenizers: Ariete (GEA); for mixer-dispersers: 700-X (Silverson); for pin mills: Contraplex (Hosakawa); for impact mills: Condux (Netzsch).

[0032] The first part (a1) of the lysis is carried out under the usual conditions recommended by the prior art of cell lysis, in particular with regard to the duration of the enzymatic lysis or grinding cycle.

[0033] The continued dissolution step (a2) completes the dissolution by changing the operating conditions without prior extraction of the dissolved biomass. The change in operating conditions is a change in temperature, but does not include a substantial change in the suspension medium that partially contains the dissolved biomass, such as the addition of salts, acids or bases. A substantial change in the suspension medium before or at the same time as lowering the temperature is not an a priori "continuation of dissolution" according to the present invention. The above-mentioned substantial change in the suspension medium after dissolution is described in US2011 / 295028 and US2015 / 176042, but does not allow efficient extraction of oil with high PUFA content.

[0034] Advantageously, the enzymatic lysis consists essentially of two parts (a1) and (a2), which are substantially differentiated by a decrease in temperature when passing to the second part of the lysis (a2).

[0035] The dissolution temperature in the second part is at least 10°C lower than the dissolution temperature in the first part. Preferably, the dissolution temperature of the second part is 40°C or lower, advantageously between 5°C and 40°C, more advantageously between 10°C and 35°C, even between 15°C and 30°C, preferably between 20°C and 30°C. The dissolution of the second part (a2) at or at the end of the dissolution at a lower temperature is advantageously carried out for at least 30 minutes and up to 30 hours. The prior art teaches to maintain the temperature above 40°C, whether the starting biomass is a microorganism (WO2018011275) or an oilseed (Rosenthal et al., 1996), or even to increase the temperature to above 80°C to improve extraction during or after the dissolution step. Surprisingly, the inventors have found that the opposite (lower temperature) gives better results, especially for oils with high PUFA content. The temperature of step (a2) is not increased at the end of the dissolution prior to the separation step (b).

[0036] The mechanical separation of oil from the dissolved biomass (b) is also well known to the skilled artisan as gravity separation, in particular centrifugation as described in WO 01 / 53512. It is also possible to use continuous separation, in particular centrifugal plate separators. Such separators are known to continuously extract oil from a complex medium containing solid residues and water, as described in WO 2010 / 096002, and are commercially available, in particular by Alfa Laval, Flottweg or GEA Westfalia. This continuous separation process is preferred in the process for obtaining oil according to the invention.

[0037] Depending on the PUFA content of the production organism, the crude oil obtained by the method according to the invention may contain a very high content of PUFAs, in particular DHA, of more than 60% relative to the total mass of lipids extracted, advantageously at least 62% PUFAs, even more advantageously at least 65% PUFAs, preferably more than 67% PUFAs, more preferentially at least 70% PUFAs and even more preferentially 75% PUFAs relative to the total mass of lipids extracted.

[0038] The oil obtained by the method according to the invention is substantially in the form of triglycerides. The triglycerides preferably represent at least 80%, more preferably at least 90%, even more preferably at least 93% of the total lipid mass. The triglyceride content is analyzed, for example, by thin layer chromatography (Jouet et al., 2003).

[0039] In general, the extraction of oil from biomass using the method according to the invention results in a slight increase in the content of PUFA, especially DHA and DPA, and promotes the extraction of these PUFAs compared to low molecular weight saturated fatty acids.However, this enrichment does not substantially change the inherent properties of the oil contained in the biomass, especially the triglyceride content.In any case, the oil according to the invention is an oil whose fatty acid content has not been substantially modified by the addition of PUFA, for example in the form of esters, enrichment, and / or removal of saturated fatty acids such as palmitic acid.

[0040] The resulting oil, commonly called crude oil, can be used as is or can be refined, in particular to facilitate storage by suppressing rancidity or to change the color to make it more acceptable to the consumer. Refining processes, in particular degumming, neutralization of free fatty acids, bleaching, and deodorization, are well known to those skilled in the art. These processes remove (completely or partially) phospholipids, pigments, volatile substances and free fatty acids. In fact, these methods do not substantially change the relative content of saturated or unsaturated fatty acids or triglycerides in the resulting refined oil compared to the crude oil.

[0041] Therefore, the present invention also relates to a method for extracting an oil rich in PUFAs as previously described, comprising a refining step (c).

[0042] Some methods of the prior art have a so-called "winterization" step carried out on crude or refined oils, particularly to remove saturated fatty acids, with the effect of increasing the PUFA content (WO02 / 10322). The oils extracted according to the present invention, whether crude or refined, do not require the use of "winterization" a priori. However, those skilled in the art may choose to add such a "winterization" step if they find commercial benefits in the final product.

[0043] Depending on the PUFA-rich oil-producing microorganism used, in particular the DHA-producing microorganism, the oil obtained by the method according to the invention may contain more than 10 mg / kg of natural carotenoids, even more than 30 mg / kg, preferentially more than 40 mg / kg, even more preferentially more than 60 mg / kg, or even 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 of astaxanthin, even more preferentially more than 30 mg / kg, even more preferentially more than 40 mg / kg. Canthaxanthin is also contained, but in small amounts. Other carotenoids such as lutein and zeaxanthin may be present, but at the detection limit of the method used. The term "natural carotenoids" means that no carotenoids have been added, i.e. they are obtained from the same biomass from which the oil is obtained and are extracted from this biomass at the same time as the extraction of the oil. Natural carotenoids are produced by the strain under heterotrophic fermentation conditions without any special stimulation. Natural carotenoids are therefore present throughout the process according to the invention and protect the fatty acids, especially DHA, from oxidation. Because the refining process may remove pigments, refined oils may contain less carotenoids, if any at all.

[0044] The crude or refined oil may be diluted for further use.

[0045] Therefore, the present invention also relates to a method for extracting an oil rich in PUFAs as previously described, further comprising a step (d) of diluting the crude oil obtained in the mechanical separation step (b) or the refined oil obtained in the refining step (c).

[0046] The oil used to dilute the PUFA-rich oil obtained by the method according to the invention is generally and preferably a vegetable oil suitable for human and animal consumption.In particular, it can be sunflower oil, rapeseed oil, soybean oil, walnut oil, sesame oil, hemp oil, hazelnut oil, argan oil, olive oil, linseed oil or other oil suitable for food use.The oil added can also be other PUFA-containing oils, in particular DHA, ARA and / or EPA-containing oils, in particular other oils of microbial origin or fish oils.

[0047] The present invention also relates to compositions comprising a PUFA-rich oil obtained by the method according to the present invention, whether it is a crude oil, a refined oil or a diluted oil.

[0048] The compositions according to the present invention may contain one or more excipients. An excipient is an ingredient or mixture of ingredients used in the present invention to impart to said compositions, including food, pharmaceutical, cosmetic and industrial compositions, the desired properties for the storage and use of said compositions. An excipient is described as "pharmaceutical acceptable" if it can be used in contact with human and animal tissues without causing excessive toxicity, irritation, allergic reactions or other complications, and is added to pharmaceutical compositions whose properties are known by the pharmacopoeia. A variety of excipients may be used, such as organic or inorganic bases, organic or inorganic acids, pH buffers, stabilizers, antioxidants, adhesives, release agents, coating agents, external phase components, controlled release components, surfactants, humectants, fillers, softeners, or combinations thereof.

[0049] Depending on the purpose, the composition according to the invention is in particular a pharmaceutical composition, a cosmetic composition, a nutraceutical composition or a food product.

[0050] Food products are intended for both human and animal consumption and include solid, pasty and liquid compositions, in particular liquid products including general foods, milk, beverages, therapeutic and nutritional drinks, functional foods, supplements, dietary supplements, newborn formulas including premature infant formulas, foods for pregnant or lactating women, foods for adults, foods for the elderly and animal feed.

[0051] The PUFA-rich oil obtained by the method according to the invention, whether crude or refined, or the biomass containing said oil, can be used directly as it is or can be added as an additive to oils, spreads, other lipid ingredients, beverages, soy-based sauces, dairy products (milk, yogurt, cheese, ice cream), bakery products, nutritional products, e.g. in the form of nutritional supplements (in capsule or tablet form), vitamin supplements, food supplements, powders to be diluted for beverages such as energy drinks or milk powders for infant formulas, finished or semi-finished powdered foods, etc., according to applications known to the person skilled in the art.

[0052] Pet foods are also known to those skilled in the art and are intended in particular for domestic animals such as cows, pigs, chickens, sheep, goats, or for fish farming, such as seafood or farmed fish.

[0053] Pharmaceutical compositions containing oils rich in PUFAs are also known to those of skill in the art, either used alone or in combination with other medical products.

[0054] The crude or refined oil rich in PUFAs obtained by the method according to the invention, or the biomass containing said oil, may be formulated in the form of a single dose composition, in particular in the form of tablets, capsules, powders, granules suitable for oral administration.

[0055] The present invention also relates to the use of the PUFA-rich crude, refined or diluted oil obtained by the method according to the invention for food products for human or animal consumption, in particular food products for newborns, children, or pregnant or lactating women.

[0056] Such uses are well known to those skilled in the art and are described, inter alia, in WO2010 / 107415 and on the DSM website (https: / / www.dsm.com / markets / foodandbeverages / en_US / products / nutritional-lipids / life-dha.html). EXAMPLES

[0057] Example 1: Fermentation of high DHA content strain The cultivation was carried out in fermenters (bioreactors) with a useful volume of 1-5 L, equipped with dedicated automation and monitoring by a computer station. The cultivation was carried out with two strains of Aurantiochytrium mangrovei, with two different cultivation protocols. The system was adjusted to pH 5 by addition of base (NH4OH for examples b1 and b2, NaOH for example a) and pH adjustment was carried out during the whole cultivation period under nitrogen feeding (for examples b1 and b2). The cultivation temperature was 30°C, then 22°C and finally 18°C ​​at the end of the cultivation.

[0058] The CCAP4062 / 7 strain was used for examples a and b1, and the FCCB1897 strain was used for example b2.

[0059] The composition of the medium is shown in Table 1.

[0060] [Table 1]

[0061] Glucose was added in the form of an enriched solution with a carbon:nitrogen:phosphorus (CNP) molar ratio of 533:11:1 (example a) or in a solution containing glucose only (examples b1 and b2).

[0062] Culture monitoring: Total biomass concentration was monitored by measuring the dry mass (filtered through Whatman GF / F filters and then oven dried at 105 °C for a minimum of 24 h before measurement). Fatty acid analysis was performed for the biomass according to a method conforming to the ISO 12966-2 standard and for the oil according to the European Pharmacopoeia 9.0 (2.4.29.).

[0063] The fatty acid profiles of biomass obtained under conditions a, b1 and b2 are shown as percentages of total fatty acids in Table 2. SFA: saturated fatty acids.

[0064] [Table 2]

[0065] Example 2: Cultivation of high DHA content strains under industrial conditions 10m 3 (Example d) or 180m 3 When grown under similar conditions in industrial size fermentors such as the tank of Example e, with glucose addition in the form of medium b and an enriched solution with a carbon:nitrogen:phosphorus (CNP) molar ratio of 533:0.4:1, the high DHA Aurantiochytrium mangrovei strains produce biomass with similar fatty acid composition.

[0066] The fatty acid profiles of the biomass of Examples d and e are shown in Table 3 as a percentage of total fatty acids.

[0067] [Table 3]

[0068] Example 3: Oil extraction from biomass of high DHA strains: Comparison of yields with different methods Oil was extracted from the same biomass, produced under the conditions of Example 2, containing over 60% DHA (Table 4).

[0069] [Table 4]

[0070] Extraction was carried out according to various methods known to those skilled in the art, including, inter alia, chemical lysis (WO2015 / 095688A1) or enzymatic lysis and addition of sodium sulfate (WO2011 / 153246) or change in pH (WO2015 / 095694). The lipid extraction yields related to the abundance in the biomass are shown in Table 5. Tests 1, 2 and 3 were carried out according to the teachings of the prior art. Test 4 was carried out according to the present invention.

[0071] [Table 5]

[0072] Example 4: Oil extraction from biomass of strains with high DHA content Oil was extracted from the biomass produced in Example 2 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 decreasing the temperature from 5 to 40°C. (c) Mechanical separation of oil by centrifugal plate separator.

[0073] The extraction yield is 60% of the lipids extracted from the biomass.

[0074] [Table 6]

[0075] References ·EP 0 223 960; EP 1 001 034 ·WO 1994 / 008467; WO 1997 / 037032; WO 2001 / 054510; WO 02 / 10322; WO 03 / 049832; WO 2010 / 107415; WO2011 / 153246; WO 2012 / 035262; WO 2013 / 136025; WO 2013 / 136028; WO 2014 / 146098; WO 2015 / 004402; WO 2015 / 004403; WO2015 / 095688; WO2015 / 095694; WO 2015 / 150716; WO 2016 / 030631 ·US 6,750,048, US 2011 / 295028, US 2015 / 176042, US 2014 / 350222 ·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. ·Maged P. Mansour,1,* Pushkar Shrestha,2 Srinivas Belide,2 James R. Petrie,2 Peter D. Nichols,1 and Surinder P. Singh2 (2014). Characterization of Oilseed Lipids from “DHA-Producing Camelina sativa”: A New Transformed Land Plant Containing Long-Chain Omega-3 Oils. Nutrients. 2014 Feb; 6(2): 776-789. ·Ruiz-Lopez N, Haslam RP, Napier JA, Sayanova O (2014) Successful high-level accumulation of fish oil omega-3 long chain polyunsaturated fatty acids in a transgenic oilseed crop. Plant J. 77(2):198-208 ·Rosenthal A, Pyle DL, and Niranjan K (1996) Aqueous and enzymatic processes for edible oil extraction. Enzyme and Microbial Technology 19:402-420.

Claims

1. 1. A method for extracting oil rich in PUFAs from a biomass of an oil producing organism comprising the steps of: The method comprises: (a) enzymatically lysing cells of a biomass of said oil producing organism; (b) mechanically separating the oil from the dissolved biomass and recovering the crude oil; Including, The step (a) of lysing the cells comprises the following two parts: (a1) a first part carried out at a first temperature between 50° C. and 95° C.; then (a2) a second part that continues to carry out dissolution at a second temperature that is equal to or lower than 40° C.; Part (a1) and part (a2) are carried out without adding salts, acids or bases to the medium in which the lysis of the cells is carried out; The method of claim 1, wherein the second temperature is at least 10° C. lower than the first temperature.

2. 2. The method according to claim 1, characterized in that the biomass of an oil-producing organism is a biomass of a PUFA-producing microorganism.

3. 3. The method of claim 2, wherein the biomass is a suspension of the microorganisms.

4. 4. The method according to any one of claims 1 to 3, characterized in that the oil produced by the oil-producing organism comprises more than 50% PUFAs.

5. 5. The method according to claim 1, wherein the PUFA is selected from docosahexaenoic acid (DHA, C22:6n3), docosapentaenoic acid (DPA, C22:5n6), arachidonic acid (ARA, C20:4n6), eicosapentaenoic acid (EPA, C20:5n3) and mixtures thereof.

6. The method according to claim 2 or 3, wherein the PUFA-producing microorganism is selected from filamentous fungi and protists.

7. 4. The method according to claim 2 or 3, wherein the PUFA producing microorganisms are selected from Thraustochytrids, Dinophyceae, Diatoms, Eustigmatophyceae.

8. The method according to any one of claims 1 to 7, wherein the first temperature is between 50°C and 90°C.

9. The method of any one of claims 1 to 8, wherein the second temperature is between 5°C and 40°C.

10. The method according to any one of claims 1 to 9, characterized in that the mechanical separation of oil from the dissolved biomass (b) is a continuous separation by means of a centrifugal plate separator.

11. The method according to any one of claims 1 to 10, characterized in that it comprises a step (c) of refining the crude oil obtained in step (b).

12. 12. The method according to any one of claims 1 to 11, characterized in that it comprises a step (d) of diluting the crude oil obtained in step (b) or the refined oil obtained in step (c).

Images