WO2017150723A1 - Procédé de production de stérol - Google Patents

Procédé de production de stérol Download PDF

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WO2017150723A1
WO2017150723A1 PCT/JP2017/008586 JP2017008586W WO2017150723A1 WO 2017150723 A1 WO2017150723 A1 WO 2017150723A1 JP 2017008586 W JP2017008586 W JP 2017008586W WO 2017150723 A1 WO2017150723 A1 WO 2017150723A1
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sterol
sterols
acid
algae
culture
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Japanese (ja)
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鈴木　茂雄
喬 柿山
宣博 平塚
康博 立山
雅弘 林
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味の素株式会社
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/02—Algae
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J1/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, not substituted in position 17 beta by a carbon atom, e.g. estrane, androstane
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J13/00—Normal steroids containing carbon, hydrogen, halogen or oxygen having a carbon-to-carbon double bond from or to position 17
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J17/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, having an oxygen-containing hetero ring not condensed with the cyclopenta(a)hydrophenanthrene skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J5/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane and substituted in position 21 by only one singly bound oxygen atom, i.e. only one oxygen bound to position 21 by a single bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J51/00—Normal steroids with unmodified cyclopenta(a)hydrophenanthrene skeleton not provided for in groups C07J1/00 - C07J43/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J7/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of two carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
    • 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
    • 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
    • C12P33/00—Preparation of steroids
    • 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
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group

Definitions

  • the present invention relates to algae that produce sterols and a method for producing sterols using the algae.
  • Sterols are steroid derivatives having a hydroxyl group at the C-3 position and are found in various organisms such as animals, plants and fungi. Sterols are produced, for example, by extraction from biological resources that contain those sterols. Moreover, what produces sterols, such as cholesterol, is known in the Labyrinthula which are heterotrophic algae (nonpatent literature 1).
  • An object of the present invention is to provide an algae that produces sterol and a method for producing sterol using the algae.
  • the present invention can be exemplified as follows. [1] Culturing an algae selected from the group consisting of AJ7867 strain (FERM BP-22304) and its derivatives in a medium; Recovering sterols from algal bodies produced by the culture, A method for producing a sterol, comprising: [2] The method, wherein the sterol is one or more sterols selected from the group consisting of animal sterols, plant sterols, and fungal sterols.
  • the sterol is cholesterol, 7-dehydrocholesterol, glycolic acid, taurocholic acid, cholic acid, estradiol, estrone, ethinylestradiol, estriol, dehydroepiandrosterone, methylandrostenediol, 5 ⁇ -pregnane-3 ⁇ , 20 ⁇ -diol, Pregnenolone, chenodeoxycholic acid, dehydrocholic acid, dihydroxycholesterol, digitoxigenin, desmosterol, lasosterol, stigmasterol, ⁇ -sitosterol, brassicasterol, campesterol, diosgenin, oleanolic acid, betulinic acid, ursolic acid, hecogenin, salsasapogenin , Isofucosterol, avenasterol, ⁇ 7-stigmastenol, ⁇ 7-campestenol, fucos Roll, monkey gas Te roll is one or more sterols selected from
  • the sterol is cholesterol.
  • the algae is an Aurantiochytrium algae having a base sequence represented by SEQ ID NO: 3.
  • a method for producing a sterol comprising: [7] The method, wherein the sterol is one or more sterols selected from the group consisting of animal sterols, plant sterols, and fungal sterols.
  • the sterol is cholesterol, 7-dehydrocholesterol, glycolic acid, taurocholic acid, cholic acid, estradiol, estrone, ethinylestradiol, estriol, dehydroepiandrosterone, methylandrostenediol, 5 ⁇ -pregnane-3 ⁇ , 20 ⁇ -diol, Pregnenolone, chenodeoxycholic acid, dehydrocholic acid, dihydroxycholesterol, digitoxigenin, desmosterol, lasosterol, stigmasterol, ⁇ -sitosterol, brassicasterol, campesterol, diosgenin, oleanolic acid, betulinic acid, ursolic acid, hecogenin, salsasapogenin , Isofucosterol, avenasterol, ⁇ 7-stigmastenol, ⁇ 7-campestenol, fucos Roll, monkey gas Te roll is one or more sterols selected from
  • the algae is an algae selected from AJ7867 strain (FERM BP-22304) and its derivatives.
  • the algae of the present invention has high sterol production ability.
  • the algae of the present invention may have one sterol-producing ability or may have two or more sterol-producing ability.
  • the algae of the present invention may be, for example, algae selected from AJ7867 strain (FERM BP-22304) and its derivatives.
  • AJ7867 shares were entrusted to the Patent Biological Depositary Center (Postal Code 292-0818, Kisarazu Kazusa Kamashi 2-5-8, Room 120, Chiba, Japan) on January 8, 2016 Deposited under the number FERM P-22304. Thereafter, the AJ7867 strain was transferred to an international deposit under the Budapest Treaty on December 22, 2016, and was given the accession number FERM BP-22304.
  • the AJ7867 strain is considered to be related to the Aurantiochytrium genus by molecular phylogenetic analysis using the 18S rDNA sequence as an index.
  • the base sequence of 18S rDNA of AJ7867 strain is shown in SEQ ID NO: 3. That is, the algae of the present invention may be algae such as auranthiochytrium algae having the base sequence shown in SEQ ID NO: 3, for example.
  • the algae of the present invention is specifically an algae selected from, for example, AJ7867 strain (FERM BP-22304) and its derivative strain, and may have the base sequence shown in SEQ ID NO: 3.
  • the algae of the present invention are specifically algae selected from, for example, the AJ7867 strain (FERM BP-22304) and its derivative strains, and may be auranthiochytrium algae.
  • the alga of the present invention is an algae selected from, for example, the AJ7867 strain (FERM BP-22304) and its derivative strain, and has the base sequence shown in SEQ ID NO: 3 It may be what is.
  • derived strain refers to a strain constructed with the AJ7867 strain as a parent strain (ancestor strain) and having a sterol-producing ability equal to or higher than that of the parent strain (ancestral strain).
  • the derivative strain may be bred by artificial modification, for example.
  • Artificial alteration includes alteration by genetic engineering techniques and alteration by mutation treatment. Mutation treatments include X-ray irradiation, UV irradiation, and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), ethylmethanesulfonate (EMS), and methylmethanesulfonate (MMS). ) And the like.
  • stock may arise naturally, for example in the case of use of a parent strain (ancestor strain).
  • parent strain ancestor strain
  • examples of such derivative strains include mutant strains that are naturally generated when the AJ7867 strain is cultured.
  • a derivative strain may be constructed by one type of modification or by a combination of two or more types of modification.
  • the induced strain has a sterol production ability equal to or higher than that of the parent strain” means that when the induced strain is cultured in a medium, the sterol is produced (ie accumulated) when the parent strain is cultured under the same conditions per dry weight of algal bodies. It may mean producing (accumulating) sterols in an amount of 70% or more, 80% or more, 90% or more, or 95% or more of the amount.
  • the derivative strain has, for example, a total sterol amount of 2.5% (w / w-DCW) or more, 2.7% (w / w-DCW) or more, 3.0% (w / w-DCW) or more, Alternatively, sterol may be generated (accumulated) in an amount of 3.3% (w / w-DCW) or more.
  • the derivative strain is, for example, in an amount of 1.2% (w / w-DCW) or more, 1.5% (w / w-DCW) or more, or 2.0% (w / w-DCW) or more. Cholesterol may be generated (accumulated).
  • the derivative strain is, for example, 0.95% (w / w-DCW) or more, 1.0% (w / w-DCW) or more, or 1.1% (w / w-DCW) or more.
  • Stigmasterol may be generated (accumulated). “% (W / w-DCW)” indicates the percentage of the weight of an object (such as sterol) relative to the dry alga body weight (DCW).
  • the culture conditions are not particularly limited as long as the algae of the present invention can sufficiently grow. Examples of such culture conditions include the culture conditions described below. Specific examples of such culture conditions include the culture conditions described in the examples.
  • the amount of sterol can be measured, for example, by a method described later.
  • the “sterol amount” means an amount converted to the weight of free sterol unless otherwise specified.
  • Steprol is a general term for steroid derivatives having a hydroxyl group at the C-3 position.
  • Step 2 is a general term for compounds having a cyclopentanohydrophenanthrene ring skeleton.
  • sterol is a general term for compounds having a cyclopentanohydrophenanthrene ring skeleton having a hydroxyl group at the C-3 position.
  • the basic skeleton of sterol that is, a cyclopentanohydrophenanthrene ring skeleton having a hydroxyl group at the C-3 position is represented by the following formula (1).
  • Sterols may have various substituents in the basic skeleton. Typical substituents that sterol has include a methyl group at the C-10 position, a methyl group at the C-13 position, and an alkyl group at the C-17 position.
  • the alkyl group may be, for example, linear, branched or cyclic, and may be saturated or unsaturated.
  • the cyclopentanohydrophenanthrene ring skeleton may contain one or more double bonds.
  • Typical skeletons possessed by steroids include cholestane, cholane, pregnane, androstane, and estrane. These representative steroid skeletons are respectively represented by the following formulas (2-1) to (2-5). That is, typical sterols include compounds having a skeleton having a hydroxyl group introduced at the C-3 position of the steroid skeleton represented by the following formulas (2-1) to (2-5).
  • sterols examples include animal sterols, plant sterols, and fungal sterols.
  • Animal sterols”, “plant sterols” and “fungal sterols” refer to sterols found in animals, plants and fungi, respectively.
  • the “plant” mentioned here includes algae.
  • animal sterols include cholesterol, 7-dehydrocholesterol, glycolic acid, taurocholic acid, cholic acid, estradiol, estrone, ethinylestradiol, estriol, dehydroepiandrosterone, methylandrostenediol, 5 ⁇ -pregnane-3 ⁇ , 20 ⁇ -diol , Pregnenolone, chenodeoxycholic acid, dehydrocholic acid, dihydroxycholesterol, digitoxigenin, desmosterol, lasosterol.
  • Plant sterols include stigmasterol, ⁇ -sitosterol, brassicasterol, campesterol, diosgenin, oleanolic acid, betulinic acid, ursolic acid, hecogenin, sarsasapogenin, isofucosterol, abenasterol, ⁇ 7-stigmastenol, ⁇ 7-campestenol, fucosterol, salgasterol can be mentioned.
  • ergosterol As the fungal sterol, ergosterol can be mentioned.
  • sterols examples include cholesterol and stigmasterol. More particularly, sterols include cholesterol.
  • Sterols can exist in the form of, for example, a free form, a fatty acid ester form, a glycoside form, and the like. That is, “sterol” may mean a free sterol, a sterol fatty acid ester, a sterol glycoside, or a mixture thereof, unless otherwise specified.
  • the algae of the present invention can be used, for example, for the production of sterols. That is, the method of the present invention is a method for producing sterol, comprising culturing the algae of the present invention in a medium and recovering sterol from the algal bodies produced by the culture.
  • “algae” refers to algae cells obtained by culturing algae in a medium. In the method of the present invention, one sterol may be produced, or two or more sterols may be produced.
  • the medium to be used is not particularly limited as long as the algae of the present invention can grow and sterols are produced.
  • a normal medium used for culturing heterotrophic algae such as Labyrinthula can be used.
  • the medium may contain a component selected from a carbon source, a nitrogen source, a phosphoric acid source, a sulfur source, and other various organic and inorganic components as necessary.
  • Specific examples of the medium include GPY medium (Example) prepared with 0 to 1 ⁇ artificial seawater.
  • the carbon source examples include glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, waste molasses, starch hydrolysate, biomass hydrolyzate and other sugars, and organic acids such as acetic acid and citric acid. Alcohols such as ethanol, glycerol and crude glycerol, and fatty acids.
  • one type of carbon source may be used, or two or more types of carbon sources may be used in combination.
  • the nitrogen source include ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate, organic nitrogen sources such as peptone, yeast extract, meat extract, soybean protein degradation product, ammonia, and urea. Ammonia gas or ammonia water used for pH adjustment may be used as a nitrogen source. As the nitrogen source, one kind of nitrogen source may be used, or two or more kinds of nitrogen sources may be used in combination.
  • the phosphoric acid source examples include phosphates such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and phosphate polymers such as pyrophosphoric acid.
  • phosphates such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate
  • phosphate polymers such as pyrophosphoric acid.
  • the phosphoric acid source one type of phosphoric acid source may be used, or two or more types of phosphoric acid sources may be used in combination.
  • the sulfur source include inorganic sulfur compounds such as sulfate, thiosulfate, and sulfite, and sulfur-containing amino acids such as cysteine, cystine, and glutathione.
  • the sulfur source one kind of sulfur source may be used, or two or more kinds of sulfur sources may be used in combination.
  • organic and inorganic components include, for example, inorganic salts such as sodium chloride and potassium chloride; trace metals such as iron, manganese, magnesium and calcium; vitamin B1, vitamin B2, vitamin B6 and nicotine Examples include vitamins such as acid, nicotinamide, and vitamin B12; amino acids; nucleic acids; and organic components such as peptone, casamino acid, yeast extract, and soybean protein degradation products containing these.
  • inorganic salts such as sodium chloride and potassium chloride
  • trace metals such as iron, manganese, magnesium and calcium
  • vitamin B1, vitamin B2, vitamin B6 and nicotine include vitamins such as acid, nicotinamide, and vitamin B12; amino acids; nucleic acids; and organic components such as peptone, casamino acid, yeast extract, and soybean protein degradation products containing these.
  • vitamins such as acid, nicotinamide, and vitamin B12
  • amino acids amino acids
  • nucleic acids amino acids
  • organic components such as peptone, casamino acid, yeast extract, and soybean
  • Culture conditions are not particularly limited as long as the algae of the present invention can grow and sterols are produced.
  • the culture can be performed under normal conditions used for culture of heterotrophic algae such as Labyrinthula.
  • Cultivation can be performed using a liquid medium.
  • a liquid medium may be directly inoculated with the algae of the present invention cultured in a solid medium such as an agar medium, or a seed culture of the algae of the present invention in a liquid medium.
  • the medium may be inoculated. That is, the culture may be performed separately for seed culture and main culture. In that case, the culture conditions of the seed culture and the main culture may or may not be the same.
  • the amount of the alga of the present invention contained in the medium at the start of culture is not particularly limited.
  • the main culture may be performed, for example, by inoculating 1-50% (v / v) of the seed culture solution in the main culture medium.
  • Culture can be performed by batch culture, fed-batch culture, continuous culture, or a combination thereof.
  • the medium at the start of the culture is also referred to as “initial medium”.
  • a medium supplied to a culture system (fermentor) in fed-batch culture or continuous culture is also referred to as “fed-batch medium”.
  • supplying a feeding medium to a culture system in fed-batch culture or continuous culture is also referred to as “fed-batch”.
  • cultivation is performed by dividing into seed culture and main culture, for example, both seed culture and main culture may be performed by batch culture. Further, for example, seed culture may be performed by batch culture, and main culture may be performed by fed-batch culture or continuous culture.
  • Culture can be performed, for example, under aerobic conditions.
  • the aerobic condition means that the dissolved oxygen concentration in the liquid medium is 0.33 ppm or more, which is the detection limit of the oxygen membrane electrode, and preferably 1.5 ppm or more.
  • the oxygen concentration may be controlled to, for example, about 5 to 50%, preferably about 10% of the saturated oxygen concentration.
  • culture under aerobic conditions can be performed by aeration culture, shaking culture, agitation culture, or a combination thereof.
  • the pH of the medium may be, for example, pH 3 to 10, preferably pH 4.0 to 9.5. During the culture, the pH of the medium can be adjusted as necessary.
  • the pH of the medium is various alkaline or acidic substances such as ammonia gas, aqueous ammonia, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, calcium hydroxide, magnesium hydroxide, hydrochloric acid, sulfuric acid, etc. Can be adjusted.
  • the culture temperature may be, for example, 20 to 35 ° C, preferably 25 ° C to 35 ° C.
  • the culture period may be, for example, 10 hours to 120 hours.
  • the culture may be continued, for example, until the carbon source in the medium is consumed or until the activity of the algae of the present invention is lost. By culturing the algae of the present invention under such conditions, algal bodies containing sterols are produced.
  • Sterols can be collected from algal cells as appropriate. That is, sterols can be extracted from algal bodies and recovered. Algal bodies may be used for extraction of sterol while contained in the medium, or may be collected from the medium and then used for extraction of sterol.
  • the algal bodies that is, the culture medium containing the algal bodies and the algal bodies recovered from the culture medium may be appropriately subjected to treatments such as dilution, concentration, freezing, thawing, and drying, and then subjected to sterol extraction. These treatments may be performed alone or in appropriate combination. These treatments can be appropriately selected according to various conditions such as the type of sterol extraction method.
  • the method for recovering the algal cells from the medium is not particularly limited, and for example, a known method (Grima, E. M. et al. 2003. Biotechnol. Advances 20: 491-515) can be used.
  • algal bodies can be recovered from the culture medium by methods such as natural sedimentation, centrifugation, and filtration. At that time, a flocculant may be used.
  • the collected algal bodies can be appropriately washed using an appropriate medium.
  • the collected alga bodies can be appropriately resuspended using an appropriate medium.
  • Examples of the medium that can be used for washing and suspension include an aqueous medium (aqueous solvent) such as water and an aqueous buffer, an organic medium (organic solvent) such as methanol, and a mixture thereof.
  • aqueous medium such as water and an aqueous buffer
  • organic medium organic solvent
  • the medium can be appropriately selected according to various conditions such as the type of sterol extraction method.
  • the method for extracting sterol is not particularly limited, and for example, a known method can be used.
  • a technique for example, a technique of extracting lipids from general algae can be mentioned.
  • Specific examples of such methods include organic solvent treatment, ultrasonic treatment, bead crushing treatment, acid treatment, alkali treatment, enzyme treatment, hydrothermal treatment, supercritical treatment, microwave treatment, electromagnetic field treatment, and compression treatment. Is mentioned. These methods can be used alone or in appropriate combination.
  • the organic solvent used for the organic solvent treatment is not particularly limited as long as it can extract sterols from algal bodies.
  • the organic solvent include alcohols such as methanol, ethanol, 2-propanol, butanol, pentanol, hexanol, heptanol, and octanol, ketones such as acetone, ethers such as dimethyl ether and diethyl ether, methyl acetate, and ethyl acetate. Such as esters, alkanes such as n-hexane, and chloroform.
  • Examples of lipid extraction methods using organic solvents include the Bligh-Dyer method and the Folch method.
  • the organic solvent one kind of organic solvent may be used, or two or more kinds of organic solvents may be used in combination.
  • the pH of the alkali treatment is not particularly limited as long as it is a pH at which sterols can be extracted from algal cells.
  • the pH of the alkali treatment is usually pH 8.5 or higher, preferably pH 10.5 or higher, more preferably pH 11.5 or higher, and pH 14 or lower.
  • the temperature for the alkali treatment is usually 30 ° C. or higher, preferably 50 ° C. or higher, more preferably 70 ° C. or higher.
  • the temperature of the alkali treatment may be preferably 120 ° C. or lower.
  • the alkali treatment time may be usually 10 minutes or longer, preferably 30 minutes or longer, more preferably 50 minutes or longer.
  • the alkali treatment time may be preferably 150 minutes or less.
  • An alkaline substance such as NaOH or KOH can be used for the alkali treatment.
  • the recovery of the extracted sterol can be performed by a known method used for separation and purification of compounds. Examples of such a method include an ion exchange resin method and a membrane treatment method. These methods can be used alone or in appropriate combination.
  • the collected sterol may contain components such as algal bodies, medium components, moisture, components used for extraction processing, and metabolic byproducts of the algae of the present invention in addition to sterols.
  • the sterol may be purified to the desired degree. Sterol purity is, for example, 1% (w / w) or higher, 2% (w / w) or higher, 5% (w / w) or higher, 10% (w / w) or higher, 30% (w / w) It may be 50% (w / w) or more, 70% (w / w) or more, 90% (w / w) or more, or 95% (w / w) or more.
  • the type and amount of sterol can be determined by a known method used for detection or identification of a compound. Examples of such a method include HPLC, LC / MS, GC / MS, and NMR. These methods can be used alone or in appropriate combination.
  • the recovered sterol may be, for example, free sterol, sterol fatty acid ester, sterol glycoside, or a mixture thereof.
  • a free sterol can also be produced by hydrolysis. Hydrolysis can be carried out by a conventional method.
  • sterol fatty acid esters can be hydrolyzed enzymatically by esterases
  • sterol glycosides can be hydrolyzed enzymatically by glycosidases.
  • Sterol can be used for various purposes.
  • the use of sterol is not particularly limited.
  • the sterol can be used, for example, as it is, alone or in combination with other components, or as a raw material for other components.
  • Applications of sterols include food additives, feed additives, health foods, pharmaceuticals, pharmaceutical raw materials, chemical products, chemical raw materials, cosmetic ingredients, and cosmetic raw materials.
  • Examples of feed include livestock feed and aquatic feed.
  • Example 1 Acquisition of a sterol accumulating strain of Labyrinthula A sample of leaf septic containing water from a river in Okinawa was collected. An appropriate amount of pine pollen (collected in Miyazaki Prefecture) was added to the sample and allowed to stand at room temperature for 8 days. Penicillin G calcium and streptomycin sulfate were added to 0.1 ⁇ Raihimani preservation medium (Nissui) prepared with 0.5 ⁇ artificial seawater to a final concentration of 50 mg / L, respectively, and final concentrations of 2 mg / L and 0.01 mg / L, respectively.
  • Raihimani preservation medium Nisui
  • ⁇ 1 x artificial seawater composition > NaCl 30 g KCl 0.7 g MgCl 2 ⁇ 6H 2 O 10.8 g MgSO 4 ⁇ 7H 2 O 5.4 g CaCl 2 ⁇ 2H 2 O 1 g 1000 ml of ultrapure water
  • Example 2 Molecular phylogenetic analysis of sterol-accumulating strains
  • the 18S rDNA region of the 18S rDNA region was amplified using the universal primer for 18S rDNA region amplification (SEQ ID NOs: 1 and 2) of Labyrinthula.
  • SEQ ID NOs: 1 and 2 the universal primer for 18S rDNA region amplification
  • Molecular phylogenetic analysis was performed using the base sequence as an index.
  • the nucleotide sequence of the 18S rDNA region of the AJ7867 strain determined is shown in SEQ ID NO: 3.
  • each node indicates the support rate by 1000 bootstrap re-sampling.
  • the AJ7867 strain was related to the Aurantiochytrium genus, so the strain was named Aurantiochytrium sp. AJ7867.
  • Example 3 Sterol production culture
  • the sterol production ability of the AJ7867 strain was compared using the Thraustochytrium sp. ATCC26185 strain, which is an existing sterol production strain of Labyrinthula, as a control strain.
  • Vitamin B1 (Nacalai) with AJ7867 strain and ATCC26185 strain in 0.2 ⁇ Raihimani preservation medium (Nissui) prepared with 0.5 ⁇ artificial seawater so that the final concentrations are 2 mg / L, 0.01 mg / L and 0.01 mg / L, respectively.
  • the cells on the plate medium were scraped into 6 platinum ears, inoculated into a 300 ml baffled Erlenmeyer flask containing 30 ⁇ mL of GPY medium prepared with 0.25 x artificial seawater of the following composition, culture temperature 25 ° C, stirring 120 rpm (Rotary), and cultured for 24 hours. 5 ml of the obtained culture solution is inoculated into a 500 ml baffled Erlenmeyer flask containing 45 ml of GTY medium prepared with 0.25 x artificial seawater, and cultured for 51 hours at a culture temperature of 25 ° C and stirring of 120 rpm (rotary). It was.
  • GPY medium ⁇ Composition of GPY medium> (A ward) Glucose 20 g / L (B ward) Polypeptone 10 g / L Yeast extract 5 g / L (C ward) Vitamin B1 2 mg / L Vitamin B2 0.01 mg / L Vitamin B12 0.01 mg / L (District D) MES 8.79 g / L
  • the B section was adjusted to pH 6.0 using HCl, and then the A section was autoclaved at 120 ° C. for 10 minutes with no pH adjustment. In the D section, pH was adjusted to 6.0 with NaOH, and then in the C section, the pH was not adjusted and the filter filtration was performed. After the A and B sections were cooled to room temperature, the 4 sections were mixed.
  • ⁇ Composition of GTY medium> (A ward) Glucose 50 g / L (B ward) Tryptone 10 g / L Yeast extract 5 g / L (C ward) Vitamin B1 2 mg / L Vitamin B2 0.01 mg / L Vitamin B12 0.01 mg / L (District D) MES 8.79 g / L
  • the B section was adjusted to pH 6.0 using HCl, and then the A section was autoclaved at 120 ° C. for 10 minutes with no pH adjustment. In the D section, pH was adjusted to 6.0 with NaOH, and then in the C section, the pH was not adjusted and the filter filtration was performed. After the A and B sections were cooled to room temperature, the 4 sections were mixed.
  • lipid was extracted from the obtained precipitate using the Bligh-Dyer method to obtain a lipid extract.
  • a fatty acid methylation kit Nacalai
  • the obtained hexane layer was applied to gas chromatography to quantify each sterol.
  • the dry algal body weight (DCW) was calculated by centrifuging 0.5 ml of the culture solution and drying the obtained precipitate at 50 ° C. for 3 days, and then subtracting the weight of the Eppendorf tube from the weight measurement value.
  • the present invention provides algae that produce sterols. Sterols can be produced using the algae.

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Abstract

Cette invention concerne des algues qui génèrent un stérol et un procédé de production de stérol les utilisant. Selon la présente invention, le stérol est produit par culture d'algues, telles que la souche AJ7867 (FERM BP-22304) et les souches dérivées de celles-ci, qui a la capacité de générer du stérol et collecte du stérol à partir de la masse algale ainsi obtenue.
PCT/JP2017/008586 2016-03-03 2017-03-03 Procédé de production de stérol WO2017150723A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019078359A1 (fr) * 2017-10-19 2019-04-25 味の素株式会社 Procédé de production de stérol

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060160203A1 (en) * 1992-10-16 2006-07-20 Martek Biosciences Corporation Process for the heterotrophic production of microbial products with high concentrations of omega-3 highly unsaturated fatty acids
JP2017073998A (ja) * 2015-10-14 2017-04-20 味の素株式会社 ステロールの製造法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060160203A1 (en) * 1992-10-16 2006-07-20 Martek Biosciences Corporation Process for the heterotrophic production of microbial products with high concentrations of omega-3 highly unsaturated fatty acids
JP2017073998A (ja) * 2015-10-14 2017-04-20 味の素株式会社 ステロールの製造法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
NAKAZAWA, ATSUSHI ET AL.: "TLC screening of thraustochytrid strains for squalene production", JOURNAL OF APPLIED PHYCOLOGY, vol. 26, 2014, pages 29 - 41, XP055374842 *
WANG, G.: "Characterization of Lipid Components in Two Microalgae for Biofuel Application", J OURNAL OF THE AMERICAN OIL CHEMISTS' SOCIETY, vol. 89, no. 1, January 2012 (2012-01-01), pages 135 - 143, XP019995521 *
WEETE, J. D. ET AL.: "Lipids and Ultrastructure of Thraustochytrium sp. ATCC 26185", LIPIDS, vol. 32, no. 8, 1997, pages 839 - 845, XP009077836 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019078359A1 (fr) * 2017-10-19 2019-04-25 味の素株式会社 Procédé de production de stérol
JPWO2019078359A1 (ja) * 2017-10-19 2021-01-07 味の素株式会社 ステロールの製造法
JP7205480B2 (ja) 2017-10-19 2023-01-17 味の素株式会社 ステロールの製造法

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