PREPARATION COMPRISING A PROBIOTIC STRAIN OF THE GENUS BACILLUS MEGATERIUM AND A POLYUNSATURATED FATTY ACID COMPONENT

Abstract

The invention relates to preparation comprising at least one probiotic strain belonging to the genus Bacillus megaterium, and a polyunsaturated fatty acid component comprising at least one omega-3 or omega-6 fatty acid selected from eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), arachidonic acid (ARA), alpha linolenic acid, stearidonic acid, eicosatetraenoic acid, docosapentaenoic acid, linoleic acid, γ-linolenic acid, wherein the polyunsaturated fatty acid component comprises an omega-3 or omega-6 fatty acid salt. The invention further relates to use of the inventive preparation as a feed or food supplement, pharmaceutical product, feed-or foodstuff compositions and for topical applications.

Claims

1. A preparation, comprising: a probiotic strain of a genus Bacillus megaterium, and a polyunsaturated fatty acid component comprising an omega-3 or omega-6 fatty acid 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, and γ-linolenic acid, wherein the polyunsaturated fatty acid component comprises an omega-3 or omega-6 fatty acid salt.

2. The preparation according to claim 1, wherein the probiotic strain is present in a dormant form or as vegetative cells.

3. The preparation according to claim 1, wherein the omega-3 or omega-6 fatty acids are in a form of free fatty acids, salts, natural triglycerides, fish oil, phospholipid esters or ethyl esters.

4. The preparation according to claim 1, wherein the omega-3 or omega-6 fatty acid is EPA, DHA or ARA.

5. The preparation according to claim 1, further comprising 5-Aminolevulinic Acid.

6. The preparation according to claim 1, wherein the probiotic strain is at least one selected from the group consisting of Bacillus megaterium DSM 32963, Bacillus megaterium DSM 33296, and Bacillus megaterium DSM 33299.

7. The preparation according to claim 1, wherein the polyunsaturated fatty acid component comprises an omega-3 fatty acid salt.

8. The preparation according to claim 1, wherein the polyunsaturated fatty acid component comprises a preparation comprising a dispersion of at least one phospholipid and at least one omega-3 or omega-6 fatty acid.

9. The preparation according to claim 1, further comprising a coating for delayed release or enteric or colonic release.

10. A feed or food supplement, comprising the preparation according to claim 1.

11. A pharmaceutical product, comprising the preparation according to claim 1.

12. A feed- or foodstuff composition comprising: the preparation according to claim 1 and an additional feed or food ingredient.

13. The feed- or foodstuff composition according to claim 12, wherein the feed- or foodstuff composition is suitable for improving a health status, in gut health, cardiovascular health, cardio-metabolic health, lung health, joint health, eye health, mental health, oral health or immune health of an animal or a human being.

14. The feed- or foodstuff composition according to claim 13 for improving the health status of an animal or a human being by increasing a total amount of one or more lipid mediators in a host via their production by gastrointestinal microorganisms wherein the one or more lipid mediators are selected from the group consisting of 17-HDHA, 14-HDHA, 13-HDHA, 7-HDHA, 4-HDHA, 18-HEPE, 15-HEPE, 12-HEPE, 11-HEPE, 5-HEPE, 15-HETE, 12-HETE, 11-HETE, 8-HETE, 5-HETE, 9-HODE, 13-HODE, PDX, PD1, AT-PD1, MaR1, MaR2, LTB4, t-LTB4, RvD1-5, AT-RvD1, RvE1, RvE3, LXA4, LXA5, LXB4, and LXB5, increasing a total amount of EPA in the host, and/or increasing a total amount of DHA in the host.

15. A topical application on the skin, the eye, or the oral cavity employing suitable matrices or carriers, the topical application comprising the preparation according to claim 1.

16. The topical application according to claim 15, wherein the preparation is loaded on and/or in pre-synthesized multiphase biomaterials comprising nanocellulose, wherein the nanocellulose is bacterially synthesized nanocellulose (BNC) selected from the group consisting of BNC comprising a network of cellulose fibers or nanowhiskers, BNC comprising two or more different layers of cellulose fibrils, wherein each layer consists of BNC from a different microorganism or from microorganisms cultivated under different conditions, BNC comprising of at least two different cellulose networks, and a BNC composite material further comprising a polymer.

17. A method, comprising: biotechnologically producing a specialized pro-resolving lipid mediator with a Bacillus megaterium strain in a dormant form or as vegetative cells, exhibiting a) a 16S rDNA sequence with a sequence identity of at least 99.5%, above all 100%, to the polynucleotide sequence according to SEQ ID NO: 1 or SEQ ID NO:2, SEQ ID NO:13 or SEQ ID NO:14, SEQ ID NO: 25 or SEQ ID NO: 26; b) a yqfD sequence with a sequence identity of at least 99.5%, above all 100%, to the polynucleotide sequence according to SEQ ID NO: 3, SEQ ID NO: 15 or SEQ ID NO: 27; c) a gyrB sequence with a sequence identity of at least 99.5%, above all 100%, to the polynucleotide sequence according to SEQ ID NO: 4, SEQ ID NO: 16, SEQ ID NO: 28; d) an rpoB sequence with a sequence identity of at least 99.5%, above all 100%, to the polynucleotide sequence according to SEQ ID NO: 5, SEQ ID NO: 17, SEQ ID NO: 29; and/or e) a groEL sequence with a sequence identity of at least 99.5%, above all 100%, to the polynucleotide sequence according to SEQ ID NO: 6, SEQ ID NO: 18, SEQ ID NO: 30.

Description

WORKING EXAMPLES

Example 1: The Strains Bacillus megaterium DSM 32963, DSM 33296 and DSM 33299 Each Possess Genetic Sequences for a Cytochrome P450 Monooxygenases (CYP450)

[0102] The strains Bacillus megaterium DSM 32963, DSM 33296 and DSM 33299 were isolated each from a soil sample from a pristine garden in east Westphalia. They have been deposited with the DSMZ on Nov. 27, 2018 (DSM 32963) and on Oct. 17, 2019 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.

[0103] The genome sequence of B. megaterium DSM 32963 contains a gene [SEQ ID No: 7] encoding a protein with an identity of 97,9% at the amino acid level to P450 BM3 (CYP102A1) of B. megaterium ATCC 14581 (AAA87602.1). This enzyme incorporates both, a P450 oxygenase and a NADPH:P-450 reductase[46]. The natural substrates of P450 BM3 were analyzed to be long chain fatty acids (C12 to C20), which are exclusively hydroxylated at the subterminal positions (ω-1 to ω-3)[47].

[0104] Lower sequence similarities on amino acid level ranging from 21,1% to 30,5% on partial sequence hits were observed to sequences identified within the genome sequence of B. megaterium DSM 32963 compared to P450 BM3. These further potential cytochrome genes might have similar functions compared to P450 BM3 [Seq ID No: 8-12] (see table 1).

TABLE-US-00001 TABLE 1 BLASTp of P450 BM3 against protein sequences of Bacillus megaterium DSM 32963 (SEQ ID in the hit column refers to the corresponding nucleotide sequence) Function E- HSP % % based on Query sequence Hit value Score length Identity Gaps BLAST CYP102_Bm_AAA87602 DSM 0 5.461,00 1.049,00 97.9 0 Seq ID 7 32963_00731 [SEQ ID No 7] CYP102_Bm_AAA87602 DSM 8.35E−62 558 568 30.49 7.87 sulfite 32963_00276 reductase [SEQ ID No (NADPH) 8] flavoprotein alpha- component CYP102_Bm_AAA87602 DSM 4.03E−12 161 187 26.9 6.6 cytochrome 32963_02107 P450 [SEQ ID No 9] CYP102_Bm_AAA87602 DSM 1.08E−10 149 285 24.56 18.42 cytochrome 32963_01384 P450 [SEQ ID No 10] CYP102_Bm_AAA87602 DSM 2.17E−08 130 210 24.38 14.05 cytochrome 32963_02065 P450 [SEQ ID No 11] CYP102_Bm_AAA87602 DSM 5.04E−04 94 292 21.08 21.08 cytochrome 32963_02258 P450 [SEQ ID No (BM1) 12]

[0105] The genome sequence of B. megaterium DSM 33296 contains a gene [SEQ ID No: 19] encoding a protein with an identity of 98,9% at the amino acid level to P450 BM3 (CYP102A1) of B. megaterium ATCC 14581 (AAA87602.1). This enzyme incorporates both, a P450 oxygenase and a NADPH:P-450 reductase[46]. The natural substrates of P450 BM3 were analyzed to be long chain fatty acids (C12 to C20), which are exclusively hydroxylated at the subterminal positions (ω-1 to ω-3)[47].

[0106] Lower sequence similarities on amino acid level ranging from 21,5% to 30,7% on partial sequence hits were observed to sequences identified within the genome sequence of B. megaterium DSM 33296 compared to P450 BM3. These further potential cytochrome genes might have similar functions compared to P450 BM3 [Seq ID No: 20-24] (see table 2).

TABLE-US-00002 TABLE 2 BLASTp of P450 BM3 against protein sequences of Bacillus megaterium DSM 33296 (SEQ ID in the hit column refers to the corresponding nucleotide sequence) E- HSP Query Hit value Score length % Identity % Gaps Function CYP102_Bm_AAA87 DSM 0.00 5.506,00 1.049,00 98.86 0.00 cytochrome 602 33296_54_CDS_D P450 SM 33296_02126 [SEQ ID no 19] CYP102_Bm_AAA87 DSM 1.48E−61 556.00 556.00 30.70 7.72 sulfite 602 33296_54_CDS_D reductase SM 33296_02261 (NADPH) [SEQ ID no 20] flavoprotein alpha- component CYP102_Bm_AAA87 DSM 2.74E−12 162.00 195.00 26.34 6.34 cytochrome 602 33296_46_CDS_D SM_33296_01053 [SEQ ID no 21] CYP102_Bm_AAA87 DSM 1.06E−10 149.00 272.00 24.22 17.39 cytochrome 602 33296_70_CDS_D P450 SM_33296_03886 [SEQ ID no 22] CYP102_Bm_AAA87 DSM 1.26E−8 132.00 226.00 24.91 15.85 cytochrome 602 33296_46_CDS_D P450 SM_33296_01002 [SEQ ID no 23] CYP102_Bm_AAA87 DSM 1.03E−3 91.00 288.00 21.45 21.16 cytochrome 602 33296_46_CDS_D P450 SM_33296_01221 [SEQ ID no 24]

[0107] The genome sequence of B. megaterium DSM 33299 contains a gene [SEQ ID No: 31] encoding a protein with an identity of 96,1% at the amino acid level to P450 BM3 (CYP102A1) of B. megaterium ATCC 14581 (AAA87602.1). This enzyme incorporates both, a P450 oxygenase and a NADPH:P-450 reductase[46]. The natural substrates of P450 BM3 were analyzed to be long chain fatty acids (C12 to C20), which are exclusively hydroxylated at the subterminal positions (ω-1 to ω-3)[47].

[0108] Lower sequence similarities on amino acid level ranging from 20,9% to 30,5% on partial sequence hits were observed to sequences identified within the genome sequence of B. megaterium DSM 33299 compared to P450 BM3. These further potential cytochrome genes might have similar functions compared to P450 BM3 [Seq ID No: 32-37] (see table 3).

TABLE-US-00003 TABLE 3 BLASTp of P450 BM3 against protein sequences of Bacillus megaterium DSM 33299 (SEQ ID in the hit column refers to the corresponding nucleotide sequence) Function E- HSP based on Query sequence Hit value Score length % Identity % Gaps BLAST CYP102_Bm_AAA87 DSM 0.00 5.390,00 1.049,00 96.09 0.00 cytochrome 602 33299_122_CDS_D P450 SM 33299_05006 [SEQ ID no 31] CYP102_Bm_AAA87 DSM 6.77E−62 558.00 568.00 30.49 7.87 sulfite 602 33299_58_CDS_D reductase SM 33299_02626 (NADPH) [SEQ ID no 32] flavoprotein alpha- component CYP102_Bm_AAA87 DSM 2.56E−12 162.00 195.00 25.85 6.34 cytochrome 602 33299_123_CDS_D P450 SM 33299_05123 [SEQ ID no 33] CYP102_Bm_AAA87 DSM 1.55E−11 156.00 272.00 25.16 17.39 cytochrome 602 33299_15_CDS_D P450 SM 33299_00711 [SEQ ID no 34] CYP102_Bm_AAA87 DSM 4.26E−8 127.00 210.00 23.65 13.28 cytochrome 602 33299_123_CDS_D P450 SM 33299_05165 [SEQ ID no 35] CYP102_Bm_AAA87 DSM 2.54E−6 113.00 192.00 25.56 14.35 cytochrome 602 33299_6_CDS_DS P450 M 33299_00407 [SEQ ID no 36] CYP102_Bm_AAA87 DSM 7.19E−5 101.00 280.00 20.94 21.53 cytochrome 603 33299_87_CDS_D P450 SM 33299_03167 [SEQ ID no 37]

Example 2: Preparation of Omega-3 Fatty Acid Dispersions with Dioleylphosphatidylcholine (DOPC) in Phosphate Buffer

[0109] 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) or lysine salt of free omega-3 fatty acid in form of omega-3 lysine salt (AvailOm®, Evonik) were added and dissolved. In the case of free omega-3 fatty acid salt, 20 μl of distilled water were added to dissolve the product completely.

[0110] The lysine salt of 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.

[0111] 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. Afterwards the dispersion was put on ice and sonified for 15 minutes to generate nanometer scale dispersions, presumably liposomes (Branson Sonifier, 100% amplitude, 50% impuls). Finally, the dispersions were sterile filtered through 0.2 μm syringe filters. The resulting dispersions were characterized with regards to particle size in via dynamic light scattering (DLS) measurements (Zetasizer Nano ZS, Malvern). The dispersions contained 40 g/I phospholipids and 10 g/I omega-3 fatty acids or esters.

Example 3: The Strain B. megaterium DSM 32963 is Able to Produce Intracellularly 18-Hydroxy-Eicosapentaenoic Acid (18-HEPE)

[0112] For B. megaterium DSM 32963 an associated intracellularly activity of SPM-producing enzyme(s) could be demonstrated. From 10 ml 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 a 100 ml flask. The complete culture was transferred to a 200 ml main culture in LBG. The 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 and 2 ml of supplements (table 2), respectively. These cultures were incubated in 100 ml shaking flasks for 16 h at 30° C. and 200 rpm.

[0113] Different forms of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) sources were added to the B. megaterium DSM 32963 cell cultures to a final concentration of 0.4 g/I in form of omega-3 lysine salt (AvailOm® by Evonik), fish oil (Omega-3 1400 by Doppelherz®), and omega-3 ethyl ester (PronovaPure® 500:200 EE by BASF). These substances were added as sonificated emulsions and as dispersion formulations (preparation described in example 2), respectively.

[0114] DOPC formulation without omega-3 fatty acid content, and PBS buffer were used as controls without EPA.

TABLE-US-00004 TABLE 2 Supplements, preparation of stock solutions, and its calculated EPA content (g/l) EPA content Preparation of calculated Supplement stock solution (g/l) DOPC formulation undiluted 0 Omega-3 lysine salt 0.6 g in 100 ml PBS 2.04 buffer Fish oil 1 g in 100 ml PBS 2.04 buffer Omega-3 ethyl ester 0.408 g in 100 ml 2.04 PBS buffer Preparation of omega-3 lysine 6 ml + 4 ml PBS buffer 2.04 sal tdispersion with DOPC Preparation of fish oil dispersion undiluted 2.04 withDOPC Preparation of omega-3 ethyl 4.08 ml + 5.92 ml PBS 2.04 ester dispersion with DOPC buffer PBS buffer undiluted 0

[0115] The supernatants were separated by centrifugation (15 min, 4000 rpm, room temperature), and the cell cultures were then each harvested. Afterwards, 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). Pellets were freeze dried overnight and resuspended in a solvent consisting of a water/acetonitrile mixture (ratio pellet: solvent was 1:2, solvent composition: 65% H.sub.2O, pH8 and 35% MeCN). The cell disruption was carried out in Lysing Matrix tubes (0.1 mm silica spheres) in a Ribolyser.

[0116] The cell homogenate (and the diluted supernatant) was filtered and then used for the detection of 18-hydroxy-eicosapentaenoic acid (18-HEPE) by LC/ESI-MS analysis (Agilent QQQ 6420, Gemini 3μ C6-Phenyl) in positive SIM-Mode at m/z 318 as well as the precursor compound EPA at m/z 302.

[0117] The addition of EPA, in form of omega-3 lysine salt, omega-3 ethyl ester, omega-3 lysine salt dispersions with DOPC, or fish oil dispersions with DOPC to the B. megaterium cells resulted in a cell associated (=intracellular+extracellular adsorbed) accumulation of 18-HEPE (table 3). By far the highest value of intracellular 18-HEPE was achieved by the addition of omega-3 lysine salt; it was tenfold higher than in the other approaches.

TABLE-US-00005 TABLE 3 Concentration of cell associated 18-HEPE (mg/ml) in cellular extract of Bacillus megaterium DSM 32963 cells Cell associated 18- HEPE in cellular Supplement added extract (mg/ml) PBS buffer 0 DOPC formulation 0 Omega-3 lysine salt >1.5 Fish oil 0 Omega-3 ethyl ester >0.01 Preparation of omega-3 lysine salt dispersion >0.15 with DOPC Preparation of fish oil dispersion with DOPC >0.10 Preparation of omega-3 ethyl ester dispersion 0 with DOPC

Example 4: The Synbiotic Combination of Bacillus megaterium DSM 32963 and Dispersion Formulation of Omega-3 Fatty Acid Salt AvailOm® Leads to Extracellular Amounts of 18-HEPE

[0118] To investigate the amount of extracellularly appearing 18-HEPE, the B. megaterium DSM 32963 cells were cultivated as described in example 1. The cells were resuspended in 10 ml LBG or LBG containing 9.76 g/I FeSSIF-V2 (biorelevant.com), which is a mixture of taurocholate, phospholipids and other components designed to simulate bile surfactants, and 2 ml of supplements (table 2) were added, respectively. Additionally, the supplements were also added respectively to the different media in shaking flasks without cells and treated under the same conditions (controls). The 18-HEPE concentrations of the culture supernatants and controls were determined after incubation at 16 h, 30° C. and 200 rpm (table 4). It could be shown that the Bacillus megaterium DSM 32963 cells are able to synthesize 18-HEPE from omega-3 lysine salt (AvailOm®) dispersions, which is extracellularly detectable. Of note, the omega-3 4 18-HEPE conversion rate detected by this method is up to 0.075, which exceeds the basal content of 18-HEPE of 0.0005% in an esterified fish oil, disclosed by WO 2017/041094. More importantly, we discovered that the omega-3 lysine salt is converted by Bacillus megaterium strains to a multitude of (final) SPM products at even higher concentrations than 18-HEPE (see example 6), which is of physiological relevance.

TABLE-US-00006 TABLE 4 measured 18-HEPE concentrations (mg/l) of culture supernatants and controls 18-HEPE content (mg/l) super- control -netto- bile natant without cellular Supplement added acids of culture cells produced PBS buffer − 0.0 0.0 0.0 DOPC formulation − 0.0 0.0 0.0 Omega-3 lysine salt − >0.2 >0.3 0.0 Fish oil − 0.0 0.0 0.0 Omega-3 ethyl ester − 0.0 0.0 0.0 Preparation of omega-3 lysine − >0.5 >0.1; <0.2 >0.3 salt dispersion with DOPC Preparation of fish oil dispersion − >0.05 0.0 >0.05 with DOPC Preparation of omega-3 ethyl − 0.0 >0.1 0.0 ester dispersion with DOPC PBS buffer + 0.0 0.0 0.0 DOPC formulation + 0.0 0.0 0.0 Omega-3 lysine salt + >0.4 >0.4 0.0 Fish oil + 0.0 0.0 0.0 Omega-3 ethyl ester + 0.0 0.0 0.0 Preparation of omega-3 lysine + >1.2 >0.5; <1 >0.2 salt dispersion with DOPC Preparation of fish oil dispersion + 0.0 0.0 0.0 with DOPC Preparation of omega-3 ethyl + 0.0 0.0 0.0 ester dispersion with DOPC

Example 5: Biotransformation of EPA by Different Bacillus Species

[0119] To investigate the ability of different Bacillus species to produce intracellularly 18-HEPE, cells of different species were cultivated as described in example 1. The cells were resuspended in 10 ml LBG containing 9.76 g/I FeSSIF-V2 (biorelevant.com), which is a mixture of taurocholate, phospholipids and other components designed to simulate bile surfactants, and 1.2 ml of the omega-3 lysine salt dispersion with DOPC were added, respectively. The internal 18-HEPE concentrations of the cells after incubation for 16 hat 30° C. and 200 rpm were determined as described in example 3.

[0120] Only the Bacillus megaterium cells were able to synthesize 18-HEPE internally from omega-3 lysine salt (AvailOm®) dispersions, whereby B.subtilis, B. amyloliquefaciens, B. pumilus and B. licheniformis were not.

TABLE-US-00007 TABLE 5 Intracellularly measured 18-HEPE content (mg/ml) of B. megaterium DSM 32963 cells Internal 18-HEPE content Strain Species (mg/ml in 10 mg pellet) DSM23778 B. subtilis 0 (wildtype 168) B. subtilis 0 B. amyloliquefaciens 0 B. pumilus 0 B. licheniformis 0 DSM 32963 B. megaterium >0.15

Example 6: The Production of SPM by Bacillus megaterium DSM 32963 from Omega-3 Fatty Acid Salt AvailOm® Under Different Culture Conditions

[0121] Lipidometabolomics:

[0122] Bacterial supernatant samples were subjected to lipid extraction using RP-phase solid phase extraction and subsequently analyzed by ultra performance liquid chromatography ESI tandem mass spectrometry (UPLC-MS/MS) according to a published procedure [48]. Under these conditions approximately 40 different LM, including 5-hydroxy-eicosapentaenoic acid 5-HEPE, 8-HEPE, 11-HEPE, 12-HEPE, 15-HEPE, 18-HEPE, 5-hydroxy-eicosatetraenoic acid (5-HETE), 8-HETE, 11-HETE, 12-HETE, 15-HETE, 4-hydroxy-DHA (4-HDHA), 7-HDHA, 13-HDHA, 14-HDHA, 17-HDHA, lipoxin A.sub.4 (LXA4), LXB4, resolvin E1 (RvE1), RvE3, resolvin D1-5 (RvD1-5), AT-RvD1, RvD2, protectin D1 (PD1), AT-PD1, maresin 1(MaR1), MaR2, plus 4 fatty acid substrates can be detected with a lower limit of detection of 1 pg.

[0123] To investigate if Bacillus megaterium is capable of producing other PUFA oxygenation products in addition to 18-HEPE, a lipidometabolomics screening of supernatants from Bacillus megaterium strain 32963 cultured as in example 4 with AvailOm® was performed, either dissolved or in a dispersion formulation, with or without bile acids. Cell-free preparations of AvailOm® formulations were treated and analyzed in parallel and served as controls for non-enzymatic, spontaneous formation of oxygenation products. Values given in table 6 display net concentrations of products formed, i.e. after subtraction of control values. As can be seen, numerous oxygenation products including SPM have been formed by the bacteria. Concentrations of several SPM exceed by far the concentrations of SPM found in human plasma samples, which are typically in a range of ˜20-100 pg/ml [27, 40, 41]. For comparison, human breast milk contains ˜6.000 pg/ml RvE1 and ˜10.000 pg/ml RvD1 [42]. Given the fact that SPM exert receptor-mediated effects in vitro and in vivo in rodents in the nM or even pM range [6], findings displayed in table 6 strongly imply the physiological and therapeutic importance of our invention.

[0124] The type of PUFA formulation had a great impact on product levels, which were generally higher in the presence of PUFA dispersion formulations and/or the addition of bile acids as solubilizers. In parallel, abundance of mono-hydroxylated SPM precursors 5-HEPE, 11-HEPE, 12-HEPE, 15-HEPE, 18-HEPE, 5-HETE, 8-HETE, and 9-NODE was lower in these samples compared to samples treated with AvailOm in absence of dispersion formulation and bile acids. This can be explained by an increased conversion of these precursors to di- and trihydroxylated fatty acids.

TABLE-US-00008 TABLE 6 Extracellular concentrations of PUFA oxygenation products of Bacillus megaterium DSM 32963 cells − bile acids + bile acids − bile acids AvailOm + bile acids AvailOm AvailOm dispersion AvailOm dispersion Substance pg/ml pg/ml pg/ml pg/ml 7-HDHA 397 18-HEPE 24463 15-HEPE 7536 12-HEPE 4413 11-HEPE 4659 5-HEPE 2253 8-HETE 148 5-HETE 13 1834 9-HODE 43 PDX 1 109 19542 16623 27541 PD1 1 113 3841 3666 6564 AT-PD1 1 96 247 RvD1 598 1863 AT-RvD1 519 4932 22 732 RvD2 87 99 40 27 RvD3 178 8409 1115 906 RvD4 3681 RvD5 68 9016 4750 7071 RvE1 88 57826 22930 34786 RvE3 33092 769 17772 MaR1 114 145 48 189 MaR2 120 197 LTB4 171 43 71 t-LTB4 360 667 LXA4 LXA5 7767 41263 26620 69960 LXB4 76301 26356 7986 LXB5 2376 669042 299105 492508

Example 7: The Production of SPM from a Dispersion Formulation of Omega-3 Fatty Acid Salt AvailOm® by Other Bacillus megaterium Strains

[0125] 47 additional Bacillus megaterium strains sourced from various habitats were screened for their SPM production capacity to determine if this is a general phenomenon of the species Bacillus megaterium and to detect strain-specific differences in types and quantities of SPM being produced. Cells were cultured as detailed in example 4, with dispersion formulation of AvailOm® serving as omega-3 fatty acid source. Cell-free preparations of AvailOm® were treated and analyzed in parallel and served as controls for non-enzymatic, spontaneous formation of oxygenation products. It was observed that all tested strains produced measurable (>1pg/ml) amounts of various SPM and precursors thereof, that these amounts were hugely different between the strains (up to 2.000 fold), and that concentrations of RvE3 were particularly high (up to 1.3 μg/ml) in most of the strains.

[0126] Values given in table 7 display net concentrations of PUFA oxygenation products formed by two of the top performing strains, Bacillus megaterium DSM 33296 and Bacillus megaterium DSM 33299.

TABLE-US-00009 TABLE 7 Extracellular concentrations of PUFA oxygenation products of Bacillus megaterium DSM 33296 and Bacillus megaterium DSM 33299 cells. Bacillus megaterium Bacillus megaterium DSM 33296 DSM 33299 Substance pg/ml pg/ml 7-HDHA 202129 243080 18-HEPE 391397 217725 15-HEPE 256797 151168 12-HEPE 124701 79699 11-HEPE 124860 63306 5-HEPE 395188 989691 8-HETE 24207 15941 5-HETE 15434 9356 PDX 1 78345 138918 PD1 1 99029 75342 AT-PD1 1 4558 10317 RvD1 3626 4389 AT-RvD1 9612 48243 RvD2 265 428 RvD3 381 941 RvD4 1986 2523 RvD5 15079 34168 RvE1 5840 28379 RvE3 1322555 880739 MaR1 1161 1576 MaR2 1522 1233 LTB4 1769 2206 t-LTB4 1780 4181 LXA4 1157 1430 LXA5 4693 128825

Example 8: Capsules Comprising EPA-DHA Amino Acid Salts and Bacillus megaterium Strain(s) as Food Supplement or as Drug

[0127] The following components were filled in HPMC capsules (size 00).

TABLE-US-00010 TABLE 7 Preparations for filling into HPMC capsules. Compound Capsule I Capsule II Capsule III Omega-3 amino acid* 250 mg 50 mg 800 mg salt Bacillus megaterium 1 × 10.sup.7 CFU- 1 × 10.sup.7 CFU- 1 × 10.sup.7 CFU- strain.sup.# 1 × 10.sup.11 CFU 1 × 10.sup.11 CFU 1 × 10.sup.11 CFU *Amino acids are selected from L-ornithine, L-lysine and L-arginine. .sup.#Strain selected from Bacillus megaterium DSM 32963, DSM 33296, DSM 33299.

[0128] The capsules may further contain amino acids selected from L-ornithine, L-aspartate, L-lysine and L-arginine.

[0129] The capsules may further contain further carbohydrate ingredients, selected from arabinoxylans, barley grain fibre, oat grain fibre, rye fibre, wheat bran fibre, inulins, fructooligosaccharides (FOS), galactooligosaccharides (GOS), resistant starch, beta-glucans, glucomannans, galactoglucomannans, guar gum and xylooligosaccharides.

[0130] The capsules may further contain one or more plant extracts, selected from ginger, cinnamon, grapefruit, parsley, turmeric, curcuma, olive fruit, panax ginseng, horseradish, garlic, broccoli, spirulina, pomegranate, cauliflower, kale, cilantro, green tea, onions, and milk thistle.

[0131] The capsules may further contain astaxanthin, charcoal, chitosan, glutathione, monacolin K, plant sterols, plant stanols, sulforaphane, collagen, hyalurone, phosphatidylcholine.

[0132] The capsules may comprise further vitamins selected from biotin, vitamin A, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid), vitamin B9 (folic acid or folate), vitamin C (ascorbic acid), vitamin D (calciferols), vitamin E (tocopherols and tocotrienols) and vitamin K (quinones) or minerals selected from sulfur, iron, chlorine, calcium, chromium, cobalt, copper, magnesium, manganese, molybdenum, iodine, selenium, and zinc.

REFERENCES

[0133] 1. Balk E M, Lichtenstein A H: Omega-3 Fatty Acids and Cardiovascular Disease: Summary of the 2016 Agency of Healthcare Research and Quality Evidence Review. Nutrients 2017, 9(8). [0134] 2. Calder P C: Marine omega-3 fatty acids and inflammatory processes: Effects, mechanisms and clinical relevance. Biochim Biophys Acta 2015, 1851(4):469-484. [0135] 3. Papanikolaou Y, Brooks J, Reider C, Fulgoni V L, 3rd: U.S. adults are not meeting recommended levels for fish and omega-3 fatty acid intake: results of an analysis using observational data from NHANES 2003-2008. Nutr J 2014, 13:31. [0136] 4. Clarke T C, Black L I, Stussman B J, Barnes P M, Nahin R L: Trends in the use of complementary health approaches among adults: United States, 2002-2012. Natl Health Stat Report 2015(79):1-16. [0137] 5. Schunck W H, Konkel A, Fischer R, Weylandt K H: Therapeutic potential of omega-3 fatty acid-derived epoxyeicosanoids in cardiovascular and inflammatory diseases. Pharmacol Ther 2018, 183:177-204. [0138] 6. Serhan Conn., Krishnamoorthy S, Recchiuti A, Chiang N: Novel anti-inflammatory—pro-resolving mediators and their receptors. Curr Top Med Chem 2011, 11(6):629-647. [0139] 7. Serhan Conn., Jain A, Marleau S, Clish C, Kantarci A, Behbehani B, Colgan S P, Stahl G L, Merched A, Petasis N A et al: Reduced inflammation and tissue damage in transgenic rabbits overexpressing 15-lipoxygenase and endogenous anti-inflammatory lipid mediators. J Immunol 2003, 171(12):6856-6865. [0140] 8. Prescott D, McKay D M: Aspirin-triggered lipoxin enhances macrophage phagocytosis of bacteria while inhibiting inflammatory cytokine production. Am J Physiol Gastrointest Liver Physiol 2011, 301(3):G487-497. [0141] 9. Rajasagi N K, Reddy P B, Mulik S, Gjorstrup P, Rouse B T: Neuroprotectin D1 reduces the severity of herpes simplex virus-induced corneal immunopathology. Invest Ophthalmol Vis Sci 2013, 54(9):6269-6279. [0142] 10. Baillie J K, Digard P: Influenza—time to target the host? N Engl J Med 2013, 369(2):191-193. [0143] 11. Morita M, Kuba K, Ichikawa A, Nakayama M, Katahira J, Iwamoto R, Watanebe T, Sakabe S, Daidoji T, Nakamura S et al: The lipid mediator protectin D1 inhibits influenza virus replication and improves severe influenza. Cell 2013, 153(1):112-125. [0144] 12. Zhu M, Wang X, Sun L, Schultzberg M, Hjorth E: Can inflammation be resolved in Alzheimer's disease? Ther Adv Neurol Disord 2018, 11:1756286418791107. [0145] 13. Kasikara C, Doran A C, Cai B, Tabas I: The role of non-resolving inflammation in atherosclerosis. J Clin Invest 2018, 128(7):2713-2723. [0146] 14. Gonzalez-Periz A, Horrillo R, Ferre N, Gronert K, Dong B, Moran-Salvador E, Titos E, Martinez-Clemente M, Lopez-Parra M, Arroyo V et al: Obesity-induced insulin resistance and hepatic steatosis are alleviated by omega-3 fatty acids: a role for resolvins and protectins. FASEB J 2009, 23(6):1946-1957. [0147] 15. Jin Y, Arita M, Zhang Q, Saban D R, Chauhan S K, Chiang N, Serhan Conn., Dana R: Anti-angiogenesis effect of the novel anti-inflammatory and pro-resolving lipid mediators. Invest Ophthalmol Vis Sci 2009, 50(10):4743-4752. [0148] 16. Connor K M, SanGiovanni JP, Lofqvist C, Aderman C M, Chen J, Higuchi A, Hong S, Pravda E A, Majchrzak S, Carper D et al: Increased dietary intake of omega-3-polyunsaturated fatty acids reduces pathological retinal angiogenesis. Nat Med 2007, 13(7):868-873. [0149] 17. Livne-Bar I, Wei J, Liu H H, Alqawlaq S, Won G J, Tuccitto A, Gronert K, Flanagan J G, Sivak J M: Astrocyte-derived lipoxins A.sub.4 and B4 promote neuroprotection from acute and chronic injury. J Clin Invest 2017, 127(12):4403-4414. [0150] 18. Arita M, Yoshida M, Hong S, Tjonahen E, Glickman J N, Petasis N A, Blumberg R S, Serhan Conn.: Resolvin E I, an endogenous lipid mediator derived from omega-3 eicosapentaenoic acid, protects against 2,4,6-trinitrobenzene sulfonic acid-induced colitis. Proc Natl Acad Sci USA 2005, 102(21):7671-7676. [0151] 19. Barnig C, Frossard N, Levy B D: Towards targeting resolution pathways of airway inflammation in asthma. Pharmacol Ther 2018, 186:98-113. [0152] 20. Aoki H, Hisada T, Ishizuka T, Utsugi M, Kawata T, Shimizu Y, Okajima F, Dobashi K, Mori M: Resolvin E1 dampens airway inflammation and hyperresponsiveness in a murine model of asthma. Biochem Biophys Res Commun 2008, 367(2):509-515. [0153] 21. Perretti M, Norling L V: Actions of SPM in regulating host responses in arthritis. Mol Aspects Med 2017, 58:57-64. [0154] 22. Dalli J, Zhu M, Vlasenko N A, Deng B, Haeggstrom J Z, Petasis N A, Serhan Conn.: The novel 13S,14S-epoxy-maresin is converted by human macrophages to maresin 1 (MaR1), inhibits leukotriene A4 hydrolase (LTA4H), and shifts macrophage phenotype. FASEB J 2013, 27(7):2573-2583. [0155] 23. Liu Y, Fang X, Zhang X, Huang J, He J, Peng L, Ye C, Wang Y, Xue F, Ai D et al: Metabolic profiling of murine plasma reveals eicosapentaenoic acid metabolites protecting against endothelial activation and atherosclerosis. Br J Pharmacol 2018, 175(8):1190-1204. [0156] 24. Endo J, Sano M, Isobe Y, Fukuda K, Kang J X, Arai H, Arita M: 18-HEPE, an n-3 fatty acid metabolite released by macrophages, prevents pressure overload-induced maladaptive cardiac remodeling. J Exp Med 2014, 211(8):1673-1687. [0157] 25. Pochard C, Coquenlorge S, Jaulin J, Cenac N, Vergnolle N, Meurette G, Freyssinet M, Neunlist M, Rolli-Derkinderen M: Defects in 15-HETE Production and Control of Epithelial Permeability by Human Enteric Glial Cells From Patients With Crohn's Disease. Gastroenterology 2016, 150(1):168-180. [0158] 26. Tang Y, Zhang M J, Hellmann J, Kosuri M, Bhatnagar A, Spite M: Proresolution therapy for the treatment of delayed healing of diabetic wounds. Diabetes 2013, 62(2):618-627. [0159] 27. Barden A E, Mas E, Croft K D, Phillips M, Mori T A: Specialized proresolving lipid mediators in humans with the metabolic syndrome after n-3 fatty acids and aspirin. Am J Clin Nutr 2015, 102(6):1357-1364. [0160] 28. Miyata J, Arita M: Role of omega-3 fatty acids and their metabolites in asthma and allergic diseases. Allergol Int 2015, 64(1):27-34. [0161] 29. Mangino M J, Brounts L, Harms B, Heise C: Lipoxin biosynthesis in inflammatory bowel disease. Prostaglandins Other Lipid Mediat 2006, 79(1-2):84-92. [0162] 30. Wang C W, Colas R A, Dalli J, Arnardottir H H, Nguyen D, Hasturk H, Chiang N, Van Dyke T E, Serhan Conn.: Maresin 1 Biosynthesis and Proresolving Anti-infective Functions with Human-Localized Aggressive Periodontitis Leukocytes. Infect Immun 2015, 84(3):658-665. [0163] 31. Ringholz F C, Buchanan P J, Clarke D T, Millar R G, McDermott M, Linnane B, Harvey B J, McNally P, Urbach V: Reduced 15-lipoxygenase 2 and lipoxin A4/leukotriene B4 ratio in children with cystic fibrosis. Eur Respir J 2014, 44(2):394-404. [0164] 32. Robertson R C, Seira Oriach C, Murphy K, Moloney G M, Cryan J F, Dinan T G, Paul Ross R, Stanton C: Omega-3 polyunsaturated fatty acids critically regulate behaviour and gut microbiota development in adolescence and adulthood. Brain Behav Immun 2017, 59:21-37. [0165] 33. Mujico J R, Baccan G C, Gheorghe A, Diaz L E, Marcos A: Changes in gut microbiota due to supplemented fatty acids in diet-induced obese mice. Br J Nutr 2013, 110(4):711-720. [0166] 34. Vance R E, Hong S, Gronert K, Serhan Conn., Mekalanos J J: The opportunistic pathogen Pseudomonas aeruginosa carries a secretable arachidonate 15-lipoxygenase. Proc Natl Acad Sci USA 2004, 101(7):2135-2139. [0167] 35. Furuya T, Shibata D, Kino K: Phylogenetic analysis of Bacillus P450 monooxygenases and evaluation of their activity towards steroids. Steroids 2009, 74(12):906-912. [0168] 36. Bernhardt R: Cytochromes P450 as versatile biocatalysts. J Biotechnol 2006, 124(1):128-145. [0169] 37. Capdevila J H, Wei S, Helvig C, Falck J R, Belosludtsev Y, Truan G, Graham-Lorence S E, Peterson J A: The highly stereoselective oxidation of polyunsaturated fatty acids by cytochrome P450BM-3. J Biol Chem 1996, 271(37):22663-22671. [0170] 38. Levitt Md.: Volume and composition of human intestinal gas determined by means of an intestinal washout technic. N Engl J Med 1971, 284(25):1394-1398. [0171] 39. Albenberg L, Esipova T V, Judge C P, Bittinger K, Chen J, Laughlin A, Grunberg S, Baldassano R N, Lewis J D, Li H et al: Correlation between intraluminal oxygen gradient and radial partitioning of intestinal microbiota. Gastroenterology 2014, 147(5):1055-1063 e1058. [0172] 40. Polus A, Zapala B, Razny U, Gielicz A, Kiec-Wilk B, Malczewska-Malec M, Sanak M, Childs C E, Calder P C, Dembinska-Kiec A: Omega-3 fatty acid supplementation influences the whole blood transcriptome in women with obesity, associated with pro-resolving lipid mediator production. Biochim Biophys Acta 2016, 1861(11):1746-1755. [0173] 41. See VHL, Mas E, Prescott S L, Beilin L J, Burrows S, Barden A E, Huang R C, Mori T A: Effects of prenatal n-3 fatty acid supplementation on offspring resolvins at birth and 12 years of age: a double-blind, randomised controlled clinical trial. Br J Nutr 2017, 118(11):971-980. [0174] 42. Weiss Ga., Troxler H, Klinke G, Rogler D, Braegger C, Hersberger M: High levels of anti-inflammatory and pro-resolving lipid mediators lipoxins and resolvins and declining docosahexaenoic acid levels in human milk during the first month of lactation. Lipids Health Dis 2013, 12:89. [0175] 43. Piewngam P, Zheng Y, Nguyen T H, Dickey S W, Joo H S, Villaruz A E, Glose Kans., Fisher E L, Hunt R L, Li B et al: Pathogen elimination by probiotic Bacillus via signalling interference. Nature 2018, 562(7728):532-537. [0176] 44. Al-Thubiani A S A, Maher Y A, Fathi A, Abourehab M A S, Alarjah M, Khan M S A, Al-Ghamdi S B: Identification and characterization of a novel antimicrobial peptide compound produced by Bacillus megaterium strain isolated from oral microflora. Saudi Pharm J 2018, 26(8):1089-1097. [0177] 45. Miura M, Ito K, Hayashi M, Nakajima M, Tanaka T, Ogura S: The Effect of 5-Aminolevulinic Acid on Cytochrome P450-Mediated Prodrug Activation. PLoS One 2015, 10(7):e0131793. [0178] 46. Ruettinger R T, Wen L P, Fulco A J: Coding nucleotide, 5′ regulatory, and deduced amino acid sequences of P-450BM-3, a single peptide cytochrome P-450:NADPH-P-450 reductase from Bacillus megaterium. J Biol Chem 1989, 264(19):10987-10995. [0179] 47. Kang J Y, Kim S Y, Kim D, Kim D H, Shin S M, Park S H, Kim K H, Jung H C, Pan J G, Joung Y H et al: Characterization of diverse natural variants of CYP102A1 found within a species of Bacillus megaterium. AMB Express 2011, 1(1):1. [0180] 48. Werz O, Gerstmeier J, Libreros S, De la Rosa X, Werner M, Norris P C, Chiang N, Serhan Conn.: Human macrophages differentially produce specific resolvin or leukotriene signals that depend on bacterial pathogenicity. Nat Commun 2018, 9(1):59.