METHOD OF CULTURING MICROORGANISMS FOR THE ACCUMULATION OF LIPIDS

Abstract

The present invention relates to the industrial cultivation of protists for the production of lipids containing polyunsaturated fatty acids (PUFA), in particular eicosapentaenoic acid (EPA) and arachidonic acid (ARA).

Description

Title of the invention: METHOD FOR CULTURING MICROORGANISMS FOR THE ACCUMULATION OF LIPIDS FIELD OF THE INVENTION

[0001] The present invention relates to the industrial cultivation of protists for the production of lipids containing polyunsaturated fatty acids (PUFA), in particular eicosapentaenoic acid (EPA) and arachidonic acid (ARA). BACKGROUND OF THE INVENTION.

[0002] Protists of the genus Pythium are filamentous microorganisms belonging to the class Oomycetes with similarities to fungi. Pythium has the particularity of synthesizing and accumulating reserve lipids containing Polyunsaturated Fatty Acids (PUFA) such as Eicosapentaenoic Acid (EPA) and Arachidonic Acid (ARA). The interest of this protist lies in the majority accumulation of EPA among other PUFA, in particular TARA.

[0003] Some have therefore considered using Pythium as an industrial strain for the production of EPA-rich oil (EP 1 001 034; WO 2014 / 137894; Lio J, Wang T. (2013); Liang Y, Zhao X, Strait M, Wen Z. (2012); Athalye SK, Garcia RA, Wen Z. (2009); Stinson EE, Kwoczak R, Kurantz MJ. (1991); Gandhi SR, Weete JD. (1991)).

[0004] Pythium is a non-photosynthetic microorganism that grows in an aqueous medium or in the soil, often as a parasite of wild or cultivated plants (cereals and vegetable crops). Pythium is grown heterotrophically, i.e. in the absence of light. This is one of the advantages identified in the state of the art: not having to depend on light (WO 2009 / 143007).

[0005] However, the fat content in strains cultivated by these cultivation methods, less than 30% of the dry matter, remains too low for industrial exploitation.

[0006] The present invention makes it possible to solve this problem with a new cultivation method which makes it possible to increase the fat content to levels of percentages of dry matter compatible with industrial exploitation. BRIEF DESCRIPTION OF THE INVENTION.

[0007] The present invention relates to a method for preparing a biomass of protists of the genus Pythium comprising PUFA-rich lipids, said method comprising (a) culturing protists of the genus Pythium on a culture medium comprising a carbon source, and (b) recovering the biomass from the culture medium, the culture step comprising a lighting phase.

[0008] The invention also relates to a method for preparing PUFA-rich lipid compositions which comprises culturing from a) culturing protists of the genus Pythium on a culture medium comprising a carbon source, (b) recovering biomass from the culture medium and (c) extracting PUFA-rich lipids from the recovered biomass, the culturing step (a) comprising an illumination phase.

[0009] The invention also relates to a process for preparing a pharmaceutical, cosmetic, nutraceutical or food composition comprising a biomass of protists of the genus Pythium comprising lipids rich in PUFA, said process comprising (a) the culture of protists of the genus Pythium on a culture medium comprising a carbon source, (b) the recovery of the biomass from the culture medium and (d) the formulation of a composition by adding the biomass recovered in (b) to usual components of pharmaceutical, cosmetic, nutraceutical or food compositions, the culture step (a) comprising a lighting phase.

[0010] The invention also relates to a process for preparing a pharmaceutical, cosmetic, nutraceutical or food composition comprising a lipid composition rich in PUFA, said process comprising (a) the culture of protists of the genus Pythium on a culture medium comprising a carbon source, (b) the recovery of the biomass from the culture medium, (c) the extraction of the lipids rich in PUFA from the recovered biomass and (d) the formulation of a composition by adding the lipid composition extracted in (c) to usual components of pharmaceutical, cosmetic, nutraceutical or food compositions, the culture step (a) comprising a lighting phase.

[0011] The invention also relates to a biomass of protists of the genus Pythium comprising lipids rich in PUFA, the lipid content being at least 30% relative to the dry matter (% DM), preferably at least 50% DM, more preferably at least 55% DM, in particular at least 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65% DM.

[0012] Protists of the genus Pythium are advantageously protists of the species Pythium irregulare. DETAILED DESCRIPTION OF THE INVENTION.

[0013] i. Definitions

[0014] By "biomass" is meant a set of protist cells of the genus Pythium produced by their culture and separated from the culture medium (also called fermentation juice). The cells may or may not have retained their physical integrity. It is therefore understood that said biomass may comprise a quantity of degraded protist cells ranging from 0% to 100%. By "degraded" is meant that the physical integrity of said microorganism cells may have been altered, such as for example lysed microorganisms, resulting for example from a homogenization process or enzymatic lysis. Once produced, this biomass can be raw, just separated from its culture medium, dried or not, degraded or not.

[0015] A raw biomass extracted from the culture medium can have a humidity level of 70% to 90%, generally 80% to 85%.

[0016] Dried biomass or “dry biomass” has a moisture content of 1% to 10%, generally 2% to 7%.

[0017] By "protists of the genus Pythium" is meant all the protists designated under the class Oomycetes and of the Genus Pythium, producer of EPA, and capable of being cultivated industrially. This also includes strains with improved performances obtained by mutagenesis and selection, or by modification of the genome. This set of designated protists includes in particular the species Pythium insidiuosum, Pythium irregulare, Pythium intermedium, Pythium splendens, Pythium ultimum. According to a preferred alternative of the invention, the protists of the genus Pythium are of the species Pythium irregulare.

[0018] By "industrial", "industrial culture" or "industrially cultivated" is meant a culture of protists in a culture medium appropriate for their growth and the production of PUFA and in a volume appropriate for the production of sufficient quantities to address a market. These industrial cultures are carried out by fermentation in discontinuous mode called "batch", in semi-continuous mode called "fed batch" or in continuous mode. The fermenters have volumes which can range from 1000 L to more than 200 m3.

[0019] By "culture medium" is meant an aqueous composition comprising the nutrients necessary for the growth of protists of the genus Pythium, in particular a source of carbon, a source of nitrogen, a source of phosphorus, but also mineral salts, and / or vitamins, trace elements, etc. well known to those skilled in the art.

[0020] The culture medium may be a chemically defined medium, of known and reproducible composition, in which the content of each element is known and not comprising rich or complex organic and / or mineral matter.

[0021] The culture medium may alternatively comprise components of variable composition, such as rich or complex organic and / or mineral materials.

[0022] By rich or complex organic matter is meant unpurified organic matter, in the form of mixtures for which the exact composition and concentrations of the various components of the mixture are not known exactly, not controlled, and may exhibit significant variability from one batch to another. As an example of rich or complex organic matter, we can cite yeast extracts or peptones which are products of a protein hydrolysis reaction or even infusions of plants such as potatoes or rich mineral materials such as marine mineral salts or other complex growth agents, not having a fixed concentration of each of their components.

[0023] The composition of the culture medium can vary over time with the consumption of nutrients by the strains of protists of the genus Pythium. Depending on the chosen culture method, it is possible to supplement the culture medium during the culture by feeding it with one or more complementary culture media which contain all or part of the nutrients present in the initial culture medium.

[0024] By "carbon source" is meant more particularly a complex carbon source which is not CO2, in particular chosen from sugars, organic acids or polyols, such as glucose, cellulose derivatives, lactate, starch, lactose, sucrose, acetate, glycerol, fructose, xylose, any product or co-product rich in one or more of the above-mentioned compounds and mixtures thereof.

[0025] "PUFA", abbreviation for "Poly Unsaturated Fatty Acid" or Poly Unsaturated Fatty Acids, are polyunsaturated fatty acids comprising at least 16 carbon atoms and at least 2 unsaturations, in particular fatty acids identified by the signs co3 and co6, such as α-linolenic acid (ALA or C18:3n3), β-linolenic acid (AGA or (C18:3n3), arachidonic acid (ARA or C20:4n6), eicosapentaenoic acid (EPA or C20:5n3), docosahexaenoic acid (DHA or C22:6n3) or docosapentaenoic acid (DPA or C22:5n6).

[0026] By "PUFA-rich lipids" is meant a PUFA-rich oil comprising at least 10% of PUFAs chosen from EPA and TARA relative to the total mass of lipids, preferably at least 15%. The total mass of lipids represents at least 50% of the dry biomass, advantageously at least 60%, more advantageously at least 65%,

[0027] The PUFA-rich 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 and to the oil extracted from this biomass, whether crude or purified.

[0028] A so-called "crude" oil is an oil extracted from biomass after separation of the lipids from the aqueous phase and the insolubles, in particular the proteins. Methods for extracting lipids from biomass are well known to those skilled in the art, described in particular WO 2020 / 053375, WO 2001 / 053512, WO 2011 / 153246, US 2014 / 350222, WO 2015 / 095694, WO 01 / 53512, WO 2010 / 096002.

[0029] A so-called "refined" oil is a purified oil obtained after purification of the crude oil, in particular refined according to its intended use. The methods for refining crude lipid compositions are well known to those skilled in the art, described in particular by Manjula et al. (2006), GB 2,031,290, US 5,310,487 or US 4,971,660.

[0030] By "modified oil" is meant a crude or refined oil whose fatty acid composition has been modified. This modification may include a concentration of PUFA, by elimination of other saturated or unsaturated fatty acids, or even a dilution by addition of oils with a different lipid profile.

[0031] By "lipid composition" is meant a mixture of lipids comprising at least one PUFA-rich oil according to the invention, extracted from biomass, raw, refined and / or modified.

[0032] Unless otherwise indicated, percentages are given by mass.

[0033] ii. Biomass cultivation

[0034] The present invention relates to a method for preparing a biomass of protists of the genus Pythium comprising PUFA-rich lipids, said method comprising (a) culturing protists of the genus Pythium on a culture medium comprising a carbon source, and (b) recovering the biomass from the culture medium, the culture step comprising an illumination phase.

[0035] The cultivation of protists of the genus Pythium in heterotrophy on a culture medium comprising a carbon source is well known to those skilled in the art. These cultures can be in discontinuous mode called "batch", in semi-continuous mode called "fed batch" or in continuous mode.

[0036] The method according to the invention is advantageously implemented for industrial production of biomass.

[0037] Whether in batch, semi-continuous or continuous mode, the cultivation step (a) consists of producing a fermentation must comprising the fermentation juice and the biomass, with a density of at least 20 g / L of dry matter. Advantageously, the cultivation will be carried out so as to achieve a density of at least 30 g / L, advantageously at least 40 g / L.

[0038] Once the desired culture density has been reached, the biomass is recovered from the culture medium, separated from the fermentation juice.

[0039] The cultivation of protists of the genus Pythium is well known to those skilled in the art who will be able to determine the conditions for implementing these cultures in aerobic mode to obtain the desired density. In particular, the cultivation methods described in Stinson et al. (1991), US2014256973, which the person skilled in the art can adapt to an industrial culture.

[0040] The inocula used for the cultivation of Pythium can be obtained by two methods.

[0041] The first consists of carrying out a culture on agar favorable to the production of spores, for example PDA medium (Potato Dextrose Agar) or Czapek Dox agar. After a suitable growth time, the spores are harvested in a liquid medium according to a method known to those skilled in the art. For example, water or a suitable saline medium is used to flood the aerial part of the mycelium which has developed on the agar. The liquid loaded with spores is then recovered and the spores counted.

[0042] Inoculation takes place in a liquid culture in a baffled Erlenmeyer flask containing a medium suitable for growth such as PDB (Potato Dextrose Broth) or Czapek Dox. From 100 to 10,000 spores per milliliter of medium are added to the culture medium. Inoculation with a suitable number of spores limits growth in the form of pellets, which is a characteristic of the growth of filamentous microorganisms.

[0043] A second method consists of recovering the wet biomass from a culture in a flask and then homogenizing this biomass. This biomass can be in the form of balls. Homogenization is carried out in a blender-type device or other homogenizer. 5 to 15% of this homogenate is used to inoculate a culture in a baffled Erlenmeyer flask and in a medium suitable for growth such as PDB (Potato Dextrose Broth) or Czapek Dox. Homogenization limits the formation of balls during the culture thus inoculated.

[0044] Growth is carried out in a shaking incubator at a temperature generally ranging from 20 to 30°C. The orbital shaking is set between 100 and 200 revolutions per minute. After a growth time of the order of 5 to 15 days, generally 7 days, the biomass can be recovered to inoculate another culture of a larger volume, such as a fermenter, or to be harvested or analyzed.

[0045] The invention consists of illuminating the culture during all or part of the culture stage (mixotrophy).

[0046] Usually, the culture of Pythium is carried out in heterotrophy, that is to say without a light source, since this microorganism is not photosynthetic and therefore does not need it for its growth. The inventors unexpectedly found that lighting the culture made it possible to increase the lipid content in the cells of protists of the genus Pythium compared to a heterotrophic culture described in the state of the art.

[0047] The wavelengths suitable for illuminating Pythium cultures according to the invention are preferably in a spectrum which goes from UV to near infrared via the visible, generally between 300 nm and 750 nm. This may be so-called "white" light whose radiation spectrum generally ranges from 400 to 750 nm, Ultra-Violet (UV) radiation whose radiation spectrum is between 300 and 430 nm, so-called "blue" light whose radiation spectrum generally ranges from 430 to 500 nm, so-called "green" light whose radiation spectrum generally ranges from 500 to 570 nm, so-called "yellow" light whose radiation spectrum generally ranges from 570 to 590 nm, or so-called "red" light whose radiation spectrum generally ranges from 590 to 750 nm.

[0048] The light intensity may be between 20 and 5000 pmoles of photons / m2 / sec, preferably between 50 and 3000 pmoles of photons / m2 / sec, more preferably between 50 and 2000 pmoles of photons / m2 / sec. In particular, the light intensity may be at least 200 pmoles of photons / m2 / sec, from 200 to 1000 pmoles of photons / m2 / sec, in particular approximately 500 pmoles of photons / m2 / sec.

[0049] The light intensity used for so-called “white” light illumination is advantageously between 100 and 1000 pmoles of photons / m2 / sec.

[0050] The light intensity used for UV lighting is advantageously between 50 and 500 pmoles of photons / m2 / sec.

[0051] The light intensity used for blue light illumination is advantageously between 50 and 500 pmoles of photons / m2 / sec.

[0052] The light intensity used for green light illumination is advantageously between 50 and 500 pmoles of photons / m2 / sec.

[0053] The light intensity used for yellow light illumination is advantageously between 100 and 1000 pmoles of photons / m2 / sec.

[0054] The light intensity used for red light illumination is advantageously between 50 and 1000 pmoles of photons / m2 / sec.

[0055] The composition of the light spectrum will depend in particular on the lamps used for lighting the crops. These are in particular fluorescent or neon tube type lamps, sodium lamp type or even LED type lamps, preferably LED type lamps.

[0056] For so-called "white" light illumination, LED type lamps will advantageously be used, the emission spectrum of which is between 400 and 750 nm. These white LED type sources generally have a main emission peak in the blue at around 470 nm and a more diffuse secondary emission between 500 and 700 nm.

[0057] For lighting with so-called “UV” light, LED type lamps will advantageously be used, the emission spectrum of which is between 380 and 400 nm.

[0058] For lighting in so-called “blue” light, LED type lamps will advantageously be used, the emission spectrum of which is between 450 and 500 nm.

[0059] For so-called “green” light, LED type lamps will advantageously be used, with an emission spectrum between 500 and 570 nm.

[0060] For lighting in so-called “yellow” light, LED type lamps will advantageously be used, the emission spectrum of which is between 570 and 590 nm.

[0061] For lighting in so-called “red” light, LED type lamps will advantageously be used, with an emission spectrum between 590 and 750 nm.

[0062] The lighting can be implemented throughout the duration of the cultivation step (a), or partially. Those skilled in the art will know when to start the lighting and when to stop it at the end of the cultivation before the biomass recovery step (b).

[0063] The duration of the lighting can range from 10 to 100% of the time of implementation of step (a) of culture, preferably between 30 and 100%, more preferably between 50 and 100%.

[0064] Partial lighting may be provided during a phase conducive to lipid accumulation, generally from the middle or end of the exponential growth phase.

[0065] The lighting may be continuous, or discontinuous with alternating periods of lighting and periods of darkness. The successive phases of lighting may be between 1 second and 10 minutes, for example between 10 seconds and 2 minutes, or between 20 seconds and 1 minute. They are spaced by phases of darkness of a duration equal to or different from the duration of each lighting phase.

[0066] Industrial mixotrophic culture devices allowing lighting to be varied, both in wavelength, intensity and duration, are for example described in patent applications WO 2009 / 069967, US 2010 / 005711, WO 2014 / 174182 or WO 2019 / 034792.

[0067] When the light intensity to be delivered is chosen at a high value, in particular beyond 500 pmoles of photons / m2 / sec, the electrical consumption and the heat release can be limited by providing the light in the form of a high-frequency flash, generally between 0.5 and 150 kHz, advantageously between 1 and 100 kHz, which results in an alternation of illuminated phases and dark phases in a period of between 0.001 and 0.00001 seconds. The duration of the illuminated phase can be from 1 to 90% of each period, depending on the desired reductions in electrical consumption and heat release. The use of electrical equipment and the configuration thereof are known to those skilled in the art.

[0068] According to a preferred embodiment of the invention, the lighting will be provided by a LED type light source delivering red lighting whose emission spectrum will be between 600 and 750 nm and whose duration will be between 50 and 100% of the culture step.

[0069] iii. Recovery of biomass

[0070] Step (b) of recovering the biomass from the culture medium comprises separating the biomass from the fermentation juice.

[0071] The recovery methods are well known to those skilled in the art, in particular by centrifugation (plate centrifuge or sedicant), or by filtration (plate filter, press filter, ceramic or organic tangential filtration).

[0072] The recovered raw biomass may be washed to remove some solubles (e.g., by filtration to remove the fermentation medium and washing with water).

[0073] The raw or washed biomass can also be dried by usual methods, in particular by atomization or freeze-drying.

[0074] It can also be treated by adding components useful for its preservation and storage, such as for example antioxidant agents, before or after drying, or sugars to avoid spontaneous heating of the biomass.

[0075] The invention also relates to a biomass of protists of the genus Pythium comprising lipids rich in PUFA, the lipid content being at least 30% relative to the dry matter (% DM), preferably at least 50% DM, more preferably at least 55% DM, in particular at least 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65% DM, up to 75% DM.

[0076] PUFAs are essentially composed of a mixture of ARA and EPA. The ARA / EPA weight ratio is advantageously 0.5 to 1, more particularly 0.6 to 0.9, in particular approximately 0.7.

[0077] The PUFA (ARA + EPA) content is at least 10% of the sum of the fatty acids, advantageously at least 15%, in particular 15 to 40%. According to a particular embodiment, the total content of ARA and EPA is at least 20%.

[0078] The ARA content is at least 5%, preferably at least 6%, more preferably 7.5% or more than 7.5%, in particular at least 7.7%, at least 8% or at least 9%.

[0079] The EPA content is preferably at least 12% of the sum of the fatty acids, more preferably at least 13%, in particular at least 14%, at least 15%, at least 16% or at least 17%.

[0080] The fatty acid content in the oils according to the invention, crude or refined, is advantageously as follows:

[0081] myristic acid (C14:0) 5% to 10%

[0082] palmitic acid (C16:0) 10% to 20%

[0083] palmitoleic acid (C16:1 n-7) 6% to 10%

[0084] oleic acid (Cl8:1 n-9c) 15% to 25%

[0085] linoleic acid (C18:2 n-6c) 10% to 25%

[0086] arachidonic acid (C20:4 n-6) 5% to 15%

[0087] eicosapentaemoic acid (C20:5 n-3) 7% to 20%

[0088] More advantageously, the fatty acid content in the oils according to the invention, crude or refined, is as follows:

[0089] myristic acid (C14:0) 6% to 7%

[0090] palmitic acid (C16:0) 11% to 16%

[0091] palmitoleic acid (C16:1 n-7) 6% to 8.5%

[0092] oleic acid (Cl8:1 n-9c) 16% to 25%

[0093] linoleic acid (C18:2 n-6c) 13% to 22%

[0094] arachidonic acid (C20:4 n-6) 6% to 10%

[0095] eicosapentaemoic acid (C20:5 n-3) 10% to 20%

[0096] In particular, the content of the main fatty acids is of the order of 9% for myristic acid (C14:0); 12% for palmitic acid (C16:0); 7% for palmitoleic acid (C16:1 n-7); 17% for oleic acid (C18:1 n-9c); 20% for linoleic acid (C18:2 n-6c); 9% for arachidonic acid or ARA (C20:4 n-6) and 18% for eicosapentaemoic acid or EPA (C20:5 n-3).

[0097] According to a particular embodiment, the biomass according to the invention has been “stabilized” for its conservation, storage and / or transport.

[0098] This may involve the addition of stabilizing components not found associated with protist cells in nature, such as sugars (WO 2020 / 036814), antioxidants such as tocopherol, rosemary extract or ascorbic acid.

[0099] According to a particular embodiment, the biomass is no longer able to develop on a culture medium, the vital functions of the cells being altered. This is particularly the case for degraded biomass, in particular degraded and dry biomass.

[0100] iv. Lipid extraction

[0101] The invention also relates to a method for preparing PUFA-rich lipid compositions which comprises culturing from a) culturing protists of the genus Pythium on a culture medium comprising a carbon source, (b) recovering biomass from the culture medium and (c) extracting PUFA-rich lipids from the recovered biomass, the culturing step (a) comprising an illumination phase.

[0102] Several industrial methods for extracting PUFA-rich lipids from microalgal biomass cultivated to produce said oils are described in the literature and known to those skilled in the art. Lipid extraction includes lysis of the biomass cells to release the PUFA-rich oils they contain, then the separation of lipids from solid fractions and water-soluble fractions.

[0103] Lysis can be mechanical (pressing or grinding) or enzymatic with proteases or cellulases.

[0104] Mechanical lysis methods are well known, in particular by ball mill, mixer-disperser, high pressure homogenizer, pin mill or impact mill, ultrasound, pulsed electric fields. As devices for the implementation of these mechanical lysis methods, we will mention in particular (name of the manufacturer in parentheses) for the ball mill: Discus-1000 (Netzsch); ECM-AP60 (WAB); for the high pressure homogenizer: Ariete (GEA); for the mixer-disperser: 700-X (Silverson), for the pin mill: Contraplex (Hosakawa); for the impact mill: Condux (Netzsch).

[0105] The enzymes that can be used are known, in particular described in WO2015 / 095688, WO2011 / 153246, US6750048 and WO2015 / 095694, in particular proteases or cellulases such as the enzymes marketed by the company Novozyme under the names Alcalase 2.5 L, Alcalase 2.4 L, Alcalase 3.0 T, Novozym 37071, Flavourzyme 1000 L, Novozym FM 2.4 L, Protamex, Viscozyme. The conditions of use 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, enzymatic lysis is carried out in an oxygen-poor atmosphere. Generally, the oxygen concentration is less than 1% by mass.

[0106] Mention will be made in particular of the lysis methods described in application WO 2020 / 053375, WO 2001 / 053512.

[0107] The extraction of oils from lysed biomass can be carried out by various known methods, such as the addition of sodium in the form of sodium sulfate or sodium chloride (WO 2011 / 153246), solvent extraction (US 2014 / 350222) and / or high temperatures for several hours (WO 2015 / 095694), or by numerous other methods described in the literature (WO 2019 / 219396, WO 2019 / 219443, WO 2019 / 121752, WO 2018 / 122057, WO 2018 / 013670, WO 2018 / 011286, WO 2018 / 011275, WO 2018 / 013670, WO 2018 / 011286, WO 2018 / 011275, WO 2015 / 095696, WO 2011 / 153246, WO 2002 / 010423).

[0108] Preferably, the extraction of oils from lysed biomass is carried out by mechanical separation, also well known to those skilled in the art, such as gravity separation, in particular by centrifugation as described in patent application WO 01 / 53512. Continuous separation may also be used, in particular by a plate centrifugal separator. Such separators are known for continuously extracting oils from complex media comprising solid residues and water, as described in patent application WO 2010 / 096002, notably marketed by the companies Alfa Laval, Flottweg or GEA Westfalia, in particular. This continuous separation step is preferred in the process used to obtain the oil according to the invention.

[0109] In certain cases, the lysis and extraction steps are simultaneous, under mechanical action, generally by one or more centrifugations (WO 2019 / 032880) in particular with the centrifugation devices described above.

[0110] Extraction of oil from biomass may favor the extraction of these PUFAs compared to lower molecular weight saturated fatty acids. Advantageously, this concentration does not substantially modify the intrinsic properties of the oil contained in the biomass, in particular the triglyceride content. Preferably, the oil according to the invention is an oil which has not undergone substantial modifications of its fatty acid content by the addition of PUFAs, for example in the form of esters, by concentration and / or by the elimination of saturated fatty acids such as palmitic acid.

[0111] The oil obtained is generally an oil called crude oil, which can be used as is or be refined, in particular to facilitate its conservation, by preventing it from going rancid, or to modify its color or its odor so as to make it more acceptable to a consumer. These purification and refining steps are well known to those skilled in the art, described in patent applications (WO 2002 / 010322, WO 2017 / 035403), in particular steps of degumming, neutralization of free fatty acids, bleaching 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 purified oil.

[0112] The process according to the invention may also comprise a step of modifying the oils obtained previously and described above, whether they are crude, purified or refined.

[0113] Certain processes of the state of the art have a so-called “winterization” step implemented on crude or purified oils, in particular to eliminate saturated fatty acids, with the effect of increasing the PUFA content (WO 02 / 10322). The oils extracted according to the invention, crude or refined, do not a priori require “winterization” to be exploited. However, those skilled in the art may choose to add such a “winterization” step if they find any commercial advantage for their final product.

[0114] Some processes include modification of the oils recovered from the biomass, for example to modify their composition in certain saturated fatty acids. or unsaturated, in particular to promote the concentration of PUFA. Such methods known to those skilled in the art include in particular enzymatic treatments by enzymes such as lipases (CN 105349587, WO 2019 / 219904, WO 2019 / 219903).

[0115] Other components may be added to the crude, purified or refined oils according to the invention, such as antioxidant agents, in particular those described in applications WO 2019 / 185942, WO 2019 / 185940, WO 2019 / 185939, WO 2019 / 185910, WO 2019 / 185894, WO 2019 / 185889 and WO 2019 / 185888.

[0116] Crude or refined oils, or oils of modified composition can also be diluted for their subsequent use. The oils used to dilute the PUFA-rich oil obtained by the process according to the invention are generally and preferably vegetable oils suitable for human or animal food consumption. Mention will be made in particular of sunflower, rapeseed, soybean, walnut, sesame, hemp, hazelnut, argan, olive, linseed, or any other oil suitable for food use. The added oil can also be an oil comprising other PUFAs, in particular DHA, in particular other oils of microbial origin or even fish oils.

[0117] The process according to the invention may further comprise such a step of diluting a crude or refined oil obtained previously.

[0118] The invention also relates to an oil rich in PUFA obtained by the process according to the invention, crude or refined or capable of being obtained by the process according to the invention.

[0119] The composition of the oil according to the invention, in particular the content of PUFA and other fatty acids, is given above for the composition of the oil contained in the biomass.

[0120] v. Compositions

[0121] The invention also relates to a process for preparing a pharmaceutical, cosmetic, nutraceutical or food composition comprising a biomass of protists of the genus Pythium comprising lipids rich in PUFA, said process comprising (a) the culture of protists of the genus Pythium on a culture medium comprising a carbon source, (b) the recovery of the biomass from the culture medium and (d) the formulation of a composition by adding the biomass recovered in (b) to usual components of pharmaceutical, cosmetic, nutraceutical or food compositions, the culture step (a) comprising a lighting phase.

[0122] The invention also relates to a process for preparing a pharmaceutical, cosmetic, nutraceutical or food composition comprising a lipid composition rich in PUFA, said process comprising (a) the culture of protists of the genus Pythium on a culture medium comprising a carbon source, (b) recovering the biomass from the culture medium, (c) extracting the PUFA-rich lipids from the recovered biomass and (d) formulating a composition by adding the lipid composition extracted in (c) to usual components of pharmaceutical, cosmetic, nutraceutical or food compositions, the culture step (a) comprising a lighting phase.

[0123] The invention also relates to a composition which comprises a biomass or an oil rich in PUFA capable of being obtained by the process according to the invention as described above.

[0124] A composition according to the invention may 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 preservation and use, including foods and pharmaceutical, cosmetic and industrial compositions. An excipient may be described as a "pharmaceutically acceptable" excipient when it is added to a pharmaceutical composition whose properties are known to the pharmacopoeia for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reaction or other complications.Various excipients may be used such as an organic or mineral base, an organic or mineral acid, a pH buffer, a stabilizer, an antioxidant, an adhesion promoter, a separating agent, a coating agent, an outer phase component, a controlled release component, a surfactant, a humectant, a filler, an emollient or combinations thereof.

[0125] Depending on their destination, the compositions according to the invention are in particular pharmaceutical, cosmetic, nutraceutical or food compositions.

[0126] Foods are intended for both humans and animals and include solid, pasty or liquid compositions. Particular mention may be made of common foods, liquid products, including milks, drinks, therapeutic drinks and nutritional drinks, functional foods, supplements, neutraceuticals, infant formulas, including formulas for premature infants, foods for pregnant or breastfeeding women, foods for adults, geriatric foods and animal foods.

[0127] The PUFA-rich oil obtained by the process according to the invention, whether crude or refined, or the biomass containing it, can be used directly as or added as an additive in an oil, a spread, another fatty ingredient, a beverage, 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 capsule or tablet form), vitamin supplements, food supplements, powders to be diluted for drinks, such as energy drinks or milk powders for infant formulas, finished or semi-finished powdered food products, etc., according to the uses known to those skilled in the art.

[0128] Animal feeds are also known to those skilled in the art. They are in particular intended for farm animals, such as cows, pigs, chickens, sheep, goats or in fish farming for crustaceans or farmed fish.

[0129] Pharmaceutical compositions comprising a PUFA-rich oil are also known to those skilled in the art, the oil being used alone or in combination with other medicaments.

[0130] The PUFA-rich oil obtained by the process according to the invention, crude or refined, 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 oral administration.

[0131] The invention also relates to the use of an oil rich in PUFA obtained by the process according to the invention, crude, refined or diluted, for human or animal food, in particular for feeding newborns, children, or pregnant or breastfeeding women.

[0132] Such uses are well known to those skilled in the art, notably described in patent application WO 2010 / 107415 and on the DSM company website.

[0133] EXAMPLES

[0134] Example 1

[0135] The Pythium irregulare strain from the NB RC collection No. 30346 is cultivated in a 250 mL baffled Erlenmeyer flask in 50 mL of PDB (Potato Dextrose Broth) culture medium composed of 200 g of potato infusion and 20 g of dextrose. The wet biomass is recovered by centrifugation at 10,000 g for 5 minutes and then homogenized by vortexing in the presence of glass beads with a diameter of 1 mm and a quantity representing a volume of 1 to 2 mL. 5 mL of this homogenate is used to inoculate a 250 mL flask containing 45 mL of PDB medium. The flasks are incubated at a temperature of 26°C and with magnetic stirring by a magnetic bar at 150 rpm. Light is provided from below via a device comprising several LEDs of the same type, continuously throughout the entire cultivation stage. Each of the magnetic stirring cultivation stations can be equipped with a different type of lighting device.The lighting intensity is set at 200 pmoles of photons / m2 / sec. A heterotrophic growth control is carried out under the same experimental conditions except for the absence of light. The cultures are incubated under these conditions for 7 days. The biomass is harvested by centrifugation at 10,000 g for 5 minutes and the supernatant discarded. After washing the biomass with distilled water, it is lyophilized. The lipid content of the biomass is analyzed by GC-FID after a transesterification step on an aliquot of approximately 2 mg of this freeze-dried biomass.

[0136] The results of the lipid analysis are presented in Table 1. The percentages of fatty acids are given relative to the total mass of fatty acid.

[0137] [Tables 1] UV Indicator 385 nm Blue 455 nm Green 521 nm Yellow Red 593 nm Red 660 nm White MG / MS% 23.6 61.6 60.2 43.3 52.3 36.7 68.10 30.3 C14:0 6.1 7.1 7.0 6.7 6.3 6.9 8.5 6.6 C16:0 14.6 14.3 13.0 14.3 16.1 13.2 15.2 13.4 C16:1 5.7 8.2 7.7 7.0 8.6 7.9 7.1 6.4 C18:1 15.9 22.3 19.9 21.2 23.7 21.0 23.2 19.0 C18:2 18.2 15.2 14.7 13.5 14.8 13.5 15.3 15.0 ARA 7.0 6.7 7.5 6.4 5.5 6.4 7.7 7.8 EPA 11.7 10.8 11.8 13.8 8.1 12.5 10.9 12.7

[0138] Depending on the light used, with the cultivation method according to the invention, compared to a heterotrophic cultivation (without light) of the state of the art, an ARA production in relation to the dry matter multiplied by a factor of at least 1.5, on average by a factor of approximately 2.4, up to a factor of approximately 3.7 is observed.

[0139] The production of EPA relative to dry matter is multiplied by a factor of at least 2.5, on average by a factor of about 3.6, up to a factor of about 4.7.

[0140] The total production of ARA and EPA relative to dry matter is multiplied by a factor of at least 2.1, on average by a factor of about 3.1, up to a factor of about 4.2.

[0141] Example 2

[0142] The Pythium irregulare strain from the NB RC collection No. 100109 is cultivated in a 250 mL baffled Erlenmeyer flask in 50 mL of PDB (Potato Dextrose Broth) culture medium composed of 200 g of potato infusion and 20 g of dextrose. The wet biomass is recovered by centrifugation at 10,000 g for 5 minutes and then homogenized by vortexing in the presence of glass beads with a diameter of approximately 1 mm and a quantity representing a volume of 1 to 2 mL. 5 mL of this homogenate is used to inoculate a 250 mL flask containing 45 mL of PDB medium. The flasks are incubated at a temperature of 26°C and with magnetic stirring by a magnetic bar at 150 rpm. Light is provided from below via a device comprising several LEDs with an emission peak at 660 nm and this continuously throughout the duration of the culture stage. The lighting intensity is set at 200, 500 or 2000 pmoles of photons / m2 / sec. A heterotrophic growth control is carried out under the same experimental conditions except for the absence of light. The cultures are incubated under these conditions for 7 days. The biomass is harvested after 7 days by centrifugation at 10,000 g for 5 minutes and the supernatant discarded. After washing the biomass with distilled water, it is freeze-dried. The lipid content of the biomass is analyzed by GC-FID after a transesterification step on an aliquot of approximately 2 mg of this freeze-dried biomass.

[0143] The results of the lipid analysis are presented in Table 2.

[0144] The percentages of fatty acids are given relative to the total mass of fatty acid.

[0145] [Tables2] Control 200 g E 500 g E 2000 g E MG / MS% 19.1 61.5 73.3 66.9 C14:0 9.5 8.7 8.8 9.2 C16:0 18.1 12.8 12.5 11.3 C16:1 12.0 7.2 8.1 6.7 C18:1 20.8 19.6 19.3 16.9 C18:2 18.6 19.4 18.2 21.0 ARA 7.5 9.2 9.3 9.9 EPA 8.2 14.7 13.7 17.6

[0146] Depending on the light intensity, with the cultivation method according to the invention, compared to a heterotrophic cultivation (without light) of the state of the art, an ARA production is observed relative to the dry matter multiplied by a factor of at least 3.4, on average by a factor of approximately 3.9, up to a factor of approximately 4.

[0147] The production of EPA relative to dry matter is multiplied by a factor of at least 3.3, on average by a factor of about 3.7, up to a factor of about 4.3.

[0148] The total production of ARA and EPA relative to dry matter is multiplied by a factor of at least 3.3, on average by a factor of about 3.8, up to a factor of about 4.2.

[0149] Example 3

[0150] The Pythium irregulare strain from NBRC collection No. 100109 is grown in a 250 mL baffled Erlenmeyer flask in 50 mL of PDB (Potato Dextrose Broth) culture medium composed of 200 g of potato infusion and 20 g of dextrose. The wet biomass is recovered by centrifugation at 10,000 g for 5 minutes and then homogenized by vortexing in the presence of glass beads with a diameter of approximately 1 mm and a quantity representing a volume of 1 to 2 mL. 5 mL of this homogenate is used to inoculate a 250 mL flask containing 45 mL of PDB medium. The flasks are incubated at a temperature of 26°C in an incubator with shaking set at 140 rpm. Light is provided by a series of daylight fluorescent tubes at 6000°K. The lighting intensity is around 50 pmoles of photons / m2 / sec. The cultures are incubated under these conditions for 7 days.The biomass is harvested after 7 days by centrifugation at 10,000 g for 5 minutes and the supernatant discarded. After washing the biomass with distilled water, it is freeze-dried. The lipid content of the biomass is analyzed by GC-FID after a transesterification step on an aliquot of approximately 2 mg of this freeze-dried biomass.

[0151] The results of the lipid analysis are presented in Table 3.

[0152] The percentages of fatty acids are given relative to the total mass of fatty acid.

[0153] [Tables3] AB AVERAGE HETEROTROPHY MG / MS% 45.8 47.1 21.3 C14:0 9.0 8.6 7.8 C16:0 16.2 15.5 16.4 C16:1 16.9 14.7 8.9 C18:1 21.4 20.1 18.8 C18:2 15.8 16.5 18.4 ARA 4.1 8.8 7.2 EPA 5.8 5.7 10.0

[0154] With the cultivation method according to the invention, a clear increase in the lipid content in the dry biomass is observed compared to the generally observed heterotrophic cultures (without light). Compared to the dry matter, an increase of more than 60% in PUFA is observed compared to the values ​​generally observed in heterotrophic cultures. REFERENCES

[0155] Athalye SK, Garcia RA, Wen Z., Use of biodiesel-derived crude glycerol for producing eicosapentaenoic acid (EPA) by the fungus Pythium irregulare, J Agric Food Chem. 2009 Apr 8;57(7):2739-44. doi:10.1021 / jf803922w.

[0156] Gandhi SR, Weete JD., Production of the polyunsaturated fatty acids arachidonic acid and eicosapentaenoic acid by the fungus Pythium ultimum, J Gen Microbiol. 1991 Aug;137(8):1825-30.

[0157] Liang Y, Zhao X, Strait M, Wen Z., Use of dry-milling derived thin stillage for producing eicosapentaenoic acid (EPA) by the fungus Pythium irregulare, Bioresour Technol. 2012 May;l 11:404-9. doi: 10.1016 / j.biortech.2012.02.035.

[0158] Ren, Liang; Zhou, Pengpeng; Zhu, Yuanmin; Zhang, Ruijiao; Yu, Lo — Improved eicosapentaenoic acid prod; Applied Microbiology and Biotechnology Volume 101 issue 9 2017 [doi 10.1007_s00253-016-8044-0]

[0159] Lio J, Wang T., Pythium irregulare fermentation to produce arachidonic acid (ARA) and eicosapentaenoic acid (EPA) using soybean processing co-products as substrates, Appl Biochem Biotechnol. 2013 Jan;169(2):595-611. doi: 10.1007 / sl2010-012-0032-y-

[0160] Manjula S, Subramanian R. Membrane technology in degumming, dewaxing, deacidifying, and decolorizing edible oils. Crit Rev Food Sci Nutr. 2006;46(7):569-92. Review. PubMed PMID: 16954065.

[0161] Stinson EE, Kwoczak R, Kurantz MJ., Effect of cultural conditions on production of eicosapentaenoic acid by Pythium irregulare. Journal of Industrial Microbiology & Biotechnology Volume 8 issue 3 1991 [doi 10.1007_bf01575850]

[0162] Wu L, Roe CL, Wen Z., The safety assessment of Pythium irregulare as a producer of biomass and eicosapentaenoic acid for use in dietary suppléments and food ingrédients, Appl Microbiol Biotechnol. 2013 Sep;97(17):7579-85. doi: 10.1007 / s00253-013-5114-

[0163] CN 105349587, GB 2 031 290, US 4,971,660, US 5,310,487, US 6,750,048, US 2010 / 005711, US 2014 / 256973, US 2014 / 350222, WO 2001 / 053512, WO 2002 / 010423, WO 2002 / 10322, WO 2009 / 069967, WO 2009 / 143007, WO 2010 / 096002, WO 2010 / 107415, WO2011 / 153246, WO 2014 / 174182, WHERE 2015 / 095688, WO2015 / 095694, WO 2015 / 095696, WO 2017 / 035403, WO 2018 / 011275, WO 2018 / 011286, WO 2018 / 013670, WO 2018 / 122057, WO 2019 / 034792, WO 2019 / 032880, WO 2019 / 121752, WO 2019 / 185888, WO 2019 / 185889, WO 2019 / 185894, WO 2019 / 185910, WO 2019 / 185939, WO 2019 / 185940, WO 2019 / 185942, WO 2019 / 219903, WO 2019 / 219904, WO 2019 / 219396, 2019 / 219443, WO 2020 / 053375

Claims

Claims

1. A method for preparing a biomass of protists of the genus Pythium comprising PUFA-rich lipids, said method comprising (a) culturing protists of the genus Pythium on a culture medium comprising a carbon source, and (b) recovering the biomass from the culture medium, the culture step comprising an illumination phase.

2. Method according to claim 1, characterized in that the protists of the genus Pythium are protists of the species Pythium irregulare.

3. Method according to one of claims 1 or 2, characterized in that the lighting phase is implemented during all or part of the cultivation step (a).

4. Method according to claim 3, characterized in that the duration of the lighting ranges from 10 to 100% of the time of implementation of step (a) of culture, preferably between 50 and 100%.

5. Method according to one of claims 1 to 4, characterized in that the lighting intensity is between 50 and 2000 pmoles of photons / m2 / sec, preferably between 100 and 1000 pmoles of photons / m2 / sec.

6. Method according to one of claims 1 to 5, characterized in that the lighting phase is implemented by lighting with white light, UV light, blue light, green light, yellow light or red light.

7. Method according to one of claims 1 to 6, characterized in that it comprises a step (c) of extracting the lipids rich in PUFA from the biomass recovered in (b).

8. Process for the preparation of a pharmaceutical, cosmetic, nutraceutical or food composition comprising a biomass of protists of the genus Pythium comprising lipids rich in PUFA, characterized in that it comprises the steps: (a) the culture of protists of the genus Pythium, the culture step comprising a lighting phase, (b) the recovery of the biomass from the culture medium according to one of claims 1 to 6, and (d) the formulation of a pharmaceutical, cosmetic, nutraceutical or food composition by adding the biomass recovered in step (b) to usual components of pharmaceutical, cosmetic, nutraceutical or food compositions.

9. Process for the preparation of a pharmaceutical, cosmetic, nutraceutical or food composition comprising PUFA-rich lipids, characterized in that it comprises the steps: (a) the culture of protists of the genus Pythium, the culture step comprising a lighting phase, (b) the recovery of the biomass from the culture medium according to one of claims 1 to 6, (c) the extraction of the PUFA-rich lipids from the biomass recovered in (b), and (d) the formulation of a pharmaceutical, cosmetic, nutraceutical or food composition by adding the lipid composition extracted in (c) to usual components of pharmaceutical, cosmetic, nutraceutical or food compositions.

10. Method according to one of claims 1 to 9, characterized in that the biomass of protists of the genus Pythium comprising lipids rich in PUFA obtained has a lipid content of at least 30% by weight relative to the total weight of dry matter (% DM) and at least 15% by weight of PUFA chosen from EPA and TARA relative to the total weight of fatty acids.

11. Process according to one of claims 1 to 10, characterized in that the biomass oil obtained has a PUFA content of at least 15% by weight relative to the total weight of fatty acids and chosen from EPA and TARA and whose triglyceride content is greater than 90% by weight relative to the total weight of fat.

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