PREPARATION COMPRISING A DISPERSION OF PHOSPHOLIPIDS AND FATTY ACID SALTS

Abstract

A preparation containing a mixture of at least one phospholipid, and at least one fatty acid salt of a cation with an anion derived from a fatty acid. A method for preparing such preparation and the use of such preparation to provide polyunsaturated fatty acids to cells, tissues, organs or organisms, for example in the field of cell and tissue culture, organ preservation, human or animal nutrition, or cosmetics.

Claims

1. A preparation, comprising a dispersion of at least one phospholipid, and at least one fatty acid salt of a cation with an anion derived from an omega-3 or omega-6 fatty acid.

2. The preparation according to claim 1, wherein the fatty acid is selected from the group consisting of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), arachidonic acid (ARA), alpha linolenic acid, stearidonic acid, eicosatetraenoic acid, docosapentaenoic acid, linoleic acid, γ-linolenic acid and a derivative thereof.

3. The preparation according to claim 1, wherein the cation is at least one organic cation derived from the group consisting of lysine, arginine, ornithine and choline.

4. The preparation according to claim 1, wherein the phospholipid is a deoiled phospholipid comprising a phosphatidylcholine content of greater than 40 weight % and a phosphatidylethanolamine content of lower than 5 weight %.

5. The preparation according to claim 1, wherein the phospholipid is a non-hydrogenated phospholipid having an oleic and/or linoleic acid content of greater than 70 weight % of total fatty acids.

6. The preparation according to claim 1, wherein a mass ratio of the phospholipid to the fatty acid salt is greater than 0.005.

7. The preparation according to claim 1, wherein the preparation is in the form of a powder or a liquid that results in colloidal dispersions with mean particle sizes of smaller than 1 μm when mixed with water at a pH value of between pH 6.5 and 7.5.

8. The preparation according to claim 1 wherein components are finely dispersed in each other so that both phospholipid and fatty acid salts are present and detectable in amounts of 100 μg and smaller.

9. A culture medium, comprising the preparation according to claim 1.

10. The culture medium according to claim 9, wherein said culture medium is in liquid form, in form of a gel, a powder, a granulate, a pellet or in form of a tablet.

11. A method for preparing the preparation according to claim 1, the method comprising: a. dissolving a phospholipid and a fatty acid salt together in a water-miscible solvent and adding small amounts of water to fully dissolve the salt, thereby obtaining a solution; b. adding the solution to an aqueous system to prepare a lipid dispersion; c. reducing a particle size of the lipid dispersion to a mean particle size of smaller than 500 nm via sonification or homogenization; and d. optionally drying the preparation.

12. A method for preparing the preparation according to claim 1, the method comprising: a. dissolving a phospholipid in a water-miscible solvent, thereby obtaining a phospholipid solution; b. dissolving the fatty acid salt in an aqueous system and adding the phospholipid solution to the aqueous system to prepare a lipid dispersion; c. reducing a particle size of the lipid dispersion to a mean particle size of smaller than 500 nm via sonification or homogenization; and d. optionally drying the preparation.

13. The method according to claim 11, wherein the water-miscible solvent is at least one selected from the group consisting of ethanol, glycerol and propylene glycol.

14. A method for providing polyunsaturated fatty acids to cells, tissues, organs or organisms, the method comprising: contacting the preparation of claim 1 with cells, tissues, organs or organisms.

15. A method for cultivating or stimulating expansion of mesenchymal stem cells, the method comprising: contacting the preparation of claim 1 with mesenchymal stem cells.

16. A feed or food supplement or a pharmaceutical product, comprising the preparation of claim 1.

Description

WORKING EXAMPLES

[0048] The preparations of omega-3 fatty acids were characterized with the following methods:

[0049] 1.1 Particle Size Determination

[0050] Particle size was determined via dynamic light scattering (DLS) measurements (Zetasizer Nano ZS, Malvern).

[0051] 1.2 Turbidity Measurement

[0052] Turbidity of the preparations was measured after 100× dilution with water in a photometer at 600 nm in cuvettes with 1 cm light path.

[0053] 1.3 Sterile Filtration Properties

[0054] 5 ml of liquid preparations were filtered through a 0.2 μm sterile syringe filter. Ease of filtration was evaluated via increasing counter pressure or complete clogging. Turbidity measurement via photometry at 600 nm was evaluated before and after filtration. Higher turbidity is correlated with larger particle sizes. Reduction of turbidity is a sign of material loss through filtration, which is undesirable since the composition of the sample should be changed as little as possible.

[0055] 1.4 Drying and Evaluation of Powder Properties

[0056] The preparations were freeze dried and the dry preparations were evaluated qualitatively with regard to consistency and flow behavior.

Example 1: Preparation and Characterization of Omega-3 Fatty Acid Dispersions with Dioleylphosphatidylcholine (DOPC) in Phosphate Buffer

[0057] To prepare formulations of omega-3 fatty acid dispersions 0.8 g of dioleylphosphatidylcholine (DOPC, Lipoid GmbH) were dissolved in 1 ml ethanol. 0.2 g of fish oil (Omega-3 1400, Doppelherz®), omega-3 ethyl ester (PronovaPure® 500:200 EE, BASF), lysine salt of free omega-3 fatty acid in form of omega-3 lysine salt (AvailOm®, Evonik), omega-3 fatty acid ornithine salt, or omega-3 fatty acid arginine salt were added and dissolved. The lysine salt did not dissolve in ethanol, but it was discovered that it could be dissolved when additionally 20 μl of distilled water were added.

[0058] The lysine salt of the free omega-3 fatty acid in form of omega-3 lysine salt (AvailOm®, Evonik) contains around 67% of fatty acids and high amounts of the omega-3 fatty acids EPA and DHA and small amounts of the omega-3 fatty acid docosapentaenoic acid and the omega-6 fatty acids arachidonic acid, docosatetraenoic acid and docosaenoic acid isomer.

[0059] 1 ml of the respective solutions was added dropwise to 20 ml of a 0.1 M phosphate buffer, pH=8, at a temperature of 45° C. and under intense stirring. pH was adjusted to pH=8 with NaOH if necessary. Afterwards the dispersion was put on ice and sonified (Branson Sonifier, 100% amplitude, 50% impulse) for 15 minutes to generate nanometer scale dispersions, presumably liposomes. The dispersions were sterile filtered through 0.2 μm syringe filters. Turbidity and particle size were measured before and after sterile filtration as described in 1.2. The preparation contained 40 g/l phospholipids and 10 g/l omega-3 fatty acids or esters. In a last step, the dispersions were freeze-dried and the appearance of the preparation obtained was analyzed visually.

[0060] Surprisingly it was found, that the preparations obtained with the omega-3 fatty acid lysine, arginine or ornithine salt had a smaller particle size, showed the best filtration properties and resulted a substantially less sticky powder after freeze-drying. The results for the triglyceride and ethyl ester as comparative example (comp.) and the fatty acid lysine, arginine and ornithine salts (according to the invention) are summarized in table 1.

TABLE-US-00001 TABLE 1 Properties of various omega-3 fatty acid dispersions with DOPC Composition 1.1 (comp.) 1.2 (comp.) 1.3 (inv.) 1.4 (inv.) 1.5 (inv.) Phospholipid DOPC DOPC DOPC DOPC DOPC Omega-3 fatty acid Triglyceride Ethyl ester Fatty acid Fatty acid Fatty acid form lysine salt arginine salt ornithine salt Mean particle size 168 148 132 97 95 (nm) before sterile filtration Turbidity before 0.42 0.38 0.05 n.d. n.d. sterile filtration (AU) Turbidity after 0.25 0.17 0.04 n.d. n.d. sterile filtration (AU) Reduction of 0.17 0.21 0.01 n.d. n.d. turbidity by sterile filtration (AU) Filtration filter filter easy to easy to easy to performance clogging clogging filter filter filter Properties of dried 1 1 3 n.d. n.d. product (1 = sticky- 4 = free flowing)

Example 2: Preparation of Omega-3 Fatty Acids and Omega-3 Fatty Acid Lysine Salt Dispersions with DOPC in Water

[0061] Formulations were prepared as described in example 1 except that water was used instead of phosphate buffer. In addition to omega-3 fatty acid lysine salt (AvailOm®, Evonik) the corresponding omega-3 free fatty acid was used to determine differences between the two forms. pH measurement with a pH electrode after addition of lipids to water showed that formulations with the free acid were rather acidic and had to be adjusted with NaOH before sonification to obtain proper dispersion whereas addition of the respective salts resulted in preparations with an appropriate pH that could be dispersed via sonification without addition of base. Surprisingly, the freeze-dried product containing the omega-3 fatty acid lysine salt (2.2) was better flowing and less sticky than the powder obtained from processing the omega-3 free fatty acid (2.1 comparative) and thus provides benefits regarding processing of such powders. The results are shown in table 2.

TABLE-US-00002 TABLE 2 Differences between compositions prepared with omega-3 free fatty acid and omega-3 fatty acid lysine salt. Composition 2.1 (comp.) 2.2 (inventive) Phospholipid DOPC DOPC Omega-3 fatty acid form Free fatty acid Fatty acid lysine salt pH after addition of ethanolic 4.6 9.1 lipid solution to water Addition of NaOH (base) Yes No required to obtain neutral or alkaline pH Properties of dried product 2   3   (1 = oily, sticky-5 = free flowing)

Example 3: Preparation and Characterization of Omega-3 Fatty Acid Lysine Salt Dispersions with DOPC at Various Ratios of Fatty Acid Salt to Phospholipid

[0062] Formulations were prepared as described in example 1 except that water instead of phosphate buffer was used and different ratios of omega-3 fatty acid salt to phospholipid were used. The obtained aqueous dispersions all contained 10 g/l of omega-3 fatty acid salt but different concentrations of DOPC. An aqueous colloidal solution of omega-3 fatty acid salt without phospholipid was also prepared for comparison. 5 ml of the obtained dispersions were diluted in 45 ml of 20 mM phosphate buffer, pH=7, to evaluate dispersion properties at physiological pH via particle size and turbidity measurements. Turbidity of the diluted dispersions was measured in 96-well microtiter plates at 600 nm with a liquid volume of 100 μl in a microplate reader (Tecan Infinite 200 PRO). Surprisingly it was found, that already small amounts of DOPC were sufficient to improve dispersion of omega-3 fatty acid salt compared to the omega-3 fatty acid salt without added phospholipid. This could be demonstrated by reduced turbidity and smaller particle sizes. The results are summarized in table 3.

TABLE-US-00003 TABLE 3 Compositions with various DOPC omega-3 fatty acid lysine salt ratios, obtained particle size and turbidity after dilution of compositions at pH = 7 (n. d.: not determined) Composition 3.1 3.2 3.3 3.4 3.5 3.6 DOPC (g/l) 40 20 10 4 1 0 Omega-3 fatty acid lysine salt 10 10 10 10 10 10 (g/l) Ratio DOPC/omega-3 4 2 1 0.4 0.1 — Mean particle diameter (nm) 95 194 226 157 388 n. d. after dilution at pH = 7 Absorbance (AU) at 600 nm 0.2 0.2 0.3 0.3 0.4 0.9 after dilution at pH = 7

Example 4: Preparation and Characterization of Omega-3 Fatty Acid Dispersions with Lecithin in Water

[0063] Formulations were prepared as described in example 3, except that 0.8 g of deoiled sunflower lecithin/phosphatidylcholine with a phosphatidylcholine content >90 weight % (Lipoid H 100) was used instead of DOPC. Dilutions in phosphate buffer at pH=7 were prepared and turbidity was measured as described in example 3. The results are shown in table 4 and were comparable to those obtained with DOPC. Already small amounts of phospholipid resulted in a much finer dispersion of the fatty acid salts at this pH value as indicated by a lower turbidity. The differences were also easy to observe visually. Whereas 4.6 appeared rather milky, the phospholipid containing compositions were almost transparent and only slightly turbid.

TABLE-US-00004 TABLE 4 Compositions with various lecithin omega-3 fatty acid lysine salt ratios, obtained particle size and turbidity after dilution of compositions at pH = 7 Composition 4.1 4.2 4.3 4.4 4.5 4.6 Phosphatidylcholine Lipoid 40 20 10 4 1 0 H100 (g/l) Omega-3 fatty acid lysine 10 10 10 10 10 10 salt (g/l) Ratio Lipoid H100/omega-3 4 2 1 0.4 0.1 Absorbance (600 nm) after 0.21 0.18 0.26 0.13 0.29 0.75 dilution at pH = 7

Example 5: Alternative Method to Prepare Omega-3 Fatty Acid Salt Dispersions with Dioleylphosphatidylcholine in Water

[0064] It was found that preparations according to the invention can be prepared in a mixing sequence that simplifies the process and is not suitable for omega-3 fatty free fatty acid form or respective esters. Instead of dissolving the omega-3 fatty acid lysine salt with the phospholipid in ethanol it was directly dissolved in water at a concentration of 10 g/l and the ethanolic phospholipid solution was added subsequently. Afterwards the dispersion was sonified and sterile filtered as described in the previous examples.

Example 6: Stimulation of Production of 18-Hydroxy-Eicosapentaenoic Acid (18-HEPE) in the Strain B. megaterium DSM 32963

[0065] Due to their low polarity, the bioavailability of omega-3 fatty acids often is not sufficient and only low amounts are actually used and converted by cells in biochemical reactions. It could be shown, that the preparations described in this invention enhance bioavailability and metabolic conversion of omega-3 fatty acids of microbial cells, which is relevant in nutritional applications such as microbiome utilization and modulation.

[0066] Bacillus megaterium DSM 32963 has been identified by screening of naturally occurring isolates. It has been deposited with the DSMZ on Nov. 27, 2018 under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure under the Accession Number as mentioned before in the name of Evonik Degussa GmbH.

[0067] The preparations described in table 1 were added to microbial cultures of Bacillus megaterium DSM 32963 in liquid cultures in shake flasks. For comparison purposes, an omega-3 fatty acid lysine salt solution in water with the same omega-3 fatty acid concentration was also added. Bacillus megaterium DSM 32963 was found to catalyze the conversion of eicosapentaenoic acid (EPA) into bioactive 18-hydroxy-eicosapentaenoic acid (18-HEPE).

[0068] From 10 ml auf Luria Bertami broth (LB, Thermo Fisher Scientific) with 0.1% Glucose (LBG) a culture of B. megaterium DSM 32963 was grown for 24 h at 30° C. and 200 rpm in 100 ml flask. The complete culture was transferred to 200 ml main culture in LBG. Main culture was grown for 6 h at 30° C. and 200 rpm in a 2 l flask. The cell culture was then harvested in 10 ml portions, the supernatant removed by centrifugation (15 min, 4000 rpm, room temperature) and the cell pellet resuspended in 10 ml LBG or LBG containing 9.76 g/l FeSSIF-V2 (biorelevant.com), which is a mixture of taurocholate, phospholipids and other components designed to simulate bile surfactants, and 2 ml of lipid stock solutions were added, that were prepared from the sterile filtered preparations in example 2 to obtain the same EPA concentration in each experiment (table 5), respectively. Additionally, the supplements were also added respectively to the different media in shaking flasks without cells, and treated under the same conditions to control for non-biochemical product formation. These cultures & respective controls were incubated in 100 ml shaking flasks for 16 h at 30° C. and 200 rpm.

TABLE-US-00005 TABLE 5 Supplements, preparation of stock solutions, and its calculated EPA content (g/l) EPA content Preparation Preparation of stock solution calculated (g/l) Omega-3 lysine salt (preparation 4.6) 0.6 g in 100 ml PBS buffer 2.04 Preparation of fish oil with DOPC undiluted 2.04 (preparation 1.1) Preparation of omega-3 ethyl ester 4.08 ml + 5.92 ml PBS buffer 2.04 with DOPC (preparation 1.2) Preparation of omega-3 lysine salt with 6 ml + 4 ml PBS buffer 2.04 DOPC (preparation 1.3)

[0069] Subsequently, the cells were separated by centrifugation (15 min, 4000 rpm, room temperature), and supernatants were withdrawn to analyze for the presence of omega-3 metabolites. The supernatants were diluted with a solvent consisting of a water/acetonitrile mixture (ratio supernatants:solvent was 1:2, solvent composition: 65% H.sub.2O, pH8 and 35% MeCN).

[0070] The diluted supernatant samples were filtered and then used for the detection of 18-hydroxy-eicosapentaenoic acid (18-HEPE) by LC/ESI-MS analysis (Agilent QQQ 6420, Gemini 3p C6-Phenyl) in positive SIM-Mode at m/z 318 as well as the precursor compound EPA at m/z 302.

[0071] In the presence and absence of bile salts, 18-HEPE was formed and detected in the supernatant most effectively, when omega-3 fatty acid was provided to Bacillus megaterium DSM 32963 cells as omega-3 lysine salt formulated with phospholipids. The results are summarized in table 6.

TABLE-US-00006 TABLE 6 measured 18-HEPE concentrations (mg/l) of culture supernatants in absence and presence of bile acids 18-HEPE content (mg/l) control -netto- supernatant without cellular Supplement added bile acids of culture cells produced PBS buffer − 0.0 0.0 0.0 Omega-3 lysine salt (4.6) − >0.2 >0.3  0.0 Omega-3 lysine salt with DOPC (1.3) − >0.5 >0.1; <0.2 >0.3 Fish oil with DOPC (1.1) − >0.05 0.0 >0.05 Omega-3 ethyl ester with DOPC (1.2) − 0.0 >0.1  0.0 PBS buffer + 0.0 0.0 0.0 Omega-3 lysine salt with DOPC (1.3) + >1.2 >0.5; <1   >0.2 Fish oil with DOPC (1.1) + 0.0 0.0 0.0 Omega-3 ethyl ester with DOPC (1.2) + 0.0 0.0 0.0

Example 7: Stimulated Expansion of Human Bone Marrow Mesenchymal Stromal Cells by Addition of Phospholipid Dispersed Omega-3 Fatty Acid Salts

[0072] Human bone marrow mesenchymal stromal cells (MSCs) are used for medical purposes in the form of cell therapies and to derive specific cell and tissue types for tissue engineering, e.g. for the generation of chondrocytes to make in vitro cartilage. In order to do so, suitable medium compositions are required that allow efficient expansion/multiplication of isolated cells. In general, it is assumed that lipids need to be supplemented if serum free, chemically defined media are used. The suitability of the lipid formulations described in this invention to efficiently expand stromal cells was evaluated as described in the following passage.

[0073] A chemically defined cell culture medium was prepared as described by Jung et al. (Cytotherapy, 2010; 12: 637-657). The composition of the medium is shown in Table 7.

TABLE-US-00007 TABLE 7 composition of chemically defined medium for cultivation of mesenchymal stromal cells Ingredient Concentration applied DMEM/Ham's F-12 medium, 1:1 L-glutamine 1.5 mM Chemically defined lipid concentrate 0.1 % Sodium bicarbonate 1.725 g/L HEPES 4.90 mM Insulin 23 mg/L Human apo- transferrin 25 mg/L Putrescine dihydrochloride 9 mg/L Progesterone 5.66 μg/L Human serum albumin (HSA) 4 g/L bFGF 20 μg/L TGF-β1 10 μg/L Ascorbic acid 50 mg/L Hydrocortisone 100 nM Fetuin 1 g/L Attachment substrate 0.1% Gelatine

[0074] The chemically defined lipid concentrate was obtained by Thermo Fisher Scientific (Catalog no. 11905031) and consisted of a mixture of fatty acids and cholesterol formulated with non-ionic surfactants (Polysorbate 80 and Pluronic F-68).

[0075] Two additional batches of this medium were prepared, one without chemically defined lipid concentrate and one where the chemically defined lipid concentrate was replaced by the preparation of omega-3 lysine salt formulated with DOPC (example 1.3). 0.1 ml of liquid preparation was added to 100 ml of cell culture medium, corresponding to a 1000× dilution and resulting in a fatty acid concentration of about 10 mg/l.

[0076] Human bone marrow mesenchymal stromal cells derived in chemically defined medium from bone marrow mononuclear cells were obtained from StemCell Technologies, thawed as recommended by the supplier and expanded in the three different medium versions. The medium was inoculated at a cell density of 150 000 cells/ml and cells were expanded in T25 cell culture flasks in a CO.sub.2 incubator. The medium was replaced every 2-3 days and cells were passaged before becoming confluent. Cells were detached with Accutase™ (StemCell Technologies), viable cell concentration was determined and fresh medium was inoculated with 150 000 cell/ml at each passage.

[0077] Cumulated population doublings (CPD) as an indicator of growth performance, were calculated from the number of passages and the measured cell concentrations. Surprisingly it was found, that the chemically defined lipid concentrate as described in the literature did not have a positive effect on CPD (CPD=4.63) when compared to the medium without added lipids (CPD=4.77). In contrast to that, addition of omega-3 lysine salt formulated with DOPC did have positive effects and resulted in increased CPD of 7.37.