Method for extracting oil-soluble molecule

Abstract

The present invention addresses the problem of providing a method for stably and easily extracting oil-soluble molecules with high efficiency without using an amphiphilic organic solvent such as isopropanol or a lipophilic surfactant which need to be removed from a hydrophobic organic solvent.　Provided is a method for extracting oil-soluble molecules in cells or cell disruption products thereof into a hydrophobic organic solvent, the method comprising: a dispersion step for dispersing an aqueous solution including cells or cell disruption products thereof into a hydrophobic organic solvent to form a water-in-oil solution, by using at least one method selected from the group consisting of a high-speed rotation method, a pressure method, and an ultrasonic method; a dispersion maintaining step for maintaining the dispersed state of the water-in-oil solution; and a separation step for stopping the dispersing and separating the aqueous phase and the oil phase of the water-in-oil solution. The dispersion step is performed under conditions that do not substantially include 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 effects. 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 extracting oil-soluble molecules efficiently, stably, and simply without using amphiphilic organic solvents such as isopropanol or lipophilic surfactants, which need to be removed from hydrophobic organic solvents.

[0011] In order to solve the above problems, the inventors conducted intensive research and found that by forming a water-in-oil solution from an aqueous solution containing cell disruptors and an organic solvent, 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: a dispersion step of dispersing an aqueous solution containing the cells or cell lysates in a hydrophobic organic solvent using one or more methods selected from the group consisting of a high-speed rotation method, a pressure method and an ultrasonic method to form a water-in-oil solution; a dispersion maintenance step of maintaining the dispersion state of the water-in-oil solution; and a separation step of stopping the dispersion process and separating the aqueous phase and oil phase of the water-in-oil solution, wherein the dispersion 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 dispersion maintenance step is carried out for a time of 40 minutes or less. [3] The method according to [1] or [2], wherein the dispersion step comprises mixing the aqueous solution with the hydrophobic organic solvent in a volume of 0.01 to 4 times the volume of the hydrophobic organic solvent. [4] The method according to any one of [1] to [3], wherein the dispersion step includes adding the aqueous solution to the hydrophobic organic solvent at a rate of 60% by volume / s or less relative to the volume of the hydrophobic organic solvent. [5] The method according to any one of [1] to [4], wherein the dispersion step includes an addition step of adding the aqueous solution to the hydrophobic organic solvent in a volume of 60% or less of the hydrophobic organic solvent, and a dispersion step of dispersing the added aqueous solution in the hydrophobic organic solvent, and the addition step and the dispersion step are each performed two or more times. [6] The method according to any one of [1] to [5], wherein the SP value of the hydrophobic organic solvent is 7 to 10. [7] The method according to any one of [1] to [6], wherein the oil-soluble molecule is coenzyme Q. [8] The method according to any one of [1] to [7], wherein the cell or its cell disruptor is a microbial cell or its cell disruptor.[9] A method for preparing an oil-soluble molecule-containing solution containing oil-soluble molecules in cells or cell lysates, comprising: a dispersion step of dispersing an aqueous solution containing the cells or cell lysates in a hydrophobic organic solvent using one or more methods selected from the group consisting of a high-speed rotation method, a pressure method and an ultrasonic method to form a water-in-oil solution; a dispersion maintenance step of maintaining the dispersion state of the water-in-oil solution; a separation step of stopping the dispersion process and separating the aqueous phase and oil phase of the water-in-oil solution; and a recovery step of recovering the separated oil phase as an oil-soluble molecule-containing solution, wherein the dispersion step is carried out under conditions that substantially do not contain amphiphilic organic solvents and / or lipophilic surfactants.

[10] A method for preparing oil-soluble molecules in cells or cell lysates thereof, comprising: a dispersion step of dispersing an aqueous solution containing the cells or cell lysates in a hydrophobic organic solvent using one or more methods selected from the group consisting of high-speed rotation, pressure, and ultrasonic methods to form a water-in-oil solution; a dispersion maintenance step of maintaining the dispersion state of the water-in-oil solution; a separation step of stopping the dispersion process and separating the aqueous and oil phases of the water-in-oil solution; 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 dispersion step is carried out under conditions substantially free of amphiphilic organic solvents and / or lipophilic surfactants.

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

[10] , further comprising an alkali treatment step of increasing the pH in the water-in-oil solution. This specification incorporates the disclosures of Japanese Patent Application No. 2025-053405, 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 lysates can be easily 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 easily 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 lysates can be easily prepared without using amphiphilic organic solvents or lipophilic surfactants.

[0016] This figure shows the results of Example 1.

[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 a dispersion step, a dispersion maintenance step, and a separation 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 and convenience 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] In this specification, "dispersion" refers to a system in which one liquid phase is dispersed in the other phase as fine particles. In particular, the dispersion in this specification refers to a dispersion composed of two liquid phases with low affinity for each other and which does not have storage stability as it immediately separates after dispersion treatment.

[0022] An "emulsion" refers to a system in which one liquid phase is dispersed in particulate form between two liquid phases with low affinity for the other. In particular, the emulsion as used herein refers to an emulsion with high storage stability that does not immediately separate after emulsification. The method described in this specification does not form an emulsion composed of an aqueous solution containing cells or their cell lysates and a hydrophobic organic solvent.

[0023] The "aqueous phase" refers to the liquid phase of a dispersion or emulsion whose solvent is water. On the other hand, the "oil phase" refers to the liquid phase other than the aqueous phase of a dispersion or emulsion. In particular, in this specification, the oil phase broadly refers to the organic phase, and its solvent is a hydrophobic organic solvent.

[0024] In this specification, "water in oil (w / o) solution" refers to a dispersion in which the oil phase is the continuous phase and the aqueous phase is the dispersed phase.

[0025] "Dispersion" or "dispersion treatment" refers to a process of mixing an aqueous phase and an oil phase to form a dispersion.

[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" refers to the phase separation of an emulsion or dispersion into two liquid phases. Through this separation, the emulsion or dispersion appears to separate into two liquid phases, each of which can be isolated.

[0029] 1-3. Process The method of this embodiment includes a dispersion step, a dispersion maintenance step, and a separation step as essential steps, and a raw material solution preparation step and an alkali treatment 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 content 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, and the like.

[0039] At least a portion of the cells used for preparing the aqueous solution contains oil-soluble molecules. Usually, the cells 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.), and sterol esters having a fatty acid or the like ester-bonded to the terminal thereof (sterol fatty acid esters, etc.). Specific examples of coenzyme Q include coenzyme Q9, Q10, Q11, or combinations thereof. Coenzyme Q10 exists in an oxidized form and a reduced form, and both can be used in the method of the present invention.

[0041] The concentration of the oil-soluble molecules 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 in the case of an acidic aqueous solution, it can be, for example, 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. Furthermore, in the case of neutral or basic aqueous solutions, the values ​​can be, for example, 7-12, 7.5-11, 7.5-10, 7.7-10, 7.8-10, 7.9-10, 8-10, 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, 8-9.5, 8-9, 8.2-9, 8.3-9, 8.4-9, 8.5-9, 7.5-8, etc.

[0043] Surfactants may be used in this process, but in particular, lipophilic surfactants are preferably removed before the next dispersion step.

[0044] The aqueous solutions, cells, and cell lysates prepared in this process may be subjected to any treatment before the dispersion 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 dispersion 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, centrifugation, 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. Dispersion Step The "dispersion 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 dispersed in a hydrophobic organic solvent by one or more methods selected from the group consisting of high-speed rotation, pressure, and ultrasonic methods to form a water-in-oil solution, and 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 does not include emulsification treatment. 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 / cm3 ) 1 / 2 , 7 (cal / cm 3 ) 1 / 2 to 8 (cal / cm 3 ) 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, for example, ketones having 3 to 6 carbon atoms 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. Furthermore, the number of carbon atoms is not particularly limited, but for example, nitriles with 2-20, 2-12, or 2-8 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, a water-in-oil solution is prepared from an aqueous solution and a hydrophobic organic solvent, but the ratio of the aqueous solution to the hydrophobic organic solvent is not particularly limited. For example, the volume of the aqueous solution mixed with the hydrophobic organic solvent can be 0.01 times or more, 0.05 times or more, 0.07 times or more, 0.1 times or more, 0.15 times or more, 0.2 times or more, 0.25 times or more, 0.3 times or more, 0.35 times or more, 0.4 times or more, 0.45 times or more, 0.5 times or more, etc., compared to the volume of the hydrophobic organic solvent. Furthermore, for example, the volume of the aqueous solution mixed with the hydrophobic organic solvent can be 10 times or less, 9 times or less, 7 times or less, 5 times or less, 4 times or less, 3 times or less, 2 times or less, 1.5 times or less, 1.2 times or less, 1.1 times or less, 1 time or less, 0.8 times or less, 0.7 times or less, 0.65 times or less, 0.6 times or less, 0.55 times or less, 0.5 times or less, etc. While there are no specific limits on the range, for example, the volume of the aqueous solution mixed with the hydrophobic organic solvent is 0.01 to 10 times, 0.05 to 9 times, 0.07 to 7 times, 0.1 to 5 times, 0.01 to 4 times, 0.05 to 4 times, 0.07 to 4 times, 0.1 to 4 times, 0.15 to 4 times, 0.2 to 4 times, 0.25 to 4 times, 0.3 to 4 times, 0.35 to 4 times, 0.4 to 4 times, 0 .45-4x, 0.5-4x, 0.01-3x, 0.05-3x, 0.07-3x, 0.1-3x, 0.15-3x, 0.2-3x, 0.25-3x, 0.3-3x, 0.35-3 times, 0.4-3x, 0.45-3x, 0.5-3x, 0.01-2x, 0.05-2x, 0.07-2x, 0.1-2x, 0.15-2x, 0.2-2x, 0.25-2x, 0.3 ~2x, 0.35-2x, 0.4-2x, 0.45-2x, 0.5-2x, 0.01-1.5x, 0.05-1.5x, 0.07-1.5x, 0.1-1.5x, 0.15-1.5 times, 0.2 to 1.5 times, 0.25 to 1.5 times, 0.3 to 1.5 times, 0.35 to 1.5 times, 0.4 to 1.5 times, 0.45 to 1.5 times, 0.5 to 1.5 times, 0.01 to 1.2 times, 0.0 5-1.2x, 0.07-1.2x, 0.1-1.2x, 0.15-1.2x, 0.2-1.2x, 0.25-1.2x, 0.3-1.2x, 0.35-1.2x, 0.4-1.2 times, 0.45 to 1.2 times, 0.5 to 1.2 times, 0.01 to 1.1 times, 0.05 to 1.1 times, 0.07 to 1.1 times, 0.1 to 1.1 times, 0.15 to 1.1 times, 0.2 to 1.1 times, 0.25-1.1x, 0.3-1.1x, 0.35-1.1x, 0.4-1.1x, 0.45-1.1x, 0.5-1.1x, 0.01-1x, 0.05-1x, 0.07-1x, 0.1-1x, 0.15-1x, 0.2-1x, 0.25-1 times, 0.3-1x, 0.35-1x, 0.4-1x, 0.45-1x, 0.5-1x, 0.01-0.8x, 0.05-0.8x, 0.07-0.8x, 0.1-0.8x, 0.15-0.8x, 0.2-0.8x, 0.25-0.8x, 0 .3~0.8x, 0.35~0.8x, 0.4~0.8x, 0.45~0.8x, 0.5~0.8x, 0.01~0.7x, 0.05~0.7x, 0.07~0.7x, 0.1~0.7x, 0.15~0.7x, 0.2~0.7x, 0.2 5-0.7 times, 0.3-0.7 times, 0.35-0.7 times, 0.4-0.7 times, 0.45-0.7 times, 0.5-0.7 times, 0.01-0.65 times, 0.05-0.65 times, 0.07-0.65 times, 0.1-0.65 times, 0.15-0.65 times, 0.2-0.65 times, 0.25-0.65 times, 0.3-0.65 times, 0.35-0.65 times, 0.4-0.65 times, 0.45-0.65 times, 0.5-0.65 times, 0.01-0.6 times, 0.05-0.6 times, 0.07-0.6 times, 0.1- 0.6x, 0.15-0.6x, 0.2-0.6x, 0.25-0.6x, 0.3-0.6x, 0.35-0.6x, 0.4-0.6x, 0.45-0.6x, 0.5-0.6x, 0.01-0.55x, 0.05-0.55x, 0.07- The multiplier can be set to 0.55, 0.1-0.55, 0.15-0.55, 0.2-0.55, 0.25-0.55, 0.3-0.55, 0.35-0.55, 0.4-0.55, 0.45-0.55, 0.5-0.55, 0.01-0.5, 0.05-0.5, 0.07-0.5, 0.1-0.5, 0.15-0.5, 0.2-0.5, 0.25-0.5, 0.3-0.5, 0.35-0.5, 0.4-0.5, 0.45-0.5, etc.

[0061] The volume of the aqueous solution may be determined based on the concentration of the aqueous solution. The criteria in this case are not particularly limited, but for example, the volume of the hydrophobic organic solvent to be extracted can be increased as the concentration increases.

[0062] Specifically, for example, the concentration can be set to a certain level when all oil-soluble molecules in an aqueous solution are extracted into a hydrophobic organic solvent. Specifically, the concentration when all oil-soluble molecules are extracted into a hydrophobic organic solvent can be set to, for example, 1.0 × 10⁻⁶ -4 M to 1M, 1.0 x 10 -4 M to 0.1M, 1.0 x 10 -4 M to 0.06M, 5.0 x 10 -4 M to 0.05M, 5.0 x 10 -4 M to 0.04M, 5.0 x 10 -4 M to 0.03M, 1.0 x 10 -3 M to 0.03M, 2.0 x 10 -3 M to 0.03M, 3.0 x 10 -3 M to 0.03M, 4.0 x 10 -3 M to 0.03M, 5.0 x 10 -3 M to 0.03M, 5.0 x 10 -4 M to 0.02M, 2.0 x 10 -3 M to 0.02M, 3.0 x 10 -3 M to 0.02M, 4.0 x 10 -3 M to 0.02M, 5.0 x 10 -3 M to 0.02M, 5.0 x 10 -4 M to 0.01M, 2.0 x 10 -3 M to 0.01M, 3.0 x 10 -3 M to 0.01M, 4.0 x 10 -3 M to 0.01M, 5.0 x 10 -3 The mixing ratio of aqueous solution to hydrophobic organic solvent can be determined so that the concentration is M to 0.01M, etc.

[0063] In this process, an aqueous solution and a hydrophobic organic solvent are dispersed together. The method of addition is not particularly limited. Specific addition methods include, for example, dropwise addition, flow addition, spraying, or a combination thereof. For example, the hydrophobic organic solvent may be added to the aqueous solution, the aqueous solution may be added to the hydrophobic organic solvent, 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 aqueous solution is added to the hydrophobic organic solvent (dropwise, etc.). When addition is performed multiple times, the method may be the same for all additions, or it may differ for one or more additions.

[0064] The dispersion treatment in this step may be performed after the addition of the aqueous solution and / or hydrophobic organic solvent, or it may be performed simultaneously with the addition. Preferably, when the dispersion treatment is performed simultaneously with the addition of the aqueous solution and / or hydrophobic organic solvent, it can be performed, for example, simultaneously with the addition of the aqueous solution to the hydrophobic organic solvent.

[0065] The addition rate when adding continuously and / or multiple times is not particularly limited. For example, an aqueous solution can be added to a hydrophobic organic solvent at a rate of 80 vol% / s or less, 70 vol% / s or less, 60 vol% / s or less, 50 vol% / s or less, 40 vol% / s or less, 30 vol% / s or less, 20 vol% / s or less, 18 vol% / s or less, 17 vol% / s or less, or 15 vol% / s or less relative to the volume of the hydrophobic organic solvent. Alternatively, for example, an aqueous solution can be added to a hydrophobic organic solvent at a rate of 0.005 vol% / s or more, 0.001 vol% / s or more, 0.01 vol% / s or more, 0.05 vol% / s or more, 0.1 vol% / s or more, 0.5 vol% / s or more, 1 vol% / s or more, 5 vol% / s or more, 10 vol% / s or more, 15 vol% / s or more, or 16 vol% / s or more relative to the volume of the hydrophobic organic solvent.

[0066] When multiple additions are performed, this process includes an addition step of adding an aqueous solution to a hydrophobic organic solvent, and a dispersion step of dispersing the added aqueous solution in the hydrophobic organic solvent, with the addition and dispersion steps being repeated. In this case, each addition is treated as a separate addition step, and the dispersion process between each addition is treated as a single dispersion step. Here, the dispersion process may be stopped when one or more additions are made, or additions may be made intermittently during a continuously performed dispersion process. Specifically, examples include embodiments in which additions (dropwise, etc.) are made repeatedly while the dispersion process is being performed.

[0067] The amount of aqueous solution added in each addition step is not particularly limited. For example, it can be added to a hydrophobic organic solvent in volumes of 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 1% or less of the hydrophobic organic solvent. Alternatively, for example, it can be added to a hydrophobic organic solvent in volumes of 0.005% or more, 0.001% or more, 0.01% or more, 0.05% or more, 0.1% or more, 0.5% or more, 1% or more, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, or 50% or more of the hydrophobic organic solvent.

[0068] There are no particular limitations on the number of times the addition step and dispersion step can be performed, but for example, they can be performed 2 or more times, 3 or more times, 4 or more times, 5 or more times, 6 or more times, 7 or more times, 8 or more times, 9 or more times, 10 or more times, 20 or more times, 25 or more times, 50 or more times, 75 or more times, 100 or more times, 500 or more times, 1,000 or more times, 5,000 or more times, 10,000 or more times, etc.

[0069] The number of times the addition step and the dispersion step are performed may be the same or different. Typically, the dispersion step is performed after the addition step, so the number of times the addition step and dispersion step are the same, or the number of times the dispersion step is one more than the number of addition steps.

[0070] This process and the dispersion treatment in this process are carried out under conditions that are substantially free of amphiphilic organic solvents and / or lipophilic surfactants. Examples of amphiphilic organic solvents and / or 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 components and amounts originally present in the cells or their cell lysates used. Typically, the oil-in-water solution 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 cell lysates in a water-in-oil solution 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.

[0071] There are no particular limitations on the capacity of the container in which dispersion processing and agitation are performed. 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 connected by piping, the capacity including the piping within the range affected by the external force applied for dispersion processing (for example, within the range of the space partitioned during dispersion processing if partitions are provided during dispersion processing) should be as stated above.

[0072] The distributed processing in this process is carried out by one or more methods selected from the group consisting of high-speed rotation, pressure, and ultrasonic methods.

[0073] The "high-speed rotation method" is a method of dispersion that utilizes the high shear force generated by the high-speed rotation of a rotating body (rotating blade or rotor). While there are no specific limitations on the type of equipment used, examples include installing a homomixer Mark II (PRIMIX), an UltraTax homogenizer T25 (IKA), or a Creamix (M-Technic) in a container. The homomixer Mark II is a device that disperses 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 disperses fluid discharged from a high-speed rotating rotor as it passes through a minute slit. The conditions for using such dispersion equipment can be appropriately set for each model and are not particularly limited. For example, the tip speed of the rotating body (diameter of the rotating body × 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.

[0074] For dispersion using a high-speed rotation method, equipment capable of continuously processing large volumes of fluid, such as in-line 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 dispersers also disperse fluid 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 dispersion tends to be higher. The various conditions of an in-line 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.

[0075] The "pressure method" is a method of dispersing a liquid by applying high pressure and forcing it to flow 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 dispersion 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.

[0076] For pressure-based dispersion, inline equipment such as inline static fluid mixing devices can be suitably used. Specifically, this includes methods of applying pressure to a static mixer (manufactured by Noritake), an MSE static mixer (manufactured by Iris Inc.), an OHR mixer (manufactured by OHR Fluid Engineering Laboratory), etc., and flowing the fluid through it. 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 dispersion 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 dispersion using these devices can be appropriately set 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, 0.7 MPa or higher, 0.8 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 2 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, more preferably 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 The pressure can be set to 0.4MPa to 1MPa, 0.5MPa to 3MPa, 0.5MPa to 2.5MPa, 0.5MPa to 2MPa, 0.5MPa to 1.5MPa, 0.5MPa to 1MPa, 0.6MPa to 3MPa, 0.6MPa to 2.5MPa, 0.6MPa to 2MPa, 0.6MPa to 1.5MPa, 0.6MPa to 1MPa, 0.7MPa to 3MPa, 0.7MPa to 2.5MPa, 0.7MPa to 2MPa, 0.7MPa to 1.5MPa, 0.7MPa to 1MPa, 0.8MPa to 3MPa, 0.8MPa to 2.5MPa, 0.8MPa to 2MPa, 0.8MPa to 1.5MPa, 0.8MPa to 1MPa, etc.

[0077] The "ultrasonic method" is a method of dispersion 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 dispersion using 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.

[0078] There is no particular 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, 2.5 minutes or more, 3 minutes 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, for example, 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 9 minutes, 30 seconds to 8 minutes, 30 seconds to 7 minutes, 30 seconds to 6 minutes, 30 seconds to 5 minutes, 30 seconds to 4 minutes, 30 seconds to 3 minutes, 30 seconds to 2.5 minutes, 30 seconds to 2 minutes, 1 minute to 60 minutes, 1 minute to 45 minutes, 1 minute to 30 minutes, 1 minute to 20 minutes, 1 minute to 15 minutes, 1 minute to 10 minutes, 1-9 minutes, 1-8 minutes, 1-7 minutes, 1-6 minutes, 1-5 minutes, 1-4 minutes, 1-3 minutes, 1-2.5 minutes, 1-2 minutes, 1.5-60 minutes, 1.5-45 minutes, 1.5-30 minutes, 1.5-20 minutes, 1.5-15 minutes, 1.5-10 minutes, 1.5-9 minutes, 1.5-8 minutes, 1.5-7 minutes, 1.5-6 minutes, 1.5-5 minutes, 1.5-4 minutes, 1.5-3 minutes, 1.5-2.5 minutes, 1.5-2 minutes, 2-60 minutes, 2-45 minutes, 2-30 minutes, 2-20 minutes, 2-15 minutes, 2-10 minutes, 2-9 minutes, 2-8 minutes, 2-7 minutes, 2-6 minutes, 2-5 minutes, 2-4 minutes, 2-3 minutes, 2-2.5 minutes, 2.5-60 minutes, 2.5-45 minutes, 2.5-30 minutes, 2.5-20 minutes, 2.5-15 minutes The intervals can be set to minutes, 2.5 to 10 minutes, 2.5 to 9 minutes, 2.5 to 8 minutes, 2.5 to 7 minutes, 2.5 to 6 minutes, 2.5 to 5 minutes, 2.5 to 4 minutes, 2.5 to 3 minutes, 3 to 60 minutes, 3 to 45 minutes, 3 to 30 minutes, 3 to 20 minutes, 3 to 15 minutes, 3 to 10 minutes, 3 to 9 minutes, 3 to 8 minutes, 3 to 7 minutes, 3 to 6 minutes, 3 to 5 minutes, 3 to 4 minutes, etc.

[0079] Specifically, for example, the conditions could be: tip velocity of 2.4 m / s or more for 40 minutes or less; tip velocity of 2.4 m / s or more for 30 seconds to 40 minutes; tip velocity of 2.4 m / s or more for 1 minute to 30 minutes; tip velocity of 2.4 m / s or more for 1.5 minutes to 20 minutes; tip velocity of 2.4 m / s or more for 1.5 minutes to 10 minutes; tip velocity of 2.4 m / s or more for 1.5 minutes to 5 minutes; tip velocity of 2.4 m / s or more for 1.5 minutes to 3 minutes; tip velocity of 2.4 m / s or more for 1.5 minutes to 2 minutes; tip velocity of 2.4 m / s or more for 2 minutes to 10 minutes, etc. Furthermore, for example, when performing dispersion using a pressure method, conditions such as pressure of 0.3 MPa or higher for 40 minutes or less, pressure of 0.6 MPa or higher for 40 minutes or less, pressure of 0.6 MPa or higher for 30 seconds to 40 minutes, pressure of 0.6 MPa or higher for 1 minute to 30 minutes, pressure of 0.6 MPa or higher for 2 minutes to 20 minutes, pressure of 0.6 MPa or higher for 3 minutes to 15 minutes, pressure of 0.6 MPa or higher for 4 minutes to 10 minutes, and pressure of 0.6 MPa or higher for 5 minutes or more The test conditions can be set to 10 minutes, 40 minutes or less at a pressure of 0.8 MPa or higher, 30 seconds to 40 minutes at a pressure of 0.8 MPa or higher, 1 minute to 30 minutes at a pressure of 0.8 MPa or higher, 2 minutes to 20 minutes at a pressure of 0.8 MPa or higher, 3 minutes to 15 minutes at a pressure of 0.8 MPa or higher, 1 minute to 5 minutes at a pressure of 0.8 MPa or higher, 1 minute to 4 minutes at a pressure of 0.8 MPa or higher, 1 minute to 3 minutes at a pressure of 0.8 MPa or higher, etc.

[0080] The temperature conditions during the dispersion process are not particularly limited, as long as the dispersion process 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 aqueous solution and the hydrophobic organic solvent. 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.

[0081] 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.

[0082] The pH conditions during dispersion processing are not particularly limited. For example, the process can be carried out using the pH conditions described above for aqueous solutions.

[0083] 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.

[0084] 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. When this process is performed multiple times, at least this process and the separation process will each be performed multiple times.

[0085] The method according to this embodiment can be performed using either batch extraction or continuous extraction.

[0086] 1-3-3. Dispersion Maintenance Step The "dispersion maintenance step" is an essential step in the method of this embodiment and is a step in which the dispersion state of the water-in-oil solution is maintained. This step can be performed simultaneously with or after the dispersion step. This step does not include emulsification.

[0087] The water-in-oil solution only needs to remain in a dispersed state, and the conditions for this are not particularly limited. For example, the dispersed state can be maintained by continuing the dispersion process under the same conditions as the dispersion process. The dispersion conditions in this process can be within the range of the conditions detailed in the dispersion process, and may be the same as or different from those in the dispersion process.

[0088] The time for this process, and the combined time for the distributed process and this process, are not particularly limited, but for example, they can be the times exemplified in the distributed process. In particular, for example, the time for this process, and the combined time for the distributed process and this process, are 30 seconds to 60 minutes, 30 seconds to 45 minutes, 30 seconds to 40 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, 1 minute to 60 minutes, 1 minute to 45 minutes, 1 minute to 40 minutes, 1 minute to 30 minutes, 1 minute to 20 minutes, 1 minute to 15 minutes, 1 minute to 10 minutes, 1 minute to 5 minutes, 1.5 minutes to 60 minutes, 1.5 minutes to 45 minutes, 1.5 minutes to 40 minutes, 1.5 minutes to 30 minutes, 1.5 minutes to The intervals can be 20 minutes, 1.5 to 15 minutes, 1.5 to 10 minutes, 1.5 to 5 minutes, 2 to 60 minutes, 2 to 45 minutes, 2 to 40 minutes, 2 to 30 minutes, 2 to 20 minutes, 2 to 15 minutes, 2 to 10 minutes, 2 to 5 minutes, 5 to 60 minutes, 5 to 45 minutes, 5 to 40 minutes, 5 to 30 minutes, 5 to 20 minutes, 5 to 15 minutes, 5 to 10 minutes, 10 to 60 minutes, 10 to 45 minutes, 10 to 40 minutes, 10 to 30 minutes, 10 to 20 minutes, 10 to 15 minutes, etc.

[0089] This process can be performed multiple times in conjunction with the distributed process. When this process is 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.

[0090] 1-3-4. Alkali Treatment Step The "alkali treatment step" is an optional step in the method of this embodiment, and is a step in which the pH of the water-in-oil solution is increased. This step can be performed simultaneously with the dispersion step, and / or simultaneously with the dispersion maintenance step if one is performed.

[0091] In this process, the pH of the water-in-oil solution is increased. There are no particular limitations on the range of pH increase in this process. For example, if the aqueous solution used as a raw material is an acidic solution, the pH of the water-in-oil solution can be increased until it becomes alkaline. For example, in this process, the pH can be increased in ranges such as 1 or higher, 1.5 or higher, 2 or higher, 2.5 or higher, 2.6 or higher, 2.7 or higher, 2.8 or higher, 2.9 or higher, 3 or higher, 3 or higher, 3.1 or higher, 3.2 or higher, 3.3 or higher, 3.4 or higher, 3.5 or higher, 3.6 or higher, 3.7 or higher, 3.8 or higher, 3.9 or higher, 4 or higher, 4.1 or higher, 4.2 or higher, 4.3 or higher, 4.4 or higher, 4.5 or higher, 4.6 or higher, etc. Specific ranges of pH increase include, for example, 1-9, 1.5-8, 2-7.5, 2-7, 2-6.5, 2-6, 2-5.5, 2-5, 2-4.9, 2-4.8, 2-4.7, 2-4.6, 2.5-7.5, 2.5-7, 2.5-6.5, 2.5-6, 2.5-5.5, 2.5-5, 2.5-4.9, 2.5-4.8, 2.5-4.7, 2.5-4.6, 2.7-7, 2.8-7, 2.8-6.5, 2.8-6, 2.8-5.5, 2.8-5, 2.8-4.9, 2.8-4.8, 2.8-4.7, 2.8- It can be 4.6, 2.9-7, 3-6.5, 3-6, 3-5.5, 3-5, 3-4.9, 3-4.8, 3-4.7, 3-4.6, 3.2-6.5, 3.2-6, 3.2-5.5, 3.2-5, 3.2-4.9, 3.2-4.8, 3.2-4.7, 3.2-4.6, 3.4-6, 3.5-6, 3.7-5.5, 3.7-5, 3.7-4.9, 3.7-4.8, 3.7-4.7, 3.7-4.6, 3.9-5.5, 4-5, 4.2-5, 4-5, 4.2-7, 4.4-7, 4.5-7, 4.6-7, etc.

[0092] The pH after the rise in this process is not particularly limited. For example, the pH can be 7 or higher, 7.1 or higher, 7.5 or higher, 8 or higher, 8.1 or higher, 8.2 or higher, 8.3 or higher, 8.4 or higher, 8.5 or higher, 8.6 or higher, 8.7 or higher, 8.8 or higher, 8.9 or higher, 9 or higher, 9.1 or higher, 9.2 or higher, 9.3 or higher, 9.5 or higher, 9.7 or higher, 9.9 or higher, 10 or higher, etc. Also, for example, the pH can be 14 or lower, 13 or lower, 12 or lower, 11 or lower, 10.5 or lower, 10.4 or lower, 10.3 or lower, 10.2 or lower, 10.1 or lower, etc. The specific pH ranges after the pH increase in this process are, for example, 7-14, 7.1-14, 7.5-14, 7.5-13, 8-14, 8-13, 8-12, 8-11, 8-10.5, 8-10.4, 8-10.3, 8-10.2, 8-10.1, 8.1-14, 8.1-13, 8.1-12, 8.1-11, 8.1-10.5, 8.1-10.4, and 8.1-10. 3, 8.1-10.2, 8.1-10.1, 8.2-14, 8.2-13, 8.2-12, 8.2-11, 8.2-10.5, 8.2-10.4, 8.2-10.3, 8.2-10.2, 8.2-10.1, 8.3-14, 8.3-13, 8.3-12, 8.3-11, 8.3-10.5, 8.3-10.4, 8.3-10.3, 8.3-10.2, 8.3-1 0.1, 8.5-14, 8.5-13, 8.7-14, 8.7-13, 8.7-12, 8.7-11, 8.7-10.5, 8.7-10.4, 8.7-10.3, 8.7-10.2, 8.7-10.1, 8.9-14, 8.9-13, 9-14, 9-13, 9-12, 9-11, 9-10.5, 9-10.4, 9-10.3, 9-10.2, 9-10.1, 9. The ranges can be 2-14, 9.2-13, 9.2-12, 9.2-11, 9.2-10.5, 9.2-10.4, 9.2-10.3, 9.2-10.2, 9.2-10.1, 9.5-14, 9.5-13, 9.5-12, 9.7-14, 9.7-13, 9.7-12, 9.9-14, 9.9-13, 9.9-12, 10-14, 10-13, 10-12, etc.

[0093] The method for increasing the pH in this process is not particularly limited, but typically the pH can be increased by adding an alkalizing agent. The alkalizing agent may be added in solid form or in liquid form. The type of alkalizing agent used in this case is not particularly limited, and for example, alkaline compounds known in the art can be used. The alkalizing agent may be an inorganic compound, an organic compound, or a combination thereof. For example, an inorganic alkalizing agent can be suitably used. Specific alkalizing agents include, for example, sodium hydroxide, potassium hydroxide, calcium hydroxide, rubidium hydroxide, cesium hydroxide, barium hydroxide, calcium carbonate, calcium bicarbonate, calcium oxide, magnesium oxide, magnesium carbonate, potassium carbonate, sodium carbonate, sodium bicarbonate, trisodium phosphate, sodium pyrophosphate, sodium tripolyphosphate, sodium tetrapolyphosphate, tripotassium phosphate, potassium pyrophosphate, potassium tripolyphosphate, sodium orthosilicate, sodium metasilicate, potassium silicate, or a combination thereof.

[0094] The timing of this process is not particularly limited and may be simultaneous with the distribution process and / or the distribution maintenance process. For example, it may be performed simultaneously with the distribution maintenance process. The timing of this process in each of the above processes is not particularly limited. For example, it may be performed simultaneously with the start of each process (distribution maintenance process, etc.) or a certain period of time after the start. If this process is performed a certain period of time after the start of each process, the specific timing is not particularly limited. For example, it may be performed 30 seconds or more after the start of each process, 1 minute or more after the start of each process, 1.5 minutes or more after the start of each process, 2 minutes or more after the start of each process, 2.5 minutes or more after the start of each process, 3 minutes or more after the start of each process, 5 minutes or more after the start of each process, or 10 minutes or more after the start of each process. Specifically, for example, between 30 seconds and 30 minutes after the start of each process, between 30 seconds and 20 minutes, between 30 seconds and 15 minutes, between 30 seconds and 10 minutes, between 30 seconds and 5 minutes, between 1 minute and 30 minutes, between 1 minute and 20 minutes, between 1 minute and 15 minutes, between 1 minute and 10 minutes, between 1 minute and 5 minutes, between 1.5 minutes and 30 minutes, between 1.5 minutes and 20 minutes, between 1.5 minutes and 15 minutes, between 1.5 minutes and 10 minutes, between 1.5 minutes and 5 minutes, between 2 minutes and 30 minutes, 2 minutes and It can be done at intervals of 20 minutes, 2 to 15 minutes, 2 to 10 minutes, 2 to 5 minutes, 2.5 to 30 minutes, 2.5 to 20 minutes, 2.5 to 15 minutes, 2.5 to 10 minutes, 2.5 to 5 minutes, 3 to 30 minutes, 3 to 20 minutes, 3 to 15 minutes, 3 to 10 minutes, 3 to 5 minutes, 5 to 30 minutes, 5 to 20 minutes, 5 to 15 minutes, 5 to 10 minutes, etc.

[0095] 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.

[0096] 1-3-5. Separation Step The "separation step" is an essential step in the method of this embodiment, and is a step of stopping the dispersion process and separating the aqueous phase and oil phase of the water-in-oil solution. This step can be performed after the dispersion maintenance step.

[0097] In this process, the aqueous phase and the oil phase are separated by stopping the dispersion process. "Stopping the dispersion process" means reducing the dispersion conditions to a level where the aqueous phase and the oil phase can be separated, and includes completely stopping the external force used in the dispersion process. In particular, the aqueous phase and the oil phase are separated by reducing the dispersion conditions used in the dispersion process (tip speed of the rotating body in the case of a high-speed rotation method, pressure in the case of a pressure method, frequency in the case of an ultrasonic method) to below their lower limits (for example, less than 1 m / s (less than 10 m / s for an in-line method), less than 40 MPa (less than 0.1 MPa for an in-line method), or less than 15 kHz).

[0098] In the case of high-speed rotation systems, especially non-inline high-speed rotation systems, the tip speed of the rotating body only needs to be less than 1 m / s and is not particularly limited. For example, it can be 0.9 m / s or less, 0.8 m / s or less, 0.7 m / s or less, 0.6 m / s or less, 0.5 m / s or less, 0.4 m / s or less, 0.3 m / s or less, 0 m / s, etc.

[0099] In the case of an in-line high-speed rotation system, the tip speed of the rotating body only needs to be less than 10 m / s and is not particularly limited. For example, in addition to the tip speeds exemplified for non-in-line high-speed rotation systems, it can be 9 m / s or less, 8 m / s or less, 7 m / s or less, 6 m / s or less, 5 m / s or less, 4 m / s or less, 3 m / s or less, 2 m / s or less, 1 m / s or less, etc.

[0100] The pressure in the pressure system, especially the in-line pressure system, only needs to be less than 0.1 MPa and is not particularly limited. For example, it can be 0.09 MPa or less, 0.08 MPa or less, 0.07 MPa or less, 0.06 MPa or less, 0.05 MPa or less, 0.04 MPa or less, 0.03 MPa or less, 0.02 MPa or less, 0.01 MPa or less, 0 MPa, etc.

[0101] In the case of a non-inline pressure system, the tip speed of the rotating body only needs to be less than 40 MPa and is not particularly limited. For example, in addition to the pressures exemplified for the inline pressure system, the pressures can be 39 MPa or less, 35 MPa or less, 30 MPa or less, 20 MPa or less, 15 MPa or less, 10 MPa or less, 5 MPa or less, 1 MPa or less, 0.5 MPa or less, 0.1 MPa or less, etc.

[0102] In the case of the ultrasonic method, the frequency does not need to be more than 15 kHz, and is not particularly limited. It can be 14 kHz or less, 12 kHz or less, 11 kHz or less, 10 kHz or less, 8 kHz or less, 6 kHz or less, 5 kHz or less, 3 kHz or less, 1 kHz or less, etc.

[0103] The method described herein does not require any additional treatment for separation. However, any additional methods used to facilitate the separation of the aqueous and oil phases may be added as appropriate.

[0104] The additional processing in this case is not particularly limited. For example, it may be used to add inorganic salts, acids, alkalis, surfactants, change the temperature, apply centrifugal force, filter, change the volume ratio of the continuous phase to the dispersed phase, apply a high-voltage AC electric field, irradiate with ultrasound, or a combination thereof.

[0105] The processing time for this step is not particularly limited. Typically, it can be carried out until the dispersion conditions decrease to the ranges mentioned above. Specific times can be, for example, 1 minute to 2 hours, 5 minutes to 2 hours, 10 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, 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, 30 minutes to 1 hour, 1 minute to 45 minutes, 5 minutes to 45 minutes, 10 minutes to 45 minutes, 30 minutes to 45 minutes, 1 minute to 30 minutes, 5 minutes to 30 minutes, 10 minutes to 30 minutes, 1 minute to 15 minutes, 5 minutes to 15 minutes, 10 minutes to 15 minutes, 1 minute to 10 minutes, 5 minutes to 10 minutes, 1 minute to 5 minutes, etc. For example, the dispersion conditions may be reduced over the aforementioned period of time, and the separation state can be stabilized by maintaining the reduced conditions for the aforementioned period of time after the dispersion conditions have been reduced.

[0106] When this process is performed multiple times, it may be carried out under the same conditions each time, or the conditions may differ in one or more trials.

[0107] 1-4. Effects According to the method of this embodiment, oil-soluble molecules can be easily extracted from an aqueous solution into the oil phase without using amphiphilic organic solvents such as isopropyl alcohol.

[0108] 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.

[0109] 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, 96% or more, 97% or more, 97.5% or more, 98% or more, 98.5% or more, 99% or more, etc. The extraction efficiency may be improved by performing the method of this embodiment multiple times.

[0110] 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 a dispersion step, a dispersion maintenance step, a separation step, and a recovery step as essential steps, and in the case of a method for preparing oil-soluble molecules, the method includes a dispersion step, a dispersion maintenance step, a separation 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 from cells or their cell lysates with high efficiency and in a simple manner without using amphiphilic organic solvents or lipophilic surfactants.

[0111] 2-2. Process The method of this embodiment, in the case of a method for preparing an oil-soluble molecule-containing solution, includes a dispersion step, a dispersion maintenance step, a separation step, and a recovery step as essential steps, and includes a raw material solution preparation step and an alkali treatment step as optional steps. In the case of a method for preparing oil-soluble molecules, it includes a dispersion step, a dispersion maintenance step, a separation step, a recovery step, and a purification step as essential steps, and includes a raw material solution preparation step and an alkali treatment step as optional steps. Each step will be described in detail below.

[0112] 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.

[0113] 2-2-2. Dispersion Step The "dispersion 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 dispersed in a hydrophobic organic solvent by one or more methods selected from the group consisting of high-speed rotation, pressure, and ultrasonic methods to form a water-in-oil solution, and 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.

[0114] 2-2-3. Dispersion Maintenance Step The "dispersion maintenance step" is an essential step in the method of this embodiment and is a step in which the dispersion state of the water-in-oil solution is maintained. This step can be performed simultaneously with or after the dispersion step. This step may be performed in accordance with the description in the first embodiment.

[0115] 2-2-4. Alkali Treatment Step The "alkali treatment step" is an optional step in the method of this embodiment, and is a step in which the pH of the water-in-oil solution is increased. This step can be performed simultaneously with the dispersion step, and / or simultaneously with the dispersion maintenance step if one is performed. This step may be performed in accordance with the description in the first embodiment.

[0116] 2-2-5. Separation Step The "separation step" is an essential step in the method of this embodiment, and is a step in which the dispersion process is stopped and the aqueous phase and oil phase of the water-in-oil solution are separated. This step can be performed after the dispersion maintenance step. This step may be performed in accordance with the description in the first embodiment.

[0117] 2-2-6. 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 a solution containing oil-soluble molecules. This process can be performed simultaneously with or after the separation process.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 2-2-7. 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.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] 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.

[0129] The present invention will now be described in detail with reference to examples, but the present invention is not limited to these examples.

[0130] <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.

[0131] (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).

[0132] To the obtained microbial cell lysate, twice the volume of n-hexane was added and mixed to prepare a mixed solution.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] 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.

[0138] On the other hand, when half the volume of microbial cell lysate was added to n-hexane over a period of approximately 3 seconds, and dispersed 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 microbial cell lysate phase dispersed into the hexane phase, but it did not form a stable emulsion, and when the dispersion process was stopped, the mixture immediately separated into oil and water.

[0139] The hexane phase, obtained by separating oil and water, was allowed to stand for 30 minutes before being collected as an extract and subjected to HPLC analysis to calculate the extraction rate.

[0140] The extraction rate of coenzyme Q10 was 80%, which was an improvement compared to the conditions under which the sample was dispersed at a low tip velocity of approximately 0.29 m / s (Figure 1).

[0141] This demonstrates that by performing dispersion under highly dispersed conditions, oil-soluble molecules can be extracted easily, efficiently, and without the use of amphiphilic organic solvents.

[0142] <Example 2: Effect of Different Dispersion Methods on Extraction Rate> (Objective) To investigate the effect of different dispersion methods on the extraction rate of oil-soluble molecules.

[0143] (Method and Results) A mixture of microbial cell lysate (pH: approximately 5.5) prepared in the same manner as in Example 1 and n-hexane was subjected to a dispersion treatment for 3 minutes using an OHR mixer (manufactured by OHR Fluid Engineering Laboratory) under flow conditions of 20°C and 0.8 MPa. Although the microbial cell lysate phase dispersed in the hexane phase during this dispersion treatment, it did not form a stable emulsion, and the mixture immediately separated into oil and water when the dispersion treatment was stopped.

[0144] The hexane phase, obtained by separating oil and water, was allowed to stand for 30 minutes before being collected as an extract and subjected to HPLC analysis to calculate the extraction rate.

[0145] The calculated extraction rate of coenzyme Q10 was 88.4%. This indicates that, regardless of the dispersion method used, and especially when starting from a mixed solution, oil-soluble molecules can be extracted easily and efficiently without the use of amphiphilic organic solvents by performing dispersion under high dispersion conditions.

[0146] <Example 3: Effect of pH of microbial cell lysate used in dispersion treatment on extraction rate> (Objective) To investigate the effect of pH of microbial cell lysate used in dispersion treatment on the extraction rate of oil-soluble molecules.

[0147] (Methods and Results) The experiment was conducted in the same manner as in Example 2, except that a solution was used in which a microbial cell lysate (pH: approximately 5.5) prepared in the same manner as in Example 1 was adjusted to pH 9 by adding NaOH solution. The microbial cell lysate phase was dispersed in the hexane phase by dispersion treatment with an OHR mixer, but it did not form a stable emulsion, and when the dispersion treatment was stopped, the mixture immediately separated into oil and water.

[0148] The hexane phase, obtained by separating oil and water, was allowed to stand for 30 minutes before being collected as an extract and subjected to HPLC analysis to calculate the extraction rate.

[0149] The calculated extraction rate of coenzyme Q10 was 90.9%. This confirms that the pH of the microbial cell lysate used in the dispersion process does not significantly affect the extraction rate.

[0150] <Example 4: Effect of pH on extraction rate in dispersion maintenance process> (Objective) To investigate the effect of pH in the dispersion maintenance process on the extraction rate of oil-soluble molecules.

[0151] (Method and Results) First, the dispersion treatment was carried out in the same manner as in Example 2. Through this dispersion treatment, the microbial cell lysate phase was dispersed in the hexane phase.

[0152] By stopping the OHR mixer and simultaneously continuing stirring at 350 rpm using a disc-type stirring blade, the dispersion state of the water-in-oil solution, in which the microbial cell crushing liquid phase was dispersed in the hexane phase, was maintained.

[0153] Alkalination treatment was performed immediately after the OHR mixer was stopped. As shown in Table 1, NaOH was added to the water-in-oil solution (pH: approximately 5.5) to adjust the pH to 8.3-10.1.

[0154] After alkaline treatment, the dispersion was maintained for 15 minutes. Then, when stirring with the disc-type impeller was stopped, the mixture immediately separated into oil and water.

[0155] The hexane phase, obtained by separating oil and water, was allowed to stand for 30 minutes before being collected as an extract and subjected to HPLC analysis to calculate the extraction rate.

[0156] The calculated extraction rates of coenzyme Q10 are shown in Table 1.

[0157]

[0158] As shown in Table 1, it was demonstrated that the extraction rate can be improved by providing a dispersion maintenance step to maintain the dispersed state after the dispersion treatment, and by performing an alkaline treatment to raise the pH in the dispersion maintenance step.

[0159] <Example 5: Effect of Dispersion Maintenance Step Time on Extraction Rate> (Objective) To investigate the effect of the dispersion maintenance step time on the extraction rate of oil-soluble molecules.

[0160] (Methods and Results) The experiment was conducted in the same manner as in Example 4, except that the alkaline treatment was performed 5 minutes after the OHR mixer was stopped, the pH was adjusted to 9.0 by the alkaline treatment, and stirring was continued for 1 minute, 5 minutes, 10 minutes, or 30 minutes after the alkaline treatment. The microbial cell lysate phase was dispersed into the hexane phase by the dispersion treatment using the OHR mixer, and when stirring with the disc-type impeller was stopped, the mixture immediately separated into oil and water.

[0161] The hexane phase, obtained by separating oil and water, was allowed to stand for 30 minutes before being collected as an extract and subjected to HPLC analysis to calculate the extraction rate.

[0162] The calculated extraction rates of coenzyme Q10 were 97.5% after 1 minute of continuous stirring, 96.4% after 5 minutes of continuous stirring, 97.3% after 10 minutes of continuous stirring, and 97.8% after 30 minutes of continuous stirring.

[0163] This confirms that the extraction rate is not significantly affected by the duration of the dispersion maintenance process or the timing of the alkaline treatment. 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: a dispersion step of dispersing an aqueous solution containing the cells or cell lysates in a hydrophobic organic solvent using one or more methods selected from the group consisting of a high-speed rotation method, a pressure method and an ultrasonic method to form a water-in-oil solution; a dispersion maintenance step of maintaining the dispersion state of the water-in-oil solution; and a separation step of stopping the dispersion process and separating the aqueous phase and oil phase of the water-in-oil solution, wherein the dispersion step is carried out under conditions that substantially do not contain amphiphilic organic solvents and / or lipophilic surfactants.

2. The method according to claim 1, wherein the dispersion maintenance step is performed for a period of 40 minutes or less.

3. The method according to claim 1, wherein the dispersion step comprises mixing the aqueous solution with the hydrophobic organic solvent in a volume of 0.01 to 4 times the volume of the hydrophobic organic solvent.

4. The method according to any one of claims 1 to 3, wherein the dispersion step includes adding the aqueous solution to the hydrophobic organic solvent at a rate of 60 volume% / s or less relative to the volume of the hydrophobic organic solvent.

5. The method according to any one of claims 1 to 3, wherein the dispersion step comprises an addition step of adding the aqueous solution to the hydrophobic organic solvent in a volume of 60% or less of the hydrophobic organic solvent, and a dispersion step of dispersing the added aqueous solution in the hydrophobic organic solvent, and the addition step and the dispersion step are each performed two or more times.

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

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

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

9. The method according to claim 1, further comprising an alkali treatment step of increasing the pH of the water-in-oil solution.

10. A method for preparing an oil-soluble molecule-containing solution containing oil-soluble molecules in cells or cell lysates, comprising: a dispersion step of dispersing an aqueous solution containing the cells or cell lysates in a hydrophobic organic solvent using one or more methods selected from the group consisting of high-speed rotation, pressure, and ultrasonic methods to form a water-in-oil solution; a dispersion maintenance step of maintaining the dispersion state of the water-in-oil solution; a separation step of stopping the dispersion process and separating the aqueous phase and oil phase of the water-in-oil solution; and a recovery step of recovering the separated oil phase as an oil-soluble molecule-containing solution, wherein the dispersion step is carried out under conditions that substantially do not contain amphiphilic organic solvents and / or lipophilic surfactants.

11. A method for preparing oil-soluble molecules in cells or cell lysates thereof, comprising: a dispersion step of dispersing an aqueous solution containing the cells or cell lysates in a hydrophobic organic solvent using one or more methods selected from the group consisting of high-speed rotation, pressure, and ultrasonic methods to form a water-in-oil solution; a dispersion maintenance step of maintaining the dispersion state of the water-in-oil solution; a separation step of stopping the dispersion process and separating the aqueous phase and oil phase of the water-in-oil solution; 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 dispersion step is carried out under conditions that substantially do not contain amphiphilic organic solvents and / or lipophilic surfactants.

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