Enzymatically synthesized OMEGA-3 and OMEGA-6 structured polar lipids

Abstract

The present application provides a method of incorporating omega-3 fatty acids, such as EPA / DHA, into polar lipid molecules present in lecithin, the method comprising: (a) performing an enzymatic exchange reaction between the fatty acids present in the polar lipids of lecithin and omega-3 fatty acids present in concentrated fish oil to obtain an oil having a high content of polar lipids and omega-3 fatty acids, and (b) a step of concentrating the polar lipid content of the oil obtained in step a by supercritical fractionation or molecular distillation.

Description

[0001] This application claims priority to U.S. non-provisional application No. 17 / 951,048, filed on September 22, 2022. This application also claims the benefit of priority under 35 U.S.C. §119 to U.S. Provisional Patent Application No. 63 / 246,898, filed on September 22, 2021, entitled “Enzymatically Synthesized Omega-3 Structured Phospholipids”; the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present application relates to the production of phospholipid and glycolipid preparations rich in omega-3 and omega-6 fatty acids. The phospholipid and glycolipid preparations rich in omega-3 and omega-6 produced by the method of the present application can be used as a nutraceutical or nutraceutical additive for a functional food or pharmaceutical composition. The present application also relates to an enzymatic method for preparing phospholipids in which omega-3 and omega-6 fatty acids are incorporated. Background Art

[0003] Phospholipids are an important class of biomolecules. Phospholipids are basic building blocks of cell membranes and are a major part of surfactants, the thin films that occupy the air / liquid interface in the lungs. These molecules consist of a polar or charged head group and a pair of nonpolar fatty acid tails connected by a glycerol bond. This combination of polar and nonpolar segments is called amphipathic, a term that describes the tendency of these molecules to assemble at the interface between polar and nonpolar phases.

[0004] The term glycerophospholipid refers to any derivative of glycerophosphoric acid containing at least one O-acyl, or O-alkyl, or O-alk-1'-enyl residue attached to a glycerol moiety. The phosphate group forms an ester bond with the glycerol. In bacteria / eukaryotic organisms, long chain hydrocarbons are usually linked by ester bonds.

[0005] In bacteria and prokaryotes, lipids are usually composed of C 16 or C 18 The diesters of fatty acids are straight chain, especially for C 18 members, which may be unsaturated. The hydrocarbon chain attached to the glycerol is hydrophobic, while the polar head (primarily composed of a phosphate group attached to the third carbon of the glycerol backbone) is hydrophilic. This dual nature makes glycerophospholipids amphipathic. They are usually organized into a bilayer in the membrane, with the polar hydrophilic head sticking outward toward the aqueous environment and the nonpolar hydrophobic tail pointing inward. Glycerophospholipids are composed of a variety of different substances, usually with slightly different structures. The most basic structure is phosphatidic acid. This substance is an important intermediate in the synthesis of many phosphoglycerides.

[0006] Typically, glycerophospholipids use the "sn" notation, which stands for stereospecific numbering. When the letters "sn" appear in the nomenclature, by convention, the hydroxyl group of the second carbon of glycerol (2-sn) is on the left side of the Fischer projection. The numbering follows one of the Fischer projections, with the top carbon being 1-sn and the bottom carbon being 3-sn. The advantage of this particular notation is that the spatial configuration (D or L) of the glycerol molecule is intuitively determined by the residues at positions sn-1 and sn-3. For example, sn-glycerol-3-phosphate and sn-glycerol-1-phosphate are enantiomers. Most vegetable oils have unsaturated fatty acids at the sn-2 position and saturated fatty acids at the 1-sn and / or 3-sn positions. Animal fats typically contain saturated fatty acids in the 2-sn and unsaturated fatty acids at the 1-sn and / or sn3 positions.

[0007] Phospholipids containing polyunsaturated fatty acids (PUFA) provide important building blocks for living organisms and improve membrane fluidity, an important property for biological membrane function.

[0008] Studies on PUFA-containing phospholipids have shown that these biomaterials have many important physiological roles. They are high-energy, basic, structural and functional elements of all biological membranes such as cells, blood cells, lipoproteins and surfactants. In addition, they are indispensable for cell differentiation, proliferation and regeneration, maintaining and promoting the biological activities of many membrane-bound proteins and receptors. PUFA-containing phospholipids also play a decisive role in the activity and activation of many membrane-localized enzymes such as sodium-potassium ATPase, adenylate cyclase and lipoprotein lipase, which are important for the transport of molecules through membranes and the control of membrane-dependent metabolic processes between intracellular and intercellular spaces. In addition, some PUFAs, such as linoleic acid, are precursors of cell-protective prostaglandins and other eicosanoids.

[0009] Due to all their properties, many health benefits have been attributed to the consumption of fatty acids, especially PUFAs. For example, omega-3 and omega-6 type PUFAs have been reported to be effective in the treatment and prevention of cardiovascular disease (CVD).

[0010] In addition to its benefits against cardiovascular disease, diabetes and cancer, DHA is also important for enhancing brain function, especially for infant brain development. Nutritional studies have investigated the importance of DHA in the brain and found that low levels of DHA are associated with depression, memory loss, dementia and vision problems. All studies have shown that brain function in older adults improves significantly with increased levels of DHA in the blood.

[0011] The human body is unable to synthesize DHA in sufficient quantities. Therefore, it is necessary to obtain it from the diet. DHA is initially obtained through the placenta, then from breast milk, and later from sources such as fish, red meat, animal organ meats, and eggs. These types of fatty acids are primarily found naturally in fish and algae, randomly distributed in the s7 1, s7x-2, and sn-3 positions of the glycerol backbone of triglycerides. In particular, tuna, salmon, and sardines provide a rich source of DHA.

[0012] Additionally, the ability to enzymatically produce the omega-6 and omega-3 products of linoleic and alpha-linolenic acids declines with age. Therefore, as humans age, there is an increasing need to obtain DHA directly from the diet or from supplements. Because DHA is important for signaling in the brain, eyes, and nervous system, many consumers concerned about maintaining mental acuity seek a pure, safe way to replenish their DHA levels. Until recently, the primary source of DHA dietary supplements has been fish oil.

[0013] In view of the important physiological role of PUFA-containing phospholipids in human health and the scarcity of said compounds in organisms, there is a need for dietary supplementation of PUFA-containing phospholipids. Many PUFA-containing agents have stability and quality problems due to the high degree of oxidation of polyunsaturated fatty acids. These problems require the incorporation of antioxidants and the use of special measures that attempt to reduce this oxidation. Due to the antioxidant properties of phospholipids, the use of phospholipids as carriers of PUFAs can improve the stability of such products.

[0014] PUFA-containing phospholipids can be prepared in a variety of ways, primarily by (i) enzymatic esterification and transesterification of phospholipids, (ii) chemical synthesis of phospholipids, or (iii) enzymatic transphosphatidylserination of phospholipids.

[0015] It is worth mentioning that PS is the main acidic phospholipid component of the brain membrane. It has been the subject of many human clinical trials on memory loss, mood, cognitive performance and learning ability. Many studies have shown that PS can help patients with age-related memory impairment and even help optimize cognition in patients without cognitive impairment. Dietary PS is effectively and rapidly absorbed into the blood in the intestine and easily crosses the blood-brain barrier to reach the nerve cells of the brain.

[0016] PS can be extracted from bovine brain or plants, or produced by biocatalysis from soy lecithin. The main difference between the two sources is the type of fatty acids attached to positions 1 and 2 on the phospholipid backbone. Long-chain polyunsaturated n-3 fatty acids are characteristic of marine fats and are prevalent in phospholipids of marine species.

[0017] Phosphatidylserine can be prepared by transphosphatidylation reaction using phospholipase D (PLDs), by which the head groups of phospholipids can be easily modified. Thus, phosphatidylserine can be produced from phosphatidylcholine or any other phospholipid mixture and serine by PLD catalysis.

[0018] Phosphatide is also referred to as lecithin (e.g., as soybean lecithin and egg yolk lecithin), and has long been used in food, cosmetics, paints, lubricants, magnetic materials, animal feed, and pharmaceutical and agrochemical products. In this regard, the phosphatidyl acid derivatives produced by enzymatic transphosphatidyl transfer of phosphatidylcholine and compounds containing hydroxyl groups have shown in some cases a performance superior to that of the starting material.

[0019] The structure of the most common class of phospholipids (phosphoglycerides) is based on glycerol (a three-carbon alcohol with the molecular formula CH2OH-CHOH-CH2OH). Two fatty acid chains (each usually having an even number of carbon atoms from 14 to 20) are attached (by double esterification) to the first and second carbons of the glycerol molecule, denoted as the sn1 and sn2 positions, respectively. The third hydroxyl group of glycerol (at the sn3 position) reacts with phosphoric acid to form phosphatidic acid esters. Common phospholipids that are widely distributed in nature are produced by further reaction of the phosphate group in phosphatidic acid esters with alcohols (such as serine, ethanolamine, choline, glycerol, or inositol). The resulting lipids can be charged, such as phosphatidylserine (PS), phosphatidylinositol (PI), and phosphatidylglycerol (PG); or bipolar (having separate positively and negatively charged regions), such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The term "lecithin" refers to PC-type lipids.

[0020] A typical phospholipid arrangement is a saturated fatty acid at the sn1 position, such as palmitic acid or stearic acid, and an unsaturated or polyunsaturated fatty acid at the sn2 position, such as oleic acid or arachidonic acid (see the structure of phosphoglycerides for details). Figure 1 ).

[0021] Another class of phospholipids is sphingolipids. Sphingolipid molecules have the above-mentioned phosphatidyl-based head group structure, but (in contrast to ordinary phospholipid molecules) contain a single fatty acid and a long-chain alcohol as their hydrophobic components. In addition, the backbone of sphingolipids is sphingosine, which is an amino alcohol (rather than glycerol). The structure of a representative sphingolipid (sphingomyelin) is also shown in Figure 1 Sphingolipids are primarily found in neural tissue and are thought to form cholesterol-rich domains within lipid bilayer membranes that may be important for the function of certain membrane proteins.

[0022] Phospholipids have multiple functions in biological systems: as fuels, as membrane structural elements, as signaling agents, and as surfactants. For example, pulmonary surfactant is a mixture of lipids (primarily dipalmitoylphosphatidylcholine [DPPC]) and proteins that controls the surface tension of the inner lung fluid lining (the site of gas exchange), allowing this lining to expand and contract rapidly during the respiratory cycle. Phospholipids are the major lipid component of cell membranes, maintaining the structural integrity between the cell and its environment and providing boundaries between compartments within the cell.

[0023] Furthermore, glycolipids are lipids with carbohydrates attached via glycosidic (covalent) bonds. Their role is to maintain the stability of the cell membrane and facilitate cell recognition, which is essential for immune responses as well as the connection of cells to each other to form tissues. Glycolipids are present on the surface of all eukaryotic cell membranes, where they extend from the phospholipid bilayer to the extracellular environment.

[0024] Glycolipids include a variety of compounds: glycosphingolipids (cerebrosides, erythroglycosides, gangliosides, sulfatides, etc.), glyceroglycolipids, glycophosphospholipids (e.g., phosphatidylinositol), glycosylated prenols (e.g., dolichol-phosphoglycans), glycosylated sterols, glycosylated polyketides, and glycolipids.

[0025] The essential characteristic of glycolipids is the presence of monosaccharides or oligosaccharides bound to the lipid portion. The most common lipids in cell membranes are glycerolipids and sphingolipids, which have a glycerol or sphingosine backbone, respectively. Fatty acids are attached to this backbone, so that the lipid as a whole has a polar head and a nonpolar tail. The lipid bilayer of the cell membrane consists of two layers of lipids, the inner and outer surfaces of the membrane consisting of polar head groups and the inner part of the membrane consisting of nonpolar fatty acid tails.

[0026] Sugars attached to polar head groups on the outside of the cell are the ligand components of glycolipids and are also polar, allowing them to dissolve in the aqueous environment surrounding the cell. Lipids and sugars form glycoconjugates via glycosidic bonds, which are covalent bonds. The anomeric carbon of the sugar is attached to a free hydroxyl group on the lipid backbone. The structure of these sugars varies depending on the structure of the molecule to which they are attached.

[0027] The main function of glycolipids in vivo is to serve as recognition sites for cell-cell interactions. The sugars of the glycolipids will bind to specific complementary carbohydrates or lectins (carbohydrate binding proteins) of neighboring cells. The interaction of these cell surface markers is the basis for cell recognition and initiates cellular responses that contribute to activities such as regulation, growth, and apoptosis.

[0028] Glycolipids can modulate membrane organization. They are potential regulators of membrane physical properties. They have a number of features that distinguish them from phospholipids: (i) the ability of the hydroxylated sugar head groups to participate in extensive hydrogen bonding as donors and acceptors, (ii) long, largely saturated acyl chains that promote ordering and interdigitation, and (iii) very high main phase transition temperatures compared to the corresponding phospholipids, which also supports the idea that glycolipids promote order in their surroundings. Glycolipids form dense lateral domains together with cholesterol and / or sphingomyelin. Glycolipids play a major role in cell membranes. As lipids, they are components of lipid membranes and their physical properties determine in part the functions of membrane proteins embedded in the membrane.

[0029] Representative examples of glycolipids are Figure 2 shown.

[0030] The present application provides improved and more cost-effective methods for producing omega-3 / omega-6 rich glycerophospholipids and glycolipids. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The structures of phosphoglycerides and sphingomyelin with fatty acids are shown.

[0032] Figure 2 The chemical structures of glycolipids, glyceroglycolipids, and glycosphingolipids are shown.

[0033] Figure 3 is a schematic diagram of the batch process of the present application.

[0034] Figure 4 It is a schematic diagram of the continuous process of the present application.

[0035] Figure 5 A flow chart of the present application is shown.

[0036] Figure 6 The phospholipid process flow of the present application is shown.

[0037] Figure 7 The enzymatic incorporation of EPA and DHA into phospholipids to form concentrated phospholipids is structurally described.

[0038] Figure 8 The final product of the present application is shown treated with the antioxidant astaxanthin.

[0039] Purpose of the Invention

[0040] Therefore, an object of the present application is to provide an improved solvent-free enzymatic transesterification method for enriching phospholipids with omega-3 and omega-6 fatty acids. Transesterification includes transesterification and esterification of lecithin with omega-3 fatty acids and omega-6 fatty acids.

[0041] Another object of the present application is to provide a solvent-free enzymatic process for producing phosphatidylcholine, phosphatidylinositol, phosphatidylserine and phosphatidylethanolamine enriched in omega-3 and omega-6.

[0042] Another object of the present application is to provide a method for producing a stabilized phosphatidylserine preparation enriched with omega-3 / omega-6 acid residues. In the method proposed herein, the production is a solvent-free enzymatic incorporation of omega-3 and omega-6 fatty acids by a simple single step reaction, which can be easily carried out on an industrial scale.

[0043] These and other objects of the present application will become apparent from the foregoing description. Summary of the invention

[0044] The present application provides a method for modifying a phospholipid material, which comprises exchanging acyl groups in a phospholipid by enzymatically exchanging omega fatty acids with ethyl esters of triacylglycerols containing omega-3 fatty acids, wherein the reaction is carried out in the absence of a solvent with an enzymatic system consisting essentially of one or more phospholipases at a sufficient temperature and for a sufficient time to exchange the acyl groups in the phospholipid material and form a modified phospholipid material.

[0045] The present application is characterized by a method for incorporating omega-3 and omega-6 fatty acids into polar lipid molecules present in phospholipids and glycolipids, the method comprising the following steps: (a) an enzymatic exchange reaction between fatty acids in polar lipid molecules present in phospholipids and glycolipids and omega-3 and omega-6 fatty acids to obtain an oil with a high content of polar lipids and omega-3 and omega-6 fatty acids, (b) a stage of concentrating the polar lipids and omega-3 and omega-6 fatty acid content of the oil obtained in step (a) by supercritical fractionation or molecular distillation; (c) a new feed stage of concentrated omega-3 oil or omega-6 to increase the incorporation of omega-3 and omega-6 in polar lipids and achieve a higher omega-3 content and omega-6 content in the product; (d) an ultrafiltration stage carried out under a nitrogen atmosphere to give the final oil the desired viscosity and appearance.

[0046] The present application also relates to a method for incorporating EPA / DHA into polar lipid molecules present in lecithin, the method comprising the following steps: (a) an enzymatic exchange reaction between fatty acids present in the polar lipids of lecithin and omega-3 fatty acids present in concentrated fish oil or algae oil to obtain an oil with a high content of polar lipids and omega-3 fatty acids; (b) a stage of concentrating the polar lipids and omega-3 fatty acid content of the oil obtained in step (a) by supercritical fractionation or molecular distillation; (c) a new feed stage of concentrating Omega-3 oil (fish oil or algae oil) to increase the incorporation of Omega-3 into polar lipids and achieve a higher omega-3 content in the product; and (d) an ultrafiltration stage carried out under a nitrogen atmosphere to give the final oil the desired viscosity and appearance.

[0047] The present application also relates to a method for producing glycerophospholipids enriched in omega-3 and / or omega-6 fatty acids by enzymatic transesterification, comprising the following steps: (a) reacting the glycerophospholipids with an omega-3 and / or omega-6 fatty acid source in an ultrasonic reactor in the presence of a lipase and / or phospholipase (the lipase and / or phospholipase is capable of catalyzing the transesterification at the sn-1 and / or sn-2 positions of the glycerol part) for a suitable period of time to obtain glycerophospholipids enriched in the sn-1 and / or sn-2 positions of the omega-3 and / or omega-6 fatty acids; (b) removing and filtering the upper layer containing the enriched glycerophospholipids to separate the glycerophospholipids from the enzyme; and (c) deoiling the filtrate using a supercritical fluid to remove excess FFA.

[0048] The present application also provides soft capsules incorporating EPA / DHA-enriched phospholipids, wherein the phospholipids are prepared by enzymatic transesterification of phospholipids containing saturated fatty acids in an ultrasonic reactor. DETAILED DESCRIPTION OF THE INVENTION

[0050] The present application provides a method for preparing glycerophospholipids rich in omega-3 and / or omega-6. Such methods are essentially methods for enzymatic transesterification and esterification of glycerophospholipids, chemical synthesis, and enzymatic production of phosphatidylserine and other related analogs.

[0051] The present application provides an improved enzymatic transesterification method for enriching phospholipids with omega 3 and omega 6 fatty acids. Transesterification includes a transesterification process and an esterification process of lecithin with omega-3 and omega 6 fatty acids.

[0052] Applicants have developed synthetic pathways that enable the industrial production of the above-mentioned phospholipids, which have unique nutritional and clinical benefits.

[0053] The synthetic pathways described in this article can be divided into two major categories:

[0054] 1. Enzymatic esterification and transesterification of phospholipids with omega-3 and / or omega-6 fatty acids using lipase and phospholipase.

[0055] 2. Chemical esterification of phospholipids with omega-3 and omega-6 acyl donors.

[0056] The method according to the present application is characterized in that the reaction is carried out in the absence of a solvent using an enzyme system consisting of one or more phospholipases. The phospholipases may be immobilized.

[0057] The method of the present application can be applied to any desired type of phospholipids or glycolipids or sphingomyelins containing fatty acid acyl ester groups. The example of this naturally occurring phospholipid is lecithin, phosphatidic acid, phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine and diphosphatidylglycerol. Synthetic phospholipids, 1-alkyl-2-acyl-phospholipids and diacyl-phospholipids with various hydroxy compounds esterified to phosphate groups can also be processed.

[0058] The exchange reaction can be used to incorporate any desired fatty acid moiety into phospholipids. Of particular interest are omega-3 fatty acids. All oils and fats containing large amounts of these fatty acid moieties incorporated into triglycerides can be used as starting materials.

[0059] The method of the present application comprises first preparing ethyl esters (EE) concentrated in omega-3 (mainly EPA / DHA) by molecular distillation concentration of transesterified fish oil. Then, a treatment process is performed in a column filled with silica to reduce saturated, monounsaturated and 18-carbon fatty acids that affect the incorporation process in the subsequent enzymatic reaction process.

[0060] Once saturated fatty acids and C 18 Low-content ethyl ester oil can be chemically reacted with alkali (potassium hydroxide, sodium hydroxide or others) to saponify fatty acids, and then split with acid (citric acid, sulfuric acid, p-toluenesulfonic acid, etc.) to obtain free fatty acids (FFA). In addition, EE oil can be enzymatically esterified using glycerol and lipase as catalysts to obtain omega-3 concentrated triglycerides (TG).

[0061] The next stage consists of an enzymatic reaction in which the feed is fish oil concentrated in omega-3 in the chemical form EE, FFA or TG with lecithin with a high content of polar lipids: phospholipids and glycolipids, which can be of soy, rapeseed, sunflower, egg or other plant or animal origin. Considering that the chemical forms of omega-3 vary, several reaction mechanisms need to be considered:

[0062] 1. Hydrolysis to decompose fatty acids present in phospholipid / glycolipid molecules, followed by esterification with n-3 molecules from FFA, EE or TG.

[0063] 2. The omega-3 fatty acids from EE undergo transesterification with fatty acids at the SN-1 or SN-2 position of the phospholipid molecule or with fatty acids present in glycolipid molecules.

[0064] 3. The omega-3 fatty acids from TG undergo transesterification with fatty acids at the SN-1 or SN-2 position of the phospholipid molecule or with fatty acids present in the glycolipid molecule.

[0065] The above enzymatic reaction is carried out in an ultrasonic reactor, which is beneficial to surface contact and material transfer, and compared with traditional mechanical stirring, a better yield, a high omega-3 fatty acid incorporation rate and a shorter reaction time are obtained.

[0066] The possibility of using different chemical forms in fish oil essences allows diversification of the reaction mechanisms used in the incorporation process. In turn, products with different organoleptic properties are obtained, thus providing options for different pharmaceutical and nutraceutical applications as finished products.

[0067] The operating conditions of the reaction are:

[0068] -Liquid enzyme: 0.5-10% p / p

[0069] - Temperature: 30-80℃

[0070] -Pressure: 0.01-1013mbar

[0071] -Time: 1-48 hours

[0072] - Water content: 0.1-1%

[0073] -pH: 2-9

[0074] - Phospholipid content (PL): 20-60%

[0075] - Glycolipid content (GL): 2-10%

[0076] The incorporation of omega-3 fatty acids can be carried out at the SN-1 and SN-2 positions of the phospholipid molecules, or by replacing the fatty acids present in the glycolipid molecules. This process can be achieved by using the liquid enzymes phospholipases A1 and A2, respectively, which can be added independently (two reaction stages) or in a single dose (one reaction stage).

[0077] The use of liquid enzymes avoids the need for a filtration process after the enzymatic reaction to recover the enzyme for the next batch. It is worth mentioning that the implementation of the above-mentioned physical separation process requires special conditioning of the oil to achieve the correct treatment of the phospholipids. The high molecular weight of the phospholipids and the viscosity of the reaction mixture can cause clogging problems, product losses and long processing times in filtration, and the product must be heated to improve its fluidity; however, in particular, polyunsaturated fatty acids and EPA / DHA are thermolabile, so heating without appropriate treatment measures can cause saturated oxidation of fatty acids, thereby degrading the molecules. In a second measure, solvents can be used to increase the viscosity of the reaction mixture, however, a new stage should be added afterwards in which the solvent is evaporated and recovered from the structured phospholipids. The last option will generate two disadvantages, one from an economic point of view, since new equipment must be incorporated into the process and its operation is reflected in higher production costs. On the other hand, it must be guaranteed that the reaction mixture after evaporation does not contain traces of solvent, since, depending on the type of solvent used, it can have adverse effects on the health of the final consumer.

[0078] Lecithin contains a mixture of glycolipid, triglyceride and phospholipid (for example phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol), and can be derived from natural sources or synthetic sources. Preferably, the lecithin used in the present application's method is derived from plant sources such as soybean, egg yolk or rapeseed, using conventional processing methods to obtain. The phospholipid content of the lecithin used in the present application's method is preferably greater than 15 weight %, more preferably greater than 25 weight %, for example greater than 30 weight %. For example, the phospholipid content of lecithin can be 25 to 60 weight %, for example 30 to 40 weight %.

[0079] Commercial lecithin is an important by-product of the oil processing obtained in the degumming step. For example, soybean lecithin is a complex mixture, comprising phosphatides and triglycerides, and a small amount of other ingredients, such as plant glycolipids, phytosterols, tocopherols and fatty acids. The main phospholipids present in plant lecithin are phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol. Egg yolk lecithin contains phosphatidylcholine and phosphatidylethanolamine as main phospholipids. Lecithin has the potential as a multifunctional additive for food, medicine and industrial applications. The main use of lecithin in food is as an emulsifier.

[0080] Lecithin has the following chemical structure:

[0081]

[0082] In the method of the present application, the fatty acid portion of lecithin is substituted with omega-3 fatty acid residues such as docosahexaenoic acid and eicosapentaenoic acid.

[0083] The process mentioned in the patent is solvent-free. In other inventions, the use of solvents such as aniline, amines or ethanolates involves increasing the incorporation of polyunsaturated fatty acids in a shorter reaction time. However, since many of the ingredients are considered toxic and harmful to human health (even in small concentrations), the use of these solvents results in the effective evaporation of these ingredients.

[0084] Thus, the process of the present application has several advantages over conventional processes for fractionation of phospholipids, which generally use acetone. Some of these are: i) a product free of organic solvents, ii) elimination of a solvent recovery stage, iii) a drastic separation between phospholipids and oil, iv) creation of a sterile environment, v) deodorization of oil and phospholipids, vi) reduced oxidation and vii) short residence time.

[0085] Depending on the chemical form of the fish oil concentrated in omega-3, the post-treatment process is carried out. For example, if starting from FFA and EE, once the polyunsaturated fatty acids are incorporated into the phospholipid / glycolipid molecules, multi-stage molecular distillation can be carried out to increase the polar lipid content from 40% to 90%, separating them from FFA and EE due to the difference in volatility and molecular weight of these two molecules relative to polar lipids. On the other hand, if the raw material is in TG form, the concentration process will be based on the difference in solubility of TG and polar lipids in the fluid under supercritical conditions (supercritical fractionation). Molecular distillation is abandoned due to the high temperatures required to separate these two high molecular weight molecules. For supercritical fractionation, the use of carbon dioxide (CO2) is considered as a supercritical fluid due to its low environmental impact, high availability, easy recovery and evaporation of the target product, low cost and recognition as GRAS (Generally Recognized as Safe).

[0086] The method of the present application uses two types of systems to concentrate polar lipids and omega 3 fatty acids: a batch system and a continuous system. The process flow of the present application is as follows: Figure 5 and Figure 6 shown.

[0087] Batch Process

[0088] exist Figure 3In the figure, numeral 1 indicates an extractor, which can be loaded with rigid fillers such as ceramic, glass or metal marbles 2 to minimize the aggregation of phospholipids and promote deoiling in the extraction process. Ruby-O in the form of ethyl ester or triglyceride is added to the extractor, but ethyl ester is preferred because of its higher solubility in supercritical CO2 (SC-CO2). Ruby-O usually consists of 40-50% acetone insolubles (AI). The CO2 flow rate is first regulated by bypassing the extractor and the stop valve 12 through a two-way switching valve 11. Once a stable flow rate is achieved, supercritical CO2 is supplied to the extractor from the bottom by switching the two-way valve 11 and opening the manual valve 12, with a temperature of 40°C to 80°C and a pressure of 10MPa to 70MPa. The solvent passes through the extraction chamber 1, is discharged from the top, and is loaded with oil. The SC-CO2 is expanded to a pressure of 5MPa to 7MPa in the back pressure regulating valve 3 and passes through a water heating bath 4 before entering the separation container 5.

[0089] The separation vessel 5 has two outlets, one at the top, where the CO2 gas leaves without oil and enters the condenser 6 and the high-pressure reciprocating pump 7. Liquid CO2 is pumped at the extraction working pressure and heated to the working temperature in a water bath 8. The CO2 leaves the heater 8 in a supercritical state and is fed back to the extractor 1. The oil extracted from the Ruby-O is discharged from the separation vessel 5 through a manual valve 10. The CO2 dissolved in the oil lost during the discharge is replaced by CO2 supplied from the gas deposit 9 before the condenser 6.

[0090] Continuous process

[0091] The process of continuous extraction of oil from Ruby-O is as follows Figure 4As shown. Herein, Ruby-O is preheated to 30°C to 80°C in a storage tank 15 and pumped to a pressure higher than the extraction pressure by a reciprocating pump 16. Ruby-O is mixed with supercritical CO2 and delivered through a nozzle 14 to produce a highly dispersed Ruby-O jet, thereby increasing the area available for mass transfer. The working pressure is between 10MPa and 70MPa, and the working temperature is between 30°C and 80°C. The jet passes through the extraction column 13, where the oil is dissolved in CO2, and then is taken out of the column through the outlet in the bottom accumulator. At the same time, Ruby-O falls into the accumulator in the form of powdery particles. There is a two-way switching valve 27 before the column, which is mainly used to bypass the extraction column 13 during the start of operation, where the CO2 flow is unstable. The separation container 19 has two outlets, one at the top, where the CO2 gas leaves without oil and enters the condenser 21 and the high-pressure reciprocating pump 22. Liquid CO2 is pumped at a pressure slightly higher than the extraction working pressure and heated to the working temperature in a water heating bath 23. CO2 leaves the heater 23 in a supercritical state and mixes with Ruby-O before passing through the nozzle 17. The oil extracted from the Ruby-O is discharged from the separation container 19 through the manual valve 20. The Ruby-O without oil is taken out from the accumulator through the manual valve 24 and expanded into the cyclone 25. Ruby-O powder is obtained at the bottom of the cyclone 25 and CO2 is lost to the atmosphere. The CO2 dissolved in the oil lost during the discharge is replaced by CO2 supplied from the gas deposit 26 before the condenser 21.

[0092] The method of the present application can be applied to any desired type of phospholipids, glycerophospholipids or glycolipids containing a fatty acyl ester group at the sn-2 position, in particular 1-alkyl-2-acyl-phospholipids (ether-phospholipids) and diacyl-phospholipids.

[0093] Suitable phospholipids for carrying out the method of the present application are selected from the group consisting of phosphatidylcholine, phosphatidic acid, phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine and diphosphatidylglycerol as well as synthetic phospholipids having various hydroxyl compounds esterified on the phosphate group, 1-alkyl-2-acyl-phospholipids and diacyl-phospholipids.

[0094] Suitable glycolipids for practicing the method of the present application are selected from the group consisting of glyceroglycolipids, galactolipids, sulfolipids, glycosphingolipids, cerebrosides, galactocerebrosides, glucocerebrosides, sulfatides, gangliosides, erythroglycosides, glycophosphosphingo-lipids, phytoglycolipids, glycophosphatidylinositols and glycolipids.

[0095] The exchange reaction of the present application can be used to incorporate any desired fatty acid into phospholipids. Some examples of fatty acids that may be of particular interest are:

[0096] Long chain (C 18- C 22 ) polyunsaturated fatty acids, such as linoleic acid, arachidonic acid, α-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, docosapentaenoic acid or γ-linolenic acid. These may be incorporated to improve the physiological or nutritional value of phospholipids, especially diacyl-phospholipids. These may be incorporated to change the emulsifying properties, to change the physiological values ​​or to improve the oxidative stability of phospholipids, especially diacyl-phospholipids.

[0097] Preferred omega-3 fatty acids are selected from the group consisting of alpha-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid.

[0098] Preferred omega-6 fatty acids are selected from the group consisting of linoleic acid, arachidonic acid, gamma-linoleic acid and conjugated linoleic acid.

[0099] The enzyme catalyst used includes a phospholipase which may be of animal, plant or microbial origin and may be site-nonspecific or site-specific, for example, preferably an extracellular phospholipase A2, such as Lecitase (TM), Novo Nordisk a / s.

[0100] The enzyme used in the method of the present application can be used alone or immobilized on a particulate macroporous organic or inorganic support, and is preferably attached to the support by cross-linking with any suitable cross-linking agent, such as glutaraldehyde.

[0101] The phospholipase is chosen such that the phospholipase represents 25 to 100% by weight of the total enzymes, preferably 30 to 70% by weight, which is also the same percentage of the total activity of the enzyme system.

[0102] The transesterification process should be carried out under conditions of optimal activity and thermal stability of the given enzyme, preferably at 60-80°C for 1 to 72 hours, preferably about 23 hours. At the end of the reaction, the enzyme is separated, for example, by filtration. An advantage of this method is that phospholipids can be easily separated from triglycerides when necessary.

[0103] In case any other triglyceride is used and separation is required, classical lecithin purification methods can be applied, such as fractionation or degumming using acetone at about 90° C., about 0.3% phosphoric acid. As an alternative (which is not preferred), separation can be performed by high performance thin layer chromatography (HPTLC).

[0104] The modified lecithin obtained by the method of the present application can be used in many food and pharmaceutical applications.

[0105] Finally, the antioxidant astaxanthin is added and mixed.

[0106] The main implementation schemes of this application are:

[0107] - Considering the SN-1 and SN-2 positions, the EPA content in the phospholipid molecule is 5-70%. If the SN-1 position is taken into account, the EPA concentration will be 5-35%, while at the SN-2 position, the EPA concentration will be 5-35%.

[0108] - Taking into account the SN-1 and SN-2 positions, the DHA content in the phospholipid molecule is 5-70%. If the SN-1 position is taken into account, the EPA concentration will be 5-35%, while at the SN-2 position, the EPA concentration will be 5-35%.

[0109] - At least 90% of the phospholipid molecules will contain incorporated omega-3 fatty acids.

[0110] - A process to remove saturated fatty acids, monosaturated fatty acids and 18-carbon fatty acids by passing them through a column filled with silica. This will reduce impurities and increase the incorporation of polyunsaturated fatty acids in phospholipids.

[0111] -The mechanisms of ultrasound-catalyzed enzymatic reactions include hydrolysis, transesterification, esterification and transesterification.

[0112] -The molecular forms of phospholipids that need to be considered are: phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, phosphatidic acid, etc.

[0113] - Enzymatic reactions do not use solvents.

[0114] - For the enzymatic reaction, liquid lipases or phospholipases of any origin (vegetable, animal and / or microbial) will be used (Lecitase Ultra from Novozymes, Purifine from DSM and CALB from Novozymes).

[0115] - The phospholipid content of the obtained enzyme reaction mixture can vary between 20% and 60%.

[0116] - The phospholipid content after molecular distillation or supercritical fractionation can vary between 40% and 90%.

[0117] -It uses molecular distillation as a technique to concentrate phospholipids obtained from FFA or EE raw materials. On the other hand, supercritical fractionation is used to concentrate phospholipids in a mixture containing TG due to the high molecular weight of both components, which is why the concept of solubility is used for the separation of PL.

[0118] The products of the present application include the compositions shown in the following table.

[0119] Table 1 Phospholipids / glycolipids rich in EPA and DHA

[0120]

[0121] 1. Based on products of soy lecithin and fish oil we can find different concentrations of Omega-3, giving each reference a specific application, some examples are shown below:

[0122] Table 2. Omega-3 phospholipids (40 / 32) balance

[0123]

[0124]

[0125] Table 3. Functions of Omega-3 phospholipids (40 / 32)

[0126]

[0127]

[0128] 2. A range of “soy-free” products based on sunflower lecithin and fish oil, as shown in Table 4.

[0129] Table 4. Omega-3 phospholipids “soy-free”

[0130]

[0131] 3. A “plant-based” product made from algal oil and soy lecithin; its characteristics are shown in Table 5.

[0132] Table 5. Omega-3 phospholipids "plant-based"

[0133]

[0134]

[0135] 4 Products with higher concentrations of Omega-3 obtained using supercritical fluid extraction technology, which provides the possibility of achieving the Omega-3 concentrations listed in Table 6.

[0136] Table 6. SFE processed Omega-3 lecithin

[0137]

[0138] The products shown in Table 6 were obtained by the batch process described below:

[0139] The batch processor was loaded with 400 g of Omega-3 lecithin obtained from the enzymatic reaction, with an acetone insoluble content of 45%, an oil content of 55%, an EPA content of 177 mg / g, and a DHA content of 130 mg / g. The oil consisted of 93% ethyl esters, and the remaining 7% was a mixture of glycerides. The extractor was heated to 40°C and maintained at this temperature throughout the extraction process. The CO2 flow rate was adjusted to 295 g / min by bypassing the extractor using a two-way switching valve. Once the flow rate was stable, CO2 was supplied to the extractor. The extraction process was started when the pressure reached 20 MPa and continued for 1 hour, emptying the separator every 10 minutes; after 45 minutes, no more oil was extracted. When it was necessary to completely remove the powdered Omega-3 lecithin, the extractor was emptied and washed with hexane. The hexane was removed using a rotary evaporator. The product was mixed with a high EPA oil containing 60% triglycerides in a weight ratio of 1:1. A new step of enzymatic interesterification was performed to obtain a final product with more Omega-3 even in polar lipid molecules.

[0140] Figure 7 and 8 The diagram illustrates the enrichment product of the present application.

[0141] The product of the present application can be used for emulsification of lipid drug delivery systems, which have shown advantages in improving bioavailability and absorption of omega-3 in vivo since emulsification reduces the size of oil droplets and increases the contact area between oil and water.

[0142] All literature and similar materials cited in this application, including but not limited to patents, patent applications, articles, books, treatises, and Internet web pages, regardless of the format of such literature and similar materials, are expressly incorporated by reference in their entirety for any purpose, as if fully indicated. If one or more of the incorporated literature and similar materials defines or uses a term in a manner that contradicts the definition of that term in this application, this application controls.

[0143] Although the foregoing description contains many details, these should not be interpreted as limiting the scope of the application, but only as providing some current preferred embodiments of the description. Similarly, without departing from the spirit or scope of the application, other embodiments can be designed. Features from different embodiments can be used in combination. Therefore, the scope of the application is only indicated and limited by the attached claims and their legal equivalents rather than the foregoing description. All additions, deletions and modifications to the application disclosed herein that fall within the meaning and scope of the claims should be included therein.

Claims

1. A method for incorporating omega-3 and omega-6 fatty acids into polar lipid molecules present in phospholipids and glycolipids, the method comprising the steps of: (a) an enzymatic exchange reaction between the omega-3 and omega-6 fatty acids and the fatty acids present in the polar lipid molecules in the phospholipids and glycolipids to obtain an oil with a high content of polar lipids, omega-3 fatty acids and omega-6 fatty acids, (b) a stage of concentrating the polar lipids and the omega-3 and omega-6 fatty acid content of the oil obtained in step (a) by supercritical fractionation or molecular distillation; (c) a new feed stage of concentrated omega-3 oil or omega-6 to increase the incorporation of omega-3 and omega-6 in the polar lipids and to achieve a higher omega-3 and omega-6 content in the product; (d) an ultrafiltration stage carried out under a nitrogen atmosphere to give the final oil the desired viscosity and appearance.

2. The method according to claim 1, wherein the phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidic acid, phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine and diphosphatidylglycerol, as well as synthetic phospholipids having various hydroxyl compounds esterified on the phosphate group, 1-alkyl-2-acyl-phospholipids and diacyl-phospholipids.

3. The method of claim 1, wherein the glycolipid is selected from the group consisting of glyceroglycolipids, galactolipids, sulfatides, glycosphingolipids, cerebrosides, galactocerebrosides, glucocerebrosides, sulfatides, gangliosides, globosides, glycophospho-sphingosine-lipids, plant glycolipids, glycophosphatidylinositols, and glycolipids.

4. The method of claim 1, wherein the omega-3 fatty acid is selected from the group consisting of alpha-linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid.

5. The method of claim 4, wherein the omega-3 fatty acid is selected from the group consisting of eicosapentaenoic acid, docosahexaenoic acid, and mixtures thereof.

6. The method of claim 5, wherein the omega-3 fatty acid selected from the group consisting of eicosapentaenoic acid, docosahexaenoic acid, and mixtures thereof is derived from concentrated fish oil or algae oil.

7. The method of claim 1, wherein the omega-6 fatty acid is selected from the group consisting of linoleic acid, arachidonic acid, gamma-linoleic acid, and conjugated linoleic acid. The method according to claim 1 , wherein the enzymatic reaction is carried out in an ultrasonic reactor.

9. The method of claim 1, wherein the enzymatic reaction is performed in the absence of a solvent or with the addition of a solvent.

10. The method according to claim 1, wherein the enzymatic reaction uses a liquid enzyme selected from lipases or phospholipases of plant, animal and / or microbial origin.

11. The method of claim 6, wherein the concentrated fish oil may be in the form of free fatty acids (FFA), ethyl esters (EE) or triglycerides (TG).

12. The method according to claim 1, wherein the enzymatic reaction can be hydrolysis, transesterification, esterification or interesterification.

13. The method according to claim 12, wherein the transesterification process is carried out under conditions that maintain optimal enzyme activity and thermal stability, preferably at a temperature range of 60-80°C for 1 to 72 hours, more preferably for about 23 hours.

14. The oil obtained by the process of claim 12, comprising: 20-90% w / w phospholipids, 2-20% w / w glycolipids, 10-90% w / w omega-3 fatty acids (EPA / DHA) and 0.01-0.05% w / w astaxanthin.

15. The oil according to claim 14, comprising: 5-70% w / w EPA and 5-70% w / w DHA present in the polar lipids (phospholipids and glycolipids).

16. A method for incorporating EPA / DHA into polar lipid molecules present in lecithin, the method comprising the steps of: (a) an enzymatic exchange reaction between the fatty acids present in the polar lipids of lecithin and the omega-3 fatty acids present in concentrated fish oil or algae oil, in order to obtain an oil with a high content of polar lipids and omega-3 fatty acids; (b) a stage of concentration of the polar lipids and omega-3 fatty acid content of the oil obtained in stage (a) by supercritical fractionation or molecular distillation; (c) a new feed stage of concentrated omega-3 oil (fish oil or algae oil) in order to increase the incorporation of omega-3 in the polar lipids and to achieve a higher omega-3 content in the product; (d) an ultrafiltration stage carried out under a nitrogen atmosphere in order to give the final oil the desired viscosity and appearance.

17. A soft capsule incorporating EPA / DHA-enriched phospholipids prepared by enzymatically interesterifying phospholipids containing saturated fatty acids in an ultrasonic reactor. The soft capsule according to claim 17 , wherein the enriched phospholipid is phosphatidylcholine. The soft capsule according to claim 17 , wherein the enriched phospholipid is phosphatidylserine.

20. The soft capsule according to claim 17, wherein the enriched phospholipid is phosphatidylinositol.

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