Method for processing a separation product, containing carboxylic acids, of a separation process

Abstract

The invention relates to a method for processing a separation product, containing carboxylic acids, of a separation process, in particular a process for separating fish oil and / or algae oil, in which method polyunsaturated hydrocarbons are produced at least from some of the carboxylic acids by means of microbes. Advantageously, the polyunsaturated hydrocarbons are or comprise omega-3 fatty acids and / or omega-6 fatty acids, preferably EPA, DHA and / or DPA. In an embodiment of the invention, the microbes are or comprise yeast, fungus, bacteria and / or in particular marine protists, preferably microalgae. The yeast is preferably a yeast of the Yarrowia lipolytica species and the marine protist is preferably a microalga of the Schizochytrium genus, preferably Schizochytrium limacinum SR21. The invention further relates to biomass which is produced from microbes and comprises polyunsaturated hydrocarbons.

Description

[0001] Description:

[0002] Process for processing a separation product containing carboxylic acids from a separation process

[0003] The invention relates to a process for processing carboxylic acids.

[0004] US 2010 / 0166620 A1 discloses a system and a process for producing fatty acid methyl esters, commonly referred to as "biodiesel", which are obtained from a mixture of oils and fats of unknown composition containing triglycerides, proteins or other substances in any organic form containing sufficient fatty acids for conversion into biodiesel.

[0005] The invention is based on the object of making carboxylic acids more usable.

[0006] The invention is achieved by a method for processing a separation product containing carboxylic acids from a preferably industrial separation process, in which polyunsaturated hydrocarbons are produced from at least a portion of the carboxylic acids by means of microbes.

[0007] Surprisingly, it has been shown that microbes are particularly well-suited for processing the separation products. Using this method, byproducts from industrial separation processes, which have previously typically been disposed of, can be further processed. This not only advantageously avoids the expense of disposal and waste, but also increases the yield of the separation process. This is especially true if one goal of the separation process is the production of a product containing polyunsaturated hydrocarbons. The invention proves to be particularly advantageous if fish oil and / or algal oil is processed in the separation process.

[0008] The fish oil to be processed by means of the separation process, which is preferably a pre-processed fish oil intermediate, expediently has omega-3 fatty acid(s) of 30 - 80 wt.%, preferably having at least one of the following contents: eicosapentaenoic acid (EPA) of 0 - 80 wt.%, docosahexaenoic acid (DHA) of 0 - 70 wt.% and / or docosapentaenoic acid (DPA) of 0 - 10 wt.%.

[0009] The algal oil to be processed by means of the separation process, which is preferably a pre-processed algal oil intermediate, expediently has an omega-3 fatty acid content of 30 - 90 wt.%, preferably having at least one of the following contents: eicosapentaenoic acid (EPA) of 0 - 85 wt.%, docosahexaenoic acid (DHA) of 0 - 85 wt.% and / or docosapentaenoic acid (DPA) of 0 - 10 wt.%.

[0010] In one embodiment of the invention, a starting fish oil from which the fish oil intermediate is produced has a content of omega-3 fatty acid(s) of 10-50 wt.%, preferably having at least one of the following contents: EPA of 0-25 wt.%, DHA of 0-25 wt.% and / or DPA of 0-7 wt.%.

[0011] In one embodiment of the invention, a starting algal oil from which the algal oil intermediate is produced has an omega-3 fatty acid content of 10-80 wt.%, preferably having at least one of the following contents: EPA of 0-70 wt.%, DHA of 0-70 wt.% and / or DPA of 0-5 wt.%.

[0012] Advantageously, the starting fish oil and / or the starting algal oil has been produced by means of distillation processes, deacidification, ethylation / transesterification, saponification, winterization, mechanical separation processes and / or supercritical fluid extraction, preferably from a crude oil.

[0013] In one embodiment of the invention, the fish oil intermediate and / or the algal oil intermediate are produced from the starting fish oil and / or the starting algal oil by distillation, deacidification, ethylation / transesterification, saponification, mechanical separation processes, urea precipitation, chromatography, winterization, silver extraction processes, and / or supercritical fluid extraction. The fish oil intermediate and / or the algal oil intermediate preferably has an ethyl ester content of at least 50% by weight, preferably at least 80% by weight.

[0014] The said omega-3 fatty acid(s) are advantageously present in the intermediate product as ethyl esters, wherein the omega-3 fatty acid(s) are preferably present as ethyl esters to an extent of at least 50% by weight, preferably at least 60% by weight, particularly preferably at least 90% by weight.

[0015] In a particularly preferred embodiment of the invention, the separation product is a by-product of a separation process for producing a target product containing omega-3 and / or omega-6 fatty acid(s), preferably EPA and / or DHA and / or DPA, in high concentration.

[0016] In one embodiment of the invention, the target product, produced in particular from fish oil and / or from algal oil, has a content of omega-3 fatty acid(s) of > 50 wt.%, preferably of > 80 wt.%, particularly preferably of > 90 wt.%, wherein it preferably has at least one of the following contents: EPA from 0 to 99 wt.%, DHA from 0 to 99 wt.% and / or DPA from 0 to 20 wt.%.

[0017] The target product advantageously has an EPA content of > 80 wt.%, preferably > 90 wt.%, particularly preferably > 95 wt.% or a DHA content of > 80 wt.%, preferably > 90 wt.%, particularly preferably > 95 wt.%.

[0018] Furthermore, the target product may have a DPA content of > 50 wt.%, preferably > 60 wt.%, particularly preferably > 70 wt.%.

[0019] The by-product expediently has a saturated fatty acid content of > 10 wt.%, preferably > 15 wt.%, particularly preferably > 45 wt.%. In one embodiment of the invention, the by-product has a content of polyunsaturated hydrocarbons, in particular omega-3 and / or omega-6 fatty acid(s), preferably EPA, DHA and / or DPA, wherein the content is less than that of the target product, in particular at most 15 wt.%, preferably at most 10 wt.%, particularly preferably at most 5 wt.%, of the target product. Expediently, the content of polyunsaturated hydrocarbons, in particular omega-3 fatty acids, in the by-product is < 15 wt.%, preferably < 10 wt.%, particularly preferably < 5 wt.%.

[0020] In one embodiment of the invention, the by-product, produced in particular from fish oil and / or from algal oil, has at least one of the following contents: EPA of 0-25% by weight, preferably 0-15% by weight, DHA of 0-25% by weight, preferably 0-15% by weight, and / or DPA of 0-25% by weight, preferably 0-15% by weight.

[0021] The separation product is expediently produced by means of: a distillation process, in particular using a distillation device, preferably HPE (High-Pressure Extraction; for example as explained in WO2017005235 A1) or short-path distillation,

[0022] - processed by SFE (Supercritical Fluid Extraction) and / or by a chromatography method, preferably SFC (Supercritical fluid chromatography), high performance liquid chromatography (HPLC), true moving bed chromatography (TMB) and / or simulated moving bed (SMB), by a silver extraction method and / or urea precipitation (urea complexation).

[0023] In one embodiment of the invention, the by-product produced by short-path distillation has at least one of the following contents: EPA of 0-8 wt.%, preferably of 0-4 wt.%, DHA of 0-5 wt.%, preferably of 0-3 wt.%, and / or DPA, in particular of n-3-DPA, of 0-5 wt.%, preferably of 0-3 wt.%.

[0024] The by-product produced by HPE has one of the following contents: EPA of 0-12 wt.%, preferably 0-6 wt.%, DHA of 0-7 wt.%, preferably 0-4 wt.%, and / or DPA, in particular n-3-DPA, of 0-7 wt.%, preferably 0-4 wt.%.

[0025] The by-product produced by a silver extraction process has one of the following contents: EPA of 5-70 wt%, preferably 5-40 wt%, DHA of 5-70 wt%, preferably 5-40 wt%, and / or arachidonic acid of 0-12 wt%, preferably 0-6 wt%.

[0026] The by-product produced by means of urea precipitation has one of the following contents: EPA of 2-40 wt.%, preferably of 2-20 wt.%, DHA of 2-40 wt.%, preferably of 2-20 wt.%, DPA, in particular of n-3-DPA, of 2-20 wt.%, preferably of 2-10 wt.%, and / or monounsaturated fatty acids of 5-60 wt.%, preferably of 5-30 wt.%.

[0027] It has proven particularly advantageous that long-chain fatty acids, in particular a chain length > 12 C atoms, preferably a chain length > 20 C atoms, can be produced by means of microbes from short- or medium-chain fatty acids, in particular a chain length < 12 C atoms.

[0028] In one embodiment of the invention, the polyunsaturated hydrocarbons produced by the microbes are or comprise EPA, DHA and / or DPA.

[0029] In one embodiment of the invention, the microbes are selected from the group of yeast, fungi, bacteria, protists, in particular marine protists, preferably microalgae.

[0030] Particularly preferred microbes are selected from the group consisting of: Schizochytrium sp., Aurantiochytrium sp., Crypthecodinium cohnii, Isochrysis galbana

[0031] Phaeodactylum tricornutum, Dunaliella salina, Nannochloropsis oceanica, Chlorella vulgaris, Mucor circinelloides, Saccharomyces cerevisiae, Marine bacteria spp., Cyanobacteria spp., Myxobacteria spp.

[0032] In one embodiment of the invention, the microbes are or comprise a fat-dissolving yeast, preferably a yeast of the species Yarrowia lipolytica, particularly preferably a genetically modified yeast of the species Yarrowia lipolytica, for example Yarrowia lipolytica Af4.

[0033] The inventors' investigations have shown that yeast, in particular of the species Yarrowia lipolytica, produces polyunsaturated hydrocarbons, in particular DHA, from the carboxylic acids, in particular the separation product.

[0034] For use in the process according to the invention, the yeasts Candida tropicalis, Candida albicans, Debaryomyces hansenii and / or Trichosporon can also be used.

[0035] In a further embodiment of the invention, the microbes are or comprise a microalgae of the species Schizochytrium, preferably Schizochytrium limacinum, particularly preferably Schizochytrium limacinum SR21. Furthermore, it has been shown that the microalgae of the species Schizochytrium produces polyunsaturated hydrocarbons, particularly DHA, from the carboxylic acids, particularly the separation product.

[0036] The following marine protists, in particular microalgae or marine bacteria, can also be used in the process according to the invention: Marinobacter, Oceanospiralles, Pseudomonas and / or Alkanivorax.

[0037] It has proven particularly advantageous to produce polyunsaturated hydrocarbons from the carboxylic acids, especially the separation product, using a combination of yeast and microalgae. When produced using yeast and microalgae, the yeast's extracellular lipase activity creates an emulsion effect, which promotes the microalgae's production of polyunsaturated hydrocarbons.

[0038] Compared to yeast alone, the yield when using the combination is about four times higher. Compared to microalgae alone, the yield when using the combination is almost doubled.

[0039] The production of unsaturated hydrocarbons using the microalgae of the species Schizochyfrium limacinum, in particular Schizochyfrium limacinum SR21, together with yeast has proven particularly effective. In one embodiment of the invention, lipases are added to improve the production of polyunsaturated hydrocarbons by the microbes. The separation product is expediently fed into a reactor together with the microbes to produce the polyunsaturated hydrocarbons.

[0040] In one embodiment of the invention, biomass produced by the microbes is processed into a food or a pharmaceutical ingredient.

[0041] The food is preferably fed to aquatic organisms, preferably fish, which are kept for the production of fish oil, for example in a fish farm.

[0042] Furthermore, the biomass can be fed to living organisms to enrich their nutritional components or products (e.g., eggs) with polyunsaturated hydrocarbons, particularly omega-3 and / or omega-6 fatty acids. For example, chickens can be fed the biomass to achieve correspondingly increased levels of polyunsaturated hydrocarbons in the meat and / or eggs produced. The polyunsaturated hydrocarbons are expediently extracted from biomass produced by the microbes.

[0043] The invention further relates to biomass produced from microbes, which comprises polyunsaturated hydrocarbons. The biomass advantageously comprises yeast and / or microalgae, preferably yeast of the species Yarrowia lipolytica and / or the marine protist of the species Schizochytrium, preferably Schizochytrium limacinum SR21. The EPA, DHA, and / or DPA content of the biomass is preferably at least 5 mg / g dry weight.

[0044] The invention is explained in more detail below with reference to exemplary embodiments as well as tables and the attached graphics which relate to the exemplary embodiments.

[0045] Fig. 1 shows in a graph a content of cell dry weight and a content of DHA when carrying out the method according to the invention as a function of the cultivation time.

[0046] A. Materials used:

[0047] A pre-processed fish oil, which forms an intermediate product, was processed by means of a separation process, which is referred to below as “HPE” (High Pressure Extraction) and which is described in the publication WO2017005235A1.

[0048] The intermediate had the composition shown in Table 1:

[0049] Table 1

[0050] After performing the HPE, the target product had the following composition according to Table 2:

[0051] Table 2

[0052] From carboxylic acids from one of the fractions, which do not form the target product of the separation process but are usually disposed of as waste, polyunsaturated hydrocarbons were produced by means of microbes in the manner according to the invention, as explained in more detail below.

[0053] The fatty acid composition of the fraction used based on the total fatty acids contained is shown in Table 3: - ] 0 -

[0054] Table 3

[0055] 1MM.korQgrg aismen

[0056] For cultivation on the fraction shown in Table 3, a strain of the oleaginous yeast Yarrowia lipolytica and the marine protist Schizochytrium limacinum, a microalgae, were used. a) The following strain of the yeast Yarrowia lipolytica was used: Y. lipolytica, Pol h::SynPfaPptAf4 (Y. lipolytica Af4) [Gemperlein et al., 2019]. b) The following strain of the marine protist Schizochytrium limacinum was used: S. limacinum SR21 (ATCC MYA-1381) (renamed Aurantiochytrium limacinum in 2007). 2, Cultivation

[0057] The following media were used for the cultivation of Y. lipolytica and S. limacinum: a) YNB medium without carbon source

[0058] For the cultivation of Y. lipolytica, YNB medium was used in combination with a carbon source. All components were prepared as appropriate stock solutions in MilliQ water and either autoclaved at 120 °C for 20 min (ammonium sulfate) or sterile filtered using a PES membrane filter with a pore size of 0.2 μm (all others). The solutions were stored at room temperature (ammonium sulfate) or at 4 °C. The concentrations are listed in Table 4.

[0059] Table 4 b) Artificial seawater medium

[0060] For the cultivation of S. limacinum, an artificial seawater medium with yeast extract as a nitrogen source and a carbon source according to the composition shown in Table 5 was used. For mixed cultures of S. limacinum and Y. lipolytica, half the amount of salts was used for cultivation (see values ​​in parentheses).

[0061] Table 5 c) Carbon sources

[0062] For precultures, 10 g / L glycerol (stock concentration of 200 g / L; autoclaved at 120 °C for 20 min) was used as the carbon source. For main cultures, initial concentrations of 25 to 100 g / L of the above-mentioned fraction were used. The fraction was freshly sterile filtered for each cultivation using a PES membrane filter with a pore size of 0.2 μm. The fraction was stored at 4 °C after the air in the bottle had been replaced with nitrogen to reduce the risk of oxidation. dj Cultivation conditions

[0063] The cells were cultured in 250 or 500 mL shake flasks with 10% filling volume.

[0064] The flasks were shaken in an orbital shaker (Multitron shaker, Infers HT, Bottmingen, Switzerland) at 200 and 230 rpm for S. l / / iic / num / mixed cultures and Y. lipolytica at 28 °C.

[0065] 3. Methods a) Sampling

[0066] From each triple flask, 1.5 mL of culture broth is transferred into pre-weighed glass vials in duplicate. The exact volume is measured gravimetrically. The glass vials are centrifuged at 10,000 xg and 4 °C for 3 minutes. A further 100 μL of culture broth is collected in a reaction tube for pH measurement, microscopy, and the lipase assay. b) Supernatant

[0067] The supernatant, which still contains residual oil, is removed from the cell pellet using a syringe with a needle and filtered through a PES membrane filter (0.2 μm pore size; HPLC grade) into a new reaction vessel. The oil-free supernatant is stored at -20°C until further analysis by HPLC. c) Pellets The remaining pellet in the glass vial is resuspended in 600 μL hexane:water (1:6, v / v) and then centrifuged as described above to remove adhering oil from the cell surface and the glass wall of the vial. The water-hexane-oil mixture is discarded, or for non-quantitative residual fatty acid determination, the hexane-oil phase is collected in a separate glass vial and only the lower water phase is discarded. Both the hexane-oil phase and the wet cell pellets are stored at -20°C until further processing. d) Determination of cell dry weight

[0068] The moist cell pellets are dried in a rotary vacuum concentrator (RVC 2-33 CDplus with infrared heating, Christ, Germany) at 30 mbar, 240 rpm, and 40 °C for 30 minutes or until dry. The cell dry weight (CDW) is determined gravimetrically. e) Fatty acid analysis

[0069] The intracellular fatty acids in each pellet sample (after CDW determination) as well as the fatty acids in the remaining substrate HPE-OI (hexane-oil phase) are measured. For the next steps, the hexane-oil phase must be dried in a rotary vacuum concentrator (RVC 2-33 CDplus with infrared heating, Christ, Germany) at 30 mbar, 240 rpm, and 40 °C for 30 min or until dry. f) Fatty acid methyl ester (FAME) preparation

[0070] The FAME preparation was carried out as follows. 15 pg of the methyl ester of heneicosapentaenoic acid (HPA) was added to each sample as an internal standard for gas chromatography-mass spectroscopy (GCMS). 300 pL of a 50:50:2 methanol-toluene-sulfuric acid mixture (v / v / v) was added for acid-catalyzed transesterification at 80 °C for 24 h. The reaction mixture was cooled on ice, and the transesterification was stopped by adding 250 pL of a 0.5 M NH4 HCO3 and 2 M KCl solution, followed by vortexing for 30 s. Phase separation was achieved by centrifugation at 10,000 xg at room temperature for 3 min. 75 pL of the upper phase containing the FAME is transferred to a glass vial with an inlet for GCMS measurements. g) GCMS measurement

[0071] Gas chromatography was performed using a HP-6890 GC system (Hewlett Packard,

[0072] CA, USA) with an HP-88 column (30 m x 250 pm x 0.2 pm, Agilent Technologies) and helium (He 5.0) as the carrier gas. The system was equipped with an injection system (7683B series, Agilent Technologies, CA, USA) and an autosampler (7683 series, Agilent Technologies (CA, USA). The injection volume was set to 0.2 or 1.0 l / L, and the split mode was selected to 10:1 or 5:1, depending on the expected fatty acid concentration of the sample. Further parameters are listed in Table 6.

[0073] Table 6

[0074] FAME was detected using a mass-selective detector (mass spectrometer, MS; 5973 Network Series from Agilent Technologies, CA, USA). The MS was set to a solvent delay of 5 minutes. The mass-selective detector was set to SCAN mode and measured the total ion current (TIC) from m / z 25 to m / z 500. To identify the individual fatty acids, TIC and mass spectra were compared with the NIST08 mass spectra database (NIST / EPA / N IH Mass Spectral Library, 2008, Version 2.0). In addition, the retention times (RT) of individual fatty acid standard solutions (Sigma) were measured and compared to the RT of fatty acids of unclear identity for clarification. The MSD ChemStation Gl 701 EA software was used for data analysis and system control. h) Quantification of fatty acids

[0075] The integration function of the MSD ChemStation Gl 701 EA software was used to calculate the area under the curve for each peak. The results were paired with the retention times and exported to a spreadsheet file. The corresponding fatty acid was assigned to each measured RT. The amount of each detected FAME was then calculated relative to the AUC signal of the HPA-ME standard. Fatty acid concentrations were calculated relative to volume or CDW.

[0076] 4, Results

[0077] The fraction with the fatty acid composition shown in Table 3 was cultured with the yeast Yarrowia lipolytica (see section 1. a)), the marine protist Schizochytrium limacinum (see section 1. b)), and a mixture of the yeast and the marine protist for 108 hours in a reactor. Before cultivation, the fraction contained no DHA, no DPA, and 3.83% EPA.

[0078] As shown in Table 7, DHA was produced by culturing the fraction with yeast. After cultivation, the DHA content was 0.29% of the total fatty acids.

[0079] By cultivating the fraction containing the marine protist, an amount of DHA was produced such that the content was 0.38%, and an amount of DPA was produced such that the content was 0.07%.

[0080] By cultivating the fraction with a mixed culture of the yeast and the marine protist, an amount of DHA was produced such that the content was 6.34%, an amount of DPA was produced such that the content was 1.96%, and an amount of EPA was produced such that the content was 5.80%.

[0081] Table 7

[0082] Table 8 shows the cell dry weight (CDW) of the biomass produced, as well as the total fatty acid content of the cell dry weight and the DHA content of the cell dry weight. It can be seen that the DHA content is higher when cultivated with the marine protist than with the yeast. A significantly higher content is achieved when cultivated with the mixed culture.

[0083] Table 8

[0084] The production of higher EPA and DHA levels can be explained by a synergistic effect resulting from the yeast producing lipases that promote the production of EPA and DHA by the marine protist.

[0085] Fig. 1 shows in a graph the formation of biomass as a function of the cell dry weight (CDW) depending on the cultivation time for a occupancy with 25 g / L and 75 g / L of the fraction (“HPE”) in the yeast, protist and mixed cultures, respectively.

[0086] Furthermore, the graph shows the change in DHA content for the two occupancies as a function of the cultivation time.

[0087] It can be seen that the cell dry weight increased continuously up to a cultivation time of 108 hours. For a culture load of 25 g / L, the DHA content increased to up to 10 mg / mL up to a cultivation time of 160 hours. For a culture load of 75 g / L, the DHA content increased to up to 10 mg / mL up to a cultivation time of 1250 hours.

[0088] In a further embodiment, oil obtained from algae was processed using the separation process described above. Polyunsaturated hydrocarbons were produced from carboxylic acids from one of the fractions that did not form the target product of the separation process using microbes in the manner according to the invention. The results of a measurement of the DHA yield during the processing of a fraction of algal oil and a fraction of fish oil are shown in Table 9. It can be seen that yeast is better suited for producing DHA from fish oil, while the DHA yield is higher for the algal oil fraction when produced using microalgae.

[0089] Table 9

Claims

Patent claims:

1. Process for processing a separation product containing carboxylic acids from a separation process, in particular a process for separating fish oil and / or algal oil, in which at least some of the carboxylic acids are used to produce polyunsaturated hydrocarbons by means of microbes.

2. The method according to claim 1, characterized in that the polyunsaturated hydrocarbons are or comprise omega-3 and / or omega-6 fatty acids, preferably EPA, DMA and / or DPA.

3. Method according to claim 1 or 2, characterized in that the microbes are or comprise yeast, fungi, bacteria and / or, in particular marine, protists, preferably microalgae.

4. Method according to one of claims 1 to 3, characterized in that the yeast is a yeast of the species Yarrowia lipolytica, preferably a genetically modified variant of the species Yarrowia lipolytica.

5. Method according to one of claims 1 to 4, characterized in that the marine protist is a microalga of the species Schizochytrium, preferably Schizochytrium limacinum SR21.

6. Method according to any one of claims 1 to 5, characterized in that the product is a waste product of an industrial separation process, in particular a destination or chromatography process.

7. Method according to one of claims 1 to 6, characterized in that lipases are added to improve production using microbes.

8. A method according to any one of claims 1 to 7, characterized in that the separation product, together with the microbes and optionally the lipases, is placed in a reactor to produce the polyunsaturated hydrocarbons.

9. A method according to any one of claims 1 to 8, characterized in that biomass is formed by means of the microbes and the biomass is preferably processed into a foodstuff or a pharmaceutical ingredient.

10. A method according to claim 9, characterized in that the foodstuff is fed to aquatic organisms, preferably fish, intended for the production of fish oil.

11. A method according to any one of claims 1 to 9, characterized in that the polyunsaturated hydrocarbons are extracted from the biomass formed by the microbes.

12. Biomass produced from microbes, characterized in that the biomass comprises polyunsaturated hydrocarbons.Biomass according to claim 12, 13, characterized in that the biomass comprises yeast, fungi, bacteria and / or, in particular, marine protists, preferably microalgae.

14. Biomass according to claim 12 or 13, characterized in that the biomass comprises yeast of the species Yarrowia lipolytica and / or the marine protist of the species Schizochytrium, preferably Schizochytrium limacinum SR21.

15. Biomass according to one of claims 12 to 14, characterized by an EPA and / or DHA content of at least 5 mg / g cell dry weight.

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