Purified egg yolk lecithin and preparation method comprising extraction

Abstract

The present invention provides a purified egg yolk lecithin and a preparation method thereof comprising extraction, by using specific solvents and conditions, the PC / PE ratio is increased, and the influence on the environment is reduced. The invention also provides lipid emulsions and parenteral nutritional products comprising the purified egg yolk lecithin obtained by the methods of the invention.

Description

Technical Field

[0001] The present invention relates to a purified egg yolk lecithin and a preparation method thereof. In addition, the present invention relates to a lipid emulsion comprising the purified egg yolk lecithin obtained by the method of the present invention, and a nutritional composition containing the lipid emulsion. Background Art

[0002] Egg yolk lecithin is a natural phospholipid mixture extracted and refined from egg yolks. It is commercially available and used in certain specialty pharmaceuticals and parenteral nutrition products. It is also used as an emulsifier in the food and pharmaceutical industries.

[0003] During the extraction of phospholipids from egg yolk, impurities such as cholesterol and neutral lipids are also extracted. There are various methods for purifying egg yolk lecithin, including solvent extraction, chromatography, and low-temperature solvent precipitation. The choice of purification method depends on the intended use of the isolated lecithin. In certain applications, such as clinical nutrition, egg yolk lecithin rich in phosphatidylcholine is beneficial. Egg yolk lecithin rich in phosphatidylcholine is used as a pharmaceutical additive, especially as an emulsifier in fat emulsions. A certain ratio of phosphatidylcholine to phosphatidylethanolamine is required to form a stable emulsion.

[0004] Many of the methods described for isolating egg yolk lecithin from egg yolk are based on the use of large amounts of solvents such as n-hexane or acetone. Since egg yolk lecithin is poorly soluble in acetone, it precipitates, while impurities such as neutral lipids dissolve in the acetone and are separated.

[0005] EP2502928B1 discloses a method for producing purified egg yolk phospholipids by treating the egg yolk with water and an acetone-containing alcohol. The acetone content is 80 vol%, and the alcohol content is 0.3-3 vol%. However, in this relatively inefficient method, the yield of phosphatidylcholine-enriched phospholipids is low and insufficient for industrial application.

[0006] Current extraction methods involving the use of acetone often result in egg yolk lecithin containing high levels of residual impurities.

[0007] Luz Palacios and Tong Wang (Journal of American Oil Chemists Society (2005) 82, 565–569) disclosed a method for extracting de-oiled and non-de-oiled egg yolk lecithin using ethanol. However, the purity of the phosphatidylcholine-rich egg yolk lecithin was low (35.7–53.3%). This method also used a variety of organic solvents, including chlorinated solvents such as chloroform, making it an environmentally unsuitable method.

[0008] Another method involves using aluminum oxide to remove impurities from extracted egg yolk lecithin.

[0009] To obtain the purity levels required for purified egg yolk lecithin to produce pharmaceutical-grade emulsion ingredients, typically for parenteral nutrition products, multiple solvent extraction steps may be required, increasing the amount of organic solvent used, raising costs and reducing the yield of purified egg yolk lecithin. Furthermore, the use of organic solvents in these traditional extraction methods can negatively impact the environment if not properly managed, leading to environmental and safety concerns.

[0010] In particular, when purified egg yolk lecithin is used as an emulsifier for parenteral nutrition emulsions, it is beneficial that it does not contain residual aluminum ions and provides a sufficient phosphatidylcholine concentration.

[0011] There is a need in the art to develop a method for isolating egg yolk lecithin to a purity sufficient to prepare pharmaceutical grade emulsions required for parenteral nutrition emulsions, providing sufficient phosphatidylcholine in the resulting product, and reducing waste consisting of environmentally harmful solvents.

[0012] Purpose of the Invention

[0013] The primary object of the present invention is to address and overcome one or more of the shortcomings of prior art methods for isolating and purifying egg yolk lecithin.

[0014] Another object of the present invention is to provide a method for preparing purified egg yolk lecithin, which can obtain a sufficient amount of purified egg yolk lecithin suitable for industrial production.

[0015] Yet another object of the present invention is to provide a method for preparing purified egg yolk lecithin using environmentally friendly solvents and conditions, which allows for easy recovery of hazardous solvents and enables their recycling, thereby reducing the impact on the environment and ensuring the safety of the purified egg yolk lecithin production process.

[0016] Another object of the present invention is to provide a purified egg yolk lecithin containing a large amount of phosphatidylcholine.

[0017] Another object of the present invention is to provide a lipid emulsion comprising the purified egg yolk lecithin obtained by the method.

[0018] Another object of the present invention is to provide a method for preparing an emulsion suitable for parenteral nutrition, which uses the egg yolk lecithin obtained by the above method as an emulsifier to obtain a stable nutritional emulsion. Summary of the Invention

[0019] The inventors of this method for preparing purified egg yolk lecithin using SFE-EYP as a starting material have discovered that if egg yolk lecithin is extracted with a specific solvent mixture of ethanol and acetonitrile, sufficient amounts (high yields) of the final egg yolk lecithin containing high levels of phosphatidylcholine can be obtained. The inventors have provided a method for preparing purified egg yolk lecithin having a phosphatidylcholine content greater than 75% w / w.

[0020] In a first aspect, the present invention provides a method for preparing purified egg yolk lecithin, the method comprising the following steps:

[0021] a. providing supercritical fluid extracted egg yolk powder as a starting material,

[0022] b. treating the egg yolk powder extracted by supercritical fluid with solvent 1,

[0023] c. separating the product obtained in step b) from the solvent 1 used in step b),

[0024] d. extracting the egg yolk lecithin fraction from the product obtained in step c) with a solvent 2,

[0025] e. Optionally, treating the egg yolk lecithin fraction obtained in step d) with charcoal,

[0026] f. by cooling the solution obtained in step d) or step e) to precipitate impurities in the egg yolk lecithin fraction obtained in step d) or step e),

[0027] g. removing precipitated impurities from the product obtained in step f),

[0028] h. separating the purified egg yolk lecithin from the product obtained in step g), and

[0029] i. Optionally, drying purified egg yolk lecithin;

[0030] Wherein, in step d), solvent 2 is a solvent mixture consisting of ethanol and acetonitrile.

[0031] In a second aspect, the present invention provides purified egg yolk lecithin obtained by the above method.

[0032] In a third aspect, the present invention provides a lipid emulsion comprising the purified egg yolk lecithin obtained by the method of the present invention.

[0033] In a fourth aspect, the present invention provides a nutritional product suitable for parenteral administration, wherein the product comprises a lipid emulsion comprising the purified egg yolk lecithin obtained by the method of the present invention.

[0034] In a fifth aspect, the present invention provides a method for preparing a lipid emulsion, the method comprising the following steps:

[0035] a. providing an oil phase comprising oil, and optionally a pharmaceutically acceptable antioxidant, optionally a pharmaceutically acceptable co-emulsifier, preferably free long-chain fatty acids, wherein the fatty acids are present in triglyceride-bound form;

[0036] b. providing an aqueous phase comprising water for injection, and optionally a pharmaceutically acceptable tonicity agent (tonicityagent) and / or pH adjusting agent and / or chelating agent;

[0037] c. by mixing the oil phase provided in step a) with the aqueous phase provided in step b) to form a pre-emulsion;

[0038] d. forming an oil-in-water emulsion by high pressure homogenization of the pre-emulsion obtained in step c); and

[0039] e. sterilizing the oil-in-water emulsion obtained in step d), wherein the oil-in-water emulsion is optionally filled into a suitable container before or after sterilization;

[0040] Wherein, the purified egg yolk lecithin of the present invention is added in step a) or step b). BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The general structure of phospholipids is shown. DETAILED DESCRIPTION

[0042] Various methods for purifying egg yolk lecithin are known in the art, demonstrating the difficulty in controlling cholesterol via crystallization / precipitation methods. The inventors have discovered a method for obtaining purified egg yolk lecithin containing a high amount of phosphatidylcholine (PC) when starting with SFE-EYP. The key steps for enriching the PC content are believed to be the extraction step, followed by precipitation of PE. Depletion of phosphatidylethanolamine (PE) was achieved by determining the most suitable solvent mixture, freezing the crude phospholipid (PL) solution, and separating the precipitated PL (mainly PE) by filtration. After concentrating the filtrate, phospholipids enriched in PC were obtained.

[0043] The present invention provides a method for preparing purified egg yolk lecithin and the obtained purified egg yolk lecithin, which can be used for preparing a parenteral nutrition composition.

[0044] The resulting egg yolk lecithin is characterized by high purity and increased phosphatidylcholine content. This method offers several advantages, including the use of environmentally friendly solvents that can be easily recovered, recycled, and reused, thereby reducing the amount of hazardous waste. Therefore, the present invention focuses on a method for preparing purified egg yolk lecithin using an environmentally friendly solvent mixture of ethanol and acetonitrile, preferably in a volume / volume (vol / vol) ratio of 57:43.

[0045] definition

[0046] Unless the context indicates otherwise, the following definitions apply in this application.

[0047] As used herein, the terms "comprises," "includes," "comprising," "including," "having," "having" or any other variation thereof, are intended to cover a non-exclusive inclusion.

[0048] Dimethyl carbonate (DMC), also known as DMC, is a carbonate ester, in which both hydrogen atoms in carbonic acid are replaced by methyl groups.

[0049] As used herein, the term "treating" refers to the process of contacting a substance (e.g., supercritical fluid extracted egg yolk powder) with one or more solvents or other reagents. Such treatment may involve various methods, including but not limited to mixing, stirring, agitating, soaking, or exposure, to allow for physical interaction between the substance and the solvent or reagent. The results of the treatment may include purification, extraction, reaction, or other modification of the substance to achieve a desired outcome.

[0050] The term "separating" as used herein refers to the process of separating insoluble matter from a solvent or solution. This process can be accomplished by various techniques, including but not limited to filtration, decantation, extraction, distillation, evaporation, centrifugation, drying, or a combination thereof.

[0051] As used herein, the term "purified egg-yolk lecithin" refers to egg-yolk lecithin that is free of certain impurities and significantly reduced in other impurities. Purified egg-yolk lecithin is further characterized by being rich in phosphatidylcholine. Its purity and stability make it suitable for further application in nutritional and pharmaceutical products.

[0052] The term "phosphatidylcholine" refers to a class of phospholipids with choline as the head group. It therefore refers to a variety of compounds rather than one unique compound, as several different fatty acid chain types may be present ( Figure 1 ).

[0053] The term "supercritical fluid extracted egg-yolk powder" or "SFE-EYP" refers to egg-yolk powder that has been extracted for neutral lipids (such as cholesterol) using a technique called supercritical fluid extraction. This technique typically uses non-hazardous carbon dioxide as a fluid, above its critical point, where it behaves both like a gas and a liquid, allowing for selective extraction based on solubility. Under conditions of high pressure and high temperature, the resulting powder can have a reduced fat content while retaining valuable components such as phospholipids, depending on the extraction conditions used. Supercritical fluid can selectively extract certain lipids, such as phospholipids (primarily triglycerides and cholesterol), from egg yolk under mild conditions. Supercritical fluid-extracted egg-yolk powder is used as the starting material in the method of the present invention. It may also be referred to as defatted or de-oiled egg-yolk powder. This method is described in detail in Hietaniemi (2009) Eur Food Res Technol 228: 857-863.

[0054] Therefore, throughout this application, the term supercritical fluid extracted egg yolk powder (SFE-EYP), as used herein as the starting material for isolating egg yolk phosphatidylcholine, refers to a composition obtained by supercritical fluid extraction of egg yolk with CO2. Preferably, the composition comprises phosphatidylcholine (PC), phosphatidylethanolamine (PE), cholesterol, sphingomyelin (SHP), phosphatidylinositol (PI), lysophosphatidylethanolamine (LPE) and lysophosphatidylcholine (LPC), as well as various impurities, and more preferably, the composition has a reduced neutral lipid content, more preferably a reduced triglyceride and cholesterol content, compared to dry egg yolk powder.

[0055] The exact composition of SFE-EYP depends on the type of egg yolk used and the conditions used in the extraction method.

[0056] Preferably, the SFE-EYP used as starting material for the process of the present invention comprises less than 2% w / w cholesterol, residual triglycerides in the range of 1-2% w / w and more than 70% w / w phosphatidylcholine.

[0057] As used herein, the terms "extraction" or "extraction" refer to methods involving the use of a solvent to separate a specific substance from a mixture of components. This method typically involves dissolving the desired substance in a solvent or solvent mixture to separate it from other components of the mixture that are insoluble in the solvent or solvent mixture. The solvent can then be removed to yield a purified substance. In the methods of the present invention, the term extraction, as used in step d, refers to this understanding.

[0058] As used herein, the term "precipitation" or "precipitation" refers to the process by which a substance solidifies. It may crystallize or otherwise change from a liquid to a solid state. Precipitation can be induced by changes in temperature, pH, concentration, or the addition of another reagent or solvent.

[0059] As used herein, the term "drying" refers to the process of removing residual liquid (such as solvent) from a substance, typically using heat, air flow, vacuum, or a combination of these methods.

[0060] High-performance liquid chromatography (HPLC) is a technique that separates molecules based on properties such as size and surface charge. Ultraviolet (UV) spectroscopy combined with HPLC can determine the concentration of identified and separated molecules or simply compare the relative concentrations of the same eluents from different runs.

[0061] Combining a charged aerosol detector (CAD) with HPLC allows for the determination of the mass of compounds present in a sample by generating charged aerosol particles, which are detected using an electrometer. The method employed produces a signal proportional to the mass of the analyte present. It is often used to analyze compounds that cannot be detected using traditional UV / Vis methods due to the lack of a chromophore.

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. Although similar or equivalent methods and materials as described herein can be used in the practice or testing of embodiments of the present invention, suitable methods and materials are described below. In the event of any conflict, this specification (including definitions) shall prevail. In addition, materials, methods and examples are illustrative only and are not restrictive.

[0063] When an amount, concentration or other value or parameter is given as a range, preferred range or preferred upper value and / or preferred lower value list, this should be understood as specifically disclosing all ranges formed by any pair of any upper range limit or preferred value and any lower range limit or preferred value, whether or not a range is disclosed separately. Unless otherwise stated, the numerical ranges recited herein should include their endpoints and all integers and fractions within the range.

[0064] Egg yolk is composed of various nutrients, including protein, fat, and phospholipids. According to research by Aro et al. (Eur Food Res Technol (2009) 228:857-863), approximately 50% of egg yolk is water. After spray drying, the resulting powder contains approximately 60% lipids, which are composed of neutral lipids (65%), phospholipids (31%), and cholesterol (4%). Dry egg yolk powder is also described as containing approximately 15% phospholipids, primarily composed of approximately 70% phosphatidylcholine and 20% phosphatidylethanolamine (Burley RW, Vadehra DV, The Avian Egg Chemistry and Biology, John Wiley & Sons (1989)). It seems to be an indisputable fact that the main phospholipids in egg yolk are phosphatidylcholine (PC), which accounts for approximately 70% of the total phospholipids in the egg yolk, and phosphatidylethanolamine (PE), which accounts for approximately 15% of the total phospholipids in the egg yolk. Yolk proteins include lipoproteins and phosphoproteins, which together account for about 16% of the yolk weight. However, as reported in the literature, there is a great deal of variation in the composition of egg yolk, which may be due to differences in hen breed and diet.

[0065] When developing a suitable method for purifying PC-rich egg yolk lecithin, various factors need to be considered. One of these is the solubility profile of egg yolk lecithin in solvents. Based on solubility studies using a number of solvents (including C1-C4 alcohols, toluene, DMC, DEC, THF, ethers and ketones, n-hexane, mixtures of short aliphatic alcohols with nitriles, mixtures with dimethyl carbonate (DMC), or mixtures with diethyl carbonate (DEC)), the following solvents are considered to be the most suitable candidates for extracting egg yolk lecithin from washed SFE-EYP: ethanol, methanol, isopropanol, toluene, 1-butanol, mixtures of diethyl carbonate:ethanol (3:2, 2:3, and 1:1), mixtures of dimethyl carbonate:ethanol (3:2, 2:3, and 1:1), and a mixture of ethanol:acetonitrile (57:43). Where solvent ratios are given for a solvent mixture, they are based on volume:volume (vol:vol). All solvents and solvent mixtures in this list can be used to extract egg yolk lecithin from SFE-EYP.

[0066] Surprisingly, the inventors of the present application discovered, while testing these different solvents, that in step d) of the method according to the present invention, extracting the supercritical fluid extracted egg yolk powder with a mixture of ethanol:acetonitrile not only sufficiently extracted egg yolk lecithin from the washed SFE-EYP, but it also proved to be the most suitable solvent for the subsequent step f).

[0067] For the cryoextraction of impurities (step f), each solvent and solvent mixture has a certain optimal temperature range, which also depends on the concentration of the solution, which itself depends on the effectiveness of the extraction (step d).

[0068] When impurities are precipitated by cooling in step f) after the extraction step d) using an ethanol:acetonitrile solvent mixture, a large amount of components such as phosphatidylethanolamine can be removed. Compared with the use of other extraction solvents, the content of phosphatidylcholine in the purified egg yolk lecithin is more effectively increased.

[0069] Therefore, the method according to the present invention comprises an extraction step, wherein the extraction solvent is a mixture of ethanol and acetonitrile. In a preferred embodiment, the volume ratio of the mixture is 57% ethanol and 43% acetonitrile.

[0070] There are also additional environmental benefits, as the azeotrope, with a ratio of 57:43 ethanol to acetonitrile, has a boiling point of 72.9°C (compared to 78.4°C for ethanol and 82.0°C for acetonitrile). The azeotrope can be easily recovered from the reaction mixture by distillation while maintaining the desired ratio between the two solvents.

[0071] In a first aspect, the present invention relates to a method for preparing purified egg yolk lecithin, the method comprising the following steps:

[0072] a. providing supercritical fluid extracted egg yolk powder as a starting material,

[0073] b. treating the egg yolk powder extracted by supercritical fluid with solvent 1,

[0074] c. separating the product obtained in step b) from solvent 1,

[0075] d. extracting the egg yolk lecithin fraction from the product obtained in step c) with a solvent 2,

[0076] e. Optionally, treating the egg yolk lecithin fraction obtained in step d) with charcoal,

[0077] f. by cooling the solution obtained in step d) or step e) to precipitate impurities in the egg yolk lecithin fraction obtained in step d) or step e),

[0078] g. removing precipitated impurities from the product obtained in step f),

[0079] h. separating the purified egg yolk lecithin from the product obtained in step g), and

[0080] i. Optionally, drying the purified egg yolk lecithin;

[0081] Wherein, in step d), solvent 2 is a solvent mixture of ethanol and acetonitrile.

[0082] Thus, the present invention provides a multi-step process for preparing purified egg yolk lecithin using SFE-EYP as a starting material and, in step d), a mixture of ethanol and acetonitrile as a solvent, wherein SFE-EYP is provided in a first step and, in a second step, treated with a solvent 1 (such as, for example, dimethyl carbonate (DMC)). The treatment may involve mixing, stirring, agitating, soaking, or exposing the SFE-EYP powder to the solvent 1. Preferably, the treatment comprises adding the SFE-EYP to the solvent 1 and stirring the resulting mixture at an elevated temperature. Preferably, the temperature is in the range of 30-60°C, preferably 35-55°C, and most preferably 40-45°C. Further preferably, the stirring is maintained for a period of 50 to 70 minutes, preferably 50 to 65 minutes, and most preferably 55 to 65 minutes. In a preferred embodiment, treating the SFE-EYP powder with the solvent 1 results in a slurry. If oxidation protection is desired, the process is more efficient by adding a free radical scavenger. Preferably, steps b), d), e), and / or g) further comprise adding a free radical scavenger. The free radical scavenger is preferably DL-α-tocopherol. The free radical scavenger may be added during step b), during step d), during and after the charcoal treatment in step e), and / or during step g).

[0083] The amount of solvent 1 provided to be added to SFE-EYP in step b) may be 2-10 mL per gram of SFE-EYP, preferably 3-8 mL per gram of SFE-EYP, more preferably 4-8 mL per gram of SFE-EYP.

[0084] Step b) may further comprise adding a free radical scavenger. Preferably, the free radical scavenger is DL-α-tocopherol.

[0085] In step c), the product obtained in step b) is separated from solvent 1.

[0086] In a preferred embodiment, the desired product obtained in step b) is a solid.

[0087] The solid product can be isolated by conventional methods such as filtration, decantation, centrifugation or a combination thereof.

[0088] In certain embodiments, isolation of the product is accomplished by filtration to remove soluble impurities dissolved by the solvent.

[0089] The obtained product can be washed with solvent 1.

[0090] Before adding the extraction solvent mixture of ethanol and acetonitrile in step d), the product of step c) may be further dried to remove residual solvent 1 (washed or unwashed). Drying can be carried out under vacuum at a pressure of 200-300 mbar at a temperature in the range of 15-35° C., preferably 20-30° C., for 4-6 hours.

[0091] In step d), the egg yolk lecithin fraction is extracted from the product obtained in step c) using a solvent mixture of ethanol and acetonitrile. Preferably, the solvent mixture of ethanol and acetonitrile is provided in a volume / volume ratio of 57:43.

[0092] In a preferred embodiment, step d) may further comprise adding a free radical scavenger, preferably DL-α-tocopherol.

[0093] In another preferred embodiment, the free radical scavenger may be dissolved in the solvent of step d) above before the extraction process.

[0094] As described in Example 3, when testing different temperature ranges, the most suitable extraction temperature was determined.

[0095] Preferably, the extraction in step d) is carried out at a temperature in the range of 50-60°C. The solution is stirred for 30-60 minutes, preferably 30-40 minutes. It was found that since phospholipids are generally heat-sensitive components, the selected solvent allows for a relatively short extraction time, thereby allowing for a higher overall yield.

[0096] The mixture can be filtered to remove insoluble particles. The insoluble material can optionally be rinsed with a solvent mixture. The rinsed solution is then added to the filtrate.

[0097] Step e) is optional. It involves treating the egg yolk lecithin fraction obtained in step d) with charcoal, wherein the treatment comprises stirring a liquid solvent containing the extracted egg yolk lecithin fraction and the added charcoal. The amount of charcoal added in the process can vary from 1% w / w to 5% w / w, preferably 2% w / w to 4% w / w. In a preferred embodiment, the charcoal treatment is carried out at a temperature in the range of 30-50°C for a time period of 30 minutes to 3 hours, more preferably at a temperature in the range of 40-45°C for a time period of 1 hour to 2 hours.

[0098] During step e) of the method, different types of charcoal (strongly acidic or basic) can be used.

[0099] Step e) may comprise adding a free radical scavenger after separating the charcoal from the solution. Preferably, the free radical scavenger is DL-α-tocopherol.

[0100] The key step in the method for separating phospholipids and thus enriching them for phosphatidylcholine is step f), in which the unwanted components are separated by cooling (also known as cryoextraction). This is a simple and effective method for refining phospholipids under mild conditions, as it does not involve changing solvents or precipitation in acetone, and also avoids the use of aluminum oxide adsorbents, an alternative method that can lead to increased aluminum ion levels.

[0101] In some preferred embodiments, the methods of the present invention do not comprise the additional step of treating any solution with alumina.

[0102] Different ratios of ethanol to acetonitrile were successfully tested, however, a ratio of 57:43, representing an azeotropic ratio, was the preferred one. It also ensured the recyclability of the solvent.

[0103] For the cryoextraction of impurities (step f), each solvent and solvent mixture has an optimal temperature range, which also depends on the concentration of the solution, which itself depends on the effectiveness of the extraction (step d). When a mixture of ethanol:acetonitrile (57:43) was used in step d), different temperature ranges for step f) were evaluated. The results showed that the temperature range of -8°C to -12°C was more conducive to obtaining the preferred PC / PE ratio than the tested temperature ranges of -4°C to -6°C or -14°C to -16°C.

[0104] The method accordingly further comprises step f), i.e., the step of precipitating impurities (e.g., PE) from the egg yolk lecithin fraction obtained in the previous step. This is achieved by cooling the egg yolk lecithin fraction obtained in step d) or step e). Preferably, cooling is performed at a temperature in the range of -12°C to -8°C, more preferably in the range of -11°C to -10°C. After cooling, stirring is performed at the reduced temperature for 60 to 90 minutes, preferably 90 minutes, to precipitate more impurities.

[0105] The choice of a combination of ethanol and acetonitrile as the solvent in step d) offers the added benefit of allowing the mixture to be carried directly to step f) for precipitation of impurities by cooling. Because acetonitrile acts as an antisolvent for phospholipids, particularly PE, at lower temperatures, it selectively precipitates PE while retaining the majority of PC in solution. This avoids the need for an additional extract concentration step, simplifying downstream processing and achieving excellent yields.

[0106] Comparative experiments were conducted to determine the most suitable solvent for the winterization effect (i.e., precipitation at lower temperatures achieved by cooling). These experiments revealed that the most suitable solvent mixture was a mixture of ethanol and acetonitrile. The combination of ethanol and DMC allowed freezing at reduced temperatures before sufficient PE precipitation occurred, while the combination of ethanol and DEC resulted in a higher PE content (less desirable phospholipids). Furthermore, removing these solvents from the purified phospholipid extract (i.e., purified egg yolk lecithin) after the winterization step (cooling step) was more difficult and energy-intensive than removing ethanol and acetonitrile (especially in a 57:43 ratio).

[0107] Step g) involves separating the precipitated impurities from the egg yolk lecithin-containing solution. If optional step e) is performed before step f), charcoal adsorbed with other impurities can be removed simultaneously. The precipitated impurities can be separated from the egg yolk lecithin fraction obtained in step d) or step e) by conventional methods such as filtration or centrifugation. The precipitated impurities are preferably removed by filtration.

[0108] Step g) may involve adding a free radical scavenger after removing the precipitated impurities from the solution. Preferably, the free radical scavenger is DL-α-tocopherol.

[0109] After removal of the precipitate (cooling and filtration), the product obtained in step g) is a solution.

[0110] In step h) of the method, the dissolved purified egg yolk lecithin is separated from the product obtained in step g). This step can be carried out by conventional methods such as concentration, distillation, or evaporation. Preferably, the purified egg yolk lecithin is separated by evaporation. In this step, a viscous product is obtained.

[0111] The isolated purified egg yolk lecithin can be optionally dried. By optional step i) drying, for example using a belt freeze dryer, the product can be converted into a fluffy solid. Drying is preferably carried out at a temperature in the range of 25°C to 35°C for 1 to 4 hours.

[0112] The method obtains a good yield of purified egg yolk lecithin, in particular a better yield than when a different extraction solvent is used in step d), while achieving a PC / PE ratio of 6 or more, preferably 7 or more, more preferably 8 or more.

[0113] In a second aspect, the present invention provides purified egg yolk lecithin obtained by the above method, wherein the purified egg yolk lecithin has a phosphatidylcholine content of 75-80%.

[0114] Compared with the commercially available products tested, the purified egg yolk lecithin obtained by the method of the present invention has better lipid properties because it shows an enrichment of phosphatidylcholine (PC) and a reduction in the amounts of phosphatidylethanolamine (PE), cholesterol, sphingomyelin (SHP), phosphatidylinositol (PI), lysophosphatidylethanolamine (LPE), and lysophosphatidylcholine (LPC) (see Table 1).

[0115] In another preferred embodiment, the purified egg yolk lecithin obtained by this method has improved purity characteristics compared to products obtained using different methods. In particular, it does not contain residual traces of aluminum that would otherwise remain in the product when aluminum oxide is used to remove impurities.

[0116] Therefore, a preferred embodiment of the present invention is that the purified egg yolk lecithin is substantially free of aluminum ions. As used herein, substantially free of aluminum ions means less than 0.5 ppm as detected by ICP-OES. ICP-OES refers to inductively coupled plasma optical emission spectroscopy, also known as inductively coupled plasma atomic emission spectroscopy (ICP-AES), an analytical technique used to detect chemical elements. Preferably, the purified egg yolk lecithin contains less than 0.1 ppm of aluminum ions, and most preferably, less than 0.025 ppm.

[0117] The improved purity characteristics can be described as including a decrease in the amount of phosphatidylethanolamine and an increase in the amount of phosphatidylcholine compared to products obtained using different solvents. Preferably, the PC / PE ratio of the purified egg yolk lecithin obtained by the method is 7.5-9.0, more preferably 8.0-9.0, and most preferably 8.5-8.7.

[0118] Table 3 of Example 2 lists data disclosing the composition of an egg yolk lecithin sample, in particular its PC and PE contents, and the yield obtained by the method according to step d) of the present invention, i.e., extracting an egg yolk protein sample (SFE-EYP) using a list of selected extraction solvents (Solvent 2) of step d) of the method.

[0119] In step d), ethanol and methanol, as solvent 2, tend to extract some other components in the egg yolk powder, as evidenced by higher extraction yields and lower PC content. When using isopropyl alcohol (IPA) for extraction, larger amounts of solvent are required to achieve sufficient PC enrichment, and the PE content is consistently higher, thus eliminating it as a suitable solvent for the extraction step in the present method. Based on the favorable PC / PE ratio, an ethanol:acetonitrile mixture (57:43) was selected as the extraction solvent.

[0120] Table 1 lists the measured amounts of different components in the purified egg yolk lecithin samples of the present invention analyzed using HPLC-CAD. Batch 1 represents commercially available egg yolk lecithin; Batch 2 and Batch 3 represent purified egg yolk lecithin samples from SFE-EYP, which was purified using the following methods: using the first solvent 1 in step b) and using ethanol:acetonitrile (57:43) in step d).

[0121] Table 1:

[0122]

[0123] In a third aspect, the present invention provides a lipid emulsion comprising purified egg yolk lecithin obtained by the method of the present invention. The lipid emulsion may contain egg yolk lecithin as an emulsifier. This lipid emulsion can be used to provide a parenteral nutrition product comprising a substantially water-insoluble component.

[0124] In a fourth aspect, the present invention provides a nutritional product suitable for parenteral administration, the product comprising a lipid emulsion comprising the purified egg yolk lecithin obtained by the method of the present invention.

[0125] In a fifth aspect, the present invention provides a method for preparing a lipid emulsion, comprising the following steps:

[0126] a) providing an oil phase comprising oil, and optionally a pharmaceutically acceptable antioxidant, optionally a pharmaceutically acceptable co-emulsifier, preferably free long-chain fatty acids, wherein the fatty acids are present in triglyceride-bound form;

[0127] b) providing an aqueous phase comprising water for injection, and optionally a pharmaceutically acceptable tonicity agent and / or pH adjusting agent and / or chelating agent;

[0128] c) forming a pre-emulsion by mixing the oil phase provided in step a) with the water phase provided in step b);

[0129] d) forming an oil-in-water emulsion by high pressure homogenization of the pre-emulsion obtained in step c); and

[0130] e) sterilizing the oil-in-water emulsion obtained in step d), wherein the oil-in-water emulsion is optionally filled into suitable containers before or after sterilization,

[0131] Wherein, the purified egg yolk lecithin of the present invention is added in step a) or step b).

[0132] In a preferred embodiment, the purified egg yolk lecithin is obtained by the method of the present invention for preparing purified egg yolk lecithin starting from SFE-EYP.

[0133] In another preferred embodiment, the antioxidant is DL-α-tocopherol.

[0134] The components of the aqueous phase can be dispersed in water to form a suspension. This can be achieved by high-intensity mixing. During this process, the pH can be adjusted. Sodium hydroxide can be used for pH adjustment.

[0135] The oil phase can include any conventional oil for parenteral nutrition, including, for example, olive oil, soybean oil, fish oil and medium-chain triglycerides, and any other suitable lipophilic component. Preferably, the oil is selected from olive oil, soybean oil, fish oil and medium-chain triglycerides. More preferably, the oil phase includes 30% w / w soybean oil, 30% w / w medium-chain triglycerides, 25% w / w olive oil and 15% w / w fish oil. The oil phase can be transferred to the aqueous phase through a membrane filter. The aqueous phase ingredients and the oil phase ingredients can be mixed together to obtain a pre-emulsion.

[0136] The term "fish oil" refers to "purified fish oil" and "purified fish oil rich in omega 3 fatty acids," the latter comprising at least 9% (w / w) of the omega-3 fatty acid docosahexaenoic acid (DHA) and at least 13% (w / w) of the omega-3 fatty acid eicosapentaenoic acid (EPA), expressed as triglycerides, according to European Pharmacopoeia 6.0. Fish oils are commercially available. In the present disclosure, the term "fish oil" also refers to fish oil extracts, which may be further enriched or reduced in the content of certain fatty acids, respectively. Such fish oil extracts are commercially available, for example from Solutex SL.

[0137] The term "medium chain triglycerides" (MCT) refers to triglycerides of fatty acids with a length of 6-12 carbon atoms, including caproic acid, caprylic acid, capric acid, and lauric acid. MCTs are commercially available.

[0138] Emulsions can be obtained by high pressure homogenization.

[0139] The homogenization process can be carried out at a temperature in the range of 50-80°C and a pressure in the range of 350-400 bar. It is preferably carried out at a temperature in the range of 55-70°C. This process can be followed by sterilization. The emulsion can be filled into suitable containers before or after sterilization. The emulsion can be further diluted with an appropriate amount of water under stirring and then filtered to obtain the final emulsion.

[0140] abbreviation

[0141] SFE-EYP: Supercritical Fluid Extracted Egg Yolk Powder

[0142] PC :Phosphatidylcholine

[0143] PE :phosphatidylethanolamine

[0144] LPE: Lysophosphatidylethanolamine

[0145] LPC: lysophosphatidylcholine

[0146] SPH: sphingomyelin

[0147] PI: Phosphatidylinositol

[0148] HPLC: High Performance Liquid Chromatography

[0149] LC: Liquid chromatography

[0150] UV :Ultraviolet

[0151] w / w: weight ratio

[0152] DMC: dimethyl carbonate

[0153] DEC: diethyl carbonate

[0154] DL: right-handed and left-handed

[0155] Mbar: millibar

[0156] CAD: Charged Aerosol Detector

[0157] MTBE: 2-methoxy-2-methylpropane (also known as methyl tert-butyl ether)

[0158] ICP-OES: Inductively Coupled Plasma Optical Emission Spectroscopy

[0159] Example

[0160] The following examples provide detailed experimental parameters according to the present invention. These examples are intended to illustrate the present invention, but not to limit any possible embodiments of the present invention.

[0161] Example 1: Comparative experiment on the effect of selected solvents on the composition of egg yolk lecithin after winterization

[0162] Different solvent combinations were tested and the obtained compositions were analyzed.

[0163] The effects of using these different solvent combinations in step d) of the method for extracting egg yolk lecithin and in the subsequent step f) of winterization on the purity of the final egg yolk lecithin (i.e., the purified egg yolk lecithin after completing all the above steps) were analyzed by high performance liquid chromatography equipped with a charged aerosol detector.

[0164] Example 1.1: Preparation of purified egg yolk lecithin using ethanol:acetonitrile (57:43)

[0165] SFE-EYP (200 g) was extracted with 1190 mL of a 57:43 solvent mixture of ethanol and acetonitrile at 20-25°C. A stock solution of an ethanol:acetonitrile (57:43) mixture was prepared by mixing 1140 mL of ethanol with 860 mL of acetonitrile. A DL-α-tocopherol solution (10 mL, prepared by dissolving 50 mg of DL-α-tocopherol in 10 mL of the solvent mixture prepared above) was added and the reaction mixture was stirred at 55-60°C for 30-40 minutes. The hot slurry was filtered and the resulting solid was washed with 400 mL of the solvent mixture prepared above. The filtrate was treated with charcoal (8.0 g) at 40-45°C and then stirred for 55-65 minutes.

[0166] The charcoal-treated reaction was cooled to -12°C to -8°C and stirred for 90 minutes, whereupon impurities precipitated. The resulting mixture was filtered through a jacketed funnel while maintaining the jacket temperature at -12°C to -8°C, and a DL-α-tocopherol solution (10 mL, prepared by dissolving 50 mg of DL-α-tocopherol in 10 mL of the solvent mixture prepared above) was added to the filtered solution. The resulting solution was filtered again through a 5μ filter and then concentrated under vacuum at 35°C or less to obtain purified egg yolk lecithin (54 g, 0.27 w / w).

[0167] Example 1.2: Preparation of purified egg yolk lecithin by extraction with ethanol:DEC (40:60)

[0168] A mixture of ethanol and diethyl carbonate (40:60) (250 mL) was charged into a 1 L reactor, and SFE-EYP (50 g) was added with stirring. The mixture was stirred at 40-45°C for 80-90 minutes. The heterogeneous mixture was filtered while hot to obtain a clear solution. The filtrate was cooled to -20°C to -15°C and stirred for 7-8 hours, whereupon the impurities precipitated. The reaction was filtered through a jacketed funnel while maintaining the jacket temperature in the range of -20°C to -10°C. The filtered solution was concentrated under vacuum below 35°C to obtain (10.9 g, 0.22 w / w) purified egg yolk lecithin. The low yield, even without the additional charcoal treatment step, clearly demonstrated that the ethanol:DEC solvent mixture was not suitable.

[0169] Example 1.3: Preparation of purified egg yolk lecithin by extraction with ethanol:DMC (40:60)

[0170] SFE-EYP (200 g) and ethanol (1000 mL) were loaded into a 2 L reactor. The mixture was stirred at 40-45 ° C for 80-90 minutes, and then the heterogeneous reactants were filtered. The obtained clear filtrate was concentrated in vacuo at 40 ° C to obtain a solid residue. Under stirring, the solid residue was further dissolved in a mixture of ethanol and dimethyl carbonate (40:60) to obtain a solution. The obtained solution was cooled to -10 ° C to -5 ° C and stirred for 15-16 hours. The obtained precipitate was filtered through a jacket funnel while maintaining the jacket temperature in the range of -10 ° C to -5 ° C. The filtered solution was concentrated in vacuo below 35 ° C to obtain (42.0 g, 0.21 w / w) purified egg yolk lecithin. The results showed that the yield of ethanol: DMC solvent was insufficient.

[0171] The results of Examples 1.1-1.3 are shown in Table 2.

[0172] Table 2:

[0173] solvent Temperature (℃) PC (% w / w) PE (% w / w) Yield (w / w) Ethanol / acetonitrile (57:43) -12 to -8 79.40 06.14 0.28 Ethanol:DEC (40:60) -20 to -15 78.02 10.89 0.22 Ethanol:DMC (40:60) -10 to -5 78.16 9.70 0.21

[0174] Example 2: Comparative Experiment on Preparation of Purified Egg Yolk Lecithin Using Different Extraction Solvents

[0175] Different solvents suitable for the extraction step d) were tested and the resulting compositions were analyzed.

[0176] Different amounts of SFE-EYP (15-50 g) were suspended in various solvents listed in column 1 of Table 3 at 20-25°C. The heterogeneous suspension was stirred at 40-45°C for 60-90 minutes. The hot suspension was filtered and the residual protein was washed with the corresponding solvent (1 volume). The filtrate and washings were combined and the resulting solution was evaporated below 45°C.

[0177] The PC and PE contents of the treated samples were analyzed by HPLC with charged aerosol detection. In addition, the yields of these extracts were determined where possible.

[0178] Table 3:

[0179]

[0180]

[0181] The analysis showed that the choice of solvent had an impact on the PC content and PC / PE ratio even before the impurity cooling step.

[0182] Example 3: Comparative experiment of preparing purified egg yolk lecithin using different extraction temperatures in step d)

[0183] Different temperatures were tested for the extraction of egg yolk lecithin with an ethanol / acetonitrile (57:43) mixture.

[0184] The effects of using these different conditions to extract egg yolk lecithin in step d) of the method on the purity of the final egg yolk lecithin (i.e., the purified egg yolk lecithin after completing all the above steps) were analyzed by high performance liquid chromatography equipped with a charged aerosol detector.

[0185] Example 3.1:

[0186] SFE-EYP (180 g) was extracted with 1080 mL of a mixture of ethanol and acetonitrile (57:43) at 20-25°C, followed by the addition of DL-α-tocopherol (14.5 mg). An ethanol:acetonitrile stock solution was prepared by mixing ethanol (820.8 mL) and acetonitrile (619.2 mL). The mixture was stirred at 40-45°C for 30-40 minutes. The heterogeneous material was filtered, and the remaining solid protein was washed with 370 mL of the above-prepared stock solution. The filtrate and washings were combined, and the resulting solution was treated with charcoal (7.2 g) at 40-45°C and stirred for 60-70 minutes.

[0187] The charcoal-treated material was cooled to -12°C to -8°C and stirred for 80-90 minutes, whereupon the impurities precipitated. The reaction mixture was filtered through a jacketed funnel while maintaining the jacket temperature within the range of -12°C to -8°C, and DL-α-tocopherol (14.5 mg) was added to the filtered solution. The solution was filtered again through a 0.45μ filter and then concentrated under vacuum below 35°C until a viscous residue remained. The remaining viscous material was completely dried to obtain (46.0 g, 0.25 w / w) purified egg yolk lecithin.

[0188] Example 3.2:

[0189] SFE-EYP (120 g) was extracted with 710 mL of a 57:43 mixture of ethanol and acetonitrile at 20-25°C, followed by the addition of DL-α-tocopherol (30 mg dissolved in 10 mL of 57:43 ethanol:acetonitrile). An ethanol:acetonitrile stock solution was prepared by mixing ethanol (547.2 mL) and acetonitrile (412.8 mL). The mixture was stirred at 50-60°C for 30-40 minutes. The heterogeneous material was filtered, and the remaining solid protein was washed with 230 mL of the above-prepared stock solution. The filtrate and washings were combined, and the resulting solution was treated with charcoal (4.8 g) at 40-45°C and stirred for 55-60 minutes.

[0190] The charcoal-treated material was cooled to -12°C to -8°C and stirred for 80-90 minutes, whereupon the impurities precipitated. The reaction mixture was filtered through a jacketed funnel while maintaining the jacket temperature within the range of -12°C to -8°C, and DL-α-tocopherol (10 mg) was added to the filtered solution. The solution was filtered again through a 0.45μ filter and then concentrated under vacuum below 35°C to obtain (30 g, 0.25 w / w) purified egg yolk lecithin.

[0191] Example 3.3:

[0192] Pre-washed SFE-EYP (20 g) was extracted with 120 mL of a 57:43 mixture of ethanol and acetonitrile at 20-25° C., followed by the addition of DL-α-tocopherol (5 mg dissolved in 102.5 mL of 57:43 ethanol:acetonitrile). A stock solution of ethanol:acetonitrile was prepared by mixing ethanol (91.2 mL) and acetonitrile (68.8 mL).

[0193] The reaction was stirred at 60-70°C for 50-60 minutes. The heterogeneous material was filtered, and the remaining solid protein was washed with 60 mL of the stock solution prepared above. The filtrate was a very dark solution, and the experiment was stopped at the extraction stage without cooling (winterization). Therefore, comparable yields could not be determined.

[0194] The results of Examples 3.1-3.3 are shown in Table 4.

[0195] Table 4

[0196]

[0197] Example 4: Preparation of purified egg yolk lecithin using solvent 1 in step b), a solvent mixture of ethanol:acetonitrile (57:43) in the extraction step d), and charcoal treatment in step e).

[0198] 3540mL of dimethyl carbonate is loaded into a 5L reactor. A solution of DL-α-tocopherol (0.15g) is dissolved in dimethyl carbonate (60mL) and added. 600g of supercritical fluid extracted egg yolk powder is added and mixed with the 3600mL solution under stirring. The mixture is heated to 40-45°C and stirred at this temperature for 55-65 minutes. The hot mixture is filtered using a Buchner funnel equipped with 10μ Whatman filter paper and 10μ filter cloth. The filtered solid is washed with 1200mL (600mL×2) of dimethyl carbonate. The solid is dried at 20-30°C under a vacuum of 200-300mbar (610-535mmHg) for 4-6 hours.

[0199] The resulting DMC-washed SFE-EYP was extracted with 3540 mL of a solvent mixture of ethanol and acetonitrile (4800 mL of the solvent mixture was prepared using 2736 mL of anhydrous ethanol and 2064 mL of acetonitrile) at a temperature in the range of 20-25°C, followed by the addition of the DL-α-tocopherol solution. The mixture was heated to a temperature of 55-60°C and then stirred for 30-40 minutes. The hot slurry was filtered, and the resulting solid was washed with 1200 mL (600 mL x 2) of the solvent mixture. The filtrate was treated with charcoal at a temperature in the range of 40-45°C and then stirred for 55-65 minutes.

[0200] The solution obtained is cooled to -12 ℃ to -8 ℃, stirred for 90 minutes at 100RPM to further precipitate impurities. The mixture obtained is filtered through a jacketed funnel while the jacket temperature is maintained at -12 ℃ to -8 ℃, and then DL-α-tocopherol (prepared by dissolving 0.048g DL-α-tocopherol in the solvent mixture prepared before 30mL) is added to the filtered solution obtained. The solution obtained is filtered again through 5μ and 0.22μ polyvinylidene fluoride filters, then concentrated in vacuo below 35 ℃ until the remaining residue is viscous, and the remaining dope is dried to obtain 161.8g (0.27w / w) of purified egg yolk lecithin.

[0201] Example 5: Determination of purified egg yolk lecithin components by HPLC-CAD analysis

[0202] The contents of PC, PE, SPH, LPC, LPE, and cholesterol shown in Table 1 were determined by high-performance liquid chromatography (HPLC) equipped with CAD. CAD is used to detect nonvolatile analytes that do not absorb UV light by detecting charged aerosol particles generated when the mobile phase and analytes evaporate under the following conditions.

[0203] The mobile phase used was as follows:

[0204] Mobile phase A: Contains acetonitrile.

[0205] Mobile phase B: Prepare by accurately weighing 1.3 g of ammonium formate into a 1 L bottle containing 1000 mL of milli-q water. Ultrasonicate the mixture until homogenized. Adjust the pH to 3.30 by adding formic acid. Filter the solution through 0.22 mm filter paper.

[0206] Mobile phase C: Contains a mixture prepared by accurately transferring 500 mL of methanol and 500 mL of milli-q water to a 1 L bottle, followed by ultrasonic mixing and degassing.

[0207] Chloroform:methanol at a ratio of 2:1 was used as diluent and blank.

[0208] The chromatographic conditions used were as follows:

[0209] Table 5:

[0210] left pump

[0211]

[0212] Table 6:

[0213] Right pump

[0214]

[0215]

Claims

1. A method for preparing purified egg yolk lecithin, comprising the following steps: a) providing supercritical fluid extracted egg yolk powder as a starting material, b) treating the supercritical fluid extracted egg yolk powder with solvent 1, c) separating the product obtained in step b) from solvent 1, d) extracting the egg yolk lecithin fraction from the product obtained in step c) using solvent 2, e) optionally, treating the egg yolk lecithin fraction obtained in step d) with charcoal, f) precipitating impurities in the egg yolk lecithin fraction obtained in step d) or step e) by cooling the solution obtained in step d) or step e), g) removing precipitated impurities from the product obtained in step f), h) isolating purified egg yolk lecithin from the product obtained in step g), and i) optionally, drying the purified egg yolk lecithin; Wherein, in step d), the solvent 2 is a solvent mixture consisting of ethanol and acetonitrile.

2. The method according to claim 1, wherein step b), step d), step e) and / or step g) further comprises adding a free radical scavenger.

3. The method according to claim 2, wherein the free radical scavenger is DL-α-tocopherol.

4. A method according to any preceding claim, wherein step b) comprises adding supercritical fluid extracted egg yolk powder to solvent 1 and stirring the resulting mixture at an elevated temperature.

5. The process according to claim 4, wherein step b) is carried out at a temperature in the range of 30-60°C and preferably maintained under stirring for a time interval of 50-70 minutes.

6. The method according to any one of claims 1 to 3, wherein the volume / volume ratio of ethanol to acetonitrile is 57:

43.

7. The process according to any one of claims 1 to 3, wherein the extraction in step d) is carried out at a temperature in the range of 50-60°C.

8. The process according to any one of claims 1 to 3, wherein during the extraction in step d), the solution is stirred for a period of 30-60 minutes.

9. The method according to any one of claims 1 to 3, wherein in step e), the charcoal treatment is performed at a temperature in the range of 30-50°C for a time interval of 30 minutes to 3 hours.

10. Purified egg yolk lecithin obtained by the method according to any one of claims 1 to 9.

11. The purified egg yolk lecithin according to claim 10, wherein the ratio of phosphatidylcholine to phosphatidylethanolamine is 7.5-9.0, more preferably 8.0-9.0, and most preferably 8.5-8.

7.

12. The purified egg yolk lecithin according to claim 10 or 11, which is substantially free of aluminum ions.

13. A lipid emulsion comprising the purified egg yolk lecithin according to any one of claims 10 to 12.

14. A nutritional product suitable for parenteral administration comprising a lipid emulsion comprising the purified egg yolk lecithin according to any one of claims 10 to 12.

15. A method for preparing a lipid emulsion, comprising: a) providing an oil phase comprising oil, and optionally a pharmaceutically acceptable antioxidant, optionally a pharmaceutically acceptable co-emulsifier, preferably free long-chain fatty acids, wherein the fatty acids are present in triglyceride-bound form; b) providing an aqueous phase comprising water for injection, and optionally a pharmaceutically acceptable tonicity agent and / or pH adjusting agent and / or chelating agent; c) forming a pre-emulsion by mixing the oil phase provided in step a) with the water phase provided in step b); d) forming an oil-in-water emulsion by high pressure homogenization of the pre-emulsion obtained in step c); and e) sterilizing the oil-in-water emulsion obtained in step d), wherein the oil-in-water emulsion is optionally filled into suitable containers before or after sterilization, Wherein, the egg yolk lecithin according to any one of claims 11 to 13 is added in step a) or step b).

16. The method according to claim 15, wherein a pharmaceutically acceptable antioxidant is provided in step a), and the antioxidant is DL-α-tocopherol.

17. The method of claim 16, wherein the oil is selected from the group consisting of olive oil, soybean oil, fish oil, and medium chain triglycerides.

Images