WO2014096024A1

WO2014096024A1 - Method for producing a lipid-rich composition from microorganisms

Info

Publication number: WO2014096024A1
Application number: PCT/EP2013/077125
Authority: WO
Inventors: Miguel CARAUCÁN DÁVILA; Kurt Gasser; David Ruppen
Original assignee: Lonza Ltd
Priority date: 2012-12-19
Filing date: 2013-12-18
Publication date: 2014-06-26

Abstract

The subject matter of the invention is a method for producing a lipid-containing composition, wherein lipids are selectively extracted from microorganisms in the presence of at least one polar organic solvent and water. The invention also relates to lipid-rich compositions and uses thereof.

Description

PROCESS FOR PREPARING A LIPID-RICH MICRO-ORGANIZED COMPOSITION Field of the Invention

The invention relates to a method for producing a lipid-containing composition, wherein lipids are selectively extracted in the presence of at least one polar organic solvent and water from microorganisms. The invention also relates to lipid-rich compositions and uses.

Background Art and Background of the Invention Lipids are important components of human and animal nutrition. For human nutrition, especially fatty acids and triacylglycerides are important. Polyunsaturated fatty acids (PUFA), in particular omega-3 fatty acids (n3 fatty acids), are essential components of the human diet. They can be isolated from natural sources or absorbed through food. However, in most industrialized nations, the supply of n3-fatty acids is deficient. In contrast, the total fat content in the diet and the intake of saturated fatty acids and N6 fatty acids are too high. This is due to a change in dietary composition, especially in the last 150 or so years, which is correlated with the onset of various chronic diseases, such as cardiovascular disease, the leading cause of death in industrialized nations. Targeted intake of n3 fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), for example, can reduce cardiovascular risk. Therefore, it is recommended by health organizations such as the WHO to increase the consumption of n3-fatty acids. PUFAs and n3 fatty acids are mainly derived from marine coldwater fish. In recent years, microorganisms have increasingly been used as industrial sources. From suitable fermentation cultures, PUFAs can be isolated under controlled and inexpensive conditions. Suitable microorganisms for obtaining PUFAs are, for example, microalgae, bacteria or dinoflagellates. In recent years, attempts have been made to isolate fatty acids and other lipids in higher yields through improved fermentation conditions. WO 03/039832 discloses a process for the purification of oils and fatty acids from microalgae. In this case, dried microalgae are mixed with hexane and digested by grinding and centrifuged, whereby an oil-containing organic phase is obtained. After separation of the solvent, an oil fraction containing unsaturated fatty acids is obtained.

EP 9 515 460 B1 discloses a process for isolating an oil rich in unsaturated fatty acids from unicellulars, especially dinoflagellates. The protozoa are cultivated under defined conditions, after which the cells are harvested and freeze-dried. The extraction of the oil is then carried out with hexane with stirring at 50 ° C.

EP 0 935 667 B1 discloses a process for isolating lipids, in particular docosahexaenoic acid or docosapentaenoic acid, from microalgae of the genus Ulkenia. The algae are cultivated, harvested and freeze-dried. The extraction of the lipids is carried out with chloroform / methanol with simultaneous cell disruption by homogenization. However, the use of chloroform in industrial processes is disadvantageous for environmental reasons and generally in the field of food production. A modified process for isolating fatty acids from microalgae is disclosed in EP 2 105 506 A1. In this case, a complexing agent or a flocculant is added to the fermentation medium. The isolation of the oil takes place freeze-dried harvested cells digested by heat treatment in the presence of methanolic hydrochloric acid. In the process, the fatty acid profile is changed and the qualitative composition of the oil is improved, whereby overall a high overall yield is achieved.

In the described method it is necessary to break up the microorganisms and destroy them. This is disadvantageous if the cells grow slowly in fermentation solutions. DE 10 2006031 212 therefore proposes to carry out the extraction of ingredients in vivo in the presence of a solvent, wherein the cells are to be immobilized in porous layers of a silica gel / nanosol.

The described known methods are still in need of improvement, since they require numerous and sometimes cumbersome process steps and the yields are often not satisfactory. The methods also consume a lot of energy, in particular during drying, lyophilization and mechanical disruption of the cells. Some methods also use solvents or chemicals which are harmful to health and therefore undesirable in the production of food.

Object of the invention

The object of the invention is to provide compositions, processes and uses which overcome the disadvantages described above.

More particularly, the object of the invention is to provide simple and efficient methods for selectively isolating lipids from microorganisms. In particular, the isolation and purification of unsaturated fatty acids, such as DHA and DPA should be made possible in the highest possible yield and purity. The process should have as few process steps and thereby be easily reproducible on an industrial scale and at the same time cost. It is a further object of the invention to provide a lipid-containing composition which is extractable from microorganisms and which has an advantageous distribution of unsaturated fatty acids and at the same time a low proportion of undesired by-products. The resulting compositions should be safe for health and suitable for applications in cosmetics, pharmacy and food.

Disclosure of the invention

Surprisingly, the problem underlying the invention is solved by methods, compositions and uses according to the claims. The invention relates to a method for producing a lipid-containing composition, wherein lipids are extracted in the presence of at least one polar organic solvent and water from microorganisms.

The process according to the invention can be carried out with various microorganisms, such as eukaryotic unicellular organisms or bacteria. In a preferred embodiment of the invention, the microorganisms are microalgae. The microorganisms are preferably eukaryotic protozoa. Microalgae are often eukaryotic unicellular organisms, but can also be eukaryotic multicellular organisms. The microorganisms are in a preferred embodiment Stramenopilen {Stramenopiles, also Chromista or Heteroconta). These are eukaryotes, which possess at least in certain stages of growth two differently formed flagella. Most species are single-celled, with the brown algae but also include highly differentiated Mehrzeller.

Microorganisms which are suitable for obtaining PUFA are, for example, dinoflagellates (Dinophyta), in particular the genus Crypthecodinium, such as C. cohnii, or stramenopiles, such as the pinguiophyceae such as Glossomastix, Phaeomonas, Pinguiochrysis, Pinguiococcus and Polydochrysis.

Preference is given to microorganisms of the genera Japonochytrium, Schizochytrium, Thraustochytrium, Althornia, Labyrinthuloides, Aplanochytrium and Ulkenia. These microorganisms are particularly suitable for the purification of PUFA. Particularly preferred are those of the genus Ulkenia, Schizochytrium or Thraustochytrium.

Specifically suitable are the strains Thraustochytrium aureum (in particular ATCC 2821 1 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. Suitable strains, their uses as bioreactors, and fermentation and culture conditions are well known in the art. WO91 / 07498 discloses the production of PUFA with organisms of the genera Schizochytrium and Thraustochytrium, while WO98 / 03671 discloses the production of PUFA with microorganisms of the genus Ulkenia. The microorganisms may be wild-type strains or mutants. Suitable mutants are, in particular, those which contain elevated levels of lipids, such as storage lipids, or which contain increased levels of specific desired fatty acids, such as DHA. Mutants with particular properties are known in the art or can be obtained by conventional methods such as recombinant techniques or classical mutagenesis by means of chemicals or radiation.

The selection of the microorganism for the method according to the invention takes place with regard to the lipids to be extracted. The microorganism is selected with regard to the fatty acid type and distribution. The process conditions can be adjusted with an assay as described in the embodiments. Preferably, the microorganism is under the extraction conditions not lysed by the polar organic solvent. The microorganisms may optionally be supplemented with additives such as flocculants or complexing agents. The microorganism can be provided mixed with water, for example as cell paste or in aqueous solution, such as fermentation solution or buffer solution, or as dried biomass, for example as powder or granules.

The inventive method is used for the extraction of lipids. The process gives a lipid-containing composition. In this case, the lipid-containing composition may be the immediate product of the extraction, and thus an extract containing lipids and solvents. However, the lipid-containing composition can also be obtained after the extract has been further purified and / or concentrated, for example by removal of the solvent.

Preferably, the lipids are or include fatty acids and / or triacylglycerides. The lipids are preferably at least partially as an oil. The triacylglycerides can be fats, fatty oils and waxes. It is preferred that the lipids are storage lipids or contain storage lipids. Memory lipids are lipids from the cell interior that are not part of the cell membrane. They form accumulations in the cell interior, for example in the form of oil droplets (oleosomes). They can serve the cells as an energy reserve. These include, above all, neutral lipids (triglycerides). In a preferred embodiment, the lipids are therefore not phospholipids, which serve primarily for the formation of the cell membrane.

Preferably, according to the invention, a composition is obtained which, after separation of the solvents (organic solvents and water), contains more than 10% by weight, in particular more than 50% by weight or more than 90% by weight, of fatty acids and triacylglycerides. The proportion of membrane-forming lipids, in particular of phospholipids, is preferably less than 10% by weight, less than 5% by weight or less than 2% by weight. In a preferred embodiment of the invention, the lipids are polyunsaturated fatty acids (PUFA). Polyunsaturated fatty acids have at least two double bonds along the carbon chain. In a particularly preferred embodiment of the invention, the fatty acids are omega-3 fatty acids. These are compounds in which the first double bond is located at the C-3 position from the end of the chain (omega end). The total proportion of all unsaturated fatty acids, in particular the proportion of omega-3 fatty acids, in the extracted lipids is preferably at least 10% by weight, preferably at least 50% by weight or at least 90% by weight, based on the total weight of the lipids extracted. Preferably, according to the invention, a composition is obtained which, after separation of the solvents (organic solvents and water), contains more than 10% by weight, in particular more than 50% by weight or more than 90% by weight, of unsaturated fatty acids.

It is particularly preferred that the fatty acid is docosahexaenoic acid (DHA). The fatty acid can also be docosapentaenoic acid (DPA). It is known that these fatty acids accumulate and isolate in large quantities in microorganisms such as microalgae. Further omega-3 fatty acids which are suitable according to the invention are eicosatetraenoic acid, eicosatrienoic acid and alpha-linolenic acid. The total amount of DHA in the extracted fatty acids is preferably at least 10% by weight or at least 50% by weight, preferably at least 80% by weight or at least 90% by weight, based on the total weight of the extracted fatty acids. Preferably, according to the invention, a composition is obtained which, after separation of the solvents (organic solvents and water), contains more than 10% by weight or more than 50% by weight, in particular more than 80% by weight or more than 90% by weight of DHA.

The microorganisms are preferably selected to contain a high concentration of the desired lipids. In a preferred embodiment of the invention, the microorganisms contain at least 1% by weight or at least 5% by weight, preferably at least 10% by weight, at least 20% by weight. or at least 50% by weight of storage lipids, in each case based on the dry weight of the microorganisms (the dry weight here denotes the weight without water). Preferably, the proportion of memory lipids in the microorganisms 1 to 90 wt.%, In particular 5 to 85 wt.%, Particularly preferably 20 to 80 wt.%, Based on the dry weight. In a preferred embodiment of the invention, the microorganisms contain, based on the dry weight of the microorganisms, at least 1% by weight or at least 5% by weight, preferably at least 10% by weight, at least 15% by weight or at least 25% by weight of unsaturated fatty acids. especially DHA. Preferably, the proportion of unsaturated fatty acids, in particular DHA, in the microorganisms, based on the dry weight, 1 to 90 wt.%, In particular 5 to 85 wt.%, Particularly preferably 20 to 80 wt.%.

In the process according to the invention, more than 10% by weight or more than 50% by weight, in particular more than 80% by weight, more than 85% by weight, more than 90% by weight or more than 95% by weight of the storage lipids are preferred , in particular the unsaturated fatty acids, in particular the DHA, extracted, based on the respective total content in the microorganisms. The extraction according to the invention is carried out in the presence of water and at least one polar organic solvent. The water also serves as a solvent. The solvents contained form a mixture and / or a liquid / liquid two-phase system. The invention is based on the unexpected finding that, in the presence of polar organic solvents and water, a particularly efficient and selective extraction of lipids, in particular of unsaturated fatty acids, from microorganisms takes place. The process is so efficient that even without digestion of the microorganisms, a substantial proportion of the lipids can be extracted. It was surprising that the extraction can take place very selectively even without digestion of the cell wall of the microorganisms.

The extraction takes place in an extraction mixture which comprises or derives from the microorganisms, water and at least one polar organic solvent consists. In a particularly preferred embodiment, the extraction mixture has two liquid phases. These are a lower aqueous phase (which contains the microorganisms) and an upper organic phase. The lipids are transferred to the organic phase during extraction. The extraction mixture may alternatively comprise a single liquid phase containing a solvent mixture of the polar organic solvent and water. The lipids then go into the liquid phase during extraction. In this case, the polar organic solvent may be partially or completely miscible with water. Polar organic solvents are especially those containing oxygen as the heteroatom. Suitable examples are esters, ethers, alcohols and ketones. In a preferred embodiment of the invention, the polar organic solvent is an ester. The ester is preferably an alkylalkylate, such as ethyl acetate, methyl acetate, n-butyl acetate, isobutyl acetate, methyl propylate or ethyl methoxide. Particularly preferred is the use of ethyl acetate. The organic solvent is liquid at room temperature (23 ° C). Preferably, the boiling point of the organic solvent is above 30 ° C or above 70 ° C. The organic solvent is preferably between 2 and 15 C atoms, in particular between 3 and 10 C atoms. Preferably, the organic solvent does not lyse the microorganisms under the extraction conditions.

The water can be introduced into the extraction mixture with the microorganisms, for example as part of a cell paste, cell suspension, cell solution and / or the fermentation solution. Alternatively or additionally, water may be added separately from the microorganisms.

In a preferred embodiment, the polar organic solvent is completely or partially miscible with water. If the organic solvent is partially miscible with water, it is preferably saturated or supersaturated. The extraction mixture may contain additional water, which then forms a second liquid phase (the aqueous phase). In another embodiment, the polar organic solvent is nearly saturated with water, that is it contains a water content, for example, less than 5% or less than 1% below the saturation limit. Alternatively, the water content may for example be at least 30% below the saturation limit. In preferred embodiments, the water content in the extraction is at least 2.5% by weight, at least 5% by weight, at least 7.5% by weight or at least 10% by weight, based in each case on the sum of all in the extraction mixture at room temperature (23 ° C) liquid solvent (including water). The proportion of water may be, for example, up to 95% by weight, up to 80% by weight or up to 60% by weight. The amount of water may be, for example, 2.5 to 95% by weight, in particular 5 to 80% by weight or 10 to 60% by weight. The remaining portion is in each case polar organic solvent. The water content also includes the water which is optionally contained in the microorganisms and / or bound thereto.

In a further preferred embodiment, the proportion of water in the entire extraction mixture, ie including cell mass and solvent, is at least 2.5% by weight, at least 5% by weight, at least 7.5% by weight or at least 10% by weight. The proportion of water may be, for example, up to 90% by weight, up to 75% by weight or up to 50% by weight. The proportion of water may be, for example, 2.5 to 90% by weight, in particular 5 to 75% by weight.

Saturated ethyl acetate contains about 3.4% by weight of water at room temperature. In a preferred embodiment, the extraction is carried out in the presence of ethyl acetate and water, the ethyl acetate being at least saturated with water. The proportion of water is preferably higher than 3.4% by weight, in each case based on the sum of all liquid solvents (including water). With a significantly higher water content, there are two liquid phases. It is according to the invention not necessary that non-polar organic solvent, such as hexane or chloroform, is included. If necessary, you can but small amounts may be included, for example, up to 10 wt.% Or up to 5 wt.%, Based on the sum of all solvents contained.

The proportion by weight of the microorganisms (dry weight) in the extraction mixture can be, for example, between 2.5 and 75% by weight, in particular between 5 and 50% by weight, or between 10 and 30% by weight.

Before carrying out the extraction, the microorganisms can be cultured in a fermentation batch (culture solution). After growth, propagation and formation of a desired amount of microorganisms, they can be used for extraction.

In a preferred embodiment of the invention, the microorganisms are separated from the culture solution prior to extraction, for example by filtration or centrifugation. The isolated cells are optionally washed. Optionally, several washes are performed to remove contaminants and soluble components of the culture solution. The washing steps can be carried out, for example, with water or buffer solution. In the separation of the microorganisms, an aqueous biomass is obtained (cell paste). In this case, further water can be removed by pressing and / or filtering. The biomass is preferably used directly, ie without further processing steps, for extraction. In this case, the organic solvent and optionally additional water are added and optionally mixed. However, the separation of the microorganisms is not absolutely necessary since the extraction according to the invention can also be carried out after mixing the polar organic solvent with an aqueous fermentation mixture.

In a preferred embodiment, dried microorganisms are used for the extraction. Dried microorganisms are available, for example, by spray drying, lyophilization or heat drying. The use of dried microorganisms may be advantageous to the proportions and adjust the water content as evenly as possible. The drying preferably takes place in such a way that the cells are not disrupted.

In a preferred embodiment, the microorganisms are not dried before extraction, in particular not lyophilized. In known methods, such a step is carried out to allow extraction in the absence of water. According to the invention, however, it has been found that the drying of the microorganisms is not required, since a high yield can be achieved even in the presence of water. Since drying and, in particular, freeze-drying require large amounts of energy, the method according to the invention can therefore be carried out not only simpler and more efficiently, but also more cost-effectively.

In a particularly preferred embodiment of the method according to the invention, the microorganisms are not digested. Preferably, the microorganisms are not digested prior to extraction. Preferably, the microorganisms in the further process, especially in the extraction and / or by the extraction, not or not significantly digested. The extraction conditions are selected so gently that no cell disruption takes place.

Usually in the prior art, an extraction of ingredients in combination with a cell destruction (cell disruption). Cell disruption destroys the integrity of cell walls. As a result, the cell interior - such as: phospholipids, proteins - released and diffuses into the environment. Continuous disruption results in cell debris and agglomerates. The digestion can be followed microscopically or via suitable markers, optionally in conjunction with color reactions. For the digestion of microorganisms, mechanical, chemical, enzymatic and / or physical methods are usually used in the prior art. Mechanical and physical methods include grinding the cells, for example in ball mills, Ultrasonic treatment and homogenization. Even vigorous stirring and shaking can lead to the destruction of cell membranes.

According to the invention, the microorganisms are preferably not treated before and during the extraction in a manner which causes the destruction of the cell membranes or substantially promotes. According to the invention, it is also not necessary that a chemical or enzymatic digestion takes place, for example with lysozyme. Preferably, no pretreatment of the cells for purposes of cell disruption takes place and no additives are added to the extraction mixture for this purpose. However, even with an extraction, a small proportion of the microorganisms can regularly be destroyed, for example because individual microorganisms have a low stability for reasons of age or can already be obtained dead from the fermentation medium. This proportion can be minimized by a gentle treatment, for example, only slight shaking. Therefore, less than 10%, 5% or 2% of the cells are preferably digested without targeted cell disruption. According to the invention, a high yield can be achieved even without measures for cell disruption or for weakening the cell membrane. According to the invention, preferably at least 50% by weight, more than 70% by weight or more than 80% by weight of all storage lipids, especially of all unsaturated fatty acids, are extracted without cell disruption.

According to the invention, it has been found that extraction without cell disruption is particularly gentle and selective, and that in this way a particularly pure lipid-containing composition is obtained. This was unexpected because conventional extraction methods in the prior art require disruption of the cells. Without being bound by theory, the high purity is a consequence of the lipids selectively passing through the cell membrane into the organic phase, while the remainder of the cell interior essentially remains in the cells. In addition, the cell membrane lipids do not appear, or at least less so, to shift to the organic phase unless the membranes / cell wall structures are disrupted. Therefore, the proportion of unwanted by-products (proteins, phospholipids from the membranes) in the extract and in the lipid-rich Composition low. This is particularly advantageous in the food production area as the proportion of potentially incompatible components is reduced. In the case of unknown components, there is always the danger that these immune reactions, such as allergies, will be triggered.

In an alternative embodiment of the invention, the microorganisms are digested before and / or during the extraction. The yield may optionally be further increased by this cell disruption. The cell disruption can be carried out using the methods already mentioned above. The cell disruption can be effected mechanically and / or physically, for example by grinding, homogenizing or with ultrasound, and / or chemically or enzymatically, for example with lysozyme. It has been found that when the extraction according to the invention is combined, in particular with ethyl acetate, with cell disruption, a virtually quantitative separation of the lipids, in particular of the unsaturated fatty acids, can be achieved. Preferably, in a process with cell disruption, at least 80% by weight, preferably more than 90% by weight or more than 95% by weight, of all storage lipids, in particular all unsaturated fatty acids, are extracted. Whether the method according to the invention is combined with means for cell disruption will be decided with regard to the desired product in the context of a cost / benefit analysis. Although an even higher yield is achieved by an additional digestion of the cells, the process becomes more complex. Another disadvantage is that when a cell disruption is carried out, the proportion of contaminants from the cell interior and the membrane in the product is generally higher. In addition to the storage lipids, a higher proportion of lipids is extracted from the cell membrane. The invention thus provides opportunities to isolate a high proportion of lipids in a high purity in a fast process or to isolate an even higher proportion of lipids in a slightly more complex process. In a preferred embodiment of the invention, the microorganisms are not fixed to a support material before and during the extraction. In particular, it is not necessary to immobilize the microorganisms on a solid support or in a gel.

The extraction can be carried out with conventional process extraction equipment and tools. Depending on the scale, the extraction can be carried out, for example, in the presence of membranes, with an extraction centrifuge, an extraction decanter, as a cross-extraction with diafiltration, in a Soxhiet apparatus or with a separating funnel. The extraction can be carried out continuously, semi-batch or as a batch extraction.

In a preferred embodiment of the invention, the extraction is carried out at least twice, in particular two, three, four or five times. It is known to the person skilled in the art that with the repetition of an extraction with in each case relatively small amounts of extraction agent this can be saved and at the same time the yield can be increased (according to the Nernst distribution set). The saving of solvents is desirable in industrial processes for environmental and cost considerations.

The extraction according to the invention can be carried out at customary temperatures, for example at 0 ° C. to 100 ° C. In one embodiment of the invention, the process is carried out at low temperature, for example at 0 ° C to 40 ° C, in particular approximately at room temperature (23 ° C). The extraction is efficient even at such low temperatures. The extraction at low temperatures has the advantage that the extraction is gentle and energy-saving. The yield can be increased by multiple extraction.

In a further preferred embodiment, the extraction is carried out at elevated temperature, for example at 40 ° C to 100 ° C, in particular between 50 ° C and 90 ° C. The extraction at elevated temperature has the advantage that optionally the yield is increased. One possible variant is a combination of extraction with solvent removal.

The extraction is carried out until a desired amount of lipids has passed into the organic phase. For example, extraction with ethyl acetate may be carried out for a period of 1 minute to 10 hours, preferably between 30 minutes and 5 hours. It has been found that after 2 hours at 4 ° C already over 80% of the lipids are taken up from the cell interior into the organic phase.

The mixing of the components may each be assisted by appropriate means, such as gentle shaking or gentle suspending. If the procedure is carried out without disruption of the cells, the person skilled in the art adjusts the conditions so that damage to the cell membranes is avoided as far as possible. The method can be supplemented and optimized by known conventional means, for example, washing, centrifuging, filtering or other separation steps.

Preferably, the extraction mixture is centrifuged to achieve phase separation. The lipid-containing liquid phase is separated off. The product obtained is a mixture containing polar organic solvent and lipids, optionally containing a proportion of water and common impurities. The solvents, ie the organic solvent and water, can be removed by known methods, for example by evaporation at elevated temperature, and / or vacuum, membrane filtration and / or precipitation of fatty acids. As a result, a lipid-containing composition consisting essentially of lipids is obtained. The lipids can be processed further by customary methods, for example by chemical modification or refining. In a preferred embodiment, the polar organic solvent, such as ethyl acetate, after extraction is separated from the product, especially by distillation, and reused in the process. According to the invention was found that a recycling of the solvent without affecting the quality is possible. In this way, material and costs can be saved.

In a particularly preferred embodiment of the invention, the process is one for preparing a DHA-containing composition which, after separation of the ethyl acetate, contains more than 10% by weight of DHA, the DHA being extracted with ethyl acetate from microorganisms of the genus Ulkenia.

In a preferred embodiment of the invention, the method comprises the steps:

(a) providing microorganisms,

(b) adding ethyl acetate and suspending the microorganisms in the presence of water,

(c) extracting lipids, in particular polyunsaturated fatty acids (PUFA), by the ethyl acetate,

(d) separating a liquid phase with lipids dissolved therein, wherein the liquid phase is preferably an organic phase,

(e) optionally digesting the microorganisms,

(f) optionally repeating steps c) to e), and

(g) separating the solvents from the liquid, preferably organic, phase.

In step (b), preference is given to adding so much ethyl acetate that two aqueous phases form. The digestion in step (e) followed by repetition (f) of the extraction process serves to increase the yield.

The process according to the invention is also a process for the isolation and / or purification of lipids, in particular of polyunsaturated fatty acids, from microorganisms. The invention also provides the use of the method according to the invention for the isolation and / or purification of lipids, in particular of polyunsaturated fatty acids. The invention also provides a lipid-containing composition obtainable or obtained by a process according to the invention. The composition of the present invention differs from prior art lipid extracts in the low level of unwanted components from the cell interior and cell membrane. For example, only a small proportion of phospholipids is included. The proportion of peroxides and p-anisidine is low. In addition, no traces of problematic solvents are contained, such as hexane or chloroform. The composition is therefore particularly suitable for the production of products in cosmetics, pharmacy and food.

The composition of the lipids depends on the microorganisms used. In one embodiment, the high lipid composition is a mixture of the solvents with the extracted lipids. In a further embodiment, the composition is one from which the polar organic solvent has been completely or partially separated. The proportion of the organic solvent is then preferably less than 10% by weight, less than 1% by weight, less than 0.5% by weight or less than 0.1% by weight. The proportion of lipids is then preferably at least 90% by weight, at least 95% by weight or at least 98% by weight. In a preferred embodiment of the invention, the lipid-containing composition is an oil. The composition according to the invention preferably contains more than 50% by weight of unsaturated fatty acids, in particular more than 20% by weight or more than 50% by weight of DHA. The proportion of non-lipids is preferably less than 5% by weight, less than 2% by weight or 1% by weight.

The inventive method and the composition of the invention solve the problem underlying the invention. Surprisingly, the method can be used to carry out an efficient and at the same time simple extraction of lipids from microorganisms. Unusually high yields are achieved even without prior digestion of the microorganisms. The process can be carried out with dry or moist microorganisms. Due to the gentle extraction without disruption of the cells can be a highly pure Lipid composition containing low levels of unwanted cell components and components of the cell membrane. Combining the process with digestion can achieve near quantitative recovery of the lipids from the cells. The process is overall energy and cost efficient. Suitable solvents, such as ethyl acetate, are available in large quantities and are harmless to health, so that the purified lipids can be further processed in foods, pharmaceuticals or cosmetics. The solvent ethyl acetate can also be recycled after separation into the process without affecting the quality.

Characters:

FIG. 1 shows light micrographs of samples of ulkenia taken during the extraction process according to Example 8. The samples were taken before the first digestion 0 and after 1, 2 and 3 digestions. For each sample a 4x, 10x and 40x magnification is shown.

FIG. 2 shows the p-anisidine content of the extracts after each extraction (determined photometrically) for the extraction experiment from Example 9. Two different batches of cell material were examined (squares and circles). The 50.0 line is an acceptable threshold for cosmetic and food applications.

Figure 3 shows for the extraction experiment of Example 9, the peroxide content of the extracts after each extraction in mEq / kg. Two different batches of cell material were examined (squares and circles). The line at 15.0 [mEq / kg] is an acceptable threshold for cosmetic and food applications.

Examples: Examples 1 to 7: preliminary experiments for extraction with ethyl acetate / water In a series of experiments, DHA oil was extracted from microorganisms from various extraction mixtures without the cells having been previously disrupted. The microorganism Ulkenia SAM2179 was used as microorganism and pure EtOAc as extractant. In this case, extraction mixtures were prepared with different proportions of water, EtOAc and biomass as shown in Table 1. The biomass was moist after centrifugation, so that the sample to Example 4 contained water. The color of the organic phase with yellow DHA was assessed visually. The results are also summarized in Table 1. Table 1: Overview of sample preparations and results of Examples 1 to 7 (yellowing: ++ distinct, + light, - none).

The samples of Examples 1 to 4 were each mixed and centrifuged. The biomass was initially not susceptible to being suspended well in EtOAc. It was therefore initially not easy to handle mechanically, for example stirring or pumping. Within a few hours, however, the cell mass disintegrated and was then easily processable. The intensity of yellowing indicated whether and how much DHA oil was incorporated into the EtOAc phase. While the EtOAc phase was markedly yellowish in Example 1, the intensity was low in Examples 2 and 3. In Example 4, the EtOAc phase was yellowish and heavily cloudy. In Example 7, the biomass was easily resuspended in water. In contrast, the biomass in EtOAc in Example 4 immediately settled on the ground. The mass was tough and chewing gum-like. The EtOAc initially turned a little yellowish. The Biofume with the ethyl acetate was left in the refrigerator for two hours. Over time, the biomass lost consistency and formed small flakes. The ethyl acetate became distinctly yellow for 2 hours. Under the light microscope, it was seen that the cells of Example 4 were still intact. Therefore, the extraction was carried out through the cell membrane. The results show that, surprisingly, DHA oil can be selectively extracted with a sufficient amount of EtOAc without digestion of the cells in the presence of water from a fermentation solution. In order to show that the yellow color is indeed quantitatively correlated with the recovered DHA oil, the EtOAc phase and the biomass from Example 4 were separated by centrifugation after two hours, rotated and analyzed for fatty acids. The result showed that 81% of the total DHAs are contained in the EtOAc phase. Only a good 18% were not extracted and remained in the biomass. Since the fermentation broth was not digested, the high level of extracted DHA was unexpected.

Experiments 1 to 7 were repeated with digested biomass. No significant changes could be detected (not shown). The staining of EtOAc appeared to be slightly darker on visual inspection. Overall, the experiments show that an extraction of DHA-oil with EtOAc in the presence of water is feasible, even with no previous cell disruption very high yields can be achieved. Example 8: Extraction with ethyl acetate / water

First, 2.18 kg of fermentation broth from Ulkenia SAM2179 was centrifuged at 8000 rpm for 20 min, the supernatant (about 1800 ml) taken off and discarded. The biomass (about 380 g) was divided equally into two 1 L bottles and made up with 900 mL (810 g) EtOAc each. After two hours of storage with cooling, cell disruption was performed. For the content of a bottle was strong stirred and passed through a ball mill (RKM) as often as necessary until the cells are almost 100% broken.

Immediately before the first digestion and after each digestion, samples were taken to determine each of the DHA content in the EtOAc phase and in the biomass. The biomass was then separated by filtration from the EtOAc, rotated and analyzed for fatty acids. The biomass then had to be lyophilized because it had moisture despite strong negative pressure. As a result, it was found that even before the first digestion, 88% of the DHA was contained in the ethyl acetate phase while only 12% remained in the biomass. After completion of the digestion, 97% of the DHA was contained in the ethyl acetate phase while 3% remained in the biomass. The results show that a very high yield can already be achieved without cell disruption. The yield can be further increased by cell disruption, with the DHA being almost completely extracted.

Immediately prior to the first digestion and after each digestion, biomass was removed and examined under the light microscope. The results are shown in FIG. In the case of the sample labeled "0" (bottom line), ie before the first digestion, the individual intact cells are clearly visible after 2 hours, the cells joining together to form loose agglomerates, but after the cells have been disrupted, the biomass is agglomerated After the second digestion, there is hardly any intact cell mass left, but a large proportion of the cell mass was still in the ball mill, so the EtOAc supernatant was once again passed through the mill Throughput, the solid biomass was reduced by a total of about 300 g on any of the images are oil drops can be seen.

The EtOAc phases before and after digestion were analyzed by chromatography. The comparison showed that the number of by-products after cell disruption is increased. This also indicates that the cell interior was released only by the digestion or that membrane components in the Extract extract. In order to obtain as pure as possible a DHA preparation with little-known by-products, it may therefore be advantageous to carry out the process gently without disruption of the cells. This could, for example, be beneficial in the food sector to reduce the risk of intolerances and allergies.

Example 9: Examination of the purity of the product

Ulkenia was used as the starting material in the form of a moist press cake from the filter press with a water content of about 65% by weight. The wet biomass was charged and treated with pure EtOAc in a ratio of 4 parts EtOAc to 1 part dry biomass. The extraction thus resulted in a weight ratio of EtOAc / H ₂ O / dry biomass of approximately 4/2/1. The batch extraction was carried out under stirring and at T = 20 ° C. After 2 h extraction time, the stirring was turned off. After 5 minutes settling time (sedimentation of the suspension), the organic phase was removed by means of a dip tube and pressing with nitrogen from the system. In the organic phase, the DHA enriched. The remaining biomass was subjected to further batch extractions with the same amount of EtOAc (this time saturated with H ₂ O). A total of 5 extractions were carried out. After each extraction, the content of DHA-oil, of p-anisidine and the peroxide value were determined in the organic phase. The organic phases were combined. Finally, the EtOAc was distilled off into a rotovapor. The results are shown in FIGS. 2 and 3. The content of p-anisidine and peroxides serves as a quality marker for the purity. For p-anisidine, Figure 2 shows the photometrically determined amount of the product after each extraction (according to AOCS Official Method Cd 18-90). The usual upper limit acceptable for later use in the food and cosmetics industries is 50.0. Figure 3 shows the peroxide values of the products after each extraction (in mEq./kg, determined according to AOCS Official Method Cd 8-53). The usual upper limit is 15.0. The compositions of the invention have p-anisidine and peroxide values well below the usual thresholds and unusually low for unrefined crude oil. The products are therefore suitable for applications in the food or cosmetics sector.

Examples 10 to 16: Investigation of the importance of the water content

In order to determine the influence of the water content on the extraction, a series of experiments with different water contents was carried out. Ulkenia was used in the form of dry biomass (residual water content of 1 to 4% by weight) and pure EtOAc in Examples 10 to 15 and saturated EtOAc in Example 16 (water content about 3.4% by weight). The compositions of the extraction mixtures and the results are summarized in Table 2 below. In Examples 10 to 15, proportions of water in the indicated amounts were added. In Example 16, no additional water was added. Table 2 shows in each case the determined DHA content of the organic phase. The comparison shows that in the presence of water above the saturation limit of EtOAc a significantly higher yield is achieved.

Table 2: Compositions and results of Examples 10 to 16

(*) Extraction with H ₂ O-saturated EtOAc (3.4% H ₂ O)

Wdh. = Repetition

Claims

1 . A process for preparing a lipid-containing composition, wherein lipids are extracted from microorganisms in the presence of at least one polar organic solvent and water.

2. The method of claim 1, wherein the microorganisms are microalgae,

3. The method according to at least one of the preceding claims, wherein the microorganisms are those of the genus Ulkenia, Schizochytrium or Thraustochytrium.

4. The method according to at least one of the preceding claims, wherein the lipids contain polyunsaturated fatty acids.

5. The method according to at least one of the preceding claims, wherein the lipids contain docosahexaenoic acid, wherein the proportion of docosahexaenoic acid to the lipids is preferably at least 10% by weight.

6. The method according to at least one of the preceding claims, wherein the microorganisms, based on the dry weight, contain at least 2 wt.% Speicheriipide.

7. The method according to at least one of the preceding claims, wherein the extraction mixture comprises an aqueous liquid phase and an organic liquid phase.

8. The method according to at least one of the preceding claims, wherein the polar organic solvent is partially miscible with water and saturated with water, or wherein the polar organic solvent is completely miscible with water.

9. The method according to at least one of the preceding claims, wherein the polar organic solvent is an ester, in particular ethyl acetate.

10. The method according to at least one of the preceding claims, wherein the microorganisms are not digested prior to extraction.

1 1. Process according to at least one of the preceding claims, wherein the extraction mixture contains at least 2.5% by weight of water.

12. The method according to at least one of the preceding claims, wherein the organic solvent is separated after extraction from the lipid-containing composition.

13. The method according to at least one of the preceding claims, comprising the steps:

(a) providing microorganisms,

(e) optionally digesting the microorganisms,

(f) optionally repeating steps c) to e), and

(g) separating the solvents from the liquid, preferably organic, phase.

14. Use of a method according to any one of the preceding claims for the isolation and / or purification of lipids, in particular of polyunsaturated fatty acids.

15. Lipid-containing composition obtainable by a process according to any one of the preceding claims.