Method for producing biomass with increased polyunsaturated fatty acid content

Abstract

According to the present invention, it has been found that when the amount of sulfate used in the cultivation of PUFA-producing cells is chosen so that the sulfate concentration in the final stage of fermentation is always greater than zero but at the same time always below the saturation limit of the cells, a biomass with an increased mass proportion of polyunsaturated fatty acids (PUFAs) is obtained in the final product, the method having both high product / substrate yield and space / time yield.

Description

[0001] The present invention relates to a method for producing a biomass having an increased polyunsaturated fatty acid content and to a biomass obtainable by said method.

[0002] Methods for producing biomass containing polyunsaturated fatty acids (PUFAs) have been described in the prior art. Often used here are netzschleimpilze, which naturally accumulate polyunsaturated fatty acids as storage lipids in relatively large amounts in their cells.

[0003] These microalgae grow naturally in seawater, which means that the cultivation of the microalgae is initially carried out in a medium with a high chloride content. However, high chloride contents are not suitable for cultivation in steel bioreactors because they cause metal corrosion.

[0004] Therefore, fermentation media having a low chloride content and, conversely, a high sulfate content have been described in the prior art as alternative fermentation media.

[0005] In this case, however, the biomass obtained generally has a limited PUFA content, presumably especially because the high sulfate content means that a relatively high proportion of cell walls is formed in the final product at the expense of the amount of PUFA present.

[0006] Experiments with very low sulfate contents in the final stage of the fermentation have shown that, as a result of the adjusted addition of sulfate in the final stage of the fermentation, the relative proportion of PUFAs formed in the final product can be significantly increased, although at the same time, the cells are destabilized to a considerable extent, meaning that a partial release of the oil present may have occurred during the fermentation into the fermentation medium.

[0007] Based on this knowledge, the object of the present invention is to provide a process in which the biomass obtained has an increased mass proportion of PUFAs in the final product. At the same time, it is intended to preferably use a non-corrosive fermentation medium in the process, to achieve high product yields and space / time yields, and to prevent as far as possible a premature release of oil into the fermentation medium.

[0008] It is possible to achieve the object of the present invention by adjusting the sulfate content in the medium so that sulfate is always present in the medium in the final stage of fermentation, but the sulfate concentration is always lower than the saturation concentration of the cells (meaning that the sulfate present is immediately and completely assimilated by the cells).

[0009] Therefore, the present invention first provides a method for producing biomass containing PUFA, characterized in that the biomass is produced by culturing PUFA-producing cells in a fermentation medium, and the sulfate content in the fermentation medium is adjusted so that sulfate is always present in the medium in the final stage of fermentation, but the sulfate concentration in the medium is always lower than the saturation limit of the cells.

[0010] According to the invention, the sulfate saturation limit or sulfate saturation concentration of the cells is defined as the amount of sulfate that can be immediately and completely assimilated by the cells of the biomass under given conditions. In one embodiment of the invention that is preferred according to the invention, cells of the family Thraustochytriaceae are used, which have a sulfate saturation limit of about 5 g per kg of lipid-free biomass.

[0011] The present invention therefore preferably further provides a method for producing a biomass containing PUFAs, characterized in that the biomass is produced by culturing cells of the Thraustochytridaceae in a fermentation medium in which the sulfate content is adjusted such that in the final stage of the fermentation, the sulfate concentration in the medium is always 0.005 to 5 g sulfate per kilogram of lipid-free biomass, preferably 0.01 to 4 g and particularly preferably 0.01 to 3 g, 2 g or 1 g sulfate per kilogram of lipid-free biomass and in particular 0.01 to 0.05 g sulfate per kilogram of lipid-free biomass.

[0012] For the purpose of optimizing oil production, the method according to the invention preferably comprises a growth phase and a subsequent oil production phase. In the open growth phase, what first occurs is the cultivation and increase of the biomass of the PUFA-producing cells present in the fermentation medium - wherein oil production is suppressed to the maximum extent - with the result that the highest possible biomass density is established in the medium, while in the subsequent oil production phase (usually initiated by specific measures), oil production occurs mainly by the cells of the biomass, while the growth of the cells is at least as far as possible stopped. This means that in the oil production phase, the increase in biomass is at least mainly not due to an increase in cell count, but to the accumulation of lipids in the cell interior of the cells present. Cell growth in the growth phase is made possible by providing optimal growth conditions, while the transition to the oil production phase can be initiated by limiting single or multiple limiting factors (especially nutritional components, such as nitrogen sources).

[0013] According to the invention, "final stage of fermentation" is preferably understood to mean the oil production phase.

[0014] Therefore, the method according to the invention is preferably characterized by the fact that sulfate is present in the culture medium at least during 50% of the oil production period, preferably at least during 75% or 85% of the oil production period, particularly preferably at least during 90% or 95% of the oil production period, and in particular during the entire oil production period, but the sulfate concentration is always below the saturation limit of the cells present in the culture medium.

[0015] In this case, the preferred method is characterized by the fact that sulfate is present in the culture medium at least during 50% of the remaining time period of the oil production period, preferably at least during 75% or 85% of the remaining time period of the oil production period, particularly preferably at least during 90% or 95% of the remaining time period of the oil production period, and in particular during the entire oil production period, but the sulfate concentration is always below the saturation limit of the cells present in the culture medium; this means that during the corresponding complementary initial phase of the oil production period, the sulfate concentration may optionally still be above the saturation limit of the cells.

[0016] Accordingly, the present invention also provides a method for producing PUFA-containing biomass, characterized in that the biomass is produced by culturing cells of the Thraustochytridaceae family in a fermentation medium, wherein the sulfate content in the fermentation medium is adjusted so that at least during 50% of the oil production period, preferably at least during 75% or 85% of the oil production period, particularly preferably at least during 90% or 95% of the oil production period, and in particular during the entire oil production period, the sulfate concentration in the culture medium is always 0.005 to 5 g sulfate per kilogram of lipid-free biomass, preferably 0.01 to 4 g, and particularly preferably 0.01 to 3 g, 2 g or 1 g sulfate per kilogram of lipid-free biomass, and in particular 0.01 to 0.05 g sulfate per kilogram of lipid-free biomass.

[0017] Accordingly, the present invention also provides a method for producing PUFA-containing biomass, characterized in that the biomass is produced by culturing cells of the Thraustochytridaceae family in a fermentation medium, wherein the sulfate content in the fermentation medium is adjusted so that at least during 50% of the remaining period of the oil production period, preferably at least during 75% or 85% of the remaining period of the oil production period, particularly preferably at least during 90% or 95% of the remaining period of the oil production period, and in particular during the entire oil production period, the sulfate concentration in the culture medium is always 0.005 to 5 g sulfate per kilogram of lipid-free biomass, preferably 0.01 to 4 g, and particularly preferably 0.01 to 3 g, 2 g or 1 g sulfate per kilogram of lipid-free biomass, and in particular 0.01 to 0.05 g sulfate per kilogram of lipid-free biomass.

[0018] The present invention further provides a biomass obtainable by the method according to the invention, preferably a biomass comprising cells of the Thraustochytridaceae family.

[0019] In a preferred embodiment according to the invention a biomass is obtained having a sulfate content of 12 to 20 g sulfate per kg biomass, preferably 12 to 16 g sulfate per kg biomass.

[0020] Therefore, the present invention in particular further provides a PUFA-containing biomass having a sulfate content of 12 to 20 g sulfate per kg biomass, preferably 12 to 18 g sulfate per kg biomass and in particular 12 to 16 g sulfate per kg biomass, the biomass preferably comprising cells of the Thraustochytridaceae family.

[0021] According to the invention, "sulfate content" is understood to mean the total sulfate content, i.e. the content of free and bound (especially organically bound) sulfate relative to the biomass. It can be assumed that most of the sulfate present in the biomass is present as a component of exopolysaccharides (which participate in the formation of the cell wall of the microorganisms).

[0022] According to the present invention, the sulfate content is preferably determined by determining the sulfur content of the biomass obtained, since most of the sulfur present in the biomass can be attributed to the sulfate present. Due to the amount of sulfate present, sulfur attributable to other sources can be ignored. Therefore, the amount of sulfate present can be easily determined from the amount of sulfur determined.

[0023] In this context, the sulfur content of the biomass is preferably determined by elemental analysis in accordance with DIN EN ISO 11885. To analyze the sulfur content of the biomass, an appropriate aliquot of the sample is disrupted (preferably using nitric acid and hydrogen peroxide at 240° C. under pressure) before analysis to ensure that the sulfur present is readily available.

[0024] According to the present invention, "biomass", "dry biomass", "biomass of PUFA-producing cells" or "dry biomass of PUFA-producing cells" is generally understood to mean assayable dry biomass.

[0025] In this context, the amount of the dry biomass present in the sample is preferably determined as follows: collect a homogenized sample and centrifuge to separate the liquid component. After this, wash the biomass obtained by centrifugation with water to dissolve salts and any other soluble components, and then centrifuge again. Finally the dry biomass thus obtained is dried overnight in a drying cabinet. In this context, the percentage of the dry biomass present in the sample is derived by the quotient between the quality of the dry biomass measured after drying and the initial mass of the sample studied. The dry biomass thus confirmed also additionally comprises the oil formed by the biomass.

[0026] Therefore, according to the present invention, "lipid-free biomass", "lipid-free dry biomass", "lipid-free biomass of PUFA-producing cells" or "lipid-free dry biomass of PUFA-producing cells" is understood to mean the previously determined dry biomass minus the fat proportion present in the dry biomass.

[0027] The fat proportion present in the dry biomass is preferably determined by taking up the dry biomass in a methanol / chloroform solution and then subjecting the sample thus obtained to ultrasound treatment. The sample thus obtained is then saponified with potassium hydroxide and acidified with hydrochloric acid. Thereafter, the free fatty acids are methylated using BF3 (30% boron trifluoride in methanol) and separated by partition chromatography with a temperature gradient. Detection is then performed by flame ionization detection (FID). According to the invention, the biomass content is then determined by subtracting the fat content thus determined from the previously determined dry biomass content.

[0028] According to the present invention, the term lipid-free dry biomass is used because the sulfate saturation limit of the cells is independent of the lipid content of the cells, but the lipid content of the cells increases greatly during the oil production period, which means that the amount of sulfate addition must be determined by considering the total content of lipid-free dry biomass rather than the total content of dry biomass. Since the content of lipid-free dry biomass remains virtually stable after the start-up oil production period due to the maximum inhibition of cell proliferation, it is usually not necessary to measure the content of lipid-free dry biomass again after the start-up oil production period.

[0029] According to the invention, the desired sulfate concentration in the culture medium can be adjusted in different ways.

[0030] According to the invention, it is essential that the metering of sulfate must always be carried out in the final stage of the fermentation, preferably at the end of the oil production phase (so-called batch feed method). The start of the metering of sulfate is determined by whether and how much sulfate is present in the starting medium at the beginning of the fermentation. In principle, the amount of sulfate in the starting medium can be selected as desired, which means that the requirement to meter sulfate may occur at different times during the fermentation.

[0031] Since it is required that sulfate must be present in the fermentation medium in the final stage of fermentation, but the sulfate concentration here must be lower than the saturation limit of the cell, the sulfate concentration in the starting medium is an upper limit (which must be observed accordingly and easily calculated). In a preferred embodiment, the amount of sulfate in the starting medium is selected so that at least during the first 30% of the growth period of the cell, preferably at least during the first 40%, 50% or 60% of the growth period of the cell, and particularly during the first 70%, 80% or 90% of the time period, the sulfate concentration is higher than the saturation concentration of the cell. In this context, during a particular stage, the sulfate concentration is preferably at least 5g sulfate per kilogram of lipid-free biomass.

[0032] The metered addition of sulfate must start no later than before the sulfate concentration drops to zero. This is ensured by continuously measuring the sulfate concentration in the culture medium. Preferably, only when the sulfate concentration in the culture medium drops to less than 2g sulfate per kilogram of biomass, particularly when it drops to less than 1g sulfate per kilogram of biomass, and particularly preferably only when the sulfate concentration in the culture medium has dropped to less than 0.5g sulfate per kilogram of lipid-free biomass, the metered addition of sulfate is carried out, and according to the present invention, this is only carried out just before the oil production phase is converted to, to optimize oil production. Therefore, in an embodiment particularly preferred according to the present invention, the sulfate concentration in the culture medium is only just before the oil production phase begins, preferably before the oil production phase begins at most 3 hours, particularly at most 2 hours, and especially at most 1 hour ago drops to less than 2g, particularly less than 1g, and particularly preferably less than 0.5g sulfate per kilogram of lipid-free biomass.

[0033] Instead of initially providing a relatively large amount of sulfate in the starting medium, it is also possible to meter the sulfate in the growth phase of the cells. In this context, the sulfate is metered in the growth phase, preferably at least 5 g of sulfate per kilogram of biomass is added at least during the first phase of the growth phase, so that the amount of sulfate is always above the saturation concentration of the cells. In the subsequent oil production phase, the metered addition of sulfate is correspondingly reduced, so that the sulfate concentration in the oil production phase or just before the oil production phase drops below the saturation concentration of the cells, preferably below 5 g of sulfate per kilogram of lipid-free biomass, particularly preferably below 3 g, 2 g or 1 g of sulfate per kilogram of lipid-free biomass, and in particular below 0.5 g of sulfate per kilogram of lipid-free biomass.

[0034] In a further embodiment according to the invention, the sulfate is in a specified amount, as a result of which the sulfate concentration during the entire fermentation or at least during a majority of the time period during the entire fermentation remains below the saturation concentration of the cells, preferably below 5 g sulfate per kg of lipid-free biomass, in particular below 4 g, 3 g, 2 or 1 g sulfate per kg of lipid-free biomass.

[0035] Since it is essential for the process that the sulfate concentration does not drop to zero, it is possible to ensure that this is avoided by appropriate regulation of the amount of sulfate metered in (especially if the amount of biomass produced during the fermentation exceeds the initially calculated value).

[0036] In a preferred embodiment according to the present invention, sulfate is continuously metered into the fermentation medium in low amounts, preferably at a rate of 5 to 100 mg, preferably 10 to 50 mg per kilogram of fermentation culture, during most of the oil production period, preferably at least during 50%, 60% or 70% of the oil production period, and particularly preferably at least during the last 50%, 60% or 70% of the oil production period.

[0037] According to the invention, the sulfate used is preferably sodium sulfate, ammonium sulfate or magnesium sulfate and mixtures thereof. According to the invention, the feeding of sulfate can also be carried out alternatively or additionally by using industrial raw materials contaminated with sulfate or supplemented with sulfate.

[0038] According to the present invention, it has further surprisingly been found that low sulfate concentrations can also be combined with low chloride concentrations, meaning that non-corrosive fermentation media can be used to produce biomass.

[0039] Therefore, preferably, the process according to the invention is further characterized in that the fermentation medium used according to the invention has a chloride concentration of less than 1 g / l, in particular less than 500 mg / l and preferably less than 250 mg / l during the entire fermentation.

[0040] The biomass according to the invention preferably has a chloride content of not more than 2 g per kilogram of biomass, in particular from 0.5 to 1.8 g and particularly preferably from 0.5 to 1.5 g per kilogram of biomass.

[0041] According to the invention, "chloride content" is understood to mean the amount of measurable chlorine. For example, the amount of chlorine present can be determined by elemental analysis according to DIN EN ISO 11885. Chlorine is present in biomass in the form of salts known as "chlorides". When the present application refers to "chloride" or "chloride ions", only the amount of chloride ions or detectable chlorine is always meant, and not the amount of chloride salts (which, in addition to chloride ions, always include cationic counterions).

[0042] PUFA-producing cells are preferably cells which already produce PUFAs naturally; however, they may also be cells which are made capable of producing PUFAs by appropriate gene technology methods. In this context, the production may be autotrophic, mixotrophic or heterotrophic.

[0043] Accordingly, the biomass according to the invention comprises such cells and preferably consists essentially of such cells.

[0044] Preferably, the cell is a cell that produces PUFAs heterotrophically. According to the invention, the cell preferably takes the form of algae, fungi (especially yeast) or protists. Particularly preferably, the cell is a microorganism, especially a microbial algae or a fungus.

[0045] Suitable cells of oleaginous yeasts are, in particular, strains of the genera Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.

[0046] The cells are preferably those of the taxon Labyrinthulomycetes (Labyrinthulea, Netzschleimpilze, Schleimnetze), in particular those of the Thraustochytriaceae family. The Thraustochytriaceae family includes the following genera: Althornia, Aplanochytrium, Elnia, Japonochytrium, Schizochytrium, Thraustochytrium, Aurantiochytrium, Oblongichytrium, or Ulkenia. Particularly preferred are cells of the following genera: Thraustochytrium, Schizochytrium, Aurantiochytrium or Oblongichytrium, in particular those of the genus Aurantiochytrium. A particularly preferred strain is the Aurantiochytrium limacinum SR21 strain (IFO32693).

[0047] Biomass according to the present invention preferably takes the form of the product of fermentation culture method. Accordingly, biomass can not only contain cells to be destroyed, but also can contain the component of fermentation medium. These components can especially take the form of salt, defoamer and unreacted carbon source and / or nitrogen source. The cell content in this biomass is preferably at least 70 weight %, preferably at least 75 weight %. Optionally, before carrying out the cell disruption process, the cell content in the biomass can be increased to, for example, at least 80 weight % or at least 90 weight % by suitable washing steps. However, the biomass obtained also can be directly used in the cell disruption process.

[0048] Preferably, the cells in the biomass are characterized by the fact that they contain in each case at least 20% by weight, preferably at least 30% by weight and in particular at least 40% by weight of PUFAs, based on the dry mass of the cells.

[0049] In a preferred embodiment, the majority of lipids are present in the form of triglycerides, wherein 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 present in the cells are present in the form of triglycerides.

[0050] Preferably, at least 10% by weight, in particular at least 20% by weight, particularly preferably 20 to 60% by weight, in particular 20 to 40% by weight, of the fatty acids present in the cells are PUFAs.

[0051] According to the invention, polyunsaturated fatty acids (PUFA) are understood to mean fatty acids having at least two CC double bonds. According to the invention, highly unsaturated fatty acids (HUFA) are preferred among PUFAs. According to the invention, HUFA are understood to mean fatty acids having at least four CC double bonds.

[0052] PUFAs can be present in the cell in free form or in bound form. Examples of bound forms are phospholipids and esters of PUFAs, in particular monoacylglycerides, diacylglycerides and triacylglycerides. In a preferred embodiment, the majority of PUFAs are present in the form of triglycerides, wherein 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 PUFAs present in the cell are present in the form of triglycerides.

[0053] Preferred PUFAs are ω-3 fatty acids and ω-6 fatty acids, of which ω-3 fatty acids are particularly preferred. In this context, preferred ω-3 fatty acids are eicosapentaenoic acid (EPA, 20:5ω-3), in particular (5Z,8Z,11Z,14Z,17Z)-eicosapentaenoic 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, of which docosahexaenoic acid is particularly preferred.

[0054] Methods for producing PUFA-containing cells, in particular of the Thraustochytridales, have been described in detail in the prior art (see, for example, WO 91 / 07498, WO 94 / 08467, WO 97 / 37032, WO 97 / 36996, WO 01 / 54510). Production is usually carried out by culturing the cells in a fermenter in the presence of a carbon source and a nitrogen source. In this context, biomass densities of more than 100 g per liter and productivity rates of more than 0.5 g lipid per hour per liter can be achieved. The process is preferably carried out in a so-called fed-batch process, i.e. the carbon source and possibly the nitrogen source and the phosphate source are added stepwise during the fermentation. Once the desired biomass has been reached, lipid production can be induced by various measures, for example by limiting the nitrogen source, the phosphate source or the oxygen content or a combination thereof.

[0055] Suitable carbon sources are both alcohol 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, as well as organic acids such as acetic acid, propionic acid and medium-chain and long-chain fatty acids and their salts. The preferred method according to the present invention is characterized in that at least one carbon source is continuously metered into the culture medium during the complete fermentation process.

[0056] Suitable nitrogen sources are both inorganic and organic nitrogen sources. Examples of inorganic nitrogen sources are nitrates and ammonium salts, in particular ammonium sulfate and ammonium hydroxide. Examples of organic nitrogen sources are amino acids, in particular glutamic acid and urea.

[0057] According to the present invention, the initiation of the oil production phase is preferably performed as described in WO 01 / 54510 by limiting at least one limiting nutrient component, preferably by limiting at least one nitrogen source.

[0058] The chloride content in the fermentation medium is preferably below 2 g / l, in particular below 1 g / l, preferably below 500 mg / l and particularly preferably below 250 mg / l throughout the fermentation, at least during the oil production phase.

[0059] In addition to the sulfates and any chlorides used, further salts may optionally be used during the fermentation, in particular those selected from the group consisting of sodium carbonate, sodium bicarbonate, soda ash or inorganic phosphorus compounds. If further salts are used, these are each preferably used in an amount of less than 12 g / l, in particular less than 8 g / l and particularly preferably less than 5 g / l. At the start of the main fermentation, the total salt content in the fermentation medium is preferably 5 to 30 g / l, in particular 10 to 20 g / l.

[0060] Furthermore, organic phosphorus compounds and / or known growth-promoting substances, such as yeast extract or corn steep liquor, may be added in order to have a positive influence on the fermentation.

[0061] The cells are preferably fermented at a pH of 3 to 11, in particular 4 to 10, and preferably at a temperature of at least 20° C., in particular 20° C. to 40° C., and particularly preferably at least 30° C. A typical fermentation process takes about 100 hours.

[0062] According to the present invention, preferably the cell is fermented to a biomass density of at least 50 g / l, 60 g / l or 70 g / l, particularly 50 to 250 g / l or 60 to 220 g / l, preferably at least 80 g / l or 90 g / l, particularly 80 to 200 g / l, particularly preferably at least 100 g / l, and particularly 100 to 180 g / l. In this context, data are based on the dry biomass content of the fermentation broth total volume after the fermentation is complete. The content of dry biomass is by filtering the biomass from the fermentation broth, washing with water subsequently, then drying completely (e.g. in a microwave oven) and finally confirming that dry weight is measured.

[0063] In a preferred embodiment according to the invention, just before the start of the oil production phase, preferably after the biomass density has reached at least 50 g, particularly preferably at least 80 g, 100 g, 120 g or 140 g per liter of fermentation medium, the sulfate concentration is reduced to less than 5 g sulfate per kilogram of lipid-free biomass, preferably less than 4 g, 3 g, 2 g or 1 g sulfate per kilogram of lipid-free biomass, and in particular less than 0.5 g, 0.1 or 0.05 g sulfate per kilogram of lipid-free biomass, and preferably remains below this concentration during the remaining fermentation.

[0064] After harvesting the cells, or optionally even shortly before harvesting the cells, the cells are preferably pasteurized to kill the cells and inactivate enzymes that may promote lipid degradation.

[0065] After the fermentation is complete, the fermentation broth obtained can be subjected directly or optionally after prior concentration to an oil separation process in order to extract the oil present. Such oil separation processes are described, for example, in WO 01 / 53512 and WO 2011 / 153246.

[0066] Alternatively, the PUFA-containing biomass can also be harvested after fermentation is complete. For this purpose, the fermentation broth is preferably also first concentrated.

[0067] According to the present invention, the fermentation broth is preferably concentrated by centrifugation, filtration, decantation and / or solvent evaporation to first separate most of the fermentation medium from the biomass. Preferably, the solvent evaporation is achieved by spray drying or vacuum evaporation using a drum dryer, a tunnel dryer. In particular, a rotary evaporator, a thin film evaporator or a falling film evaporator can also be used to achieve solvent evaporation. A suitable alternative to solvent evaporation is, for example, reverse osmosis to concentrate the fermentation broth.

[0068] To obtain biomass, the concentrated fermentation broth thus obtained is preferably further dried, preferably by fluidized bed granulation. Preferably, the moisture content of the biomass is reduced to less than 15% by weight, particularly less than 10% by weight, and particularly preferably less than 5% by weight, by a subsequent drying method.

[0069] In one embodiment particularly preferred according to the invention, the biomass is dried according to the invention in a fluidized bed granulation process or a nozzle spray drying process, for example as described in WO 2015 / 052048.

[0070] In the drying process, silicon dioxide can optionally be added to the biomass as an anti-caking agent, so that the biomass can be converted into a manageable state. For this purpose, the fermentation broth containing the biomass and the silicon dioxide are preferably sprayed into a specific drying zone. Alternatively, the biomass is preferably mixed with the silicon dioxide only after the drying process. In this regard, reference is also made in particular to patent application WO 2015 / 052048.

[0071] In a preferred embodiment, after the drying process, the biomass to be used according to the invention has a silicon dioxide (especially hydrophilic or hydrophobic silicon dioxide) concentration of 0.2 to 10% by weight, in particular 0.5 to 5% by weight and especially 0.5 to 2% by weight.

[0072] Preferably, a free-flowing fine-grained or coarse-grained product, preferably granules, is obtained by the drying process. A product having the desired particle size can optionally be obtained from granules obtained by sieving or dust separation.

[0073] If a free-flowing fine-grained powder is obtained, it can optionally be converted into a coarse-grained, readily free-flowing and substantially dust-free product, which can be stored by suitable compacting or granulation methods.

[0074] Conventional organic or inorganic adjuvants or carriers, such as starch, gelatin, cellulose derivatives or similar substances, which are usually used as binders, gelling agents or thickeners in food processing or feed processing, can optionally be used in the subsequent granulation or compaction process.

[0075] According to the invention, "free-flowing" is understood to mean a powder that can flow unhindered from a range of glass outflow containers with different outlet opening sizes, at least from containers with 5 mm openings (Klein: Seifen, Fette, Wachse 94, 12 (1968)).

[0076] According to the invention, "fine particle size" is understood to mean a powder having a major fraction (>50%) of particles with a diameter of 20 to 100 micrometers.

[0077] According to the invention, "coarse-grained" is understood to mean a powder having a major fraction (>50%) of particles with a diameter of 100 to 2500 micrometers.

[0078] According to the invention, "dust-free" is understood to mean powders containing only a low fraction (<10%, preferably <5%) of particles with a diameter below 100 micrometers.

[0079] According to the invention, the particle size is preferably determined by laser diffraction spectroscopy. In the textbook "Particle size" by RH Müller and R. Schuhmann, Wissenschaftliche Verlagsgesellschaft Stuttgart (1996) Available methods are described in the textbook "Introduction to Particle Technology" by M. Rhodes, Wiley & Sons (1998). Since a variety of methods are available, it is preferred to use the available method first cited in the textbook by RH Müller and R. Schuhmann for measuring particle size.

[0080] The product obtained by the drying process according to the invention preferably has a fraction of at least 80% by weight, in particular at least 90% by weight, particularly preferably at least 95% by weight, of particles having a particle size of 100 to 3500 μm, preferably 100 to 3000 μm and in particular 100 to 2500 μm.

[0081] In this case, the product obtained by the fluidized bed granulation process according to the invention preferably has a fraction of particles having a particle size of 200 to 3500 μm, preferably 300 to 3000 μm, in particular 500 to 2500 μm, of at least 80% by weight, in particular at least 90% by weight, particularly preferably at least 95% by weight.

[0082] In contrast, the product obtained by the spray-drying process according to the invention preferably has a fraction of particles having a particle size of 100 to 500 μm, preferably 100 to 400 μm and in particular 100 to 300 μm of at least 80% by weight, in particular at least 90% by weight and particularly preferably at least 95% by weight.

[0083] The product obtained according to the invention by the spray-drying process and the subsequent granulation process preferably has a fraction of at least 80% by weight, in particular at least 90% by weight, particularly preferably at least 95% by weight, of particles having a particle size of 100 to 1000 μm.

[0084] The fraction of dust, ie particles having a particle size of less than 100 μm, is preferably at most 10% by weight, in particular at most 8% by weight, particularly preferably at most 5% by weight and in particular at most 3% by weight.

[0085] The bulk density of the product according to the invention is preferably from 400 to 800 kg / m 3 , particularly preferably 450 to 700 kg / m 3 .

[0086] Therefore, the present invention further provides a feed comprising the biomass according to the invention and further feed ingredients.

[0087] In this regard, the additional feed ingredients are preferably selected from protein-containing, carbohydrate-containing, nucleic acid-containing and fat-soluble components and, if appropriate, from other fat-containing components and, in addition, from other additives such as minerals, vitamins, pigments and amino acids. In addition to the nutrients, structurants may also be present, for example to improve the texture or appearance of the feed. In addition, adhesives may also be used, for example, to influence the consistency of the feed. A preferably used component which constitutes both a nutrient and a structurant is starch.

[0088] According to the invention, the feed according to the invention or the composition for producing the feed according to the invention is preferably characterized by the fact that it contains the biomass according to the invention in an amount of 1 to 25% by weight, preferably 2 to 20% by weight, in particular 3 to 15% by weight and especially 4 to 12% by weight.

[0089] The feed or composition for producing a feed preferably additionally has at least one, preferably all, of the following properties:

[0090] a) a total protein content of 33 to 67% by weight, preferably 39 to 61% by weight, in particular 44 to 55% by weight;

[0091] b) a total fat content of 5 to 25% by weight, preferably 8 to 22% by weight, in particular 10 to 20% by weight, especially 12 to 18% by weight;

[0092] c) a total starch content of up to 25% by weight, in particular up to 20% by weight, preferably from 6 to 17% by weight, particularly preferably from 8 to 14% by weight;

[0093] d) a polyunsaturated fatty acid (PUFA) content of 2 to 13% by weight, preferably 3 to 11% by weight, in particular 4 to 10% by weight, especially 5.5 to 9% by weight;

[0094] e) an ω-3 fatty acid content of 1 to 7% by weight, preferably 1.5 to 5.5% by weight, in particular 2 to 5% by weight, especially 2.5 to 4.5% by weight;

[0095] f) a DHA content of 0.5 to 3% by weight, preferably 0.8 to 2.8% by weight, in particular 1 to 2.8% by weight, especially 1.3 to 2.4% by weight, in particular 1.3 to 2.2% by weight.

[0096] Therefore, the present invention preferably also provides a feed or a composition suitable for producing a feed, which has at least one, preferably all, of the following properties:

[0097] a) a total protein content of 33 to 67% by weight, preferably 39 to 61% by weight, in particular 44 to 55% by weight;

[0098] b) a total fat content of 5 to 25% by weight, preferably 8 to 22% by weight, in particular 10 to 20% by weight, especially 12 to 18% by weight;

[0099] c) a total starch content of up to 25% by weight, in particular up to 20% by weight, preferably from 6 to 17% by weight, particularly preferably from 8 to 14% by weight;

[0100] d) a polyunsaturated fatty acid (PUFA) content of 2 to 13% by weight, preferably 3 to 11% by weight, in particular 4 to 10% by weight, especially 5.5 to 9% by weight;

[0101] e) an ω-3 fatty acid content of 1 to 7% by weight, preferably 1.5 to 5.5% by weight, in particular 2 to 5% by weight, especially 2.5 to 4.5% by weight;

[0102] f) a DHA content of 0.5 to 3% by weight, preferably 0.8 to 2.8% by weight, in particular 1 to 2.8% by weight, especially 1.3 to 2.4% by weight, in particular 1.3 to 2.2% by weight.

[0103] Therefore, the present invention preferably also provides a feed or a composition suitable for producing a feed, which has at least one, preferably all, of the following properties:

[0104] a) a total protein content of 33 to 67% by weight, preferably 39 to 61% by weight, in particular 44 to 55% by weight;

[0105] b) a total fat content of 5 to 25% by weight, preferably 8 to 22% by weight, in particular 10 to 20% by weight, especially 12 to 18% by weight;

[0106] c) a total starch content of up to 25% by weight, in particular up to 20% by weight, preferably from 6 to 17% by weight, particularly preferably from 8 to 14% by weight;

[0107] d) a content of biomass according to the invention, in particular of Labyrinthaceae biomass according to the invention, preferably of Thraustochytridaceae biomass according to the invention, of 2 to 24% by weight, preferably of 4 to 22% by weight, in particular of 9 to 20% by weight, especially of 11 to 18% by weight;

[0108] e) a polyunsaturated fatty acid (PUFA) content of 2 to 13% by weight, preferably 3 to 11% by weight, in particular 4 to 10% by weight, especially 5.5 to 9% by weight;

[0109] f) an ω-3 fatty acid content of 1 to 7% by weight, preferably 1.5 to 5.5% by weight, in particular 2 to 5% by weight, especially 2.5 to 4.5% by weight;

[0110] g) a DHA content of 0.5 to 3% by weight, preferably 0.8 to 2.8% by weight, in particular 1 to 2.8% by weight, especially 1.3 to 2.4% by weight, in particular 1.3 to 2.2% by weight.

[0111] Therefore, the present invention preferably also provides a feed or a composition suitable for producing a feed, which has at least one, preferably all, of the following properties:

[0112] a) a total protein content of 33 to 67% by weight, preferably 39 to 61% by weight, in particular 40 to 50% by weight;

[0113] b) a total fat content of 5 to 25% by weight, preferably 8 to 22% by weight, in particular 10 to 20% by weight, especially 12 to 18% by weight;

[0114] c) a total starch content of up to 25% by weight, in particular up to 20% by weight, preferably from 6 to 17% by weight, particularly preferably from 8 to 14% by weight;

[0115] d) a content of 1 to 25% by weight, preferably 2 to 20% by weight, in particular 3 to 15% by weight, especially 4 to 12% by weight of Aurantiochytrium or Schizochytrium biomass according to the invention, preferably Aurantiochytrium limacinum biomass according to the invention, in particular Aurantiochytrium limacinum SR21 biomass according to the invention;

[0116] e) a polyunsaturated fatty acid (PUFA) content of 2 to 13% by weight, preferably 3 to 11% by weight, in particular 4 to 10% by weight, especially 5.5 to 9% by weight;

[0117] f) an ω-3 fatty acid content of 1 to 7% by weight, preferably 1.5 to 5.5% by weight, in particular 2 to 5% by weight, especially 2.5 to 4.5% by weight;

[0118] g) a DHA content of 0.5 to 3% by weight, preferably 0.8 to 2.8% by weight, in particular 1 to 2.8% by weight, especially 1.3 to 2.4% by weight, in particular 1.3 to 2.2% by weight.

[0119] By extruding the above composition, an extrudate can be obtained. The present invention preferably provides such an extrudate. In this regard, the extrusion of the feed is preferably carried out with an energy input of 12 to 28 Wh / kg, in particular 14 to 26 Wh / kg, particularly preferably 16 to 24 Wh / kg, in particular 18 to 22 Wh / kg.

[0120] In this regard, a screw extruder or a twin-screw extruder is preferably used in the extrusion process. The extrusion process is preferably carried out at a temperature of 80 to 220° C., in particular 80 to 130° C., especially 95 to 110° C., a pressure of 10 to 40 bar and a shaft rotation speed of 100 to 1000 rpm, in particular 300 to 700 rpm. The residence time of the starting mixture is preferably 5 to 30 seconds, in particular 10 to 20 seconds.

[0121] The extrusion process may optionally include a densification step and / or a compression step.

[0122] The components are preferably mixed intimately with one another before the extrusion process is carried out. This is preferably carried out in a drum equipped with blades. In a preferred embodiment, this mixing step comprises the injection of steam, in particular in order to cause swelling of the starch which is preferably present. In this regard, the injection of steam is preferably carried out at a pressure of 1 to 5 bar, particularly preferably at a pressure of 2 to 4 bar.

[0123] Before mixing with the algal biomass, the other feed ingredients are preferably pulverized, if necessary, in order to ensure that a homogeneous mixture is obtained during the mixing step. The pulverization of the other feed ingredients can be carried out, for example, using a hammer mill.

[0124] The produced extrudate preferably has a diameter of 1 to 14 mm, preferably 2 to 12 mm, in particular 2 to 6 mm, and preferably also has a length of 1 to 14 mm, preferably 2 to 12 mm, in particular 2 to 6 mm. During extrusion, the length of the extrudate is set using a cutting tool. The length of the extrudate is preferably selected so that it corresponds approximately to the diameter of the extrudate. The diameter of the extrudate is defined by selecting the screen diameter.

[0125] In one preferred embodiment according to the invention, the extrudate obtained is loaded with oil after the extrusion process. For this purpose, the extrudate is preferably first dried to a moisture content of not more than 5% by weight. According to the invention, the extrusion product can be loaded with oil by, for example, placing the extrudate in oil or spraying the extrudate with oil; however, according to the invention, vacuum coating is preferred.

[0126] In this way, a feed is obtained which contains the biomass according to the invention in an amount of preferably 1 to 25% by weight, in particular 2 to 20% by weight, particularly preferably 3 to 15% by weight and in particular 4 to 12% by weight.

[0127] Accordingly, the feed preferably additionally has at least one, preferably all, of the following characteristics:

[0128] a) a total protein content of 30 to 60% by weight, preferably 35 to 55% by weight, in particular 40 to 50% by weight;

[0129] b) a total fat content of 15 to 35% by weight, preferably 18 to 32% by weight, in particular 20 to 30% by weight, especially 22 to 28% by weight;

[0130] c) a total starch content of up to 25% by weight, in particular up to 20% by weight, preferably from 5 to 15% by weight, particularly preferably from 7 to 13% by weight;

[0131] d) a polyunsaturated fatty acid (PUFA) content of 2 to 12% by weight, preferably 3 to 10% by weight, in particular 4 to 9% by weight, especially 5 to 8% by weight;

[0132] e) an ω-3 fatty acid content of 1 to 6% by weight, preferably 1.5 to 5% by weight, in particular 2 to 4.5% by weight, especially 2.5 to 4% by weight;

[0133] f) a DHA content of 0.5 to 3% by weight, preferably 0.8 to 2.5% by weight, in particular 1 to 2.5% by weight, especially 1.2 to 2.2% by weight, in particular 1.2 to 2.0% by weight.

[0134] Therefore, the present invention preferably also provides a feed, in particular an extrudate, having at least one, preferably all, of the following properties:

[0135] a) a total protein content of 30 to 60% by weight, preferably 35 to 55% by weight, in particular 40 to 50% by weight;

[0136] b) a total fat content of 15 to 35% by weight, preferably 18 to 32% by weight, in particular 20 to 30% by weight, especially 22 to 28% by weight;

[0137] c) a total starch content of up to 25% by weight, in particular up to 20% by weight, preferably from 5 to 15% by weight, particularly preferably from 7 to 13% by weight;

[0138] d) a polyunsaturated fatty acid (PUFA) content of 2 to 12% by weight, preferably 3 to 10% by weight, in particular 4 to 9% by weight, especially 5 to 8% by weight;

[0139] e) an ω-3 fatty acid content of 1 to 6% by weight, preferably 1.5 to 5% by weight, in particular 2 to 4.5% by weight, especially 2.5 to 4% by weight;

[0140] f) a DHA content of 0.5 to 3% by weight, preferably 0.8 to 2.5% by weight, in particular 1 to 2.5% by weight, especially 1.2 to 2.2% by weight, in particular 1.2 to 2.0% by weight.

[0141] Therefore, the present invention preferably also provides a feed, in particular an extrudate, having at least one, preferably all, of the following properties:

[0142] a) a total protein content of 30 to 60% by weight, preferably 35 to 55% by weight, in particular 40 to 50% by weight;

[0143] b) a total fat content of 15 to 35% by weight, preferably 18 to 32% by weight, in particular 20 to 30% by weight, especially 22 to 28% by weight;

[0144] c) a total starch content of up to 25% by weight, in particular up to 20% by weight, preferably from 5 to 15% by weight, particularly preferably from 7 to 13% by weight;

[0145] d) a content of 1 to 25% by weight, preferably 2 to 20% by weight, in particular 3 to 15% by weight, especially 4 to 12% by weight of biomass according to the invention, in particular Labyrinthaceae biomass according to the invention, preferably Thraustochytridaceae biomass according to the invention;

[0146] e) a polyunsaturated fatty acid (PUFA) content of 2 to 12% by weight, preferably 3 to 10% by weight, in particular 4 to 9% by weight, especially 5 to 8% by weight;

[0147] f) an ω-3 fatty acid content of 1 to 6% by weight, preferably 1.5 to 5% by weight, in particular 2 to 4.5% by weight, especially 2.5 to 4% by weight;

[0148] g) a DHA content of 0.5 to 3% by weight, preferably 0.8 to 2.5% by weight, in particular 1 to 2.5% by weight, especially 1.2 to 2.2% by weight, in particular 1.2 to 2.0% by weight.

[0149] Therefore, the present invention preferably also provides a feed, in particular an extrudate, having at least one, preferably all, of the following properties:

[0150] a) a total protein content of 30 to 60% by weight, preferably 35 to 55% by weight, in particular 40 to 50% by weight;

[0151] b) a total fat content of 15 to 35% by weight, preferably 18 to 32% by weight, particularly 20 to 30% by weight, especially 22 to 28% by weight;

[0152] c) a total starch content of up to 25% by weight, in particular up to 20% by weight, preferably from 5 to 15% by weight, particularly preferably from 7 to 13% by weight;

[0153] d) a content of 1 to 25% by weight, preferably 2 to 20% by weight, in particular 3 to 15% by weight, especially 4 to 12% by weight of Aurantiochytrium or Schizochytrium biomass according to the invention, preferably Aurantiochytrium limacinum biomass according to the invention, in particular Aurantiochytrium limacinum SR21 biomass according to the invention;

[0154] e) a polyunsaturated fatty acid (PUFA) content of 2 to 12% by weight, preferably 3 to 10% by weight, particularly 4 to 9% by weight and especially 5 to 8% by weight;

[0155] f) an ω-3 fatty acid content of 1 to 6% by weight, preferably 1.5 to 5% by weight, in particular 2 to 4.5% by weight, especially 2.5 to 4% by weight;

[0156] g) a DHA content of 0.5 to 3% by weight, preferably 0.8 to 2.5% by weight, in particular 1 to 2.5% by weight, especially 1.2 to 2.2% by weight, in particular 1.2 to 2.0% by weight.

[0157] According to the invention, in addition to the biomass to be used according to the invention, the fat-containing components used may be fats, in particular oils, of both animal and vegetable origin. According to the invention, suitable fat-containing components are in particular vegetable oils, for example soybean oil, rapeseed oil, sunflower oil, linseed oil or palm oil and mixtures thereof. In addition, fish oils can also optionally be used as fat-containing components in small amounts.

[0158] According to the invention, the protein-containing component used may be, for example, soy protein, pea protein, wheat gluten or corn gluten and mixtures thereof.

[0159] The following examples can be used as protein-containing components that additionally contain fat: fish meal, krill meal, mussel meal, squid meal or shrimp shells. These are hereinafter grouped under the term "marine meal". In a preferred embodiment, the feed according to the invention comprises marine meal, preferably fish meal, in an amount of 3 to 18% by weight, in particular 5 to 15% by weight, especially 7 to 13% by weight.

[0160] The carbohydrate-containing component used may be, for example, wheat flour, sunflower flour or soya flour and mixtures thereof.

[0161] When using the feed according to the invention, in particular the oil-coated extrudates according to the invention, in animal farming, it has become increasingly apparent that this in particular promotes the growth of the animals and improves the stress level of the animals.

[0162] The present invention further provides a method for breeding animals, characterized in that the feed according to the present invention is administered thereto.

[0163] In this regard, the present invention provides in particular a method for increasing the growth of animals, characterized in that a feed according to the invention is administered to them.

[0164] The present invention further provides, in particular analogously, a method for increasing the fraction of ω-3 fatty acids, in particular DHA, in the muscle tissue of an animal, characterized in that a feed according to the invention is administered thereto.

[0165] Preferably, in the method according to the invention the feed is administered at least once every two days, preferably at least once a day.

[0166] The invention further similarly provides the use of a feed according to the invention for increasing the growth of an animal.

[0167] The invention likewise further provides the use of a feed according to the invention for increasing the ω-3 fatty acid fraction in the muscle tissue of an animal.

[0168] The present invention also further provides the use of the feed according to the present invention for improving the physical condition of an animal, in particular for improving the stress level of an animal.

[0169] The invention likewise further provides for the use of a feed according to the invention in farming which allows reduced stress for the animals.

[0170] The farmed animals fed with the feed of the present invention are preferably poultry, pigs or cattle.

[0171] However, the farmed animals are particularly preferably marine animals, particularly preferably finfish or crustaceans. These include in particular carp, tilapia, catfish, tuna, salmon, trout, barramundi, bream, golden perch, cod, shrimp, lobster, crab, prawns and crayfish. The farmed animals are particularly preferably salmon. In this context, preferred types of salmon are Atlantic salmon, sockeye salmon, masusalmon, king salmon, king salmon, coho salmon, Danube salmon, Pacific salmon and pink salmon.

[0172] The farmed animals can also be fish, which are subsequently processed into fishmeal or fish oil. In this context, the fish are preferably herring, pollock, menhaden, anchovies, capelin or cod. The fishmeal or fish oil thus obtained can in turn be used in aquaculture to farm food fish or crustaceans.

[0173] However, farmed animals may also be small organisms used as feed in aquaculture. These small organisms may take the form of, for example, nematodes, crustaceans or rotifers.

[0174] The culture of marine animals can be carried out in ponds, tanks, basins or in isolated areas of the sea or lake, especially in this case in cages or net cages. Culture can be used to grow finished food fish, but it can also be used to raise fry that are subsequently released to replenish wild fish stocks.

[0175] In salmon farming, the fish are preferably first grown as smolts in freshwater tanks or artificial raceways and then continue to grow in cages or nets that float in the sea and are preferably anchored in bays or fjords.

[0176] Accordingly, the feed according to the present invention is preferably a feed for breeding of the above-mentioned animals.

[0177] Exemplary embodiments

[0178] Example 1 Production of biomass by fermenting Aurantiochytrium limacinum SR21 in a medium with a high sulfate content and subsequently drying the biomass

[0179] The cells were cultured for about 70 hours in a fed process using a steel fermenter with a fermenter volume of 2 liters, with a total starting mass of about 700 g and a final total mass of about 1.5 kg obtained. During the process, a glucose solution (570 g / kg glucose) was metered in ("fed-batch process").

[0180] The composition of the starting medium is as follows:

[0181] Medium 1: 20 g / kg glucose; 4 g / kg yeast extract; 2 g / kg ammonium sulfate; 12 g / kg or 16 g / kg sodium sulfate; 2.46 g / kg magnesium sulfate (heptahydrate); 0.45 g / kg potassium chloride; 4.5 g / kg potassium dihydrogen phosphate; 0.1 g / kg thiamine (HCl); 5 g / kg trace element solution.

[0182] The composition of the trace element solution is as follows: 35 g / kg hydrochloric acid (37%); 1.86 g / kg manganese chloride (tetrahydrate); 1.82 g / kg zinc sulfate (heptahydrate); 0.818 g / kg sodium EDTA; 0.29 g / kg boric acid; 0.24 g / kg sodium molybdate (dihydrate); 4.58 g / kg calcium chloride (dihydrate); 17.33 g / kg ferrous sulfate (heptahydrate); 0.15 g / kg cupric chloride (dihydrate).

[0183] Cultivation was carried out under the following conditions: culture temperature 28°C; aeration rate 0.5 vvm, stirrer speed 600 to 1950 rpm, and pH during the growth phase was controlled to 4.5 using aqueous ammonia (25% v / v). Before the oil production phase was started by limiting phosphate and nitrogen, fermentation was carried out until the biomass density reached 80 g / l. Due to the high sulfate content in the starting medium until the end of the fermentation (i.e., including until the end of the oil production phase), the sulfate concentration was always higher than the saturation limit of the cells, which was about 5 g per kilogram of dry biomass. The biomass obtained will be referred to as "biomass 1" (12 g / kg sodium sulfate in the starting medium) and "biomass 2" (16 g / kg sodium sulfate in the starting medium) hereinafter.

[0184] Example 2: Production of biomass by fermentation of Aurantiochytriumlimacinum SR21 in a medium with a low sulfate content and subsequent drying of the biomass

[0185] The cells were cultured in a fed process using a steel fermenter with a fermenter volume of 2 liters for about 70 hours, with a total starting mass of about 712 g and a final total mass of about 1.5 kg obtained. During the process, a glucose solution (570 g / kg glucose) was metered in ("fed-batch process").

[0186] The composition of the starting medium is as follows:

[0187] Medium 1: 20 g / kg glucose; 4 g / kg yeast extract; 0 g / kg ammonium sulfate; 2.46 g / kg magnesium sulfate (heptahydrate); 0.45 g / kg potassium chloride; 4.5 g / kg potassium dihydrogen phosphate; 0.1 g / kg thiamine (HCl); 5 g / kg trace element solution.

[0188] The composition of the trace element solution is as follows: 35 g / kg hydrochloric acid (37%); 1.86 g / kg manganese chloride (tetrahydrate); 1.82 g / kg zinc sulfate (heptahydrate); 0.818 g / kg sodium EDTA; 0.29 g / kg boric acid; 0.24 g / kg sodium molybdate (dihydrate); 4.58 g / kg calcium chloride (dihydrate); 17.33 g / kg ferrous sulfate (heptahydrate); 0.15 g / kg cupric chloride (dihydrate).

[0189] The cultivation was carried out under the following conditions: cultivation temperature 28°C; aeration rate 0.5 vvm, stirrer speed 600 to 1950 rpm, and ammonia water (25% v / v) was used to control the pH during the growth period to 4.5. Before starting the oil production period by limiting phosphate and nitrogen, the fermentation was carried out until the biomass density reached 80 g / l. After starting the oil production period, the sulfate concentration had dropped to below the detection limit of 0.05 g per kilogram of fermentation medium, and the sulfate concentration was correspondingly below the detection limit during the entire oil production period. And since sulfate was not added in a metered manner, the sulfate concentration dropped to zero during the oil production period. The biomass obtained will be referred to as "biomass 3" below.

[0190] Example 3: Production of biomass by fermentation of Aurantiochytriumlimacinum SR21 in a medium with low sulfate content, with addition of sulfate during fermentation and subsequent drying of the biomass

[0191] The cells were cultured for about 70 hours in a feed process using a steel fermenter with a fermenter volume of 2 liters, with a total starting mass of about 712 g and a final total mass of about 1.5 kg obtained. During the process, a glucose solution (570 g / kg glucose) was metered in ("batch feeding process"), while sodium sulfate solutions of different concentrations (7.4, 14.8 or 22.2 g / kg sodium sulfate) or ammonium sulfate solutions of different concentrations (6.8, 13.6 or 20.4 g / kg ammonium sulfate) were metered in 6 different batches at a constant feed rate of 3 g / h. The feed rate of the sulfate solution was selected so that the sulfate content in the storage phase remained below the detection limit of sulfate (0.05 g / l).

[0192] In addition, to exclude the "dilution effect", water at the same feed rate (3 g / h) was used in one batch as a negative control.

[0193] The composition of the starting medium is as follows:

[0194] Medium 1: 20 g / kg glucose; 4 g / kg yeast extract; 2 g / kg ammonium sulfate; 2.46 g / kg magnesium sulfate (heptahydrate); 0.45 g / kg potassium chloride; 4.5 g / kg potassium dihydrogen phosphate; 0.1 g / kg thiamine (HCl); 5 g / kg trace element solution.

[0195] The composition of the trace element solution is as follows: 35 g / kg hydrochloric acid (37%); 1.86 g / kg manganese chloride (tetrahydrate); 1.82 g / kg zinc sulfate (heptahydrate); 0.818 g / kg sodium EDTA; 0.29 g / kg boric acid; 0.24 g / kg sodium molybdate (dihydrate); 4.58 g / kg calcium chloride (dihydrate); 17.33 g / kg ferrous sulfate (heptahydrate); 0.15 g / kg cupric chloride (dihydrate).

[0196] The cultivation was carried out under the following conditions: cultivation temperature 28°C; aeration rate 0.5 vvm, stirrer speed 600 to 1950 rpm, and the pH during the growth period was controlled to 4.5 using aqueous ammonia (25% v / v). Fermentation was carried out until the biomass density reached 50 g / l before the oil production period was started by limiting phosphate and nitrogen. After the oil production period was started, the sulfate concentration had dropped to below the detection limit of 0.05 g per kilogram of fermentation medium, and the sulfate concentration was correspondingly below the detection limit throughout the oil production period. However, due to the continuous metering addition of sulfate, the sulfate concentration in the culture medium was prevented from dropping to zero. In the following, the biomass with sodium sulfate feed is referred to as "biomass 3, 4 and 5", and the biomass with ammonium sulfate feed is referred to as "biomass 6, 7 and 8".

[0197] Example 4: Examination and comparison of obtained biomass and methods

[0198] After the incubation process, the fermentation broth was heated to 60°C for 20 minutes to prevent further cell activity.

[0199] This is followed by a two-stage drying of the biomass: First, the fermentation broth is concentrated by evaporation to a dry mass of about 20 wt%. Thereafter, the production of TM The concentrated fermentation broth was spray-dried in a spray dryer (GEA NIRO) at a drying air inlet temperature of 340° C. Spray drying thus gave a powder having a dry mass of more than 95% by weight.

[0200] Table 1: Comparison of biomass obtained

[0201]

[0202] Biomasses 1 and 2 are biomasses obtained by fermentation at high sulfate content according to Example 1, biomass 3 is biomass obtained by fermentation at low sulfate content according to Example 2, and biomasses 4-9 are obtained by fermentation at low sulfate content and continuous feeding of sulfate at a low metering rate according to Example 3.

[0203] It can be seen that the fermentation process producing biomasses 4-9 gave significantly higher DHA yields and also gave more product and purer product than the process carried out at low sulfate content without continuous sulfate feed (biomass 3) or at high sulfate content (biomass 1 and 2). The preferred biomass according to the invention obtained according to the process of Example 3 contained sulfate in an amount of 12 to 16 g per kg of biomass.

Claims

1. A method for producing a biomass containing PUFA by culturing PUFA-producing cells in a fermentation medium, the method comprising a growth phase and an oil production phase, characterized in that: Sulfate is present in the fermentation medium during the entire period of the oil production phase, but the sulfate concentration is below the saturation limit of the cells during at least 50% of the period of the oil production phase, and during the period of the oil production phase when the sulfate concentration is below the saturation limit, sulfate is continuously metered in an amount to give a sulfate concentration in the fermentation medium below the detection limit of 0.05 g per kg of fermentation medium, wherein the PUFA-producing cells are those of the genus: Aurantiochytrium or Schizochytrium.

2. The method of claim 1, characterized in that The sulfate concentration is below the saturation limit of the cells during at least the last 50%, 70%, 80% or 90% of the oil production period, or during the entire oil production period.

3. The method according to claim 1 or 2, characterized in that The cells are cultured during the growth phase until the biomass density is at least 50 g per liter of fermentation medium.

4. The method according to claim 1 or 2, characterized in that The cells are cultured during the growth phase until the biomass density is between 50 and 250 g per liter of fermentation medium.

5. The method according to claim 1 or 2, characterized in that The cells are cultured during the growth phase until the biomass density is at least 80 g, 100 g, 120 g, or 140 g per liter of fermentation medium.

6. The method according to claim 1 or 2, characterized in that The cells are cultured during the growth phase until the biomass density is between 80 g, 100 g, 120 g, or 140 g and 200 g or 220 g per liter of fermentation medium.

7. The method according to claim 1 or 2, characterized in that The oil production phase is initiated by limiting at least one limiting nutrient component.

8. The method according to claim 1 or 2, characterized in that The oil production period is initiated by limiting the nitrogen source.

9. The method of claim 1 or 2, characterized in that Sulfate is metered continuously into the fermentation medium at a rate of 5 to 100 mg per kg of fermentation medium during at least 50% of the oil production period.

10. The method according to claim 1 or 2, characterized in that Sulfate is continuously metered into the fermentation medium at a rate of 10 to 50 mg per kg of fermentation medium during at least 50% of the oil production period.

11. The method according to claim 1 or 2, characterized in that Sulfate was initially provided via the starter medium and sulfate metering was started as soon as the sulfate concentration had fallen below 2 g per kg of fermentation medium.

12. The method according to claim 1 or 2, characterized in that Sulfate was first provided via the starter medium and sulfate metering was started as soon as the sulfate concentration had fallen below 1 g per kg of fermentation medium.

13. The method according to claim 1 or 2, characterized in that Sulfate was initially provided via the starter medium and sulfate metering was started as soon as the sulfate concentration had fallen below 0.5 g per kg of fermentation medium.

14. The method according to claim 1 or 2, characterized in that Until the start of sulfate metering, the sulfate concentration was below 0.5 g per kg of fermentation medium.

15. The method of claim 1 or 2, characterized in that During the entire fermentation period, a fermentation medium is used having a chloride concentration of less than 1 g / l, less than 500 mg / l, or less than 250 mg / l.

16. The method of claim 1 or 2, characterized in that The cells are those of the species Aurantiochytrium limacinum or Schizochytrium limacinum.

17. The method of claim 1 or 2, characterized in that The PUFAs are omega-3 fatty acids.

18. The method of claim 1 or 2, characterized in that The PUFA is DHA and / or EPA.

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