Method for extracting oil-soluble molecule

Abstract

The problem addressed by the present invention is to provide a method capable of efficiently and stably extracting oil-soluble molecules without using an amphiphilic organic solvent such as isopropanol or a lipophilic surfactant, both of which need to be removed from a hydrophobic organic solvent.　Provided is a method for extracting oil-soluble molecules from cells or cell lysates thereof into a hydrophobic organic solvent. The method comprises: an emulsification step that emulsifies an aqueous solution containing cells or cell lysate thereof and a hydrophobic organic solvent to form an oil-in-water emulsion solution; and a demulsification step that separates a water phase and an oil phase of the oil-in-water emulsion solution. The emulsification step is performed under conditions that do not substantially contain an amphiphilic organic solvent and / or a lipophilic surfactant.

Description

Method for extracting oil-soluble molecules

[0001] The present invention relates to a method for extracting oil-soluble molecules, a method for preparing an oil-soluble molecule-containing solution, and a method for preparing oil-soluble molecules.

[0002] Biomolecules, due to their high biocompatibility and the unique functions of each molecule, are attracting attention in fields such as health promotion and are widely available as supplements. For example, biomolecules with antioxidant properties, such as coenzyme Q, are considered particularly important for preventing lifestyle-related diseases and for anti-aging. Therefore, the importance of efficient industrial production of biomolecules that can be used in supplements is increasing year by year.

[0003] Coenzyme Q is an essential component widely distributed in living organisms, from bacteria to mammals, and is known as a component of the electron transport chain in mitochondria within cells. Coenzyme Q functions as a transporter in the electron transport chain by repeatedly undergoing oxidation and reduction within mitochondria, and reduced coenzyme Q is known to have antioxidant properties. In humans, coenzyme Q is mainly composed of coenzyme Q10, which has 10 repeating structures in its side chain, and in living organisms, approximately 40-90% usually exists in the reduced form. Known physiological effects of coenzyme Q include activation of energy production through mitochondrial activation, activation of cardiac function, stabilization of cell membranes, and protective effects on cells through antioxidant activity.

[0004] Various methods are known for producing oil-soluble molecules such as coenzyme Q. For example, a widely used method involves having microorganisms produce coenzyme Q and then extracting it from the cell lysates into an organic solvent. Known methods for extracting oil-soluble molecules from cells or their cell lysates include solid-liquid extraction, in which cells or their cell lysates prepared as a solid are brought into contact with a hydrophobic organic solvent for extraction, and liquid-liquid extraction, in which a suspension of cells or their cell lysates is brought into contact with a hydrophobic organic solvent for extraction.

[0005] Known solid-liquid extraction methods include dehydrating an aqueous suspension of cells or cell lysates to obtain wet cells and then contacting them with a hydrophobic organic solvent, and dehydrating the aqueous suspension, drying it further, and then contacting the dried cells with a hydrophobic organic solvent (Patent Document 1). However, solid-liquid extraction methods require the dehydration and drying of the aqueous suspension, and depending on the remaining moisture content, a sufficient extraction rate may not be obtained. In addition, there are problems such as high equipment costs and operating costs.

[0006] On the other hand, when using liquid-liquid extraction, the steps of dehydrating and drying the aqueous suspension, which are required in solid-liquid extraction, are unnecessary. Therefore, liquid-liquid extraction is widely used as a more practical method compared to solid-liquid extraction. Generally, when extracting oil-soluble molecules from cells or their cell lysates into a hydrophobic organic solvent by liquid-liquid extraction, amphiphilic organic solvents such as isopropanol are used in combination to achieve both high extraction efficiency and high static separation between the aqueous phase (cell or cell lysate phase) and the oil phase (hydrophobic organic solvent phase) after the extraction operation (Patent Document 2). However, since the amphiphilic organic solvent used here moves and mixes into both the aqueous and oil phases during the extraction operation, it is necessary to remove, recover, and reuse it from the hydrophobic organic solvent from which the oil-soluble molecules have been extracted in a subsequent step. This requires additional steps such as a washing step to remove the amphiphilic solvent from the hydrophobic organic solvent, and a solvent recovery step using an evaporator or distillation column to recover the amphiphilic solvent from the washing solution, resulting in complicated operations and increased costs. Furthermore, in terms of extraction efficiency, the amphiphilic organic solvent resulted in excessive contact between the aqueous and oil phases, leading to the extraction of impurities other than the target oil-soluble molecules into the hydrophobic organic solvent. Additionally, insufficient separation of the aqueous and oil phases resulted in the oil phase containing the target oil-soluble molecules remaining in the aqueous phase, leading to a decrease in yield.

[0007] Therefore, a method using a surfactant instead of isopropanol has been proposed (Patent Document 3).

[0008] WO 2004 / 011660 Japanese Patent Publication No. 2008-253271 WO 2012 / 011589

[0009] In methods utilizing surfactants, lipophilic surfactants are used to achieve both high extraction efficiency and high static separation between the aqueous phase (cells or their cell disrupted product phase) and the oil phase (hydrophobic organic solvent phase) after extraction. While the use of such specific surfactants showed improvements in terms of residual oil in the aqueous phase and reduced yield, problems remained, such as the need for a step to remove the surfactant from the hydrophobic organic solvent after extraction and the extraction of impurities other than the target oil-soluble molecules.

[0010] Therefore, the object of the present invention is to provide a method for stably and efficiently extracting oil-soluble molecules without using amphiphilic organic solvents such as isopropanol or lipophilic surfactants, which need to be removed from hydrophobic organic solvents.

[0011] To solve the above problems, the inventors conducted intensive research and found that by emulsifying an aqueous solution containing cell disruptors and an organic solvent to form an oil-in-water emulsion, it is possible to efficiently extract oil-soluble molecules using only an aqueous solution containing cell disruptors and an organic solvent, without using amphiphilic organic solvents or lipophilic surfactants.

[0012] The present invention is based on the aforementioned novel findings and provides the following: [1] A method for extracting oil-soluble molecules in cells or cell lysates into a hydrophobic organic solvent, comprising an emulsification step of emulsifying an aqueous solution containing the cells or cell lysates with a hydrophobic organic solvent to form an oil-in-water emulsion, and a deemulsification step of separating the aqueous phase and the oil phase of the oil-in-water emulsion, wherein the emulsification step is carried out under conditions that substantially do not contain amphiphilic organic solvents and / or lipophilic surfactants. [2] The method according to [1], wherein the emulsification is carried out by one or more methods selected from the group consisting of high-speed rotation, pressure, and ultrasonic methods. [3] The method according to [1] or [2], wherein the separation in the deemulsification step is carried out by one or more of the addition of an alkaline agent, centrifugation, and heating. [4] The method according to any one of [1] to [3], wherein the content ratio of the aqueous solution in the oil-in-water emulsion is 15% to 90% by volume. [5] The method according to any one of [1] to [4], wherein the particle size of the oil phase in the oil-in-water emulsion is 5 μm to 100 μm. [6] The method according to any one of [1] to [5], wherein the pH of the aqueous solution is 1.5 to 6. [7] The method according to any one of [1] to [6], wherein the emulsification step is performed below the boiling point of the hydrophobic organic solvent. [8] The method according to any one of [1] to [7], wherein the SP value of the hydrophobic organic solvent is 7 to 10. [9] The method according to any one of [1] to [8], wherein the oil-soluble molecule is coenzyme Q.

[10] The method according to any one of [1] to [9], wherein the cell or its cell lysate is a microbial cell or its cell lysate.

[11] The method according to any one of [1] to

[10] , wherein the separation in the demulsification step is performed by any two or more selected from the group consisting of adding an alkaline agent, centrifugation, and heating.

[12] The method according to any one of [1] to

[11] , wherein the separation in the demulsification step is performed by adding an alkaline agent and / or centrifugation and heating.

[13] The method according to any one of [3] to

[12] , wherein the pH of the aqueous phase is 8 to 10 by adding the alkaline agent.

[14] The method according to any one of [3] to

[13] , wherein the heating is performed to a temperature of 50°C or higher and 65°C or lower.

[15] The method according to any one of [3] to

[14] , wherein the centrifugation is performed with a centrifugal force of 3,000 × g or more for 20 minutes or more.

[16] A method for preparing an oil-soluble molecule-containing solution containing oil-soluble molecules in cells or cell lysates thereof, comprising: an emulsification step of emulsifying an aqueous solution containing the cells or cell lysates thereof with a hydrophobic organic solvent to form an oil-in-water emulsion; a deemulsification step of separating the aqueous phase and the oil phase of the oil-in-water emulsion; and a recovery step of recovering the separated oil phase as an oil-soluble molecule-containing solution, wherein the emulsification step is performed under conditions that substantially do not contain amphiphilic organic solvents and / or lipophilic surfactants.

[17] A method for preparing oil-soluble molecules in cells or cell lysates thereof, comprising: an emulsification step of emulsifying an aqueous solution containing the cells or cell lysates with a hydrophobic organic solvent to form an oil-in-water emulsion; a deemulsification step of separating the aqueous phase and the oil phase of the oil-in-water emulsion; a recovery step of recovering the separated oil phase as an oil-soluble molecule-containing solution; and a purification step of purifying the oil-soluble molecules from the recovered oil-soluble molecule-containing solution, wherein the emulsification step is carried out under conditions substantially free of amphiphilic organic solvents and / or lipophilic surfactants. This specification encompasses the disclosures of Japanese Patent Application No. 2025-053404, which forms the basis of the priority of this application.

[0013] According to the extraction method of the present invention, oil-soluble molecules in cells or their cell disrupted products can be extracted into a hydrophobic organic solvent without using amphiphilic organic solvents or lipophilic surfactants.

[0014] According to the method for preparing an oil-soluble molecule-containing solution of the present invention, an oil-soluble molecule-containing solution containing oil-soluble molecules in cells or their cell disrupted products can be prepared without using amphiphilic organic solvents or lipophilic surfactants.

[0015] According to the present invention's method for preparing oil-soluble molecules, oil-soluble molecules in cells or their cell disrupted products can be prepared without using amphiphilic organic solvents or lipophilic surfactants.

[0016] This figure shows the results of Example 1. This figure shows the emulsion prepared in Example 1. Figure 2A shows an optical microscope image of the prepared emulsion. Figure 2B shows an example of the particle size distribution of the hexane phase of the prepared emulsion. In Figure 2B, the histogram shows the frequency of particles in the hexane phase with each particle size, and the curve shows the cumulative frequency. This figure shows the results of Example 2. In the figure, the black circles indicate the points of each data. This figure shows the results of Example 3. In the figure, the solvent ratio shows the value of volume of microbial cell lysate : volume of n-hexane. This figure shows the results of Example 4.

[0017] 1. Method for Extracting Oil-Soluble Molecules 1-1. Overview The first aspect of the present invention is a method for extracting oil-soluble molecules. The method of this aspect includes an emulsification step and a deemulsification step as essential steps. According to the method of this aspect, oil-soluble molecules in cells or their cell lysates can be extracted into a hydrophobic organic solvent with high efficiency without using amphiphilic organic solvents or lipophilic surfactants.

[0018] 1-2. Definitions The terms used herein are defined below. In this specification, “hydrophobic organic solvent” means an organic solvent whose solubility in water at 25°C and 1013 hPa is less than 1% by weight. In particular, hydrophobic organic solvents in this specification do not include amphiphilic organic solvents or lipophilic surfactants.

[0019] "Extraction" refers to the process of eluting specific components from raw materials into a target solvent and preparing them in a recoverable state. In particular, extraction as used herein refers to the process of eluting oil-soluble molecules in an aqueous solution into a hydrophobic organic solvent and preparing them in a recoverable state.

[0020] In this specification, "aqueous solution" means a solution using water as the solvent. In particular, aqueous solutions in this specification use water as the main solvent and contain an oil-soluble molecule as one of the solutes.

[0021] An "emulsion" refers to a system in which two liquid phases with low affinity for each other are dispersed in particulate form within the other phase. In particular, the emulsion as used herein refers to an emulsion with high storage stability that does not immediately separate after emulsification.

[0022] The "aqueous phase" refers to the liquid phase of an emulsion whose solvent is water. On the other hand, the "oil phase" refers to the liquid phase of an emulsion that is not the aqueous phase. In particular, in this specification, the oil phase broadly refers to the organic phase, and its solvent is a hydrophobic organic solvent.

[0023] In this specification, "oil-in-water (o / w) emulsion" refers to an emulsion in which the oil phase is the dispersed phase and the aqueous phase is the continuous phase.

[0024] The type of emulsion can also be determined using a drop test. A drop test involves dropping a drop of the obtained emulsion onto the surface of water and checking whether or not the emulsion disperses. In this drop test, if the emulsion does not disperse immediately in water, it can be determined to be a "water-in-oil (w / o) emulsion," and if it disperses immediately in water, it can be determined to be an "oil-in-water (o / w) emulsion."

[0025] "Emulsification" or "emulsification process" refers to the process of mixing an aqueous phase and an oil phase to form an emulsion.

[0026] In this specification, "amphiphilic organic solvent" refers to a solvent composed of an organic compound having both hydrophilic and hydrophobic groups. For example, an amphiphilic organic solvent having a solubility of 1% by weight or more in a hydrophobic organic solvent at 25°C and 1013 hPa is an example. Another example is an amphiphilic organic solvent having a solubility of 1% by weight or more in water at 25°C and 1013 hPa.

[0027] In this specification, "lipophilic surfactant" refers to a surfactant with an HLB (Hydrophilic Lipophilic Balance) value greater than 0 and less than or equal to 8. For example, surfactants with a solubility of 1% by weight or more in hydrophobic organic solvents at 25°C and 1013 hPa are included. Generally, neither amphiphilic organic solvents nor lipophilic surfactants in this specification include intrinsic cell-derived components originally present in cells or their cell lysates. The method for calculating the HLB value is not particularly limited, but examples include the Davis method, Atlas method, Griffin method, and Kawakami method. In this specification, the HLB value used shall be the value based on the Davis method.

[0028] In this specification, "separation" means the phase separation of an emulsion into two liquid phases. Through this separation, the emulsion appears to separate into two liquid phases, each of which can be isolated.

[0029] 1-3. Process The method of this embodiment includes an emulsification step and a de-emulsification step as essential steps, and includes a raw material solution preparation step and an emulsified solution maintenance step as optional steps. Each step will be described in detail below.

[0030] 1-3-1. Raw material solution preparation step The "raw material solution preparation step" is an optional step in the method of this embodiment, and is a step of preparing an aqueous solution containing cells containing oil-soluble molecules or cell lysates thereof.

[0031] The aqueous solution prepared in this process contains cells and / or cell lysates. The type of cells in this embodiment is not particularly limited, and cells of any biological origin can be used. In this case, the cells may be of one type of biological origin or multiple types of biological origin. For example, microbial cells can be used.

[0032] In this specification, "microorganism" refers to a single-celled organism, encompassing both eukaryotic single-celled organisms (such as yeast) and prokaryotes. Typically, prokaryotes are included. The type of microorganism is not limited. For example, either eukaryotic cells and / or prokaryotic cells, such as fungi including yeast, can be used. When bacteria are used, the specific type is not particularly limited, but examples include: Agrobacterium, Aspergillus, Acetobacter, Aminobacter, Agromonas, Acidiphilium, Bulleromyces, Bullera, Brevundimonas, Cryptococcus, Chionosphaera, Candida, Cerinosterus, Exisophiala, Exobasidium, Fellomyces, Filobasidiella, Filobasidium um), Geotrichum, Graphiola, Gluconobacter, Kockovaella, Kurtzmanomyces, Lalaria, Leucosporidium, Legionella, Methylobacterium, Mycoplana coplana), Oosporidium, Pseudomonas, Psedozyma, Paracoccus, Petromyces, Rhodotorula, Rhodosporidium, Rhizomonas, Rhodobium, Rhodoplanes,The genera Rhodopseudomonas, Rhodobacter, Sporobolomyces, Sporidiobolus, Saitoella, Schizosaccharomyces, Sphingomonas, Sporotrichum, Sympodiomycopsis, Sterigmatosporidium, Tapharina, Tremella, and Trichosporon Examples of microorganisms include those of the genera Tilletiaria, Tilletia, Tolyposporium, Tilletiopsis, Ustilago, Udeniomyce, Xanthophllomyces, Xanthobacter, Paecilomyces, Acremonium, Hyhomonus, Rhizobium, Phaffia, and Haematococcus. For example, bacteria such as Agrobacterium and Gluconobacter, and yeasts such as Schizosaccharomyces, Saitoella, and Phaffia can be suitably used.

[0033] The method for preparing such cells is not particularly limited. For example, they can be prepared by culturing them under culture conditions suitable for the cells to be used.

[0034] If an aqueous solution contains cells, those cells may be living cells, dead cells, or a combination of both.

[0035] In this specification, "cell disruption product" means a preparation containing cell-derived components, which include one or more of the following: cell-derived membrane components or cell contents. In this specification, cell disruption products include concentrates, disruptions, pastes, sterilized products, dissolved products, diluted products, extracts, or dried products (e.g., spray-dried products, vacuum-dried products, drum-dried products, freeze-dried products, or freeze-dried disruptions) obtained by concentrating cells or their cultures using an evaporator or the like.

[0036] The method for preparing cell lysates is not particularly limited. For example, any known disruption, dissolution, or extraction method in the art can be used. Specific methods for preparing cell lysates include, for example, homogenizing the cell culture medium using a high-pressure homogenizer, rotary-blade homogenizer, ultrasonic homogenizer, French press, ball mill, etc., or obtaining them by sonication or freeze-thaw. Specific dissolution methods include, for example, decomposition with cellular enzymes or chemical substances such as acids and bases. Enzymes usable in such methods include, for example, one or more enzymes selected from amylase, lipase, and protease. Chemical treatments include, for example, treatment using acids (preferably strong acids) such as hydrochloric acid and sulfuric acid, or treatment using bases (preferably strong bases) such as sodium hydroxide and potassium hydroxide.

[0037] The solvent in an aqueous solution is water or an aqueous solution. In the case of an aqueous solution, the type of solute is not particularly limited, but examples include organic acids, organic bases, inorganic acids, inorganic bases, or combinations thereof. The aqueous solution may also contain culture medium components, such as water, physiological saline, buffer solutions, and culture media.

[0038] The concentration of cells or cell-derived solid contents contained in the aqueous solution is not particularly limited. Examples include 0.1 g / L to 500 g / L, 1 g / L to 400 g / L, 10 g / L to 300 g / L, 10 g / L to 200 g / L, 10 g / L to 150 g / L, 25 g / L to 150 g / L, preferably 30 g / L to 150 g / L, 40 g / L to 150 g / L, 50 g / L to 150 g / L, 60 g / L to 140 g / L, 70 g / L to 130 g / L, 80 g / L to 120 g / L, 80 g / L to 110 g / L, 80 g / L to 100 g / L, 90 g / L to 110 g / L, 90 g / L to 100 g / L, etc.

[0039] At least a part of the cells used for preparing the aqueous solution contains oil-soluble molecules. Generally, the cells to be used include at least cells capable of producing oil-soluble molecules. The cells capable of producing oil-soluble molecules in this case may be natural cells, or may be cells genetically modified by genome editing or the like.

[0040] The oil-soluble molecule is not particularly limited as long as it is a biomolecule soluble in hydrophobic organic solvents. Specific examples of the oil-soluble molecule include coenzyme Q, sterol derivatives (ergosterol, dihydroergosterol, etc.), sterol esters having a fatty acid or the like ester-bonded to the terminal thereof (sterol fatty acid esters, etc.), and the like. Specific examples of coenzyme Q include coenzyme Q9, Q10, Q11 or a combination thereof. Coenzyme Q10 exists in oxidized form and reduced form, and both can be used in the method of the present invention.

[0041] The concentration of the oil-soluble molecule in the aqueous solution is not particularly limited, and for example, 1.0×10 -4 M to 1 M, 1.0×10 -4 M to 0.1 M, 1.0×10 -4 M to 0.05 M, 5.0×10 -4 M to 0.01 M, 5.0×10 -4 M to 9.0×10 -3 M, 5.0×10 -4 M to 8.0×10 -3 M, 5.0×10 -4 M to 7.0×10 -3 M, 6.0×10 -4 M to 7.0×10-3 M, 7.0×10 -4 M~7.0×10 -3 M, 1.0×10 -3 M to 0.03M, 5.0 x 10 -3 M to 0.03M, 6.0 x 10 -3 M to 0.03M, 7.0 x 10 -3 M to 0.03M, 1.0 x 10 -3 M to 0.02M, 5.0 x 10 -3 M to 0.02M, 6.0 x 10 -3 M to 0.02M, 7.0 x 10 -3 M to 0.02M, 1.0 × 10 -3 M to 0.01M, 5.0 x 10 -3 M to 0.01M, 6.0 x 10 -3 M to 0.01M, 7.0 x 10 -3 It can be set to M to 0.01M, etc.

[0042] The aqueous solution may be acidic or basic, but for example, an acidic aqueous solution can be used. The specific pH of the aqueous solution is not particularly limited, but for example, it can be 1.5-6.5, 2-6.5, 2.5-6.5, 3-6.5, 3.5-6.5, 4-6.5, 4.5-6.5, 5-6.5, 5.5-6.5, 1.5-6, 2-6, 2.5-6, 3-6, 3.5-6, 4-6, 4.5-6, 5-6, 5.5-6, etc.

[0043] Surfactants may be used in this process, but in the case of lipophilic surfactants in particular, it is preferable that they be removed before the next emulsification step.

[0044] The aqueous solutions, cells, and cell lysates prepared in this process may be subjected to any treatment before the emulsification process. Specific treatments are not limited, but examples include freezing, thawing, separation (such as solid-liquid separation), purification, culture, washing, sorting, transformation, genetic manipulation, or combinations thereof. For example, multiple aqueous solutions prepared in this process may be mixed and used in the emulsification process. Concentration methods are not limited, but examples include evaporation concentration, membrane concentration, freeze concentration, reduced-pressure concentration, ultrasonic atomization separation, or combinations thereof. Solid-liquid separation methods are not limited, but examples include filtration using filter paper, filter cloth, cylindrical filters, natural sedimentation separation, centrifugal separation, membrane separation, vibrating membrane separation, liquid cyclone, rotary filter, adsorption separation, or combinations thereof.

[0045] This process, one or more steps of the method according to this embodiment, or the method according to this embodiment may be carried out under a deoxygenated atmosphere. A deoxygenated atmosphere can be achieved by displacement with an inert gas, reduced pressure, boiling, or a combination thereof. When using an inert gas, the specific type of gas is not particularly limited. Examples include nitrogen gas, helium gas, argon gas, hydrogen gas, carbon dioxide gas, or a combination thereof.

[0046] The aqueous solution may contain additional components such as antioxidants including zinc, hyposulfites, and ascorbic acid.

[0047] 1-3-2. Emulsification Step The "emulsification step" is an essential step in the method of this embodiment, and is a step in which an aqueous solution containing cells or cell lysates is emulsified with a hydrophobic organic solvent to form an oil-in-water emulsion solution. This step is carried out under conditions that substantially do not contain amphiphilic organic solvents and / or lipophilic surfactants. This step can be carried out after the raw material solution preparation step if one is performed. The aqueous solution is as described in detail in the raw material solution preparation step.

[0048] The type of hydrophobic organic solvent is not particularly limited, as long as it is a hydrophobic organic solvent capable of dissolving oil-soluble molecules. For example, if the SP (Solubility Parameter) value is 5.5 (cal / cm³). 3 ) 1 / 2 ~11 (cal / cm 3 ) 1 / 2、6(cal / cm 3 ) 1 / 2 ~11(cal / cm 3 ) 1 / 2 、6(cal / cm 3 ) 1 / 2 ~10(cal / cm 3 ) 1 / 2 、6(cal / cm 3 ) 1 / 2 ~9(cal / cm 3 ) 1 / 2 、6(cal / cm 3 ) 1 / 2 ~8(cal / cm 3 ) 1 / 2 、6(cal / cm 3 ) 1 / 2 ~7.5(cal / cm 3 ) 1 / 2 、6.5(cal / cm 3 ) 1 / 2 ~11(cal / cm 3 ) 1 / 2 、6.5(cal / cm 3 ) 1 / 2 ~10(cal / cm 3 ) 1 / 2 、6.5(cal / cm 3 ) 1 / 2 ~9(cal / cm 3 ) 1 / 2 、6.5(cal / cm 3 ) 1 / 2 ~8(cal / cm 3 ) 1 / 2 、6.5(cal / cm 3 ) 1 / 2 ~7.5(cal / cm 3 ) 1 / 2 、7(cal / cm 3 ) 1 / 2 ~11(cal / cm 3 ) 1 / 2 、7(cal / cm 3 ) 1 / 2 ~10(cal / cm 3 ) 1 / 2 、7(cal / cm 3 ) 1 / 2 ~9(cal / cm 3 ) 1 / 2 、7(cal / cm 3 ) 1 / 2 ~8(cal / cm3 ) 1 / 2 , 7 (cal / cm 3 ) 1 / 2 to 7.5 (cal / cm 3 ) 1 / 2 , 7.2 (cal / cm 3 ) 1 / 2 to 11 (cal / cm 3 ) 1 / 2 , 7.2 (cal / cm 3 ) 1 / 2 to 10 (cal / cm 3 ) 1 / 2 , 7.2 (cal / cm 3 ) 1 / 2 to 9 (cal / cm 3 ) 1 / 2 , 7.2 (cal / cm 3 ) 1 / 2 to 8 (cal / cm 3 ) 1 / 2 , 7.2 (cal / cm 3 ) 1 / 2 to 7.5 (cal / cm 3 ) 1 / 2 , 7.3 (cal / cm 3 ) 1 / 2 to 11 (cal / cm 3 ) 1 / 2 , 7.3 (cal / cm 3 ) 1 / 2 to 10 (cal / cm 3 ) 1 / 2 , 7.3 (cal / cm 3 ) 1 / 2 to 9 (cal / cm 3 ) 1 / 2 , 7.3 (cal / cm 3 ) 1 / 2 to 8 (cal / cm 3 ) 1 / 2 , 7.3 (cal / cm 3 ) 1 / 2 to 7.5 (cal / cm 3 ) 1 / 2 , a hydrophobic organic solvent can be used.

[0049] The specific type of hydrophobic organic solvent is not limited. For example, hydrocarbons, fatty acid esters, ethers, alcohols, fatty acids, ketones, nitrogen compounds (including nitriles and amides), sulfur compounds, or combinations thereof can be used. For example, n-hexane, dichlorodifluoromethane, ethane, n-butane, n-pentane, 1,3-butadiene, amyl ether, ethyl caprylate, diamyl ether, n-heptane, isoprene, n-octane, 1-bromopentane, n-octane, diisobutylene, 2-undecanone, ethylhexyl acrylate, vinyl chloride, isoamyl acetate, n-isobutyl acetate, diisobutyl ketone, methyl isovalerate, ethyl isobutyrate, methyl valerate, isoamyl formate, diisopropyl ketone, ethylamyl ketone, 3-octanone, ethyl benzoate, sec-butyl acetate, cyclohexane, 1-chloropentane, methyl isobutyrate, triethyl orthoformate, amyl acetate, isobutyl acetate, butyl acetate, n-butyrate Examples include ropil, 2-bromobutane, 1-iodopentane, 2-ethylhexyldiphenyl phosphate, n-butyl acetate, 1-chloropropane, n-amyl formate, amyl acetate, n-amyl acetate, 1,1,1-trichloroethane, carbon tetrachloride, 1-bromobutane, dioctyl adipate, cyclopentane, n-butyl acrylate, ethylbenzene, allyl chloride, p-xylene, p-chlorotoluene, 2,6-dimethyl-4-heptanone, n-butyl formate, 1,2-dichloropropane, ethyl chloride, chloroform, chlorostyrene, chlorobenzene, 1,1,2,2-tetrachloroethane, methyl chloride, 2-bromostyrene, anthracene, 1,2-dichlorobenzene, 1,2-dichlorobenzene, or combinations thereof. For example, n-hexane can be suitably used.

[0050] Aliphatic hydrocarbons can be cyclic, acyclic, saturated, or unsaturated, and are not particularly limited. For example, saturated aliphatic hydrocarbons can be suitably used. The number of carbon atoms is not particularly limited, but for example, aliphatic hydrocarbons with 3 to 20, 5 to 12, or 5 to 8 carbon atoms can be used. Specifically, examples include propane, butane, isobutane, pentane, 2-methylbutane, hexane, 2-methylpentane, 2,2-dimethylbutane, 2,3-dimethylbutane, heptane, heptane isomers (e.g., 2-methylhexane, 3-methylhexane, 2,3-dimethylpentane, 2,4-dimethylpentane), octane, 2,2,3-trimethylpentane, isooctane, nonane, 2,2,5-trimethylhexane, decane, dodecane, 2-pentene, 1-hexene, 1-heptene, 1-octene, 1-nonene, 1-decene, cyclopentane, methylcyclopentane, cyclohexane, methylcyclohexane, ethylcyclohexane, p-menthane, cyclohexene, etc. Preferably, examples include pentane, 2-methylbutane, hexane, 2-methylpentane, 2,2-dimethylbutane, 2,3-dimethylbutane, heptane, 2-methylhexane, 3-methylhexane, 2,3-dimethylpentane, 2,4-dimethylpentane, octane, 2,2,3-trimethylpentane, isooctane, nonane, 2,2,5-trimethylhexane, decane, dodecane, cyclopentane, methylcyclopentane, cyclohexane, methylcyclohexane, ethylcyclohexane, p-menthane, and the like. Furthermore, for example, pentane, 2-methylbutane, hexane, 2-methylpentane, 2,2-dimethylbutane, 2,3-dimethylbutane, heptane, 2-methylhexane, 3-methylhexane, 2,3-dimethylpentane, 2,4-dimethylpentane, octane, 2,2,3-trimethylpentane, isooctane, cyclopentane, methylcyclopentane, cyclohexane, methylcyclohexane, ethylcyclohexane, etc. can be suitably used, and pentane, hexane, cyclohexane, methylcyclohexane, etc. can be suitably used, and heptane, hexane, methylcyclohexane, and especially heptane and hexane can be suitably used due to their particularly high protective effect against oxidation and their versatility.

[0051] The specific aromatic hydrocarbons are not particularly limited. Furthermore, the number of carbon atoms is not particularly limited, but for example, aromatic hydrocarbons with 6-20, 6-12, 7-10 carbon atoms can be used. Specifically, examples include benzene, toluene, xylene, o-xylene, m-xylene, p-xylene, ethylbenzene, cumene, mesitylene, tetralin, butylbenzene, p-cymene, cyclohexylbenzene, diethylbenzene, pentylbenzene, dipentylbenzene, dodecylbenzene, styrene, etc. Preferably, examples include toluene, xylene, o-xylene, m-xylene, p-xylene, ethylbenzene, cumene, mesitylene, tetralin, butylbenzene, p-cymene, cyclohexylbenzene, diethylbenzene, pentylbenzene, etc. Also, for example, toluene, xylene, o-xylene, m-xylene, p-xylene, cumene, tetralin, etc. can be suitably used, and cumene, etc., can be suitably used.

[0052] The halogenated hydrocarbon may be cyclic, acyclic, saturated, or unsaturated, and is not particularly limited. For example, acyclic halogenated hydrocarbons can be suitably used. The number of carbon atoms is not particularly limited, but for example, halogenated hydrocarbons with 1 to 6, 1 to 4, or 1 to 2 carbon atoms can be used. The type of halogen is not particularly limited. For example, chlorinated hydrocarbons, fluorinated hydrocarbons, etc., can be suitably used. Specifically, examples include dichloromethane, chloroform, carbon tetrachloride, 1,1-dichloroethane, 1,2-dichloroethane, 1,1,1-trichloroethane, 1,1,2-trichloroethane, 1,1,1,2-tetrachloroethane, 1,1,2,2-tetrachloroethane, pentachloroethane, hexachloroethane, 1,1-dichloroethylene, 1,2-dichloroethylene, trichloroethylene, tetrachloroethylene, 1,2-dichloropropane, 1,2,3-trichloropropane, chlorobenzene, 1,1,1,2-tetrafluoroethane, and the like. Preferably, examples include dichloromethane, chloroform, carbon tetrachloride, 1,1-dichloroethane, 1,2-dichloroethane, 1,1,1-trichloroethane, 1,1,2-trichloroethane, 1,1-dichloroethylene, 1,2-dichloroethylene, trichloroethylene, chlorobenzene, 1,1,1,2-tetrafluoroethane, etc. In addition, for example, dichloromethane, chloroform, 1,2-dichloroethylene, trichloroethylene, chlorobenzene, 1,1,1,2-tetrafluoroethane, etc. can be suitably used.

[0053] The specific fatty acid esters are not particularly limited. Examples include propionic acid esters, acetate esters, and formic acid esters. Preferably, acetate esters and formic acid esters are used, and for example, acetate esters can be suitably used. The number of carbon atoms in the ester group is not particularly limited, but for example, alkyl esters with 1 to 8 or 1 to 6 carbon atoms, or aralkyl esters with 7 to 12 carbon atoms can be used, and for example, alkyl esters with 1 to 4 carbon atoms can be suitably used. The specific propionic acid esters are not particularly limited, but examples include methyl propionate, ethyl propionate, butyl propionate, and isopentyl propionate, and for example, ethyl propionate can be suitably used. The specific acetate esters are not particularly limited, but examples include methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate, isobutyl acetate, sec-butyl acetate, pentyl acetate, isopentyl acetate, sec-hexyl acetate, cyclohexyl acetate, and benzyl acetate. Preferably, examples include methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate, isobutyl acetate, sec-butyl acetate, pentyl acetate, isopentyl acetate, sec-hexyl acetate, cyclohexyl acetate, etc. For example, methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate, isobutyl acetate, etc., with ethyl acetate being particularly suitable. Specific formate esters are not particularly limited, but examples include methyl formate, ethyl formate, propyl formate, isopropyl formate, butyl formate, isobutyl formate, sec-butyl formate, pentyl formate, etc. Preferably, examples include methyl formate, ethyl formate, propyl formate, butyl formate, isobutyl formate, pentyl formate, etc., with ethyl formate being particularly suitable.

[0054] The ether may be cyclic, acyclic, saturated, or unsaturated, and is not particularly limited. For example, saturated ethers can be suitably used. The number of carbon atoms is not particularly limited, but for example, ethers with 3 to 20, 4 to 12, or 4 to 8 carbon atoms can be used. Specifically, examples include diethyl ether, methyl tert-butyl ether, dipropyl ether, diisopropyl ether, dibutyl ether, dihexyl ether, ethyl vinyl ether, butyl vinyl ether, anisole, phenethole, butylphenyl ether, methoxytoluene, dioxane, furan, 2-methylfuran, tetrahydrofuran, tetrahydropyran, ethylene glycol dimethyl ether, ethylene glycol diethyl ether, ethylene glycol dibutyl ether, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, and ethylene glycol monobutyl ether. Preferably, diethyl ether, methyl tert-butyl ether, dipropyl ether, diisopropyl ether, dibutyl ether, dihexyl ether, anisole, phenethole, butylphenyl ether, methoxytoluene, dioxane, 2-methylfuran, tetrahydrofuran, tetrahydropyran, ethylene glycol dimethyl ether, ethylene glycol diethyl ether, ethylene glycol dibutyl ether, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, etc. are also examples, such as diethyl ether, methyl tert-butyl ether, anisole, dioxane, tetrahydrofuran, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, etc., and for example, diethyl ether, methyl tert-butyl ether, anisole, etc., and especially methyl tert-butyl ether, etc. can be suitably used.

[0055] While the specific ketone is not particularly limited, ketones with 3 to 6 carbon atoms, for example, can be suitably used. Specifically, examples include acetone, methyl ethyl ketone, methyl butyl ketone, and methyl isobutyl ketone. For example, acetone, methyl ethyl ketone, and especially acetone can be suitably used.

[0056] Nitriles can be cyclic, acyclic, saturated, or unsaturated, and are not particularly limited. For example, saturated nitriles can be suitably used. The number of carbon atoms is not particularly limited, but for example, nitriles with 2 to 20, 2 to 12, or 2 to 8 carbon atoms can be used. Specifically, for example, acetonitrile, propionitrile, malononitrile, butyronitrile, isobutyronitrile, succinonitrile, valeronitrile, glutalonitrile, hexanonitrile, heptyl cyanide, octyl cyanide, undecanenitrile, dodecanenitrile, tridecanenitrile, pentadecanenitrile, stearonitrile, chloroacetonitrile, bromoacetonitrile, chloropropionitrile, bromopropionitrile, methoxyacetonitrile, methyl cyanoacetate, ethyl cyanoacetate, tolunitrile, benzonitrile, chlorobenzonitrile, bromobenzonitrile, cyanobenzoic acid, Examples include nitrobenzonitrile, anisonitrile, phthalonitrile, bromotolunitrile, methylcyanobenzoate, methoxybenzonitrile, acetylbenzonitrile, naphthonitrile, biphenylcarbonitride, phenylpropionitrile, phenylbutyronitrile, methylphenylacetonitrile, diphenylacetonitrile, naphthylacetonitrile, nitrophenylacetonitrile, chlorobenzyl cyanide, cyclopropanecarbonitride, cyclohexanecarbonitride, cycloheptanecarbonitride, phenylcyclohexanecarbonitride, tolylcyclohexanecarbonitride, etc. Preferably, examples include acetonitrile, propionitrile, succinonitrile, butyronitrile, isobutyronitrile, valeronitrile, methyl cyanoacetate, ethyl cyanoacetate, benzonitrile, tolunitrile, chloropropionitrile, etc., and for example, acetonitrile, propionitrile, butyronitrile, isobutyronitrile, and especially acetonitrile can be suitably used.

[0057] Other nitrogen compounds besides nitriles may be used, and are not particularly limited, but examples include amides such as formamide, N-methylformamide, N,N-dimethylformamide, N,N-dimethylacetamide, and N-methylpyrrolidone, as well as nitromethane, triethylamine, and pyridine.

[0058] The specific sulfur compounds are not limited, but examples include dimethyl sulfoxide and sulfolane.

[0059] Among the solvents mentioned above, one may be selected based on properties such as being able to be heated to a suitable temperature, having a boiling point that facilitates drying and removal of the solvent from wet materials or recovery of the solvent from crystallization filtrate (approximately 30-150°C, 50-150°C, 60-150°C, and 65-150°C at 1 atm), having a melting point that does not solidify easily when handled at room temperature or when cooled below room temperature (approximately 20°C or below, approximately 10°C or below, and approximately 0°C or below), and having low viscosity (approximately 10 cp or less at 20°C, etc.).

[0060] In this process, an oil-in-water emulsion is prepared from an aqueous solution and a hydrophobic organic solvent, but the ratio of aqueous solution to hydrophobic organic solvent is not particularly limited. For example, the content ratio of aqueous solution in the oil-in-water emulsion is preferably 10% to 95% by volume, 15% to 90% by volume, 20% to 90% by volume, 25% to 90% by volume, 30% to 90% by volume, 33% to 90% by volume, 20% to 80% by volume, 25% to 80% by volume, 30% to 80% by volume, 33% to 80% by volume, 20% to 70% by volume, 25% to 70% by volume, 30% to 70% by volume, 33% to 70% by volume, It can be 20% to 60% by volume, 25% to 60% by volume, 30% to 60% by volume, 33% to 60% by volume, 20% to 50% by volume, 25% to 50% by volume, 30% to 50% by volume, 33% to 50% by volume, 20% to 45% by volume, 25% to 45% by volume, 30% to 45% by volume, 33% to 45% by volume, 20% to 40% by volume, 25% to 40% by volume, 30% to 40% by volume, 33% to 40% by volume, etc.

[0061] In this process, an aqueous solution and a hydrophobic organic solvent are emulsified together. The method of addition is not particularly limited. For example, the hydrophobic organic solvent may be added to the aqueous solution, or the aqueous solution may be added to the hydrophobic organic solvent, or both may be added to the container simultaneously, or both or one of the hydrophobic organic solvent and the aqueous solution may be added continuously and / or multiple times. Preferably, the hydrophobic organic solvent is added to the aqueous solution. If addition is performed multiple times, the method may be the same for all additions, or it may differ for one or more additions.

[0062] This process, and the emulsification in this process, are carried out under conditions that are substantially free of amphiphilic organic solvents and / or lipophilic surfactants. Preferably, they are carried out under conditions that do not contain either amphiphilic organic solvents or lipophilic surfactants. Examples of amphiphilic organic solvents and lipophilic surfactants here include amphiphilic molecules with an HLB value greater than 0 and 8 or less (for example, 8 or less, 7.5 or less, or 1 or more, 3 or more, 5 or more, 6 or more, 7 or more, 7.5 or more), such as hydrophilic alcohols (e.g., lower alcohols with 6 or fewer, 5 or fewer, 4 or fewer, or 3 or fewer carbon atoms, such as isopropyl alcohol), polyoxyethylene-polyoxypropylene block copolymer type surfactants, sucrose fatty acid esters, polyether type surfactants (e.g., polyether polyol type surfactants), and nonionic surfactants such as alkyl ether type surfactants. Substantially free of amphiphilic organic solvents and / or lipophilic surfactants means that the amount of amphiphilic organic solvents and / or lipophilic surfactants does not exceed the amount of components originally present in the cells or their cell lysates used. Typically, the oil-in-water emulsion does not contain amphiphilic organic solvents and / or lipophilic surfactants added from outside the cells or their cell lysates. For example, the content ratio of amphiphilic organic solvents and / or lipophilic surfactants added from outside the cells or their cell lysates in the oil-in-water emulsion can be 1% by volume or less, 0.5% by volume or less, 0.1% by volume or less, 0.05% by volume or less, 0.01% by volume or less, 0.005% by volume or less, 0.001% by volume or less, 0.0005% by volume or less, 0.0001% by volume or less, 0.00001% by volume or less, etc.

[0063] There are no particular limitations on the capacity of the container in which emulsification takes place. For example, it can be 1L or more, 2L or more, 5L or more, 10L or more, 15L or more, 20L or more, 25L or more, 50L or more, 75L or more, 100L or more, 150L or more, 200L or more, 250L or more, 500L or more, 750L or more, 1,000L or more, 2,000L or more, 5,000L or more, 10,000L or more, 15,000L or more, 20,000L or more, 50,000L or more, 100,000L or more, etc. If the containers are connected by piping, the capacity including the piping within the range affected by the external force applied for emulsification (for example, within the range of the space partitioned during emulsification if a partition is provided during emulsification) should be as stated above.

[0064] The emulsification method is not particularly limited. For example, one or more methods selected from the group consisting of high-speed rotation, pressure, and ultrasonic methods may be used.

[0065] The "high-speed rotation method" is a method of emulsification that uses the high shear force generated by the high-speed rotation of a rotating body (rotating blade or rotor). There are no particular limitations on the type of equipment used, but specific examples include installing a homomixer Mark II (manufactured by PRIMIX), an UltraTax homogenizer T25 (manufactured by IKA), or a CreaMix (manufactured by M-Technic) in a container. The homomixer Mark II is a device that emulsifies fluid as it passes through a minute clearance between a high-speed rotating turbine and a fixed outer cylinder (stator), while the CreaMix is ​​a device that emulsifies fluid discharged from a high-speed rotating rotor as it passes through a minute slit. The conditions for using such emulsification equipment can be set appropriately for each model and are not particularly limited. For example, the tip speed of the rotating body (diameter of the rotating body × number of rotations per second) can be 1 m / s or more, 2 m / s or more, 2.5 m / s or more, 3 m / s or more, 4 m / s or more, 5 m / s or more, etc. Specifically, for example, the tip speed of the rotating body can be set to 0m / s, 2m / s~40m / s, 2.5m / s~40m / s, 3m / s~40m / s, 4m / s~40m / s, 5m / s~40m / s, 1m / s~30m / s, 2m / s~30m / s, 2.5m / s~30m The speed can be 3 m / s to 30 m / s, 4 m / s to 30 m / s, 5 m / s to 30 m / s, preferably 1 m / s to 20 m / s, 2 m / s to 20 m / s, 2.5 m / s to 20 m / s, 3 m / s to 20 m / s, 4 m / s to 20 m / s, 5 m / s to 20 m / s, 1 m / s to 10 m / s, 2 m / s to 10 m / s, 2.5 m / s to 10 m / s, 3 m / s to 10 m / s, 4 m / s to 10 m / s, 5 m / s to 10 m / s, etc.

[0066] For emulsification using a high-speed rotation method, equipment capable of continuously processing large volumes of fluid, such as in-line emulsifiers and dispersers, may be used. While there are no specific limitations on the type of such equipment, examples include Cavitron (manufactured by Eurotech) and Silverson (manufactured by Silverson). In-line emulsifiers also emulsify using a high-speed rotating body and shear force at a small clearance, similar to the above method. However, to process large volumes in a short time, the tip speed required for emulsification tends to be higher. The various conditions of an in-line emulsifier and disperser can be appropriately set for each model and are not particularly limited. For example, the tip speed of the rotating body can be set to 10 m / s or more, 15 m / s or more, 20 m / s or more, etc., in addition to the range mentioned above. Specifically, for example, the tip velocity of the rotating body can be set to 5m / s to 100m / s, 5m / s to 90m / s, 5m / s to 80m / s, 5m / s to 70m / s, 5m / s to 60m / s, 5m / s to 50m / s, preferably 10m / s to 100m / s, 10m / s to 90m / s, 10m / s to 80m / s, 10m / s to 70m / s, 10m / s to 60m / s, or 10m / s to 50m / s. The speeds can be 15 m / s to 100 m / s, 15 m / s to 90 m / s, 15 m / s to 80 m / s, 15 m / s to 70 m / s, 15 m / s to 60 m / s, 15 m / s to 50 m / s, more preferably 20 m / s to 100 m / s, 20 m / s to 90 m / s, 20 m / s to 80 m / s, 20 m / s to 70 m / s, 20 m / s to 60 m / s, 20 m / s to 50 m / s, etc.

[0067] The "pressure method" is a method of emulsifying a liquid by applying high pressure to it and forcing it through a narrow channel. There are no particular restrictions on the type of equipment used (also called a high-pressure homogenizer, etc.), but specific examples include the Gorin homogenizer (manufactured by SMT Co., Ltd.), microfluidizer (manufactured by Powrec Co., Ltd.), Starburst (manufactured by Sugino Machine Co., Ltd.), and Nanomizer (manufactured by Nanomizer Co., Ltd.). The conditions for high-pressure emulsification using these devices can be set appropriately for each model and are not particularly limited. For example, the pressure conditions can be 40 MPa or higher, 50 MPa or higher, 60 MPa or higher, 70 MPa or higher, 80 MPa or higher, etc. Specifically, for example, the pressure conditions can be set to 40 MPa to 300 MPa, 40 MPa to 250 MPa, 40 MPa to 200 MPa, 40 MPa to 150 MPa, 40 MPa to 100 MPa, 50 MPa to 300 MPa, 50 MPa to 250 MPa, 50 MPa to 200 MPa, 50 MPa to 150 MPa, 50 MPa to 100 MPa, 60 MPa to 300 MPa, 60 MPa to 250 MPa, 60 MPa to 200 MPa, 60 MPa to 150 MPa, 60 MPa to 100 MPa, 70 MPa to 300 MPa, 70 MPa to 250 MPa, 70 MPa to 200 MPa, 70 MPa to 150 MPa, 70 MPa to 100 MPa, preferably 80 MPa to 300 MPa, 80 MPa to 250 MPa, 80 MPa to 200 MPa, 80 MPa to 150 MPa, 80 MPa to 100 MPa, etc.

[0068] For pressure-based emulsification, inline equipment such as inline static fluid mixing devices can be suitably used. Specifically, this includes methods of applying pressure to a static mixer (Noritake), an MSE static mixer (Iris Ohyama), an OHR mixer (OHR Fluid Engineering Laboratory), etc., to flow the fluid. A static mixer is a fluid mixing device in which spiral elements with different twist directions are arranged alternately in a pipe, with one end perpendicular to the other. An MSE static mixer is a fluid mixing device in which a stack of mixing elements having numerous small through-holes and a large through-hole in the center is arranged inside a pipe, or a fluid mixing device in which such mixing elements are installed inside a pipe. An OHR mixer is a fluid mixing device that promotes mixing and emulsification by increasing cavitation in the fluid through the provision of multiple protrusions on the inner circumferential wall surface of the pipe. The pressure required when using such inline static fluid mixing devices tends to be lower than that required for the high-pressure homogenizer. The conditions for emulsification with these devices can be set appropriately for each model and are not particularly limited. For example, the pressure conditions can be set to 0.1 MPa or higher, 0.2 MPa or higher, 0.3 MPa or higher, 0.4 MPa or higher, 0.5 MPa or higher, 0.6 MPa or higher, etc. Specifically, for example, the pressure conditions can be set to 0.1 MPa to 3 MPa, 0.1 MPa to 2.5 MPa, 0.1 MPa to 2 MPa, 0.1 MPa to 1.5 MPa, 0.1 MPa to 1 MPa, preferably 0.2 MPa to 3 MPa, 0.2 MPa to 2.5 MPa, 0.2 MPa to 1.5 MPa, 0.2 MPa to 1 MPa, 0.3 MPa to 3 MPa, 0.3 MPa to 2.5 MPa, 0.3 MPa to 2 MPa, 0.3 MPa to 1.5 MPa, 0.3 MPa to 1 MPa. a. More preferably, the pressure can be 0.4 MPa to 3 MPa, 0.4 MPa to 2.5 MPa, 0.4 MPa to 2 MPa, 0.4 MPa to 1.5 MPa, 0.4 MPa to 1 MPa, 0.5 MPa to 3 MPa, 0.5 MPa to 2.5 MPa, 0.5 MPa to 2 MPa, 0.5 MPa to 1.5 MPa, 0.5 MPa to 1 MPa, 0.6 MPa to 3 MPa, 0.6 MPa to 2.5 MPa, 0.6 MPa to 2 MPa, 0.6 MPa to 1.5 MPa, 0.6 MPa to 1 MPa, etc.

[0069] The "ultrasonic method" is a method of emulsification that generates cavitation by applying ultrasonic vibrations to a liquid. There are no particular restrictions on the type of equipment used (also called an ultrasonic homogenizer, etc.), but examples include the ultrasonic homogenizer UX series (manufactured by Mitsui Electric Seiki Co., Ltd.) and the ultrasonic homogenizer LUH150 (manufactured by Yamato Chemical Co., Ltd.). The conditions for emulsification with an ultrasonic homogenizer can be set appropriately for each model and are not particularly limited. For example, the frequency can be 15kHz or higher, 20kHz or higher, 25kHz or higher, 30kHz or higher, etc. Specifically, the frequency can be, for example, 15kHz to 100kHz, 20kHz to 80kHz, 25kHz to 60kHz, 30kHz to 50kHz, preferably 20kHz to 40kHz, etc.

[0070] There is no specific time limit for this process. For example, this process can be performed for 30 seconds or more, 1 minute or more, 1.5 minutes or more, 2 minutes or more, 5 minutes or more, 15 minutes or more, 16 minutes or more, etc. Specifically, for example, 30 seconds to 120 minutes, 30 seconds to 100 minutes, 30 seconds to 90 minutes, 30 seconds to 75 minutes, 30 seconds to 60 minutes, 30 seconds to 45 minutes, 30 seconds to 30 minutes, 30 seconds to 20 minutes, 30 seconds to 15 minutes, 30 seconds to 10 minutes, 30 seconds to 5 minutes, 30 seconds to 4 minutes, 30 seconds to 3 minutes, 1 minute to 120 minutes, 1 minute to 100 minutes, 1 minute to 90 minutes, 1 minute to 75 minutes. 1-60 minutes, 1-45 minutes, 1-30 minutes, 1-20 minutes, 1-15 minutes, 1-10 minutes, 1-5 minutes, 1-4 minutes, 1-3 minutes, 1.5-120 minutes, 1.5-100 minutes, 1.5-90 minutes, 1.5-75 minutes, 1.5-60 minutes, 1.5-45 minutes, 1.5-30 minutes, 1.5-20 minutes, 1.5-15 minutes minutes, 1.5-10 minutes, 1.5-5 minutes, 1.5-4 minutes, 1.5-3 minutes, 2-120 minutes, 2-100 minutes, 2-90 minutes, 2-75 minutes, 2-60 minutes, 2-45 minutes, 2-30 minutes, 2-20 minutes, 2-15 minutes, 2-10 minutes, 2-5 minutes, 2-4 minutes, 2-3 minutes, 5-120 minutes, 5-100 minutes, 5 The timer can be set to 15-90 minutes, 5-75 minutes, 5-60 minutes, 5-45 minutes, 5-30 minutes, 5-20 minutes, 15-120 minutes, 15-100 minutes, 15-90 minutes, 15-75 minutes, 15-60 minutes, 16-120 minutes, 16-100 minutes, 16-90 minutes, 16-75 minutes, 16-60 minutes, etc.

[0071] Specifically, for example, the conditions could be: tip velocity of 1 m / s or more for 30 seconds or more; tip velocity of 2.4 m / s or more for 1 minute or more; tip velocity of 2.4 m / s or more for 1.5 minutes or more; tip velocity of 2.4 m / s or more for 2 minutes or more; tip velocity of 2.4 m / s or more for 20 minutes or more; tip velocity of 2.4 m / s or more for 30 minutes or more; tip velocity of 2.4 m / s or more for 40 minutes or more; tip velocity of 2.4 m / s or more for 50 minutes or more; or tip velocity of 2.4 m / s or more for 60 minutes or more. Furthermore, for example, when performing emulsification using a pressure method, conditions such as a pressure of 0.3 MPa or higher for 1 minute or more, a pressure of 0.6 MPa or higher for 1 minute or more, a pressure of 0.6 MPa or higher for 1.5 minutes or more, a pressure of 0.6 MPa or higher for 2 minutes or more, a pressure of 0.6 MPa or higher for 3 minutes or more, a pressure of 0.6 MPa or higher for 4 minutes or more, a pressure of 0.6 MPa or higher for 5 minutes or more, etc., can be used.

[0072] The temperature conditions for emulsification are not particularly limited, as long as the emulsification is carried out at a temperature below the boiling point of the lower of the hydrophobic organic solvent and the aqueous solution, and above the melting point of the higher of the two. For example, 0-65°C, 5-65°C, 10-65°C, 15-65°C, 20-65°C, 25-65°C, 30-65°C, 35-65°C, 40-65°C, 45-65°C, 0-60°C, 5-60°C, 10-60°C, 15-60°C, 20-60°C, 25-60°C, 30-60°C, 35-60°C, 40-60°C, 45-60°C, 0-55°C, 5-55°C, 10-55°C, 15-55°C, 20-55°C, The temperature can be set to 25-55°C, 30-55°C, 35-55°C, 40-55°C, 45-55°C, 0-50°C, 5-50°C, 10-50°C, 15-50°C, 20-50°C, 25-50°C, 30-50°C, 35-50°C, 40-50°C, 45-50°C, 0-45°C, 5-45°C, 10-45°C, 15-45°C, 20-45°C, 25-45°C, 30-45°C, 35-45°C, 40-45°C, etc.

[0073] Furthermore, for example, this process can be carried out under conditions such as below the boiling point, below -5°C, below -10°C, below -15°C, below -20°C, below -21°C, below -22°C, below -23°C, etc., with respect to the boiling point of the lower of the aqueous solution and the hydrophobic organic solvent. Alternatively, for example, this process uses the following with respect to the lower boiling point of the aqueous solution and the hydrophobic organic solvent: boiling point -65°C or higher but less than boiling point, boiling point -60°C or higher but less than boiling point, boiling point -50°C or higher but less than boiling point, boiling point -45°C or higher but less than boiling point, boiling point -40°C or higher but less than boiling point, boiling point -35°C or higher but less than boiling point, boiling point -30°C or higher but less than boiling point, boiling point -25°C or higher but less than boiling point, boiling point -24°C or higher but less than boiling point, boiling point -23°C or higher but less than boiling point, boiling point -65°C or higher but less than or equal to boiling point -5°C, boiling point -60°C or higher but less than or equal to boiling point -5°C, boiling point -50°C or higher but less than or equal to boiling point , boiling point -45°C or higher, boiling point -5°C or lower, boiling point -40°C or higher, boiling point -5°C or lower, boiling point -35°C or higher, boiling point -5°C or higher, boiling point -30°C or higher, boiling point -5°C or lower, boiling point -25°C or higher, boiling point -5°C or lower, boiling point -24°C or higher, boiling point -5°C or lower, boiling point -23°C or higher and boiling point -5 ℃ or less, boiling point -65℃ or more and boiling point -10℃ or less, boiling point -60℃ or more and boiling point -10℃ or less, boiling point -50℃ or more and boiling point -10℃ or less, boiling point -45℃ or more and boiling point -10℃ or less, boiling point -40℃ or more and boiling point -10℃ or less, boiling point -35℃ or more and boiling point -10℃ or less, boiling point -3 Boiling point -10℃ or higher, Boiling point -25℃ or higher, Boiling point -10℃ or higher, Boiling point -24℃ or higher, Boiling point -10℃ or higher, Boiling point -23℃ or higher, Boiling point -10℃ or higher, Boiling point -65℃ or higher, Boiling point -15℃ or higher, Boiling point -60℃ or higher, Boiling point -15℃ or higher, Boiling point -50℃ or higher 15℃ or less, boiling point -45℃ or more, boiling point -15℃ or less, boiling point -40℃ or more, boiling point -15℃ or less, boiling point -35℃ or more, boiling point -15℃ or less, boiling point -30℃ or more, boiling point -15℃ or less, boiling point -25℃ or more, boiling point -15℃ or less, boiling point -24℃ or more, boiling point -15℃ or less, boiling point Boiling point -23°C or higher, boiling point -15°C or lower, boiling point -65°C or higher, boiling point -20°C or higher, boiling point -60°C or higher, boiling point -20°C or higher, boiling point -50°C or higher, boiling point -20°C or higher, boiling point -45°C or higher and boiling point -20°C, boiling point -40°C or higher and boiling point -20°C or lower, boiling point -35°C or higher -20℃ or less, boiling point -30℃ or more, boiling point -20℃ or less, boiling point -25℃ or more, boiling point -20℃ or less, boiling point -24℃ or more, boiling point -20℃ or less, boiling point -23℃ or more, boiling point -20℃ or less, boiling point -65℃ or more, boiling point -21℃ or less, boiling point -60℃ or more, boiling point -21℃ or less,This can be done under conditions such as boiling point above -50°C and below -21°C, boiling point above -45°C and below -21°C, boiling point above -40°C and below -21°C, boiling point above -35°C and below -21°C, boiling point above -30°C and below -21°C, boiling point above -25°C and below -21°C, boiling point above -24°C and below -21°C, boiling point above -23°C and below -21°C, boiling point above -65°C and below -23°C, boiling point above -60°C and below -23°C, boiling point above -50°C and below -23°C, boiling point above -40°C and below -23°C, boiling point above -35°C and below -23°C, boiling point above -30°C and below -23°C, and boiling point above -25°C and below -23°C.

[0074] The pH conditions during emulsification are not particularly limited. For example, the process can be carried out using the pH conditions described above for aqueous solutions.

[0075] The composition of the oil-in-water emulsion is not particularly limited, but typically the median particle size of the oil phase is 1 μm to 50 μm, 5 μm to 40 μm, 9 μm to 30 μm, 10 μm to 20 μm, 13 μm to 18 μm, 14 μm to 18 μm, 13 μm to 17 μm, 13 μm to 16 μm, etc. Also, the particle size is typically distributed in the ranges of 1 μm to 150 μm, 5 μm to 120 μm, 5 μm to 100 μm, and 10 μm to 100 μm. The particle size distribution may be unimodal or bimodal.

[0076] During this process, the conditions may remain constant, or they may vary within the range described above. The variation in conditions may be achieved through proactive modification, through spontaneous change, or a combination thereof.

[0077] This process can be performed multiple times. When performed multiple times, the conditions may be the same for all of them, or the conditions may differ for one or more of the executions.

[0078] The method according to this embodiment can be performed using either batch extraction or continuous extraction.

[0079] 1-3-3. Emulsified Solution Maintenance Step The "emulsified solution maintenance step" is an optional step in the method of this embodiment, and is a step in which the emulsified state of the oil-in-water emulsion is maintained. This step can be performed simultaneously with or after the emulsification step.

[0080] The oil-in-water emulsion solution only needs to be maintained in an emulsified state, and the conditions for this are not particularly limited. For example, mixing may be continued under the same conditions as in the emulsification process, it may be maintained while mixing under more relaxed conditions, or it may be maintained without mixing. Since the oil-in-water emulsion solution prepared in the emulsification process is a stable emulsion, the emulsified state will be maintained even if left standing for, for example, 1 hour or more, 2 hours or more, 3 hours or more, 5 hours or more, 6 hours or more, 10 hours or more, 12 hours or more, 15 hours or more, 16 hours or more, 18 hours or more, or 20 hours or more.

[0081] The duration of this process is not particularly limited, but can be, for example, 1 minute to 24 hours, 5 minutes to 24 hours, 10 minutes to 24 hours, 30 minutes to 24 hours, 1 hour to 24 hours, 2 hours to 24 hours, 6 hours to 24 hours, 12 hours to 24 hours, 18 hours to 24 hours, 20 hours to 24 hours, 1 hour to 20 hours, 2 hours to 18 hours, 6 hours to 12 hours, 1 hour to 6 hours, 1 hour to 3 hours, 30 minutes to 2 hours, 30 minutes to 1 hour, etc.

[0082] When mixing under the same conditions as the emulsification process, the mixing conditions may be the same as or different from those of the emulsification process. For example, the emulsification process and this process may be performed alternately multiple times. If this process is performed multiple times, the conditions may be the same in all of them, or the conditions may differ in one or more of them.

[0083] 1-3-4. Demulsification Step The "demulsification step" is an essential step in the method of this embodiment, and is a step in which the aqueous phase and oil phase of the oil-in-water emulsion solution are separated. This step can be performed after the emulsification step.

[0084] The separation method used in this process is not particularly limited as long as it is capable of separating the aqueous phase and the oil phase. Specifically, applicable methods include the addition of inorganic salts, acids, alkalis, surfactants, temperature changes, centrifugal force, filtration, changes in the volume ratio of the continuous phase to the dispersed phase, loading of a high-pressure AC electric field, ultrasonic irradiation, or a combination thereof. For example, this can be suitably carried out by adding an alkaline agent, centrifugal separation, and heating, or by using one or more of these methods, or by using a combination of all of them. Furthermore, for example, heating, the addition of an alkaline agent, and / or centrifugal separation may be combined.

[0085] When adding an alkaline agent, the method of addition is not particularly limited. For example, the alkaline agent may be added to the oil-in-water emulsion solution, or the oil-in-water emulsion solution may be added to the alkaline agent, or the oil-in-water emulsion solution and the alkaline agent may be added to the container simultaneously, or both or one of them may be added continuously and / or multiple times.

[0086] The alkaline agent used is not particularly limited, as long as it can raise the pH of the aqueous phase. For example, sodium hydroxide, potassium hydroxide, ammonia, sodium methoxide, lithium hydroxide, sodium carbonate, sodium bicarbonate, magnesium oxide, calcium hydroxide, sodium acetate, or combinations thereof can be used.

[0087] The pH increases with the addition of an alkaline agent, but the extent of this increase is not particularly limited. For example, the addition of an alkaline agent can raise the pH by 1-8, 1.1-6, 1.5-5, 1.5-4, 2-4, 2-3.5, and 2.5-3.5. In addition, the addition of an alkaline agent can raise the pH of the aqueous phase to, for example, 7-12, 7.5-11, 7.5-10, 7.7-10, 7.8-10, 7.9-10, 8-10, 8-9.5, 8.2-10, 8.3-10, 8.4-10, 8.5-10, 9-10, 7.5-9, 7.7-9, 7.8-9, 7.9-9, 8-9, 8.2-9, 8.3-9, 8.4-9, 8.5-9, 7.5-8, etc.

[0088] The specific method of centrifugation is not particularly limited. For example, centrifugation can be performed using a centrifuge known in the art.

[0089] The conditions for centrifugal separation are not particularly limited, as long as a certain level of centrifugal force is applied for a certain period of time or longer to separate the aqueous and oil phases. For example, the centrifugal force can be set to 1,000 × g or more, 2,000 × g or more, 3,000 × g or more, 4,000 × g or more, 5,000 × g or more, 6,000 × g or more, 7,000 × g or more, 8,000 × g or more, etc. The duration of centrifugal separation is also not particularly limited, but can be set to 1 minute or more, 5 minutes or more, 10 minutes or more, 15 minutes or more, 20 minutes or more, 30 minutes or more, 40 minutes or more, etc. Specifically, the process can be carried out under conditions such as: centrifugal force of 3,000 × g or more for 10 minutes or more; centrifugal force of 5,000 × g or more for 10 minutes or more; centrifugal force of 8,000 × g or more for 10 minutes or more; centrifugal force of 3,000 × g or more for 15 minutes or more; centrifugal force of 5,000 × g or more for 15 minutes or more; centrifugal force of 8,000 × g or more for 15 minutes or more; centrifugal force of 3,000 × g or more for 20 minutes or more; centrifugal force of 5,000 × g or more for 20 minutes or more; centrifugal force of 8,000 × g or more for 20 minutes or more.

[0090] When heating is performed, the method is not particularly limited. Although heating will raise the temperature of the oil-in-water emulsion solution, the amount of this increase is not particularly limited. For example, the temperature increase ranges are 5°C to 50°C, 10°C to 50°C, 15°C to 50°C, 16°C to 50°C, 18°C ​​to 50°C, 19°C to 50°C, 20°C to 50°C, 5°C to 45°C, 10°C to 45°C, 15°C to 45°C, 16°C to 45°C, 18°C ​​to 45°C, 19°C to 45°C, 20°C to 45°C, 5°C to 40°C, 10°C to 40°C, 15°C to 40°C, 16°C to 40°C, 19°C to 40°C, 20°C to 40°C, 5°C to 35°C, 10°C to 35°C, 15°C to 35°C, 16°C to 35°C, 18°C ​​to 35°C, and 19°C. The temperature can be set to ~35℃, 20℃~35℃, 5℃~30℃, 10℃~30℃, 15℃~30℃, 16℃~30℃, 18℃~30℃, 19℃~30℃, 20℃~30℃, 5℃~25℃, 10℃~25℃, 15℃~25℃, 16℃~25℃, 18℃~25℃, 19℃~25℃, 20℃~25℃, 5℃~21℃, 10℃~21℃, 15℃~21℃, 16℃~21℃, 18℃~21℃, 19℃~21℃, 5℃~20℃, 10℃~20℃, 15℃~20℃, 16℃~20℃, 18℃~20℃, etc.

[0091] Furthermore, the temperature of the oil-in-water emulsion solution after heating is not particularly limited, as long as it is below the boiling point of the lower of the aqueous solution and the hydrophobic organic solvent. For example, heating can be to 10°C or higher, 15°C or higher, 20°C or higher, 25°C or higher, 30°C or higher, 35°C or higher, 40°C or higher, 45°C or higher, 50°C or higher, 55°C or higher, 60°C or higher, 61°C or higher, 62°C or higher, 63°C or higher, 64°C or higher, 65°C or higher, etc. For example, heating ranges are 10-80°C, 15-80°C, 20-80°C, 25-80°C, 30-80°C, 35-80°C, 40-80°C, 45-80°C, 50-80°C, 55-80°C, 60-80°C, 61-80°C, 62-80°C, 63-80°C, 64-80°C, 65-80°C, 10-75°C, 15-75°C, 20-75°C, 25-75°C, 30-75°C, 35-75°C, 40-75°C, 45-75°C, 50-75°C, 55-75°C, 60-75°C, 61-75°C, 62-75°C, 63-75°C, 64-75°C, and 65-75°C. It can be heated to temperatures such as 10-70°C, 15-70°C, 20-70°C, 25-70°C, 30-70°C, 35-70°C, 40-70°C, 45-70°C, 50-70°C, 55-70°C, 60-70°C, 61-70°C, 62-70°C, 63-70°C, 64-70°C, 65-70°C, 10-65°C, 15-65°C, 20-65°C, 25-65°C, 30-65°C, 35-65°C, 40-65°C, 45-65°C, 50-65°C, 55-65°C, 60-65°C, 61-65°C, 62-65°C, 63-65°C, 64-65°C, etc.

[0092] Furthermore, for example, this process can be carried out under conditions such as below the boiling point, below -1°C, below -2°C, or below -3°C, with respect to the lower boiling point of the aqueous solution or the hydrophobic organic solvent. Alternatively, for example, this process can be carried out under conditions such as above -50°C and below the boiling point, above -40°C and below the boiling point, above -35°C and below the boiling point, above -30°C and below the boiling point, above -25°C and below the boiling point, above -24°C and below the boiling point, above -23°C and below the boiling point, above -50°C and below -1°C, above -40°C and below -1°C, above -35°C and below -1°C, above -30°C and below -1°C, above -25°C and below -1°C, above -24°C and below -1°C, and above -23°C and below -1°C. It can be carried out under conditions such as below ℃, boiling point -50℃ or higher and boiling point -2℃ or lower, boiling point -40℃ or higher and boiling point -2℃ or lower, boiling point -35℃ or higher and boiling point -2℃ or lower, boiling point -30℃ or higher and boiling point -2℃ or lower, boiling point -25℃ or higher and boiling point -2℃ or lower, boiling point -24℃ or higher and boiling point -2℃ or lower, boiling point -23℃ or higher and boiling point -2℃ or lower, boiling point -50℃ or higher and boiling point -3℃ or lower, boiling point -40℃ or higher and boiling point -3℃ or lower, boiling point -35℃ or higher and boiling point -3℃ or lower, boiling point -30℃ or higher and boiling point -3℃ or lower, boiling point -25℃ or higher and boiling point -3℃ or lower, boiling point -24℃ or higher and boiling point -3℃ or lower, boiling point -23℃ or higher and boiling point -3℃ or lower.

[0093] This process is carried out at a temperature higher than the melting point of the aqueous solution or the hydrophobic organic solvent, whichever is higher.

[0094] The processing time for this step is not particularly limited. When using a method other than centrifugal separation, the processing time can be, for example, 1 minute to 5 hours, 1 minute to 3 hours, 5 minutes to 3 hours, 10 minutes to 3 hours, 15 minutes to 3 hours, 20 minutes to 3 hours, 30 minutes to 3 hours, 40 minutes to 3 hours, 1 hour to 3 hours, 1 minute to 2 hours, 5 minutes to 2 hours, 10 minutes to 2 hours, 15 minutes to 2 hours, 20 minutes to 2 hours, 30 minutes to 2 hours, 1 hour to 2 hours, 1 minute to 1.5 hours, 5 minutes to 1.5 hours, 10 minutes to 1.5 hours, 15 minutes to 1.5 hours, 20 minutes to 1.5 hours, 30 minutes to 1.5 hours, 1 hour to 1.5 hours, 1 minute to 1 hour, 5 minutes to 1 hour, 10 minutes to 1 hour, 15 minutes to 1 hour, 20 minutes to 1 hour, 30 minutes to 1 hour, etc.

[0095] When multiple methods are used in combination in this process, they may be applied simultaneously or separately. Specifically, for example, an oil-in-water emulsion solution to which an alkaline agent has been added may be heated, or centrifuged while being heated.

[0096] During this process, the conditions may remain constant, or they may vary within the range described above. The variation in conditions may be achieved through proactive modification, through spontaneous change, or a combination thereof.

[0097] This process may be performed multiple times, or it may be performed continuously by heating the piping through which the liquid passes. When this process is performed multiple times, it may be performed under the same conditions each time, or the conditions may differ in one or more trials.

[0098] 1-4. Effects According to the method of this embodiment, oil-soluble molecules can be easily extracted into the oil phase without using amphiphilic organic solvents such as isopropyl alcohol.

[0099] This reduces the costs associated with the disposal and recovery of amphiphilic organic solvents used as extraction aids, and also reduces the transfer of impurities to the oil phase due to excessive contact between the oil and aqueous phases.

[0100] The extraction rate (%) of oil-soluble molecules by the method of this embodiment (= (concentration of oil-soluble molecules contained in the aqueous phase) / (concentration of oil-soluble molecules contained in the aqueous solution) × 100) is, for example, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, etc. The extraction efficiency may be improved by performing the method of this embodiment multiple times.

[0101] 2. Preparation Method 2-1. Overview The second aspect of the present invention is a preparation method. In the case of a method for preparing an oil-soluble molecule-containing solution, the method of this aspect includes an emulsification step, a demulsification step, and a recovery step as essential steps, and in the case of a method for preparing oil-soluble molecules, the method includes an emulsification step, a demulsification step, a recovery step, and a purification step as essential steps. According to the method of this aspect, an oil-soluble molecule-containing solution or oil-soluble molecules can be prepared with high efficiency from cells or cell lysates without using amphiphilic organic solvents or lipophilic surfactants.

[0102] 2-2. Process The method of this embodiment includes an emulsification step, a de-emulsification step, and a recovery step as essential steps, and includes a raw material solution preparation step and an emulsified solution maintenance step as optional steps. In the case of a method for preparing oil-soluble molecules, a purification step is included as an additional essential step. Each step will be described in detail below.

[0103] 2-2-1. Raw Material Solution Preparation Step The "raw material solution preparation step" is an optional step in the method of this embodiment, and is a step of preparing an aqueous solution containing cells containing oil-soluble molecules or cell lysates thereof. This step may be carried out in accordance with the description in the first embodiment.

[0104] 2-2-2. Emulsification Step The "emulsification step" is an essential step in the method of this embodiment, and is a step of emulsifying an aqueous solution containing cells or cell lysates with a hydrophobic organic solvent to form an oil-in-water emulsion solution. This step is carried out under conditions that substantially do not contain amphiphilic organic solvents and / or lipophilic surfactants. This step can be carried out after the raw material solution preparation step if one is performed. This step may be carried out in accordance with the description of the first embodiment.

[0105] 2-2-3. Emulsified Solution Maintenance Step The "emulsified solution maintenance step" is an optional step in the method of this embodiment, and is a step in which the emulsified state of the oil-in-water emulsion is maintained. This step can be performed simultaneously with or after the emulsification step. This step may be performed in accordance with the description of the first embodiment.

[0106] 2-2-4. Demulsification Step The "demulsification step" is an essential step in the method of this embodiment, and is a step in which the aqueous phase and oil phase of the oil-in-water emulsion solution are separated. This step can be performed after the emulsification step. This step may be performed in accordance with the description in the first embodiment.

[0107] 2-2-5. Recovery Process The "recovery process" is an essential step in the method of this embodiment, and is a process of recovering the separated oil phase as an oil-soluble molecule-containing solution. This process can be performed simultaneously with or after the deemulsification process.

[0108] The recovery method is not particularly limited, as long as it is a method that can recover the oil phase. Examples include a method of separating the oil phase, a method of removing the aqueous phase, or a combination thereof.

[0109] During this process, the conditions may remain constant or may vary. Variations in conditions may be achieved through proactive modifications, spontaneous changes, or a combination of both.

[0110] This process may be performed multiple times or consecutively. If this process is performed multiple times, all trials may be conducted under the same conditions, or the conditions may differ in one or more trials.

[0111] In the method described herein, washing may be performed as appropriate. The washing solution used in this case is not particularly limited, but for example, water, aqueous solution, etc., can be used.

[0112] Furthermore, in the method described herein, solid impurities can be removed as appropriate by solid-liquid separation or the like. Specific methods for this include, for example, the solid-liquid separation method exemplified in the raw material solution preparation step of the first embodiment.

[0113] 2-2-6. Purification Step The "purification step" is an essential step in the method of this embodiment when it is a method for preparing oil-soluble molecules, and is a step of purifying the oil-soluble molecules from the recovered oil-soluble molecule-containing solution. This step can be performed simultaneously with or separately from the recovery step.

[0114] The method used in this process is not particularly limited, as long as it is capable of separating and / or purifying oil-soluble molecules. Examples include methods utilizing solubility such as salt precipitation, organic solvent precipitation, and crystallization; methods utilizing differences in molecular weight such as dialysis, ultrafiltration, and gel filtration; methods utilizing differences in charge such as ion exchange chromatography; methods utilizing specific binding such as affinity chromatography; methods utilizing hydrophobicity such as hydrophobic chromatography and reversed-phase chromatography; solvent substitution or a combination thereof.

[0115] Furthermore, methods such as oxidation and reduction, which increase the proportion of oil-soluble molecules in the desired state, can be used individually or in combination.

[0116] When the purpose is to prepare and / or extract oxidized oil-soluble molecules, oxidation treatment can be performed as appropriate. The method in this case is not particularly limited, but can be carried out by, for example, aeration with an oxygen-containing gas into the oil-soluble molecule-containing solution, addition of an oxidizing agent, or a combination thereof. In this case, stirring or mixing can be carried out in combination. Examples of oxidizing agents include manganese dioxide and hydrogen peroxide. For example, oxidation by aeration can be suitably used.

[0117] When the purpose is to prepare and / or extract reduced oil-soluble molecules, a reduction treatment can be performed as appropriate. The method in this case is not particularly limited, but can be carried out by adding a reducing agent to the oil-soluble molecule-containing solution, applying electricity, heating, or a combination thereof. In this case, for example, stirring or mixing can also be carried out. Examples of reducing agents include ascorbic acids such as L-ascorbic acid, D-arabo-ascorbic acid, L-ascorbyl palmitate, and L-ascorbyl stearate, sodium borohydride, sodium hydrosulfite (sodium hypochlorite), retinal, β-carotene, tocotrienol, NADH, cyanocobalamin, octyl gallate, dodecyl gallate, sesamol, and thiamine hydrochloride, and preferably sodium hydrosulfite (sodium hypochlorite), sodium borohydride, and ascorbic acids. Natural products (natural extracts, natural pigments, etc.) containing such compounds may also be used as reducing agents, and such specific natural products are not particularly limited. Specific examples of natural products include extracts such as royal jelly, black vinegar, acerola extract, pine bark extract, Scutellaria baicalensis leaf extract, Houttuynia cordata extract, enzyme-treated rutin, and pigments such as cocoa pigment, gardenia pigment, grape skin pigment, and red yeast rice pigment.

[0118] When both the concentration of oil-soluble molecules and the increase in the proportion of molecules in the desired state are performed, they may be carried out simultaneously, sequentially, or separately, for example, alternately multiple times. When carried out multiple times, the same conditions and methods may be used for all trials, or the conditions and methods may differ in one or more trials.

[0119] The present invention will now be described in detail with reference to examples, but the present invention is not limited to these examples.

[0120] <Example 1. Investigation of a method for extracting oil-soluble molecules without using amphiphilic organic solvents> (Objective) To investigate whether it is possible to extract oil-soluble molecules without using amphiphilic organic solvents, and to determine the extraction rate.

[0121] (Methods and Results) Saitoella (Schizosaccharomyces) complicata strain IFO10748, which produces coenzyme Q10, was cultured aerobically at 25°C for 72 hours in 10 L of culture medium (5 g / L peptone, 3 g / L yeast extract, 3 g / L malt extract, 20 g / L glucose, pH 6.0). The resulting culture solution containing the microbial cells (solid content concentration: approximately 100 g / L) was pressure-disrupted to prepare a microbial cell lysate containing coenzyme Q10 (pH: approximately 5.5).

[0122] To the obtained microbial cell lysate, twice the volume of n-hexane was added and mixed to prepare a mixed solution.

[0123] When the prepared mixture was mixed using a disc-type stirring blade at 45°C and 350 rpm (tip speed: equivalent to approximately 0.29 m / s) for 60 minutes, the mixture did not become an emulsion even after 60 minutes of stirring, and oil-water separation was immediately confirmed after the mixing stopped.

[0124] The hexane phase, separated from the oil-water mixture, was collected as an extract 30 minutes after the mixing was stopped, and the concentration of coenzyme Q10 was measured by HPLC analysis. The following conditions were used for the HPLC analysis: Column: YMC-Pack OSD-A / S-5μm / 12nm; Mobile phase: methanol / n-hexane = 80 / 20; Flow rate: 1 mL / min; Detection: UV 275 nm.

[0125] Based on the measured concentration of coenzyme Q10, the extraction rate was calculated using the following formula: Extraction rate (%) = (Concentration of coenzyme Q10 in the extract) / (Concentration of coenzyme Q10 in the microbial cell lysate before extraction) × 100.

[0126] The concentration of coenzyme Q10 contained in the microbial cell lysate before extraction was measured by the following method. First, a 3:1 mixture of methanol and chloroform was added to 2 mL of the microbial cell lysate before extraction to prepare a total volume of 50 mL of the measurement solution. After shaking the measurement solution at 25°C for 30 minutes, solid matter derived from the microbial cells was removed, and the liquid phase was subjected to HPLC analysis under the above conditions to measure the concentration of coenzyme Q10.

[0127] The concentration of coenzyme Q10 in the microbial cell lysate before extraction was approximately 6 g / L. Furthermore, the calculated extraction rate of coenzyme Q10 was 63% (Figure 1). This indicates that while oil-soluble molecules can be extracted, the extraction rate is low and therefore impractical.

[0128] On the other hand, when mixing was performed using a homomixer MARK II 2.5 (PRIMIX) at 45°C and 10,000 rpm (tip speed: equivalent to approximately 5 m / s) for 2 minutes, the mixture became a stable emulsion with the hexane phase dispersed, and no oil-water separation occurred even after mixing was stopped.

[0129] The particle size distribution of the emulsion after stirring was analyzed using Partica LA-960 (HORIBA). As a result, the measured particle size of the hexane phase was distributed in the range of 5 μm to 100 μm (Figure 2), with a median of approximately 16 μm.

[0130] Subsequently, the emulsion was deemulsified by adjusting its pH to 9 using an aqueous sodium hydroxide solution. After pH adjustment, the emulsion was allowed to stand at 65°C, and oil-water separation was confirmed. The separated hexane phase was collected as an extract 60 minutes after standing and subjected to HPLC analysis for calculation.

[0131] The extraction rate of coenzyme Q10 was 94%, a dramatic improvement compared to the conditions under which the mixture was mixed under non-emulsified conditions (Figure 1).

[0132] This demonstrates that oil-soluble molecules can be extracted with high efficiency without using amphiphilic organic solvents by mechanical emulsification.

[0133] <Example 2. Effect of pH during demulsification on extraction rate> (Objective) To investigate the effect of pH during demulsification on the extraction rate of oil-soluble molecules.

[0134] (Methods and Results) Microbial cell lysate was prepared in the same manner as in Example 1 (pH: approximately 5.5). The pH of the emulsion was adjusted to 5 using an aqueous sulfuric acid solution, and to 7, 8, or 9 using an aqueous sodium hydroxide solution. After pH adjustment, the emulsion was allowed to stand at 25°C, and oil-water separation was confirmed. The hexane phase separated 60 minutes after standing was collected as an extract and subjected to HPLC analysis.

[0135] The calculated extraction rates of coenzyme Q10 were 61% under pH 5 conditions, 63% under pH 7 conditions, 74% under pH 8 conditions, and 90% under pH 9 conditions (Figure 3).

[0136] This suggests that the extraction rate increases when the pH during demulsification is higher than that during emulsification, indicating more basic conditions.

[0137] <Example 3. Effect of Mixing Ratio on Extraction Rate> (Objective) To investigate the effect of the mixing ratio of microbial cell lysate and n-hexane on the extraction rate of oil-soluble molecules.

[0138] (Methods and Results) The emulsion preparation and deemulsification treatment were carried out in basically the same manner as in Example 1. The mixture was prepared by adding an equal volume of n-hexane to the microbial cell lysate and mixing.

[0139] The calculated extraction rate of coenzyme Q10 was 84.6% (Figure 4). This suggests that while the mixing ratio does not significantly affect the extraction rate of oil-soluble molecules, a higher proportion of hydrophobic organic solvent tends to result in a higher extraction rate.

[0140] <Example 4. Influence of differences in emulsification time and deemulsification treatment on extraction rate> (Objective) To investigate the influence of differences in emulsification time and deemulsification treatment on the extraction rate of oil-soluble molecules.

[0141] (Methods and Results) The mixture was prepared in the same manner as in Example 1. The mixture was stirred using a homomixer MARK II 2.5 (PRIMIX) at 45°C and 10,000 rpm (tip speed: equivalent to approximately 5 m / s) for 2 minutes, 15 minutes, or 30 minutes. Under all conditions, the mixture formed a stable emulsion with the hexane phase as the dispersed phase.

[0142] Subsequently, the emulsion was demulsified by centrifugation at 8,000 × g for 20 minutes. After centrifugation, oil-water separation was confirmed. The separated hexane phase was collected as an extract, and the concentration of coenzyme Q10 was measured by HPLC analysis to calculate the extraction rate.

[0143] The calculated extraction rates of coenzyme Q10 were 91.2% under the 2-minute condition, 94.0% under the 15-minute condition, and 92.6% under the 30-minute condition (Figure 5).

[0144] From this, it was first suggested that the demulsification treatment under the conditions of Example 1 and the demulsification treatment by centrifugation did not have a significant effect on the extraction rate. Furthermore, it was suggested that the length of the emulsification time did not have a significant effect on the extraction rate as long as it was above a certain level.

[0145] <Example 5. Effect of different emulsification methods on extraction rate (1)> (Objective) To investigate the effect of different emulsification methods on the extraction rate of oil-soluble molecules.

[0146] (Methods and Results) The emulsion preparation and demulsification process were basically carried out in the same manner as in Example 4. Emulsification was performed using an UltraTax homogenizer T25 (manufactured by IKA) at 45°C and 8,000 rpm (tip speed: equivalent to 2.4 m / s) for 60 minutes.

[0147] The mixture formed a stable emulsion with the hexane phase as the dispersed phase. The extraction rate of coenzyme Q10 calculated after demulsification was 94.3%.

[0148] This suggests that, regardless of the equipment used, the method of the present invention enables highly efficient extraction of oil-soluble molecules.

[0149] <Example 6. Effect of different emulsification methods on extraction rate (2)> (Objective) To investigate the effect of different emulsification methods on the extraction rate of oil-soluble molecules.

[0150] (Methods and Results) The preparation and demulsification of the emulsion were carried out in basically the same manner as in Example 4. Emulsification was performed using an OHR mixer (manufactured by OHR Fluid Engineering Laboratory) under flow conditions of 45°C and a pressure of 0.6 MPa for 5 minutes.

[0151] The mixture formed a stable emulsion with the hexane phase as the dispersed phase. The extraction rate of coenzyme Q10 calculated after demulsification was 92%.

[0152] This suggests that, regardless of the emulsification method used, the method of the present invention enables highly efficient extraction of oil-soluble molecules. All publications, patents, and patent applications cited herein are incorporated herein by direct reference.

Claims

1. A method for extracting oil-soluble molecules from cells or cell lysates into a hydrophobic organic solvent, comprising: an emulsification step of emulsifying an aqueous solution containing the cells or cell lysates with a hydrophobic organic solvent to form an oil-in-water emulsion; and a deemulsification step of separating the aqueous phase and the oil phase of the oil-in-water emulsion, wherein the emulsification step is carried out under conditions substantially free of amphiphilic organic solvents and / or lipophilic surfactants.

2. The method according to claim 1, wherein the emulsification is performed by one or more methods selected from the group consisting of a high-speed rotation method, a pressure method, and an ultrasonic method.

3. The method according to claim 1 or 2, wherein the separation in the demulsification step is carried out by adding an alkaline agent, centrifugal separation, and heating, one or more of these.

4. The method according to claim 1, wherein the content ratio of the aqueous solution in the oil-in-water emulsion is 15% to 90% by volume.

5. The method according to claim 1, wherein the particle size of the oil phase in the oil-in-water emulsion solution is 5 μm to 100 μm.

6. The method according to claim 1, wherein the pH of the aqueous solution is 1.5 to 6.

7. The method according to claim 1, wherein the emulsification step is performed below the boiling point of the hydrophobic organic solvent.

8. The method according to claim 1, wherein the SP value of the hydrophobic organic solvent is 7 to 10.

9. The method according to claim 1, wherein the oil-soluble molecule is coenzyme Q.

10. The method according to claim 1, wherein the cell or cell lysate thereof is a microbial cell or cell lysate thereof.

11. The method according to claim 1, wherein the separation in the demulsification step is carried out by any two or more selected from the group consisting of adding an alkaline agent, centrifugal separation, and heating.

12. The method according to claim 1, wherein the separation in the demulsification step is carried out by adding an alkaline agent and / or centrifugation and heating.

13. The method according to claim 3, wherein the pH of the aqueous phase becomes 8 to 10 upon addition of the alkaline agent.

14. The method according to claim 3, wherein the heating is to a temperature of 50°C or higher and 65°C or lower.

15. The method according to claim 3, wherein the centrifugation is performed with a centrifugal force of 3,000 × g or more for 20 minutes or more.

16. A method for preparing an oil-soluble molecule-containing solution containing oil-soluble molecules in cells or cell lysates, comprising: an emulsification step of emulsifying an aqueous solution containing the cells or cell lysates with a hydrophobic organic solvent to form an oil-in-water emulsion; a deemulsification step of separating the aqueous phase and the oil phase of the oil-in-water emulsion; and a recovery step of recovering the separated oil phase as an oil-soluble molecule-containing solution, wherein the emulsification step is carried out under conditions substantially free of amphiphilic organic solvents and / or lipophilic surfactants.

17. A method for preparing oil-soluble molecules in cells or cell lysates thereof, comprising: an emulsification step of emulsifying an aqueous solution containing the cells or cell lysates thereof with a hydrophobic organic solvent to form an oil-in-water emulsion; a deemulsification step of separating the aqueous phase and the oil phase of the oil-in-water emulsion; a recovery step of recovering the separated oil phase as an oil-soluble molecule-containing solution; and a purification step of purifying the oil-soluble molecules from the recovered oil-soluble molecule-containing solution, wherein the emulsification step is carried out under conditions that substantially do not contain amphiphilic organic solvents and / or lipophilic surfactants.

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