WO2013168617A1 - 脂質の製造方法 - Google Patents
脂質の製造方法 Download PDFInfo
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- WO2013168617A1 WO2013168617A1 PCT/JP2013/062441 JP2013062441W WO2013168617A1 WO 2013168617 A1 WO2013168617 A1 WO 2013168617A1 JP 2013062441 W JP2013062441 W JP 2013062441W WO 2013168617 A1 WO2013168617 A1 WO 2013168617A1
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B1/00—Production of fats or fatty oils from raw materials
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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
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6409—Fatty acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/09—Preparation of carboxylic acids or their salts, halides or anhydrides from carboxylic acid esters or lactones
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/03—Preparation of carboxylic acid esters by reacting an ester group with a hydroxy group
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/08—Preparation of carboxylic acid esters by reacting carboxylic acids or symmetrical anhydrides with the hydroxy or O-metal group of organic compounds
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
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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
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6436—Fatty acid esters
- C12P7/6445—Glycerides
- C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil
Definitions
- the present invention relates to a method for producing a lipid using algae of the dinoflagellate class.
- Middle chain fatty acid represented by lauric acid is a main fatty acid contained in a large amount in palm oil and palm kernel oil, and is used as a raw material for various surfactants and foods.
- the source of lauric acid is limited to palm and palm kernels, which limits the cultivation area.
- the use of arable land as a raw material for medium chain fatty acids is also a concern for future competition with biodiesel and food applications.
- Non-Patent Document 1 Crypthecodinium nicohnii, which is a dinoflagellate, has a high content of lauric acid as an organism supplying lauric acid.
- the present invention relates to a lipid production method (hereinafter sometimes referred to as “the production method of the present invention”) comprising the following steps (1) and (2).
- a step of culturing dinoflagellate algae in a medium containing glycerin to obtain a culture hereinafter sometimes referred to as “step 1”
- step 2 A step of collecting lipid from the obtained culture
- the present invention also includes a method for producing a medium-chain fatty acid ester comprising a step of transesterifying the lipid obtained by the production method of the present invention with an alcohol (hereinafter sometimes referred to as “the ester production method of the present invention”). About.
- the present invention relates to a method for producing a medium chain fatty acid (hereinafter sometimes referred to as “the fatty acid production method of the present invention”), which comprises a step of hydrolyzing the lipid obtained by the production method of the present invention.
- the present invention relates to a method for improving the productivity of medium-chain fatty acids of the algae, wherein the algae of the dinoflagellate class is cultured in a medium containing glycerin.
- the present invention relates to a method for producing algae with improved productivity of medium chain fatty acids, wherein algae of dinoflagellate is cultured in a medium containing glycerin.
- the present invention relates to providing a method for producing a lipid capable of efficiently producing a lipid having a high content of medium chain fatty acids, preferably an oil or fat.
- the present inventors further examined the production of lipids using algae of the dinoflagellate class, and using glycerin among various carbon sources usually added to the medium as a nutrient source for growing cells.
- the amount of fatty acids increases remarkably without affecting cell growth, and the content of medium-chain fatty acids in the total constituent fatty acids in the obtained lipids (hereinafter referred to as “medium-chains”). It has been found that lipids containing a high amount of medium-chain fatty acids can be efficiently produced by significantly improving the "productivity of fatty acids”.
- the production method of the present invention it is possible to efficiently produce a lipid containing a high content of medium chain fatty acids. Moreover, according to the ester manufacturing method of this invention, the ester body of medium chain fatty acid can be manufactured efficiently. Furthermore, according to the method for producing a medium chain fatty acid of the present invention, the medium chain fatty acid can be efficiently produced.
- lipids include simple lipids (fats, waxes, etc.) that are esters of fatty acids and fatty acids and various alcohols, and complex lipids (phospholipids / glycolipids) comprising fatty acids, alcohols, phosphoric acids, sugars, etc. Etc.), and the above-mentioned hydrolysis products that are insoluble in water (fatty acids, higher alcohols, sterols, etc.) and derivatized lipids such as terpenes and fat-soluble vitamins.
- simple lipids fats, waxes, etc.
- complex lipids phospholipids / glycolipids
- hydrolysis products that are insoluble in water (fatty acids, higher alcohols, sterols, etc.) and derivatized lipids such as terpenes and fat-soluble vitamins.
- the lipid is preferably a simple lipid or a complex lipid, more preferably a simple lipid, and still more preferably an oil or fat.
- the fat and oil means an ester of a fatty acid and glycerin, and specifically means a neutral lipid such as monoglyceride, diglyceride, and triglyceride.
- the medium chain fatty acid refers to a monovalent carboxylic acid having a hydrocarbon group having 10 to 14 carbon atoms, and specific examples include capric acid, lauric acid, and myristic acid.
- the fatty acid ester refers to an ester of a fatty acid and a lower or higher alcohol other than the triglyceride
- the medium chain fatty acid ester refers to an ester of the medium chain fatty acid and a lower or higher alcohol.
- the production method of the present invention includes a step of culturing dinoflagellate algae in a medium containing glycerin.
- the dinoflagellate algae used in the present invention is a group of unicellular algae that have photoflagiated and flagella in taxonomy, have vertical and horizontal grooves on the surface of the cells, and are secondary to red alga in the process of evolution.
- symbiosis it refers to algae with a characteristic form through multiple symbiosis.
- Specific examples of dinoflagellate algae include the order of Noctilucales, Prorocentrales, Dinophysales, Gymnodiniales, Peridiniales, and Goniolacales ), Blastodiniales, and Phytodiniales. Among these, from the viewpoint of improving productivity of medium chain fatty acids, algae of the order of Gymnodiniales is preferable.
- Examples of the algae of the genus Gymnodiniales include, for example, Amphidinium, Cochlodinium, Gymnodinium, Gyrodinium, Erythropsodinium, Hemidinium, Katodinium, Nematodinium, Oxyrrhis, Polykrikos, Torodinium, Symodidinium, Wols Can do.
- algae belonging to Symbiodinium is preferable from the viewpoint of improving productivity of medium chain fatty acids.
- Examples of the algae belonging to the Symbiodinium include Symbiodinium microadriaticum, Symbiodinium goreaui, Symbiodinium linucheae, Symbiodinium bermudense, Symbiodinium meandrinae, Symbiodinium califorbio, Symbiodinium kawagutium, Symbiodinium coribiobiodin Can be mentioned.
- Symbiodinium microadriaticum is preferable, and Symbiodinium microadriaticum LB2281 strain and Symbiodinium sp. NIES-2638 strain are more preferable from the viewpoint of improving productivity of medium-chain fatty acid-containing lipids.
- the algae of the dinoflagellate class is The culture collection of algae at University of Texas at Austin (UTEX), National Institute for Environmental Studies (NIES), National Center for Marine Algae and Microbiota (NCMA: former CCMP) , And can be obtained from public institutions such as the Culture Collection of Algae and Protozoa (CCAP).
- CCAP Culture Collection of Algae and Protozoa
- (Culture medium) As the medium used for culturing algae in the step 1, a conventionally known medium can be used, and one based on natural seawater or artificial seawater may be used, or a commercially available culture medium is used. May be.
- preferable medium include Daigo IMK medium, f / 2 medium, ESM medium, L1 medium, and MNK medium.
- f / 2 medium, ESM medium, and Daigo IMK medium are preferable, ESM medium and Daigo IMK medium are more preferable, and Daigo IMK medium is more preferable.
- the medium used in Step 1 contains glycerin from the viewpoint of improving the productivity of medium chain fatty acids.
- the glycerin may include polyglycerin obtained by condensation polymerization of glycerin.
- the content of glycerin in the medium is preferably 0.01% by mass or more, more preferably 0.02% by mass or more, further preferably 0.05% by mass or more, from the viewpoint of improving the productivity of medium chain fatty acids.
- it is 0.07 mass% or more, More preferably, it is 0.08 mass% or more, More preferably, it is 0.09 mass% or more, More preferably, it is 0.1 mass% or more.
- the content is preferably 10% by mass or less, more preferably 5% by mass or less, further preferably 4% by mass or less, further preferably 3% by mass or less, and further preferably 2% by mass or less. More preferably, it is 1 mass% or less, More preferably, it is 0.5 mass% or less.
- the content of glycerin in the medium is 0.01 to 10% by mass, preferably 0.02 to 5% by mass, more preferably 0.05 to 4% by mass, from the viewpoint of improving the productivity of medium chain fatty acids. More preferably, 0.07 to 3% by mass, more preferably 0.08 to 2% by mass, more preferably 0.09 to 2.0% by mass, still more preferably 0.1 to 2.0% by mass, and still more preferably. Is 0.1 to 1% by mass, more preferably 0.1 to 0.5% by mass.
- the medium used in Step 1 is appropriately added with a nitrogen source, a phosphorus source, a metal salt, vitamins, a carbon source other than the glycerin, a trace metal, etc. can do.
- a nitrogen source include NaNO 3 , KNO 3 , Ca (NO 3 ) 2 , NH 4 NO 3 , (NH 4 ) 2 SO 4 and the like.
- the phosphorus source include K 2 HPO 4 , KH 2 PO 4 , Na 2 HPO 4 , NaH 2 PO 4 , sodium glycerophosphate, and the like.
- Examples of the metal salt include NaCl, KCl, CaCl 2 , MgCl 2 , Na 2 SO 4 , K 2 SO 4 , MgSO 4 , Na 2 CO 3 , NaHCO 3 , Na 2 SiO 3 , H 3 BO 3 , MnCl. 2 , MnSO 4 , FeCl 3 , FeSO 4 , CoCl 2 , ZnSO 4 , CuSO 4 , Na 2 MoO 4 and the like.
- Examples of the vitamins include biotin, vitamin B12, Thiamine-HCl, nicotinic acid, inositol, folic acid, thymine and the like.
- the content of the nitrogen source in the medium is preferably 2 mg / L or more, more preferably 5 mg / L or more, more preferably 10 mg / L or more in terms of nitrogen atom equivalent from the viewpoint of improving the productivity of lipids and medium chain fatty acids. More preferably, it is 15 mg / L or more, more preferably 20 mg / L or more, more preferably 50 mg / L or more, more preferably 100 mg / L or more. From the same viewpoint, the content is preferably a nitrogen atom equivalent.
- the content of the nitrogen source in the medium is 2 to 700 mg / L, preferably 5 to 600 mg / L, more preferably 10 to 500 mg in terms of nitrogen atom equivalent.
- / L more preferably 15 to 400 mg / L, more preferably 20 to 300 mg / L, more preferably 50 to 250 mg / L, and still more preferably 100 to 250 mg / L.
- the content of the phosphorus source in the medium is preferably 0.5 mg / L or more, more preferably 1 mg / L or more, more preferably, in terms of phosphorus atom equivalent, from the viewpoint of improving the productivity of lipids and medium chain fatty acids. 2 mg / L or more, more preferably 4 mg / L or more, more preferably 8 mg / L or more, and from the same viewpoint, the content is 100 mg / L or less, more preferably 50 mg / L or less in terms of phosphorus atom equivalent, More preferably, it is 25 mg / L or less, More preferably, it is 20 mg / L or less.
- the content of the phosphorus source in the medium is 0.5 to 100 mg / L, more preferably 1 to 50 mg / L, more preferably phosphorus atom equivalent. It is 2 to 25 mg / L, more preferably 4 to 20 mg / L, still more preferably 8 to 20 mg / L.
- Examples of carbon sources other than glycerin include carbon dioxide, acetic acid, sodium acetate, glucose, sucrose, fructose, starch, and fatty acids.
- the carbon source other than glycerin in the medium is glucose
- the content of glucose in the medium is preferably 5% by mass or less, more preferably 2% by mass or less from the viewpoint of improving the productivity of medium chain fatty acids. More preferably, it is 1 mass% or less, More preferably, it is 0.5 mass% or less, More preferably, it is 0.1 mass% or less.
- the pH of the medium used in step 1 is preferably selected as appropriate depending on the type of algae used, and is preferably in the range of 5 to 10 from the viewpoint of suppressing the growth of various bacteria, promoting the growth of algae, and improving the productivity of medium chain fatty acids. More preferably, it is in the range of 6-9, more preferably in the range of 7-8. Further, the pH of the medium during the culture is preferably 6 to 9, more preferably 7 to 8, from the same viewpoint. The pH can be appropriately adjusted to a desired range by adding an acid or a base to the medium.
- the medium to be used is preferably sterilized by autoclaving, filter filtration, or the like from the viewpoints of suppressing the propagation of various bacteria, promoting the growth of algae, and improving the productivity of medium chain fatty acids.
- the amount of algae inoculated into the medium is not particularly limited, but is preferably 1 to 10% (vol / vol) per medium and more preferably 1 to 5% (vol / vol) from the viewpoint of growth.
- the culture temperature in step 1 is not particularly limited as long as it does not adversely affect the growth of the algae used, but is usually in the range of 5 to 40 ° C., and promotes the growth of algae, improves the productivity of medium chain fatty acids, From the viewpoint of reducing production costs, the temperature is more preferably 10 to 30 ° C, and further preferably 15 to 25 ° C.
- the production method of the present invention from the viewpoint of promoting the growth of algae and improving the productivity of medium-chain fatty acids, it is preferable to carry out the culture of algae under light irradiation.
- the light irradiation may be performed under conditions that allow photosynthesis, and may be artificial light or sunlight.
- the illuminance at the time of light irradiation is preferably in the range of 100 to 50000 lux, more preferably in the range of 300 to 10,000 lux, and still more preferably in the range of 1000 to 6000 lux, from the viewpoint of promoting the growth of algae and improving the productivity of medium chain fatty acids. Range.
- the light irradiation interval is not particularly limited, but from the viewpoint of promoting the growth of algae and improving the productivity of medium chain fatty acids, it is preferably performed in a light-dark cycle, and the cycle is preferably from the same viewpoint as described above. It is 8 to 24 hours, more preferably 10 to 18 hours, and further preferably 12 hours.
- the culture in step 1 is not particularly limited as long as algal bodies that accumulate lipids at a high concentration grow at a high concentration, and may be performed for a long period of time, for example, about 150 days.
- the culture period after the addition of glycerin is preferably 3 days or more, more preferably 7 days or more, and preferably 90 days. Below, more preferably 56 days or less, still more preferably 49 days or less, still more preferably 35 days or less, and even more preferably 30 days or less.
- the period is 3 to 90 days, preferably 3 to 30 days, more preferably 7 to 30 days.
- the culture may be any of aeration and agitation culture, shaking culture or stationary culture, but from the viewpoint of improving aeration, shaking culture is preferred.
- the culture in Step 1 means an algal body and a medium after culturing algae. From the viewpoint of improving the productivity of medium chain fatty acids and reducing the production cost, the culture is preferably an algal body after culturing algae.
- the production method of the present invention includes a step of collecting lipid from the culture obtained in the above-mentioned step 1.
- the method for collecting lipid from the culture is not particularly limited. For example, after completion of the culture, the culture medium and the algal bodies are separated, and the obtained algal bodies are crushed, followed by solvent extraction with an organic solvent to collect lipids.
- Examples of the method for separating the culture medium and the algal cells include filtration and centrifugation, but filtration is preferable from the viewpoint of reducing production costs.
- Examples of the method for crushing algal bodies include pressing, ultrasonic crushing, enzyme treatment, and chemical treatment, and pressing is preferable from the viewpoint of reducing production costs.
- Examples of the organic solvent used for the solvent extraction include chloroform, hexane, butanol, methanol, and ethyl acetate.
- chloroform, hexane, butanol, methanol, and ethyl acetate are preferable, and more preferably, ethyl acetate and hexane are included. It is 1 type or 2 types or more chosen from a group, More preferably, it is hexane.
- the weight ratio of the culture to the organic solvent during the extraction is preferably from 1/1 to 1/20, more preferably from 1/1 to 20 from the viewpoints of improving lipid extraction efficiency and reducing production costs. 1/10, more preferably 1/1 to 1/5.
- the lipid obtained by the production method of the present invention from the viewpoint of improving the productivity of the medium chain fatty acid, the content of the medium chain fatty acid in the total fatty acid in the obtained lipid is preferably 15% by mass or more, More preferably, it is 20% by mass or more, more preferably 30% by mass or more, further preferably 40% by mass or more, and further preferably 50% by mass or more. For example, 15 to 100% by mass, 30 to 85% by mass, 50 to 75%. The mass% etc. are mentioned.
- the content of lauric acid in the total constituent fatty acids in the obtained lipid is preferably 6% by mass or more, more preferably 10% by mass or more, and still more preferably from the viewpoint of improving the productivity of lauric acid.
- the lipid obtained by the production method of the present invention has a content ratio of fatty acids having 16 to 22 carbon atoms in the total constituent fatty acids in the lipid, preferably 90% or less, from the viewpoint of improving the productivity of medium chain fatty acids.
- it is 80 mass% or less, More preferably, it is 70 mass% or less, More preferably, it is 60 mass% or less, More preferably, it is 50 mass% or less, More preferably, it is 40 mass% or less, More preferably, it is 30 mass%
- it is more preferably 20% by mass or less, and still more preferably 10% by mass or less.
- the content of medium-chain fatty acids in the total constituent fatty acids in lipids is about 10% by mass without addition of glycerin (medium-chain fatty acid Productivity) can be improved to 15 to 100% by mass. That is, according to the present invention, it is possible to provide a method for improving the productivity of medium chain fatty acids to 15 to 100% by mass, preferably 30 to 85% by mass, more preferably 50 to 75% by mass by adding glycerin. it can.
- the content ratio of lauric acid in all the constituent fatty acids in the lipid (the productivity of lauric acid), which is about 5% by mass without addition of glycerin, can be improved to 6 to 85% by mass. That is, according to the present invention, it is possible to provide a method for improving the productivity of lauric acid to 6 to 85% by mass, preferably 10 to 80% by mass, more preferably 15 to 75% by mass by adding glycerin. .
- the content of medium chain fatty acids in the total constituent fatty acids in the lipid is preferably 15% by mass or more, or the content ratio of lauric acid in the total constituent fatty acids in the lipid is 6%. More preferably, it is at least mass%.
- the method of culturing the dinoflagellate algae in the glycerol-containing medium is a method for improving the productivity of the algal medium-chain fatty acid and / or improving the production amount, or the content of lauric acid and / or Or it is useful as a productivity improvement method accompanying the improvement of a production amount.
- the method for culturing dinoflagellate algae in a glycerin-containing medium is a method for producing algae with improved productivity accompanying an increase in the content and / or production amount of medium-chain fatty acids, or the inclusion of lauric acid. It is useful as a method for producing algae with improved productivity due to an improvement in the ratio and / or production amount.
- the ester production method of the present invention includes a step of transesterifying the lipid obtained by the above-described production method of the present invention with an alcohol (hereinafter sometimes referred to as “step 3”). According to the ester production method of the present invention, a medium-chain fatty acid ester can be produced efficiently.
- the transesterification reaction can be carried out by a known method.
- the reaction system may be either a batch system or a continuous system, but a continuous system is preferred from the viewpoint of improving productivity and reducing production costs.
- a tank reactor having a stirrer or a fixed bed reactor filled with a catalyst may be used, but it is preferable to use a fixed bed reactor that does not require catalyst separation from the viewpoint of reducing the purification load.
- the alcohol used in Step 3 it is preferable to use a lower alcohol having 1 to 5 carbon atoms from the viewpoint of improving productivity and reducing production cost, and examples thereof include methanol, ethanol, propanol, and butanol. Industrially, methanol is preferred from the viewpoint of low cost and easy recovery.
- the molar ratio of the alcohol to the lipid is preferably 1.5 times the stoichiometric amount or more, more preferably from the viewpoint of obtaining a good reaction rate. It is 2 times mole or more, more preferably 5 times mole or more. Further, from the viewpoint of economically reacting while suppressing the recovered amount of raw material alcohol, it is preferably 50 times mol or less, more preferably 30 times mol or less, and still more preferably 15 times mol or less.
- the molar ratio of raw alcohol to lipid is preferably 1.5 to 50 times mole, more preferably 2 It is -30 times mole, more preferably 5-15 times mole.
- Step 3 is preferably performed in the presence of a catalyst from the viewpoint of improving productivity.
- a catalyst a homogeneous alkaline catalyst such as sodium hydroxide, potassium hydroxide or sodium alcoholate is generally used, but solid catalysts such as ion exchange resin, hydrous zirconium oxide, aluminum phosphate, sulfuric acid-supported zirconia, titanosilicate, etc. Can also be used.
- the amount of the catalyst used in the transesterification reaction is preferably 1% by mass or more, more preferably 3% by mass or more, and further preferably 5% by mass or more based on the lipid. Further, from the viewpoint of maintaining a sufficient suspended state by stirring, the content is preferably 20% by mass or less, more preferably 17% by mass or less, and still more preferably 15% by mass or less based on the lipid. Therefore, from the above viewpoint, the amount of the catalyst used is preferably 1 to 20% by mass, more preferably 3 to 17% by mass, and further preferably 5 to 15% by mass.
- the reaction temperature in the transesterification reaction is preferably 50 to 220 ° C., more preferably 60 to 200 ° C., still more preferably 80 to 200 ° C., and further preferably 130 from the viewpoint of improving reaction efficiency and suppressing by-products. ⁇ 200 ° C.
- the reaction pressure is preferably 0.1 to 10 MPa, more preferably 0.5 to 8 MPa, and further preferably 2 to 6 MPa from the viewpoint of improving reaction efficiency.
- the method for producing a medium chain fatty acid of the present invention includes a step of hydrolyzing the lipid obtained by the above-described production method of the present invention.
- the method for hydrolyzing lipids is not particularly limited, and a conventionally known method can be used (for example, see “New Edition Fatty Acid Chemistry” (Shoshobo)).
- Industrially preferable hydrolysis methods include a high-temperature and high-pressure decomposition method (for example, see JP-A No. 2003-113395) and an enzymatic decomposition method (for example, see JP-A No. 2000-160188).
- separation and purification of medium chain fatty acid or medium chain fatty acid ester from the obtained mixed fatty acid ester or mixed fatty acid can be performed by a conventional method, for example, using a column chromatography method, distillation or the like.
- a method for producing lipid comprising the following steps (1) and (2).
- Step of collecting lipid from the obtained culture ⁇ 2>
- the lipid is a simple lipid
- the content ratio of the medium chain fatty acid in the total constituent fatty acids in the lipid is 15% by mass or more, preferably 20% by mass or more, more preferably 30% by mass or more, further preferably 40% by mass or more, and further The method for producing a lipid according to ⁇ 1> or ⁇ 2>, which is preferably 50% by mass or more.
- the content of lauric acid in all the constituent fatty acids in the lipid is 6% by mass or more, preferably 10% by mass or more, more preferably 15% by mass or more, further preferably 20% by mass or more, and further preferably. Is 30% by mass or more, more preferably 40% by mass or more, and the method for producing a lipid according to any one of ⁇ 1> to ⁇ 3>.
- the content of the fatty acid having 16 to 22 carbon atoms in the total constituent fatty acids in the lipid is 90% or less, preferably 80% by mass or less, more preferably 70% by mass or less, and still more preferably 60% by mass. Or less, more preferably 50% by mass or less, more preferably 40% by mass or less, further preferably 30% by mass or less, further preferably 20% by mass or less, and further preferably 10% by mass or less. Production method.
- the algae of the dinoflagellate are Noctilucales, Prorocentrales, Dinophysales, Gymnodiniales, Peridiniales and Gonyaulacales
- Gymnodinium algae is Amphidinium, Cochlodinium, Gymnodinium, Gyrodinium, Erythropsodinium, Hemidinium, Katodinium, Nematodinium, Oxyrrhis, Polykrikos, Torodinium, Symbiodinium, Warnowia, Woloszynskiella, or ooszynskiella
- the method for producing a lipid according to ⁇ 6> which is an algae belonging to the present invention.
- the algae belonging to Symbiodinium are Symbiodinium microadriaticum, Symbiodinium goreaui, Symbiodinium linucheae, Symbiodinium bermudense, Symbiodinium meandrinae, Symbiodinium californium, Symbiodinium kawagutii, Symbiodinium kawagutii, Symbiodinium corium biodin
- the method for producing lipid according to ⁇ 7> preferably Symbiodinium microadriaticum, more preferably Symbiodinium microadriaticum LB2281 strain, Symbiodinium sp. NIES-2638 strain.
- the medium used for culturing the algae in step 1 is Daigo IMK medium, f / 2 medium, ESM medium, L1 medium, or MNK medium, preferably f / 2 medium, ESM medium, or Digo.
- the content of glycerin in the medium used in Step 1 is preferably 0.01% by mass or more, more preferably 0.02% by mass or more, further preferably 0.05% by mass or more, and further preferably 0.
- the content of the nitrogen source in the medium used in Step 1 is preferably 2 mg / L or more, more preferably 5 mg / L or more, further preferably 10 mg / L or more, and further preferably 15 mg / L in terms of nitrogen atom equivalent.
- L or more more preferably 20 mg / L or more, more preferably 50 mg / L or more, more preferably 100 mg / L or more, and preferably at a nitrogen atom equivalent of 700 mg / L or less, more preferably 600 mg / L or less, more preferably Is 500 mg / L or less, more preferably 400 mg / L or less, more preferably 300 mg / L or less, more preferably 250 mg / L or less, and a nitrogen atom equivalent of 2 to 700 mg / L, preferably 5 to 600 mg / L, More preferably 10 to 500 mg / L, still more preferably 15 to 400 mg / L, Et to preferably 20 ⁇ 300mg / L, more preferably 50 ⁇ 250mg / L, more preferably is 100 ⁇ 250mg / L ⁇ 1> ⁇ any of the lipid producing method of ⁇ 10>.
- the content of the phosphorus source in the medium used in Step 1 is preferably 0.5 mg / L or more, more preferably 1 mg / L or more, still more preferably 2 mg / L or more, more preferably in terms of phosphorus atom equivalent. 4 mg / L or more, more preferably 8 mg / L or more, and phosphorus atom equivalent is preferably 100 mg / L or less, more preferably 50 mg / L or less, still more preferably 25 mg / L or less, further preferably 20 mg / L or less.
- the content of glucose in the medium used in Step 1 is 5% by mass or less, preferably 2% by mass or less, more preferably 1% by mass or less, still more preferably 0.5% by mass or less, and further The method for producing a lipid according to any one of ⁇ 1> to ⁇ 12>, preferably 0.1% by mass or less.
- the pH of the medium used in Step 1 is in the range of 5 to 10, preferably in the range of 6 to 9, more preferably in the range of 7 to 8, any one of ⁇ 1> to ⁇ 13>
- ⁇ 17> The method for producing a lipid according to any one of ⁇ 1> to ⁇ 16>, wherein the culture in the step (1) is performed under light irradiation.
- ⁇ 18> The method for producing a lipid according to ⁇ 17>, wherein the illuminance at the time of light irradiation is in the range of 100 to 50000 lux, preferably in the range of 300 to 10000 lux, more preferably in the range of 1000 to 6000 lux.
- ⁇ 19> The method for producing a lipid according to ⁇ 17> or ⁇ 18>, wherein the light irradiation is performed in a light-dark cycle, and the cycle is 8 to 24 hours, preferably 10 to 18 hours, more preferably 12 hours. .
- ⁇ 20> The method for producing a lipid according to any one of ⁇ 1> to ⁇ 17>, wherein the culture period after the addition of glycerin is 3 to 90 days, preferably 3 to 30 days, more preferably 7 to 30 days.
- a method for producing a medium-chain fatty acid ester comprising a step of transesterifying the lipid obtained by any one of the production methods of ⁇ 1> to ⁇ 20> with an alcohol.
- ⁇ 22> The method for producing a medium-chain fatty acid ester of ⁇ 21>, wherein the alcohol used in the transesterification reaction is a lower alcohol having 1 to 5 carbon atoms, preferably methanol.
- the molar ratio of the alcohol to the lipid is 1.5 to 50 times mol, preferably 2 to 30 times mol, more preferably 5 to 15 times mol.
- ⁇ 21> or ⁇ 22> a method for producing a medium-chain fatty acid ester, which is a mole.
- a method for producing a medium chain fatty acid comprising a step of hydrolyzing a lipid obtained by the production method of any one of ⁇ 1> to ⁇ 20>.
- a method for improving the productivity of medium-chain fatty acids of the alga comprising culturing algae of the dinoflagellate class in a medium containing glycerin.
- the content of medium-chain fatty acids (productivity of medium-chain fatty acids) in the total constituent fatty acids in the lipid is 15 to 100% by mass, preferably 30 to 85% by mass, more preferably 50 to 75% by mass
- a method for improving the productivity of medium chain fatty acids of ⁇ 24> algae which is improved.
- ⁇ 28> A method for producing algae with improved productivity of medium-chain fatty acids, wherein algae of the dinoflagellate class are cultured in a medium containing glycerin.
- Identification of fatty acid ester, total fatty acid content, and measurement method of fatty acid content were carried out under the following conditions by gas chromatography (GC) analysis. The identification of the fatty acid ester was judged based on whether or not the retention time was the same as that of a later-described standard substance. The amount of fatty acid ester detected by GC analysis was calculated based on the internal standard, and the total amount was defined as the total amount of fatty acid. The total fatty acid amount was divided by the dry algal body amount and multiplied by 100 to obtain the fatty acid content (%).
- GC gas chromatography
- Counting the number of cells For counting the number of cells, using a counting chamber (As One Co., Ltd .: Toma hemocytometer standard), 1/50 volume Lugol's solution (50 mg / mL iodine, 100 mg / ml) was used for a moderately diluted sample. After the cells were fixed by adding (mL potassium iodide), measurement was performed using a stereomicroscope (CKX31, Olympus) or a biological microscope (ECLIPSE 80i, Nikon).
- CKX31 stereomicroscope
- Olympus Olympus
- ECLIPSE 80i biological microscope
- Zooxanthella microadriatica LB2281 strain (Symbiodinium microadriaticum LB2281 strain) obtained from UTEX (The culture collection of algae at University of Texas at Austin) was used.
- Glycerin was added to a medium that had passed 3 weeks after the start of culture so that the final concentration was 0.5%, and further cultured for 1 week, and then the amount of fatty acid and fatty acid species were measured by GC analysis. The analysis results are shown in Table 2.
- Example 2 in which glycerin was added to the medium, a marked increase in the amount of fatty acids and an improvement in the ratio of medium chain fatty acids in the total fatty acids were observed.
- Example 6 The LB2281 strain sterilized by the micropipette method was used and cultured under the same conditions as in Example 1 except that glycerin was added to a final concentration of 1.0% from the start of culture and cultured for 4 weeks. .
- the analysis results are shown in Table 4.
- Example 7 The culture was performed under the same conditions as in Example 4 except that the culture period after addition of glycerin was 4 weeks, 5 weeks, 7 weeks, 8 weeks, or 12 weeks. At 4 weeks, 5 weeks, 7 weeks, 8 weeks, or 12 weeks of culture, a culture solution (4 mL) was collected and subjected to GC analysis. The analysis results are shown in Table 5.
- Example 8 The cells were cultured under the same conditions as in Example 6 except that the cells were cultured for 6 weeks. The analysis results are shown in Table 6.
- Example 9 Change the sodium nitrate (NaNO 3 ) in IMK medium (Table 1) to 1000 mg / L, disodium phosphate (Na 2 HPO 4 ) to 14 mg / L, and dipotassium phosphate (K 2 HPO 4 ) to 50 mg / L.
- the nitrogen source content in the culture medium is 165 mg / L in terms of nitrogen atom equivalent, and the phosphorus source content is 12 mg / L in terms of phosphorus atom equivalent).
- the culture was performed under the conditions of The analysis results are shown in Table 6.
- Example 10 Symbiodinium sp. NIES-2638 strain obtained from NIES (National Institute for Environmental Studies) was used as the algae of the dinoflagellate class, and the final concentration of 0. Glycerin was added so as to be 5%. After further culturing for 17 days, the amount of fatty acid and the fatty acid species were measured by GC analysis. The analysis results are shown in Table 7.
- ⁇ Comparative example 14> The cells were cultured under the same conditions as in Example 10 except that glycerin was not added. The analysis results are shown in Table 7.
- Example 11 As the algae of the dinoflagellate class, Heterocapsa nadol NIES-420 strain belonging to Peridiniales obtained from NIES (National Institute for Environmental Studies) was used under the same conditions as in Example 10 except that it was cultured for 7 days after addition of glycerin. Cultured. The analysis results are shown in Table 8.
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Abstract
Description
(1)渦鞭毛藻綱の藻類を、グリセリンを含有する培地中で培養して培養物を得る工程(以下、「工程1」ということがある)
(2)得られた培養物から脂質を採取する工程(以下、「工程2」ということがある)
(脂質)
本発明の製造方法において、脂質としては、脂肪酸及び脂肪酸と各種アルコールとのエステルである単純脂質(油脂・蝋など)、脂肪酸・アルコール・リン酸・糖などから成る複合脂質(リン脂質・糖脂質など)、および以上二者の加水分解生成物で水に不溶の物質(脂肪酸・高級アルコール・ステロールなど)やテルペン・脂溶性ビタミンなどの誘導脂質が挙げられる。このうち、脂質としては、中鎖脂肪酸の生産性向上の観点から、単純脂質、複合脂質であることが好ましく、単純脂質がより好ましく、油脂がさらに好ましい。
(油脂)
本発明の製造方法において、油脂とは、脂肪酸とグリセリンとのエステルを意味し、具体的には、モノグリセリド、ジグリセリド、及びトリグリセリドのような中性脂質をいう。また、中鎖脂肪酸とは、炭素数10~14の炭化水素基を有する1価のカルボン酸をいい、具体的には、カプリン酸、ラウリン酸、ミリスチン酸が挙げられる。このうち、ミリスチン酸、ラウリン酸が好ましく、ラウリン酸がより好ましい。また、脂肪酸エステルとは、前記トリグリセリド以外の、脂肪酸と低級又は高級アルコールとのエステルをいい、中鎖脂肪酸エステルとは、前記中鎖脂肪酸と低級又は高級アルコールとのエステルをいう。
本発明の製造方法は、渦鞭毛藻綱の藻類を、グリセリンを含有する培地中で培養する工程を含む。
本発明に用いられる渦鞭毛藻綱の藻類とは、分類学上、光合成を行い鞭毛を持つ単細胞藻類の一群であり、細胞の表面に縦横の溝を持ち、進化の過程で紅藻の二次共生のほか、複数の共生を経た特徴的な形態を有す藻類をいう。渦鞭毛藻綱の藻類の具体例としては、ヤコウチュウ目 (Noctilucales) 、プロロケントルム目 (Prorocentrales) 、ディノフィシス目 (Dinophysales)、ギムノディニウム目 (Gymnodiniales)、ペリディニウム目 (Peridiniales)、ゴニオラクス目 (Gonyaulacales)、ブラストディニウム目 (Blastodiniales)、有柄鞭毛藻目 (Phytodiniales)等の藻類を挙げることができる。このうち、中鎖脂肪酸の生産性向上の観点から、ギムノディニウム目 (Gymnodiniales)の藻類が好ましい。
なお、前記渦鞭毛藻綱の藻類は、The culture collection of algae at University of Texas at Austin (UTEX)、独立行政法人国立環境研究所(NIES)、National Center for Marine Algae and Microbiota (NCMA:旧CCMP)、Culture Collection of Algae and Protozoa (CCAP)等の公的機関から入手することができる。
工程1において藻類を培養するために用いられる培地には、従来公知のものを使用することができ、天然海水又は人工海水をベースにしたものを使用してもよいし、市販の培養培地を使用してもよい。好ましい培地としては、例えば、ダイゴIMK培地、f/2培地、ESM培地、L1培地、MNK培地等を挙げることができる。このうち、中鎖脂肪酸の生産性向上の観点、栄養成分濃度の観点から、f/2培地、ESM培地、ダイゴIMK培地が好ましく、ESM培地、ダイゴIMK培地がより好ましく、ダイゴIMK培地がさらに好ましい。
前記窒素源としては、例えば、NaNO3、KNO3、Ca(NO3)2、NH4NO3、(NH4)2SO4等が挙げられる。前記リン源としては、例えば、K2HPO4、KH2PO4、Na2HPO4、NaH2PO4、グリセロリン酸ナトリウム等が挙げられる。前記金属塩としては、例えば、NaCl、KCl、CaCl2、MgCl2、Na2SO4、K2SO4、MgSO4、Na2CO3、NaHCO3、Na2SiO3、H3BO3、MnCl2、MnSO4、FeCl3、FeSO4、CoCl2、ZnSO4、CuSO4、Na2MoO4等が挙げられる。前記ビタミン類としては、例えば、ビオチン、ビタミンB12、Thiamine-HCl、ニコチン酸、イノシトール、葉酸、チミン等が挙げられる。
また、培地中における前記リン源の含有量は、脂質及び中鎖脂肪酸の生産性向上の観点から、好ましくはリン原子当量で0.5mg/L以上、より好ましくは1mg/L以上、さらに好ましくは2mg/L以上、さらに好ましくは4mg/L以上、さらに好ましくは8mg/L以上であり、同様の観点から、前記含有量は、リン原子当量で100mg/L以下、より好ましくは50mg/L以下、さらに好ましくは25mg/L以下、さらに好ましくは20mg/L以下である。また、脂質及び中鎖脂肪酸の生産性向上の観点から、培地中における前記リン源の含有量は、リン原子当量で0.5~100mg/L、より好ましくは1~50mg/L、さらに好ましくは2~25mg/L、さらに好ましくは4~20mg/L、さらに好ましくは8~20mg/Lである。
培地に接種する藻類の量は、特に限定されないが、生育の観点から、培地当り1~10%(vol/vol)が好ましく、1~5%(vol/vol)がより好ましい。
工程1において培養物とは、藻類を培養した後の藻体及び培地を意味する。中鎖脂肪酸の生産性向上、及び生産コストの低減の観点から、培養物は藻類を培養した後の藻体であることが好ましい。
本発明の製造方法は、前述の工程1により得られた培養物から脂質を採取する工程を含む。
培養物から脂質を採取する方法は特に限定されない。例えば、培養終了後、培地と藻体を分離し、得られた藻体を破砕した後に、有機溶剤により溶剤抽出して脂質を採取することができる。
抽出時の培養物と有機溶剤の重量比(培養物/有機溶剤)は、脂質の抽出効率向上、生産コスト低減の観点から、1/1~1/20が好ましく、より好ましくは1/1~1/10、より好ましくは1/1~1/5である。
また、本発明の製造方法により得られる脂質は、中鎖脂肪酸の生産性向上の観点から、脂質中の全構成脂肪酸に占める炭素数16~22の脂肪酸の含有割合が、好ましくは90%以下、より好ましくは80質量%以下、さらに好ましくは70質量%以下、よりさらに好ましくは60質量%以下、よりさらに好ましくは50質量%以下、よりさらに好ましくは40質量%以下、よりさらに好ましくは30質量%以下、よりさらに好ましくは20質量%以下、よりさらに好ましくは10質量%以下である。
また、グリセリン無添加では5質量%程度である脂質中の全構成脂肪酸に占めるラウリン酸の含有割合(ラウリン酸の生産性)を、6~85質量%に向上させることができる。すなわち、本発明によれば、グリセリン添加により、ラウリン酸の生産性を6~85質量%、好ましくは10~80質量%、より好ましくは15~75質量%に向上させる方法も提供することができる。
本発明のエステル製造方法は、前述の本発明の製造方法により得られた脂質をアルコールでエステル交換反応する工程を含む(以下、「工程3」ということがある)。本発明のエステル製造方法によれば、中鎖脂肪酸エステルを効率よく製造することができる。
本発明の中鎖脂肪酸の製造方法は、前述の本発明の製造方法により得られた脂質を加水分解する工程を含む。
脂質の加水分解方法は、特に限定されず、従来公知の方法を使用することができる(例えば、「新版 脂肪酸化学」(幸書房)参照)。工業的に好ましい加水分解方法としては、高温高圧分解法(例えば、特開2003-113395号公報参照)や、酵素分解法(例えば、特開2000-160188号公報参照)が挙げられる。
<1>下記(1)及び(2)の工程を含む、脂質の製造方法。
(1)渦鞭毛藻綱の藻類を、グリセリンを含有する培地中で培養して培養物を得る工程
(2)得られた培養物から脂質を採取する工程
<2>前記脂質が、単純脂質、複合脂質又は誘導脂質であり、好ましくは単純脂質又は複合脂質であり、さらに好ましくは単純脂質であり、さらに好ましくは油脂である、<1>記載の脂質の製造方法。
<3>前記脂質中の全構成脂肪酸に占める中鎖脂肪酸の含有割合が15質量%以上であり、好ましくは20質量%以上、より好ましくは30質量%以上、さらに好ましくは40質量%以上、さらに好ましくは50質量%以上である、<1>又は<2>の脂質の製造方法。
<4>前記脂質中の全構成脂肪酸に占めるラウリン酸の含有割合が6質量%以上であり、好ましくは10質量%以上、より好ましくは15質量%以上、さらに好ましくは20質量%以上、さらに好ましくは30質量%以上、さらに好ましくは40質量%以上である、<1>~<3>のいずれかの脂質の製造方法。
<5>前記脂質中の全構成脂肪酸に占める炭素数16~22の脂肪酸の含有割合が90%以下であり、好ましくは80質量%以下、より好ましくは70質量%以下、さらに好ましくは60質量%以下、さらに好ましくは50質量%以下、さらに好ましくは40質量%以下、さらに好ましくは30質量%以下、さらに好ましくは20質量%以下、さらに好ましくは10質量%以下である、<1>の脂質の製造方法。
<6>渦鞭毛藻綱の藻類が、ヤコウチュウ目 (Noctilucales) 、プロロケントルム目 (Prorocentrales) 、ディノフィシス目 (Dinophysales)、ギムノディニウム目 (Gymnodiniales)、ペリディニウム目 (Peridiniales)、ゴニオラクス目 (Gonyaulacales)、ブラストディニウム目 (Blastodiniales)、又は有柄鞭毛藻目 (Phytodiniales)藻類であり、好ましくはギムノディニウム目藻類である、<1>~<5>のいずれかの脂質の製造方法。
<7>ギムノディニウム目藻類が、Amphidinium、Cochlodinium、Gymnodinium、Gyrodinium、Erythropsodinium、Hemidinium、Katodinium、Nematodinium、Oxyrrhis、Polykrikos、Torodinium、Symbiodinium、Warnowia、Woloszynskia、又はZooxanthellaに属する藻類であり、好ましくはSymbiodiniumに属する藻類である、<6>の脂質の製造方法。
<8>Symbiodiniumに属する藻類が、Symbiodinium microadriaticum、Symbiodinium goreaui、Symbiodinium linucheae、Symbiodinium bermudense、Symbiodinium meandrinae、Symbiodinium californium、Symbiodinium kawagutii、Symbiodinium corculorum、Symbiodinium consortia、Symbiodinium muscatinei、Symbiodinium freudenthal、Symbiodinium pulchrorum、Symbiodinium pilosumであり、好ましくはSymbiodinium microadriaticumでありさらに好ましくはSymbiodinium microadriaticum LB2281株、Symbiodinium sp. NIES-2638株である、<7>の脂質の製造方法。
<9>工程1において藻類を培養するために用いられる培地が、ダイゴIMK培地、f/2培地、ESM培地、L1培地、又はMNK培地であり、好ましくはf/2培地、ESM培地、又はダイゴIMK培地であり、より好ましくは、ESM培地、又はダイゴIMK培地であり、さらに好ましくはダイゴIMK培地である<1>~<8>のいずれかの脂質の製造方法。
<10>工程1に用いられる培地中のグリセリンの含有量が、好ましくは0.01質量%以上、より好ましくは0.02質量%以上、さらに好ましくは0.05質量%以上、さらに好ましくは0.07質量%以上、さらに好ましくは0.08質量%以上、さらに好ましくは0.09質量%以上、さらに好ましくは0.1質量%以上であり、好ましくは10質量%以下、より好ましくは5質量%以下、さらに好ましくは4質量%以下、さらに好ましくは3質量%以下、さらに好ましくは2質量%以下、さらに好ましくは1質量%以下、さらに好ましくは0.5質量%以下であり、0.01~10質量%、好ましくは0.02~5質量%、より好ましくは0.05~4質量%、さらに好ましくは0.07~3質量%、さらに好ましくは0.08~2質量%、さらに好ましくは0.09~2.0質量%、さらに好ましくは0.1~2.0質量%、さらに好ましくは0.1~1質量%、さらに好ましくは0.1~0.5質量%である、<1>~<9>のいずれかの脂質の製造方法。
<11>工程1に用いられる培地中の窒素源の含有量が、窒素原子当量で好ましくは2mg/L以上、より好ましくは5mg/L以上、さらに好ましくは10mg/L以上、さらに好ましくは15mg/L以上、さらに好ましくは20mg/L以上、さらに好ましくは50mg/L以上、さらに好ましくは100mg/L以上であり、窒素原子当量で好ましくは700mg/L以下、より好ましくは600mg/L以下、さらに好ましくは500mg/L以下、さらに好ましくは400mg/L以下、さらに好ましくは300mg/L以下、さらに好ましくは250mg/L以下であり、窒素原子当量で2~700mg/L、好ましくは5~600mg/L、より好ましくは10~500mg/L、さらに好ましくは15~400mg/L、さらに好ましくは20~300mg/L、さらに好ましくは50~250mg/L、さらに好ましくは100~250mg/Lである<1>~<10>のいずれかの脂質の製造方法。
<12>工程1に用いられる培地中のリン源の含有量が、リン原子当量で好ましくは0.5mg/L以上、より好ましくは1mg/L以上、さらに好ましくは2mg/L以上、さらに好ましくは4mg/L以上、さらに好ましくは8mg/L以上であり、リン原子当量で好ましくは100mg/L以下、より好ましくは50mg/L以下、さらに好ましくは25mg/L以下、さらに好ましくは20mg/L以下であり、リン原子当量で0.5~100mg/L、より好ましくは1~50mg/L、さらに好ましくは2~25mg/L、さらに好ましくは4~20mg/L、さらに好ましくは8~20mg/Lである<1>~<11>のいずれかの脂質の製造方法。
<13>工程1に用いられる培地中のグルコースの含有量が、5質量%以下であり、好ましくは2質量%以下、より好ましくは1質量%以下、さらに好ましくは0.5質量%以下、さらに好ましくは0.1質量%以下である、<1>~<12>のいずれかの脂質の製造方法。
<14>工程1に用いる培地のpHが、5~10の範囲であり、好ましくは6~9の範囲、さらに好ましくは7~8の範囲である、<1>~<13>のいずれかの脂質の製造方法。
<15>培地に接種する藻類の量が、培地当り1~10%(vol/vol)であり、好ましくは1~5%(vol/vol)である、<1>~<14>のいずれかの脂質の製造方法。
<16>工程1における培養温度が、5~40℃の範囲であり、好ましくは10~30℃、より好ましくは15~25℃である、<1>~<15>のいずれかの脂質の製造方法。
<17>工程(1)の培養を光照射下で行う、<1>~<16>のいずれかの脂質の製造方法。
<18>光照射時の照度が、100~50000ルクスの範囲であり、好ましくは300~10000ルクスの範囲、より好ましくは1000~6000ルクスの範囲である、<17>の脂質の製造方法。
<19>光照射を明暗周期で行い、その周期が、8時間~24時間であり、好ましくは10~18時間、より好ましくは12時間である、<17>又は<18>の脂質の製造方法。
<20>グリセリン添加後の培養期間が、3~90日間、好ましくは3~30日間、より好ましくは7~30日間である、<1>~<17>のいずれかの脂質の製造方法。
<21><1>~<20>のいずれかの製造方法により得られた脂質をアルコールでエステル交換反応する工程を有する、中鎖脂肪酸エステルの製造方法。
<22>エステル交換反応に用いられるアルコールが、炭素数1~5の低級アルコールであり、好ましくはメタノールである、<21>の中鎖脂肪酸エステルの製造方法。
<23>エステル交換反応における、脂質に対する前記アルコールのモル比(脂質を全てトリグリセリドとして換算)は、1.5~50倍モルであり、好ましくは2~30倍モル、より好ましくは5~15倍モルである、<21>又は<22>の中鎖脂肪酸エステルの製造方法。
<24><1>~<20>のいずれかの製造方法により得られた脂質を加水分解する工程を含む、中鎖脂肪酸の製造方法。
<25>渦鞭毛藻綱の藻類を、グリセリンを含有する培地中で培養する、当該藻類の中鎖脂肪酸の生産性向上方法。
<26>脂質中の全構成脂肪酸に占める中鎖脂肪酸の含有割合(中鎖脂肪酸の生産性)を、15~100質量%、好ましくは30~85質量%、より好ましくは50~75質量%に向上させる、<24>の藻類の中鎖脂肪酸の生産性向上方法。
<27>中鎖脂肪酸がラウリン酸である<25>又は<26>の生産性向上方法。
<28>渦鞭毛藻綱の藻類を、グリセリンを含有する培地中で培養する、中鎖脂肪酸の生産性が向上された藻類の製造方法。
<29>中鎖脂肪酸がラウリン酸である<28>の藻類の製造方法。
藻類の培養には、ダイゴIMK培地(日本製薬製)を用いた。培地組成の詳細を下記表1に示す。培養容器に滅菌した100mL容三角フラスコ(PYREX(登録商標)製)と綿栓(アズワン製CS-28)を使用し、フィルターユニット(ナルジェヌンク製)を用いろ過滅菌した培地を50mL分注した。予め同液体培地により継代培養していた藻類の培養液1mLを新しい培地に接種し、20℃、蛍光灯下、照度約3000ルクス、12時間明暗条件下で静置培養した。
培養により得られた培養物4mLを、3000rpm、30分の条件で遠心分離して、沈殿画分を得た。得られた沈殿画分を80℃にて約3時間から16時間乾燥させ、乾燥藻体とした。
得られた乾燥藻体の重量を測定後、0.5mLの1%食塩水にて懸濁し、内部標準として1mg/mLの7-ペンタデカノンを50μL添加後、0.5mLのクロロホルムおよび1mLのメタノールを培養液に添加して激しく攪拌後30分間放置し、その後さらに0.5mLのクロロホルムおよび0.5mLの1.5%KClを添加して攪拌後、3000rpm、15分遠心分離を行い、パスツールピペットにてクロロホルム層(下層)を回収した。
得られたクロロホルム層約0.5mLに窒素ガスを吹き付けて乾固し、0.5N水酸化カリウム/メタノール溶液700μLを添加し、80℃で30分恒温した。続いて1mLの14%三フッ化ホウ素溶液(SIGMA社製)を添加し、80℃にて20分恒温し、その後ヘキサン、飽和食塩水を各1mL添加し室温にて30分放置後、上層であるヘキサン層を回収し、乾固して脂肪酸エステルを得た。
脂肪酸エステルの同定、総脂肪酸量、脂肪酸含有量は、ガスクロマトグラフィー(GC)分析により、下記の条件で行った。
脂肪酸エステルの同定は、後述の標準物質とのリテンションタイムと同一かどうかにより判断した。またGC分析にて検出した脂肪酸エステル量を、内部標準を基準に算出し、その総量を総脂肪酸量とした。また総脂肪酸量を乾燥藻体量で除し100を乗じた値を脂肪酸含量(%)とした。
カラム:DB-1 MS 30m×200μm×0.25μm(J&W scientific製)
移動相:高純度ヘリウム(流量1mL/分)
昇温プログラム:150℃(0.5分)、40℃/分、320℃(4分)
注入口検出器温度:300℃
注入法:スプリットモード (スプリット比=75:1)
サンプル注入量:5 μl
カラム流速:0.3 mL/分(コンスタント)
検出器:FID
キャリアガス:水素
メイクアップガス:ヘリウム
標準物質:SIGMA社製の下記脂肪酸エステルを用いた。
ラウリン酸メチル(C12)、ミリスチン酸メチル(C14)、パルミチン酸メチル(C16)、ステアリン酸メチル(C18)を、不飽和脂肪酸はパルミトレイン酸メチル(C16:1)、オレイン酸メチル(C18:1)、リノール酸メチル(C18:2)、リノレン酸メチル(C18:3)、エイコサペンタエン酸メチル(C20:5)、ドコサヘキサエン酸メチル(C22:6)
細胞数測定は、カウンティング・チャンバー(アズワン社:トーマ血球計算盤スタンダード)を用い、適度に希釈した試料に対して1/50容のルゴール液(50mg/mLヨウ素、100mg/mL ヨウ化カリウム)を添加し細胞を固定した後に、実体顕微鏡(CKX31、オリンパス社製)または生物顕微鏡(ECLIPSE 80i、ニコン社製)を用いて計測した。
渦鞭毛藻綱の藻類として、UTEX(The culture collection of algae at University of Texas at Austin)より入手した、Zooxanthella microadriatica LB2281株(Symbiodinium microadriaticum LB2281株)を用いた。
グリセリンを添加しなかった、又はグリセリンに代えてグルコースを終濃度0.5%となるように添加した以外は実施例1と同様の条件で培養した。分析結果を表2に示す。
グリセリンを終濃度0.1%、0.5%、1.0%、2.0%となるように添加し、さらに2週間培養した以外は実施例1と同様の条件で培養した。分析結果を表3に示す。
グリセリンを添加しなかった、又はグリセリンに代えてグルコースを終濃度0.5%となるように添加した以外は実施例2と同様の条件で培養した。分析結果を表3に示す。
マイクロピペット法により無菌化した前記LB2281株を用い、培養開始段階から、終濃度が1.0%となるようにグリセリンを添加して4週間培養した以外は実施例1と同様の条件で培養した。分析結果を表4に示す。
グリセリンを添加しなかった(比較例5)、又は、グリセリンに代えてグルコース(比較例6)、フルクトース(比較例7)、スクロース(比較例8)、キシロース(比較例9)、ソルビトール(比較例10)、又はマンニトール(比較例11)を添加した以外は実施例6と同様の条件で培養した。分析結果を表4に示す。
グリセリン添加後の培養の期間を、4週間、5週間、7週間、8週間、又は12週間とした以外は実施例4と同様の条件で培養した。培養4週間目、5週間目、7週間目、8週間目、又は12週間目に培養液(4mL)を採取してGC分析を行った。分析結果を表5に示す。
6週間培養した以外は実施例6と同様の条件で培養した。分析結果を表6に示す。
IMK培地(表1)中の硝酸ナトリウム(NaNO3)を1000mg/L、リン酸2ナトリウム(Na2HPO4)を14mg/L、リン酸2カリウム(K2HPO4)を50mg/Lに変更し(培地中の窒素源の含有量は、窒素原子当量で165mg/L、リン源の含有量は、リン原子当量で12mg/Lである。)、6週間培養した以外は実施例6と同様の条件で培養した。分析結果を表6に示す。
グリセリンを添加しなかった以外は実施例8と同様の条件で培養した。分析結果を表6に示す。
グリセリンを添加しなかった以外は実施例9と同様の条件で培養した。分析結果を表6に示す。
渦鞭毛藻綱の藻類として、NIES(国立環境研究所)より入手したSymbiodinium sp. NIES-2638株を用い、実施例1と同様に培養開始後4週間が経過した培地に、終濃度が0.5%となるようにグリセリンを添加した。更に17日間培養した後、GC分析により脂肪酸量および脂肪酸種を測定した。分析結果を表7に示す。
グリセリンを添加しなかった以外は実施例10と同様の条件で培養した。分析結果を表7に示す。
渦鞭毛藻綱の藻類として、NIES(国立環境研究所)より入手したペリディニウム目 (Peridiniales)に属するHeterocapsa niei NIES-420株を用いグリセリン添加後7日間培養した以外は、実施例10と同条件で培養した。分析結果を表8に示す。
グリセリンを添加しなかった以外は実施例11と同様の条件で培養した。分析結果を表8に示す。
Claims (17)
- 下記(1)及び(2)の工程を含む、脂質の製造方法。
(1)渦鞭毛藻綱の藻類を、グリセリンを含有する培地中で培養して培養物を得る工程
(2)得られた培養物から脂質を採取する工程 - 前記脂質が油脂である、請求項1記載の脂質の製造方法。
- 前記油脂中の全構成脂肪酸に占める中鎖脂肪酸の含有割合が15質量%以上である、請求項2記載の脂質の製造方法。
- 前記油脂中の全構成脂肪酸に占めるラウリン酸の含有割合が6質量%以上である、請求項2記載の脂質の製造方法。
- 渦鞭毛藻綱の藻類が、ギムノディニウム目藻類である、請求項1~4のいずれか1項に記載の脂質の製造方法。
- ギムノディニウム目藻類が、Symbiodinium に属する藻類である、請求項5に記載の脂質の製造方法。
- Symbiodinium に属する藻類が、Symbiodinium microadriaticumである、請求項6に記載の脂質の製造方法。
- 培地中のグリセリンの含有量が、0.01~10質量%である、請求項1~7のいずれか1項に記載の脂質の製造方法。
- 前記培地が窒素源を含有し、培地中における窒素源の含有量が、窒素原子当量で10~500mg/Lである、請求項1~8のいずれか1項に記載の脂質の製造方法。
- 前記培地がリン源を含有し、培地中におけるリン源の含有量が、リン原子当量で1~50mg/Lである、請求項1~9のいずれか1項に記載の脂質の製造方法。
- 工程(1)の培養を光照射下で行う、請求項1~10のいずれか1項に記載の脂質の製造方法。
- 請求項1~11のいずれか1項記載の製造方法により得られた脂質をアルコールでエステル交換反応する工程を有する、中鎖脂肪酸エステルの製造方法。
- 請求項1~11のいずれか1項記載の製造方法により得られた脂質を加水分解する工程を含む、中鎖脂肪酸の製造方法。
- 渦鞭毛藻綱の藻類を、グリセリンを含有する培地中で培養する、当該藻類の中鎖脂肪酸の生産性向上方法。
- 中鎖脂肪酸がラウリン酸である請求項14に記載の生産性向上方法。
- 渦鞭毛藻綱の藻類を、グリセリンを含有する培地中で培養する、中鎖脂肪酸の生産性が向上された藻類の製造方法。
- 中鎖脂肪酸がラウリン酸である請求項16に記載の藻類の製造方法。
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