EP1685241A2

EP1685241A2 - Method for the cultivation of microorganisms of the genus i thraustochytriales /i by using an optimized low salt medium

Info

Publication number: EP1685241A2
Application number: EP04818134A
Authority: EP
Inventors: Matthias RÜSING; Markus Luy
Original assignee: Nutrinova Nutrition Specialties and Food Ingredients GmbH
Current assignee: Lonza AG
Priority date: 2003-11-10
Filing date: 2004-11-10
Publication date: 2006-08-02
Also published as: EA011858B1; EA200600947A1; US20070054384A1; EP2816104A1; IL175520A0; SG148157A1; US20150104557A1; DE10352838A1; KR101238613B1; ZA200603854B; WO2005045003A2; CA2545410A1; BRPI0416360A; AU2004287954A1; AU2004287954B2; US8900831B2; WO2005045003A3; CN1977038B; CN104073442A; KR20070042115A

Abstract

The invention relates to an optimized method for cultivating microorganisms of the genus thraustochytriales, according to which the microorganisms are cultivated in a low salt medium without adding sodium salts and chloride salts, the total salt content being less than 3.5g/L (corresponding to less than 10 percent of sea water content), whereupon the PUFAs are isolated from the microorganisms and/or the medium. The invention especially relates to novel optimized media having a substantially reduced total salt content, above all a particularly reduced NaCl content. The production of PUFAs can be substantially improved and significantly simplified by using a novel combination of different salts as a media composition in which the overall weight ratios of Na<+> and Cl<-> ions do not exceed 1.75 g/L. Furthermore, said medium preferably contains no added sodium salt and chloride salt at all, which helps prevent environmental damages caused by wastewaters containing salt.

Description

Process for the cultivation of microorganisms of the genus Thraustochytriales using an optimized low salt medium

Various polyunsaturated fatty acids (PUFAs for polyunsaturated fatty acids) and especially omega-3 fatty acids (n3 fatty acids) are essential components of human nutrition.

However, it is known that the supply of n3 fatty acids is poor in most industrialized countries. In contrast, the total fat content in the diet and the intake of saturated fatty acids and n6 fatty acids is too high. This is due to a change in our near-composition, which has mainly taken place in the past approx. 150 years and which is correlated with the occurrence of various chronic diseases of civilization, such as cardiovascular diseases - the main cause of death in industrialized nations (Simopoulos, AP, 1999, Am. J Clin Nutr 70, 560-569). A large number of studies have now shown that by specifically increasing the intake of n3 fatty acids, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), the cardiovascular risk can be significantly reduced (GISSI-Prevenzione Investigators (Gruppo Italiano per lo Studio della Sopravvivenza nell'Infarto miocardico), 1999, Dietary suplementation with n-3 polyunsaturated fatty acids and vitamin E after myocardial infarction: results of the GISSI-pevenzione trial., Lancet 354, 447-455; Burr et al, 1989, Effects of changes in fat, fish, and fiber intake on death and myocardial reinfarction: diet and reinfarction trial (DART). Lancet 2, 757-761). Accordingly, many different organizations (WHO, FAO, AHA; ISSFAL, British Nutrition Foundation and many others) recommend 'significantly increasing the intake of n3 fatty acids (Kris-Eherton et al., Fish Consumption, Fish Oil, Omega-3 Fatty Acids, and Cardiovascular Disease, Circulation 2002, 21M -2151).

Sources for the production of PUFAs and in particular n3 fatty acids are primarily marine cold water fish and oils derived from them, but also marine microorganisms, which have the advantage over fish that they can be used in fermenters to produce PUFAs under inexpensive and controlled conditions. In fermentative production there is no risk of contamination, as is often described for fish or fish oils obtained from them (Olsen SF. Int J Epidemiol. 2001: 1279-80). In addition, the composition of the oils obtained can be positively influenced by the selection of the organism and the culture conditions and is not subject to seasonal fluctuations, as is also described for fish and fish products (Gamez-Meza et al. Lipids 1999: 639-42).

Microorganisms that are suitable for the production of n3-PUFA can be found, for example, in the bacteria under the Vibrio genus (e.g. Vibrio marinus) or under the Dinoflageliaten (Dinophyta), there in particular the genus Crypthecodinium, such as C. cohnii or among the stramenopiles, such as the Pinguiophyceae such as Glossomastix, Phaeomonas, Pinguiochrysis, Pingiüococcus and Polydochrysis. Preferred microorganisms for the fermentative production of PUFA belong to the stramenopiles (or labyrinthulomycota), in particular to the order Thraustochytriales, (Thraustchytriideä) and there again in particular to the genera Japonochytrium, Schizochytrium, Thraustochytrium, Althornia, and Labyrinthochoidium.

It is known that some of the microorganisms mentioned can be used for the industrial production of fatty acids, corresponding processes have been described. For example, international patent application WO 91/07498 A1 discloses the production of PUFAs with organisms of the genera Thraustochytrium and Schizochytrium. WO 91/11918 AI discloses the production of PUFAs with Crypthecodinium cohnii, WO 96/33263 AI and the corresponding European patent application EP 0 823 475 AI describe the production of PUFAs with microorganisms of the genus Schizochytrium, while the patent application WO 98/03671 describes the production of PUFAs with microorganisms of the genus Ulkenia are disclosed.

The natural habitat of the described microorganisms and especially the labyrinthulomycota are marine habitats. Conventionally, these microorganisms are therefore cultivated in correspondingly saline media, the salinity of seawater for the purposes of the present invention being defined as 32-35 g / L and a proportion of 90-95% of sodium and chloride. Typical media for the cultivation of marine microorganisms such as Thraustochytrium or Schizochytrium are based on sea water (e.g. ATCC (American Type ^' Culture Collection) 790 By + Medium [yeast extract 1.0 g, peptone 1.0 g, D + glucose 5.0 g, sea water 1 L]). But it is also known that Microorganisms of the order Thraustochytriales can survive with very low salinity in the culture medium. However, their growth below a limit of 7.5 - 15 g salt / L, corresponding to a salinity of 7.5 - 15% o, is described as very low and without intermediate maxima in the low salinity range. Optimal growth rates are only achieved above the salinity limit specified above (Fan et al. Botanica Marina 45, 2002, pp. 50-57).

For the commercial fermentation of euryhaline microorganisms, however, reduced salt contents of about 50-60% of the sea water have also been described. According to Henderson's "Dictionary of biological terms", marine organisms that can adapt to a wide range of salinity are referred to as euryhalin (Henderson WD, Lawrence, E., Henderson's dictionary of biological terms, 10 ^th ed. 1992 p. 173 ).

It has been described that struryopile (or labyrinthulomycota) euryhaline microorganisms can produce larger amounts of PUFA in fermentation media containing a reduced content of sodium ions (60% of sea water) (U.S. Pat. No. 6,451,567). Also described is the use of low chloride culture media to reduce the corrosive effects of chloride on fermentation equipment (U.S. Pat. No. 6,410,281). This has been shown, for example, for microorganisms of the genera Thraustochytrium and Schizochytrium with fermentation media which contain chloride in a concentration of not more than 3 g / L (US Pat. No. 5,340,742, US Pat. No. 6,451,567, US Pat. No. 6,410,281), Es it is also known that cultivation is also possible under conditions with a reduced salt content compared to sea water. Particular reference is made here to the patent specifications WO 98/03671 AI, EP 0 823 475 AI, U.S. Pat. No 6,451,567, U.S. Pat. No 6,410,281.

It is also known that for the fermentation of a microorganism of the genus Schizochytrium {Schizochytrium sp. S31; ATTC 20888) a maximum of the relative yield of fatty acids is achieved at a sodium chloride concentration of 1.75 g / L. The total amount of salt used is less than 10% of the seawater salt content, but mainly consists of sodium and chloride ions (EP 0 512 997 Bl and US Pat. No. 5,518,918) However, all of the methods described so far have disadvantages. The effectiveness of fenrientative processes is particularly limited by the achievable biomass and the content of product per biomass. Furthermore, the oils formed can in some cases have fatty acid spectra which do not necessarily correspond to the desired products, but which first have to be changed in terms of process technology. Due to the sometimes low content of product per biomass, processing is often considerably complicated, since relatively large amounts of the biomass have to be processed in order to obtain relatively small amounts of product. Furthermore, all the processes described so far have their own relatively high total salt contents in the culture media. This not only leads to massive problems in the processing of the products, but is also extremely disadvantageous from an environmental point of view, since not only large amounts of biomass are produced, but also very saline wastewater that has to be disposed of.

In view of the prior art, it was therefore an object of the present invention to provide a new, simple and economical process for the cultivation of Thraustochytriales, media with reduced salt contents being used. Apart from the cost efficiency, the process should be easy to carry out and enable the production of high purity PUFAs or products containing PUFA in high yield.

These and other tasks, which are not explicitly mentioned, but which can easily be derived or inferred from the contexts discussed at the outset, are achieved by the subject matter defined in the claims of the present invention. ,

An advantageous method for the cultivation of Thraustochytriales is provided by the method defined in claim 1. This method involves culturing microorganisms of the order Thraustochytriales in a low salt medium without the addition of sodium or chloride ions in solid or dissolved form, with a total salt content of less than 10% based on sea water, i.e. less than approx. 3.5 g / L total salts.

The invention further comprises a method for producing high-purity PUFAs.

Preferred PUFAs according to the invention are DHA, DPA and EPA. In particular, the microorganisms cultured in the abovementioned process have a production of more than 10%, preferably more than 14%, and very particularly preferably more than 18% o DHA per dry biomass.

In particular, the microorganisms cultured in the abovementioned process show a production of more than 5%, preferably more than 7%, and very particularly preferably more than 10%> DPA per dry biomass.

By isolating the PUFAs from the microorganisms (biomass) and / or the culture medium following the cultivation, the PUFAs can be obtained in high yield and purity.

Furthermore, the present invention also includes a method for producing biomass, the biomass being made available by the cultivation method according to the invention.

This biomass can be used in all conceivable ways. In particular, this biomass, e.g. in the dried state (dry organic matter), used directly as food or as feed.

Furthermore, the invention also includes an oil which is obtained in that the invention. Cultivation process is carried out, and the oil is isolated from the microorganisms (biomass) and / or the culture medium.

In particular, it is an oil which, in addition to many other preferred uses, can advantageously be used for human nutrition.

The microorganisms show a production of more than 30% by weight of oil, preferably of more than 35% by weight of oil, per unit weight of dry organic matter under the conditions according to the invention.

According to the invention, oil is understood to mean a proportion of at least 70% neutral lipids and at least 2% phospholipids, which corresponds to the normal fatty acid spectrum of Thraustochytriales known to the person skilled in the art. The neutral lipids consist of at least 80%> triglycerides and other compounds such as diacylglycerides, sterols etc. Furthermore, the weight fraction of the triglycerides consists of approximately 95% fatty acids and 5% glycerin. The possibility of a marine microorganism with such a low salt concentration, which corresponds to less than 10% of the typical sea salt content, to be able to ferment, and in particular the fact that the addition of the predominant ions Na and Cl ^" , which normally make up about 90% of the ions present in the sea water, was completely dispensed with, was completely surprising.

Surprisingly, not only was the fermentation itself possible, but the proportion of PUFA in the biomass also increased significantly when the low salt medium according to the invention was used. It is even more surprising that this effect not only compensates for a slight decrease in the biomass produced, but even surpasses it (example 2). The proportion of the predominant PUFA DHA per dry biomass increased by more than 10% compared to the comparison fermentation in medium 1 (50% sea salt content). The processing of the products is simplified by the higher product concentration and the lower contamination by salts and this with an overall increased space-time yield.

Up to the present invention, there was no known fermentation process for the production of n3 fatty acids in microorganisms from the order Thraustochytriales using a medium with such an extremely low salt concentration and without sodium and chloride salt additives.

According to the invention, PUFAs are polyunsaturated long-chain fatty acids with a chain length> C12 with at least two double bonds. PUFAs which can be produced by the process according to the invention are in particular n3 fatty acids and n6 fatty acids.

Under n3 fatty acids (omega-3 fatty acid, ω3 fatty acids) in the sense of the invention are understood to be polyunsaturated long-chain fatty acids with a chain length> C12 with at least two or more double bonds, the first of the double bonds between the carbon atoms C3 and C4 being constituted starting from the alkyl end , Accordingly, with n6 fatty acids, the first double bond is between the carbon atoms C6 and C7 starting from the alkyl end.

Microorganisms from the group of the labyrinthulomycota can be used for the production of the PUFAs by the process according to the invention. Microorganisms of the order Thraustochytriales (Thraustchytriidea) are preferred (Lewis, TE, Nichols, PD, McMeekin, TA, The Biotechnological Potential of Thraustochytrids, Marine Biotechnology, 1999, pp. 580-587 and Porter, D. Phyl m Labyrinthulomycota in the Handbook of protoctista : the structure, cultivation, habitats, and life histories of the eukaryotic microorgamsms and their descendants exclusive of animals, plants, and fungi: a guide to the algae, ciliates, foraminifera, sprorozoa, water molds, and other protoctists. Editors: Margulis, L, Corliss, JO, Melkonian, M. and Chapman, DJ., Editorial coordmator, McKhann, HJ., Jones and Bartlett Publishers, ISBN 0-86720-052-9 1990, pp. 388-398). Microorganisms of the genera Japonochytrium, Labyrinthuloides, Aplanochytrium Althomia, Schizochytrium, Thraustochytrium and Ulkenia are particularly preferred. Of these, Schizochytrium, Thraustochytrium and 'Ulkenia are very particularly preferred. The following are particularly preferred: Japonochytrium sp. ATCC 28207, Thraustochytrium aureum (in particular ATCC 28211 or ATTC 34304), Thraustochytrium roseum ATCC 28210 Thraustochytrium sp. ATCC 20890, ATTC 20891, ATTC 20892 and ATTC 26185, Schizochytrium aggregatum ATTC 28209, Schizochytrium sp. ATCC 20888 and ATTC 20889, Schizochytrium SR21, and Ulkenia spec. SAM 2179 and SAM 2180.

Microorganisms suitable for the process according to the invention are both wild-type forms and also mutants and strains derived therefrom and recombinant strains of the corresponding organisms. The present invention particularly includes mutants or recombinant strains for increasing the production of PUFA.

The microorganisms in the sense of the present invention are cultivated by inoculating a liquid or a solid medium with a preculture of these organisms.

Culture techniques suitable for microorganisms of the Thraustochytriales order are well known to those skilled in the art. Typically, but not exclusively, the culture is carried out by means of aqueous fermentation in an appropriate container. ^'Examples for a typical vessel for such fermentation include shaker flasks or bioreactors, such as STRs (stirred tank reactors), or bubble columns. ^" The culture is typically carried out at temperatures between 10.degree. C. and 40.degree. C., preferably between 20.degree. C. and 35.degree. C., particularly preferably between 25.degree. C. and 30.degree. C., very particularly preferably between 27.degree. C. and 29.degree and especially at 28 ° C.

In a further embodiment of the present invention, the low salt medium comprises less than 1.5 g / L total salts.

In a further preferred embodiment of the present invention, the total salt content of the low salt medium corresponds to a value <15% of the salt content of Sea water, preferably <12% and particularly preferably <10%>. A total salt content of <8% of the salt content of sea water is very particularly preferred.

No sodium salts are added to the low salt medium. No chloride salts are further added to the low salt medium according to the invention.

According to the invention, addition is understood to mean addition both in dissolved and in solid form. For example, the addition of sea water, even in the smallest amounts, would be an addition of sodium or chloride salts according to the invention. The addition of unusual media components to the ^'medium of the invention, must, if unusual .Medienbestandteile corresponding sodium or chloride ions, to be understood as addition of these salts. However, it is clear to the person skilled in the art that the usual (and mostly necessary, ie indispensable) media components are water (tap water), yeast extract, corn steep liquor and the like. Ä. Have a very low, unavoidable proportion of sodium and chloride. The addition of such customary media components is therefore not to be understood according to the invention as the addition of sodium or chloride salts.

For example, yeast extract contains less than 2% by weight NaCl. Therefore, yeast extract is added to the medium in the usual way, i.e. between 10 and 20 g / L, the NaCl content increases by less than 0.2 g / L. According to the invention, this is not understood to be the addition of NaCl.

In a particularly preferred embodiment, this medium is therefore free of sodium and / or chloride salt additives.

The total sodium content of the low salt medium is very particularly preferably below 2 g / L, preferably below 500 mg / L and very particularly preferably below 150 mg / L. The total chloride content of the low salt medium is preferably below 2 g / L, preferably below 500 mg / L and very particularly preferably below 250 mg / L.

The sum of the proportions by weight of Na and Cl ions is particularly preferably less than 1.75 g / L.

The low salt medium further preferably comprises one or more carbon sources, as well as one or more nitrogen sources. Substances which can be used as sources of carbon and nitrogen are well known to the person skilled in the art for the cultivation of microorganisms of the order Thraustochytriales. Carbon sources that can be used are, for example, carbohydrates such as glucose, fructose, xylose, sucrose, maltose, soluble starch, fucose, glucosamine, dextran, glutamic acid, molasses, glycerin or mannitol or fats and oils or vegetable hydrolysates.

Usable natural nitrogen sources are, for example, peptone, yeast extract, malt extract, meat extract, casamino acids, corn steep liquor or soybeans, usable organic nitrogen sources are, for example, glutamate and urea, but also inorganic nitrogen sources such as, for example, ammonium acetate, ammonium hydrogen carbonate, ammonium sulfate or ammonium nitrate can be used as the nitrogen source.

The low salt medium can contain all further components which are beneficial to the person skilled in the art for the cultivation of microorganisms of the order Thraustochytriales, in particular inorganic salts of, for example, Ca, Mg, K, Fe, Ni, Co, Cu, Mn, Mo or Zn. Examples include phosphates such as potassium dihydrogen phosphate or Carbonates such as calcium carbonate, sulfates such as ammonium sulfate, magnesium sulfate, iron sulfate or copper sulfate. Other inorganic salts that can be used are, for example, halides, such as potassium bromide or potassium iodide.

Possibly. the medium may include additional macro or micronutrients such as amino acids, purines, pyrimidme, com steep liquor, protein hydrolyzates, vitamins (water soluble and / or water insoluble) and other media components well known to those skilled in the art. Anti-foaming agents can be added if necessary. The medium can contain complex components or be chemically defined.

The amount of the individual components can vary as long as there is no negative effect on the growth or productivity of the microorganisms. The person skilled in the art can easily determine the composition according to the needs of the microorganism in the individual case. Generally, the carbon source is added in a concentration of 50 to 300 g / L and the nitrogen source in a concentration of 1 to 30 g / L. The nitrogen content is preferably made dependent on the carbon content of the medium.

A particularly preferred low salt medium optionally comprises, in addition to further constituents such as, for example, nutritional constituents, at least one salt selected from the group consisting of magnesium sulfate, calcium carbonate and potassium phosphate, where the salt (s) are preferably added at a maximum of 3 g / L each, particularly preferably at a maximum of 1 g / L each, without the total salt content according to the invention being exceeded. It is particularly preferred if magnesium sulfate, calcium carbonate and potassium phosphate are added to the medium.

Preferred nutritive components are glucose, yeast extract and / or corn steep liquor (Com Steep Liqour [CSL]) in the usual amounts and other nutritional components familiar to the person skilled in the art.

The pH of the medium is adjusted to a range from 3 to 10, preferably 4 to 8, particularly preferably 5 to 7 and very particularly preferably 6 by adding an appropriate acid or alkali.

The medium is then sterilized. Techniques for sterilizing media are well known to the person skilled in the art, for example autoclaving and sterile filtration.

The cultivation can take place in batch, fed-batch or in a continuous manner, as is generally known to the person skilled in the art.

Batch or fed-batch cultivation is usually carried out over 1 to 12 days, preferably 2-10 days, particularly preferably for 3-9 days.

The media components can be added to the low salt medium individually or as a mixture, and a pre-prepared mixture is also conceivable. The components, in particular the carbon and nitrogen source (s) or certain medium additives, can be added before or during the cultivation. The addition can be repeated one or more times or continuously.

The PUFAs produced are generally in the form of neutral fats, for example as triacylglycerides or polar lipids such as, for example, phosphatidylcholine, phosphatidylethanolamine or phosphatidylinositol.

However, for the purposes of the present invention, the terms PUFA, n3 fatty acid or n3 active substances are understood to mean all possible forms in which the corresponding fatty acids can be present, ie both as free fatty acids, esters, triglycerides, phospholipids or other derivatives. All these substances are summarized below and the terms are used synonymously. Furthermore, the PUFAs can be converted and enriched by chemical or biocatalytic transesterification, for example with the aid of suitable enzymes (lipases), before or after isolation from the culture.

The isolation of PUFAs from the fermented microorganisms or the medium and the analysis of the fatty acid spectrum is carried out by methods known and customary to the person skilled in the art [Wanasundara JN, Wanasundara, L, Shahidi, F., Omega-3 fatty acid concentrates: a review of production technologies, Seafoods - Quality, Technology and Nutraceutical Applications, 2002, pp. 157-174].

FIG. 1 shows the product formation of DHA as a function of the salt concentration. The maximum can be clearly seen in the area according to the invention (data from Example 1)

Figure 2 shows biomass and DHA content depending on the salt concentration. The maximum in the range according to the invention can also be clearly seen here (data from Example 1).

The fermentation medium on which the process according to the invention is based is described below with the aid of a few examples. However, the fermentation medium and the invention are not restricted to these examples.

Example 1: Influence of different amounts of salt in the medium on the production of PUFA by Ulkenia sp. SAM 2179.

Strain SAM 2179 (Ulkenia spec BP-5601; WO9803671) was cultivated in 300 ml Erlenmeyer flasks with a chicane in 50 ml medium (temperature: 28 ° C, shaking rate: 150 rpm).

Medium 1: DH1 medium

Glucose monohydrate (g / L): 56.25

Yeast extract (g / L): 12.5 [Difco]

Tropic Marin (g / L): 16.65 [Dr. Biener GmbH, Wartenberg, Germany] pH adjusted to 6.0 with HC1

Medium 2: DH2 medium (without salt) glucose monohydrate (g / L): 56.25

Yeast extract (g / L): 12.5 [Difco] pH adjusted to 6.0 with HC1

The salts of Medium 1 (Tropic Marin) were used in the following concentrations: IX (Medium 1), 0.75X (Medium 1.1), 0.5X (Medium 1.2), 0.25X (Medium 1.3) and 0.1X (Medium 1.4).

The cells were harvested after 48 h of cultivation by centrifugation. The cells were then freeze-dried and the dry biomass was determined. The cells were disrupted and the fatty acids were determined by heat treatment for 2 hours in 10% strength methanolic hydrochloric acid at 60 ° C. (with stirring). The esters were then analyzed in a gas chromatograph to determine the fatty acid composition (Wanasundara, UN, Wanasundara, J., Shahidi, F., Omega-3 fatty acid concentrates: a review of production technologies, Seafoods - Quality, Technology and Nutraceutical Applications, 2002, Pp. 157-174). Table 1: Influence of different salt contents on fermentation parameters Total salt content NaCl time BTM DHA '' Amount of DHA- from sea water based on BTM DHA room-line- seawater yield (approx.%. Approx. G (h) (g / L) (%) (&^' D fg / Lxd)

Medium 1 50 15 48 25.4 16.6 4.20 2.11

Medium 1.1 37.5 11.25 48 23.7 16.3 3.87 1.94

Medium 1.2 25 7.5 48 22.5 18.9 4.26 2.13 VB

Medium 1.3 12.5 3.75 48 19.9 21.2 4.22 2.11

Medium 1.4 5 1.5 48 21.0 23.9 5.02 2.51

Medium 2 0 0 48 17.2 19.0 3.25 1.63

^* 'Mean values from two experiments each.

BTM: dry organic matter; DHA / BTM:% by weight DHA (docosahexaenoic acid) per unit weight BTM; g / Lxd space-time yield in grams per liter per day; left: hour; VB: comparative example.

Table 2: Influence of different salt contents on the fatty acid spectrum 14: 0 15: 0 16: 0 22: 5 22: 6 Other PA DPAωf. DHAω3 fatty acids (%) (%) {%) <%) (%) (%)

Medium 1 2.9 3.7 32.2 10.2 45.7 5.5

Medium 1.1 2.6 3.8 31.8 10.4 45.2 6.2

Medium 1.2 2.0 3.6 29.5 11.2 47.4 6.3 VB

Medium 1.3 2.0 3.9 30.5 10.8 46.3 6.5

Medium 1.4 2.6 4.8 32.4 11.1 43.5 5.6

Medium 2 2.2 8.2 25.4 12.0 46.4 6.0

^* 'Medium wave from two experiments each. 14: 0 myristic acid; 15: 0 pentadecanoic acid; 16: 0 palmitic acid; DPAωό: docosapentaenoic acid (omegaό); DHAω3: docosahexaenoic acid (omega3)

The fermentation of Ulkenia sp. Strain SAM 2179 at different concentrations. from Tropic Marin, starting from about 50% to 0% seawater salt content, tends to show a decrease in biomass with decreasing salt content in the medium. However, surprisingly, fermentation is a significant exception with a low salt content of around 5% of sea water. Here, the course of biomass production surprisingly shows an intermediate maximum at which higher values are achieved again. In addition, the proportion of the predominant fatty acid DHA per dry biomass increases with decreasing salt concentration and also has its highest value at 5% of the sea water salt content, while being at an even lower salt content decreases again. This results in a maximum for the space-time yield of the essential PUFA DHA, at about 5% seawater salt content (see Table 1). This maximum leads to an increase in the DHA space-time yield of more than 15%. In relation to the entire fatty acid spectrum, the proportion of DHA at around 5% sea salt content is somewhat lower than at higher or lower salt contents (see Table 2), but the overall productivity of DHA and generally fatty acids or oil is greatest here ( see table 1). Based on these surprising results, the low salt medium on which the invention is based was developed.

Example 2: Production of PUFA by Ulkenia sp. SAM 2179 in various fermentation media.

Strain SAM 2179 was cultivated in 300 ml Erlerrmeyer flasks with a chicane in 50 ml medium (temperature: 28 ° C, shaking rate: 150 rpm).

Medium 1: PH 1 medium

Glucose monohydrate (g / L): 56.25

Yeast extract (g / L): 12.5 [Difco]

Medium 2: DH2 medium (without salt): glucose monohydrate (g / L): 56.25 yeast extract (g / L): 12.5 [Difco] pH adjusted to 6.0 with HC1

Medium 3: DH3 medium (with salt supplement without sodium and without chloride addition):

Glucose monohydrate (g / L): 56.25

Yeast extract (g / L): 12.5 [Difco]

Magnesium sulfate (g / L): 1

Calcium carbonate (g / L) 1

Potassium phosphate (g / L) 1 pH adjusted to 6.0 with H ₂ SO ₄

The cells were harvested after 48 h of cultivation by centrifugation. The cells were then freeze-dried and the dry biomass was determined. Disruption of the cells and The fatty acids were determined by heat treatment for 2 hours in 10% strength methanolic hydrochloric acid at 60 ° C. (with stirring). The esters were then analyzed in a gas chromatograph to determine the fatty acid composition.

Table 3: Influence of different salt contents on fermentation parameters total NaCl time BTM DHA / amount of DHA salt content related to BTM in space-time of MeerMeerDHA Ausbeυ .e water water approx.% Approx. G (h) (g / L). (%) (g / L) (g / Lxd)

Medium 1 ^* 50 15 48 25.4 16.6 4.20 2.11 Comparative example

Medium 2 ^* 0 0 48 17.2 ^• 19.0 3.25 1.63

Medium 3 ^** 10 0 48 22.7 18.9 4.29 2.14 ^J Average values from two experiments each. ^J Mean values from three experiments each.

The low salt medium according to the invention without sodium and chloride addition was first used in the fermentation in a concentration of about 10% of the sea water salt content (medium 3). The biomass obtained is somewhat lower than that of fermentations with about 50% sea water salt content, but the DHA content per dry biomass is larger and surprisingly leads to a total. same or even increased space-time yields (see Table 3). This is of great advantage for later processing of the biomass for the production of DHA. Surprisingly, it was found that the addition of sodium and / or chloride (the main salts of sea water) can advantageously be completely dispensed with for the fermentation.

Example 3: Influence of different amounts of salt in the medium without sodium and chloride salt addition on the production of PUFA by Ulkenia sp. SAM 2179.

Strain SAM 2179 was cultivated in 300 ml Erlenmeyer flasks with a chicane in 50 ml medium (temperature: 28 ° C, shaking rate: 150 rpm). Medium 3: DH3 medium (with salt supplement without sodium and without chloride addition) glucose monohydrate (g / L): 56.25

Yeast extract (g / L): 12.5 [Difco] magnesium sulfate (g / L): 1 calcium carbonate (g / L) 1. IX salts of potassium phosphate (g / L) 1 pH adjusted to 6.0 with H ₂ SO ₄

The salts of medium 3 were used in the following concentrations: 10X with 10 g / L each, 2X with 2 g / L each, IX with 1 g / L each, 0.5X with 0.5 g / L each or 0.25X with 0 each, 25g / L.

The cells were harvested after 48 hours of cultivation by centrifugation. The cells were then freeze-dried and the dry biomass was determined. The cells were disrupted and the fatty acids were determined by heat treatment for 2 hours in 10% strength methanolic hydrochloric acid at 60 ° C. (with stirring). The esters were then analyzed in gas chromatograph to determine the fatty acid composition.

Table 4: Influence of different salt contents on production parameters total NaCl time BTM ^' DHA / amount DH ^' A- salt content based on BTM ^' of space- from sea-sea water DHA cell water yield approx.% Approx. G (h) (g ^/ i-) ( %) (g / L) (g / Lxd)

Medium 3 (10X) 100 0 48 32.5 12.5 4.05 2.03 Comparative example

Medium 3 (2X) 20 0 48 23.8 20.3 4.82 2.41

Medium 3 (IX) ^** 10 0 48 22.7 18.9 4.29 2.14

Medium 3 (0.5X) 5 0 48 22.7 21.2 4.82 2.41

Medium 3 (0 _; 25X) 2.5 0 48 21.2 19.7 4.18 2.09

^-, mean values from three experiments each.

The microorganism Ulkenia spec. Was used to determine the optimum salt concentration of the fermentation medium without the addition of sodium or chloride. Strain 2179 fermented in the above medium with different salt concentrations. In this case too, the DHA content per dry biomass is highest with a salt content of 5% of the sea water (see also Example 1). The productivity in terms of space-time yield is also at an optimal value at this salinity (see Table 4). Example 4: Production of PUFA by Schizochytrium strain SR21 (Schizochytrium spec, MYA-1381; EP0823475) in different fermentation media:

Schizochytrium strain SR21 was cultivated in 300 ml Erlenmeyer flasks with a chicane in 50 ml medium (temperature: 28 ° C, shaking rate: 150 rprα).

Medium 1: DHI medium

Glucose monohydrate (g / L): 56.25

Yeast extract (g / L): 12.5 [Difco]

Medium 2: DH2 medium (without salt) glucose monohydrate (g / L): 56.25 yeast extract (g / L): 12.5 [Difco] pH adjusted to 6.0 with HC1

Medium 3: DH3 medium (with salt supplement without sodium and without chloride addition)

Glucose monohydrate (g / L): 56.25

Yeast extract (g / L): 12.5 [Difco]

Magnesium sulfate (g / L): 1

Calcium carbonate (g / L) 1

Potassium phosphate (g / L) 1 pH adjusted to 6.0 with H ₂ SO ₄

The cells were harvested after 48 hours of cultivation by centrifugation. The cells were then freeze-dried and the dry biomass was determined. The cells were disrupted and the fatty acids were determined by heat treatment for 2 hours in 10% strength methanolic hydrochloric acid at 60 ° C. (with stirring). The esters were then analyzed in a gas chromatograph to determine the fatty acid composition. Table 5: Influence of different salt contents on fermentation parameters total NaCl time BTM DHA / amount of DHA salt content based on ^■ BTM DHA Ra m- from MeerMeenvasser time water yield approx.% Approx. G (h) (g / L) (% ) (g / L) (g / Lxd)

Medium 1 SR 21 50 15 48 • 27.1 13.7 3.61 1.81 Comparative example

Medium2 SR 21 0 0 48 19.5 18.2 3.56 1.78 Medium 3 SR 21 10 0 48 22.3 18.7 4.17 2.09

Table 6: Influence of different salt contents on the fatty acid spectrum 14: 0 15: 0 16: 0 22: S 22: 6 Other VA DPAωö DHÄω3 fatty acids (%) (%) (%) (%) (%) (%)

Medium 1 SR 21 3.5 3.3 43.4 7.5 37.5 4.8 Comparative example

Medium 2 SR 21 3.5 5.3 43.1 7.4 36.5 4.2

Medium 3 SR 21 3.5 4.1 44.4 7.1 36.1 4.8

The low salt medium described in the invention also leads to an optimization of the production of PUFA in other labyrinthulomycota organisms. For example, the microorganism Schizochytrium spec. Strain SR21 can be fermented in low salt medium without sodium or chloride addition. The DHA content based on the dry matter also has an optimum in this case with a salt content of 10% of the sea water. In addition, there is an even stronger effect on the space-time yield of DHA and thus on the productivity of the fermentation (see Table 5). The DHA content based on the entire fatty acid spectrum is also somewhat lower in this case (see Table 6), but this does not harm a surprisingly high space-time yield of DHA (see Table 5). The optimized low salt medium on which the invention is based leads to a general increase in the production of PUFAs in various members of the labyrinthulomycota.

Claims

claims:

1. Process for the cultivation of microorganisms of the genus Thraustochytriales, the microorganisms being cultivated in a fermentation medium without the addition of sodium salts and chloride salts, with a total salt content of less than 3.5 g / L total salts.

2. The method of claim 1, wherein the microorganisms produce a production of more than 30 wt .-% oil per unit weight of dry biomass, preferably more than 35 wt .-% oil per dry matter mass.

3. The method according to claim 1 or 2, wherein up to 3g / L CaCO _{3 are} added to the medium, preferably 1g / L.

4. The method according to any one of the preceding claims, wherein the microorganisms produce more than 10%, preferably more than 14%), and very particularly preferably more than 18% DHA per dry biomass.

5. The method according to any one of the preceding claims, wherein the microorganisms produce a production of more than 5%, preferably more than 1%, and very particularly preferably more than 10% DPA per dry biomass.

6. The method according to any one of the preceding claims, characterized by the use of a low salt medium, the total salt content of which is in the range <15% of the salt content of sea water, preferably <12%>, particularly preferably <10% and very particularly preferably <8%.

7. The method according to any one of the preceding claims, characterized in that the sum of the proportions by weight of Na ⁺ - and Cl ^" ions in the low salt medium comprises less than 1.75 g / L.

8. The method according to any one of the preceding claims, characterized in that the total sodium content of the low salt medium is below 150 mg / L.

9. The method according to any one of the preceding claims, characterized in that the total chloride content of the low salt medium is less than 250 mg / L.

10. The method according to any one of the preceding claims, characterized in that the low salt medium comprises glucose, yeast extract, magnesium sulfate, calcium carbonate and potassium phosphate.

11. The method according to any one of claims 1 to 9, characterized in that the low salt medium comprises glucose, corn steep liquor, magnesium sulfate, calcium carbonate and potassium phosphate.

12. The method according to claim 10 or 11, characterized in that the low salt medium comprises magnesium sulfate, calcium carbonate and potassium phosphate each at most 3 g / L, particularly preferably at most 1 g / L each.

13. The method according to any one of the preceding claims, characterized in that the low salt medium has a pH between 3 and 10, preferably between 5 and 7.

14. The method according to any one of the preceding claims, characterized in that the cultivation takes place between 10 ° C and 40 ° C, preferably between 25 ° C and 35 ° C.

15. The method according to any one of the preceding claims, characterized in that the cultivation takes place for 1 to 10 days, preferably for 3 to 9 days.

16. The method according to any one of the preceding claims, characterized in that the microorganism belongs to the genus Schizochytrium, Thraustochytrium or Ulkenia.

17. The method according to any one of the preceding claims, characterized in that the microorganism Ulkenia spec. SAM 2179 is.

18. The method according to any one of the preceding claims, characterized in that the microorganism Schizochytrium spec. SR 21 is.

19. Oil with a content of at least 10% DHA, produced using a method according to one of claims 1 to 18 and subsequent isolation of the oil from the culture broth and / or the biomass contained therein.

20. Oil with a content of at least 5% DPA, produced using a method according to one of claims 1 to 18 and subsequent isolation of the oil from the culture broth and / or the biomass contained therein.

21. DHA with at least 90% purity, produced using a method according to one of claims 1 to 18 and subsequent isolation of the DHA from the culture broth and / or the biomass contained therein.

22. DPA with at least 90% purity, produced using a method according to one of claims 1 to 18 and subsequent isolation of the DPA from the culture broth and / or the biomass contained therein.

23. Biomass obtainable by a method according to one of claims 1 to 18 and subsequent separation of the biomass from the culture broth.

24. Feed comprising biomass according to claim 23.

25. Food for human consumption comprising biomass according to claim 23.