WO2012011589A1 - 脂溶性生理活性物質の製造方法 - Google Patents
脂溶性生理活性物質の製造方法 Download PDFInfo
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- WO2012011589A1 WO2012011589A1 PCT/JP2011/066764 JP2011066764W WO2012011589A1 WO 2012011589 A1 WO2012011589 A1 WO 2012011589A1 JP 2011066764 W JP2011066764 W JP 2011066764W WO 2012011589 A1 WO2012011589 A1 WO 2012011589A1
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- fat
- extraction
- organic solvent
- physiologically active
- coenzyme
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
- C07C45/78—Separation; Purification; Stabilisation; Use of additives
- C07C45/80—Separation; Purification; Stabilisation; Use of additives by liquid-liquid treatment
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/66—Preparation of oxygen-containing organic compounds containing the quinoid structure
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/10—Foods or foodstuffs containing additives; Preparation or treatment thereof containing emulsifiers
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D11/00—Solvent extraction
- B01D11/02—Solvent extraction of solids
- B01D11/0288—Applications, solvents
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B61/00—Dyes of natural origin prepared from natural sources, e.g. vegetable sources
Definitions
- the present invention relates to a method for producing a fat-soluble physiologically active substance. Specifically, the present invention relates to a method for producing a fat-soluble physiologically active substance by extracting a fat-soluble physiologically active substance from an aqueous suspension of a microbial cell containing a fat-soluble physiologically active substance or a microbial cell disruption product thereof.
- coenzyme Q is an essential component widely distributed in the living body from bacteria to mammals, and is known as a component of mitochondrial electron transport system in cells in the living body.
- Coenzyme Q plays a role as a transfer component in the electron transport system by repeating oxidation and reduction in mitochondria, and it is known that reduced coenzyme Q of coenzyme Q has an antioxidant effect. It has been.
- coenzyme Q10 having 10 repeating side chains of coenzyme Q is the main component, and in vivo, about 40 to 90% is usually present as a reduced form.
- Examples of the physiological action of coenzyme Q include activation of energy production by mitochondrial activation action, activation of cardiac function, stabilization effect of cell membrane, protection effect of cells by antioxidant action, and the like.
- the oxidized coenzyme Q10 has been conventionally used as a congestive heart failure drug or a health food.
- a reduced coenzyme Q10 having a higher physiological activity has become known.
- a fat-soluble physiologically active substance such as coenzyme Q10 can be obtained, for example, by synthesis, fermentation, extraction from a natural product, or the like. If necessary, the obtained extract can be purified by chromatography or crystallized by crystallization to obtain a product with higher purity.
- a method is generally employed in which a microorganism that produces coenzyme Q10 is cultured and coenzyme Q10 in the microorganism is extracted from the suspension of the microorganism using an organic solvent. .
- an aqueous suspension of cultured microorganisms is dehydrated to form wet cells, and then contacted with an organic solvent.
- an organic solvent There are known a method of further drying after dehydration and bringing the cells into contact with an organic solvent as dried cells, and a method of bringing an aqueous suspension into contact with an organic solvent as it is and extracting between the liquid and the liquid.
- Patent Document 1 a suspension of cultured Phaffia yeast is centrifuged to recover the cells, and the recovered cells are spray-dried, and then astaxanthin in the cells while being crushed with a mixed solvent such as hexane and ethanol.
- a mixed solvent such as hexane and ethanol.
- Other examples include culturing Mortierella cells, dehydrating and drying the cell suspension, and then extracting arachidonic acid-containing oil with hexane (Patent Document 2), culturing Mucor cells
- Patent Document 3 An example (Patent Document 3) is known in which ⁇ -linolenic acid is extracted with a solvent such as hexane after the liquid is crushed and freeze-dried. In these extraction methods, dry cells and an organic solvent that is an extraction solvent are mixed, and after the extraction operation is completed, solid-liquid separation is performed to remove cell residues, thereby obtaining an organic phase containing the target substance. it can.
- Patent Document 4 after wet cells or dried cells of a coenzyme Q10-containing microorganism are brought into contact with methanol at a low temperature and removing impurities inside and outside the cells, the next is brought into contact with methanol at a high temperature, An example in which coenzyme Q10 is extracted is disclosed.
- a solid-liquid extraction operation since the specific gravity difference between the bacterial cells and the extraction solvent is large, solid-liquid separation after extraction is easy, and there is an advantage that extraction with high efficiency is possible with little loss of the target substance. .
- the disrupted suspension of cultured microorganisms was directly contacted with an organic solvent such as hexane or 2-propanol to extract coenzyme Q10 in the cells.
- an organic solvent such as hexane or 2-propanol
- the target substance can be extracted with a high yield and a large throughput without dehydrating and drying the microorganisms.
- the present invention reduces the yield of a lipid-soluble physiologically active substance from microbial cells containing a fat-soluble physiologically active substance without using special dehydration and drying equipment, and by the deterioration of the separation between the solvent and the bacterial cell component. It aims at providing the manufacturing method which can extract without incurring and can industrially produce efficiently.
- a specific surfactant is present in an aqueous suspension of microbial cells or microbial cell disruptions containing a fat-soluble physiologically active substance, and an organic solvent is used.
- an organic solvent is used.
- the oil-water separation proceeds rapidly after mixing and standing, and the fat-soluble physiologically active substance can be extracted efficiently.
- the present invention was completed by finding that it is suitable for industrial production.
- the present invention is as follows.
- the addition amount of the nonionic surfactant is 0.01% by weight or more with respect to the aqueous suspension of microbial cells or microbial cell disruptions
- the conventional method for extracting useful components from microbial cells has problems in equipment, production cost, and operational stability, and has the same problem as a method for producing fat-soluble physiologically active substances.
- the fat-soluble physiologically active substance can be efficiently extracted by the liquid-liquid extraction operation, which is suitable for industrial production.
- the preferred specific surfactant found in the present invention during the liquid-liquid extraction operation, the dispersed phase containing the target substance at the time of extraction is finely dispersed in the extraction solvent, and the target substance is extracted into the extraction solvent.
- the oil-water separation when the organic phase and the aqueous phase are allowed to stand still can be performed quickly.
- Loss of the extraction solvent due to the transfer of the organic phase to can also be suppressed. Further, conventionally, in order to obtain a target substance with a higher yield, even when a plurality of types of extraction solvents are required for the extraction operation, the present invention performs a high yield operation using only a single solvent. Therefore, the apparatus and operation can be simplified, and effects such as reduction of energy required for solvent recovery and reduction of environmental load can be obtained.
- an organic solvent is mixed from an aqueous suspension of microbial cells or microbial cell disruptions containing a fat-soluble physiologically active substance in the presence of a specific nonionic surfactant described later, and the organic solvent phase is mixed with fat.
- a method for producing a fat-soluble physiologically active substance characterized by extracting a soluble physiologically active substance.
- the fat-soluble physiologically active substance to be extracted is a physiologically active substance that is produced in microbial cells, has affinity for organic solvents (lipid soluble), and is useful for living organisms. If there is, it will not be specifically limited.
- Specific examples thereof include coenzymes Q such as coenzyme Q10, vitamins such as vitamin A, vitamin D, vitamin E and vitamin K, carotenoids such as carotene, astaxanthin and fucoxanthin, fat-soluble polyphenols, Examples include flavonoids, sterols such as ergosterol, ⁇ -lipoic acid, L-carnitine and the like. Of these, coenzyme Q10, astaxanthin, ergosterol and the like are preferable, and coenzyme Q10 is particularly preferable.
- coenzyme Q10 has an oxidized type and a reduced type.
- the present invention targets both the oxidized coenzyme Q10 and the reduced coenzyme Q10 as the coenzyme Q10, but the coenzyme Q10 containing the reduced coenzyme Q10, ie, the reduced coenzyme Q10 alone, It is preferable to target coenzyme Q10 which is a mixture of reduced coenzyme Q10 and oxidized coenzyme Q10.
- coenzyme Q10 which is a mixture of reduced coenzyme Q10 and oxidized coenzyme Q10.
- the microorganism containing the fat-soluble physiologically active substance used in the present invention is a microorganism that produces the target fat-soluble physiologically active substance or its precursor in the cell or a microorganism originally containing a certain amount or more of the substance. Any of bacteria, yeast and mold can be used without limitation. Of these, microorganisms that produce the fat-soluble physiologically active substance in the cells are preferred.
- microorganism examples include, for example, the genus Agrobacterium, the genus Aspergillus, the genus Acetobacter, the genus Aminobacter, the genus Agromonas, the acidiphyllum ( Acidiphilium, Bulleromyces, Bullera, Brevundimonas, Cryptococcus, Chionosphaera, Candida, Cerinosterus, Cerinosterus, Cerinosterus, Cerinosterus Exisophiala genus, Exobasidium genus, Fellomyces genus, Filobabasidiella genus, Filobasidium genus, Geotrichum genus, Graphiola genus, Gluconobacter genus , Kokkobaela Kockovaella genus, Kurtzmanomyces genus, Lalaria genus, Leucosporidium genus, Legionella genus, Methylobacterium genus
- bacteria or yeasts are preferable, and bacteria are more preferably non-photosynthetic bacteria.
- Agrobacterium, Gluconobacter, and Methylobacterium are particularly preferably the genus Schizosaccharomyces, the genus Saitoella, the Phaffia genus, etc.
- a microorganism that produces a fat-soluble physiologically active substance outside the cells, that is, produces the substance in the culture solution is also encompassed in the present invention.
- microorganisms that produce fat-soluble physiologically active substances include not only wild strains of the above-mentioned microorganisms, but also, for example, transcription and translational activity or expression of genes involved in the biosynthesis of the target lipid-soluble physiologically active substances of the above-mentioned microorganisms. Mutants and recombinants in which the enzyme activity of the protein is modified or improved can also be preferably used. By culturing the microorganism, microbial cells containing a fat-soluble physiologically active substance such as coenzyme Q10 can be obtained.
- the culture method is not particularly limited, and a culture method suitable for the target microorganism or suitable for production of the target fat-soluble physiologically active substance can be appropriately selected.
- the culture period is not particularly limited as long as a desired amount of the desired fat-soluble physiologically active substance is produced in the microbial cells.
- the production amount (content) of the fat-soluble physiologically active substance in that case is not particularly limited depending on the purpose.
- the content of the fat-soluble physiologically active substance per medium is, for example, 0.5 ⁇ g / mL or more, preferably 1 ⁇ g. / ML or more, more preferably 2 ⁇ g / mL or more.
- the fat-soluble physiologically active substance when extracting the fat-soluble physiologically active substance from the microorganism cell containing the fat-soluble physiologically active substance, it can be directly extracted from the microorganism cell. It can also be used as a microbial cell disruption product and extracted from the disruption product.
- Cell disruption contributes to efficient extraction of fat-soluble bioactive substances produced and accumulated in microbial cells.
- the cell disruption treatment may not always be necessary for bacteria, but when using yeast or mold cells, it is particularly preferable to perform the cell disruption treatment. When yeast or mold cells are used, if the cells are not disrupted, the recovery efficiency of the fat-soluble physiologically active substance produced and accumulated in the cells is lowered. Needless to say, cell disruption and extraction may be performed simultaneously.
- the surface structure such as the cell wall is damaged to the extent that extraction of the desired fat-soluble physiologically active substance is possible, and the microbial cells are not necessarily broken or fragmented. There is no need.
- microbe cell or microbial cell disruption aqueous suspension refers to a microbial cell or microbial cell disruption suspended in an aqueous solvent such as water, physiological saline, buffer, or culture medium. Preferably, it is suspended in water and / or medium.
- the form of the microbial cell to be subjected to cell disruption is an aqueous suspension of a microbial cell, a culture solution, a concentrated culture solution, a microbial cell collected from the culture solution as a wet cell, or a washed product thereof.
- a solvent for example, including water, physiological saline, buffer solution, etc.
- dried cells obtained by drying the wet cells dried cells with a solvent (for example, water, (Including physiological saline, buffer solution, etc.) may be used, but preferably, an aqueous suspension of microbial cells, a culture solution, a concentrated culture solution, or a washed product thereof.
- a culture solution, a solution obtained by concentrating the culture solution, or a solution obtained by washing them are preferably, a culture solution, a solution obtained by concentrating the culture solution, or a solution obtained by washing them.
- the disruption of the microbial cells is performed by performing one or several of the following disruption methods in an arbitrary order.
- the crushing method include physical treatment, chemical treatment, enzymatic treatment, heat treatment, autolysis, osmotic pressure dissolution, protoplast dissolution, and the like.
- Examples of the physical treatment include the use of a high-pressure homogenizer, a rotary blade homogenizer, an ultrasonic homogenizer, a French press, a ball mill, or a combination thereof.
- Examples of the chemical treatment include treatment using an acid such as hydrochloric acid and sulfuric acid (preferably a strong acid), treatment using a base such as sodium hydroxide and potassium hydroxide (preferably a strong base), and combinations thereof. Can be mentioned.
- an acid such as hydrochloric acid and sulfuric acid (preferably a strong acid)
- a base such as sodium hydroxide and potassium hydroxide (preferably a strong base)
- combinations thereof can be mentioned.
- Examples of the enzymatic treatment include methods using lysozyme, zymolyase, glucanase, novozyme, protease, cellulase, etc., and these may be used in combination as appropriate.
- Examples of the heat treatment include a treatment at 60 to 140 ° C. for about 30 minutes to 3 hours.
- Examples of the self-digestion include treatment with a solvent such as ethyl acetate.
- treatment with a solution different from the intracellular salt concentration can cause osmotic lysis or protoplast lysis of the cells.
- this method since this method alone often has an insufficient cell disruption effect, it is preferably used in combination with the physical treatment, chemical treatment, enzymatic treatment, heat treatment, self-digestion and the like as described above.
- the cell disruption method as a pretreatment for extraction / recovery of a fat-soluble physiologically active substance includes physical treatment, chemical treatment (especially acid treatment, preferably strong acid (for example, in an aqueous solution) among the disruption methods described above. (Acid having a pKa of 2.5 or less) and heat treatment are preferred, and physical treatment is more preferred from the viewpoint of crushing efficiency.
- a microbial cell containing a fat-soluble physiologically active substance, or a pulverized product of a microbial cell containing a fat-soluble physiologically active substance obtained as described above, in the state of an aqueous suspension is fat-soluble. Extract physiologically active substances.
- the method for preparing an aqueous suspension of microbial cells or microbial cell disruptions is not particularly limited.
- a culture solution after culturing a microorganism that produces a fat-soluble physiologically active substance It is prepared by suspending washed or wet cells or dried cells of the microbial cells in water or an aqueous solvent.
- an aqueous suspension of microbial cells can be prepared by crushing by the above method.
- the concentration of microbial cells in the aqueous suspension of microbial cells or microbial cell disruptions to be extracted is not particularly limited, but is usually 1 to 25 weights in terms of the dry weight of the microbial cells. It is preferable to carry out in the range of 10 to 20% by weight economically.
- an aqueous suspension of microbial cells or microbial cell disruptions containing the fat-soluble physiologically active substance and an organic solvent are mixed, and a liquid-
- a liquid- By extracting the fat-soluble physiologically active substance into the organic solvent phase by liquid extraction, preferably without leaving the step of forced oil-water separation, the mixture is left to stand for oil-water separation, and the fat is separated from the separated organic solvent phase.
- the soluble physiologically active substance is collected. That is, the substance can be efficiently obtained by continuously performing a step of extracting the fat-soluble physiologically active substance from the mixed solution of the aqueous suspension and the organic solvent, and a step of allowing the mixture to stand and separating the oil and water. You can also.
- glycerin fatty acid esters sucrose fatty acid esters, sorbitan fatty acid esters, polyether polyol type surfactants, polyoxyethylene alkyl ether type interfaces are used as nonionic surfactants used during extraction. It is necessary to use an activator, a polyoxyethylene-polyoxypropylene block copolymer type surfactant or an alkyl ether type surfactant. Two or more of these nonionic surfactants can be used in combination, or these nonionic surfactants and other surfactants may be used in combination.
- glycerin fatty acid esters include partial glycerides of fatty acids, polyglycerin fatty acid esters, polyglycerin condensed ricinoleic acid esters, and the like.
- partial glycerides of fatty acids include monoglycerol monocaprylate, monoglycerol monocaprate, monoglycerol dicaprylate, monoglycerol dicaprate, monoglycerol dilaurate, monoglycerol dimyristate, Monoglycerin fatty acid esters such as monoglyceryl distearate, monoglycerin dioleate, monoglycerin dierucate, monoglycerin dibehenate; monoglycerin caprylic acid succinate, monoglycerin stearate citrate, Monoglycerol stearate acetate, monoglycerol stearate succinate, monoglycerol stearate lactate, monoglycerol ester Phosphoric
- polyglycerin fatty acid ester examples include polyglycerin mainly composed of polyglycerin having a polymerization degree of 2 to 10, and fatty acid having 6 to 22 carbon atoms each esterified to one or more hydroxyl groups of polyglycerin. Is mentioned.
- hexaglycerin monocaprylic acid ester hexaglycerin dicaprylic acid ester, decaglycerin monocaprylic acid ester, triglycerin monolauric acid ester, tetraglycerin monolauric acid ester, pentaglycerin monolauric acid ester, hexaglycerin monolauric acid ester , Decaglycerol monolaurate, triglycerol monomyristate, pentaglycerol monomyristate, pentaglycerol trimyristate, hexaglycerol monomyristate, decaglycerol monomyristate, diglycerol monooleate, tri Glycerol monooleate, tetraglycerin monooleate, pentaglycerin Monooleic acid ester, hexaglycerin monooleic acid ester, decaglycerin monooleic acid ester,
- polyglycerin condensed ricinoleic acid ester examples include those having an average degree of polymerization of polyglycerin of 2 to 10 and an average degree of condensation of polyricinoleic acid (average of the number of condensation of ricinoleic acid) of 2 to 4, for example. Tetraglycerin condensed ricinoleic acid ester, pentaglycerin condensed ricinoleic acid ester, hexaglycerin condensed ricinoleic acid ester and the like.
- sucrose fatty acid esters examples include those obtained by esterifying one or more hydroxyl groups of sucrose with fatty acids having 6 to 18, preferably 6 to 12 carbon atoms. Specific examples include sucrose palmitate and sucrose stearate.
- sorbitan fatty acid esters examples include those obtained by esterifying one or more hydroxyl groups of sorbitans with fatty acids having 6 to 18 carbon atoms, preferably 6 to 12 carbon atoms. Specific examples include sorbitan monostearate and sorbitan monooleate.
- polyether polyol type surfactant examples include Adecanol LG series (LG-109, LG-126, LG-294, LG-295S, LG-299, LG-805) manufactured by ADEKA Corporation. It is done.
- polyoxyethylene alkyl ether type surfactants those obtained by addition polymerization of ethylene oxide to aliphatic alcohols having 12 to 22 carbon atoms are preferable.
- Emulgen series (103, 104P) manufactured by Kao Corporation. , 105, 106, 108, 109P, 120, 123P, 147, 150, 210, 220, 306P, 320P, 350, 404, 408, 409PV, 420, 430, 705, 707, 709, 1108).
- polyoxyethylene-polyoxypropylene block copolymer type surfactant examples include propylene oxide (PO) between ethylene oxide (EO) chains, which are block copolymers obtained by adding ethylene oxide to both ends of polypropylene glycol.
- PO propylene oxide
- EO ethylene oxide
- EOxPOyEOz reverse type polyoxyethylene-polyoxypropylene block copolymer
- ethylenediamine type polyoxyethylene-polyoxypropylene block copolymer and the like are also included.
- Examples of the polyoxyethylene-polyoxypropylene block copolymer having a propylene oxide (PO) chain between the ethylene oxide (EO) chains include, for example, Pluronic L series (L-31, L-34, L-44, L-61, L-62, L-64, L-71, L-72, L-101, L-121), Pluronic P series (P-65, P-84, P-85, P -103, P-105, P-123), Pluronic F series (F-68, F-108, F-127), and Pluronic PE series manufactured by BASF.
- Pluronic L series L-31, L-34, L-44, L-61, L-62, L-64, L-71, L-72, L-101, L-121
- Pluronic P series P-65, P-84, P-85, P -103, P-105, P-123
- Pluronic F series F-68, F-108, F-127
- Examples of the reverse type polyoxyethylene-polyoxypropylene block copolymer include Pluronic R series (25R-1, 25R-2, 17R-2, 17R-3, 17R-4) manufactured by ADEKA Corporation, and BASF Corporation. Examples include the Pluronic RPE series made by the company.
- ethylenediamine-type polyoxyethylene-polyoxypropylene block copolymer examples include, for example, Pluronic TR series (TR-701, TR-702, TR-704) manufactured by ADEKA Corporation, and Tetronic series (poloxamine) manufactured by BASF Corporation. Is mentioned.
- a triblock copolymer type surfactant having a butylene oxide (BO) chain between two ethylene oxide (EO) chains having a structure similar to that of a polyoxyethylene-polyoxypropylene block copolymer type surfactant.
- the agent EOxBOyEOz
- the agent can also be used in the production method of the present invention.
- surfactants having a weight average molecular weight in the range of 500 to 8000 are preferred, and a weight average molecular weight of 1000 to A surfactant in the range of 4000 is more preferred.
- alkyl ether type nonionic surfactant examples include ADEKA LB series (LB-53B, LB-720, LB-820, LB-54C, LB-83, LB-93, LB) manufactured by ADEKA Corporation. -103, LB-1220, LB-1520), Adekator LA series (LA-675B, LA-775, LA-875, LA-975, LA-1275) and the like.
- nonionic surfactants two or more of the nonionic surfactants shown here may be used in combination.
- the nonionic surfactants it is preferable to use at least a polyoxyethylene-polyoxypropylene block copolymer type surfactant.
- one polyoxyethylene-polyoxypropylene block copolymer type surfactant can be used alone, but two or more polyoxyethylene-polyoxypropylene block copolymer type surfactants are used in combination.
- Nonionic surfactants to be combined with polyoxyethylene-polyoxypropylene block copolymer type surfactants include the nonionic surfactants described above, that is, glycerin fatty acid esters, sucrose fatty acid esters, sorbitan Examples include fatty acid esters, polyether polyol type surfactants, polyoxyethylene alkyl ether type surfactants, and alkyl ether type surfactants. Among them, a combination of two types of polyoxyethylene-polyoxypropylene block copolymer type surfactants and a combination of polyoxyethylene-polyoxypropylene block copolymer type surfactants and sucrose fatty acid esters are preferable.
- More preferred is a combination of two polyoxyethylene-polyoxypropylene block copolymer type surfactants, and a polyoxyethylene-polyoxypropylene block copolymer having a propylene oxide (PO) chain between ethylene oxide (EO) chains and
- PO propylene oxide
- EO ethylene oxide
- the combination of ethylenediamine type polyoxyethylene-polyoxypropylene block copolymer is particularly preferred.
- the amount of polyoxyethylene-polyoxypropylene block copolymer type surfactant used is the other amount. More than nonionic surfactants are preferred, for example, preferably 50% by weight or more, more preferably 60% by weight or more, and more preferably 75% by weight or more based on the total amount of nonionic surfactants used. preferable.
- a dispersed phase containing a fat-soluble physiologically active substance as a target substance can be finely dispersed in an organic solvent as an extraction solvent during extraction. it can.
- the contact efficiency between the extraction solvent and the fat-soluble physiologically active substance is improved, and the transfer of the fat-soluble physiologically active substance to the organic solvent phase is promoted.
- a surfactant is conventionally used for liquid-liquid extraction, the affinity between the aqueous phase and the organic solvent phase increases due to the interfacial effect.
- a solvent for dissolving the surfactant when a paste-like or flake-like nonionic surfactant is used, it is preferable to use a solvent for dissolving the surfactant. Even when a liquid surfactant is used, it is preferable to use a solvent when the viscosity is high.
- a solvent used in that case water and alcohols are desirable, and they may be used alone or as a mixed solvent of water and alcohol.
- the amount of the specific nonionic surfactant used during the extraction operation is 0.01% by weight or more as the concentration with respect to the aqueous suspension of microbial cells or microbial cell disruptions. Preferably, it is in the range of 0.01 to 10% by weight, more preferably in the range of 0.1 to 5% by weight, and in the range of 0.5 to 5% by weight. It is particularly preferred.
- the amount of the surfactant added is 0.01% by weight or less, the fine dispersion of the aqueous suspension in the organic solvent does not proceed and sufficient extraction efficiency cannot be ensured.
- the amount of the surfactant added exceeds 10% by weight, the affinity between the organic solvent and the aqueous suspension becomes higher than necessary, so that the fine dispersion of the aqueous suspension in the organic solvent is promoted.
- the oil-water separation property may be deteriorated when the organic solvent and the aqueous suspension in a mixed state are left standing.
- the method for performing extraction in the presence of the specific nonionic surfactant is not particularly limited, and a predetermined amount of surfactant is contained in the mixture of the aqueous suspension and the organic solvent at the time of extraction.
- a method of adding a surfactant to an aqueous suspension of microbial cells or microbial cell disruption before extraction, and a surfactant to an organic solvent used for extraction In addition to the method of adding a surfactant to a mixture of an organic solvent and an aqueous suspension, a surfactant is added in advance during or before preparation of an aqueous suspension of microbial cells or microbial cell disruptions. Or a method in which the surfactant used for disrupting the microbial cells is used as it is during extraction.
- organic solvents used for extraction include hydrocarbons, fatty acid esters, ethers, alcohols, fatty acids, ketones, nitrogen compounds (including nitriles and amides), sulfur compounds And the like.
- the hydrocarbons are not particularly limited, and examples thereof include aliphatic hydrocarbons, aromatic hydrocarbons, and halogenated hydrocarbons. Of these, aliphatic hydrocarbons and aromatic hydrocarbons are preferable, and aliphatic hydrocarbons are more preferable.
- the aliphatic hydrocarbon is not particularly limited regardless of whether it is cyclic or non-cyclic, or saturated or unsaturated, but saturated hydrocarbons are generally preferably used. Usually, those having 3 to 20 carbon atoms, preferably 5 to 12 carbon atoms, more preferably 5 to 8 carbon atoms are used.
- the aromatic hydrocarbon is not particularly limited, but usually an aromatic hydrocarbon having 6 to 20 carbon atoms, preferably 6 to 12 carbon atoms, more preferably 7 to 10 carbon atoms is used. Specific examples include, for example, benzene, toluene, xylene, o-xylene, m-xylene, p-xylene, ethylbenzene, cumene, mesitylene, tetralin, butylbenzene, p-cymene, cyclohexylbenzene, diethylbenzene, pentylbenzene, dipentylbenzene. , Dodecylbenzene, styrene and the like.
- the halogenated hydrocarbon is not particularly limited regardless of whether it is cyclic or non-cyclic, or saturated or unsaturated, but generally non-cyclic hydrocarbons are preferably used. More preferred are chlorinated hydrocarbons and fluorinated hydrocarbons, and even more preferred are chlorinated hydrocarbons. Further, those having 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, more preferably 1 to 2 carbon atoms are suitably used. Specific examples include, for example, dichloromethane, chloroform, carbon tetrachloride, 1,1-dichloroethane, 1,2-dichloroethane, 1,1,1-trichloroethane, 1,1,2-trichloroethane, 1,1,1,2, and the like.
- 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 and the like. More preferred are dichloromethane, chloroform, 1,2-dichloroethylene, trichloroethylene, chlorobenzene, 1,1,1,2-tetrafluoroethane and the like.
- the fatty acid esters are not particularly limited, and examples thereof include propionic acid esters, acetic acid esters, and formic acid esters. Preferred are acetate esters and formate esters, and more preferred are acetate esters.
- the ester group is not particularly limited, but is usually an alkyl ester having 1 to 8 carbon atoms, an aralkyl ester having 7 to 12 carbon atoms, preferably an alkyl ester having 1 to 6 carbon atoms, more preferably 1 to 1 carbon atom. 4 alkyl esters are used.
- propionic acid ester examples include, for example, methyl propionate, ethyl propionate, butyl propionate, isopentyl propionate, and the like. Preferred is ethyl propionate.
- acetate ester examples include, for example, methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate, isobutyl acetate, sec-butyl acetate, pentyl acetate, isopentyl acetate, sec-hexyl acetate, cyclohexyl acetate, benzyl acetate and the like. Can be mentioned.
- formate ester examples include, for example, methyl formate, ethyl formate, propyl formate, isopropyl formate, butyl formate, isobutyl formate, sec-butyl formate, pentyl formate, and the like.
- Preferred are methyl formate, ethyl formate, propyl formate, butyl formate, isobutyl formate, pentyl formate, and the like. Most preferred is ethyl formate.
- Ethers are not particularly limited, regardless of whether they are cyclic or non-cyclic, and saturated or unsaturated. In general, saturated ones are preferably used. Usually, those having 3 to 20 carbon atoms, preferably 4 to 12 carbon atoms, more preferably 4 to 8 carbon atoms are used.
- Specific examples include, for example, diethyl ether, methyl tert-butyl ether, dipropyl ether, diisopropyl ether, dibutyl ether, dihexyl ether, ethyl vinyl ether, butyl vinyl ether, anisole, phenetole, butyl phenyl ether, methoxy toluene, dioxane, furan, 2 -Methyl furan, tetrahydrofuran, tetrahydropyran, ethylene glycol dimethyl ether, ethylene glycol diethyl ether, ethylene glycol dibutyl ether, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether and the like.
- diethyl ether methyl tert-butyl ether, dipropyl ether, diisopropyl ether, dibutyl ether, dihexyl ether, anisole, phenetol, butyl phenyl 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, and the like.
- Alcohols are not particularly limited regardless of whether they are cyclic or non-cyclic, or saturated or unsaturated, but saturated alcohols are generally preferably used. Usually, it has 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, more preferably 1 to 6 carbon atoms. Of these, monohydric alcohols having 1 to 5 carbon atoms, dihydric alcohols having 2 to 5 carbon atoms, and trihydric alcohols having 3 carbon atoms are preferable.
- these alcohols include, for example, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutyl alcohol, tert-butyl alcohol, 1-pentanol, 2-pentanol, 3 -Pentanol, 2-methyl-1-butanol, isopentyl alcohol, tert-pentyl alcohol, 3-methyl-2-butanol, neopentyl alcohol, 1-hexanol, 2-methyl-1-pentanol, 4-methyl- 2-pentanol, 2-ethyl-1-butanol, 1-heptanol, 2-heptanol, 3-heptanol, 1-octanol, 2-octanol, 2-ethyl-1-hexanol, 1-nonanol, 1-decanol, 1 -Undecanol, 1-Dodecano Monohydric alcohols such as sodium, allyl alcohol,
- the monohydric alcohol is preferably methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutyl alcohol, tert-butyl alcohol, 1-pentanol, 2-pentanol, 3-pen Tanol, 2-methyl-1-butanol, isopentyl alcohol, tert-pentyl alcohol, 3-methyl-2-butanol, neopentyl alcohol, 1-hexanol, 2-methyl-1-pentanol, 4-methyl-2- Pentanol, 2-ethyl-1-butanol, 1-heptanol, 2-heptanol, 3-heptanol, 1-octanol, 2-octanol, 2-ethyl-1-hexanol, 1-nonanol, 1-decanol, 1-undecanol 1-Dodecanol, Be Benzyl alcohol, cyclohexanol, 1-methylcyclo
- Particularly preferred are methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutyl alcohol, 2-methyl-1-butanol, isopentyl alcohol, etc.
- 2-propanol. is there.
- 1,2-ethanediol, 1,2-propanediol, 1,3-propanediol and the like are preferable, and 1,2-ethanediol is most preferable.
- trihydric alcohol glycerin is preferable.
- fatty acids examples include formic acid, acetic acid, propionic acid, and the like. Preferred are formic acid and acetic acid, and most preferred is acetic acid.
- the ketones are not particularly limited, and those having 3 to 6 carbon atoms are preferably used. Specific examples include acetone, methyl ethyl ketone, methyl butyl ketone, methyl isobutyl ketone, and the like. Preferred are acetone and methyl ethyl ketone, and most preferred is acetone.
- Nitriles are not particularly limited regardless of whether they are cyclic or non-cyclic, saturated or unsaturated, but saturated ones are generally preferably used. Usually, those having 2 to 20 carbon atoms, preferably 2 to 12 carbon atoms, more preferably 2 to 8 carbon atoms are used.
- Specific examples include, for example, acetonitrile, propionitrile, malononitrile, butyronitrile, isobutyronitrile, succinonitrile, valeronitrile, glutaronitrile, hexanenitrile, heptyl cyanide, octyl cyanide, undecane nitrile, dodecane nitrile, tridecane.
- nitrogen compounds other than nitriles include amides such as formamide, N-methylformamide, N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, nitromethane, triethylamine, pyridine and the like. be able to.
- sulfur compounds include dimethyl sulfoxide and sulfolane.
- the boiling point can be moderately heated to increase the solubility, and from the viewpoint of easy removal of the solvent from the wet body and recovery of the solvent from the crystallization filtrate, etc.
- the melting point is preferably in the range of 30 to 150 ° C., and the melting point is about 20 ° C. or less, preferably about 10 ° C. or less, more preferably from the viewpoint of being hard to solidify when handled at room temperature and when cooled to below room temperature. Is about 0 ° C. or lower, and the viscosity is preferably as low as about 10 cp at 20 ° C., for example.
- organic solvents for the purpose of extracting and recovering fat-soluble physiologically active substances from aqueous suspensions of microbial cells or microbial cell disruptions, extraction is performed from the viewpoint of performing liquid-liquid extraction in a two-phase system. It is preferable to use a hydrophobic organic solvent or a solvent containing a hydrophobic organic solvent as the solvent.
- the hydrophobic organic solvent used in this case is not particularly limited, and among the above-mentioned organic solvents, a hydrophobic solvent which is not completely miscible with water and becomes a two-phase system can be used.
- a hydrophobic solvent which is not completely miscible with water and becomes a two-phase system can be used.
- Fatty acid esters, ethers and other hydrophobic organic solvents more preferably hydrocarbons, more preferably aliphatic hydrocarbons. Of the aliphatic hydrocarbons, those having 5 to 8 carbon atoms are preferably used.
- aliphatic hydrocarbons having 5 to 8 carbon atoms include, 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. Particularly preferred are hexane, heptane and methylcyclohexane, and most preferred are hexane and heptane.
- a hydrophilic organic solvent can be supplementarily used in combination to further improve the aqueous suspension in the organic solvent. It is more preferable since it can promote the refinement and can improve the stability of high oil / water separation at the time of standing.
- the hydrophilic organic solvent used in combination with the hydrophobic organic solvent is not particularly limited, and a hydrophilic one of the above-mentioned organic solvents can be used. is there.
- the alcohols monohydric alcohols having 1 to 5 carbon atoms are preferably used. Specific examples thereof include, for example, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutyl alcohol, tert-butyl alcohol, 1-pentanol, 2-pentanol, and 3-pen.
- Examples include butanol, 2-methyl-1-butanol, isopentyl alcohol, tert-pentyl alcohol, 3-methyl-2-butanol, neopentyl alcohol, and the like. Particularly preferred are methanol, ethanol, 1-propanol and 2-propanol, and most preferred is 2-propanol.
- the amount of the hydrophobic organic solvent used as the extraction solvent is not particularly limited, but the concentration at the time of extraction may be the total solution of the extraction system (microorganism cells or aqueous suspension of the cell debris). It is preferably used in the range of 25 to 80% by volume, more preferably in the range of 50 to 75% by volume with respect to the volume of the mixed solution of turbid liquid, extraction solvent, nonionic surfactant, etc. preferable.
- the amount of the hydrophilic organic solvent used when the hydrophilic organic solvent is used in combination as described above is not particularly limited as long as the two-phase system can be maintained.
- the hydrophilic organic solvent used is preferably used in the range of 50% by volume, more preferably in the range of 0.1 to 10% by volume, and still more preferably in the range of 0.2 to 5% by volume. Even if the amount of the hydrophilic organic solvent used is a very small amount such as 0.2 to 2% by volume with respect to the total solution volume, the effect can be exhibited in the present invention.
- the method of adding each solvent when using a hydrophilic organic solvent and a hydrophobic organic solvent in combination is not particularly limited, and the extraction solvent is obtained by appropriately mixing the hydrophilic organic solvent and the hydrophobic organic solvent.
- a hydrophobic organic solvent may be added after adding a hydrophilic organic solvent to an aqueous suspension of microorganism cells or disrupted microorganism cells, or vice versa.
- the temperature at the time of extraction is not particularly limited, but it can be generally 0 to 60 ° C., preferably 20 to 50 ° C.
- the extraction method either batch extraction or continuous extraction can be performed, but industrially preferable is continuous extraction in terms of productivity, and countercurrent multistage extraction is particularly preferable among continuous extractions.
- the stirring time for batch extraction is not particularly limited, but is usually 5 minutes or longer, and the average residence time for continuous extraction is not particularly limited, but is usually 10 minutes or longer.
- an aqueous suspension of a microbial cell or cell disrupted product, an organic solvent, and the surfactant described above are mixed for extraction for a predetermined time, and then the mixture is allowed to stand. , Separate the water phase from the organic solvent phase containing the fat-soluble physiologically active substance, but if separation at the oil-water interface is extremely slow, forcefully use a centrifuge, continuous centrifuge, hydrocyclone, etc. It can also be separated.
- the extraction rate in the production method of the present invention is usually 70% or more, more preferably 80% or more, and further preferably 90% or more.
- the extraction rate here refers to the fat-soluble physiologically active substance contained in the extract after the completion of the extraction operation with respect to the total amount of the fat-soluble physiologically active substance contained in the aqueous suspension of microbial cells or microbial cell crushed material before extraction. It can be calculated
- a fat-soluble physiologically active substance is obtained by extracting a fat-soluble physiologically active substance in an organic solvent from a microbial cell or a microbial cell lysate containing the fat-soluble physiologically active substance by the above operation. Can be isolated and recovered.
- the obtained organic solvent solution containing the fat-soluble physiologically active substance can be used as it is or can be further purified by subjecting it to a conventional purification method. For example, after purification with an adsorbent such as activated carbon or clay, the organic solvent can be distilled off to obtain an extract containing a fat-soluble physiologically active substance or a purified product of a fat-soluble physiologically active substance.
- the target fat-soluble physiologically active substance can also be obtained as a crystal body by crystallization operation.
- coenzyme Q10 when the target fat-soluble physiologically active substance is coenzyme Q10, coenzyme Q10 is introduced into an organic solvent from a microbial cell containing coenzyme Q10 or a microbial cell lysate thereof by the production method of the present invention.
- the extract containing the coenzyme Q10 obtained by extraction can be purified by a method known per se to obtain coenzyme Q10.
- purification is performed using an adsorbent treatment such as activated carbon or white clay, column chromatography, etc., and before or after that, oxidation or reduction treatment is performed as necessary, and crystallization operation is used to obtain high-purity coenzyme Q10. Crystals can be obtained.
- an aqueous suspension of a microbial cell containing a fat-soluble physiologically active substance or a microbial cell disruption product thereof is used as a polyoxyethylene-polyoxypropylene block copolymer type surfactant, sucrose fatty acid ester. , Glycerin fatty acid esters, sorbitan fatty acid esters, polyether polyol type surfactants, polyoxyethylene alkyl ether type surfactants and alkyl ether type surfactants.
- a method for purifying a fat-soluble physiologically active substance is characterized by mixing with an organic solvent in the presence of a surfactant and extracting the fat-soluble physiologically active substance.
- Example 1 Saitoella complicata IFO10748 strain producing coenzyme Q10 is used in 10 L medium (peptone 5 g / L, yeast extract 3 g / L, malto extract 3 g / L, glucose 20 g / L, pH 6.0). And aerobically cultured at 25 ° C. for 72 hours. The obtained culture solution containing microbial cells was crushed twice with a pressure homogenizer (manufactured by Runny) at a crushing pressure of 80 MPa to prepare a microbial cell disruption solution containing coenzyme Q10.
- a polyoxyethylene-polyoxypropylene block copolymer type surfactant (Pluronic L-62, manufactured by ADEKA) was added to a concentration of 3.3% by weight. Then, 70 parts by volume of hexane was mixed, and batch extraction was performed at 45 ° C. for 60 minutes. When the mixture was allowed to stand after mixing for a predetermined time, rapid oil-water separation was confirmed, and the volume ratio of the extraction residue (the aqueous phase portion containing the lower microorganism-derived solid matter) to the total amount of the mixture was 0.37. there were. When the separated hexane phase was collected as an extract and analyzed by HPLC, the extraction rate of coenzyme Q10 was 90.8%.
- Example 2 A polyoxyethylene-polyoxypropylene block copolymer type surfactant (Pluronic L-62, manufactured by ADEKA) was added to the cell disruption solution prepared in the same manner as in Example 1 to a concentration of 1.3% by weight. 30 parts by volume of hexane was mixed with 69 parts by volume of hexane and 1 part by volume of 2-propanol, and batch extraction was performed at 45 ° C. for 60 minutes. When the mixture was allowed to stand after mixing for a predetermined time, rapid oil-water separation was confirmed, and the volume ratio of the extraction residue to the total liquid amount was 0.33. When the separated hexane phase was collected as an extract and analyzed by HPLC, the extraction rate of coenzyme Q10 was 92.5%.
- Pluronic L-62 manufactured by ADEKA
- Example 3 A polyether polyol type surfactant (Adecanol LG-126, manufactured by ADEKA) was added to the cell disruption solution prepared in the same manner as in Example 1 to a concentration of 3.3% by weight. Then, 70 parts by volume of hexane was mixed, and batch extraction was performed at 45 ° C. for 60 minutes. When the mixture was allowed to stand after mixing for a predetermined time, rapid oil / water separation was confirmed, and the volume ratio of the extraction residue to the total liquid volume was 0.39. The separated hexane phase was collected as an extract and analyzed by HPLC. As a result, the extraction rate of coenzyme Q10 was 79.5%.
- Example 4 To 30 parts by volume of a cell disruption solution prepared in the same manner as in Example 1, an alkyl ether type surfactant (Adecatol LA-775, manufactured by ADEKA) was added to a concentration of 3.3% by weight. 70 parts by volume of hexane was mixed, and batch extraction was performed at 45 ° C. for 60 minutes. When the mixture was allowed to stand after mixing for a predetermined time, rapid oil-water separation was confirmed, and the volume ratio of the extraction residue to the total liquid volume was 0.35. The separated hexane phase was collected as an extract and analyzed by HPLC. As a result, the extraction rate of coenzyme Q10 was 85.3%.
- an alkyl ether type surfactant (Adecatol LA-775, manufactured by ADEKA) was added to a concentration of 3.3% by weight. 70 parts by volume of hexane was mixed, and batch extraction was performed at 45 ° C. for 60 minutes. When the mixture was allowed to stand after mixing for a predetermined time, rapid
- Example 5 30 parts by volume of a microbial cell disruption solution prepared in the same manner as in Example 1 was added with an alkyl ether type surfactant (Adecatol LA-1275, manufactured by ADEKA) to a concentration of 0.3% by weight. Then, 70 parts by volume of hexane was mixed, and batch extraction was performed at 45 ° C. for 60 minutes. After mixing for a predetermined time, the mixture was allowed to stand, and then forced oil / water separation was performed using a centrifuge. When the separated hexane phase was collected as an extract and analyzed by HPLC, the extraction rate of coenzyme Q10 was 71.3%.
- an alkyl ether type surfactant Alkyl LA-1275, manufactured by ADEKA
- Example 6 To a cell disruption solution prepared in the same manner as in Example 1, 1.3% by weight of polyoxyethylene-polyoxypropylene block copolymer type surfactant (Pluronic L-62, manufactured by ADEKA), sucrose stearate ( S-1670 (manufactured by Mitsubishi Chemical Foods) to a concentration of 0.3% by weight was mixed with 30 parts by volume of hexane, 70 parts by volume of hexane, and batch extraction was performed at 45 ° C. for 60 minutes. . When the mixture was allowed to stand after mixing for a predetermined time, rapid oil-water separation was confirmed, and the volume ratio of the extraction residue to the total liquid volume was 0.32. When the separated hexane phase was collected as an extract and analyzed by HPLC, the extraction rate of coenzyme Q10 was 84.6%.
- Pluronic L-62 polyoxyethylene-polyoxypropylene block copolymer type surfactant
- S-1670 manufactured by Mitsubishi Chemical Foods
- Example 7 In a cell disruption solution prepared in the same manner as in Example 1, 1.3% by weight of polyoxyethylene-polyoxypropylene block copolymer type surfactant (Pluronic L-62, manufactured by ADEKA), ethylenediamine-type polyoxyethylene- Polyoxypropylene block polymer type surfactant (Pluronic TR-702, manufactured by ADEKA) was added to a concentration of 0.3% by weight, and 30 parts by volume of hexane was mixed with 45 parts by volume. A batch extraction operation for 60 minutes was performed. When the mixture was allowed to stand after mixing for a predetermined time, rapid oil-water separation was confirmed, and the volume ratio of the extraction residue to the total liquid volume was 0.30. The separated hexane phase was collected as an extract and analyzed by HPLC. As a result, the extraction rate of coenzyme Q10 was 83.0%.
- Pluronic L-62 polyoxyethylene-polyoxypropylene block copolymer type surfactant
- Pluronic TR-702
- Example 8 In a cell disruption solution prepared in the same manner as in Example 1, 1.3% by weight of polyoxyethylene-polyoxypropylene block copolymer type surfactant (Pluronic L-62, manufactured by ADEKA), ethylenediamine-type polyoxyethylene- Polyoxypropylene block polymer type surfactant (Pluronic TR-701, manufactured by ADEKA) was added to a concentration of 0.3% by weight, and 30 parts by volume of hexane was mixed with 45 parts by volume at 45 ° C. A batch extraction operation for 60 minutes was performed. When the mixture was allowed to stand after mixing for a predetermined time, rapid oil-water separation was confirmed, and the volume ratio of the extraction residue to the total liquid volume was 0.31. The separated hexane phase was collected as an extract and analyzed by HPLC. As a result, the extraction rate of coenzyme Q10 was 89.9%.
- Pluronic L-62 polyoxyethylene-polyoxypropylene block copolymer type surfactant
- Example 9 Saccharomyces cerevisiae IFO0309 strain producing ergosterol is used with 10 L medium (peptone 5 g / L, yeast extract 3 g / L, malto extract 3 g / L, glucose 20 g / L, pH 6.0). Cultured aerobically at 28 ° C. for 72 hours. The obtained microbial cells were crushed twice with a crushing pressure of 80 MPa using a pressure homogenizer (manufactured by Runny) to prepare a microbial cell disruption solution containing ergosterol.
- 10 L medium peptone 5 g / L, yeast extract 3 g / L, malto extract 3 g / L, glucose 20 g / L, pH 6.0. Cultured aerobically at 28 ° C. for 72 hours. The obtained microbial cells were crushed twice with a crushing pressure of 80 MPa using a pressure homogenizer (manufactured by Runny) to prepare a microbial cell disruption solution containing ergosterol.
- a polyoxyethylene-polyoxypropylene block copolymer type surfactant (Pluronic L-62, manufactured by ADEKA) was added to a concentration of 3.3% by weight. Then, 70 parts by volume of hexane was mixed, and batch extraction was performed at 45 ° C. for 60 minutes. When the mixture was allowed to stand after mixing for a predetermined time, rapid oil-water separation was confirmed, and the volume ratio of the extraction residue to the total liquid volume was 0.35. The separated hexane phase was collected as an extract and analyzed by HPLC. As a result, the extraction rate of ergosterol was 89.1%.
- Comparative Example 1 70 parts by volume of hexane was mixed with 30 parts by volume of the cell disruption solution prepared in the same manner as in Example 1, and batch extraction was performed at 45 ° C. for 60 minutes. When the mixture was allowed to stand after mixing for a predetermined time, rapid oil-water separation was confirmed, and the volume ratio of the extraction residue to the total liquid volume was 0.35, but the separated hexane phase was collected as an extract, and HPLC As a result, the extraction rate of coenzyme Q10 was 60.2%.
- Comparative Example 2 70 parts by volume of hexane was mixed with 30 parts by volume of lysolecithin (manufactured by Degussa) to a concentration of 0.7% by weight, which was prepared in the same manner as in Example 1. A batch extraction operation for 60 minutes was performed. After mixing for a predetermined time, the mixture was allowed to stand, but oil / water separation did not proceed. The separated hexane phase was collected as an extract and analyzed by HPLC. As a result, the extraction rate of coenzyme Q10 was 62.2%.
- Table 1 shows the charging conditions, extraction rates of fat-soluble physiologically active substances, and volume ratios of extraction residues in Examples 1 to 9, Comparative Examples 1 to 3, and Reference Example.
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Abstract
Description
補酵素Q10などの脂溶性生理活性物質は、例えば、合成、発酵、天然物から抽出することなどによって得ることができる。また必要に応じ、得られた抽出物を、クロマトグラフィーにより精製したり、晶析により結晶化してより純度の高いものを得ることもできる。例えば、補酵素Q10を得るにあたっては、補酵素Q10を産生する微生物を培養し、その微生物の懸濁液から有機溶媒を用いて微生物中の補酵素Q10を抽出する方法が一般的となっている。
しかし、これらの方法では、抽出前に、培養した微生物の水性懸濁液から大量の水分を、遠心分離、スプレードライヤー、凍結乾燥機等の装置により脱水、乾燥する工程が必要となるうえ、菌体中に残存する含水率によっては十分な抽出率が得られない場合があるほか、装置コスト、運転コストが大きくなる等の問題があった。
また従来、界面活性剤を液-液抽出に使用した場合では、界面効果により水相と有機溶媒相の親和性が高くなるため、均相化が生じやすく、水相と有機溶媒相の分離に長時間を要したり、又は時間をかけても分離しないことが多く、場合によっては遠心分離等の操作により強制的に分離させる必要があった。そのため、一般的な液-液抽出では、抽出工程における界面活性剤の使用は好ましくないと考えられていた。
本発明は、脂溶性生理活性物質を含有する微生物細胞中から、脂溶性生理活性物質を、特殊な脱水、乾燥設備を用いることなく、また溶媒と菌体成分との分離性悪化による収率低下を招くことなく抽出し、効率的に工業生産できる製造法を提供することを目的とする。
[1]脂溶性生理活性物質を含有する微生物細胞又はその微生物細胞破砕物の水性懸濁液を、
ポリオキシエチレン-ポリオキシプロピレンブロックコポリマー型界面活性剤、ショ糖脂肪酸エステル類、グリセリン脂肪酸エステル類、ソルビタン脂肪酸エステル類、ポリエーテルポリオール型界面活性剤、ポリオキシエチレンアルキルエーテル型界面活性剤及びアルキルエーテル型界面活性剤からなる群より選択される少なくとも1種類の非イオン性界面活性剤存在下に、
有機溶媒と混合し、
脂溶性生理活性物質を抽出することを特徴とする脂溶性生理活性物質の製造方法。
[2]脂溶性生理活性物質が、補酵素Q10であることを特徴とする[1]に記載の製造方法。
[3]補酵素Q10が、還元型補酵素Q10、又は、還元型補酵素Q10と酸化型補酵素Q10の混合物であることを特徴とする[2]に記載の製造方法。
[4]非イオン性界面活性剤が、少なくともポリオキシエチレン-ポリオキシプロピレンブロックコポリマー型の界面活性剤であることを特徴とする[1]~[3]のいずれか1項に記載の製造方法。
[5]非イオン性界面活性剤の添加量が、微生物細胞又は微生物細胞破砕物の水性懸濁液に対して、0.01重量%以上であることを特徴とする[1]~[4]のいずれか1項に記載の製造方法。
[6]有機溶媒が、疎水性有機溶媒であることを特徴とする[1]~[5]のいずれか1項に記載の製造方法。
[7]さらに親水性有機溶媒を併用することを特徴とする[6]に記載の製造方法。
[8]抽出は、連続抽出であることを特徴とする[1]~[7]のいずれか1項に記載の製造方法。
[9]脂溶性生理活性物質を含有する微生物細胞又はその微生物細胞破砕物の水性懸濁液を、
ポリオキシエチレン-ポリオキシプロピレンブロックコポリマー型界面活性剤、ショ糖脂肪酸エステル類、グリセリン脂肪酸エステル類、ソルビタン脂肪酸エステル類、ポリエーテルポリオール型界面活性剤、ポリオキシエチレンアルキルエーテル型界面活性剤及びアルキルエーテル型界面活性剤からなる群より選択される少なくとも1種類の非イオン性界面活性剤存在下に、
有機溶媒と混合し、
脂溶性生理活性物質を抽出することを特徴とする脂溶性生理活性物質の精製方法。
[10]脂溶性生理活性物質が、補酵素Q10であることを特徴とする[9]に記載の精製方法。
[11]補酵素Q10が、還元型補酵素Q10、又は、還元型補酵素Q10と酸化型補酵素Q10の混合物であることを特徴とする[10]に記載の精製方法。
[12]非イオン性界面活性剤が、少なくともポリオキシエチレン-ポリオキシプロピレンブロックコポリマー型の界面活性剤であることを特徴とする[9]~[11]のいずれか1項に記載の精製方法。
[13]非イオン性界面活性剤の添加量が、微生物細胞又は微生物細胞破砕物の水性懸濁液に対して、0.01重量%以上であることを特徴とする[9]~[12]のいずれか1項に記載の精製方法。
[14]有機溶媒が、疎水性有機溶媒であることを特徴とする[9]~[13]のいずれか1項に記載の精製方法。
[15]さらに親水性有機溶媒を併用することを特徴とする[14]に記載の精製方法。
[16]抽出は、連続抽出であることを特徴とする[9]~[15]のいずれか1項に記載の精製方法。
更に、本発明で見出した好ましい特定の界面活性剤を液-液抽出操作時に用いることで、抽出時に目的物質を含んだ分散相を抽出溶媒中に微細分散化させ、目的物質の抽出溶媒中への移動を促進するとともに、有機相と水相を静置した際の油水分離も速やかに行えるため、安定的に高い収率で脂溶性生理活性物質の製造が可能となるだけでなく、水相への有機相の移行による抽出溶媒の損失も抑制できる。また、従来、より高収率で目的物質を得るためには抽出操作に複数種の抽出溶媒が必要であった場合に対しても、本発明では単一溶媒のみでも高収率の操作を行うことができるため、装置、操作の簡素化が図れるうえ、溶媒回収にかかるエネルギーの削減、環境負荷低減等の効果も得ることができる。
本発明は、脂溶性生理活性物質を含有する微生物細胞又は微生物細胞破砕物の水性懸濁液から、後述する特定の非イオン性界面活性剤存在下に有機溶媒を混合し、有機溶媒相に脂溶性生理活性物質を抽出することを特徴とする脂溶性生理活性物質の製造方法である。
なお脂溶性生理活性物質を菌体外に産生する、すなわち培養液中に当該物質を産生する微生物も本発明に包含される。
上記微生物を培養することで、補酵素Q10などの脂溶性生理活性物質を含有する微生物細胞を得ることができる。培養方法は特に限定されず、対象となる微生物に適した、あるいは目的とする脂溶性生理活性物質の産生に適した培養方法が適宜選択しうる。培養期間も特に限定されず、微生物細胞中に所望の量の目的とする脂溶性生理活性物質が産生されればよい。その場合の脂溶性生理活性物質の産生量(含有量)としては、目的により特に限定されないが、例えば培地あたりの脂溶性生理活性物質の含有量として、例えば0.5μg/mL以上、好ましくは1μg/mL以上、より好ましくは2μg/mL以上である。
なお、本発明の「破砕」においては、目的とする脂溶性生理活性物質の抽出が可能となる程度に細胞壁等の表面構造が損傷を受ければよく、必ずしも微生物細胞が破れる、あるいは断片化される必要はない。
また、細胞内の塩濃度と異なる溶液で処理することにより、細胞の浸透圧溶解や原形質溶解を引き起こすこともできる。但し、この方法のみでは細胞破砕効果が不十分な場合が多いため、上記のような物理的処理、化学的処理、酵素的処理、加熱処理、自己消化等と合わせて用いるのが好ましい。
上記非イオン性界面活性剤の中でも、ポリオキシエチレン-ポリオキシプロピレンブロックコポリマー型の界面活性剤を少なくとも使用するのが好ましい。その場合、ポリオキシエチレン-ポリオキシプロピレンブロックコポリマー型の界面活性剤1種を単独で使用することもできるが、ポリオキシエチレン-ポリオキシプロピレンブロックコポリマー型の界面活性剤を2種以上組み合わせて使用するか、ポリオキシエチレン-ポリオキシプロピレンブロックコポリマー型の界面活性剤とそれ以外の非イオン性界面活性剤を組み合わせて使用するのが特に好ましい。ポリオキシエチレン-ポリオキシプロピレンブロックコポリマー型の界面活性剤に組み合わせる非イオン性界面活性剤としては、上述したような非イオン性界面活性剤、すなわち、グリセリン脂肪酸エステル類、ショ糖脂肪酸エステル類、ソルビタン脂肪酸エステル類、ポリエーテルポリオール型界面活性剤、ポリオキシエチレンアルキルエーテル型界面活性剤、アルキルエーテル型界面活性剤が挙げられる。そのなかでも、2種のポリオキシエチレン-ポリオキシプロピレンブロックコポリマー型の界面活性剤の組み合わせや、ポリオキシエチレン-ポリオキシプロピレンブロックコポリマー型の界面活性剤とショ糖脂肪酸エステル類の組み合わせが好ましく、2種のポリオキシエチレン-ポリオキシプロピレンブロックコポリマー型の界面活性剤の組み合わせがより好ましく、エチレンオキサイド(EO)鎖の間にプロピレンオキサイド(PO)鎖を有するポリオキシエチレン-ポリオキシプロピレンブロックコポリマーとエチレンジアミン型ポリオキシエチレン-ポリオキシプロピレンブロックコポリマーの組み合わせが特に好ましい。
好ましくは、アセトニトリル、プロピオニトリル、スクシノニトリル、ブチロニトリル、イソブチロニトリル、バレロニトリル、シアノ酢酸メチル、シアノ酢酸エチル、ベンゾニトリル、トルニトリル、クロロプロピオニトリルであり、より好ましくは、アセトニトリル、プロピオニトリル、ブチロニトリル、イソブチロニトリルであり、最も好ましくは、アセトニトリルである。
ここでいう抽出率は、抽出前の微生物細胞又は微生物細胞破砕物の水性懸濁液中に含まれる脂溶性生理活性物質の総量に対する、抽出操作終了後の抽出液に含まれる脂溶性生理活性物質の量の割合であり、具体的には後述する実施例のようにして求めることができる。
例えば、活性炭や白土等の吸着剤で精製した後、有機溶媒を留去して、脂溶性生理活性物質を含む抽出物あるいは脂溶性生理活性物質の精製物とすることもできる。また通常使用されるカラムクロマトグラフィーや液-液分配、その他水や有機溶媒による洗浄などの精製処理に付してもよい。なお、これらの精製処理は、単独もしくは数種組み合わせて行うこともできる。また必要に応じて、鹸化、酸化、還元、その他合成反応処理などの工程を加えることもできる。また晶析操作などで目的とする脂溶性生理活性物質を結晶体として得ることもできる。
抽出率(%)=抽出液に含まれる補酵素Qの重量/抽出前の微生物細胞破砕液中に含まれていた補酵素Qの重量×100
カラム:YMC-Pack4.6×250mm(YMC.Co.,Ltd.製)
移動相:メタノール/n-ヘキサン=85/15
流速:1mL/分
検出:UV275nm
補酵素Q10を産生するサイトエラ・コンプリカタ(Saitoella complicata)IFO10748株を、10Lの培地(ペプトン5g/L、酵母エキス3g/L、マルトエキス3g/L、グルコース20g/L、pH6.0)を用いて、好気的に25℃で72時間培養した。得られた微生物菌体を含む培養液を、圧力式ホモジナイザー(ラニー社製)により破砕圧力80MPaで2回破砕し、補酵素Q10を含有する微生物細胞破砕液を調製した。
得られた微生物細胞破砕液に、ポリオキシエチレン-ポリオキシプロピレンブロックコポリマー型界面活性剤(プルロニックL-62、ADEKA社製)を3.3重量%の濃度となるよう添加したもの30容量部に、ヘキサン70容量部を混合し、45℃で60分間の回分抽出操作を行った。所定時間混合の後、静置させたところ、速やかな油水分離が確認され、混合液の全液量に対する抽出残渣(下層の微生物由来固形物を含む水相部分)の容量比は0.37であった。分離したヘキサン相を抽出液として採取し、HPLCによる分析を行ったところ、補酵素Q10の抽出率は90.8%であった。
実施例1と同様に調製した菌体破砕液に、ポリオキシエチレン-ポリオキシプロピレンブロックコポリマー型界面活性剤(プルロニックL-62、ADEKA社製)を1.3重量%の濃度となるよう添加したもの30容量部に、ヘキサン69容量部、2-プロパノール1容量部をそれぞれ混合し、45℃で60分間の回分抽出操作を行った。所定時間混合の後、静置させたところ、速やかな油水分離が確認され、全液量に対する抽出残渣の容量比は0.33であった。分離したヘキサン相を抽出液として採取し、HPLCによる分析を行ったところ、補酵素Q10の抽出率は92.5%であった。
実施例1と同様に調製した菌体破砕液に、ポリエーテルポリオール型の界面活性剤(アデカノールLG-126、ADEKA社製)を3.3重量%の濃度となるよう添加したもの30容量部に、ヘキサン70容量部を混合し、45℃で60分間の回分抽出操作を行った。所定時間混合の後、静置させたところ、速やかな油水分離が確認され、全液量に対する抽出残渣の容量比は0.39であった。分離したヘキサン相を抽出液として採取し、HPLCによる分析を行ったところ、補酵素Q10の抽出率は79.5%であった。
実施例1と同様に調製した菌体破砕液に、アルキルエーテル型の界面活性剤(アデカトールLA-775、ADEKA社製)を3.3重量%の濃度となるよう添加したもの30容量部に、ヘキサン70容量部を混合し、45℃で60分間の回分抽出操作を行った。所定時間混合の後、静置させたところ、速やかな油水分離が確認され、全液量に対する抽出残渣の容量比は0.35であった。分離したヘキサン相を抽出液として採取し、HPLCによる分析を行ったところ、補酵素Q10の抽出率は85.3%であった。
実施例1と同様に調製した菌体破砕液に、アルキルエーテル型の界面活性剤(アデカトールLA-1275、ADEKA社製)を0.3重量%の濃度となるよう添加したものを30容量部に、ヘキサン70容量部を混合し、45℃で60分間の回分抽出操作を行った。所定時間混合の後、静置させたのちに、遠心分離機による強制油水分離を実施した。分離したヘキサン相を抽出液として採取し、HPLCによる分析を行ったところ、補酵素Q10の抽出率は71.3%であった。
実施例1と同様に調製した菌体破砕液に、ポリオキシエチレン-ポリオキシプロピレンブロックコポリマー型界面活性剤(プルロニックL-62、ADEKA社製)を1.3重量%、ショ糖ステアリン酸エステル(S-1670、三菱化学フーズ社製)を0.3重量%の濃度となるようそれぞれ添加したもの30容量部に、ヘキサン70容量部を混合し、45℃で60分間の回分抽出操作を行った。所定時間混合の後、静置させたところ、速やかな油水分離が確認され、全液量に対する抽出残渣の容量比は0.32であった。分離したヘキサン相を抽出液として採取し、HPLCによる分析を行ったところ、補酵素Q10の抽出率は84.6%であった。
実施例1と同様に調製した菌体破砕液に、ポリオキシエチレン-ポリオキシプロピレンブロックコポリマー型界面活性剤(プルロニックL-62、ADEKA社製)を1.3重量%、エチレンジアミン型ポリオキシエチレン-ポリオキシプロピレンブロックポリマー型界面活性剤(プルロニックTR-702、ADEKA社製)を0.3重量%の濃度となるようそれぞれ添加したもの30容量部に、ヘキサン70容量部を混合し、45℃で60分間の回分抽出操作を行った。所定時間混合の後、静置させたところ、速やかな油水分離が確認され、全液量に対する抽出残渣の容量比は0.30であった。分離したヘキサン相を抽出液として採取し、HPLCによる分析を行ったところ、補酵素Q10の抽出率は83.0%であった。
実施例1と同様に調製した菌体破砕液に、ポリオキシエチレン-ポリオキシプロピレンブロックコポリマー型界面活性剤(プルロニックL-62、ADEKA社製)を1.3重量%、エチレンジアミン型ポリオキシエチレン-ポリオキシプロピレンブロックポリマー型界面活性剤(プルロニックTR-701、ADEKA社製)を0.3重量%の濃度となるようそれぞれ添加したもの30容量部に、ヘキサン70容量部を混合し、45℃で60分間の回分抽出操作を行った。所定時間混合の後、静置させたところ、速やかな油水分離が確認され、全液量に対する抽出残渣の容量比は0.31であった。分離したヘキサン相を抽出液として採取し、HPLCによる分析を行ったところ、補酵素Q10の抽出率は89.9%であった。
エルゴステロールを産生するサッカロミセス・セレビシエ(Saccharomyces cerevisiae)IFO0309株を、10Lの培地(ペプトン5g/L、酵母エキス3g/L、マルトエキス3g/L、グルコース20g/L、pH6.0)を用いて、好気的に28℃で72時間培養した。得られた微生物菌体を、圧力式ホモジナイザー(ラニー社製)により破砕圧力80MPaで2回破砕し、エルゴステロールを含有する微生物細胞破砕液を調製した。
得られた微生物細胞破砕液に、ポリオキシエチレン-ポリオキシプロピレンブロックコポリマー型界面活性剤(プルロニックL-62、ADEKA社製)を3.3重量%の濃度となるよう添加したもの30容量部に、ヘキサン70容量部を混合し、45℃で60分間の回分抽出操作を行った。所定時間混合の後、静置させたところ、速やかな油水分離が確認され、全液量に対する抽出残渣の容量比は0.35であった。分離したヘキサン相を抽出液として採取し、HPLCによる分析を行ったところ、エルゴステロールの抽出率は89.1%であった。
実施例1と同様に調製した菌体破砕液30容量部に、ヘキサン70容量部を混合し、45℃で60分間の回分抽出操作を行った。所定時間混合の後、静置させたところ、速やかな油水分離を確認され、全液量に対する抽出残渣の容量比は0.35であったが、分離したヘキサン相を抽出液として採取し、HPLCによる分析を行ったところ、補酵素Q10の抽出率は60.2%であった。
実施例1と同様に調製した菌体破砕液に、リゾレシチン(デグサ社製)を0.7重量%の濃度となるよう添加したもの30容量部に、ヘキサン70容量部を混合し、45℃で60分間の回分抽出操作を行った。所定時間混合の後、静置させたが、油水分離が進まなかったため、遠心分離機による強制油水分離を実施した。分離したヘキサン相を抽出液として採取し、HPLCによる分析を行ったところ、補酵素Q10の抽出率は62.2%であった。
実施例1と同様に調製した菌体破砕液に、ポリビニルアルコール(和光純薬社製)を3.3重量%の濃度となるよう添加したものを30容量部に、ヘキサン70容量部を混合し、45℃で60分間の回分抽出操作を行った。所定時間混合の後、静置させたところ、速やかな油水分離が確認され、全液量に対する抽出残渣の容量比は0.35であったが、分離したヘキサン相を抽出液として採取し、HPLCによる分析を行ったところ、補酵素Q10の抽出率は61.9%であった。
実施例1と同様に調製した菌体破砕液30容量部に、ヘキサン52容量部、2-プロパノール18容量部の割合で混合し、45℃で60分間の回分抽出操作を行った。所定時間混合の後、静置させたところ、速やかな油水分離が確認されたが、全液量に対する抽出残渣の容量比は0.48と、使用した抽出溶媒の水相への移行量がかなり多い結果となった。分離したヘキサン相を抽出液として採取し、HPLCによる分析を行ったところ、補酵素Q10の抽出率は91.5%であった。
本出願は日本で出願された特願2010-164531を基礎としており、その内容は本明細書に全て包含されるものである。
Claims (8)
- 脂溶性生理活性物質を含有する微生物細胞又はその微生物細胞破砕物の水性懸濁液を、
ポリオキシエチレン-ポリオキシプロピレンブロックコポリマー型界面活性剤、ショ糖脂肪酸エステル類、グリセリン脂肪酸エステル類、ソルビタン脂肪酸エステル類、ポリエーテルポリオール型界面活性剤、ポリオキシエチレンアルキルエーテル型界面活性剤及びアルキルエーテル型界面活性剤からなる群より選択される少なくとも1種類の非イオン性界面活性剤存在下に、
有機溶媒と混合し、
脂溶性生理活性物質を抽出することを特徴とする、
脂溶性生理活性物質の製造方法。 - 脂溶性生理活性物質が、補酵素Q10であることを特徴とする請求項1に記載の製造方法。
- 補酵素Q10が、還元型補酵素Q10、又は、還元型補酵素Q10と酸化型補酵素Q10の混合物であることを特徴とする請求項2に記載の製造方法。
- 非イオン性界面活性剤が、少なくともポリオキシエチレン-ポリオキシプロピレンブロックコポリマー型の界面活性剤であることを特徴とする請求項1~3のいずれか1項に記載の製造方法。
- 非イオン性界面活性剤の添加量が、微生物細胞又は微生物細胞破砕物の水性懸濁液に対して、0.01重量%以上であることを特徴とする請求項1~4のいずれか1項に記載の製造方法。
- 有機溶媒が、疎水性有機溶媒であることを特徴とする請求項1~5のいずれか1項に記載の製造方法。
- さらに親水性有機溶媒を併用することを特徴とする請求項6に記載の製造方法。
- 抽出は、連続抽出であることを特徴とする請求項1~7のいずれか1項に記載の製造方法。
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JP2015508906A (ja) * | 2012-03-02 | 2015-03-23 | バーグ エルエルシー | コエンザイムq10を検出するための方法及びキット |
JP2016208909A (ja) * | 2015-05-08 | 2016-12-15 | 株式会社カネカ | 脂溶性生理活性物質の製造方法 |
US9872112B2 (en) | 2013-01-31 | 2018-01-16 | Invensense, Inc. | Noise mitigating microphone system |
JPWO2018003974A1 (ja) * | 2016-07-01 | 2019-04-25 | 株式会社カネカ | 補酵素q10の製造方法 |
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CN102659652A (zh) * | 2012-04-12 | 2012-09-12 | 西北农林科技大学 | 从雨生红球藻中提取总虾青素的固相萃取方法 |
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CN103025881B (zh) | 2016-03-30 |
US9006493B2 (en) | 2015-04-14 |
ES2676369T3 (es) | 2018-07-19 |
DK2597156T3 (en) | 2018-10-08 |
US20130225868A1 (en) | 2013-08-29 |
EP2597156B1 (en) | 2018-06-27 |
EP2597156A1 (en) | 2013-05-29 |
CN103025881A (zh) | 2013-04-03 |
JP5016734B2 (ja) | 2012-09-05 |
EP2597156A4 (en) | 2016-08-17 |
JPWO2012011589A1 (ja) | 2013-09-09 |
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