Method for separating lipids from lipid-containing biomass

Abstract

The present invention relates to a method for separating lipids containing polyunsaturated fatty acids from lipid-containing biomass.

Description

Technical Field

[0001] The present invention relates to a method for separating lipids containing polyunsaturated fatty acids from lipid-containing biomass. Background Art

[0002] Lipids containing PUFAs (polyunsaturated fatty acids) are highly regarded in the feed, food, and pharmaceutical industries. Due to overfishing, there is a high demand for alternative sources of lipids containing PUFAs other than fish oil. In addition, certain yeast and algal strains, especially microalgal cells such as those of the order Thraustochytriales, are very good sources of lipids containing PUFAs.

[0003] Previously, the separation of oil from the cells was carried out by using organic solvents such as hexane. However, the use of organic solvents results in dangerous operating conditions, requires the use of expensive explosion-proof equipment, and requires the implementation of expensive solvent recovery methods to avoid environmental pollution.

[0004] Therefore, as an alternative method for separating oil, the salting out of oil with a large amount of sodium chloride has been developed. However, the use of a large amount of sodium chloride results in a defatted biomass by-product that cannot be used as a feed ingredient due to its high salt content, so this method is not very sustainable. In addition, the high salt concentration causes rapid corrosion of the steel equipment used.

[0005] Therefore, there is a need to develop a method for efficiently separating oil from biomass that is carried out without the use of organic solvents and without the use of a large amount of salt. Such methods are described in WO 2018 / 011275, WO 2018 / 011286, WO 2018 / 013670, and WO 2018 / 122057. It is shown here in particular that if an appropriate amount of base equivalent is added to a biomass suspension containing lipids containing PUFAs, effective separation of the oil from the biomass can be achieved without adding organic solvents or a large amount of salt.

[0006] Based on this prior art, the object of the present invention is to further improve the efficiency of the oil separation method. Summary of the Invention

[0007] Surprisingly, it has been found that if the ratio of oil to total dry matter (TDM) is at least 0.5, preferably 0.5 to 0.9, before the mechanical separation of the oil phase and the water phase, the overall efficiency of the oil separation method is further improved.

[0008] Accordingly, a first subject of the present invention is a method for separating lipids containing polyunsaturated fatty acids (PUFAs) from biomass, the method comprising the following steps:

[0009] a) To provide a demulsified composition containing PUFA, characterized in that the ratio of oil to total dry matter (TDM) in the suspension is at least 0.5, preferably 0.5 to 0.9, more preferably 0.5 to 0.7 or 0.5 to 0.65, especially 0.5 to 0.6;

[0010] b) To separate the light phase containing the oil from the heavy phase containing water, salt, residual oil and cell debris by mechanical means.

[0011] Preferably, the suspension according to (a) has a TDM content of 20 to 60% by weight, more preferably 25 to 55% by weight, especially 30 to 50% by weight. If necessary, the TDM content can be adjusted either by concentrating the demulsified composition containing PUFA itself or, preferably, by concentrating the cell-containing suspension before demulsification. Detailed implementation

[0012] There are mainly two different ways to implement the subject matter of the present invention.

[0013] One way to implement the present invention is to prepare a demulsified composition containing PUFA showing a ratio of oil to TDM of at least 0.5, preferably 0.5 to 0.9, by performing a demulsification method mainly disclosed in the prior art, but adding oil during the process of the demulsification method to increase the ratio of oil to TDM in the composition before the mechanical separation of the oil-containing phase and the water phase occurs.

[0014] Therefore, in a preferred embodiment of the present invention, the preparation of the demulsified composition containing PUFA comprises the following steps:

[0015] a) To provide a suspension of biomass containing cells, the cells containing lipids containing PUFA;

[0016] b) To at least partially lyse the cells in the biomass;

[0017] c) To concentrate the suspension to a total dry matter (TDM) content of 20 to 60% by weight if the suspension has a lower TDM content;

[0018] d) To adjust the temperature in the suspension to 20°C to 100°C, preferably 25°C, 30°C, 40°C, 50°C or 60°C to 100°C, more preferably 65°C to 95°C, especially 70°C to 90°C;

[0019] e) Maintain the temperature within the range as described in (d) for at least 0.5 hours, preferably for at least 1, 2, 3, 4, 5, 6, 7 or 8 hours, more preferably for 1 to 36 hours, especially for 2, 3, 4, 5, 6, 7 or 8 hours to 36 hours, more preferably for 10 to 24 hours, while adding 7.5 to 25 moles, preferably 8.5 to 22 moles, especially 10 to 20 moles, more preferably 11 to 18 moles, especially 12 to 17 moles of base equivalents to 10 kg of the total dry matter contained in the suspension;

[0020] f) Adjust the pH value to 5.0 to 8.5, preferably 5.0 to 7.5, more preferably 5.0 to 6.5, and incubate for 0.1 to 6 hours, preferably 0.5 to 4 hours;

[0021] It is characterized in that, after step (b), preferably after step (e) or after step (f), more preferably at some time after step (f), oil is added to the suspension such that the ratio of oil to total dry matter (TDM) reaches at least 0.5, preferably 0.5 to 0.9 or 0.5 to 0.8, especially 0.5 to 0.7 or 0.5 to 0.65, more preferably 0.5 to 0.6.

[0022] The oil added to increase the ratio of oil to TDM can be any kind of edible oil, especially microbial oil, vegetable oil, oil of animal origin or a mixture thereof. In a preferred embodiment of the present invention, the oil is also an oil containing PUFA.

[0023] The oil of animal origin is preferably the oil of marine organisms. According to the present invention, "oil of marine organisms" or "marine oil" generally should be understood to mean an oil obtained from marine organisms, preferably from marine animals. In addition to fish oil which is preferred according to the present invention, it also means an oil separated from other marine organisms, especially from marine animals, such as from krill, bivalves, squid or shrimp. Preferably, the marine oil to be used according to the present invention is fish oil, especially the fatty oil from fish, particularly preferably the fatty oil from the following fish families, Engraulidae, Carangidae, Clupeidae, Osmeridae, Scombridae and / or Ammodytidae.

[0024] The use of marine oil, especially fish oil, and vegetable oil, especially canola oil and soybean oil, has proven to be very suitable for improving the oil separation of the present invention.

[0025] Since the biomass of the present invention is preferably microbial biomass, in particular the biomass of the class Labyrinthulea, the addition of marine oil preferably results in a product comprising a mixture of: microbial oil containing PUFAs, in particular oil of the class Labyrinthulea, and marine oil. Accordingly, a further subject of the present invention is an oil composition which comprises microbial oil, in particular oil of the class Labyrinthulea, preferably of the genus Aurantiochytrium or the genus Schizochytrium, and marine oil, in particular fish oil, in a (weight / weight) ratio of from 30:1 to 1:30, preferably in a (weight / weight) ratio of from 20:1 to 1:20, more preferably in a (weight / weight) ratio of from 10:1 to 1:10, 5:1 to 1:5 or 2:1 to 1:2.

[0026] Accordingly, a further subject of the present invention is also a lipid which comprises: 5 to 95% by weight, in particular 10 to 90% by weight or 20 to 80% by weight, especially 30 to 70% by weight of microbial oil, in particular oil of the Stramanopiles, preferably oil of the class Labyrinthulea, more preferably oil of the genus Aurantiochytrium or the genus Schizochytrium; and 5 to 95% by weight, in particular 10 to 90% by weight or 20 to 80% by weight, especially 30 to 70% by weight of oil of marine organisms, preferably fish oil, or vegetable oil, in particular canola oil or soybean oil.

[0027] In a preferred embodiment of the present invention, the added oil is a vegetable oil, in particular a vegetable oil selected from the group consisting of canola oil, soybean oil, rapeseed oil, palm oil, palm kernel oil, coconut oil, corn oil, olive oil, sunflower oil, cottonseed oil, linseed oil and mixtures thereof, with canola oil and soybean oil being particularly preferred.

[0028] Since the biomass of the present invention is preferably microbial biomass, in particular the biomass of the class Labyrinthulea, the addition of vegetable oil preferably results in a product comprising a mixture of: microbial oil containing PUFAs, in particular oil of the class Labyrinthulea, and vegetable oil. Accordingly, a further subject of the present invention is an oil composition which comprises microbial oil, in particular oil of the class Labyrinthulea, preferably of the genus Aurantiochytrium or the genus Schizochytrium, and vegetable oil, in particular canola oil, in a (weight / weight) ratio of from 30:1 to 1:30, preferably in a (weight / weight) ratio of from 20:1 to 1:20, more preferably in a (weight / weight) ratio of from 10:1 to 1:10, 5:1 to 1:5 or 2:1 to 1:2.

[0029] Accordingly, a further subject of the present invention is also lipids, said lipids comprising: 5 to 95% by weight, in particular 10 to 90% by weight or 20 to 80% by weight, especially 30 to 70% by weight of microbial oil, especially the oil of protalveolates, preferably the oil of Rhizochytrium, more preferably the oil of Aurantiochytrium or Schizochytrium; and 5 to 95% by weight, in particular 10 to 90% by weight or 20 to 80% by weight, especially 30 to 70% by weight of vegetable oil, especially canola oil and soybean oil.

[0030] The oil product of the present invention preferably contains PUFA in an amount of at least 10% by weight, preferably in an amount of at least 15% by weight, 20% by weight or 25% by weight, more preferably in an amount of at least 30% by weight, 40% by weight or 50% by weight.

[0031] In a preferred embodiment of the present invention, especially when preparing a mixture of microbial oil and marine oil, the amount of DHA is substantially the same as the amount of EPA. That is, a preferred embodiment of the present invention is such a lipid that contains DHA and EPA in a ratio of 2:1 to 1:2, more preferably 3:2 to 2:3, especially 4:3 to 3:4, wherein the lipid is preferably such a mixture: a mixture of microbial oil, especially the oil of Rhizochytrium, more preferably the oil of Aurantiochytrium or Schizochytrium, and marine oil, especially fish oil.

[0032] The oil product of the present invention preferably additionally exhibits at least one, more preferably at least 3, 5, 7 or 10 of the following characteristics: a) a peroxide value of less than 0.5, preferably less than 0.3, especially less than 0.15; b) an aniline value of less than 15, preferably less than 10; c) a TOTOX value of less than 25, preferably less than 20, more preferably less than 15; d) a content of free fatty acids of less than 5% by weight, preferably less than 3% by weight; e) a content of moisture and impurities of less than 1% by weight, preferably less than 0.5% by weight; f) a viscosity of less than 250 cps, more preferably less than 200 cps, especially less than 160 cps; g) a flash point of at least 300 °C, more preferably at least 350 °C, especially at least 400 °C, particularly at least 450 °C; h) a content of ω-3 fatty acids of at least 35% by weight, preferably at least 40 or 45% by weight, especially at least 50% by weight, particularly the content of DHA and EPA; i) preferably, DHA and EPA each in an amount of at least 8% by weight, preferably at least 10% by weight, especially at least 15% by weight; j) preferably, an amount of organic solvent of less than 0.5% by weight, more preferably less than 0.1% by weight, especially less than 0.05% by weight, particularly less than 0.01% by weight; k) preferably, an amount of chloride of less than 0.1% by weight, more preferably less than 0.05% by weight, especially less than 0.01% by weight; l) preferably, a content of crude fat of more than 90% by weight.

[0033] The aniline value (AV) is determined according to the AOCS official method Cd 18-90. The AV is a measure of the amount of secondary reaction products such as aldehydes and ketones of fatty acids that appear during the oxidation of the oil.

[0034] The peroxide value (PV) is determined according to the AOCS official method CD 8-53. The PV is a measure of the amount of primary reaction products such as peroxides and hydroperoxides that appear during the oxidation of the oil. According to the present invention, the PV is measured in meq / kg.

[0035] The content of free fatty acids is determined according to the AOCS official method AOCS Ca 5a-40. The content of moisture is determined according to the AOCS official methods AOAC 930.15, 935.29. The content of insoluble impurities is determined according to the AOCS official method AOCS 3a-46. The amounts of DHA and EPA are determined according to the AOCS official method AOCS Ce 1b-89. The amount of total fat is determined according to the AOCS official method AOCS 996.06. The amount of crude fat is determined according to the AOCS official methods AOAC 920.39, 954.02.

[0036] In a further preferred embodiment of the present invention, the oil is an oil containing PUFA, and the oil containing PUFA is from the same type of microorganism as the oil contained in the (demulsified) suspension.

[0037] In a highly preferred embodiment of the present invention, the added oil is the oil from the method itself being applied, i.e., it is the oil previously prepared by the said method. In other words, the oil obtained by mechanically separating the oil phase from the water phase is partially recycled to a previous step of the separation method to increase the ratio of oil to TDM.

[0038] Accordingly, a further preferred subject matter of the present invention is a method for separating lipids containing polyunsaturated acids (PUFA) from biomass, the method comprising the following steps:

[0039] a) providing a demulsified biomass composition containing PUFA;

[0040] b) separating the light phase containing oil from the heavy phase containing water, salts, residual oil and cell debris by mechanical means;

[0041] c) partially recycling the separated oil to increase the ratio of oil to TDM.

[0042] Accordingly, a particularly preferred subject matter of the present invention is also a method for separating lipids containing polyunsaturated fatty acids (PUFA) from biomass, the method comprising the following steps:

[0043] a) providing a suspension of biomass containing cells, the cells containing lipids containing PUFA;

[0044] b) at least partially lysing the cells in the biomass;

[0045] c) concentrating the suspension to a total dry matter (TDM) content of 20 to 60% by weight if the suspension has a lower TDM content;

[0046] d) adjusting the temperature in the suspension to 20°C to 100°C, preferably 25°C, 30°C, 40°C, 50°C or 60°C to 100°C, more preferably 65°C to 95°C, especially 70°C to 90°C;

[0047] e) Maintain the temperature within the range as described in (d) for at least 0.5 hours, preferably at least 1, 2, 3, 4, 5, 6, 7 or 8 hours, more preferably for 1 to 36 hours, especially for 2, 3, 4, 5, 6, 7 or 8 hours to 36 hours, more preferably 10 to 24 hours, while adding 7.5 to 25 moles, preferably 8.5 to 22 moles, especially 10 to 20 moles, more preferably 11 to 18 moles, especially 12 to 17 moles of base equivalents to 10 kg of the total dry matter contained in the suspension;

[0048] f) Adjust the pH value to 5.0 to 8.5, preferably 5.0 to 7.5, more preferably 5.0 to 6.5, and incubate for 0.1 to 6 hours, preferably 0.5 to 4 hours;

[0049] g) Separate the light phase containing the oil from the heavy phase containing water, salts, residual oil and cell debris by mechanical means;

[0050] It is characterized in that, after step (b), preferably after step (e) or after step (f), more preferably at some time after step (f), a part of the separated oil obtained in step (g) is added to the suspension, so that the ratio of oil to total dry matter (TDM) reaches at least 0.5, preferably 0.5 to 0.9 or 0.5 to 0.8, especially 0.5 to 0.7 or 0.5 to 0.65, more preferably a value of 0.5 to 0.6.

[0051] In a particularly preferred embodiment of the present invention, the recycling of the oil is carried out as a continuous process, that is, the oil obtained after the mechanical separation of the aqueous phase in the last step of the process is partially and continuously recycled into the process, preferably after step (b), more preferably after step (e) or at some time after step (f).

[0052] Alternatively, the preparation of the demulsified PUFA-containing composition is carried out by fermenting the PUFA-producing cells until the ratio of oil to TDM reaches at least 0.5, preferably at least 0.5 to 0.9.

[0053] Therefore, in another preferred embodiment of the present invention, the preparation of the demulsified PUFA-containing composition comprises the following steps:

[0054] a) Ferment the PUFA-producing cells until the ratio of oil to total dry matter (TDM) in the fermentation broth reaches at least 0.5, preferably 0.5 to 0.9 or 0.5 to 0.8, especially 0.5 to 0.7 or 0.5 to 0.65, more preferably a value of 0.5 to 0.6;

[0055] b) at least partially lyse the cells in the biomass;

[0056] c) concentrate the suspension to a total dry matter (TDM) content of 20 to 60% by weight if the suspension has a lower TDM content;

[0057] d) adjust the temperature in the suspension to 20°C to 100°C, preferably 25°C, 30°C, 40°C, 50°C or 60°C to 100°C, more preferably 65°C to 95°C, especially 70°C to 90°C;

[0058] e) maintain the temperature within the range as described in (d) for at least 0.5 hours, preferably for at least 1, 2, 3, 4, 5, 6, 7 or 8 hours, more preferably for 1 to 36 hours, especially for 2, 3, 4, 5, 6, 7 or 8 hours to 36 hours, more preferably 10 to 24 hours, while adding a total of 7.5 to 25 moles, preferably 8.5 to 22 moles, especially 10 to 20 moles, more preferably 11 to 18 moles, especially 12 to 17 moles of base equivalents to 10 kg of total dry matter contained in the suspension;

[0059] f) adjust the pH value to 5 to 8.5, preferably 5 to 7.5, more preferably 5 to 6.5, and incubate for 0.1 to 6 hours, preferably 0.5 to 4 hours.

[0060] In the method as disclosed previously, step (e), i.e., the step of adding base equivalents, causes the emulsion contained in the suspension to break into a light phase containing oil and a heavy phase containing water, cell debris and salts. In the context of the present application, such breaking of the emulsion is also referred to as "demulsification".

[0061] If not otherwise stated, the pH according to the present invention is preferably maintained below 11.5, more preferably below 11, especially below 10.5, particularly 7 to 11.5, more preferably 8 to 11 or 9 to 11, especially 10 to 11 during the demulsification step. This is achieved by adding the base not all at once but continuously or step by step during the demulsification step, so that exceeding the specified pH value can be avoided.

[0062] In a specific embodiment of the present invention, the demulsification is carried out at a relatively low pH below 9.0, especially at a pH between 7.0 and below 9.0, preferably at a pH between 7.0 and 8.5, especially at a pH between 7.5 and 8.5.

[0063] In a further specific embodiment of the present invention, the demulsification is carried out at a temperature below 80 °C, in particular at a temperature of 20 °C to below 80 °C, preferably at a temperature of 25 to 75 °C, 30 to 75 °C, 40 to 75 °C, 50 to 75 °C or 60 to 75 °C. That is, in particular, the temperature as in the method disclosed previously is adjusted accordingly in step (d).

[0064] In a further specific embodiment of the present invention, the demulsification step is carried out for less than 12 hours, in particular for 0.5, 1, 2 or 4 to 10 hours, preferably 0.5, 1, 2 or 4 to 8 hours, or 0.5, 1, 2 or 4 to 6 hours.

[0065] In a further specific embodiment of the present invention, the demulsification is carried out without prior lysis of the cells in the biomass, i.e., step (b) of the above method is omitted.

[0066] Preferably, in order to prepare the demulsified composition, the suspension is continuously mixed by using a stirrer and / or an agitator. In particular, low shear stirring and / or axial flow stirring can be applied, especially as disclosed in WO 2015 / 095694. Impellers suitable for stirring include straight blade impellers, Rushton blade impellers, axial flow impellers, radial flow impellers, concave blade disc impellers, high-efficiency impellers, propellers, paddles, turbines and combinations thereof.

[0067] According to the present invention, the term "alkali equivalent" takes into account the fact that not only monovalent alkalis but also divalent or polyvalent alkalis exist, and not only monovalent alkalis can be used but also divalent or polyvalent alkalis can be used. In the case of using a divalent alkali instead of a monovalent alkali or in addition to a monovalent alkali, only half the molar amount of such a divalent alkali needs to be used compared to the molar amount of the monovalent alkali to be used in order to achieve the same amount of alkali equivalent; in the case of using a trivalent alkali, only one-third of the molar amount of the monovalent alkali needs to be used; and so on. In the case of using a monovalent alkali, such as sodium hydroxide, the amount of alkali equivalent is the same as the amount of the alkali.

[0068] Based on the molar amount, the weight of the alkali can be easily calculated by using the molar weight of the alkali. For example, in the case of the highly preferred alkali sodium hydroxide, the molar weight is equal to 40 g / mol. This means that 1 mole of sodium hydroxide corresponds to 40 g of sodium hydroxide.

[0069] The preferred bases used according to the present invention are selected from: hydroxides, in particular sodium hydroxide, lithium hydroxide, potassium hydroxide and / or calcium hydroxide; carbonates, in particular sodium carbonate, potassium carbonate and / or magnesium carbonate; and / or bicarbonates, in particular lithium bicarbonate, sodium bicarbonate and / or potassium bicarbonate. In a very preferred embodiment of the present invention, the base used according to the present invention is only or almost only sodium hydroxide. Due to ease of handling, the base is preferably used in liquid form, in particular as a concentrated solution, wherein the concentration of the base in the solution is preferably 10 to 60% by weight, in particular 20 to 50% by weight.

[0070] According to the present invention, after providing a suspension containing biomass, a lysis step is preferably carried out. In one of the subsequent steps of this procedure without any explicit lysis step, the lysis step can be omitted if - for example due to the applied fermentation conditions - most of the cells or the cells have been lysed or are easily broken.

[0071] Lysis of the cells in the biomass can be carried out by methods known to those skilled in the art, in particular enzymatically, mechanically, physically or chemically, or by applying a combination thereof.

[0072] Depending on the exposure time and / or the degree of force applied, a composition containing only lysed cells or a composition containing a mixture of cell debris and intact cells can be obtained. The term "biomass containing lysed lipids" refers to such a suspension that contains water, cell debris and oil released from the cells in the biomass, but may also contain additional components, in particular salts, intact cells, additional contents of lysed cells, and components of the fermentation medium, in particular nutrients. According to the present invention, "at least partially lysing the cells" means that at least 20%, preferably at least 40%, 60% or 80% of the cells contained in the suspension are lysed.

[0073] In a preferred embodiment of the present invention, after the step of lysing the cells, only a small amount of intact cells, in particular less than 20%, preferably less than 10%, more preferably less than 5% of the intact cells (relative to the total number of intact cells present before lysing the cells in the biomass) are present in the lysed biomass or biomass suspension.

[0074] Lysis of the cells can be achieved, for example, by using the following: French cell press, sonicator, homogenizer, microfluidizer, ball mill, rod mill, pebble mill, sand mill, high-pressure grinding roll, vertical shaft impactor, industrial mixer, high-shear mixer, paddle mixer and / or polytron homogenizer.

[0075] In a preferred embodiment of the present invention, lysis of the cells comprises enzymatic treatment of the cells by applying cell wall degrading enzymes.

[0076] According to the present invention, the cell wall degrading enzymes are preferably selected from: proteases, cellulases (e.g., Cellustar CL (Dyadic), Fibrezyme G2000 (Dyadic), Celluclast (Novozymes), Fungamyl (Novozymes), Viscozyme L (Novozymes)), hemicellulases, chitinases, pectinases (e.g., Pectinex (Novozymes)), sucrases, maltases, lactases, α-glucosidases, β-glucosidases, amylases (e.g., Alphastar Plus (Dyadic); Termamyl (Novozymes)), lysozymes, neuraminidases, galactosidases, α-mannosidases, glucuronidases, hyaluronidases, pullulanases, glucocerebrosidases, galactocerebrosidases, acetylgalactosaminidases, fucosidases, hexosaminidases, iduronidases, maltase-glucoamylases, xylanases (e.g., Xylanase Plus (Dyadic), Pentopan (Novozymes)), β-glucanases (e.g., Vinoflow Max (Novozymes), Brewzyme LP (Dyadic)), mannanases and combinations thereof. The protease can be selected from serine proteases, threonine proteases, cysteine proteases, aspartic proteases, metalloproteases, glutamic proteases, alkaline proteases (subtilisin) and combinations thereof. The chitinase can be chitotriosidase. The pectinase can be selected from pectolyase, pectozyme, polygalacturonase and combinations thereof.

[0077] The appropriate pH for using the enzyme depends on the pH optimum of the enzyme.

[0078] In a preferred embodiment of the present invention, an enzyme having a pH optimum between 7.0 and 8.0, particularly a pH optimum of about 7.5, is used, so that the pH applied in this step is from 7.0 to 8.0, preferably from 7.3 to 7.7. A preferred enzyme that can be used in this pH range is alkaline protease.

[0079] The enzyme is preferably added as a concentrated enzyme solution, in particular in an amount of 0.01 to 1.5% by weight, preferably in an amount of 0.03 to 1.0% by weight, more preferably in an amount of 0.05 to 0.5% by weight, relative to the total amount of the suspension after addition of the concentrated enzyme solution.

[0080] In a highly preferred embodiment of the invention, cell lysis is carried out as follows:

[0081] i) The biomass suspension is heated to a temperature between 50 °C and 70 °C, preferably to a temperature between 55 °C and 65 °C, and a cell wall degrading enzyme is added to the suspension, in particular to the fermentation broth, and the appropriate pH value is adjusted as required, at which pH the enzyme functions properly;

[0082] ii) The temperature and pH are maintained within the ranges described in (ii) for at least 1 hour, preferably for at least 2 hours, more preferably for 2 to 4 hours.

[0083] In step (i), the enzyme can be added before or after heating the suspension and / or before or after adjusting the pH. In the same way, heating of the suspension can be carried out before or after adjusting the pH. - However, in a preferred embodiment, if adjustment of the pH is necessary, the enzyme is added after heating the suspension and after adjusting the pH. - In a highly preferred embodiment, all the methods are carried out more or less simultaneously.

[0084] Preferably, in steps (i) and (ii), the suspension is continuously mixed by using a stirrer and / or agitator.

[0085] According to the invention, demulsification is preferably carried out with a suspension having a dry matter content of 20 to 60% by weight, more preferably 25 to 60% by weight, in particular 30 to 55% by weight or 30 to 45% by weight. This can be achieved either by providing a suspension with an appropriately high amount of biomass or by concentrating the biomass suspension, in particular after lysing the cells in the biomass. Thus, in a preferred embodiment of the invention, the suspension is concentrated to a total dry matter content of 20 to 60% by weight, more preferably 25 to 60% by weight, in particular 30 to 55% by weight, especially 30 to 50% by weight or 30 to 45% by weight, optionally after lysing the cells in the biomass and before the demulsification step.

[0086] The suspension is preferably concentrated by evaporation of water at a temperature not exceeding 100 °C, preferably from 70 °C to 100 °C, more preferably from 80 °C to 90 °C, until a total dry matter content of 20 to 60% by weight, more preferably 25 to 60% by weight, in particular 30 to 55% by weight or 30 to 45% by weight is reached.

[0087] The concentration of the suspension is preferably carried out in a forced circulation evaporator (available, for example, from GEA in Germany) to allow rapid removal of water. Alternatively or additionally, concentration can be carried out by falling film evaporation, thin film evaporation and / or rotary evaporation.

[0088] Generally, according to the invention, the pH can be adjusted by using an alkali or an acid known to the person skilled in the art. The decrease in pH can in particular be carried out by using an organic acid or an inorganic acid such as sulfuric acid, nitric acid, phosphoric acid, boric acid, hydrochloric acid, hydrobromic acid, perchloric acid, hypochlorous acid, chlorous acid, fluorosulfuric acid, hexafluorophosphoric acid, acetic acid, citric acid, formic acid or a combination thereof. Since it is desirable to avoid high chloride contents, in a preferred embodiment of the invention, hydrochloric acid is not used or only a small amount of hydrochloric acid is used in the process according to the invention. According to the invention, sulfuric acid is the preferred substance for lowering the pH. - The increase in pH can in particular be carried out by using an organic base or an inorganic base such as the following: hydroxides, in particular sodium hydroxide, lithium hydroxide, potassium hydroxide and / or calcium hydroxide; carbonates, in particular sodium carbonate, potassium carbonate or magnesium carbonate; and / or bicarbonates, in particular lithium bicarbonate, sodium bicarbonate and / or potassium bicarbonate. - Due to ease of handling, acids and bases are preferably used in liquid form, in particular as concentrated solutions, wherein the concentration of the acid or base in the solution is preferably from 10 to 55% by weight, in particular from 20 to 50% by weight. In particular, sulfuric acid is also preferably used in concentrated form.

[0089] The method according to the invention includes, as a further step, the step of harvesting the PUFA-containing lipid from the demulsified composition.

[0090] The harvesting of the PUFA-containing lipid preferably includes: "neutralization" of the demulsified suspension, and subsequent separation of the resulting oil-containing light phase from the heavy phase containing water, salts, cell debris and residual oil.

[0091] The "neutralization" of the demulsified composition (step (f) of the method according to the invention) is preferably achieved by adding an acid, preferably sulfuric acid, to adjust the pH to 5.0 to 8.5, in particular 5.0 to 7.5, preferably 5.0 to 7.0. In a preferred embodiment of the invention, the pH is adjusted to below 7, preferably the pH is adjusted to 5.0 to 6.5, 5.5 to 6.5 or 5.0 to 6.0, since a higher oil yield is observed at pH values below 7.

[0092] Before the start of separation of the light and heavy phases, the neutralized composition can be stirred for several minutes up to several hours, preferably 0.1 to 6 hours, more preferably 0.5 to 4 hours, at this neutral pH.

[0093] If the demulsified composition obtained in step (e) of the process according to the invention has a pH value outside this range, only "neutralization" of the demulsified composition as described above is required.

[0094] Separation of the oil-containing light phase from the heavy phase containing water, salts and cell debris is preferably achieved by mechanical means, and preferably at a temperature of 60 - 90 °C, more preferably 70 - 80 °C, and preferably at a pH value of 6 - 9, more preferably 7 - 8.5. According to the invention, "mechanical means" particularly refers to filtration and centrifugation methods known to those skilled in the art. In a preferred embodiment of the invention, separation of the oil-containing light phase from the aqueous phase is carried out by a two-step centrifugation method, particularly as disclosed in WO2019 / 032880.

[0095] After separation of the oil-containing light phase, the oil containing PUFA thus obtained can be further processed by methods known to those skilled in the art, particularly refining, bleaching, deodorization and / or winterizing.

[0096] A particular advantage of the process of the invention is that the process of the invention can be carried out without the use of any organic solvents, particularly without the use of any polar or non-polar organic solvents. Thus, in a preferred embodiment of the invention, no organic solvents or only trace amounts of organic solvents, particularly polar or non-polar organic solvents, are used for the separation of the oil containing PUFA from the biomass. Typical organic solvents are hexane and ethanol.

[0097] In a preferred embodiment of the invention, less than 2% by weight of non-polar organic solvent is used, more preferably less than 1% by weight, 0.5% by weight or 0.1% by weight of non-polar organic solvent. In a particularly preferred embodiment of the invention, no non-polar organic solvent is used at all. In a very preferred embodiment of the invention, less than 2% by weight of organic solvent is used, generally, particularly preferably less than 1% by weight, 0.5% by weight or 0.1% by weight of organic solvent. In a particularly preferred embodiment of the invention, no organic solvent is used at all.

[0098] Another advantage of the method of the present invention is that a very effective separation of the oil from the remaining biomass can be achieved without the addition of sodium chloride which is commonly used for salting out oil from biomass. Preferably, the method can be carried out without the addition of any chloride salt at all, especially without the addition of any salt for salting out oil. However, due to the fermentation medium used for the growth of the biomass, small amounts of chloride salts, especially sodium chloride, may be present in the suspension.

[0099] Accordingly, in a preferred embodiment of the present invention, no sodium chloride or only trace amounts of sodium chloride are used to improve the separation of the oil. In a preferred embodiment of the present invention, less than 1 wt% of sodium chloride is used, more preferably less than 0.5 wt% or 0.2 wt% of sodium chloride is used for separating the oil from the biomass, especially less than 0.1 wt% or 0.05 wt% of sodium chloride, wherein the wt% is based on the total weight of the composition after the addition of sodium chloride.

[0100] In a particularly preferred embodiment of the present invention, no chloride salt or only small amounts of chloride salts are used to improve the oil separation. In this embodiment, preferably less than 1 wt% of chloride salt is used, more preferably less than 0.5 wt% or 0.2 wt% of chloride salt is used for separating the oil from the biomass, especially less than 0.1 wt% or 0.05 wt%, wherein the wt% is based on the total weight of the composition after the addition of the chloride salt.

[0101] In a highly preferred embodiment of the present invention, usually no salt or only trace amounts of salt are used to improve the oil separation. In this embodiment, preferably less than 1 wt% of salt is used, more preferably less than 0.5 wt% or 0.2 wt% of salt is used for separating the oil from the biomass, especially less than 0.1 wt% or 0.05 wt%, wherein the wt% is based on the total weight of the composition after the addition of the salt.

[0102] The method of the present invention enables a very effective separation of the oil contained in the biomass from the cell debris and other substances contained in the suspension, especially the fermentation broth. By using the method of the present invention, preferably more than 90 wt%, especially more than 95 wt%, 98 wt% or 99 wt% of the oil contained in the biomass can be separated from the biomass, and the separation is carried out under very economical and sustainable conditions.

[0103] "Chloride" according to the present invention refers to the detectable amount of chlorine. For example, by elemental analysis according to DIN EN ISO 11885, the amount of chlorine present can be determined. Chlorine is present in the form of salts referred to as "chlorides". The content of the chloride - also referred to as "chloride ion" - mentioned according to the present invention only refers to the detectable amount of chlorine, and not to the amount of the complete chloride salt that also contains a cationic counterion in addition to the chloride ion.

[0104] The total dry matter (TDM), especially the total dry matter (TDM) of a suspension, refers to the residual weight of the suspension after it has been dried, i.e., after the water has been removed. Thus, TDM relates not only to cell debris and salts, but especially also to the oil contained in the suspension.

[0105] The TDM content is preferably determined by gravimetric analysis. For this purpose, a homogeneous sample of a specific volume is weighed before and after freeze-drying. The remaining weight of the dried sample corresponds to the total dry matter contained in the specific volume. The TDM content of the sample is the quotient between the mass of the sample after freeze-drying and the mass of the sample before freeze-drying.

[0106] The amount of oil in the sample is preferably determined by fatty acid methyl ester analysis (FAME). For this purpose, first the lipids in the sample are saponified with KOH. After that, the free fatty acids are methylated with MeOH. Then, by using an internal standard, the methylated fatty acids can be determined and quantified by gas chromatography.

[0107] In a specific embodiment of the present invention, the aqueous phase containing water, salts, residual oil and cell debris obtained as a by-product in the aforementioned oil harvesting step is converted into dry biomass by drying the biomass to a total dry matter content of more than 90% by weight.

[0108] The conversion of the heavy phase containing water, salts, remaining oil and cell debris obtained as a by-product in the oil harvesting step into dry biomass by drying the biomass to a total dry matter content of more than 90% by weight can be carried out in different ways.

[0109] In a highly preferred manner, the conversion is carried out by concentrating the heavy phase to a dry matter content of 30 - 50% by weight, preferably 35 - 45% by weight, and subsequent spray granulation of the biomass by fluidized bed granulation. By doing so, biomass with favorable characteristics can be obtained in a very effective manner. Spray granulation by fluidized bed granulation is disclosed in more detail in EP13176661.0.

[0110] The concentration of the heavy phase to a dry matter content of 30 - 50 wt% is preferably carried out by solvent evaporation, in particular vacuum evaporation, and / or by using a rotary evaporator, a thin-film evaporator or a falling-film evaporator. An alternative available for solvent evaporation is the reverse osmosis method.

[0111] As an alternative to spray granulation, other drying methods for the concentrated heavy phase, in particular other convective drying methods, such as channel drying or spray drying, in particular nozzle spray drying, or contact drying methods, such as drum drying, or radiation drying methods, such as infrared drying, would be applicable alternatives, wherein by using these methods, particles with smaller or larger diameters are generally obtained.

[0112] According to the present invention, during the drying process, an anti-caking agent, in particular silica, preferably hydrophobic or hydrophilic silica, can optionally be added to the biomass to prevent caking. For this purpose, a suspension containing the biomass and the silica, in particular the fermentation broth, is preferably sprayed into a specific drying zone. Alternatively or additionally, after this drying process, the biomass can be mixed with the anti-caking agent. Regarding the use of silica as an anti-caking agent, reference can be made in particular to patent application EP13187631.0.

[0113] By a granulation method, it is possible to convert a fine-grained powder into a coarse-grained dust-free product. Conventional organic or inorganic aids or carriers, such as starch, gelatin, cellulose derivatives or the like, which are commonly used as binders, gelling agents or thickeners in food processing or feed processing, can optionally be used in this subsequent granulation method. Further aids preferably used according to the present invention are disclosed in WO2016 / 050560, wherein carboxymethyl cellulose is a particularly preferred binder.

[0114] After drying and optionally granulating and / or screening the biomass, the dried biomass is preferably stored or packaged.

[0115] The granular biomass of the present invention and the aqueous suspension of the present invention can be used in different ways. For example, they can be used to facilitate the preparation of foodstuff or feedstuff. Alternatively, they can be used directly as foodstuff or feedstuff.

[0116] Thus, similarly, a further subject of the present invention is a method for preparing a feedstuff or a foodstuff, wherein the granular biomass and / or the aqueous suspension according to the present invention are used and preferably mixed with other feedstuff or foodstuff ingredients.

[0117] The PUFA-containing cells of the biomass are preferably microbial cells or plant cells. Preferably, due to the polyketide synthase system, the cells are capable of producing PUFAs. The polyketide synthase system can be an endogenous polyketide synthase system or, due to genetic engineering, an exogenous polyketide synthase system.

[0118] The plant cells can be specifically selected from the cells of the following: Brassicaceae, Elaeagnaceae, and Fabaceae. The cells of Brassicaceae can be selected from the genus Brassica, especially Brassica napus, Brassica rapa, and Brassica juncea; the cells of Elaeagnaceae can be selected from the genus Elaeagnus, especially from the species Oleae europaea; the cells of Fabaceae can be selected from the genus Glycine, especially from the species Glycine max.

[0119] Microorganisms containing lipids with PUFAs are widely described in the prior art. Herein, the cells used can be, in particular, cells that have naturally produced PUFAs (polyunsaturated fatty acids); however, they can also be cells that, as a result of suitable genetic engineering methods or due to random mutagenesis, exhibit improved production of PUFAs or are fully capable of producing PUFAs. The production of PUFAs can be auxotrophic, mixotrophic, or heterotrophic.

[0120] The biomass preferably contains cells that produce PUFAs heterotrophically. The cells according to the invention are preferably selected from algae, fungi, especially yeasts, bacteria, or protists. The cells are more preferably microbial algae or fungi.

[0121] Suitable cells of oil-producing yeasts are especially the following strains: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon, and Lipomyces.

[0122] Suitable cells of oil-producing microalgae and similar algal microorganisms are in particular microorganisms selected from the phylum Stramenopiles (also known as Heterokonta). Microorganisms of the phylum Stramenopiles can in particular be selected from the following microorganisms: Hamatores, Proteromonads, Opalines, Developayella, Diplophrys, Labrinthulids, Thraustochytrids, Biosecids, Oomycetes, Hypochytridiomycetes, Commation, Reticulosphaera, Pelagomonas, Pelagococcus, Ollicola, Aureococcus, Parmales, Diatoms, Xanthophytes, Phaeophytes (brown algae), Eustigmatophytes, Raphidophytes, Synurids, Axodines (including Rhizochromulinales, Pedinellales, Dictyochales), Chrysomeridales, Sarcinochrysidales, Hydrurales, Hibberdiales and Chromulinales. Other preferred microalgae include members of green algae and dinoflagellates, including members of the genus Crypthecodiurn.

[0123] The biomass according to the invention preferably comprises the following cells and preferably consists essentially of such cells: the taxonomic unit Labyrinthulomycetes (Labyrinthulea, net slime fungi, slime nets), in particular those from the family Thraustochytriaceae. The family Thraustochytriaceae (thraustochytrids) includes the genera Althornia, Aplanochytrium, Aurantiochytrium, Botryochytrium, Elina, Japonochytrium, Oblongichytrium, Parietichytrium, Schizochytrium, Sicyoidochytrium, Thraustochytrium and Ulkenia. The biomass particularly preferably comprises cells from the genus Aurantiochytrium, Oblongichytrium, Schizochytrium or Thraustochytrium, in particular cells from the genus Schizochytrium.

[0124] According to the invention, the polyunsaturated fatty acid (PUFA) is preferably a highly unsaturated fatty acid (HUFA).

[0125] The cells present in the biomass are preferably distinguished by the fact that, in each case based on cell dry matter, they contain at least 20% by weight, preferably at least 30% by weight, in particular at least 35% by weight of PUFA.

[0126] According to the invention, the term "lipid" includes phospholipids, free fatty acids, esters of fatty acids, triacylglycerols, sterols and sterol esters, carotenoids, xanthophylls (such as oxidized carotenoids), hydrocarbons, compounds derived from isoprenoids and other lipids known to those skilled in the art. According to the invention, the terms "lipid" and "oil" are used interchangeably.

[0127] In a preferred embodiment, in this case, most of the lipids are present in the form of triglycerides, with preferably at least 50% by weight, in particular at least 75% by weight and in a particularly preferred embodiment at least 90% by weight of the lipids being present in the cells in the form of triglycerides.

[0128] According to the invention, the polyunsaturated fatty acid (PUFA) is understood to mean a fatty acid having at least two, in particular at least three C-C double bonds. According to the invention, highly unsaturated fatty acids (HUFA) are preferred among the PUFAs. According to the invention, HUFA is understood to mean a fatty acid having at least four C-C double bonds.

[0129] PUFAs can exist in cells in free form or in bound form. Examples of the bound form are phospholipids and esters of PUFAs, in particular monoacylglycerides, diacylglycerides and triacylglycerides. In a preferred embodiment, most PUFAs exist in the form of triglycerides, preferably in an amount of at least 50% by weight, in particular at least 75% by weight, and in a particularly preferred embodiment, at least 90% by weight of the PUFAs exist in the form of triglycerides in the cells.

[0130] Preferred PUFAs are ω-3 fatty acids and ω-6 fatty acids, with ω-3 fatty acids being particularly preferred. Preferred ω-3 fatty acids are eicosapentaenoic acid (EPA, 20:5ω-3), in particular (5Z,8Z,11Z,14Z,17Z)-eicosa-5,8,11,14,17-pentaenoic acid and docosahexaenoic acid (DHA, 22:6ω-3), in particular (4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenoic acid.

[0131] In a highly preferred embodiment of the present invention, cells that simultaneously produce large amounts of EPA and DHA are used, in particular Schizochytrium strains, wherein DHA is preferably produced in an amount of at least 20% by weight, preferably in an amount of at least 30% by weight, in particular in an amount of 30 to 50% by weight, while EPA is produced in an amount of at least 5% by weight, preferably in an amount of at least 10% by weight, in particular in an amount of 10 to 20% by weight (each calculated based on the total amount of lipids contained in the cells). Schizochytrium strains that produce DHA and EPA can be obtained by continuous mutagenesis, followed by appropriate selection of mutant strains that exhibit excellent EPA and DHA yields and a specific EPA:DHA ratio. Any chemical or non-chemical (e.g., ultraviolet (UV) radiation) reagent capable of inducing genetic alterations in yeast cells can be used as a mutagen. These reagents can be used alone or in combination with each other, and these chemical reagents can be used as pure reagents or in combination with solvents.

[0132] As previously mentioned, preferred species of Schizochytrium microorganisms that simultaneously produce large amounts of EPA and DHA are deposited under ATCC accession numbers PTA-10208, PTA-10209, PTA-10210 or PTA-10211, PTA-10212, PTA-10213, PTA-10214, PTA-10215.

[0133] The suspension of biomass according to the invention preferably has a biomass density of at least 80 or 100 g / l, in particular a biomass density of 80 to 250 g / l, in particular a biomass density of 80 to 200 g / l, more preferably at least 120 or 140 g / l, in particular a biomass density of 120 or 140 to 250 g / l, especially a biomass density of at least 160 or 180 g / l (calculated on the dry matter content), and is preferably a fermentation broth. Thus, under the conditions for the production of PUFAs by microorganisms, the suspension can be obtained by culturing and growing suitable cells in a fermentation medium.

[0134] Methods for producing biomass, in particular biomass containing cells that contain lipids, in particular cells of the order Thraustochytriales that contain PUFAs, are described in detail in the prior art (see, for example, WO91 / 07498, WO94 / 08467, WO97 / 37032, WO97 / 36996, WO01 / 54510). Generally, the production is carried out by culturing cells in a fermenter in the presence of a carbon source, a nitrogen source, and many additional substances that allow the growth of microorganisms and the production of PUFAs, such as minerals. In this case, a biomass density of more than 100 grams per liter and a production rate of more than 0.5 grams of lipid per liter per hour can be obtained. The method is preferably carried out in a method known as fed-batch, that is, the carbon source and the nitrogen source are fed in an increasing manner during fermentation. When the desired biomass has been obtained, lipid production can be induced by various measures, such as by restricting the nitrogen source, the carbon source, or the oxygen content, or a combination of these.

[0135] In a preferred embodiment of the invention, the cells are grown until they reach a biomass density of at least 80 g / l or 100 g / l, more preferably at least 120 g / l or 140 g / l, especially at least 160 g / l or 180 g / l (calculated on the total dry matter content). Such methods are disclosed, for example, in US 7,732,170.

[0136] Preferably, the cells are fermented in a medium with low salinity, especially to avoid corrosion. This can be achieved by using sodium salts without chlorine instead of sodium chloride as the sodium source, such as sodium sulfate, sodium carbonate, sodium bicarbonate, or soda ash. Preferably, chloride is used in an amount of less than 3 g / l, especially less than 500 mg / l, and particularly preferably less than 100 mg / l in the fermentation.

[0137] Suitable carbon sources are both alcohol carbon sources and non-alcohol carbon sources. Examples of alcohol carbon sources are methanol, ethanol, and isopropanol. Examples of non-alcohol carbon sources are fructose, glucose, sucrose, molasses, starch, and corn syrup.

[0138] Suitable nitrogen sources are both inorganic nitrogen sources and organic nitrogen sources. Examples of inorganic nitrogen sources are nitrates and ammonium salts, especially ammonium sulfate and ammonium hydroxide. Examples of organic nitrogen sources are amino acids, especially glutamate and urea.

[0139] In addition, inorganic phosphorus compounds or organic phosphorus compounds and / or known growth-stimulating substances such as yeast extract or corn steep liquor can be added to have a positive effect on fermentation.

[0140] The cells are preferably fermented at a pH of 3 to 11, especially 4 to 10, preferably at a temperature of at least 20 °C, especially 20 °C to 40 °C, particularly preferably at least 30 °C. A typical fermentation process takes about 100 hours.

[0141] After the fermentation has ended, the cells can be pasteurized to kill the cells and inactivate the enzymes that may promote lipid degradation. Pasteurization is preferably carried out by heating the biomass to a temperature of 50 °C to 121 °C, preferably 50 °C to 70 °C, for a period of 5 to 150 minutes, especially 20 to 100 minutes.

[0142] Similarly, after the fermentation has ended, antioxidants can be added to protect the PUFAs present in the biomass from oxidative degradation. In this case, preferred antioxidants are BHT, BHA, TBHA, ethoxyquin, β-carotene, vitamin E especially tocopherol, and vitamin C. If an antioxidant is used, it is preferably added in an amount of 0.001 wt% to 0.1 wt%, preferably 0.002 wt% to 0.05 wt%, based on the total amount of the fermentation broth after adding the antioxidant.

[0143] Examples

[0144] Example 1: Preparation of the suspension used in the demulsification test

[0145] In a stirring container, a cell culture solution containing unwashed microbial cells (Schizochiytrium sp.) with a biomass density of more than 100 g / l was heated to 60 °C. After heating the suspension, the pH was adjusted to 7.5 by using caustic soda (50 wt% NaOH solution), and then alkaline protease in liquid form ( 2.4 FG (Novozymes)) was added in an amount of 0.5 wt% (based on the weight of the culture solution). Stirring was continued at 60 °C for 3 hours. Thereafter, the lysed cell mixture was transferred to a rotary evaporator and heated to a temperature of 80 °C under a reduced pressure of 400 mbar. The mixture was concentrated in the rotary evaporator until a total dry matter content of about 35 wt% was reached.

[0146] As a next step, demulsification of the concentrated suspension was carried out. Using a stirred vessel B-DCU–Quad 2L (Sartorius, Germany), all experiments were carried out with approximately 1 liter of the enzyme-treated and subsequently concentrated fermentation broth. Demulsification was carried out at a temperature of 90 °C for 24 hours. The suspension was stirred at 300 rpm. Over a period of 3 hours, 1.5 moles of caustic soda / kg of TDM were added continuously. After 24 hours, the demulsified composition was neutralized to either pH 5.5 or pH 7.5. After neutralization, the TDM content and the oil content were determined to obtain the ratio of oil to TDM. Subsequently, different amounts of oil were added to study the effect of the ratio of oil to TDM on the final oil yield. As the oil, either Schizochytrium oil obtained previously by the method of the present invention or vegetable oil (rapeseed oil or soybean oil) was added. Approximately 2 hours after the optional addition of oil, a 50 g sample of the homogenized suspension was taken and the cell debris was separated by centrifugation at 13,500 g. Subsequently, the amounts of EPA and DHA in the supernatant were determined.

[0147] Table 1: Effect of the ratio of TDM to oil on the oil yield

[0148]

[0149] The experimental data show that when the ratio of oil to TDM exceeds a value of 0.5, the total oil yield increases significantly. Whether this value is reached by fermenting the cells until the ratio of oil to TDM exceeds this value or by recycling Schizochytrium oil during demulsification before separating the oil-containing light phase from the aqueous phase, the different ways do not play a role here. Additionally, it can be seen that the oil yield is much higher when the pH value is adjusted to 5.5 after demulsification compared to adjusting the pH value to 7.5.

[0150] Table 2: Effect of the ratio of TDM to oil on the oil yield; addition of rapeseed oil

[0151]

[0152] The experimental data show that the effect achieved with recycled Schizochytrium oil can also be achieved by adding vegetable oil, especially rapeseed oil.

[0153] Table 3: Effect of the ratio of TDM to oil on the oil yield; addition of soybean oil

[0154]

[0155] The experimental data show that the effect achieved with recycled Schizochytrium oil can also be achieved by adding soybean oil.

Claims

1. A method for separating lipids containing polyunsaturated fatty acids (PUFA) from biomass, which comprises the following steps: a) Providing a demulsified composition containing PUFA, characterized in that the ratio of oil to total dry matter (TDM) in the composition is from 0.5 to 0.7, wherein the composition has a TDM content of 20 to 60% by weight and a pH value of 5.0 to 8.5; b) Separating the light phase containing the oil from the heavy phase containing water, salts, residual oil and cell debris by mechanical means.

2. The method according to claim 1, characterized in that in step (a), the ratio of oil to total dry matter (TDM) in the composition is from 0.5 to 0.

65.

3. The method according to claim 1, characterized in that the composition according to (a) has a TDM content of 25 to 55% by weight.

4. The method according to claim 1, characterized in that the composition according to (a) has a TDM content of 30 to 50% by weight.

5. The method according to any one of the preceding claims, characterized in that the preparation of the demulsified composition containing PUFA comprises the following steps: a) Providing a suspension of biomass containing cells, the cells containing lipids containing PUFA; b) At least partially lysing the cells in the biomass; c) Concentrating the suspension to a total dry matter (TDM) content of 20 to 60% by weight, if the suspension has a lower TDM content; d) Adjusting the temperature in the suspension to 20 °C to 100 °C; e) Maintaining the temperature within the range as described in (d) for at least 0.5 hours, while adding a total of 7.5 to 25 molar equivalents of base to 10 kg of total dry matter contained in the suspension; f) Adjusting the pH value to 5.0 to 8.5 and incubating for 0.1 to 6 hours; characterized in that at some time after step (b), oil is added to the suspension such that the ratio of oil to total dry matter (TDM) reaches a value of at least 0.

5.

6. The method according to claim 5, wherein in step (d), the temperature is adjusted to 65 °C to 95 °C.

7. The method according to claim 5, wherein in step (e), the temperature is maintained within the range as described in (d) for 4 to 36 hours.

8. The method according to claim 5, wherein in step (e), 10 to 20 molar equivalents of base are added to 10 kg of total dry matter contained in the suspension.

9. The method according to claim 5, wherein the oil is added to the suspension after step (e) or after step (f).

10. The method according to claim 5, wherein the oil added to the suspension is microbial oil; oil of marine organisms; or vegetable oil; or a mixture thereof.

11. The method according to claim 10, wherein the vegetable oil is canola oil or soybean oil.

12. The method according to any one of claims 1 to 4, It is characterized in that the preparation of the demulsified PUFA-containing composition comprises the following steps: a) fermenting cells producing PUFA until the ratio of oil to total dry matter (TDM) in the fermentation broth reaches a value of at least 0.5, obtaining a suspension containing the biomass of the cells; b) at least partially lysing the cells in the biomass; c) concentrating the suspension to a total dry matter (TDM) content of 20 to 60% by weight, if the suspension has a lower TDM content; d) adjusting the temperature in the suspension to 20°C to 100°C; e) maintaining the temperature within the range as described in (d) for at least 0.5 hours, while adding a total of 7.5 to 25 molar equivalents of base to 10 kg of total dry matter contained in the suspension; f) adjusting the pH value to 5.0 to 8.5 and incubating for 0.1 to 6 hours.

13. The method according to claim 12, wherein, in step (a), the fermentation is carried out until the ratio of oil to total dry matter (TDM) in the fermentation broth reaches a value of 0.5 to 0.

9.

14. The method according to claim 12, wherein, in step (a), the fermentation is carried out until the ratio of oil to total dry matter (TDM) in the fermentation broth reaches 0.5 to 0.

65.

15. The method according to claim 12, wherein, in step (d), the temperature is adjusted to 65°C to 95°C.

16. The method according to claim 12, wherein, in step (e), the temperature is maintained within the range as described in (d) for 4 to 36 hours.

17. The method according to claim 12, wherein, in step (e), 10 to 20 molar equivalents of base are added to 10 kg of total dry matter contained in the suspension.

18. The method according to claim 12, wherein, in step (e), the pH value is maintained below 11.5 or below 11.

19. The method according to claim 12, wherein, in step (e), the pH value is maintained within the range of 6.0 to 11.

5.

20. The method according to claim 12, wherein, in step (e), the pH value is maintained within the range of 8.0 to 10.

5.

21. The method according to claim 5, wherein, the base is selected from: hydroxides; and / or carbonates; and / or bicarbonates.

22. The method according to claim 5, wherein, the suspension provided in step (a) already has a total dry matter content of 20 to 60% by weight.

23. The method according to claim 12, wherein, to lyse the cells, no salt or only a small amount of salt is used, where "a small amount" means adding salt in an amount less than 0.1 g / l of the fermentation broth.

24. The method according to claim 12, wherein, to lyse the cells, no organic solvent or only a small amount of organic solvent is used, where "a small amount" means adding an organic solvent in an amount less than 0.1 g / l of the fermentation broth.

25. The method according to claim 12, wherein, the suspension is provided as a fermentation broth having a biomass density of 80 to 250 g / l.

26. The method according to claim 5, wherein, the cells containing lipids with PUFAs are selected from fungi, protists, bacteria, plant cells, and mixtures thereof.

27. The method according to claim 5, wherein, the cells containing lipids with PUFAs are selected from algae.

28. The method according to claim 5, wherein, the cells containing lipids with PUFAs are selected from microalgae.

29. The method according to claim 28, wherein, the microalgae are selected from the family Thraustochytriaceae.

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