Method for producing concentrated therapeutic phospholipid compositions from krill extracts containing high levels of free fatty acids

Abstract

A method for producing a therapeutic enriched phospholipid composition from raw krill oil containing a high content of free fatty acids is provided. In particular, a process for producing a concentrated phospholipid composition is provided, comprising the steps of: fractionating raw krill oil (RKO) containing at least 7% free fatty acids (FFA) to obtain a fraction enriched in phospholipids and undesired phases; and separating the phospholipid-enriched fraction from the undesired phases to produce a therapeutic enriched phospholipid composition in a yield of at least 30%.

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Application No. 62 / 804,285, filed February 12, 2019, the contents of which are incorporated herein by reference in their entirety.

[0002] A method is provided for producing a concentrated therapeutic phospholipid composition using raw krill oil having a free fatty acid content greater than 7%. [Background technology]

[0003] Krill oil compositions have been described as providing beneficial effects in humans such as lowering cholesterol, inhibiting platelet adhesion, inhibiting arterial plaque formation, preventing hypertension, controlling arthritic conditions, enhancing transdermal transport, reducing symptoms of premenstrual conditions, or controlling blood glucose levels in patients (WO 02 / 102394).

[0004] It is known that the composition of the oil extracted from krill varies depending on the nature of the starting material. Factors influencing the lipid content and profile of krill oil are season, species and age. The storage and processing conditions of the starting material also affect the oil composition. For example, the harvesting technique and the storage method of the krill before processing will affect the amount of certain components in the resulting oil. Pre-processing steps such as heating, cooling (including freeze-drying) and washing are also factors that affect the final components present in the extracted oil.

[0005] Krill oil extraction protocols are described. US Patent Application No. 2010 / 0143571 discloses that when preparing lipid fractions from fresh krill, krill lipases remain active even after the krill die and hydrolyze part of the krill lipids. According to US Patent Application No. 2010 / 0143571, the hydrolysis of lipids carried out by these lipases is problematic and undesirable. As a result, methods are described that provide a low degree of hydrolysis of krill lipids and thus minimize the risk of resulting in krill oil with undesirable amounts of free fatty acids. Therefore, pretreatment steps are desirable when handling raw materials, such as thermal pretreatment of krill to inactivate the enzymatic degradation of lipids and ensure a product with low levels of free fatty acids (FFA).

[0006] US Patent No. 9,028,877 describes an extract from Antarctic krill that contains high levels of astaxanthin, concentrated amounts of phospholipids, including ether phospholipids, and omega-3 fatty acids. More specifically, US Patent No. 9,028,877 discloses a method for processing krill freshly caught at the catch site, such as on board a ship, to minimize the processing of frozen krill transported from the catch site to the processing site, which is expensive to transport and can result in degradation of the krill starting material. The krill is first subjected to a protein denaturation process, such as a heating process, to avoid the formation of enzymatically degraded oil components, such as free fatty acids.

[0007] International Application No. WO2011 / 050474, for example, discloses concentrated therapeutic phospholipid (PL) compositions comprising about 60% w / w phospholipids. These concentrated therapeutic phospholipid compositions are produced using krill oil starting material, as described in U.S. Patent Application No. 07 / 09,096 and U.S. Patent No. 9,028,877. Such krill oil starting material does not allow for the production of economically viable commercial quantities of these concentrated therapeutic PL compositions for use in a consistent manner in the pharmaceutical industry.

[0008] International Application No. WO 2019 / 111055 discloses a method for producing protein phospholipid complexes from crustaceans in which hydrolysis occurs without denaturing the proteins.

[0009] Thus, there remains a need to provide methods for processing krill to produce economically viable commercial quantities of concentrated therapeutic PL compositions in a consistent and efficient manner for use in the pharmaceutical industry. Summary of the Invention [Problem to be solved by the invention]

[0010] A method for producing an enriched therapeutic phospholipid composition is provided, comprising the steps of extracting raw krill oil (RKO) containing at least 7% free fatty acids (FFA) to obtain a phospholipid-rich fraction and an undesired layer, and separating the phospholipid-rich fraction from the undesired layer. [Means for solving the problem]

[0011] According to one embodiment, the methods described herein produce a therapeutic enriched phospholipid composition in a yield of at least 30%.

[0012] According to another embodiment, the methods described herein produce a therapeutic enriched phospholipid composition in a yield of between 30%-60%.

[0013] According to further embodiments, the total phospholipids in the therapeutic concentrated phospholipid composition are at a concentration of at least 50% (w / w (phospholipid / composition)).

[0014] According to one embodiment, a therapeutic concentrated phospholipid composition comprises free and bound docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), wherein the total free and bound EPA in the composition is present at a concentration of 15% to 25% (w / w), and the total free and bound DHA in the composition is present at a concentration of 10% to 15% (w / w).

[0015] According to a further embodiment, the concentrated therapeutic phospholipid composition has the following formula (I): [ka] wherein R1 and R2 each independently represent, optionally, a docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA) residue; and Each X is independently selected from -CH2CH2NH3, -CH2CH2N(CH3)3°, and the following formula: [ka] The compound includes a compound represented by the formula:

[0016] According to another embodiment, the RKO comprises at least 7% FFA.

[0017] According to one embodiment, the RKO comprises at least 15% FFAs.

[0018] According to a further embodiment, the RKO is fractionated by countercurrent supercritical CO2 extraction.

[0019] According to additional embodiments, the RKO is mixed with a solvent to be fractionated into a first low density layer and a first high density layer enriched in phospholipids; the first high density layer enriched in phospholipids is separated from the first low density layer; the first separated high density layer enriched in phospholipids is mixed with an organic solvent comprising at least about 85% by weight of a C3-C8 ketone solvent to obtain a mixture; the mixture is fractionated into a second low density layer and a second layer enriched in phospholipids; and the second layer enriched in phospholipids is separated from the second low density layer to obtain a concentrated phospholipid composition.

[0020] According to one embodiment, the method comprises mixing the RKO and the first organic solvent in a ratio of about 5:about 15 by volume organic solvent:kg RKO.

[0021] According to one embodiment, the concentrated phospholipid composition is mixed with one or more free fatty acids.

[0022] According to another embodiment, the organic solvent comprises at least about 85% by weight of a C3-C8 ketone solvent.

[0023] According to additional embodiments, the methods described herein include combining the phospholipid-rich second phase with a stabilizer, a viscosity-reducing agent, or a combination thereof to produce a phospholipid-rich fraction.

[0024] According to one embodiment, the method comprises fractionating at least 100 kg of RKO.

[0025] According to one embodiment, the method includes heating the RKO to a temperature of about 20° C. to about 70° C. prior to combining the RKO and the first organic solvent.

[0026] According to further embodiments, the methods described herein further comprise passing the mixture of the RKO and the organic solvent through a filter.

[0027] According to one embodiment, the filter is a 0.45 μm filter.

[0028] According to one embodiment, the method described herein further comprises passing the mixture of the RKO and the organic solvent through a first filter and a second filter.

[0029] According to one embodiment, the first filter is a 10 pm filter and the second filter is a 0.45 pm filter.

[0030] According to another embodiment, the methods described herein further comprise combining the phospholipid-rich second layer with a viscosity-lowering agent.

[0031] According to one embodiment, the phospholipid-rich second layer is combined with a viscosity-reducing agent in a ratio of about 0.1:about 0.3.

[0032] According to one embodiment, the stabilizer comprises Vitamin E.

[0033] According to one embodiment, the method is a continuous method.

[0034] According to a further embodiment, the total phospholipids in the composition are at a concentration of 55% (w / w (phospholipids / total composition)).

[0035] According to one embodiment, the total phospholipids in the composition are at a concentration of 60% (w / w (phospholipids / total composition)).

[0036] According to another embodiment, the total phospholipids in the composition are at a concentration of 90% (w / w (phospholipids / total composition)).

[0037] According to additional embodiments, the therapeutic phospholipid composition comprises triglycerides at a concentration of less than about 5% (w / w).

[0038] According to one embodiment, the RKO is extracted from frozen krill or krill meal.

[0039] Reference will now be made to the accompanying drawings, in which: [Brief description of the drawings]

[0040] [Figure 1] FIG. 1 shows the correlation between the PL content of the phospholipid-containing fraction and the PL content of the krill oil starting material.

[0041] [Diagram 2] FIG. 2 shows the correlation between the yield of the final concentrated therapeutic phospholipid composition and the PL content of the krill oil starting material extract.

[0042] [Diagram 3] FIG. 3 shows the correlation between free fatty acid containing fraction yield and free fatty acid level in percent (FFA%) in the krill oil starting material.

[0043] [Figure 4] FIG. 4 shows the correlation between the yield of the final concentrated therapeutic phospholipid composition and the level of free fatty acid percentage (FFA%) in the krill oil starting material. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] According to the present specification, a method for extracting oil from krill is provided, wherein the resulting oil contains a high content of free fatty acids (FFA).

[0045] International Publication No. WO2011 / 050474, for example, discloses concentrated therapeutic phospholipid (PL) compositions comprising about 60% w / w phospholipids, which have surprising effects in the treatment of cardiovascular disease, particularly by lowering triglyceride (TG) levels. These concentrated therapeutic phospholipid compositions are produced from krill oil starting material as described herein.

[0046] The concentrated therapeutic phospholipid composition can be produced or generated by any method known in the art. However, the yield of the obtained concentrated therapeutic phospholipid composition is low and not economically viable. Also, this composition is produced using a unique method that allows the variability of krill to be controlled and the target specification of the therapeutic product to be consistently reached. Therefore, such compositions are subject to regulatory approval, so the production method needs to be consistent and efficient.

[0047] First, the oil containing the phospholipids can be separated from a natural source and can be further processed.

[0048] Thus, according to one embodiment, there is provided a method for producing a therapeutic enriched phospholipid composition comprising the steps of extracting raw krill oil (RKO) containing at least 7% free fatty acids (FFA) to obtain a phospholipid-rich fraction and an undesired layer, and separating said phospholipid-rich fraction from the undesired layer to produce an enriched phospholipid composition.

[0049] According to one embodiment, the concentrated phospholipid composition comprises: Formula (I): [ka] wherein R1 and R2 each independently represent, optionally, a docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA) residue; and Each X is independently selected from -CH2CH2NH3, -CH2CH2N(CH3)3°, and the following formula: [ka] A compound represented by the formula: - free EPA and free DHA (wherein the total free and bound EPA in the composition is at a concentration of 15% to 25% (w / w) and the total free and bound DHA in the composition is at a concentration of 10% to 15% (w / w)), and the total phospholipids in the composition are at a concentration of at least 50% (w / w (phospholipids / composition)).

[0050] Harvesting / storage techniques that can be used to increase the FFAs of the resulting raw krill oil (RKO) include, but are not limited to, the use of enzymes on freshly caught krill to store the krill for a period of time prior to processing and to allow hydrolysis of lipid levels by lipases naturally found in the krill.

[0051] The time and temperature during freezing of the krill and addition of enzymes are some of the techniques that can be used to increase the concentration of FFAs in frozen krill.

[0052] According to another embodiment, the frozen krill described and encompassed herein may be dried and / or cooked to produce a krill meal, such as one having increased levels of FFAs.

[0053] At this stage, the krill meal or frozen krill is stored. Differences in storage temperature and storage time may affect the level of FFAs in the resulting RKO. The actual extraction process can be carried out as described in International Application No. WO 2011 / 050474, the contents of which are incorporated herein by reference in their entirety, where frozen krill is mechanically ground and incubated with a solvent of 9:1 acetone:water ratio at 8°C for 60-90 minutes to extract different ratios of lipids (PL, TG, and FFA) from the krill biomass. Lipids are separated from protein and krill material by filtration at pressure (50-60 kpa). The solid phase is discarded. The soluble extract is evaporated under vacuum by successive distillation columns to remove the solvent (acetone). Most of the aqueous (water) fraction is separated from the lipid fraction by decantation, and the remaining water is removed by evaporation under vacuum and gentle heating. These fractions are dosed, analyzed and blended to constitute an intermediate krill oil product, which is reanalyzed to achieve the desired specifications resulting in the krill oil starting material or RKO. Enzymes can be added to increase the level of FFA concentration in the RKO.

[0054] Thus, according to one embodiment, the use of an RKO having an FFA content of greater than 7%, preferably 8%, and more preferably 15% is encompassed to produce an enriched phospholipid composition.

[0055] The bulk RKO is then further processed to produce a concentrated therapeutic phospholipid composition. This krill oil starting material can be further processed using countercurrent supercritical CO2 extraction to concentrate the composition and produce a concentrated therapeutic phospholipid composition. For example, countercurrent supercritical CO2 extraction at 70°C and 30 MPa can be used to remove certain biomolecules, such as all triglycerides, from the krill oil starting material. As more TG is removed from the bulk krill oil starting material, the concentration of phospholipids increases. Once TG is removed by this method, the phospholipid composition will be at a concentration of about 60-66% (w / w (phospholipids / composition)). Included herein is a method for producing a concentrated therapeutic phospholipid composition having a phospholipid concentration of up to about 90% (w / w (phospholipids / composition)).

[0056] Alternatively, as described in International Publication No. WO2019 / 218062, the contents of which are incorporated herein by reference in their entirety, the RKO is fractionated in a vessel already containing acetone under agitation. The RKO / acetone mixture is pumped into the vessel through a solvent-resistant filtration train including a prefilter with a porosity of 10 pm and a final filter with a porosity of 0.45 pm to provide a filtered mixture of RKO / acetone. Water is added to the filtered RKO / acetone mixture under constant agitation. The mixture is then allowed to settle in a processing vessel until an upper light phase (i.e., low density layer) and a lower heavy phase (i.e., high density layer) are observed (where the light phase is clear and a clean intermediate phase is observed between the light and heavy phases). Once this stage is reached, the heavy phase is transferred to another vessel and the volume is calculated. Half the volume of the heavy phase is then added to the vessel under constant agitation, followed by acetone, followed by the remaining half of the heavy phase being transferred to the same vessel. Agitation is continued and the resulting mixture is allowed to settle in a processing vessel until two phases are present (a low density light phase and a high density heavy phase). Here, the light phase is clear and a clean interphase is observed between the light and heavy phases. Once such settling is achieved, the heavy phase (containing the purified phospholipids) is transferred to the vessel. With constant agitation, absolute ethanol is added to the vessel as a viscosity reducing agent. A vitamin E preparation (α-tocopherol) is added to the heavy phase as an antioxidant. The mixture is then agitated to provide the phospholipid-rich fraction. The total time for the batch process from RKO to phospholipid-rich fraction is approximately 24 hours or less, but can be longer as needed to allow for more time for phase separation or to allow for larger size vessels.

[0057] Thus, according to one embodiment, the method includes fractionating the RKO into a first low-density layer rich in phospholipids and a first high-density layer; separating the first high-density layer rich in phospholipids from the first low-density layer; mixing the first separated high-density layer rich in phospholipids with an organic solvent comprising at least about 85% by weight of a C3-C8 ketone solvent relative to the mixture; fractionating the mixture into a second low-density layer and a second layer rich in phospholipids; separating the second layer rich in phospholipids from the second low-density layer to obtain a concentrated phospholipid composition. According to one embodiment, the organic solvent comprises at least about 85% by weight of a C3-C8 ketone solvent and an aqueous portion. Alternatively, the second layer rich in phospholipids can be combined with a stabilizer, a viscosity reducing agent, or a combination thereof to produce a phospholipid-rich fraction. For example, the method includes fractionating at least 100 kg of RKO as starting material. The RKO may be mixed with the first organic solvent in a ratio of about 5: about 15 by volume organic solvent:kg RKO. Further, according to one embodiment, the method includes heating the RKO to a temperature of about 20° C. to about 70° C. Also included is a combination of a phospholipid-rich second phase with a viscosity reducing agent. According to one embodiment, the stabilizer includes Vitamin E.

[0058] The process may be continued as follows: Preheated RKO (between 30°C-65°C) and acetone are fed using calibrated pumps where mixing of RKO and acetone is performed using a mixing pump. The resulting RKO / acetone mixture is filtered in-line through 10 pm and 0.45 pm pore size filters. The filtered RKO / acetone feed is sent to a static mixer along with a soft water feed where each feed is fed using calibrated pumps to ensure the flow rate of water and the flow rate of the filtered RKO / acetone feed. The static mixer is fed into a horizontal settler where the light and heavy phases (containing phospholipids) are continuously collected at the tip of the settler respectively. The horizontal settler is maintained at a temperature of 20-25°C.

[0059] The heavy phase can then be directed to a second static mixer to which a simultaneous feed of acetone is also directed. From the static mixer, the resulting mixture flows to a vertical settler maintained at a temperature of 20-25°C, where the vertical settler provides a light phase and a heavy phospholipid-containing phase. Each phase is continuously withdrawn from the vertical settler.

[0060] For storage, the phospholipid-rich fraction is transferred to a container under a nitrogen blanket, sealed, and stored at 2-8°C.

[0061] Thus, when the RKO material contains more than 7% FFA content (see Table 1), the yield of further processing to obtain the final desired enriched therapeutic phospholipid composition is at least 30%, preferably 30-60%. When a krill oil starting material containing 6.9% FFA was used (starting material #6, see Table 1), the yield of the final enriched therapeutic phospholipid composition was 48.8%, compared to 50.8% or 54.1% when the krill oil starting material contained 8% or 8.4% FFA (starting material #5 or 6, respectively, see Table 1). [Table 1]

[0062] As can be seen in Figure 1, there is a strong correlation between the phospholipid (PL)-containing fraction yield and the PL content of the krill oil starting material (RKO). The yield increases with increasing PL content.

[0063] On the contrary, no correlation was observed between the yield of enriched therapeutic phospholipid compositions and the PL content of RKOs in the range of PL evaluated (Figure 2).

[0064] On the other hand, there is a strong correlation between the yield of FFA-containing fractions and the FFA level (percentage) (FFA%) of the RKO: all parameters increase with increasing FFA% content of the krill oil starting material (Figure 3).

[0065] Within the tested range of FFA%, there is a modest correlation between the yield of concentrated therapeutic PL composition and FFA% (Figure 4).

[0066] Thus, to obtain an efficient yield of extraction of a concentrated therapeutic phospholipid composition, the minimum threshold to begin to obtain a satisfactory yield of a free fatty acid-containing fraction within specifications is about 7-8% FFA or higher. Ideally, the level of FFA in the starting material should be about 15%.

[0067] Thus, according to one embodiment, the use of RKO with FFA content of more than 7%, preferably 8%, more preferably 15% is included to produce the concentrated phospholipid composition. According to one embodiment, the total phospholipids in the concentrated phospholipid composition are at a concentration of 50% (w / w (phospholipids / total composition)), preferably at a concentration of 55% (w / w (phospholipids / total composition)), more preferably at a concentration of 60% (w / w (phospholipids / total composition)). According to one embodiment, the concentrated phospholipid composition comprises triglycerides at a concentration of less than about 5% (w / w). EXAMPLES

[0068] Example I Production of RKO with high levels of free fatty acids from frozen krill

[0069] Fresh krill is harvested using pumps or trawls, transported to fishing vessels and stored in collection wells. The krill is further washed, drained of excess water, moulded and frozen on board into frozen krill blocks. The frozen krill blocks are packaged and stored on board. The frozen krill is shipped to shore and stored until it is used later in the extraction process.

[0070] Oil is extracted from frozen krill by crushing the entire block, adding acetone to the crushed krill and transferring it to a tank under agitation. The mixture is stirred constantly for about 60 minutes and then passed through a filter to separate the krill residue.

[0071] The filtrate from the acetone solution containing the extracted oil is sent to a distillation column to remove the acetone from the solution and purify it.

[0072] The resulting mixture is composed of water and extracted krill oil. The mixture is sent to a settler where the krill oil floats on top of the aqueous phase. The oil is collected by overflow and transferred to an evaporator where residual acetone and water are removed. The dried krill oil is sent to a storage tank and further packed in drums for storage. The produced oil is further used as RKO in the preparation of concentrated phospholipids composition as described in Example II. Example II Production of RKO with high levels of free fatty acids from krill meal and further processing into concentrated therapeutic phospholipid compositions

[0073] Fresh krill is harvested using pumps or trawls, transported to the fishing vessel and stored in collection wells. The krill is further washed, drained of excess water, moulded and frozen on board the vessel into frozen krill blocks. The frozen krill blocks are packaged and stored on board the vessel. The frozen krill is sent to shore and stored until it is used later in the process.

[0074] The frozen krill blocks are crushed and pumped to a cooker where the crushed krill is boiled at 90-95°C. The boiled krill is transferred to a decanter centrifuge to remove excess moisture. The resulting solid phase is sent to a dryer where the moisture content of the solid phase is reduced to 5-15% and the dried solids are cooled to a temperature below 35°C, crushed to a reduced particle size, and transported to a bagging silo. It is packaged, sealed in bags, and stored at a temperature below -18°C until it is used later in the extraction process.

[0075] The krill meal is transferred to an extraction tank containing 95% ethanol and stirred to extract the oil. The mixture is stirred constantly for approximately 60 minutes and then passed through a filter to separate the krill residue from the ethanol solution containing the extracted oil.

[0076] The resulting solution is sent to an evaporator where the ethanol is removed. Then, absolute ethanol is added to the oil to remove proteins, salts, and impurities, and it is filtered through a 0.5 pm filter. The filtrate is sent to an evaporator where it is evaporated to dryness under vacuum. The dried oil is then sterilized and heated at 70°C for 30 minutes.

[0077] The krill oil is further diluted with 3 L of 95% ethanol per kg of oil. The mixture is incubated at -20°C for 2 hours. After incubation, the mixture is allowed to return to room temperature and two layers form. The top layer is enriched in phospholipids and the bottom layer is enriched in triglycerides.

[0078] The upper layer is collected and sent to a horizontal thin film evaporator. The mixture is forced against the heated walls (105°C) of the evaporation chamber, and a countercurrent nitrogen flow enhances the drying efficiency. To ensure a stable drying performance of the device, the temperature of the composition (NMT 100°C) is recorded near the desired concentrated composition outlet.

[0079] The dried concentrate composition is collected in molds, cooled, demolded, and cut into approximately 1 Kg pieces. The pieces are wrapped in LDPE bags, wrapped in Mylar bags, sealed, and placed in fiber drums. Once labeled, the drums are stored in a cold room at 2-8°C as QC for testing. Example III Production of RKO with high levels of free fatty acids by enzymatic treatment and further processing into concentrated therapeutic phospholipid compositions

[0080] Fresh krill is harvested using pumps or trawls, transported to fishing vessels and stored in collection wells, where it is further washed and drained of excess water and sent to a feed tank.

[0081] From the feed tank, the krill is pumped to a cooker where it is boiled at 90-95 °C. The boiled krill is transferred to a decanter centrifuge to remove excess water. The resulting solid phase is sent to a dryer where the moisture content of the solid phase is reduced to 5-15%. The dried solids are cooled to a temperature below 35 °C, crushed, resulting in a reduced particle size, transported to a bagging silo, packaged, sealed in bags and stored at room temperature on board the ship. The bags of krill meal are brought to shore and stored at temperatures below -18 °C until used later in the extraction process.

[0082] The krill meal is transferred to an extraction tank containing 95% ethanol and stirred to extract the oil. The mixture is stirred constantly for approximately 60 minutes and then passed through a filter to separate the krill residue from the ethanol solution containing the extracted oil.

[0083] The resulting solution is sent to an evaporator where the ethanol is removed. Then absolute ethanol is added to the oil to remove proteins, salts and impurities, and it is filtered through a 0.5 pm filter. The filtrate is sent to an evaporator where it is evaporated to dryness under vacuum.

[0084] Lipolytic enzymes and water are added to the oil to carry out controlled enzymatic hydrolysis of the oil to produce free fatty acids. After the reaction, the oil is heated to inactivate the enzymes and stop the enzymatic reaction. The oil is further sent to an evaporator where it is evaporated to dryness under vacuum. The krill oil is then packaged and stored.

[0085] Krill oil is fed into a supercritical CO2 extraction system. CO2 is introduced into the system. When the extractor conditions reach the specified operating temperature and pressure, a stream of supercritical CO2 is passed through the oil. The heated and pressurized supercritical CO2 extracts the triglyceride-enriched oil and concentrates the phospholipids remaining in the extraction vessel. The supercritical CO2 and extracted triglyceride-enriched oil enter a separator, which operates at sufficient pressure and temperature (900 psi and 45°C) to flash the CO2 and precipitate the extracted triglyceride-enriched oil fraction from the gas. The separator contents are periodically discharged and metered. The gas continues in a recycle loop through the extraction vessel until the process reaches the established and calculated extraction endpoint range.

[0086] Once the extraction endpoint range is reached, the phospholipid-enriched material remaining in the extractor is discharged directly into a pre-weighed pail. The amount of product collected is weighed, an analytical sample is taken, and the bucket is labeled. The bucket is stored in a cold room at 2-8°C as QC for testing.

Claims

1. 1. A method for producing a therapeutic enriched phospholipid composition comprising the steps of: - treating the krill biomass to increase the content of free fatty acids (FFA); - Extracting Raw Krill Oil (RKO) containing at least 7% Free Fatty Acids (FFA) from the treated krill biomass and obtaining a phospholipids enriched fraction and an unwanted layer; and - separating said phospholipid-enriched fraction from said unwanted layer; The method comprising:

2. 2. The method of claim 1, wherein the therapeutic enriched phospholipid composition is produced in a yield of at least 30%.

3. 10. The method of claim 1, wherein said therapeutic enriched phospholipid composition is produced in a yield of 30% to 60%.

4. The method of any one of claims 1 to 3, wherein the total phospholipids in the composition are present in a concentration of at least 50% (w / w (phospholipids / composition)).

5. 5. The method of any one of claims 1 to 4, wherein the composition comprises free and bound docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), the total free and bound EPA in the composition being present in a concentration of 15% to 25% (w / w), and the total free and bound DHA in the composition being present in a concentration of 10% to 15% (w / w).

6. The therapeutic concentrated phospholipid composition has the following formula (I): 【Chemistry 1】 [In the formula, R １ and R ２ each independently optionally represents a docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA) residue; and Each X is independently -CH ２ CH ２ N.H. ３ , -CH ２ CH ２ N (CH ３ ) ３ and the following formula: 【Chemistry 2】 The method according to any one of claims 1 to 5, comprising a compound represented by the formula:

7. The method of any one of claims 1 to 6, wherein the RKO comprises at least 15% FFA.

8. The RKO is countercurrent supercritical CO ２ The method according to any one of claims 1 to 7, wherein the fractionation is carried out by extraction.

9. 8. The method of any one of claims 1 to 7, wherein the RKO is fractionated into a first low density layer and a first high density layer that is enriched in phospholipids; separating the first high density layer that is enriched in phospholipids from the first low density layer; mixing the first separated high density layer that is enriched in phospholipids with an organic solvent comprising at least 85% by weight of a C3-C8 ketone solvent to form a mixture; fractionating the mixture into a second low density layer and a second layer that is enriched in phospholipids; and separating the second layer that is enriched in phospholipids from the second low density layer to obtain an enriched phospholipid composition.

10. 10. The method of claim 9, wherein the concentrated phospholipid composition is mixed with one or more free fatty acids.

11. 11. The method of claim 9 or 10, further comprising combining the phospholipid-rich second phase with a stabilizer, a viscosity reducing agent, or a combination thereof to produce a phospholipid-rich fraction.

12. 11. The method of claim 9 or 10, comprising fractionating at least 100 kg of RKO.

13. 12. The method of any one of claims 9 to 11, comprising mixing the RKO and organic solvent in a ratio of about 5:about 15 by volume organic solvent:kg RKO.

14. 14. The method of claim 13, comprising heating the RKO to a temperature of about 20° C. to about 70° C. prior to combining the RKO and the organic solvent.

15. The method of any one of claims 9 to 14, further comprising passing the mixture of RKO and organic solvent through a filter.

16. 16. The method of claim 15, wherein the filter is a 0.45 μm filter.

17. The method of any one of claims 9 to 16, further comprising passing the mixture of RKO and organic solvent through a first filter and a second filter.

18. 18. The method of claim 17, wherein the first filter is a 10 pm filter and the second filter is a 0.45 pm filter.

19. The method of any one of claims 9 to 18, further comprising combining the phospholipid-rich second layer with a viscosity reducing agent.

20. 20. The method of claim 19, wherein the phospholipid-rich second layer is combined with a viscosity-lowering agent in a ratio of about 0.1:about 0.

3.

21. The method of claim 11 , wherein the stabilizer comprises Vitamin E.

22. The method according to any one of claims 1 to 21, which is a continuous method.

23. 23. The method of any one of claims 1 to 22, wherein the total phospholipids in the composition are at a concentration of 55% (w / w (phospholipids / total composition)).

24. 23. The method of any one of claims 1 to 22, wherein the total phospholipids in the composition are at a concentration of 60% (w / w (phospholipids / total composition)).

25. The method of any one of claims 1 to 24, wherein the composition comprises triglycerides at a concentration of less than 5% (w / w).

26. 26. The method of any one of claims 1 to 25, wherein the RKO is extracted from frozen krill or krill meal.

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