JP2016208909A - Production method of lipid-soluble physiologically active substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a production method in which a lipid-soluble physiologically active substance is easily extracted on operation from microbial cells containing the lipid-soluble physiologically active substance without using special dewatering and drying equipment, and without causing reduction of yield due to separability deterioration between solvent and fungus body components, and the substance can be stably and efficiently produced industrially.SOLUTION: In a production method of a lipid-soluble physiologically active substance, after aqueous suspension of microbial cells or microbial cell crushed products containing a lipid-soluble physiologically active substance is thermally treated with at least one nonionic surfactant selected from polyoxyethylene-polyoxypropylene block copolymer type surfactant, sucrose fatty acid esters and polyether type surfactant under the existence of alkali, the suspension is mixed with organic solvent to extract a lipid-soluble physiologically active substance. In the production method of a lipid-soluble physiologically active substance, mixture in thermal treatment has pH7.5 or more, NaOH or KOH is at least used as alkali, and treatment is performed within a temperature range of 55 to 100°C.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a method for producing a fat-soluble physiologically active substance. More 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 microbial cells containing the fat-soluble physiologically active substance or a microbial cell disruption product thereof.

Many fat-soluble physiologically active substances useful for living bodies are known. Among them, coenzyme Q is an essential component widely distributed in the living body from bacteria to mammals, and is known as a component of the 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. In humans, coenzyme Q10, in which the side chain of coenzyme Q has 10 repeating structures, is the main component, and about 40 to 90% is usually present in reduced form in vivo. The physiological action of coenzyme Q10 includes 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.
Of coenzyme Q10, oxidized coenzyme Q10 has been used conventionally as a congestive heart failure drug and health food, and in recent years, a reduced coenzyme having a higher bioactivity has become known. .

  A fat-soluble physiologically active substance such as coenzyme Q10 can be obtained by, for example, synthesis, fermentation, extraction from a natural product, or the like. Further, if necessary, the obtained extract can be purified by chromatography or crystallized by crystallization to obtain a product with higher purity. For example, in order to obtain coenzyme Q10, 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.

  Conventionally, in the operation of extracting useful components contained in microbial cells, an aqueous suspension of cultured microorganisms is dehydrated to form wet cells, and then contacted with an organic solvent. There are known a method of further drying after dehydration, 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.

  As an example of extracting an aqueous suspension of microorganisms without dehydration and drying, the cultured microorganism disruption suspension was directly contacted with an organic solvent such as hexane or 2-propanol to extract coenzyme Q10 in the cells. An example is known (Patent Document 1). In such a liquid-liquid extraction operation, the target substance can be extracted with a high yield and a large throughput without dehydrating and drying the microorganisms. When an organic solvent is used to extract a liquid, particularly an aqueous suspension of the cell disrupted product, particularly an aqueous suspension of the cell disrupted product by physical treatment, an emulsification phenomenon may occur due to the presence of some cellular components such as proteins. It is easy to occur, the organic phase containing the target substance is trapped in the disruption suspension of microbial cells, the organic phase cannot be separated efficiently, not only resulting in a decrease in yield, but also the recovery rate of the extraction solvent used is low The problem also arises. Further, in addition to the hydrophobic organic solvent, it is necessary to use a certain amount or more of the hydrophilic organic solvent, which causes problems such as complicated operation and increased cost.

  Conventionally, when a surfactant is used for liquid-liquid extraction, the affinity between the aqueous phase and the organic solvent phase is increased due to the interfacial effect. In many cases, it takes a long time or does not separate even for a long time, and in some cases, it has been necessary to forcibly separate by an operation such as centrifugation. Therefore, in general liquid-liquid extraction, it has been considered that the use of a surfactant in the extraction process is not preferable.

  On the other hand, in Patent Document 2, in the liquid-liquid extraction of a fat-soluble physiologically active substance such as coenzyme Q10, a specific surfactant such as a polyoxyethylene-polyoxypropylene block copolymer type is used. High extraction rate and operational stability are confirmed. However, although the operation stability is higher than that of the method of Patent Document 1, the amount of the surfactant necessary for performing an effective extraction operation is relatively large, and the aqueous suspension of microbial cells or the cell disruption product is relatively high. Depending on the storage state and culture conditions of the suspension, the extraction operation may become unstable, and a liquid-liquid extraction method that can be operated more reliably and at low cost has been demanded.

JP 2008-253271 A WO2012-011589

As described above, conventional extraction of useful components such as fat-soluble physiologically active substances from microbial cells requires special equipment for dehydration and drying of microorganisms before extraction, or is useful after extraction The problem is that the separation between the solvent containing the components and the bacterial cell components is poor, the yield is insufficient, and the use of multiple solvents is required, which complicates operations and increases costs. was there. Also, in liquid-liquid extraction using a surfactant, it is difficult to bear both yield and stability in the conventional method, and if the balance of surfactant addition is lost, the separation and yield are adversely affected. There was a problem that driving operation was difficult.

  The present invention provides a fat-soluble physiologically active substance from microbial cells containing a fat-soluble physiologically active substance without using a special dehydration and drying facility, and the yield due to deterioration of the separation between the solvent and the bacterial cell component. An object of the present invention is to provide a production method that can be easily extracted in operation without causing a decrease, and can be industrially produced stably and efficiently.

As a result of diligent studies to solve the above problems, an alkali and a specific surfactant were added to an aqueous suspension of microbial cells or microbial cell disruptions containing a fat-soluble physiologically active substance before extraction. Pre-treatment with warming and stirring, followed by extraction using a hydrophobic organic solvent, etc., enables fat-soluble bioactive substances to be stably and efficiently extracted even in liquid-liquid extraction, industrial production As a result, the present invention has been completed.
That is, the present invention provides an aqueous suspension of a microbial cell containing a fat-soluble physiologically active substance or a microbial cell disruption product thereof, a polyoxyethylene-polyoxypropylene block copolymer type surfactant, sucrose fatty acid ester, and poly Extracting at least one nonionic surfactant selected from the group consisting of ether type surfactants with an organic solvent after heating in the presence of an alkali and then extracting a fat-soluble physiologically active substance. A feature of the present invention is a method for producing a fat-soluble physiologically active substance. Here, coenzyme Q10 is preferable as the fat-soluble physiologically active substance to be extracted. In addition, the pH during the heating treatment is preferably 7.5 or more, and the temperature during the heating is preferably 55 to 100 ° C. The nonionic surfactant used during the extraction is at least a polyether type surfactant or polyoxy In a preferred embodiment, an ethylene-polyoxypropylene block copolymer type surfactant is used.

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. According to this production method, the fat-soluble physiologically active substance can be efficiently and reliably extracted by the liquid-liquid extraction operation, and is suitable for industrial production.
Furthermore, by carrying out a heating treatment in the presence of a preferred specific surfactant and alkali found in the present invention before the liquid-liquid extraction operation, the dispersed phase containing the target substance at the time of extraction is finely dispersed in the extraction solvent. In addition to facilitating the transfer of the target substance 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, so that the production of a fat-soluble physiologically active substance with a stable and high yield It becomes possible. Further, conventionally, in order to obtain a target substance with a higher yield, even in the case where a plurality of types of extraction solvents are required for the extraction operation, the present invention allows a high yield operation with only a single solvent. Therefore, it is possible to simplify the apparatus and operation, and to obtain effects such as reduction of energy required for solvent recovery and reduction of environmental load.

FIG. 1 is a schematic view of the apparatus used in Example 9. FIG. 2 shows the extraction results of each tank in Example 9.

  Hereinafter, embodiments of the present invention will be described in detail.

  In the present invention, an aqueous suspension of microbial cells or microbial cell disruptions containing a fat-soluble physiologically active substance is heated in the presence of a specific nonionic surfactant and an alkali described later, A method for producing a fat-soluble physiologically active substance, characterized by mixing and extracting the fat-soluble physiologically active substance in an organic solvent phase.

  In the production method of the present invention, 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, α-lipoic acid, L-carnitine, and the like. Of these, coenzyme Q10, astaxanthin, ergosterol and the like are preferable, and coenzyme Q10 is particularly preferable.

  As described above, 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. In addition, in this specification, when only describing coenzyme Q10, regardless of oxidized coenzyme Q10 and reduced coenzyme Q10, when both coexist, it represents the entire mixture.

  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. Among these, microorganisms that produce fat-soluble physiologically active substances in the cells are preferred. Specific examples of the microorganism include, for example, the genus Agrobacterium, the genus Aspergillus, the genus Acetobacter, the genus Aminobacter, the genus Agromonas, and the acidophilus. Genus, genus Bulleromyces, genus Bullera, genus Brevundimonas, genus Cryptococcus, genus Chionosphaera, genus Candida, genus Cerinosterus (Exo) Genus, Exobasidium genus, Fellomyces genus, Philobabasidiella genus, Filobasidium genus, Geotrichum genus, Graphiola genus, Gluconobacter genus (Ko genus ckovaella, genus Kurtzmanomyces, genus Lalaria, genus Leucosporidium, genus Legionella, genus Methylobacterium, genus Mycoplana, Oosporidium, Pseudomonas, Psedozyma, Paracoccus, Petromyc, Rhodotorula, Rhodosporidium, Rhodomonas, Rhizomonas Rhodobium genus, Rhodoplanes genus, Rhodopseudomonas genus, Rhodobacter genus, Sporobolomyces genus, Sporidiobolus genus, Saitoella genus Genus, Sphingomonas (Sphingomonas) genus, Sporotrichum genus, Sympodiomycopsis genus, Sterigmatosporidium genus, Tapharina genus, Tremella genus, Trichosporon genus, Tile aria ), Tilletia, Tolyposporium, Tilletiopsis, Ustilago, Udeniomyce, Xanthophllomyces, Xanthobacter And microorganisms of the genus Paecilomyces, Acremonium, Hyhomonus, Rhizobium, Phaffia, Haematococcus, and the like. From the viewpoint of ease of culture and productivity, bacteria or yeasts are preferable, non-synthetic bacteria are more preferable for bacteria, and genus Agrobacterium, Gluconobacter, etc. are preferable for yeast. Particularly preferred examples include the genus Schizosaccharomyces, the genus Saitoella, the genus Phaffia and the like.

  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.

  Examples of 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 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. For example, the content of the fat-soluble physiologically active substance per medium is, for example, 0.5 μg / ml or more, preferably 1 μg. / Ml or more, more preferably 2 μg / ml or more.

In the production method of the present invention, as a method for extracting the fat-soluble physiologically active substance from the microbial cell, it can be directly extracted from the microbial cell. It can also be extracted from the crushed material. 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.
In the “fracturing” of the present invention, it is sufficient that the surface structure such as a cell wall is damaged to the extent that extraction of a target fat-soluble physiologically active substance is possible, and microbial cells are not necessarily broken or fragmented. There is no need.

  In the present application, the “aqueous suspension of microbial cells or microbial cell disruptions thereof” refers to a suspension of microbial cells or microbial cell disruptions thereof in an aqueous solvent such as water, physiological saline, buffer solution, or culture medium. Preferably, it is suspended in water and / or a culture 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. , Wet cells suspended in 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. From the viewpoint of operability and the like, more 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. Examples of the crushing method include physical digestion, chemical treatment, enzymatic treatment, autolysis, osmotic dissolution, protoplast dissolution, and the like.

Examples of the physical treatment include use of a high-pressure homogenizer, a rotary blade type 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.
Examples of the enzymatic treatment include a method using lysozyme, zymolyase, glucanase, novozyme, protease, cellulase, etc., and these may be used in combination as appropriate.

Examples of the self-digestion include treatment with a solvent such as ethyl acetate. In addition, treatment with a solution different from the intracellular salt concentration can cause osmotic lysis or protoplast lysis of the cells. However, since this method alone often has an insufficient cell disruption effect, it is preferably used in combination with the above physical treatment, chemical treatment, enzymatic treatment, autolysis and the like.
In the present invention, 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. The acid) in which the pKa is 2.5 or less is preferred, and the physical treatment is more preferred from the viewpoint of crushing efficiency. Moreover, when disrupting cells by physical treatment, it may be performed simultaneously with the heating treatment of the present invention described later. On the other hand, in chemical treatment, enzyme treatment, and the like, the presence of an alkali or a surfactant used in the heat treatment may affect the heat treatment, and therefore it is preferable to perform the heat treatment as a separate step after the crushing treatment.

  In the production method of the present invention, before extraction of a physiologically active substance to be used by mixing an organic solvent with an aqueous suspension of microbial cells or microbial cell disruptions containing the fat-soluble physiologically active substance, alkali and a specific non-active substance are extracted. It is characterized by carrying out a heating treatment in the presence of an ionic surfactant (hereinafter also referred to as “heating treatment of the present invention”). In the present invention, the method for preparing an aqueous suspension of microbial cells or microbial cell disruptions is not particularly limited. For example, 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. Alternatively, an aqueous suspension of microbial cells can be prepared by crushing by the above method. In the production method of the present invention, the cell concentration in the aqueous suspension of the microbial cell disrupted product to be heated or extracted is not particularly limited, but is usually 1 to 25 in terms of the dry weight of the cell. It is preferable to carry out in the range of 10% by weight to 20% by weight.

  The alkali used in the heating treatment of the present invention is not particularly limited, but aluminum hydroxide, ammonium hydroxide, potassium hydroxide, calcium hydroxide, sodium hydroxide, magnesium hydroxide, iron hydroxide, copper hydroxide (II These may be used in combination of two or more thereof, but from the viewpoint of raw material costs, at least sodium hydroxide or potassium hydroxide is preferably used, and sodium hydroxide is more preferable. Further, the amount of alkali used in the heating treatment of the present invention is not particularly limited depending on the type and concentration of the alkali used, but the aqueous suspension of microbial cells or microbial cell disruptions during the heating treatment is not limited. The amount that makes the pH 7.5 or higher is preferable, and the amount that makes the pH 8 or higher is more preferable.

  Further, in the heating treatment of the present invention, it is necessary to use a polyoxyethylene-polyoxypropylene block copolymer type surfactant, a sucrose fatty acid ester, or a polyether 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.

  Examples of the polyoxyethylene-polyoxypropylene block copolymer type surfactant include propylene oxide (PO) between ethylene oxide (EO) chains, which are block copolymers obtained by adding ethylene oxide to both ends of polypropylene glycol. In addition to those having a chain (EOxPOyEOz), reverse type polyoxyethylene-polyoxypropylene block copolymers, ethylenediamine type polyoxyethylene-polyoxypropylene block copolymers, and the like are also included.

  Examples of polyoxyethylene-polyoxypropylene block copolymers having a propylene oxide (PO) chain between the ethylene oxide (EO) chains include, for example, Pluronic L series (L-31, L-34, manufactured by ADEKA Corporation). 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.

  Examples of the reverse type polyoxyethylene-polyoxypropylene block copolymer include ADEKA Corporation's Pluronic R series (25R-1, 25R-2, 17R-2, 17R-3, 17R-4) and BASF Corporation. Examples include the Pluronic RPE series made by the company. Examples of the ethylenediamine-type polyoxyethylene-polyoxypropylene block copolymer 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. Further, 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. Similarly to the polyoxyethylene-polyoxypropylene block copolymer type surfactant, the agent (EOxBOyEOz) can also be used in the production method of the present invention.

  Among these polyoxyethylene-polyoxypropylene block copolymer type surfactants (and analogs thereof), surfactants having a weight average molecular weight in the range of 500 to 8000 are preferred, and the weight average molecular weight is 1000 to 8000. A surfactant in the range of 4000 is more preferred.

  Examples of the sucrose fatty acid esters include those obtained by esterifying one or more hydroxyl groups of sucrose with fatty acids having 6 to 18 carbon atoms, preferably 6 to 12 carbon atoms. Specific examples include sucrose palmitate and sucrose stearate.

  Examples of the polyether type surfactant include Adecanol LG series (LG-109, LG-126, LG-294, LG-295S, LG-299, LG-805) manufactured by ADEKA Corporation, and San Nopco. SN deformer PC, SN deformer 444, SN deformer 470, SN deformer 485, etc. manufactured by the company are listed.

  Among these polyether type surfactants (and analogs thereof), surfactants having a mass average molecular weight in the range of 500 to 8000 are preferable, and surfactants having a mass average molecular weight in the range of 1000 to 4000 are preferable. An agent is more preferable.

  Needless to say, the nonionic surfactants shown here can be used in combination of two or more, and can be used in combination with other surfactants as long as the effects are not impaired. In consideration of the properties and raw material costs, it is preferable to use one type. Among the nonionic surfactants, it is preferable to use a polyoxyethylene-polyoxypropylene block copolymer type surfactant or a polyether type surfactant, and a polyether type surfactant is more preferable.

  The amount of the specific nonionic surfactant used in the heating treatment of the present invention is preferably 0.01% by weight or more as a concentration with respect to the aqueous suspension of microbial cells or microbial cell disrupted materials. More preferably, it is 1% by weight or more. When the amount of the surfactant added is less than 0.01% by weight, in the subsequent extraction process, the fine dispersion of the aqueous suspension in the organic solvent does not proceed and sufficient extraction efficiency may not be ensured. The upper limit of the amount of the nonionic surfactant used is not particularly limited, but is preferably 5% by weight or less, and more preferably 1% by weight or less. When the addition amount of the surfactant exceeds 5% by weight, the affinity between the organic solvent and the aqueous suspension becomes higher than necessary in the subsequent extraction step. The oil-water separability may be deteriorated when left standing. In the present invention, by performing this heating treatment, it is ensured even when the amount of the surfactant added is as small as 1% by weight or less compared to the conventional liquid-liquid extraction method using a surfactant. Since extraction operation can be performed, it is more excellent in terms of raw material price.

The heating treatment of the present invention is performed by heating an aqueous suspension of microbial cells or microbial cell disruptions containing a fat-soluble physiologically active substance in the presence of the specific nonionic surfactant and alkali. To be implemented. In this step, the specific nonionic surfactant and alkali may be added simultaneously to the aqueous suspension of microbial cells or microbial cell disruptions, or may be added separately. From the viewpoint of operability, after adding alkali to an aqueous suspension of microbial cells or microbial cell disruptions containing a fat-soluble physiologically active substance and confirming that the pH has reached a predetermined level, It is preferable to add an ionic surfactant and further mix. The heating temperature during the heating treatment of the present invention is not particularly limited, but is usually preferably 40 ° C or higher, more preferably 50 ° C or higher, further preferably 55 ° C or higher, and particularly preferably 60 ° C or higher. Although an upper limit is not specifically limited, Usually, 100 degrees C or less, Preferably it can implement in 70 degrees C or less.
Moreover, the heating process of this invention is normally implemented by a stirring state.

  By performing such a heating treatment, not only can the extraction efficiency of the fat-soluble physiologically active substance in the extraction step described later be improved, but also by using a specific nonionic surfactant during the heating treatment. In the subsequent extraction step, it was found that not only the affinity between the organic solvent and the aqueous suspension was improved, but also the oil / water separation surely proceeded rapidly after mixing and standing. Therefore, in the production method of the present invention, the organic solvent phase in which the fat-soluble physiologically active substance, which is the target substance, is dissolved can be recovered with a stable and higher yield.

  In the production method of the present invention, after the heating treatment, an aqueous suspension of microbial cells or microbial cell disruption containing the fat-soluble physiologically active substance, a specific nonionic surfactant and an alkali mixture, Furthermore, the organic solvent is mixed, and the fat-soluble physiologically active substance is recovered by extracting the fat-soluble physiologically active substance into the organic solvent phase by liquid-liquid extraction. In the production method of the present invention, 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.

  Although it does not restrict | limit especially as hydrocarbons, For example, an aliphatic hydrocarbon, an aromatic hydrocarbon, a halogenated hydrocarbon etc. can be mentioned. 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 in general, a saturated hydrocarbon is 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. Specific examples include propane, butane, isobutane, pentane, 2-methylbutane, hexane, 2-methylpentane, 2,2-dimethylbutane, 2,3-dimethylbutane, heptane, heptane isomers (for example, 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. it can. Preferably, 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. More preferably, 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., more preferably pentane, hexane, cyclohexane, methylcyclohexane Particularly preferred are hexane, heptane and methylcyclohexane, and most preferred are hexane and heptane.

  Although it does not restrict | limit especially as an aromatic hydrocarbon, Usually, C6-C20, Preferably it is C6-C12, More preferably, a C7-C10 thing 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. Preferred are toluene, xylene, o-xylene, m-xylene, p-xylene, ethylbenzene, cumene, mesitylene, tetralin, butylbenzene, p-cymene, cyclohexylbenzene, diethylbenzene, pentylbenzene and the like. More preferred are toluene, xylene, o-xylene, m-xylene, p-xylene, cumene, tetralin and the like. Most preferred is cumene.

  The halogenated hydrocarbon is not particularly limited regardless of whether it is cyclic or non-cyclic, or saturated or unsaturated. In general, a non-cyclic hydrocarbon is 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 -Tetrachloroethane, 1,1,2,2-tetrachloroethane, pentachloroethane, hexachloroethane, 1,1-dichloroethylene, 1,2-dichloroethylene, trichloroethylene, tetrachloroethylene, 1,2-dichloropropane, 1,2,3- Examples thereof include trichloropropane, chlorobenzene, 1,1,1,2-tetrafluoroethane, and the like. Preferably, 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.

  Although it does not restrict | limit especially as fatty acid esters, For example, propionate ester, acetate ester, formate ester etc. can be mentioned. Preferred are acetate esters and formate esters, and more preferred are acetate esters. Although it does not restrict | limit especially as an ester group, Usually, a C1-C8 alkyl ester and a C7-C12 aralkyl ester, Preferably a C1-C6 alkyl ester, More preferably, it is C1-C1. 4 alkyl esters are used.

  Specific examples of the propionic acid ester include, for example, methyl propionate, ethyl propionate, butyl propionate, isopentyl propionate, and the like. Preferred is ethyl propionate.

  Specific examples of the acetate ester 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. Preferred are 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 the like. More preferred are methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate, isobutyl acetate and the like, and most preferred is ethyl acetate.

  Specific examples of the formate ester include 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 whether saturated or unsaturated. In general, saturated ethers 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 can be mentioned. Preferably, 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. More preferred are diethyl ether, methyl tert-butyl ether, anisole, dioxane, tetrahydrofuran, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether and the like. More preferred are diethyl ether, methyl tert-butyl ether, anisole and the like, and most preferred is methyl tert-butyl ether.

  The alcohol is not particularly limited regardless of whether it is cyclic or non-cyclic, and whether saturated or unsaturated. In general, a saturated alcohol is preferably used. Usually, it is C1-C20, Preferably it is C1-C12, More preferably, it is C1-C6. 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.

  Specific examples of these alcohols include 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 allyl alcohol, propargyl alcohol, benzyl alcohol, cyclohexanol, 1-methylcyclohexanol, 2-methylcyclohexanol, 3-methylcyclohexanol, 4-methylcyclohexanol; 1,2-ethanediol, 1 2,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 2,3-butanediol, 1,5-pentanediol, etc. Examples thereof include trihydric alcohols such as glycerin.

  The monohydric alcohol is preferably methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutyl alcohol, tert-butyl alcohol, 1-pentanol, 2-pentanol, or 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, Ben Alcohol, cyclohexanol, 1-methylcyclohexanol, 2-methylcyclohexanol, 3-methylcyclohexanol, 4-methylcyclohexanol, and the like. More preferably, 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- And ethyl-1-butanol, cyclohexanol and the like. More preferably, 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, and the like. Particularly preferred are methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutyl alcohol, 2-methyl-1-butanol, isopentyl alcohol and the like, most preferably 2-propanol. is there.

  As the dihydric alcohol, 1,2-ethanediol, 1,2-propanediol, 1,3-propanediol and the like are preferable, and 1,2-ethanediol is most preferable. As the trihydric alcohol, glycerin is preferable.

  Examples of fatty acids 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.

  The nitriles are not particularly limited regardless of whether they are cyclic or non-cyclic, and whether saturated or unsaturated. In general, saturated ones are 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 Nitrile, pentadecane nitrile, stearonitrile, chloroacetonitrile, bromoacetonitrile, chloropropionitrile, bromopropionitrile, methoxyacetonitrile, methyl cyanoacetate, ethyl cyanoacetate, tolunitrile, benzonitrile, chlorobenzonitrile, bromobenzonitrile, cyano Benzoic acid, nitrobenzonitrile, anisonitrile, phthalonitrile, bromotolunitrile, methyl cyanobenzoate, methoxybenzene Zonitrile, acetylbenzonitrile, naphthonitrile, biphenylcarbonitrile, phenylpropionitrile, phenylbutyronitrile, methylphenylacetonitrile, diphenylacetonitrile, naphthylacetonitrile, nitrophenylacetonitrile, chlorobenzyl cyanide, cyclopropanecarbonitrile, cyclohexanecarbonitrile, Examples include cycloheptanecarbonitrile, phenylcyclohexanecarbonitrile, tolylcyclohexanecarbonitrile and the like.

  Preferred are acetonitrile, propionitrile, succinonitrile, butyronitrile, isobutyronitrile, valeronitrile, methyl cyanoacetate, ethyl cyanoacetate, benzonitrile, tolunitrile, chloropropionitrile, more preferably acetonitrile, Pionitrile, butyronitrile, isobutyronitrile, and most preferably acetonitrile.

  Examples of 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.

  Examples of sulfur compounds include dimethyl sulfoxide and sulfolane.

  Among the organic solvents, it is preferable to select in consideration of properties such as boiling point, melting point and viscosity. For example, the boiling point is about 30 at 1 atm from the viewpoint of appropriate heating for increasing the solubility and easy removal of the solvent from the wet body and easy recovery of the solvent from the crystallization filtrate. The melting point is preferably in the range of ˜150 ° C., and the melting point is about 0 ° C. or higher, preferably about 10 ° C. or higher, more preferably from the viewpoint of being hard to solidify when handled at room temperature and when cooled to room temperature or lower. The viscosity is about 20 ° C. or higher, and the viscosity is preferably as low as about 10 cp or less at 20 ° C.

  Among the above organic solvents, for the purpose of extracting and recovering a fat-soluble physiologically active substance from an aqueous suspension 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. , Fatty acid esters, ethers and other hydrophobic organic solvents, more preferably hydrocarbons, more preferably aliphatic hydrocarbons. Among aliphatic hydrocarbons, those having 5 to 8 carbon atoms are preferably used. Specific examples of the aliphatic hydrocarbon 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.

  In the production method of the present invention, by performing extraction in the presence of a surfactant, the affinity between the aqueous suspension of microbial cells or microbial cell disruptions and the hydrophobic organic solvent is improved, and sufficient extraction efficiency is achieved. In addition to the above hydrophobic organic solvent as an organic solvent used during extraction, a hydrophilic organic solvent can be supplementarily used in combination to further improve the aqueous suspension in the organic solvent. The promotion of miniaturization and high stability improvement of oil / water separation can be added at the time of standing, which may be more preferable. In the production method of the present invention, 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. Among alcohols, monohydric alcohols having 1 to 5 carbon atoms are preferably used. Specific examples thereof include 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.

In the production method of the present invention, 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, and in the range of 50 to 75% by volume with respect to the volume of the mixed solution of turbid liquid, extraction solvent, alkali, nonionic surfactant, etc. Is more preferable. In addition, 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, but is 0.1 to 0.1% of the total solution volume. It is preferably used in the range of 50% by volume, more preferably in the range of 0.1 to 10% by volume, and further 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.
In the production method of the present invention, the temperature at the time of extraction is not particularly limited, but is usually 0 to 65 ° C, preferably 20 to 55 ° C.

  As the extraction method, either batch extraction or continuous extraction can be performed, but industrially continuous extraction is preferable in terms of productivity, and countercurrent multistage extraction as shown in FIG. Is particularly preferred. 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. In addition, when batch extraction or continuous extraction is performed, a specific nonionic surfactant used in the heat treatment of the present invention may be added as appropriate in the extraction step. For example, in the countercurrent multistage extraction, it is more preferable to add the nonionic surfactant to each extraction tank. In that case, the extraction efficiency can be further increased.

  In the production method of the present invention, usually, 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.

  By employing the production method of the present invention as described above, it is possible to extract the fat-soluble physiologically active substance contained in the aqueous suspension of microbial cells or microbial cell disrupted material with high efficiency. The extraction rate in the production method of the present invention is usually 75% or more, more preferably 85% 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. Specifically, it can be obtained as in the embodiments described later.

  In the production method of the present invention, 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. Moreover, you may attach | subject to refinement | purification processes, such as column chromatography normally used and liquid-liquid distribution. These purification treatments can be performed alone or in combination of several kinds. If necessary, steps such as saponification, oxidation, reduction, and other synthetic reaction treatments can be added. Moreover, the target fat-soluble physiologically active substance can also be obtained as a crystal body by crystallization operation or the like. For example, 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 coenzyme Q10 obtained by extraction can be purified by a method known per se to obtain coenzyme Q10. For example, if necessary, it is purified by treatment with an adsorbent such as activated carbon or white clay, or column chromatography, and before or after that, oxidation or reduction treatment is performed as necessary. Crystallization of the high-purity coenzyme Q10 is performed using crystallization operation. It can also be acquired.

EXAMPLES Next, although an Example demonstrates this invention concretely, this invention is not limited only to these Examples.

The extraction rate in each example was calculated as follows. Methanol-chloroform (3: 1) mixed solution is added to 1 ml of coenzyme Q10-containing microbial cell disruption solution before extraction to make a total volume of 50 ml, and after stirring at 25 ° C. for 30 minutes, the solid matter derived from the cells is solid-liquid. The coenzyme Q concentration in the obtained liquid layer part was measured under the following HPLC conditions, and the amount of coenzyme Q contained in the microbial cell disruption liquid to be extracted was calculated. Similarly, the coenzyme Q concentration in the extract after the extraction operation was measured under the following HPLC conditions, the amount of extracted coenzyme Q was calculated, and the extraction rate was determined by the following equation.
Extraction rate (%) = weight of coenzyme Q contained in the extract / weight of coenzyme Q contained in the microbial cell disruption solution before extraction × 100

(HPLC measurement conditions)
Column: YMC-Pack ODS-A (manufactured by YMC. Co., Ltd)
Oven temperature: 30 ℃
Mobile phase: methanol / hexane = 85/15 (volume ratio)
Liquid feed rate: 1.0 ml / min
Detection: UV275nm

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 160 hours. The obtained culture solution containing the microbial cells was concentrated by centrifugation, and crushed twice with a pressure homogenizer at a crushing pressure of 80 MPa to prepare a microbial cell disruption solution containing coenzyme Q10.

After adding 40 wt% sodium hydroxide to the obtained microbial cell disruption solution so as to have a pH of 8, a polyether type surfactant (SN deformer PC, manufactured by San Nopco) was added at a concentration of 0.5 wt%. It added so that it might become. This was stirred at 60 ° C. for 10 minutes.
72 parts by volume of hexane was mixed with 28 parts by volume of the microbial cell disruption solution after the heat treatment, and a batch extraction operation was performed at 55 ° 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 0.31. The separated hexane phase was collected as an extract and analyzed by HPLC. As a result, the extraction rate of coenzyme Q10 was 88.3%.

(Example 2)
40% by weight sodium hydroxide was added to the microbial cell disruption solution prepared in the same manner as in Example 1 so that the pH was 8, and then 0.5% by weight of a polyether surfactant (SN deformer 444, manufactured by San Nopco). % Concentration was added, and this was stirred at 60 ° C. for 10 minutes.

  72 parts by volume of hexane was mixed with 28 parts by volume of the microbial cell disruption solution after the heat treatment, and a batch extraction operation was performed at 55 ° 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 0.31. The separated hexane phase was collected as an extract and analyzed by HPLC. As a result, the extraction rate of coenzyme Q10 was 88.2%.

Example 3
40% by weight sodium hydroxide was added to the microbial cell disruption solution prepared in the same manner as in Example 1 so that the pH was 8, and then 0.5 wt. Of a polyether type surfactant (SN deformer 470, manufactured by San Nopco). % Concentration was added, and this was stirred at 60 ° C. for 10 minutes.

  72 parts by volume of hexane was mixed with 28 parts by volume of the microbial cell disruption solution after the heat treatment, and a batch extraction operation was performed at 55 ° 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 0.3. The separated hexane phase was collected as an extract and analyzed by HPLC. As a result, the extraction rate of coenzyme Q10 was 86.1%.

Example 4
40% by weight sodium hydroxide was added to the microbial cell disruption solution prepared in the same manner as in Example 1 so that the pH was 8, and then 0.5% by weight of a polyether type surfactant (SN deformer 485, manufactured by San Nopco). % Concentration was added, and this was stirred at 60 ° C. for 10 minutes.

  72 parts by volume of hexane was mixed with 28 parts by volume of the microbial cell disruption solution after the heat treatment, and a batch extraction operation was performed at 55 ° 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 0.3. When the separated hexane phase was collected as an extract and analyzed by HPLC, the extraction rate of coenzyme Q10 was 84.0%.

(Example 5)
40% by weight sodium hydroxide was added to the microbial cell disruption solution prepared in the same manner as in Example 1 so that the pH was 8, and then a polyether type surfactant (Adecanol LG126, manufactured by Adeka) was added at 0.5% by weight. The resulting solution was stirred at 60 ° C. for 10 minutes.

  72 parts by volume of hexane was mixed with 28 parts by volume of the microbial cell disruption solution after the heat treatment, and a batch extraction operation was performed at 55 ° 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 0.34. The separated hexane phase was collected as an extract and analyzed by HPLC. As a result, the extraction rate of coenzyme Q10 was 85.0%.

(Example 6)
40% by weight sodium hydroxide was added to the microbial cell disruption solution prepared in the same manner as in Example 1 so that the pH was 8, and then a polyether type surfactant (Adecanol LG-126, manufactured by Adeka) was added to 0.5%. It added so that it might become the density | concentration of a weight%, and this was stirred for 10 minutes on 60 degreeC conditions.

  72 parts by volume of hexane was mixed with 28 parts by volume of the microbial cell disruption solution after the heat treatment, and a batch extraction operation was performed at 55 ° C. for 60 minutes. After mixing for a predetermined time, the extract obtained after standing was recovered, and the remaining aqueous phase was added to the same amount of hexane and 0.5% by weight of a polyether type surfactant (Adecanol LG-) as the aqueous phase. 126, manufactured by Adeka) and extracted. This was repeated twice and a three-stage batch extraction was performed. As a result, rapid oil-water separation was confirmed in all three stages. The separated hexane phase was collected as an extract and analyzed by HPLC. As a result, the total extraction rate of coenzyme Q10 in 3 stages was 97.1%.

(Example 7)
After adding 40% by weight sodium hydroxide to a microbial cell disruption solution prepared in the same manner as in Example 1 to pH 8, a polyoxyethylene-polyoxypropylene block copolymer type surfactant (Pluronic L-62, ADEKA) Was added to a concentration of 0.5% by weight, and the mixture was stirred at 60 ° C. for 10 minutes.

  72 parts by volume of hexane was mixed with 28 parts by volume of the microbial cell disruption solution after the heat treatment, and a batch extraction operation was performed at 55 ° 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 0.34. The separated hexane phase was collected as an extract and analyzed by HPLC. As a result, the extraction rate of coenzyme Q10 was 88.4%.

(Example 8)
40% by weight of sodium hydroxide was added to the microbial cell disruption solution prepared in the same manner as in Example 1 so that the pH was 7.5, and a polyether type surfactant (Adecanol LG126, manufactured by Adeka Corporation) It added so that it might become a density | concentration of 5 weight%, and this was stirred for 10 minutes on 60 degreeC conditions.

  72 parts by volume of hexane was mixed with 28 parts by volume of the microbial cell disruption solution after the heat treatment, and a batch extraction operation was performed at 55 ° 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 0.34. The separated hexane phase was collected as an extract and analyzed by HPLC. As a result, the extraction rate of coenzyme Q10 was 79.1%.

Example 9
40% by weight sodium hydroxide was added to the microbial cell disruption solution prepared in the same manner as in Example 1 so that the pH was 8, and then a polyether type surfactant (Adecanol LG126, manufactured by Adeka) was added at 0.5% by weight. The resulting solution was stirred at 60 ° C. for 10 minutes.

  Thereafter, 28 parts by volume of the microbial cell disrupted liquid after the above heat treatment and 72 parts by volume of hexane were fixed, and polyether surfactants (Adecanol LG126, Adeka LG, and Adeka) were regularly added to the second and third stages. And the countercurrent three-stage continuous extraction was carried out for 8 hours so that the surfactant concentration in each tank was 0.5% by weight with respect to the microbial cell disruption solution during the operation period. The apparatus used at this time is shown in FIG. During operation, stable separation and extraction rate were confirmed throughout, and the average extraction rate over 8 hours was 97.0%. Moreover, the time-dependent change of the extraction rate of coenzyme Q10 in each tank is shown in FIG.

(Comparative Example 1)
A polyether type surfactant (Adecanol LG126, manufactured by Adeka) was added to the microbial cell disruption solution prepared in the same manner as in Example 1 to a concentration of 0.5% by weight. 72 parts by volume of hexane was mixed with 28 parts by volume of the microorganism culture disruption solution, and batch extraction was performed at 55 ° 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 66.1%.

(Comparative Example 2)
After adding 40 wt% sodium hydroxide to pH 8 to the microbial cell disruption solution prepared in the same manner as in Example 1, 0.5 wt% polyoxyethylene (20) sorbitan monolaurate (Tween 20) was added. It added so that it might become a density | concentration, and this was stirred for 10 minutes on 60 degreeC conditions.

  72 parts by volume of hexane was mixed with 28 parts by volume of the microbial cell disruption solution after the heat treatment, and a batch extraction operation was performed at 55 ° C. for 60 minutes. After mixing for a predetermined time, the mixture was allowed to stand, but the oil and water were not separated and the layers were stratified, and the hexane phase could not be collected as the extract.

(Comparative Example 3)
40% by weight sodium hydroxide was added to the microbial cell disruption solution prepared in the same manner as in Example 1 so as to have a pH of 8, and then 0.5% by weight of an alkyl ether surfactant (Adecatol LA775, manufactured by Adeka) And stirred for 10 minutes at 60 ° C.
72 parts by volume of hexane was mixed with 28 parts by volume of the microbial cell disruption solution after the heat treatment, and a batch extraction operation was performed at 55 ° C. for 60 minutes. After mixing for a predetermined time, the mixture was allowed to stand, but the oil and water were not separated and the layers were stratified, and the hexane phase could not be collected as the extract.

(Comparative Example 4)
After adding 40 wt% sodium hydroxide to pH 8 to the microbial cell disruption solution prepared in the same manner as in Example 1, decaglycene oleate was added to a concentration of 0.5 wt%. This was stirred at 60 ° C. for 10 minutes.

  72 parts by volume of hexane was mixed with 28 parts by volume of the microbial cell disruption solution after the heat treatment, and a one-stage extraction operation was performed at 55 ° C. for 60 minutes. After mixing for a predetermined time, the mixture was allowed to stand, but the oil and water were not separated and the layers were stratified, and the hexane phase could not be collected as the extract.

Claims (12)

Polyoxyethylene-polyoxypropylene block copolymer type surfactants, sucrose fatty acid esters, and polyether type surfactants containing aqueous suspensions of microbial cells or microbial cell disruptions containing fat-soluble physiologically active substances At least one nonionic surfactant selected from the group consisting of: and after heating in the presence of alkali, mixing with an organic solvent to extract a fat-soluble physiologically active substance, A method for producing a fat-soluble physiologically active substance. The manufacturing method according to claim 1, wherein the pH of the mixed solution during the heating treatment is 7.5 or more. The manufacturing method of Claim 1 or 2 which uses at least sodium hydroxide or potassium hydroxide as an alkali used at the time of a heating process. The temperature at the time of a heating process exists in the range of 55 to 100 degreeC, The manufacturing method of any one of Claims 1-3 characterized by the above-mentioned. The production method according to any one of claims 1 to 4, wherein the fat-soluble physiologically active substance is coenzyme Q10. 6. The production method according to claim 5, wherein the coenzyme Q10 is reduced coenzyme Q10 or a mixture of reduced coenzyme Q10 and oxidized coenzyme Q10. 7. The nonionic surfactant is at least a polyether type surfactant or a polyoxyethylene-polyoxypropylene block copolymer type surfactant. Manufacturing method. The addition amount of a nonionic surfactant is 0.01 to 1 weight% with respect to the aqueous suspension of a microbial cell or a microbial cell crushed material, The any one of Claims 1-7 characterized by the above-mentioned. The production method according to item. The manufacturing method according to claim 1, wherein the organic solvent is a hydrophobic organic solvent. Furthermore, a hydrophilic organic solvent is used together, The manufacturing method of Claim 9 characterized by the above-mentioned. Extraction is continuous extraction, The manufacturing method of any one of Claims 1-10 characterized by the above-mentioned. Furthermore, the said nonionic surfactant is added to each extraction tank in continuous extraction, The manufacturing method of Claim 11 characterized by the above-mentioned.