EP3877535A1

EP3877535A1 - Method for producing a biomass which can be easily broken down and which has an increased content of polyunsaturated fatty acids

Info

Publication number: EP3877535A1
Application number: EP19802122.2A
Authority: EP
Inventors: Horst Priefert; Jens Schneider; Joachim WINDAU; Gabriel ZÁVODSKÝ; Christian Rabe
Original assignee: Evonik Operations GmbH
Current assignee: Evonik Operations GmbH
Priority date: 2018-11-09
Filing date: 2019-11-07
Publication date: 2021-09-15
Also published as: CN112955564A; BR112021008853A2; WO2020094751A1; CA3118657A1; US20220017930A1

Abstract

According to the invention, it has been found that when cultivating PUFA-producing cells, if the quantity of sulfate to be used is selected such that the sulfate concentration is reduced to null in the last phase of the growth phase of the cells, a biomass is obtained which can be easily broken down and has an increased mass content of polyunsaturated fatty acids (PUFAs) in the final product.

Description

Process for the production of an easily digestible biomass with an increased content of polyunsaturated fatty acids

The present invention relates to a method for producing an easily digestible biomass with an increased content of polyunsaturated fatty acids and to a biomass obtainable by this method.

Processes for producing biomass containing polyunsaturated fatty acids (PUFAs) have already been described in the prior art. Network slime molds are often used, which naturally accumulate polyunsaturated fatty acids in the cell in large quantities as storage lipids.

These microalgae naturally grow in seawater, so that the microalgae were initially cultivated in media with a high chloride content. However, high chloride levels are unsuitable for cultivation in steel bioreactors because they cause corrosion of the metal.

As alternative fermentation media, therefore, those with a low chloride content and instead a high sulfate content are described in the prior art.

However, the Aigen biomass obtained generally has a limited PUFA content, possibly because the high sulfate content means that the proportion of cell wall formed in the final product is relatively high at the expense of the amount of PUFAs contained.

Due to the high sulfate content, the cell wall is also quite stable, which makes it difficult to release the oil from the cells.

Surprisingly, it has been found according to the invention that by limiting the sulfate content during the fermentation, the relative proportion of PUFAs formed can be increased significantly and that the release of the oil from the cells can be significantly facilitated by the reduction in the stability of the cell wall which is achieved at the same time by limiting the sulfate, the cell wall can be destabilized to such an extent that the oil can be released from the cells without great mechanical effort.

The object of the present invention was therefore to provide a method for providing a biomass containing PUFAs, in which the biomass obtained has an increased mass fraction of PUFAs in the final product and at the same time the release of the PUFAs contained in the final product is facilitated. At the same time, it should preferably be possible to use a non-corrosive fermentation medium in the process.

The object of the invention is achieved by a method for producing a biomass containing PUFAs, characterized in that for the production of the biomass, the cells producing PUFAs are cultivated in a fermentation medium, the sulfate content being adjusted so that the sulfate content in the last phase of fermentation falls to zero.

Methods according to the invention preferably comprise a growth phase and a subsequent oil production phase in order to optimize oil production. In the previous growth phase, the cultivation and the increase of the biomass in the Fermentation medium containing PUFAs producing cells - as far as possible

Suppression of oil production so that the highest possible biomass density is set in the medium, while in the subsequent oil production phase, which is usually initiated by special measures, oil production mainly takes place through the cells of the biomass, the growth of the cells being at least largely stopped . This means that in the oil production phase the increase in biomass is at least not primarily due to an increase in the number of cells, but rather to the accumulation of lipids in the interior of the existing cells. - The growth of the cells in the growth phase is made possible by providing optimal growth conditions, while the transition to the oil production phase is limited by limiting one or more limiting factors,

in particular nutrient components such as nitrogen sources can be introduced. According to the invention, the “last phase of the fermentation” preferably means the oil production phase.

Processes according to the invention are preferably characterized in that the sulfate concentration in the fermentation medium drops to zero in the second half of the growth phase, preferably in the last eighth of the growth phase, particularly preferably shortly before the oil production phase begins.

The present invention furthermore relates to biomasses, preferably those which contain cells of the family of the Thraustochytriaceae, which can be obtained by methods according to the invention.

It has been found to be particularly advantageous according to the invention to specify the sulfate content in the medium in such a way that a final sulfate concentration in the biomass obtained of 7 to 10 g per kg of biomass is obtained. The cells then still have sufficient stability to protect the PUFAs contained against undesired oxidative degradation. The amount of

Add sulfate can be easily calculated because the

added sulfate is completely assimilated by the cells. In this way, a biomass is obtained which is easy to digest, but is still sufficiently stable to be able to be converted into a dry biomass. Due to the relatively unstable cell wall, biomasses according to the invention can accordingly preferably use low mechanical forces, preferably already apply a force of up to 12 Wh / kg, particularly preferably up to 10 or 8 Wh / kg, in particular 1 to 12 Wh / kg, 1 up to 10 Wh / kg or 2 to 8 Wh / kg.

Another object of the present invention is also a biomass that can be obtained with a method according to the invention.

Another object of the present invention is therefore in particular a biomass containing PUFAs, which has a sulfate content of 7 to 10 g sulfate, preferably 7.5 to 9.5 g sulfate, per kg biomass.

According to the invention, “sulfate content” is understood to mean the total content of sulfate, ie the content of free and bound, in particular organically bound, sulfate in relation to the biomass. It can be assumed that the majority of the sulfate contained in the biomass is present as a component of exopolysaccharides that contribute to the formation of the cell wall

Microorganisms are involved.

According to the invention, the sulfate content is preferably determined by determining the sulfur content of the biomass obtained, since the majority of the sulfur contained in the biomass can be attributed to the sulfate contained. Sulfur, which can be traced to other sources, is negligible due to the amount of sulfate contained. The amount of sulfate contained can thus be readily determined from the amount of sulfur found.

The sulfur content of the biomass is preferably determined by elemental analysis in accordance with DIN EN ISO 11885. For the analysis of the sulfur content of the biomass

appropriate aliquots of the sample, preferably with nitric acid and

Hydrogen peroxide digested at 240 ° C under pressure to ensure free access to the contained sulfur.

Under "Biomass", "Bio dry mass", "Biomass PUFAs producing cells" or

According to the invention, “bio-dry mass of PUFAs producing cells” is generally to be understood as the determinable bio-dry mass.

The amount of dry biomass contained in a sample is preferably determined as follows: A homogeneous sample is taken and for the purpose of separating the liquid

Components centrifuged. The biomass obtained by centrifugation is then washed with water to bring salts and any other soluble constituents into solution, and then centrifuged again. - The dry biomass obtained in this way is finally in the

Drying cabinet dried overnight. The percentage of dry biomass contained in the sample results from the quotient between the mass of certain

Bio dry mass after drying and initial mass of the examined sample. The dry biomass thus determined also includes the oil formed by the biomass.

The fat content contained in the dry biomass is preferably determined by taking up the dry biomass in a methanol / chloroform solution and subsequent ultrasound treatment of the sample thus obtained. - The sample thus obtained is then saponified with potassium hydroxide and acidified using hydrochloric acid. The free fatty acids are then methylated using BF3 (30% boron trifluoride in methanol) and by partition chromatography using a

Temperature gradients separated. The detection is then carried out using flame ionization detection (FID).

According to the invention, “lipid-free biomass” is understood to mean the dry biomass minus the fat content determined in this way.

According to the invention, the desired sulfate concentration in the medium can be set in different ways.

It is essential according to the invention that the sulfate concentration drops to zero in the course of the fermentation, the sulfate concentration preferably falling to zero in the second half of the growth phase, particularly preferably in the last eighth of the time of the growth phase. It is particularly preferred here if the sulfate concentration shortly before the start of the oil production phase, in particular up to 3 hours, preferably up to 2 hours, especially up to one hour before the start of the oil production phase. - This can be achieved simply by completely introducing the required amount of sulfate in the batch medium at the start of the fermentation (so-called batch process). The amount of sulfate required can be easily calculated since the cells used to form the biomass completely assimilate the sulfate added in a relatively small amount.

If a so-called fed-batch process is used, the amount of sulfate required can alternatively be metered in during the fermentation, or a portion of the sulfate can be introduced accordingly and the rest metered in during the fermentation.

Especially if it turns out during the fermentation that the amount of biomass produced exceeds the originally calculated value, subsequent metering in of sulfate can ensure that the resulting biomass has sufficient cell stability to prevent premature release of the oil.

In a preferred embodiment, the amount of sulfate in the batch medium is selected such that the sulfate concentration changes at least during the first 30% of the time in the growth phase of the cells, preferably at least during the first 40, 50 or 60%, in particular during the first 70, 80 or 90%, the time of the growth phase of the cells, above that

Saturation concentration of the cells is located. - The sulfate concentration is preferably at least 5 g sulfate per kg lipid-free biomass during the specified phase.

According to the invention, sodium sulfate, ammonium sulfate or magnesium sulfate and mixtures thereof are preferably used as the sulfate salt. According to the invention, the sulfate can alternatively or additionally also be introduced by using technical

Raw materials are made that are contaminated or supplemented with sulfate.

It has also surprisingly been found according to the invention that the

Shutting down the sulfate concentration can also be combined with low chloride concentrations so that the biomass can be obtained with a non-corrosive fermentation medium.

A method according to the invention is therefore preferably 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, preferably less than 250 mg / l, during the entire fermentation.

Biomasses according to the invention preferably have a chloride content of at most 2 grams per kg of biomass, in particular 0.5 to 1.8 g, particularly preferably 0.5 to 1.5 g, per kg of biomass.

According to the invention, “chloride content” means the amount of determinable chlorine. The amount of chlorine present can be determined, for example, by elemental analysis according to DIN EN ISO 11885. Chlorine is present in the biomass in the form of salts called "chlorides". When “chlorides” or “chloride ions” are referred to in the present application, only the amount of chloride or detectable chlorine is meant, not the amount of chloride salts, which in addition to the chloride ion are always cationic Include counterions. The cells that produce PUFAs are preferably cells that already naturally produce PUFAs, but it can also be cells that have been enabled to produce PUFAs by appropriate genetic engineering methods. The production can be autotrophic, mixotrophic or heterotrophic.

The biomass according to the invention accordingly comprises such cells and preferably consists essentially of such cells.

The cells are preferably those which produce PUFAs heterotrophically. According to the invention, the cells are preferably algae, fungi, in particular yeasts, or protists. The cells are particularly preferably microorganisms, in particular microbial algae or fungi.

Strains of Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces are particularly suitable as cells of oil-producing yeasts.

A biomass according to the invention preferably comprises cells of the taxon Labyrinthulomycetes (Labyrinthulea, network slime molds, slime networks), in particular those of the family of

Thraustochytriaceae. The genera Althornia, Aplanochytrium, Elnia, Japonochytrium, Schizochytrium, Thraustochytrium, Aurantiochytrium, Oblongichytrium and Ulkenia belong to the family of Thraustochytriaceae. It is particularly preferably cells of the genera Thraustochytrium, Schizochytrium, Aurantiochytrium or Oblongichytrium, especially those of the genus Aurantiochytrium. The trunk is a particularly preferred trunk

Aurantiochytrium limacinum SR21 (IFO 32693).

The biomass according to the invention is preferably the product of a fermentative cultivation process. The biomass can be used in addition to

cells to be digested also contain constituents of the fermentation medium. These components can in particular be salts, anti-foaming agents and unreacted carbon sources and / or nitrogen sources. The cell content in this biomass is preferably at least 70% by weight, preferably at least 75% by weight. The cell content in the biomass can, if necessary, be carried out before the cell disruption process is carried out

corresponding washing steps can be increased, for example, to at least 80 or at least 90% by weight. However, the biomass obtained can also be used directly in the cell disruption process.

The cells of the biomass are preferably distinguished by the fact that they have a PUFA content of at least 20% by weight, preferably at least 30% by weight, in particular at least 40% by weight, in each case based on the dry cell mass.

In a preferred embodiment, a majority of the lipids are in the form of triglycerides, 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 contained in the cell Form of triglycerides are present.

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 contained in the cell are PUFAs. According to the invention, polyunsaturated fatty acids (PUFAs) are understood to mean fatty acids which have at least two CC double bonds. According to the invention, highly unsaturated fatty acids (HUFAs) are preferred among the PUFAs. HUFAs are according to the invention

Understand fatty acids that have at least four C-C double bonds.

The PUFAs can be present in the cell in free form or in bound form. Examples of the presence in bound form are phospholipids and esters of PUFAs, in particular monoacyl, diacyl and triacylglycerides. In a preferred embodiment, a majority of the PUFAs are in the form of triglycerides, 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 contained in the cell Form of triglycerides are present.

Preferred PUFAs are omega-3 fatty acids and omega-6 fatty acids, omega-3 fatty acids being particularly preferred. Preferred omega-3 fatty acids are the

Eicosapentaenoic acid (EPA, 20: 5w-3), in particular the (5Z, 8Z, 1 1 Z, 14Z, 17Z) eicosa-5,8, 1 1, 14, 17-pentaenoic acid, and the docosahexaenoic acid (DHA, 22: 6w-3), especially the

(4Z, 7Z, 10Z, 13Z, 16Z, 19Z) -Docosa-4,7, 10, 13, 16, 19-hexaenoic acid, where the

Docosahexaenoic acid is particularly preferred.

Process for the production of cells containing PUFAs, in particular of order

Thraustochytriales are described in detail in the prior art (see e.g. WO91 / 07498, WO94 / 08467, WO97 / 37032, W097 / 36996, W001 / 54510). The production is usually carried out by culturing cells in the presence of a carbon source and a nitrogen source in a fermenter. Biomass densities of more than 100 grams per liter and production rates of more than 0.5 grams of lipid per liter per hour can be achieved.

The process is preferably carried out as a so-called fed-batch process, i.e. that the carbon source and possibly also the nitrogen and phosphate sources are fed incrementally during the fermentation. After reaching the desired biomass, lipid production can be induced by different measures, for example by limiting the nitrogen source, the phosphate source or the oxygen content or combinations thereof.

Both alcoholic and non-alcoholic carbon sources can be considered as carbon sources. Examples of alcoholic carbon sources are methanol, ethanol and isopropanol. Examples of non-alcoholic carbon sources are fructose, glucose, sucrose, molasses, starch and corn syrup, as well as organic acids such as acetic acid, propionic acid and medium and long chain fatty acids and their salts. Processes which are preferred according to the invention are distinguished in that at least one carbon source is metered into the medium continuously during the complete fermentation process.

Both inorganic and organic nitrogen sources can be considered as 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. The oil production phase is preferably initiated according to the invention, as described in WO 01/54510, by limiting at least one limiting nutrient component, preferably by limiting at least one nitrogen source.

In addition to sulfates and optionally used chlorides, other salts, in particular selected from sodium carbonate, can optionally also be used in the fermentation.

Sodium bicarbonate, soda ash or inorganic phosphorus compounds can be used. If further salts are used, these are preferably used in each case in an amount of less than 12 g / l, in particular less than 8 g / l, particularly preferably less than 5 g / l. The total salt content in the fermentation medium is at the beginning of the

Main fermentation preferably 5 to 30 g / l, in particular 10 to 20 g / l.

In addition, organic phosphorus compounds and / or known ones

growth-stimulating substances, such as yeast extract or corn steep liquor, can be added to positively influence the fermentation.

The cells are preferably fermented at a pH of 4 to 11, in particular 6 to 10, and preferably at a temperature of at least 20 ° C., in particular 20 to 40 ° C., particularly preferably at least 30 ° C. A typical fermentation process takes up to about 100 hours.

According to the invention, the cells are preferably up to a biomass density of at least 50, 60 or 70 g / l, in particular 50 to 250 g / l or 60 to 220 g / l, preferably at least 80 or 90 g / l, in particular 80 to 200 g / l, particularly preferably at least 100 g / l, in particular 100 to 180 g / l, fermented. The information here relates to the dry matter content in relation to the total volume of the fermentation broth after the end of the fermentation. The dry matter content is determined by filtering the biomass from the

Fermentation broth, then washing with water, then drying completely - for example in the microwave - and finally determining the dry weight.

In a preferred embodiment according to the invention, the sulfate concentration drops to zero shortly before the start of the oil production phase, preferably after a biomass density of at least 50 g, particularly preferably at least 80, 100, 120 or 140 g, has been reached per liter of fermentation medium.

The cells are preferably pasteurized after the cells have been harvested or, if appropriate, even shortly before the cells are harvested, in order to kill the cells and inactivate enzymes which could promote the breakdown of the lipids.

After the fermentation has ended, the fermentation broth obtained can be subjected to an oil isolation process immediately or, if appropriate, after prior concentration, in order to recover the oil contained. Such oil insulation processes are described, for example, in WO 01/53512 and WO 201 1/153246.

Alternatively, the biomass containing PUFAs can also be harvested after the end of the fermentation. For this purpose, the fermentation broth is preferably first concentrated. According to the invention, the fermentation broth is preferably concentrated by centrifugation, filtration, decanting and / or solvent evaporation in order to first separate a large part of the fermentation medium from the biomass. The

Solvent evaporation is preferably carried out using a drum dryer, a tunnel dryer, spray drying or vacuum evaporation. The

Solvent evaporation can in particular also be carried out using a rotary evaporator, a thin-film evaporator or a falling film evaporator. As an alternative to

Solvent evaporation can also be used, for example, in reverse osmosis to narrow down the fermentation broth.

In order to obtain the biomass, the concentrated fermentation broth thus obtained is preferably further dried, preferably by fluid bed granulation. The subsequent drying preferably reduces the moisture content of the biomass to below 15% by weight, in particular below 10% by weight, particularly preferably below 5% by weight.

In an embodiment which is particularly preferred according to the invention, the biomass is dried in a fluidized bed granulation process or in a

Nozzle spray drying processes, as described for example in WO 2015/052048.

If necessary, silica can be added to the biomass as an anti-caking agent during drying in order to convert it into a more manageable state. The fermentation broth containing biomass and the silica are preferably sprayed into the respective drying zone for this purpose. Alternatively, the biomass is preferably mixed with the silica only after drying. In this regard, reference is also made in particular to patent application WO 2015/052048.

In a preferred embodiment, after drying, a biomass to be used according to the invention has a concentration of silica, in particular hydrophilic or hydrophobic silica, of 0.2 to 10% by weight, in particular 0.5 to 5% by weight, especially 0.5 up to 2% by weight.

Drying preferably gives a free-flowing, finely divided or coarse-grained product, preferably granules. If necessary, a product with the desired grain size can be obtained from the granules obtained by sieving or dust removal.

If a free-flowing, finely divided powder has been obtained, this can optionally be converted into a coarse-grained, free-flowing, storable and largely dust-free product by means of suitable compacting or granulating processes.

In this subsequent granulation or compacting, customary organic or inorganic auxiliaries or carriers such as starch, gelatin,

Cellulose derivatives or similar substances commonly used in food or

Feed processing can be used as a binding, gelling or thickening agent. According to the invention, “free-flowing” is to be understood as meaning a powder that consists of a series of

Glass outlet vessels with differently sized outlet openings can at least flow freely out of the vessel with the opening 5 millimeters (small: soaps, oils, fats, waxes 94, 12 (1968)). According to the invention, “finely divided” means a powder with a predominant proportion (> 50%) of a grain size of 20 to 100 micrometers in diameter.

According to the invention, “coarse-grained” means a powder with a predominant proportion (> 50%) of a grain size of 100 to 2500 micrometers in diameter.

According to the invention, “dust-free” is to be understood as a powder which contains only small proportions (<10%, preferably <5%) of particle sizes below 100 micrometers.

According to the invention, the grain or particle sizes are preferably determined by methods of laser diffraction spectrometry. The methods to be used are described in the textbook "Particle size measurement in laboratory practice" by R.H. Müller and R. Schuhmann,

Wissenschaftliche Verlagsgesellschaft Stuttgart (1996) and in the textbook "Introduction to Particle Technology" by M. Rhodes, Verlag Wiley & Sons (1998). If different methods are applicable, the first-mentioned applicable method from the textbook by R.H. Müller and R. Schuhmann applied to particle size measurement.

The products obtained by drying processes according to the invention preferably have a proportion of at least 80% by weight, in particular at least 90% by weight, particularly preferably at least 95% by weight, of particles with a particle size of 100 to 3500

Microns, preferably 100 to 3000 microns, especially 100 to 2500 microns.

The products of a fluidized bed granulation process obtained according to the invention preferably have a proportion of at least 80% by weight, in particular at least 90% by weight, particularly preferably at least 95% by weight, of particles with a particle size of 200 to 3500 micrometers, preferably 300 to 3000 microns, especially 500 to 2500 microns. By contrast, the products of a spray drying process obtained according to the invention preferably have a proportion of at least 80% by weight, in particular at least 90% by weight, particularly preferably at least 95% by weight, of particles with a particle size of 100 to 500 micrometers, preferably 100 to 400 microns, especially 100 to 300 microns.

The products obtained according to the invention of a spray drying process and subsequent granulation process preferably have a proportion of at least 80% by weight, in particular at least 90% by weight, particularly preferably at least 95% by weight, of particles with a particle size of 100 to 1000 micrometers.

The proportion of dust, i.e. Particles with a grain size of less than 100 micrometers are preferably at most 10% by weight, in particular at most 8% by weight, particularly preferably at most 5% by weight, especially at most 3% by weight.

The bulk density of the products according to the invention is preferably 400 to 800 kg / m ³ , particularly preferably 450 to 700 kg / m ³ .

Another object of the present invention is therefore also a feed that comprises a biomass according to the invention and further feed ingredients. The further feed ingredients are preferably selected from protein-containing, carbohydrate-containing, nucleic acid-containing and lipid-soluble components as well as optionally further fat-containing components and further from other additives such as minerals, vitamins, pigments and amino acids. In addition to nutritional substances, it can also contain structuring substances to improve the texture or appearance of the feed. Furthermore, binders can also be used, for example, to influence the consistency of the feed. A preferred component that is both a nutritional substance and a structuring substance is starch.

A feed according to the invention or one for producing a feed according to the invention

The composition used according to the invention is preferably characterized in that it contains a 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, especially 4 to 12 % By weight. This feed or the composition used to produce the feed preferably further has at least one, preferably all, of the following properties: a) a total protein content of 33 to 67% by weight, preferably 39 to 61% by weight , in particular 44 to 55% by weight;

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;

c) a total starch content of at most 25% by weight, in particular at most 20% by weight, preferably 6 to 17% by weight, particularly preferably 8 to 14% by weight;

d) a content of polyunsaturated fatty acids (PUFAs) 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;

e) a content of omega-3 fatty acids 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;

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. A preferred object according to the invention is therefore also a feed or a composition suitable for producing the feed, which has at least one, preferably all, of the following properties:

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

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;

d) a content of polyunsaturated fatty acids (PUFAs) of 2 to 13% by weight,

e) a content of omega-3 fatty acids 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; 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. A preferred object according to the invention is therefore also a feed or a composition suitable for producing the feed, which has at least one, preferably all, of the following properties:

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

d) a content of biomass according to the invention, in particular a labyrinthulea biomass according to the invention, preferably a Thraustochytriaceae biomass according to the invention, of 2 to 24% by weight, preferably 4 to 22% by weight, in particular 9 to 20% by weight all 11 to 18% by weight;

e) a content of polyunsaturated fatty acids (PUFAs) of 2 to 13% by weight,

f) a content of omega-3 fatty acids 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;

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. A preferred object according to the invention is therefore also a feed or a composition suitable for producing the feed, which has at least one, preferably all, of the following properties:

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

d) a content of an aurantiochytrium or schizochytrium biomass according to the invention, preferably an aurantiochytrium limacinum biomass according to the invention, especially an aurantiochytrium limacinum SR21 biomass according to the invention, 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;

e) a content of polyunsaturated fatty acids (PUFAs) of 2 to 13% by weight,

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. An extrudate can be obtained by extruding the aforementioned compositions. These extrudates represent a preferred subject of the present invention. The feed is preferably extruded with an energy input of 12-28 Wh / kg, in particular 14-26 Wh / kg, particularly preferably 16-24 Wh / kg, especially 18- 22 Wh / kg.

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

The extrusion process can optionally be a compacting and / or

Compression step include.

Before the extrusion process is carried out, the components are preferably intimately mixed with one another. This is preferably done in a drum equipped with blades. In a preferred embodiment, this mixing step is carried out by water vapor injection, in particular in order to cause the starch preferably contained to swell. The steam injection is preferably carried out at a pressure of 1 to 5 bar, particularly preferably at a pressure of 2 to 4 bar.

The other feed ingredients are preferably comminuted - if necessary - before mixing with the Aigen biomass to ensure that a homogeneous mixture is obtained in the mixing step. The other feed ingredients can be comminuted, for example, using a hammer mill.

The extrudate produced 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 . The length of the extrudate is adjusted by using a cutting tool during extrusion. The length of the extrudate is preferably chosen so that it approximately corresponds to the diameter of the extrudate. The diameter of the extrudate is determined by the choice of the sieve diameter.

In a preferred embodiment according to the invention, the extrusion process is followed by the loading of the extrudate obtained with oil. For this purpose, the extrudate is preferably first dried to a moisture content of at most 5% by weight. The loading of the

Extrusion product with oil can be carried out according to the invention, for example by placing the extrudate in oil or spraying the extrudate with oil, but it is carried out according to the invention

preferably by vacuum coating.

Feedstuffs are obtained in this way, the biomasses according to the invention preferably in an amount of 1 to 25% by weight, in particular 2 to 20% by weight, particularly preferably 3 to 15% by weight, especially 4 to 12% by weight. %, contain.

Accordingly, these feeds preferably furthermore have at least one, preferably all, of the following properties: a) a total protein content of 30 to 60% by weight, preferably 35 to 55% by weight, in particular 40 to 50% by weight;

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;

c) a total starch content of at most 25% by weight, in particular at most 20% by weight, preferably 5 to 15% by weight, particularly preferably 7 to 13% by weight;

d) a content of polyunsaturated fatty acids (PUFAs) 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;

e) a content of omega-3 fatty acids 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. -%;

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. A preferred object according to the invention is therefore also a feed, in particular an extrudate, which has at least one, preferably all, of the following properties: a) a total protein content of 30 to 60% by weight, preferably 35 to 55% by weight , in particular 40 to 50 wt .-%;

d) a content of polyunsaturated fatty acids (PUFAs) of 2 to 12% by weight,

d) a content of a biomass according to the invention, especially one

Labyrinthulea biomass according to the invention, preferably one according to the invention

Thraustochytriaceae biomass, from 1 to 25% by weight, preferably 2 to 20% by weight, in particular 3 to 15% by weight, especially 4 to 12% by weight;

e) a content of polyunsaturated fatty acids (PUFAs) 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; f) a content of omega-3 fatty acids 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. -%;

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. A preferred object according to the invention is therefore also a feed, in particular an extrudate, which has at least one, preferably all, of the following properties: a) a total protein content of 30 to 60% by weight, preferably 35 to 55% by weight. -%, in particular 40 to 50 wt .-%;

d) a content of an aurantiochytrium or schizochytria biomass according to the invention, preferably an aurantiochytrium limacinum biomass according to the invention, especially an aurantiochytrium limacinum SR21 biomass according to the invention, 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;

e) a content of polyunsaturated fatty acids (PUFAs) of 2 to 12% by weight,

f) a content of omega-3 fatty acids 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. -%;

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. In addition to the biomass to be used according to the invention, fats, in particular oils, animal and also of vegetable origin can be used as fat-containing component according to the invention. According to the invention, vegetable oils, for example soybean oil, rapeseed oil, sunflower seed oil, flaxseed oil or palm oil and mixtures thereof, are particularly suitable as the fatty component. In addition, fish oil can optionally also be used in small quantities as a fatty component.

According to the invention, the protein-containing component can be, for example, soy protein,

Pea protein, wheat gluten or corn gluten and mixtures thereof can be used.

Fish protein, krill meal, mussel meal, squid meal or shrimp shells can be used, for example, as a protein-containing component which additionally contains fats. These are summarized below under the term "marine flour". In a preferred one

Embodiment comprises a feed according to the invention marine flour, 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.

For example, wheat flour, sunflower flour or soy flour and mixtures thereof can be used as the carbohydrate-containing component. When using animal feed according to the invention, in particular an oil-coated extrudate according to the invention, in animal breeding, it has been found that this promotes the growth of the animals to a particular extent and improves the stress state of the animals.

Another object of the present invention is also a method for breeding animals, characterized in that a feed according to the invention is administered to them. The present invention relates in particular to a method for increasing the growth of animals, characterized in that a feed according to the invention is administered to them.

Another object of the present invention is in particular also a method for increasing the proportion of omega-3 fatty acids, in particular DHA, in the muscle tissue of animals, characterized in that a feed according to the invention is administered to them.

In the process according to the invention, the feed is preferably administered at least every two days, preferably at least once a day.

Another object of the present invention is also the use of a

Animal feed according to the invention for increasing the growth in animals.

Another object of the present invention is also the use of a

Animal feed according to the invention for increasing the proportion of omega-3 fatty acids in the

Muscle tissue in animals.

Another object of the present invention is also the use of a

Animal feed according to the invention for improving the health status of animals, in particular for improving the stress status of animals.

Another object of the present invention is also the use of a

Animal feed according to the invention to enable stress-reduced breeding of the animals.

The bred animals which are fed with a feed according to the invention are preferably poultry, pigs or cattle.

However, the bred animals are particularly preferably marine animals, particularly preferably finfish or crustaceans. These include carp, tilapia, catfish, tuna, salmon, trout, baramundi, bream, perch, cod, shrimp, lobster, crab, shrimp and crayfish. The animals bred are particularly preferably salmon. Preferred types of salmon are the Atlantic salmon, the sockeye salmon, the masu salmon, the king salmon, the ketal salmon, the silver salmon, the Danube salmon, the Pacific salmon and the humpback salmon.

The bred animals can in particular also be fish, which are subsequently processed into fish meal or fish oil. The fish are preferably herring, pollack, menhaden, anchovies, capelin or cod. The fish meal or fish oil thus obtained can in turn be used in aquaculture for the breeding of edible fish or crustaceans.

However, the bred animals can also be small organisms that are used as feed in aquaculture. These small creatures can be, for example, nematodes, crustaceans or rotifers. The marine animals can be grown in ponds, tanks, basins or in delimited areas in the sea or in lakes, in particular in cages or net pens. The cultivation can serve to breed the finished edible fish, but can also be used to breed juveniles, which are then released to the

Adding wild fish.

In salmon farming, it is preferred to start growing up to young salmon in

Fresh water tanks or artificial water flows and then further breeding in the sea in floating cages or net pens, which are preferably anchored in bays or fjords.

The feed according to the invention is accordingly preferably a feed for use in the breeding of the aforementioned animals.

Embodiments

Example 1: Production of the biomass by fermentation of Aurantiochytrium limacinum SR21 in a medium with a high sulfate content and subsequent drying of the

Biomass

The cells were cultivated for approx. 70 h in a feed process using a steel fermenter with a fermenter volume of 2 liters and a total starting mass of approx. 700 g and a total final mass of approx. 1.5 kg. A glucose solution (570 g / kg glucose) was metered in during the process (“fed-batch process”)

The composition of the starting medium was as follows:

Medium 1: 20 g / kg glucose; 4 g / kg yeast extract; 2 g / kg ammonium sulfate; 12 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.

The composition of the trace element solution was 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 iron sulfate (heptahydrate); 0.15 g / kg copper chloride (dihydrate).

The cultivation was carried out under the following conditions: cultivation temperature 28 ° C;

Aeration rate 0.5 vvm, stirrer speed 600 - 1950 rpm, control the pH in the growth phase at 4.5 with the help of ammonia water (25% v / v). The fermentation took place up to a biomass density of 80 g / l before the oil production phase was initiated by limiting phosphate and nitrogen. Due to the high sulfate content in the starting medium, the sulfate concentration is always above the saturation limit of the cells, which is around 5 g per kg of dry biomass, until the end of the fermentation, ie also until the end of the oil production phase. The biomass obtained is referred to below as “biomass 1” (12 g / kg sodium sulfate in the starting medium) or “biomass 2” (16 g / kg sodium sulfate in the starting medium). To determine the sulfate content of the biomass obtained, the sulfur content of the biomass was determined in accordance with DIN ISO 11885.

Example 2: Production of the biomass by fermentation of Aurantiochytrium limacinum SR21 in a medium with a low sulfate content and subsequent drying of the biomass

The cells were cultivated for approx. 70 h in a feed process using a steel fermenter with a fermenter volume of 2 liters with a total starting mass of 712 g and a total final mass of approx. 1.5 kg. A glucose solution (570 g / kg glucose) was metered in during the process (“fed-batch process”)

The composition of the starting medium was as follows:

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.

Aeration rate 0.5 vvm, stirrer speed 600 - 1950 rpm, control of the pH value in the

Growth phase at 4.5 with the help of ammonia water (25% v / v). The fermentation took place up to a biomass density of 80 g / l before the oil production phase was initiated by limiting phosphate and nitrogen. The sulfate concentration fell below the detection limit of 0.05 g per kg fermentation medium when the oil production phase was initiated, and the sulfate concentration was accordingly below the detection limit during the entire oil production phase. And since no sulfate was added, the sulfate concentration fell to zero in the course of the oil production phase. The biomass obtained is referred to below as “Biomass 3”. Example 3: Processing and comparison of the biomass or process obtained

After cultivation, the fermentation broths were heated to 60 ° C. for 20 minutes in order to prevent further cell activity.

The biomass was then dried in two stages

Fermentation broth concentrated by evaporation to a dry matter of about 20 wt .-%. The concentrated fermentation broth was then spray dried using a Production MinorTM Spray Dryer (GEA NIRO) at an inlet temperature of

Drying air at 340 ° C. Spray drying gave a powder with a dry matter of more than 95% by weight. Table 1: Comparison of the biomass obtained

Biomasses 1 and 2 are biomasses obtained by fermentation with a high sulfate content according to Example 1, while Biomass 3 is a biomass obtained by fermentation with a low sulfate content according to Example 2 has been.

It can be seen that the fermentation process, which was carried out with a low sulfate content and led to biomass 3, produced a product with a significantly higher purity, that is to say a product with a significantly increased proportion of DHA, than the process with a high Sulphate content was carried out and led to biomass 1 and 2. In addition, the sulfate content in the biomass obtained is significantly lower than in the process with a high sulfate content.

Claims

1. A process for producing a biomass containing PUFAs by culturing cells producing PUFAs in a fermentation medium, characterized in that the

Sulfate content is adjusted so that the sulfate concentration drops to zero in the course of the fermentation.

2. A method for producing a biomass containing PUFAs according to claim 1, wherein the method comprises a growth phase and an oil production phase, characterized in that the sulfate concentration in the fermentation medium drops to zero before the start of the oil production phase.

3. The method according to claim 2, characterized in that the sulfate concentration in

Fermentation medium in the second half of the growth phase, preferably in the last

Eighth of the growth phase, particularly preferably shortly before the start of the oil production phase, falls to zero.

4. The method according to any one of the preceding claims, characterized in that the amount of sulfate is predetermined so that a final sulfate concentration of 7 to 10, preferably 7.5 to 9.5 g sulfate per kg biomass.

5. The method according to any one of the preceding claims, comprising as a further step the mechanical digestion of the biomass obtained using low mechanical forces, preferably a force of up to 12 Wh / kg, in particular 1 to 10

Wh / kg.

6. The method according to any one of claims 1 to 5, characterized in that the fermentation broth thus obtained is subjected to oil isolation.

7. The method according to any one of claims 1 to 5, characterized in that the fermentation broth thus obtained - if necessary after prior concentration - is preferably further processed with a low energy input to a feed.

8. The method according to any one of the preceding claims, characterized in that the cells, preferably in the growth phase, up to a biomass density of at least 50 g per liter of fermentation medium, in particular 50 to 250 g per liter

Fermentation medium, preferably at least 80, 100, 120 or 140 g per liter Fermentation medium, in particular 80, 100, 120 or 140 g to 200 or 220 g per liter of fermentation medium, can be cultivated.

9. The method according to claim 8, characterized in that the sulfate concentration in the

Medium drops to zero after a biomass density of at least 50 g, preferably at least 80 g, in particular at least 100 or 120 g, has been reached per liter of fermentation medium.

10. The method according to any one of the preceding claims, characterized in that the oil production phase is initiated by limiting at least one limiting nutrient component, in particular by limiting a nitrogen source.

11. The method according to any one of the preceding claims, characterized in that the sulfate concentration in the medium during the entire time of the oil production phase is below 0.05 g per kg of fermentation medium, preferably zero.

12. The method according to any one of the preceding claims, characterized in that it has a chloride content of at most 3 g per kg during the entire fermentation

Contains fermentation medium.

13. The method according to any one of the preceding claims, characterized in that the microorganisms are those of the taxon Labyrinthulomycetes (Labyrinthulea, network slime molds, slime networks), in particular those of the family of

Thraustochytriaceae, preferably of the genera Althornia, Aplanochytrium, Elnia,

Japonochytrium, Schizochytrium, Thraustochytrium, Aurantiochytrium, Oblongichytrium or Ulkenia, especially those of the genera Schizochytrium or Aurantiochytrium, especially of the species Aurantiochytrium limacinum.

14. Biomass containing PUFAs, characterized in that they contain microorganisms of

Taxons Labyrinthulomycetes comprises and a sulfate content of 7 to 10 g, preferably 7.5 to

9.5 g of sulfate per kg of biomass.

15. Biomass containing PUFAs according to one of the preceding claims, characterized

characterized that the cells of the taxon Labyrinthulomycetes (Labyrinthulea, retinal slime molds, slime nets) are those of the family of the Thraustochytriaceae, preferably of the genera Althornia, Aplanochytrium, Elnia, Japonochytrium,

Schizochytrium, Thraustochytrium, Aurantiochytrium, Oblongichytrium or Ulkenia, particularly preferably those of the genera Schizochytrium or Aurantiochytrium, in particular of the species Aurantiochytrium limacinum.