WO2023167322A1 - Method for culturing alga belonging to cyanidiophyceae - Google Patents

Method for culturing alga belonging to cyanidiophyceae Download PDF

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WO2023167322A1
WO2023167322A1 PCT/JP2023/008132 JP2023008132W WO2023167322A1 WO 2023167322 A1 WO2023167322 A1 WO 2023167322A1 JP 2023008132 W JP2023008132 W JP 2023008132W WO 2023167322 A1 WO2023167322 A1 WO 2023167322A1
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glycogen
algae belonging
medium
culture
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PCT/JP2023/008132
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French (fr)
Japanese (ja)
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稔康 岨
千尋 堤端
進也 宮城島
崇之 藤原
俊亮 廣岡
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Ｅｎｅｏｓ株式会社
大学共同利用機関法人情報・システム研究機構
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Publication of WO2023167322A1 publication Critical patent/WO2023167322A1/en

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    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds

Definitions

  • the present invention relates to a method for culturing algae belonging to the class Idyucogome or a method for producing glycogen by culturing algae belonging to the class Idyucogome.
  • Algae (red algae) belonging to the class Idyucogome are unicellular algae that grow in sulfuric acid hot springs.
  • Patent Document 1 describes a medium composition for producing lipids while maintaining the growth of Cyanidial red algae, and a method for producing lipids using the medium composition.
  • the medium composition used to produce lipids contains sufficient ammonium salts such as ammonium sulfate, contains sodium salts such as sodium chloride, and further contains inorganic acids such as phosphoric acid. It is described to be a strongly acidic medium composition.
  • Patent Document 1 does not examine culture conditions for the purpose of producing glycogen (polysaccharide).
  • living cells contain various molecules such as proteins, amino acids, sugars, lipids, phosphoric acid, sodium, and potassium, and generally have a higher solution concentration than the surrounding environment. Therefore, water tends to diffuse into the cells, and the amount of water necessary for metabolism and growth is kept at an appropriate level.
  • water containing sodium ions and potassium ions in the cells leaks out of the cells. Since these ions are necessary for cell function, it is known that cells become difficult to proliferate and in some cases lead to cell death (Non-Patent Document 1). Therefore, in culturing microorganisms, increasing the concentration of saccharides in the medium at the initial stage of culture is not usually practiced, or even if it is practiced, there are many problems with cell growth.
  • lipids are produced from algae by using the medium composition, but a growth rate comparable to that of normal medium is not achieved.
  • the purpose of the present invention is to provide a method for culturing algae belonging to the class Idyucogome and a method for causing the algae belonging to the class Idyucogome to produce glycogen.
  • the present inventor has conducted extensive research and discovered a method for culturing algae belonging to the class Idyucogome and a method for producing glycogen from algae belonging to the class Idyucogome. That is, the present invention is as follows.
  • a method for culturing algae belonging to the class Idyucogome comprising culturing the algae belonging to the class Idyucogome in a medium containing 5 to 40% by weight of a carbon source.
  • the carbon source is a monosaccharide, disaccharide or sugar alcohol.
  • the method of [1] or [2], wherein the culture is batch culture.
  • [5] A method for producing glycogen by the culture method according to any one of [1] to [3].
  • [6] The method of [5], further comprising recovering glycogen from the culture.
  • [7] The method according to [5] or [6], wherein the amount of glycogen is 10 mg/g or more relative to the algae belonging to the class Idycogome in the culture.
  • a method for producing glycogen in algae belonging to the class Idyucogome comprising culturing the algae belonging to the class Idyucogome in a medium containing a carbon source of 5 to 40% by weight.
  • the present invention provides a method for culturing algae belonging to the class Idycogome.
  • the culturing method of the present invention increases the amount of algal bodies of algae belonging to the class Idyunicogome in the culture, and improves the efficiency of culturing the algae belonging to the class Idyuteicogome.
  • the culture method of the present invention increases the amount of glycogen produced by algae belonging to the class Idycogome.
  • the present invention also provides a method for producing glycogen. Therefore, according to the present invention, the efficiency of glycogen production by algae belonging to the class Idycogome is improved.
  • a dried culture of algae belonging to the class Idycogome Since the dried matter contains a large amount of glycogen, the present invention enables efficient production of glycogen that can be used as a raw material for biomass fuel derived from algae.
  • the present invention relates to a method for culturing algae belonging to the class Cyanidiophyceae or a method for causing algae belonging to the class Cyanidiophyceae to produce glycogen.
  • the algae culture efficiency is surprisingly increased, We found that it efficiently produced glycogen.
  • a medium further containing 0.05 to 10 mM iron ions In order to produce glycogen with high efficiency, it is preferable to use a medium further containing 0.05 to 10 mM iron ions.
  • the class Cyanidiophyceae is taxonomically classified as Cyanidiophyceae in the phylum Rhodophyta, and is further classified into three genera: the genus Cyanidioschyzon, the genus Cyanidium, and the genus Galdieria. be.
  • algae belonging to any of the genus Cyanidioschyzon, Cyanidium and Galdieria may be used as the algae belonging to the class Cyanidioschyzon.
  • algae belonging to the genus Galdieria are preferably algae belonging to the genus Galdieria.
  • algae belonging to the genus Cyanidioschizon such as Cyanidioschyzon merolae
  • algae belonging to the genus Cyanidium such as Cyanidium caldarium, Galdieria sulphuraria, etc.
  • algae belonging to the genus Galderia or variants thereof, or combinations thereof.
  • algae belonging to the class Idycogome may have a haploid cell form or a diploid cell form.
  • strains of algae belonging to the class Idycogome to be used are not limited.
  • G 127 diploid, having a strong cell wall, available from CCCryo
  • G 108 (1n) diploid described in JP 2020-072698
  • the haploid is induced according to the method of , and its single culture strain is prepared and used.It does not have a strong cell wall. Diploids of G 127 and G 108, respectively, were obtained from public institutions (e.g.
  • G127 CCCryo 127-00 is CCCryo (Culture Collection of Cryophilic Algae)
  • G108 SAG 108.79
  • SAG The Culture Collection of Algae at readily available from Gottingen University
  • a mutant strain can be easily obtained by a person skilled in the art by subjecting the strain to mutation treatment.
  • Mutation treatment includes, for example, treatment with a drug having a mutagenic action or high-energy beam irradiation treatment.
  • Agents with mutagenic activity include base analogs such as, for example, ethyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine and 5-bromouracil.
  • High energy rays also include UV, gamma rays, X-rays and heavy ion beams.
  • Mutation treatment also includes a method of mutating a specific gene using a gene recombination method. Mutants also include transformants.
  • Algae belonging to the class Ideucogome may be collected from high-temperature, high-sulfur, low-pH environments, such as sulfate springs, according to previous reports (De Luca P. et al., 1978, Webbia, 33, 37-44). Algae belonging to the class Idyucogome can be maintained and cultured in Allen medium or modified Allen (MA) medium (Allen, M. B., 1959, Arch. Mikrobiol., 32, 270-277; Kuroiwa, T. et al. ., 1993, Protoplasma, 175, 173-177; Ohnuma M. et al., 2008, Plant Cell Physiol., 117-120; Kuroiwa T. et al., 2012, Cytologia, 77(3), 289-299) .
  • Allen medium or modified Allen (MA) medium Allen, M. B., 1959, Arch. Mikrobiol., 32, 270-277; Kuroiw
  • the present invention includes a medium for culturing algae belonging to the class Idyucogome or a medium for making algae belonging to the class Idyucogome produce glycogen.
  • the medium for culturing algae belonging to the class Idyucogome of the present invention or the medium for making the algae belonging to the class Idyucogome to produce glycogen is characterized by containing 5 to 40% by weight of a carbon source.
  • the carbon source can be contained in a basal medium commonly used for culturing algae belonging to the class Idycogome.
  • Medium containing 5-40% by weight carbon source may further contain 0.07-10 mM iron ions.
  • a medium containing a carbon source can be used for culturing algae belonging to the class Idycogome, and can also be used to improve the efficiency of culturing algae belonging to the class Idycogome.
  • the present invention includes a method for culturing algae belonging to the class Idycogome or a method for improving the culture efficiency, which comprises culturing the algae belonging to the class Idycogome in a medium containing 5 to 40% by weight of a carbon source.
  • the efficiency of culturing algae belonging to the class Idyucogome can be evaluated based on conventionally known cell growth evaluation methods such as the turbidity method and dry alga body weight.
  • the turbidity method the turbidity (optical density) at a wavelength of 750 nm of a culture sampled from a culture vessel is measured using a spectrophotometer or the like.
  • a dilution series may be prepared with water or medium to dilute the culture to a concentration suitable for measurement.
  • the weight of the dried algal body can be measured by drying a certain amount of the culture collected from the culture vessel and then measuring the weight.
  • Algae belonging to the class Idycogome store glycogen in the cytosol as storage polysaccharides.
  • the amount of algal bodies in the culture is increased, and a large amount of the culture containing algal bodies in which glycogen is accumulated is obtained. can get to Therefore, by culturing algae belonging to the class Idycogome using the medium of the present invention, it is possible to increase the amount of glycogen produced by the algae belonging to the class Idycogome.
  • the medium of the present invention containing 5 to 40% by weight of carbon source can also be used as a medium for producing glycogen in algae belonging to the class Idycogome.
  • a method for producing glycogen in algae belonging to the class Idyucogome, or a method for improving the production efficiency of glycogen, comprising culturing algae belonging to the class Idyucogome in a medium containing 5 to 40% by weight of a carbon source is included in the present invention.
  • the amount of glycogen produced can be evaluated by the amount of glycogen contained in algae belonging to the class Idyucogome in the culture.
  • the amount of glycogen contained in algae belonging to the class Idyucogome can be determined by extracting glycogen from cultured alga bodies and measuring the amount of extracted glycogen. For example, after drying a certain amount of culture material collected from a culture vessel, water is added to the dried material (dried algal body), mixed with a mixer, heat-treated and centrifuged, and the resulting supernatant is extracted with glycogen. It can be liquid.
  • the present invention also includes dried cultures of algae belonging to the class Idycogome obtained by the culture method of the present invention or the method of producing glycogen of the present invention.
  • glycogen extract from cultured algae belonging to the class Idycogome by making appropriate modifications.
  • the amount of glycogen contained in the glycogen extract can be quantified using a commercially available kit. Upon quantification, a dilution series may be prepared with water or a solvent to dilute the glycogen extract to a concentration suitable for measurement. Also, the amount of glycogen contained in the glycogen extract may be quantified by chromatography such as HPLC.
  • Algae belonging to the class Cocogoma before being cultured in the medium of the present invention or algae belonging to the class Cocogoma that have been cultured in a medium other than the medium of the present invention, such as a basal medium, can be used as a control.
  • the medium of the present invention containing 5-40% by weight of carbon source may further contain 0.05-10 mM iron ions.
  • a medium containing a carbon source and iron ions can be used for culturing algae belonging to the class Idycogome, or can be used to improve the efficiency of culturing algae belonging to the class Idycogome.
  • a method for culturing algae belonging to the class Idycogome comprising culturing algae belonging to the class Idycogome, in a medium containing 5 to 40% by weight of a carbon source and 0.05 to 10 mM iron ions, or a method for improving the culture efficiency, Included in the present invention.
  • Evaluation of the efficiency of culturing algae belonging to the class Idycogome when using the medium of the present invention containing 5 to 40% by weight of carbon source and 0.05 to 10 mM iron ions can be performed in the same manner as described above.
  • the amount of algae in the culture is increased and glycogen is accumulated. It is possible to obtain a large amount of culture containing the treated algae.
  • the glycogen concentration in the alga body is increased, and the culture containing the alga body in which a large amount of glycogen is accumulated. can be obtained.
  • the amount of algae in the culture is increased, and the glycogen concentration in the algae is is increased, and a large amount of culture containing algal bodies in which a large amount of glycogen is accumulated can be obtained.
  • the amount of glycogen produced by the algae belonging to the class Idycogome can be increased.
  • a medium containing 5 to 40% by weight of a carbon source and 0.05 to 10 mM of iron ions is a medium for producing glycogen in algae belonging to the class Idycogome or improving the production efficiency of glycogen by algae belonging to the class Idyucogome. It can also be used as a medium for A method for producing glycogen in algae belonging to the class Idycogome, comprising culturing algae belonging to the class Idycogome in a medium containing 5 to 40% by weight of a carbon source and 0.05 to 10 mM iron ions, or improving the production efficiency of glycogen. Methods of remediation are included in the present invention.
  • any sugar that can be used by algae belonging to the class Idycogome can be used as a carbon source.
  • monosaccharides such as glucose, fructose, galactose and mannose
  • Sugar alcohols such as glycerol
  • preferred carbon sources are monosaccharides or disaccharides, more preferably glucose or sucrose.
  • the carbon source is glucose.
  • the carbon source is sucrose.
  • the carbon source can be contained in the basal medium at a concentration of 5-40% by weight.
  • the carbon source concentration can vary within the range of 5-40% by weight.
  • the concentration of the carbon source in the medium is, for example, 7 to 40% by weight, 7 to 35% by weight, 8 to 35% by weight, 9 to 35% by weight, 10 to 35% by weight, 15 to 35% by weight.
  • any iron ion source that produces iron ions in a medium and is commonly used for culturing microorganisms can be used .
  • ) 3 , FeCl 2 , FeCl 3 or their hydrates or combinations thereof can be used.
  • the source of iron ions is FeSO4 heptahydrate ( FeSO4.7H2O ).
  • the source of iron ions is FeCl 3 hexahydrate (FeCl 3 .6H 2 O).
  • iron ions may be divalent or trivalent. Also, the number of hydrates is not particularly limited.
  • iron ions can be contained in the basal medium at a concentration of 0.05 to 10 mM.
  • concentration of iron ions can vary within the range of 0.05-10 mM, such as within the range of 0.07-10 mM.
  • the concentration of iron ions in the medium is, for example, 0.07 to 10 mM, 0.1 to 10 mM, 0.15 to 10 mM, 0.2 to 10 mM, 0.25 to 10 mM, 0.3 to 10 mM.
  • 0.4-10 mM 0.5-10 mM, 0.07-9 mM, 0.1-9 mM, 0.12-9 mM, 0.15-9 mM, 0.2-9 mM, 0.25-9 mM, 0 .3-9 mM, 0.4-9 mM, 0.5-9 mM, 0.07-8 mM, 0.1-8 mM, 0.12-8 mM, 0.15-8 mM, 0.2-8 mM, 0.25 ⁇ 8mM, 0.3-8mM, 0.4-8mM, 0.5-8mM, 0.07-7mM, 0.1-7mM, 0.12-7mM, 0.15-7mM, 0.2-7mM , 0.25-7 mM, 0.3-7 mM, 0.4-7 mM, 0.5-7 mM, 0.07-6 mM, 0.1-6 mM, 0.12-6 mM, 0.15-6 mM, 0 .2-6 mM, 0.25-6 mM, 0.3-6 mM, 0.4-6 mM, 0.
  • the basal medium can be used without particular limitation as long as it is a medium known to be capable of culturing algae belonging to the class Idyucogome.
  • examples of the basal medium include Allen medium or modified Allen medium (Allen, M. B., 1959, Arch. Mikrobiol., 32, 270-277; Kuroiwa, T. et al., 1993, Protoplasma, 175, 173-177; Ohnuma M. et al., 2008, Plant Cell Physiol., 117-120; Kuroiwa T. et al., 2012, Cytologia, 77(3), 289-299). , but not limited to.
  • the basal medium is preferably liquid medium.
  • the medium contains, in addition to the above components, known additives such as trace metal elements such as selenium, antibiotics such as penicillin, streptomycin and gentamicin, and pH indicators such as phenol red and bromophenol blue.
  • the carbon source or carbon source and iron ions are contained in the basal medium and the algae belonging to the class Idyucogome are seeded, it is desirable not to supply the medium into the culture system. That is, in one aspect of the present invention, necessary components in the medium, such as nutrients, are not continuously or intermittently supplied into the system during cultivation.
  • batch culture can be carried out in which all necessary nutrients, carbon sources and optionally iron ions are contained in the medium at the start of the culture. Dissolved oxygen may be adjusted.
  • the present invention relates to a method for culturing algae belonging to the class Idycogome using the medium described above. Specifically, the present invention relates to a method for culturing algae belonging to the class Idyllicogome, comprising culturing the algae belonging to the class Idyllicogome in a medium containing 5 to 40% by weight of a carbon source. The cultured algae belonging to the class Idycogome can be recovered. In addition, by culturing algae belonging to the class Idyunicogome by the culture method of the present invention, it is possible to cause the algae belonging to the class Idyuteicogome to produce glycogen.
  • the present invention also relates to a method for producing glycogen in algae belonging to the class Idycogome using the medium described above. Specifically, the present invention relates to a method for producing glycogen in algae belonging to the class Idyucogome, comprising culturing the algae belonging to the class Idyucogome in a medium containing 5 to 40% by weight of a carbon source. The method for producing glycogen may further include a step of recovering glycogen derived from the cultured algae belonging to the class Idycogome.
  • the medium may further contain 0.07 to 10 mM iron ions.
  • the concentration of the carbon source e.g., monosaccharides, disaccharides or sugar alcohols
  • concentration of iron ions are appropriately selected from the concentration ranges described herein. , can be combined.
  • the medium in the present invention is: A medium containing 5-40 wt% carbon source and 0.07-10 mM iron ions; A medium containing 5-30 wt% carbon source and 0.15-5 mM iron ions; A medium containing 7-25% by weight carbon source and 0.15-2 mM iron ions; A medium containing 7-18% by weight of carbon source and 0.3-1 mM iron ions; A medium containing 7-10% by weight of carbon source and 0.3-1 mM of iron ions; A medium containing 7-9% by weight of carbon source and 0.3-1 mM of iron ions; A medium containing 8-10% by weight of a carbon source and 0.3-1 mMmM of iron ions; A medium containing 7-18% by weight of carbon source and 0.3-0.9 mM of iron ions; A medium containing 7-10 wt% carbon source and 0.3-0.9 mMmM iron ion; A medium containing 7-9% by weight of carbon source and 0.3-0.9 mM of iron ions; and
  • the culture conditions for algae belonging to the class Idyucogome may be any method as long as the algae belonging to the class Idyucogome grow, and those skilled in the art can appropriately select and change them from known methods. The description can also be referred to as appropriate.
  • the temperature for culturing algae belonging to the class Idycogome can be 37°C to 50°C, and may be 40°C to 45°C. Both aerobic conditions and anaerobic conditions can be used as ventilation conditions.
  • the light irradiation condition both the light condition and the dark condition can be used.
  • a culture tank commonly used for culturing algae can be used.
  • test tube a test tube, a Sakaguchi flask, an Erlenmeyer flask, a round pond, a raceway pond, a column type culture tank, a flat panel type culture tank, a tube type culture tank, a tank such as a jar fermenter, etc. can be used, but these are limited. It is not.
  • the culture period can also be appropriately set by those skilled in the art.
  • the culture period for algae belonging to the class Idycogome can be 1 day to 6 months, 1 day to 3 months, 1 day to 2 months, 1 day to 4 weeks, 1 to 4 weeks, for example 1 to 2 weeks. can.
  • a person skilled in the art can appropriately select a method for collecting cultured algae belonging to the class Idyunicogome or a method for collecting glycogen derived from cultured algae belonging to the class Idyunicogome.
  • the cultured algae belonging to the class Idyucogome contain glycogen in their cells
  • the culture containing the algal body may be used as it is as the glycogen derived from the algae belonging to the class Idyucogome.
  • a concentrate obtained by concentrating the culture by centrifugation, filtration, or the like may be used as glycogen derived from algae belonging to the class Idycogome.
  • the dried product obtained by drying the culture, concentrate, or crushed product may be used as glycogen derived from algae belonging to the class Idycogome. Drying may be performed by any method such as freeze drying, heat drying, spray drying, and vacuum drying, but freeze drying is preferred.
  • the dried matter may be processed to extract or purify glycogen.
  • the culture, concentrate, or dried product may be subjected to treatments such as heating, irradiation with ultraviolet rays and/or radiation, and pulverization using a mill or glass beads.
  • the method for producing glycogen in the present invention may include the above-described step of recovering glycogen.
  • the present invention also includes cultures, concentrates, dried products, or processed products obtained by the culture method or glycogen production method of the present invention.
  • the culture method of the present invention achieves an alga body amount of algae belonging to the class Idycogome in the culture solution (per culture solution) of about 10 g/L or more, about 15 g/L or more, or about 20 g/L or more. be done.
  • the glycogen production method of the present invention provides about 10 mg/g or more, about 15 mg/g or more, about 20 mg/g or more, about 30 mg/g or more, about 40 mg/g or more in the culture (algal body) (per culture) glycogen concentration is achieved.
  • Garuderia was placed in a test tube and cultured under the following conditions. Galderia was used as deposited at the Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalytics and Bioprocesses IZI-BB under strain number CCCryo 127-00 Galdieria sulphuraria (Galdieri) Merola 1982.
  • Experiment B used 1 ⁇ MA medium.
  • a 10 ⁇ MA(-) medium supplemented with 1 to 100 volumes of Fe (0.0752 mM EDTA.2Na and 0.0592 mM FeSO.sub.4.7H.sub.2O ) was used.
  • the 10 ⁇ MA(-) medium is a medium containing medium components at ten times the concentration of 1 ⁇ MA, but not containing EDTA.2Na and FeSO.sub.4.7H.sub.2O .
  • Amount of algae in culture medium (g/L) (Amount of algae/culture medium) 1 mL of the culture solution was dispensed into a 1.5 mL microtube and centrifuged (TOMY, high-speed microcentrifuge MX-307) (15000 ⁇ g, 10 minutes). After removing the supernatant, 1 mL of 1 ⁇ MA medium was added to the microtube and suspended for 5 minutes with a direct mixer (manufactured by AS ONE, Direct Mixer DM-301). Centrifugation was performed again and the supernatant was removed (washing). The washing operation was performed once more, and the obtained alga bodies were frozen at -20°C. It was dried for several hours with a freeze dryer (manufactured by EYELA, freeze dryer FDU-1200 model) and weighed (g/L).
  • Amount of glycogen in alga (mg/L) (amount of glycogen/culture solution)
  • Glycogen extraction was performed on a sample obtained by freezing alga bodies in the same manner as the amount (g/L) of alga bodies in the culture medium. 400 ⁇ L of ion-exchanged water was added to the frozen algal body, mixed with a direct mixer, and heated for 10 minutes in a 95° C. aluminum block (Aluminum block constant temperature bath CB-100A, manufactured by AS ONE). After centrifugation at 15000 ⁇ g for 10 minutes, the resulting supernatant was used as a glycogen extract.
  • a commercially available kit was used for glycogen quantification (Glycogen Assay Kit manufactured by BioVision). The extract was serially diluted 10- to 1000-fold, and the diluted solution with an appropriate concentration (mg/L) was used for the measurement. Data on glycogen content are not suitable for comparison between experimental series.
  • Amount of glycogen in alga body/Amount of alga body (mg/g) Glycogen concentration in alga body Amount of glycogen in alga body (mg/L) obtained in (3) (amount of glycogen/culture solution) was divided by the algal mass (g/L) in the culture solution obtained in (2) (alga mass/culture solution). The obtained value means the glycogen concentration in the algal body.
  • the amount of algal bodies in the culture solution increased when the culture was performed at a concentration of glucose, which is a carbon source, of 5.0% or higher.
  • a concentration of glucose which is a carbon source
  • the amount of algae in the culture solution exceeded 20 g/L, and the increase in the amount of algae in the culture solution was remarkable.
  • the amount of algal bodies in the culture solution was large.
  • the amount of glycogen per algal mass increased when the algae were cultured at a glucose concentration of 5.0% or more, which is a carbon source.
  • Experiment B had similar OD750 values when cultured using glucose (Example 7) or sucrose (Example 8) as the carbon source. This result indicates that algae belonging to the class Idycogome grew to the same extent in Examples 7 and 8. It was also shown that the use of glycerol (9%) as a carbon source maintained the growth of algae belonging to the class Idycogome. The results of Experiment B indicated that the effect of the carbon source on the growth efficiency of algae belonging to the class Idyucogome is considered to be small, if at all, due to differences in the carbon source.
  • the amount of glycogen in the alga per unit amount of alga increased when the culture was carried out at an iron ion concentration of 0.0592 mM or higher in the medium.
  • the amount of glycogen increased significantly.
  • the results of Experiment C showed that culturing in a medium containing an iron ion concentration of 0.0592 mM or more can improve the glycogen production efficiency of algae belonging to the class Idycogome.
  • the present invention provides a method for culturing algae belonging to the class Idycogome.
  • the culturing method of the present invention increases the amount of algal bodies of algae belonging to the class Idyunicogome and improves the efficiency of culturing the algae belonging to the class Idyuteicogome.
  • the culture method of the present invention increases glycogen production.
  • the present invention also provides a method for producing glycogen. Therefore, according to the present invention, the efficiency of glycogen production by algae belonging to the class Idycogome is improved.
  • a dried culture of algae belonging to the class Idycogome Since the dried matter contains a large amount of glycogen, the present invention enables efficient production of glycogen that can be used as a raw material for biomass fuel derived from algae.

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Abstract

The purpose of the present invention is to provide a method for culturing an alga belonging to Cyanidiophyceae and a method for producing glycogen. The present invention provides a method for culturing an alga belonging to Cyanidiophyceae or a method for producing glycogen, each method comprising culturing an alga belonging to Cyanidiophyceae in a medium that contains a preset concentration of a carbon source optionally together with a preset concentration of iron ions.

Description

イデユコゴメ綱に属する藻類の培養方法Method for culturing algae belonging to the class Idyucogome
　本発明は、イデユコゴメ綱に属する藻類の培養方法又はイデユコゴメ綱に属する藻類を培養してグリコーゲンを生産する方法に関する。 The present invention relates to a method for culturing algae belonging to the class Idyucogome or a method for producing glycogen by culturing algae belonging to the class Idyucogome.
　イデユコゴメ綱に属する藻類（紅藻）は、硫酸酸性温泉に生育する単細胞性の藻類である。 Algae (red algae) belonging to the class Idyucogome are unicellular algae that grow in sulfuric acid hot springs.
　特許文献１には、紅藻シアニジウム目の増殖を維持しながら、脂質を生産させるための培地組成物、及び当該培地組成物を用いる脂質の生産方法が記載される。特許文献１には、脂質を生産するために用いられる培地組成物は、硫酸アンモニウム等のアンモニウム塩を十分に含み、塩化ナトリウム等のナトリウム塩を含み、さらにリン酸等の無機酸の含量を調節した強酸性の培地組成物であることが記載されている。一方で、特許文献１では、グリコーゲン（多糖類）の生産を目的とする培養条件の検討は行われていない。 Patent Document 1 describes a medium composition for producing lipids while maintaining the growth of Cyanidial red algae, and a method for producing lipids using the medium composition. In Patent Document 1, the medium composition used to produce lipids contains sufficient ammonium salts such as ammonium sulfate, contains sodium salts such as sodium chloride, and further contains inorganic acids such as phosphoric acid. It is described to be a strongly acidic medium composition. On the other hand, Patent Document 1 does not examine culture conditions for the purpose of producing glycogen (polysaccharide).
　ところで、生物の細胞（細胞質）はタンパク質、アミノ酸、糖類、脂質、リン酸、ナトリウム、カリウムなどの様々な分子を含み、一般的に細胞周囲の環境よりも高い溶液濃度を有する。そのため、水は細胞の中へと拡散する傾向があり、代謝・増殖などに必要な水分量が適正に保たれる。しかし、微生物の培養において、糖類等を高濃度で含む培地中で細胞を培養すると、細胞中のナトリウムイオンやカリウムイオンを含んだ水分が細胞外に流出してしまう。これらのイオンは細胞機能に必要であることから、細胞は増殖が困難になり、場合よっては細胞死に至ることが知られている（非特許文献１）。したがって、微生物の培養においては培養初期に培地中の糖類の濃度を高くすることは通常行われないか、仮に行われたとしても細胞の増殖に問題があることが多い。 By the way, living cells (cytoplasm) contain various molecules such as proteins, amino acids, sugars, lipids, phosphoric acid, sodium, and potassium, and generally have a higher solution concentration than the surrounding environment. Therefore, water tends to diffuse into the cells, and the amount of water necessary for metabolism and growth is kept at an appropriate level. However, in culturing microorganisms, when cells are cultured in a medium containing a high concentration of sugars and the like, water containing sodium ions and potassium ions in the cells leaks out of the cells. Since these ions are necessary for cell function, it is known that cells become difficult to proliferate and in some cases lead to cell death (Non-Patent Document 1). Therefore, in culturing microorganisms, increasing the concentration of saccharides in the medium at the initial stage of culture is not usually practiced, or even if it is practiced, there are many problems with cell growth.
　特許文献１においては、培地組成物を用いることにより藻類から脂質が生産されるが、通常の培地程度の増殖速度は達成されていない。 In Patent Document 1, lipids are produced from algae by using the medium composition, but a growth rate comparable to that of normal medium is not achieved.
特開２０１５－１９２５９８JP 2015-192598
　本発明は、イデユコゴメ綱に属する藻類の培養方法及びイデユコゴメ綱に属する藻類にグリコーゲンを生産させる方法の提供を目的とする。 The purpose of the present invention is to provide a method for culturing algae belonging to the class Idyucogome and a method for causing the algae belonging to the class Idyucogome to produce glycogen.
　本発明者は、鋭意研究を行い、イデユコゴメ綱に属する藻類の培養方法及びイデユコゴメ綱に属する藻類からグリコーゲンを生産する方法を見出した。すなわち、本発明は以下のとおりである。
［１］
　イデユコゴメ綱に属する藻類を、５～４０重量％の炭素源を含む培地で培養することを含む、イデユコゴメ綱に属する藻類を培養する方法。
［２］
　前記炭素源が、単糖類、二糖類又は糖アルコールである、［１］に記載の方法。
［３］
　培養が回分培養である、［１］又は［２］に記載の方法。
［４］
　培地が０．０７～１０ｍＭの鉄イオンをさらに含む、［１］～［３］のいずれか一項記載の方法。
［５］
　［１］～［３］いずれか一項記載の培養方法によりグリコーゲンを生産する方法。
［６］
　培養物からグリコーゲンを回収することをさらに含む、［５］に記載の方法。
［７］
　グリコーゲンが、培養物中のイデユコゴメ綱に属する藻類に対し１０ｍｇ／ｇ以上含まれる、［５］又は［６］に記載の方法。
［８］
　［１］～［７］のいずれか一項の方法で得られるイデユコゴメ綱に属する藻類の培養物を乾燥した、イデユコゴメ綱に属する藻類の乾燥物。
［９］
　イデユコゴメ綱に属する藻類を、５～４０重量％の炭素源を含む培地で培養することを含む、イデユコゴメ綱に属する藻類にグリコーゲンを生産させる方法。
［１０］
　前記炭素源が、単糖類、二糖類又は糖アルコールである、［９］に記載の方法。
［１１］
　培地が０．０７～１０ｍＭの鉄イオンをさらに含む、［９］又は［１０］に記載の方法。
［１２］
　グリコーゲンが、培養物中のイデユコゴメ綱に属する藻類に対し１０ｍｇ／ｇ以上含まれる、［９］～［１１］のいずれか一項に記載の方法。
［１３］
　５～４０重量％の炭素源を含む、イデユコゴメ綱に属する藻類を培養するための培地。
［１４］
　０．０７～１０ｍＭの鉄イオンをさらに含む、［１３］に記載の培地。 The present inventor has conducted extensive research and discovered a method for culturing algae belonging to the class Idyucogome and a method for producing glycogen from algae belonging to the class Idyucogome. That is, the present invention is as follows.
[1]
A method for culturing algae belonging to the class Idyucogome, comprising culturing the algae belonging to the class Idyucogome in a medium containing 5 to 40% by weight of a carbon source.
[2]
The method according to [1], wherein the carbon source is a monosaccharide, disaccharide or sugar alcohol.
[3]
The method of [1] or [2], wherein the culture is batch culture.
[4]
The method according to any one of [1] to [3], wherein the medium further contains 0.07 to 10 mM iron ions.
[5]
A method for producing glycogen by the culture method according to any one of [1] to [3].
[6]
The method of [5], further comprising recovering glycogen from the culture.
[7]
The method according to [5] or [6], wherein the amount of glycogen is 10 mg/g or more relative to the algae belonging to the class Idycogome in the culture.
[8]
A dried product of algae belonging to the class Idyllicogome, obtained by drying the culture of algae belonging to the class Idyllicogome obtained by the method according to any one of [1] to [7].
[9]
A method for producing glycogen in algae belonging to the class Idyucogome, comprising culturing the algae belonging to the class Idyucogome in a medium containing a carbon source of 5 to 40% by weight.
[10]
The method of [9], wherein the carbon source is a monosaccharide, disaccharide or sugar alcohol.
[11]
The method of [9] or [10], wherein the medium further contains 0.07-10 mM iron ions.
[12]
The method according to any one of [9] to [11], wherein the amount of glycogen is 10 mg/g or more for algae belonging to the class Ideucogome in the culture.
[13]
A medium for culturing algae belonging to the class Idycogome, containing 5 to 40% by weight of a carbon source.
[14]
The medium of [13], further comprising 0.07-10 mM iron ions.
　本発明により、イデユコゴメ綱に属する藻類の培養方法が提供される。本発明の培養方法により、培養物中のイデユコゴメ綱に属する藻類の藻体量が増加し、イデユコゴメ綱に属する藻類の培養効率が改善する。 The present invention provides a method for culturing algae belonging to the class Idycogome. The culturing method of the present invention increases the amount of algal bodies of algae belonging to the class Idyunicogome in the culture, and improves the efficiency of culturing the algae belonging to the class Idyuteicogome.
　別の態様において、本発明の培養方法により、イデユコゴメ綱に属する藻類によるグリコーゲンの生産量が増加する。また、本発明により、グリコーゲンの生産方法が提供される。したがって、本発明によれば、イデユコゴメ綱に属する藻類によるグリコーゲンの生産効率が改善する。 In another aspect, the culture method of the present invention increases the amount of glycogen produced by algae belonging to the class Idycogome. The present invention also provides a method for producing glycogen. Therefore, according to the present invention, the efficiency of glycogen production by algae belonging to the class Idycogome is improved.
　本発明の別の態様において、イデユコゴメ綱に属する藻類の培養効率を改善させつつ、かつ、イデユコゴメ綱に属する藻類によるグリコーゲンの生産効率を改善させることができる。 In another aspect of the present invention, it is possible to improve the efficiency of culturing algae belonging to the class Idyucogome, and at the same time improve the efficiency of glycogen production by algae belonging to the class Idyucogome.
　本発明の別の態様において、イデユコゴメ綱に属する藻類の培養物を乾燥した乾燥体が提供される。乾燥体はグリコーゲンを多く含むことから、本発明により、藻類由来のバイオマス燃料の原料になり得るグリコーゲンの効率の良い製造が可能になる。 In another aspect of the present invention, there is provided a dried culture of algae belonging to the class Idycogome. Since the dried matter contains a large amount of glycogen, the present invention enables efficient production of glycogen that can be used as a raw material for biomass fuel derived from algae.
　以下、本発明をさらに詳細に説明する。本発明の範囲はこれらの説明に限定されることはなく、以下の例示以外についても、本発明の趣旨を損なわない範囲で適宜変更し実施し得る。なお、本明細書に記載した全ての文献および刊行物は、その目的にかかわらず参照によりその全体を本明細書に組み込むものとする。また、本明細書は、本願の優先権主張の基礎となる日本国特許出願2022-033751（2022年3月4日出願）の特許請求の範囲、明細書、および図面の開示内容を包含する。 The present invention will be described in further detail below. The scope of the present invention is not limited to these explanations, and other than the following examples may be appropriately changed and implemented without impairing the gist of the present invention. All documents and publications mentioned herein are hereby incorporated by reference in their entirety for any purpose. In addition, this specification includes the disclosure contents of the claims, specification, and drawings of Japanese Patent Application No. 2022-033751 (filed on March 4, 2022), which is the basis for claiming priority of this application.
　本発明は、イデユコゴメ綱（Cyanidiophyceae）に属する藻類の培養方法又はイデユコゴメ綱（Cyanidiophyceae）に属する藻類にグリコーゲンを生産させる方法に関する。 The present invention relates to a method for culturing algae belonging to the class Cyanidiophyceae or a method for causing algae belonging to the class Cyanidiophyceae to produce glycogen.
　微生物の培養において、糖類等を高濃度で含む培地中で細胞を培養すると、細胞中のナトリウムイオンやカリウムイオンを含んだ水分が細胞外に流出してしまう。これらのイオンは細胞機能に必要であり、細胞は増殖が困難になり、場合よっては細胞死に至ることが知られている。このように、微生物の培養においては培養初期に糖類の濃度を高くすることは通常行われない。本発明においては、イデユコゴメ綱に属する藻類の培養において、培養開始時に５～４０重量％の濃度の炭素源（糖）を含む培地で培養することにより、驚くべきことに藻類の培養効率が高まり、グリコーゲンを効率的に産生することを見出した。グリコーゲンを高効率で産生するためには、０．０５～１０ｍＭの鉄イオンをさらに含む培地を用いることが好ましい。 In culturing microorganisms, if cells are cultured in a medium containing a high concentration of sugars, etc., water containing sodium ions and potassium ions in the cells will flow out of the cells. These ions are required for cell function and are known to make cells difficult to proliferate and in some cases to cell death. Thus, in culturing microorganisms, it is not usually practiced to increase the concentration of saccharides at the initial stage of culture. In the present invention, in culturing algae belonging to the class Idyucogome, by culturing in a medium containing a carbon source (sugar) at a concentration of 5 to 40% by weight at the start of the culture, the algae culture efficiency is surprisingly increased, We found that it efficiently produced glycogen. In order to produce glycogen with high efficiency, it is preferable to use a medium further containing 0.05 to 10 mM iron ions.
　イデユコゴメ綱は、分類学上、紅色植物門（Rhodophyta）イデユコゴメ綱（Cyanidiophyceae）として分類され、シアニディオシゾン（Cyanidioschyzon）属、シアニジウム（Cyanidium）属及びガルデリア（Galdieria）属の３属にさらに分類される。本発明において、イデユコゴメ綱に属する藻類として、シアニディオシゾン（Cyanidioschyzon）属、シアニジウム（Cyanidium）属及びガルデリア（Galdieria）属のいずれの属に属する藻類を用いてもよい。本発明において、イデユコゴメ綱に属する藻類は、ガルデリア（Galdieria）属に属する藻類が好ましい。 The class Cyanidiophyceae is taxonomically classified as Cyanidiophyceae in the phylum Rhodophyta, and is further classified into three genera: the genus Cyanidioschyzon, the genus Cyanidium, and the genus Galdieria. be. In the present invention, algae belonging to any of the genus Cyanidioschyzon, Cyanidium and Galdieria may be used as the algae belonging to the class Cyanidioschyzon. In the present invention, algae belonging to the genus Galdieria are preferably algae belonging to the genus Galdieria.
　本発明において、例えば、シアニディオシゾン・メローラエ（Cyanidioschyzon merolae）等のシアニディオシゾン属に属する藻類、シアニジウム・カルダリウム（Cyanidium caldarium）等のシアニジウム属に属する藻類、ガルデリア・スルフラリア（Galdieria sulphuraria）等のガルデリア属に属する藻類、又はこれらの変異体又はこれらの組合せを用いることができる。 In the present invention, for example, algae belonging to the genus Cyanidioschizon such as Cyanidioschyzon merolae, algae belonging to the genus Cyanidium such as Cyanidium caldarium, Galdieria sulphuraria, etc. algae belonging to the genus Galderia, or variants thereof, or combinations thereof.
　本発明において、イデユコゴメ綱に属する藻類は、一倍体の細胞形態であっても、二倍体の細胞形態であってもよい。 In the present invention, algae belonging to the class Idycogome may have a haploid cell form or a diploid cell form.
　本発明において、用いるイデユコゴメ綱に属する藻類の株は限定されない。例えば、ガルデリア・スルフラリア（Galdieria sulphuraria）の株としては、G 127（二倍体。強固な細胞壁を有する、CCCryoから入手可能）、G 108（1n）（二倍体から特開2020-072698に記載の方法に従って一倍体を誘導し、その単独培養株を作製してから利用。強固な細胞壁を有さない。）又はその変異株が挙げられる。G 127及びG 108のそれぞれの二倍体は、公的機関（例えば、G127（CCCryo 127-00）はCCCryo（Culture Collection of Cryophilic Algae）、G108（SAG 108.79）はSAG（The Culture Collection of Algae at Goettingen University））から容易に入手することができる。 In the present invention, strains of algae belonging to the class Idycogome to be used are not limited. For example, as strains of Galdieria sulphuraria, G 127 (diploid, having a strong cell wall, available from CCCryo), G 108 (1n) (diploid described in JP 2020-072698) , the haploid is induced according to the method of , and its single culture strain is prepared and used.It does not have a strong cell wall. Diploids of G 127 and G 108, respectively, were obtained from public institutions (e.g. G127 (CCCryo 127-00) is CCCryo (Culture Collection of Cryophilic Algae), G108 (SAG 108.79) is SAG (The Culture Collection of Algae at readily available from Gottingen University)).
　変異株は、当業者であれば、株に変異処理を行うことによって容易に得ることができる。変異処理としては、例えば、変異原作用を有する薬剤による処理又は高エネルギー線照射処理が挙げられる。変異原作用を有する薬剤として、例えば、エチルメタンスルホネート、N-メチル-N′-ニトロ-N-ニトロソグアニジン及び5-ブロモウラシル等の塩基類似体が挙げられる。また、高エネルギー線としては、ＵＶ、ガンマ線、Ｘ線及び重イオンビームが挙げられる。変異処理には、遺伝子組換え法を利用して特定の遺伝子を変異させる方法も含まれる。変異株には、形質転換体も含まれる。 A mutant strain can be easily obtained by a person skilled in the art by subjecting the strain to mutation treatment. Mutation treatment includes, for example, treatment with a drug having a mutagenic action or high-energy beam irradiation treatment. Agents with mutagenic activity include base analogs such as, for example, ethyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine and 5-bromouracil. High energy rays also include UV, gamma rays, X-rays and heavy ion beams. Mutation treatment also includes a method of mutating a specific gene using a gene recombination method. Mutants also include transformants.
　イデユコゴメ綱に属する藻類は、高温、高硫黄、低ｐＨの環境、たとえば硫酸塩泉から、既報に従って採集してもよい（De Luca P. et al., 1978, Webbia, 33, 37-44）。イデユコゴメ綱に属する藻類は、アレン培地又は改変アレン（MA）培地において維持培養することができる（Allen, M. B., 1959, Arch. Mikrobiol., 32, 270-277； Kuroiwa, T. et al., 1993, Protoplasma, 175, 173-177； Ohnuma M. et al., 2008, Plant Cell Physiol., 117-120； Kuroiwa T. et al., 2012, Cytologia, 77(3), 289-299）。 Algae belonging to the class Ideucogome may be collected from high-temperature, high-sulfur, low-pH environments, such as sulfate springs, according to previous reports (De Luca P. et al., 1978, Webbia, 33, 37-44). Algae belonging to the class Idyucogome can be maintained and cultured in Allen medium or modified Allen (MA) medium (Allen, M. B., 1959, Arch. Mikrobiol., 32, 270-277; Kuroiwa, T. et al. ., 1993, Protoplasma, 175, 173-177; Ohnuma M. et al., 2008, Plant Cell Physiol., 117-120; Kuroiwa T. et al., 2012, Cytologia, 77(3), 289-299) .
　本発明は、イデユコゴメ綱に属する藻類を培養するための培地又はイデユコゴメ綱に属する藻類にグリコーゲンを生産させるための培地を含む。 The present invention includes a medium for culturing algae belonging to the class Idyucogome or a medium for making algae belonging to the class Idyucogome produce glycogen.
　本発明のイデユコゴメ綱に属する藻類を培養するための培地又はイデユコゴメ綱に属する藻類にグリコーゲンを生産させるための培地は、５～４０重量％の炭素源を含むことを特徴とする。炭素源は、イデユコゴメ綱に属する藻類を培養するのに通常用いられる基礎培地に含有させることができる。５～４０重量％の炭素源を含む培地は、０．０７～１０ｍＭの鉄イオンをさらに含有してもよい。 The medium for culturing algae belonging to the class Idyucogome of the present invention or the medium for making the algae belonging to the class Idyucogome to produce glycogen is characterized by containing 5 to 40% by weight of a carbon source. The carbon source can be contained in a basal medium commonly used for culturing algae belonging to the class Idycogome. Medium containing 5-40% by weight carbon source may further contain 0.07-10 mM iron ions.
　５～４０重量％の炭素源を含む本発明の培地を用いてイデユコゴメ綱に属する藻類を培養することにより、培養物中の藻体量が増加し、イデユコゴメ綱に属する藻類の培養効率が改善する。したがって、炭素源を含む培地は、イデユコゴメ綱に属する藻類の培養のために用いることができるし、イデユコゴメ綱に属する藻類の培養効率を改善するためにも用いることができる。５～４０重量％の炭素源を含む培地でイデユコゴメ綱に属する藻類を培養することを含む、イデユコゴメ綱に属する藻類の培養方法又は培養効率を改善する方法は、本発明に含まれる。 By culturing algae belonging to the class Idycogome using the medium of the present invention containing a carbon source of 5 to 40% by weight, the amount of algal bodies in the culture increases, and the efficiency of culturing the algae belonging to the class Idyuteicogome is improved. . Therefore, a medium containing a carbon source can be used for culturing algae belonging to the class Idycogome, and can also be used to improve the efficiency of culturing algae belonging to the class Idycogome. The present invention includes a method for culturing algae belonging to the class Idycogome or a method for improving the culture efficiency, which comprises culturing the algae belonging to the class Idycogome in a medium containing 5 to 40% by weight of a carbon source.
　イデユコゴメ綱に属する藻類の培養効率は、濁度法や乾燥藻体重量などの従来公知の細胞増殖の評価方法に基づき評価することができる。例えば、濁度法では、培養容器から採取した培養物の波長750 nmにおける濁度（光学密度）を分光光度計等を用いて測定する。測定の際に、水又は培地で希釈系列を作製し、測定に適する濃度まで培養物を希釈してもよい。また、培養容器から採取した一定量の培養物を乾燥させた後、重量を測定することにより乾燥藻体の重量を測定することができる。 The efficiency of culturing algae belonging to the class Idyucogome can be evaluated based on conventionally known cell growth evaluation methods such as the turbidity method and dry alga body weight. For example, in the turbidity method, the turbidity (optical density) at a wavelength of 750 nm of a culture sampled from a culture vessel is measured using a spectrophotometer or the like. At the time of measurement, a dilution series may be prepared with water or medium to dilute the culture to a concentration suitable for measurement. Also, the weight of the dried algal body can be measured by drying a certain amount of the culture collected from the culture vessel and then measuring the weight.
　対照の値と比較して、濁度が大きい又は乾燥藻体量が多い場合、イデユコゴメ綱に属する藻類の藻体量が増加した、又は、イデユコゴメ綱に属する藻類の培養効率が改善したと評価することができる。本発明の培地で培養する前のイデユコゴメ綱に属する藻類又は基礎培地などの本発明の培地以外の培地で培養したイデユコゴメ綱に属する藻類を対照として用いることができる。 When the turbidity is large or the amount of dry algae is large compared to the control value, it is evaluated that the amount of algae belonging to the class Idyunicogome has increased or the culture efficiency of the algae belonging to the class Idyunicogome has improved. be able to. Algae belonging to the class Cocogoma before being cultured in the medium of the present invention or algae belonging to the class Cocogoma that have been cultured in a medium other than the medium of the present invention, such as a basal medium, can be used as a control.
　イデユコゴメ綱に属する藻類は、グリコーゲンを貯蔵多糖として細胞質基質中に貯蔵する。５～４０重量％の炭素源を含む本発明の培地でイデユコゴメ綱に属する藻類を培養することにより、培養物中の藻体量が増加し、グリコーゲンが蓄積された藻体を含む培養物を多量に得ることができる。したがって、本発明の培地を用いてイデユコゴメ綱に属する藻類を培養することにより、イデユコゴメ綱に属する藻類によるグリコーゲンの生産量を増加させることができる。また、５～４０重量％の炭素源を含む本発明の培地は、イデユコゴメ綱に属する藻類にグリコーゲンを生産させるための培地としても用いることができる。５～４０重量％の炭素源を含む培地でイデユコゴメ綱に属する藻類を培養することを含む、イデユコゴメ綱に属する藻類にグリコーゲンを生産させる方法又はグリコーゲンの生産効率を改善する方法は、本発明に含まれる。 Algae belonging to the class Idycogome store glycogen in the cytosol as storage polysaccharides. By culturing algae belonging to the class Idycogome in the medium of the present invention containing 5 to 40% by weight of the carbon source, the amount of algal bodies in the culture is increased, and a large amount of the culture containing algal bodies in which glycogen is accumulated is obtained. can get to Therefore, by culturing algae belonging to the class Idycogome using the medium of the present invention, it is possible to increase the amount of glycogen produced by the algae belonging to the class Idycogome. The medium of the present invention containing 5 to 40% by weight of carbon source can also be used as a medium for producing glycogen in algae belonging to the class Idycogome. A method for producing glycogen in algae belonging to the class Idyucogome, or a method for improving the production efficiency of glycogen, comprising culturing algae belonging to the class Idyucogome in a medium containing 5 to 40% by weight of a carbon source is included in the present invention. be
　グリコーゲンの生産量は、培養物中のイデユコゴメ綱に属する藻類に含まれるグリコーゲン量によって評価することができる。イデユコゴメ綱に属する藻類に含まれるグリコーゲン量は、培養した藻体からグリコーゲンを抽出し、抽出したグリコーゲンの量を測定すればよい。例えば、培養容器から採取した一定量の培養物を乾燥させた後、乾燥物（乾燥藻体）に水を加えてミキサーで混合した後に熱処理及び遠心処理をして得られた上清をグリコーゲン抽出液とすることができる。本発明は、本発明の培養方法又は本発明のグリコーゲンを生産する方法により得られるイデユコゴメ綱に属する藻類の培養物を乾燥した乾燥体も含む。当業者であれば適宜改変を実施して培養したイデユコゴメ綱に属する藻類からグリコーゲン抽出液を得ることができる。グリコーゲン抽出液中に含まれるグリコーゲン量は、市販のキットを用いて定量することができる。定量の際に、水又は溶媒で希釈系列を作製し、測定に適する濃度までグリコーゲン抽出液を希釈してもよい。また、グリコーゲン抽出液中に含まれるグリコーゲン量は、ＨＰＬＣ等のクロマトグラフィーによって定量してもよい。 The amount of glycogen produced can be evaluated by the amount of glycogen contained in algae belonging to the class Idyucogome in the culture. The amount of glycogen contained in algae belonging to the class Idyucogome can be determined by extracting glycogen from cultured alga bodies and measuring the amount of extracted glycogen. For example, after drying a certain amount of culture material collected from a culture vessel, water is added to the dried material (dried algal body), mixed with a mixer, heat-treated and centrifuged, and the resulting supernatant is extracted with glycogen. It can be liquid. The present invention also includes dried cultures of algae belonging to the class Idycogome obtained by the culture method of the present invention or the method of producing glycogen of the present invention. A person skilled in the art can obtain a glycogen extract from cultured algae belonging to the class Idycogome by making appropriate modifications. The amount of glycogen contained in the glycogen extract can be quantified using a commercially available kit. Upon quantification, a dilution series may be prepared with water or a solvent to dilute the glycogen extract to a concentration suitable for measurement. Also, the amount of glycogen contained in the glycogen extract may be quantified by chromatography such as HPLC.
　対照の値と比較して、培養物中のイデユコゴメ綱に属する藻類に含まれるグリコーゲン量が多い場合、グリコーゲンの生産量が増加した、又はグリコーゲンの生産効率が改善したと評価することができる。本発明の培地で培養する前のイデユコゴメ綱に属する藻類又は基礎培地などの本発明の培地以外の培地で培養したイデユコゴメ綱に属する藻類を対照として用いることができる。 When the amount of glycogen contained in algae belonging to the class Idyucogome in the culture is large compared to the control value, it can be evaluated that the amount of glycogen produced has increased or the production efficiency of glycogen has improved. Algae belonging to the class Cocogoma before being cultured in the medium of the present invention or algae belonging to the class Cocogoma that have been cultured in a medium other than the medium of the present invention, such as a basal medium, can be used as a control.
　本発明の別の態様において、５～４０重量％の炭素源を含む本発明の培地は、０．０５～１０ｍＭの鉄イオンをさらに含むこともできる。 In another aspect of the present invention, the medium of the present invention containing 5-40% by weight of carbon source may further contain 0.05-10 mM iron ions.
　本発明の一態様において、５～４０重量％の炭素源及び０．０５～１０ｍＭの鉄イオンを含む本発明の培地を用いてイデユコゴメ綱に属する藻類を培養することにより、培養物中の藻体量が増加し、イデユコゴメ綱に属する藻類の培養効率が改善する。したがって、炭素源と鉄イオンを含む培地は、イデユコゴメ綱に属する藻類の培養のために用いることができるし、あるいはイデユコゴメ綱に属する藻類の培養効率を改善するために用いることもできる。５～４０重量％の炭素源及び０．０５～１０ｍＭの鉄イオンを含む培地でイデユコゴメ綱に属する藻類を培養することを含む、イデユコゴメ綱に属する藻類の培養方法又は培養効率を改善する方法は、本発明に含まれる。 In one embodiment of the present invention, by culturing algae belonging to the class Idycogome using the medium of the present invention containing 5 to 40% by weight of a carbon source and 0.05 to 10 mM of iron ions, alga bodies in the culture The amount is increased, and the efficiency of culturing algae belonging to the class Idycogome is improved. Therefore, a medium containing a carbon source and iron ions can be used for culturing algae belonging to the class Idycogome, or can be used to improve the efficiency of culturing algae belonging to the class Idycogome. A method for culturing algae belonging to the class Idycogome, comprising culturing algae belonging to the class Idycogome, in a medium containing 5 to 40% by weight of a carbon source and 0.05 to 10 mM iron ions, or a method for improving the culture efficiency, Included in the present invention.
　５～４０重量％の炭素源及び０．０５～１０ｍＭの鉄イオンを含む本発明の培地を用いる場合のイデユコゴメ綱に属する藻類の培養効率の評価は、前述と同様に実施することができる。 Evaluation of the efficiency of culturing algae belonging to the class Idycogome when using the medium of the present invention containing 5 to 40% by weight of carbon source and 0.05 to 10 mM iron ions can be performed in the same manner as described above.
　また、本発明の別の態様において、５～４０重量％の炭素源及び０．０５～１０ｍＭの鉄イオンを含む培地で培養することにより、培養物中の藻体量が増加し、グリコーゲンが蓄積された藻体を含む培養物を多量に得ることができる。また、炭素源及び鉄イオンを含む本発明の培地を用いてイデユコゴメ綱に属する藻類を培養することにより、藻体中のグリコーゲン濃度が増加し、グリコーゲンが多量に蓄積された藻体を含む培養物を得ることができる。また別の態様において、炭素源及び鉄イオンを含む本発明の培地を用いてイデユコゴメ綱に属する藻類を培養することにより、培養物中の藻体量が増加し、かつ、藻体中のグリコーゲン濃度が増加し、グリコーゲンが多量に蓄積された藻体を含む培養物を多量に得ることができる。このように、炭素源及び鉄イオンを含む本発明の培地を用いてイデユコゴメ綱に属する藻類を培養することにより、イデユコゴメ綱に属する藻類によるグリコーゲンの生産量を増加させることができる。したがって、５～４０重量％の炭素源及び０．０５～１０ｍＭの鉄イオンを含む培地は、イデユコゴメ綱に属する藻類にグリコーゲンを生産させるための培地又はイデユコゴメ綱に属する藻類によるグリコーゲンの生産効率を改善するための培地としても用いることができる。５～４０重量％の炭素源及び０．０５～１０ｍＭの鉄イオンを含む培地でイデユコゴメ綱に属する藻類を培養することを含む、イデユコゴメ綱に属する藻類にグリコーゲンを生産させる方法又はグリコーゲンの生産効率を改善する方法は、本発明に含まれる。 In another aspect of the present invention, by culturing in a medium containing 5 to 40% by weight of carbon source and 0.05 to 10 mM of iron ions, the amount of algae in the culture is increased and glycogen is accumulated. It is possible to obtain a large amount of culture containing the treated algae. In addition, by culturing algae belonging to the class Idyucogome using the medium of the present invention containing a carbon source and iron ions, the glycogen concentration in the alga body is increased, and the culture containing the alga body in which a large amount of glycogen is accumulated. can be obtained. In another embodiment, by culturing algae belonging to the class Idyucogome using the medium of the present invention containing a carbon source and iron ions, the amount of algae in the culture is increased, and the glycogen concentration in the algae is is increased, and a large amount of culture containing algal bodies in which a large amount of glycogen is accumulated can be obtained. Thus, by culturing algae belonging to the class Idycogome using the medium of the present invention containing a carbon source and iron ions, the amount of glycogen produced by the algae belonging to the class Idycogome can be increased. Therefore, a medium containing 5 to 40% by weight of a carbon source and 0.05 to 10 mM of iron ions is a medium for producing glycogen in algae belonging to the class Idycogome or improving the production efficiency of glycogen by algae belonging to the class Idyucogome. It can also be used as a medium for A method for producing glycogen in algae belonging to the class Idycogome, comprising culturing algae belonging to the class Idycogome in a medium containing 5 to 40% by weight of a carbon source and 0.05 to 10 mM iron ions, or improving the production efficiency of glycogen. Methods of remediation are included in the present invention.
　５～４０重量％の炭素源及び０．０５～１０ｍＭの鉄イオンを含む本発明の培地を用いる場合のイデユコゴメ綱に属する藻類のグリコーゲン生産量又はグリコーゲン生産効率の改善の評価は、前述と同様に実施することができる。 Evaluation of improvement in glycogen production amount or glycogen production efficiency of algae belonging to the class Idycogome when using the medium of the present invention containing 5 to 40% by weight of carbon source and 0.05 to 10 mM iron ions is performed in the same manner as described above. can be implemented.
　本発明において、炭素源としてイデユコゴメ綱に属する藻類が利用できる糖類であればいずれを用いてもよく、例えば、グルコース、フルクトース、ガラクトース、マンノース等の単糖類、スクロース、マルトース、ラクトースなどの二糖類、グリセロールなどの糖アルコールを単独又は組み合わせて用いることができる。本発明において、好ましい炭素源は、単糖類又は二糖類であり、より好ましくはグルコース又はスクロースである。本発明の一態様において、炭素源はグルコースである。本発明の別の態様において、炭素源はスクロースである。 In the present invention, any sugar that can be used by algae belonging to the class Idycogome can be used as a carbon source. For example, monosaccharides such as glucose, fructose, galactose and mannose; Sugar alcohols such as glycerol can be used alone or in combination. In the present invention, preferred carbon sources are monosaccharides or disaccharides, more preferably glucose or sucrose. In one aspect of the invention, the carbon source is glucose. In another aspect of the invention, the carbon source is sucrose.
　本発明において、炭素源は５～４０重量％の濃度になるように基礎培地に含有させることができる。炭素源の濃度は、５～４０重量％の範囲内で変更することができる。本発明において、培地中の炭素源の濃度は、例えば、７～４０重量％、７～３５重量％、８～３５重量％、９～３５重量％、１０～３５重量％、１５～３５重量％、１８～３５重量％、５～３０重量％、７～３０重量％、８～３０重量％、９～３０重量％、１０～３０重量％、１５～３０重量％、１８～３０重量％、７～２５重量％、８～２５重量％、９～２５重量％、１０～２５重量％、１５～２５重量％、１８～２５重量％、１５～２４重量％、７～１８重量％、８～１８重量％、９～１８重量％、１０～１８重量％、１５～１８重量％、７～１０重量％、８～１０重量％、７～９重量％、８～９重量％、１６～２３重量％、１６～２２重量％、１６～２１重量％、１６～２０重量％、１６～１９重量％、１６～１８重量％、１７～２３重量％、１７～２２重量％、１７～２１重量％、１７～２０重量％、１７～１９重量％、１７～１８重量％、１８～２３重量％、１８～２２重量％、１８～２１重量％、１８～２０重量％、１８～１９重量％、又は１８重量％であってもよい。 In the present invention, the carbon source can be contained in the basal medium at a concentration of 5-40% by weight. The carbon source concentration can vary within the range of 5-40% by weight. In the present invention, the concentration of the carbon source in the medium is, for example, 7 to 40% by weight, 7 to 35% by weight, 8 to 35% by weight, 9 to 35% by weight, 10 to 35% by weight, 15 to 35% by weight. , 18-35% by weight, 5-30% by weight, 7-30% by weight, 8-30% by weight, 9-30% by weight, 10-30% by weight, 15-30% by weight, 18-30% by weight, 7 ~25 wt%, 8-25 wt%, 9-25 wt%, 10-25 wt%, 15-25 wt%, 18-25 wt%, 15-24 wt%, 7-18 wt%, 8-18 wt% % by weight, 9-18% by weight, 10-18% by weight, 15-18% by weight, 7-10% by weight, 8-10% by weight, 7-9% by weight, 8-9% by weight, 16-23% by weight , 16-22% by weight, 16-21% by weight, 16-20% by weight, 16-19% by weight, 16-18% by weight, 17-23% by weight, 17-22% by weight, 17-21% by weight, 17 ~20 wt%, 17-19 wt%, 17-18 wt%, 18-23 wt%, 18-22 wt%, 18-21 wt%, 18-20 wt%, 18-19 wt%, or 18 wt% %.
　本発明において、鉄イオン源として、培地中で鉄イオンを生じるものであり、微生物の培養に通常使用されるものであれば、いずれを用いることもでき、例えば、FeSO4、Fe2(SO4)3、FeCl2、FeCl3、もしくはそれらの水和物又はそれらの組合せを用いることができる。本発明の一態様において、鉄イオン源はFeSO4の７水和物（FeSO4・7H2O）である。本発明の別の態様において、鉄イオン源はFeCl3の6水和物（FeCl3・6H2O）である。本発明において、鉄イオンは２価でも３価でもよい。また、水和物の数も特に限定されない。 In the present invention, any iron ion source that produces iron ions in a medium and is commonly used for culturing microorganisms can be used . ) 3 , FeCl 2 , FeCl 3 or their hydrates or combinations thereof can be used. In one embodiment of the invention, the source of iron ions is FeSO4 heptahydrate ( FeSO4.7H2O ). In another embodiment of the invention, the source of iron ions is FeCl 3 hexahydrate (FeCl 3 .6H 2 O). In the present invention, iron ions may be divalent or trivalent. Also, the number of hydrates is not particularly limited.
　本発明において、鉄イオンは０．０５～１０ｍＭの濃度になるように基礎培地に含有させることができる。鉄イオンの濃度は、０．０５～１０ｍＭの範囲内、例えば０．０７～１０ｍＭの範囲内で変更することができる。本発明において、培地中の鉄イオンの濃度は、例えば、０．０７～１０ｍＭ、０．１～１０ｍＭ、０．１５～１０ｍＭ、０．２～１０ｍＭ、０．２５～１０ｍＭ、０．３～１０ｍＭ、０．４～１０ｍＭ、０．５～１０ｍＭ、０．０７～９ｍＭ、０．１～９ｍＭ、０．１２～９ｍＭ、０．１５～９ｍＭ、０．２～９ｍＭ、０．２５～９ｍＭ、０．３～９ｍＭ、０．４～９ｍＭ、０．５～９ｍＭ、０．０７～８ｍＭ、０．１～８ｍＭ、０．１２～８ｍＭ、０．１５～８ｍＭ、０．２～８ｍＭ、０．２５～８ｍＭ、０．３～８ｍＭ、０．４～８ｍＭ、０．５～８ｍＭ、０．０７～７ｍＭ、０．１～７ｍＭ、０．１２～７ｍＭ、０．１５～７ｍＭ、０．２～７ｍＭ、０．２５～７ｍＭ、０．３～７ｍＭ、０．４～７ｍＭ、０．５～７ｍＭ、０．０７～６ｍＭ、０．１～６ｍＭ、０．１２～６ｍＭ、０．１５～６ｍＭ、０．２～６ｍＭ、０．２５～６ｍＭ、０．３～６ｍＭ、０．４～６ｍＭ、０．５～６ｍＭ、０．０７～５ｍＭ、０．１～５ｍＭ、０．１２～５ｍＭ、０．１５～５ｍＭ、０．２～５ｍＭ、０．２５～５ｍＭ、０．３～５ｍＭ、０．４～５ｍＭ、０．５～５ｍＭ、０．０７～４ｍＭ、０．１～４ｍＭ、０．１２～４ｍＭ、０．１５～４ｍＭ、０．２～４ｍＭ、０．２５～４ｍＭ、０．３～４ｍＭ、０．４～４ｍＭ、０．５～４ｍＭ、０．０７～３ｍＭ、０．１～３ｍＭ、０．１２～３ｍＭ、０．１５～３ｍＭ、０．２～３ｍＭ、０．２５～３ｍＭ、０．３～３ｍＭ、０．４～３ｍＭ、０．５～３ｍＭ、０．１５～２ｍＭ、０．２～２ｍＭ、０．３～２ｍＭ、０．４～２ｍＭ、０．５～２ｍＭ、０．１５～１ｍＭ、０．２～１ｍＭ、０．３～１ｍＭ、０．４～１ｍＭ、０．５～１ｍＭ、０．１５～０．９ｍＭ、０．２～０．９ｍＭ、０．３～０．９ｍＭ、０．４～０．９ｍＭ、０．５～０．９ｍＭ、０．１５～０．８ｍＭ、０．２～０．８ｍＭ、０．３～０．８ｍＭ、０．４～０．８ｍＭ、０．５～０．８ｍＭ、０．１５～０．７ｍＭ、０．２～０．７ｍＭ、０．３～０．７ｍＭ、０．４～０．７ｍＭ、０．５～０．７ｍＭであってもよい。 In the present invention, iron ions can be contained in the basal medium at a concentration of 0.05 to 10 mM. The concentration of iron ions can vary within the range of 0.05-10 mM, such as within the range of 0.07-10 mM. In the present invention, the concentration of iron ions in the medium is, for example, 0.07 to 10 mM, 0.1 to 10 mM, 0.15 to 10 mM, 0.2 to 10 mM, 0.25 to 10 mM, 0.3 to 10 mM. , 0.4-10 mM, 0.5-10 mM, 0.07-9 mM, 0.1-9 mM, 0.12-9 mM, 0.15-9 mM, 0.2-9 mM, 0.25-9 mM, 0 .3-9 mM, 0.4-9 mM, 0.5-9 mM, 0.07-8 mM, 0.1-8 mM, 0.12-8 mM, 0.15-8 mM, 0.2-8 mM, 0.25 ~8mM, 0.3-8mM, 0.4-8mM, 0.5-8mM, 0.07-7mM, 0.1-7mM, 0.12-7mM, 0.15-7mM, 0.2-7mM , 0.25-7 mM, 0.3-7 mM, 0.4-7 mM, 0.5-7 mM, 0.07-6 mM, 0.1-6 mM, 0.12-6 mM, 0.15-6 mM, 0 .2-6 mM, 0.25-6 mM, 0.3-6 mM, 0.4-6 mM, 0.5-6 mM, 0.07-5 mM, 0.1-5 mM, 0.12-5 mM, 0.15 ~5mM, 0.2-5mM, 0.25-5mM, 0.3-5mM, 0.4-5mM, 0.5-5mM, 0.07-4mM, 0.1-4mM, 0.12-4mM , 0.15-4 mM, 0.2-4 mM, 0.25-4 mM, 0.3-4 mM, 0.4-4 mM, 0.5-4 mM, 0.07-3 mM, 0.1-3 mM, 0 .12-3 mM, 0.15-3 mM, 0.2-3 mM, 0.25-3 mM, 0.3-3 mM, 0.4-3 mM, 0.5-3 mM, 0.15-2 mM, 0.2 ~2mM, 0.3-2mM, 0.4-2mM, 0.5-2mM, 0.15-1mM, 0.2-1mM, 0.3-1mM, 0.4-1mM, 0.5-1mM , 0.15-0.9 mM, 0.2-0.9 mM, 0.3-0.9 mM, 0.4-0.9 mM, 0.5-0.9 mM, 0.15-0.8 mM, 0 .2-0.8mM, 0.3-0.8mM, 0.4-0.8mM, 0.5-0.8mM, 0.15-0.7mM, 0.2-0.7mM, 0.3 It may be ~0.7 mM, 0.4-0.7 mM, 0.5-0.7 mM.
　本発明において、基礎培地は、従来、イデユコゴメ綱に属する藻類を培養することができることが知られている培地であれば、特に限定されることなく用いることができる。本発明において、基礎培地の例としては、アレン培地又は改変アレン培地（Allen, M. B., 1959, Arch. Mikrobiol., 32, 270-277； Kuroiwa, T. et al., 1993, Protoplasma, 175, 173-177； Ohnuma M. et al., 2008, Plant Cell Physiol., 117-120； Kuroiwa T. et al., 2012, Cytologia, 77(3), 289-299）を挙げることができるが、これらに限定されない。本発明において、基礎培地は、好ましくは液体培地である。 In the present invention, the basal medium can be used without particular limitation as long as it is a medium known to be capable of culturing algae belonging to the class Idyucogome. In the present invention, examples of the basal medium include Allen medium or modified Allen medium (Allen, M. B., 1959, Arch. Mikrobiol., 32, 270-277; Kuroiwa, T. et al., 1993, Protoplasma, 175, 173-177; Ohnuma M. et al., 2008, Plant Cell Physiol., 117-120; Kuroiwa T. et al., 2012, Cytologia, 77(3), 289-299). , but not limited to. In the present invention, the basal medium is preferably liquid medium.
　本発明において、培地には上記成分の他に、公知の添加剤、たとえば、セレンなどの微量金属元素、ペニシリン、ストレプトマイシン、ゲンタマイシンなどの抗生物質、フェノールレッド、ブロモフェノールブルーなどのｐＨ指示薬を含んでいてもよい。 In the present invention, the medium contains, in addition to the above components, known additives such as trace metal elements such as selenium, antibiotics such as penicillin, streptomycin and gentamicin, and pH indicators such as phenol red and bromophenol blue. You can
　本発明において、炭素源又は炭素源と鉄イオンを基礎培地に含有させ、イデユコゴメ綱に属する藻類を播種した後は、培養中系内への培地の供給は行わないことが望ましい。すなわち、本発明の一態様において、培養中に栄養分などの培地中の必要な成分は、連続的又は間欠的に系内に供給されない。本発明の一態様において、培養の開始時に必要な栄養成分、炭素源及び場合により鉄イオンをすべて培地中に含有させておく、回分培養で実施することができる。溶存酸素については、調整してもよい。 In the present invention, after the carbon source or carbon source and iron ions are contained in the basal medium and the algae belonging to the class Idyucogome are seeded, it is desirable not to supply the medium into the culture system. That is, in one aspect of the present invention, necessary components in the medium, such as nutrients, are not continuously or intermittently supplied into the system during cultivation. In one aspect of the present invention, batch culture can be carried out in which all necessary nutrients, carbon sources and optionally iron ions are contained in the medium at the start of the culture. Dissolved oxygen may be adjusted.
　本発明は、上記の培地を用いたイデユコゴメ綱に属する藻類の培養方法に関する。具体的には、イデユコゴメ綱に属する藻類を、５～４０重量％の炭素源を含む培地で培養することを含む、イデユコゴメ綱に属する藻類を培養する方法に関する。培養したイデユコゴメ綱に属する藻類は回収することができる。また、本発明の培養方法によりイデユコゴメ綱に属する藻類を培養することにより、イデユコゴメ綱に属する藻類にグリコーゲンを生産させることができる。 The present invention relates to a method for culturing algae belonging to the class Idycogome using the medium described above. Specifically, the present invention relates to a method for culturing algae belonging to the class Idyllicogome, comprising culturing the algae belonging to the class Idyllicogome in a medium containing 5 to 40% by weight of a carbon source. The cultured algae belonging to the class Idycogome can be recovered. In addition, by culturing algae belonging to the class Idyunicogome by the culture method of the present invention, it is possible to cause the algae belonging to the class Idyuteicogome to produce glycogen.
　また、本発明は、上記の培地を用いたイデユコゴメ綱に属する藻類にグリコーゲンを生産させる方法に関する。具体的には、イデユコゴメ綱に属する藻類を、５～４０重量％の炭素源を含む培地で培養することを含む、イデユコゴメ綱に属する藻類にグリコーゲンを生産させる方法に関する。グリコーゲンの生産方法において、培養したイデユコゴメ綱に属する藻類に由来するグリコーゲンを回収する工程をさらに含んでもよい。 The present invention also relates to a method for producing glycogen in algae belonging to the class Idycogome using the medium described above. Specifically, the present invention relates to a method for producing glycogen in algae belonging to the class Idyucogome, comprising culturing the algae belonging to the class Idyucogome in a medium containing 5 to 40% by weight of a carbon source. The method for producing glycogen may further include a step of recovering glycogen derived from the cultured algae belonging to the class Idycogome.
　上記本発明の方法において、培地は０．０７～１０ｍＭの鉄イオンをさらに含有してもよい。炭素源と鉄イオンを基礎培地に含有させる場合、炭素源（例えば、単糖類、二糖類又は糖アルコール）の濃度及び鉄イオンの濃度は、本明細書に記載した濃度の範囲からそれぞれ適宜選択し、組み合わせることができる。例えば、本発明における培地は：
　５～４０重量％の炭素源及び０．０７～１０ｍＭの鉄イオンを含む培地；
　５～３０重量％の炭素源及び０．１５～５ｍＭの鉄イオンを含む培地；
　７～２５重量％の炭素源及び０．１５～２ｍＭの鉄イオンを含む培地；
　７～１８重量％の炭素源及び０．３～１ｍＭの鉄イオンを含む培地；
　７～１０重量％の炭素源及び０．３～１ｍＭの鉄イオンを含む培地；
　７～９重量％の炭素源及び０．３～１ｍＭの鉄イオンを含む培地；
　８～１０重量％の炭素源及び０．３～１ｍＭｍＭの鉄イオンを含む培地；
　７～１８重量％の炭素源及び０．３～０．９ｍＭの鉄イオンを含む培地；
　７～１０重量％の炭素源及び０．３～０．９ｍＭｍＭの鉄イオンを含む培地；
　７～９重量％の炭素源及び０．３～０．９ｍＭの鉄イオンを含む培地；
　８～１０重量％の炭素源及び０．３～０．９ｍＭの鉄イオンを含む培地；
　７～１８重量％の炭素源及び０．３～０．８ｍＭの鉄イオンを含む培地；
　７～１０重量％の炭素源及び０．３～０．８ｍＭｍＭの鉄イオンを含む培地；
　７～９重量％の炭素源及び０．３～０．８ｍＭの鉄イオンを含む培地；
　８～１０重量％の炭素源及び０．３～０．８ｍＭの鉄イオンを含む培地；
　７～１８重量％の炭素源及び０．５～０．７ｍＭの鉄イオンを含む培地；
　７～１０重量％の炭素源及び０．５～０．７ｍＭｍＭの鉄イオンを含む培地；
　７～９重量％の炭素源及び０．５～０．７ｍＭの鉄イオンを含む培地；又は
　８～１０重量％の炭素源及び０．５～０．７ｍＭの鉄イオンを含む培地；
が含まれるが、これに限定されない。 In the above method of the present invention, the medium may further contain 0.07 to 10 mM iron ions. When the carbon source and iron ions are contained in the basal medium, the concentration of the carbon source (e.g., monosaccharides, disaccharides or sugar alcohols) and the concentration of iron ions are appropriately selected from the concentration ranges described herein. , can be combined. For example, the medium in the present invention is:
A medium containing 5-40 wt% carbon source and 0.07-10 mM iron ions;
A medium containing 5-30 wt% carbon source and 0.15-5 mM iron ions;
A medium containing 7-25% by weight carbon source and 0.15-2 mM iron ions;
A medium containing 7-18% by weight of carbon source and 0.3-1 mM iron ions;
A medium containing 7-10% by weight of carbon source and 0.3-1 mM of iron ions;
A medium containing 7-9% by weight of carbon source and 0.3-1 mM of iron ions;
A medium containing 8-10% by weight of a carbon source and 0.3-1 mMmM of iron ions;
A medium containing 7-18% by weight of carbon source and 0.3-0.9 mM of iron ions;
A medium containing 7-10 wt% carbon source and 0.3-0.9 mMmM iron ion;
A medium containing 7-9% by weight of carbon source and 0.3-0.9 mM of iron ions;
A medium containing 8-10 wt% carbon source and 0.3-0.9 mM iron ions;
A medium containing 7-18% by weight of carbon source and 0.3-0.8 mM of iron ions;
A medium containing 7-10% by weight of carbon source and 0.3-0.8 mMmM iron ion;
A medium containing 7-9% by weight of carbon source and 0.3-0.8 mM of iron ions;
A medium containing 8-10% by weight of carbon source and 0.3-0.8 mM of iron ions;
A medium containing 7-18% by weight of carbon source and 0.5-0.7 mM of iron ions;
A medium containing 7-10% by weight of carbon source and 0.5-0.7 mMmM iron ion;
A medium containing 7-9% by weight carbon source and 0.5-0.7 mM iron ions; or a medium containing 8-10% by weight carbon source and 0.5-0.7 mM iron ions;
including but not limited to.
　イデユコゴメ綱に属する藻類の培養条件は、イデユコゴメ綱に属する藻類が生育する条件であればいずれの方法でもよく、当業者であれば、公知の方法から適宜選択、変更することができ、実施例の記載も適宜参考にすることができる。例えばイデユコゴメ綱に属する藻類の培養温度は、３７℃～５０℃とすることができ、４０℃～４５℃としてもよい。また、通気条件としては、好気条件及び嫌気条件のいずれをも用いることができる。光の照射条件としては、光条件及び暗条件のいずれをも用いることができる。培養には、藻類の培養に通常用いられる培養槽を用いることができる。例えば、試験管、坂口フラスコ、三角フラスコ、円形ポンド、レースウェイポンド、カラム型培養槽、フラットパネル型培養槽、チューブ型培養槽、ジャーファーメンターなどのタンク等を用いることができるがこれらの限定されるわけではない。 The culture conditions for algae belonging to the class Idyucogome may be any method as long as the algae belonging to the class Idyucogome grow, and those skilled in the art can appropriately select and change them from known methods. The description can also be referred to as appropriate. For example, the temperature for culturing algae belonging to the class Idycogome can be 37°C to 50°C, and may be 40°C to 45°C. Both aerobic conditions and anaerobic conditions can be used as ventilation conditions. As for the light irradiation condition, both the light condition and the dark condition can be used. For culturing, a culture tank commonly used for culturing algae can be used. For example, a test tube, a Sakaguchi flask, an Erlenmeyer flask, a round pond, a raceway pond, a column type culture tank, a flat panel type culture tank, a tube type culture tank, a tank such as a jar fermenter, etc. can be used, but these are limited. It is not.
　培養期間も当業者が適宜設定することができる。例えば、イデユコゴメ綱に属する藻類の培養期間は、１日～６月間、1日～３月間、１日～２月間、１日～４週間、１～４週間、例えば１～２週間とすることができる。 The culture period can also be appropriately set by those skilled in the art. For example, the culture period for algae belonging to the class Idycogome can be 1 day to 6 months, 1 day to 3 months, 1 day to 2 months, 1 day to 4 weeks, 1 to 4 weeks, for example 1 to 2 weeks. can.
　培養したイデユコゴメ綱に属する藻類を回収する方法又は培養したイデユコゴメ綱に属する藻類に由来するグリコーゲンを回収する方法は、当業者は適宜選択することができる。 A person skilled in the art can appropriately select a method for collecting cultured algae belonging to the class Idyunicogome or a method for collecting glycogen derived from cultured algae belonging to the class Idyunicogome.
　培養したイデユコゴメ綱に属する藻類は細胞内にグリコーゲンを含むため、藻体を含む培養物をそのままイデユコゴメ綱に属する藻類に由来するグリコーゲンとしてもよい。また、培養物を遠心分離やろ過分離等により濃縮した濃縮物をイデユコゴメ綱に属する藻類に由来するグリコーゲンとしてもよい。上記培養物や濃縮物や破砕物を必要に応じて乾燥させた乾燥物をイデユコゴメ綱に属する藻類に由来するグリコーゲンとしてもよい。乾燥は、凍結乾燥、加熱乾燥、噴霧乾燥、真空乾燥などの方法のいずれであってもよいが、凍結乾燥が好ましい。乾燥物を処理してグリコーゲンを抽出又は精製してもよい。さらに、培養物、濃縮物又は乾燥物に、加熱、紫外線及び／又は放射線等による光照射、ミル又はガラスビーズなどによる粉砕等の処理を施してもよい。本発明におけるグリコーゲン生産方法は、上記のグリコーゲンを回収する工程を含んでいてもよい。 Since the cultured algae belonging to the class Idyucogome contain glycogen in their cells, the culture containing the algal body may be used as it is as the glycogen derived from the algae belonging to the class Idyucogome. Alternatively, a concentrate obtained by concentrating the culture by centrifugation, filtration, or the like may be used as glycogen derived from algae belonging to the class Idycogome. The dried product obtained by drying the culture, concentrate, or crushed product may be used as glycogen derived from algae belonging to the class Idycogome. Drying may be performed by any method such as freeze drying, heat drying, spray drying, and vacuum drying, but freeze drying is preferred. The dried matter may be processed to extract or purify glycogen. Furthermore, the culture, concentrate, or dried product may be subjected to treatments such as heating, irradiation with ultraviolet rays and/or radiation, and pulverization using a mill or glass beads. The method for producing glycogen in the present invention may include the above-described step of recovering glycogen.
　本発明の培養方法又はグリコーゲン生産方法により得られる培養物、濃縮物、乾燥物又は処理物も本発明に含まれる。 The present invention also includes cultures, concentrates, dried products, or processed products obtained by the culture method or glycogen production method of the present invention.
　一態様において、本発明の培養方法により、約１０ｇ／Ｌ以上、約１５ｇ／Ｌ以上、約２０ｇ／Ｌ以上の、培養液中（培養液あたり）のイデユコゴメ綱に属する藻類の藻体量が達成される。 In one aspect, the culture method of the present invention achieves an alga body amount of algae belonging to the class Idycogome in the culture solution (per culture solution) of about 10 g/L or more, about 15 g/L or more, or about 20 g/L or more. be done.
　一態様において、本発明のグリコーゲン生産方法により、約１０ｍｇ／ｇ以上、約１５ｍｇ／ｇ以上、約２０ｍｇ／ｇ以上、約３０ｍｇ／ｇ以上、約４０ｍｇ／ｇ以上の、培養物（藻体）中（培養物あたり）のグリコーゲン濃度が達成される。 In one embodiment, the glycogen production method of the present invention provides about 10 mg/g or more, about 15 mg/g or more, about 20 mg/g or more, about 30 mg/g or more, about 40 mg/g or more in the culture (algal body) (per culture) glycogen concentration is achieved.
　本明細書において、数値範囲について「～」を用いた記載では、特に断りがない限り、下限値及び上限値を含むものとする。例えば、「１～４０」という記載では、下限値である「１」、上限値である「４０」のいずれも含むものとする。すなわち、「１～４０」は、「１以上４０以下」と同じ意味である。 In this specification, descriptions using "~" for numerical ranges include lower and upper limits unless otherwise specified. For example, the description “1 to 40” includes both the lower limit “1” and the upper limit “40”. That is, "1 to 40" has the same meaning as "1 or more and 40 or less".
　以下、実施例により本発明をさらに詳しく説明するが、本発明はこれら実施例のみに限定されるものではない。 The present invention will be described in more detail below with reference to examples, but the present invention is not limited only to these examples.
［培養条件］
　ガルデリアを試験管に供し、下記の条件で培養した。ガルデリアは、Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalytics and Bioprocesses IZI-BBに株番号CCCryo 127-00 Galdieria sulphuraria (Galdieri) Merola 1982として寄託されたものを用いた。 [Culture conditions]
Garuderia was placed in a test tube and cultured under the following conditions. Galderia was used as deposited at the Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalytics and Bioprocesses IZI-BB under strain number CCCryo 127-00 Galdieria sulphuraria (Galdieri) Merola 1982.
培地
　実験Ａでは、以下の組成の１×ＭＡ培地（M-Allen、国立研究開発法人国立環境研究所微生物系統保存施設https://mcc.nies.go.jp/02medium.html）の４倍濃度の培地成分を含む４×ＭＡ培地を使用した。
Figure JPOXMLDOC01-appb-T000001
 In medium experiment A, 4 times the concentration of 1 × MA medium (M-Allen, National Institute for Environmental Studies, National Institute for Environmental Studies, https://mcc.nies.go.jp/02medium.html) with the following composition A 4×MA medium containing the medium components of was used.
Figure JPOXMLDOC01-appb-T000001
　実験Ｂは、１×ＭＡ培地を使用した。
　実験Ｃでは、１０×ＭＡ（－）培地に、Fe（0.0752 mM EDTA・2Na及び0.0592 mM FeSO4・7H2O）の１～１００倍量を適宜添加した培地を用いた。１０×ＭＡ（－）培地は、１×ＭＡの１０倍濃度の培地成分を含むが、EDTA・2NaとFeSO4・7H2Oを含有しない培地である。 Experiment B used 1×MA medium.
In Experiment C, a 10×MA(-) medium supplemented with 1 to 100 volumes of Fe (0.0752 mM EDTA.2Na and 0.0592 mM FeSO.sub.4.7H.sub.2O ) was used. The 10×MA(-) medium is a medium containing medium components at ten times the concentration of 1×MA, but not containing EDTA.2Na and FeSO.sub.4.7H.sub.2O .
培養条件
（１）実験Ａ（炭素源濃度の検討、比較例１～２、実施例１～６）
　・温度：40 ℃
　・振盪：330 rpm
　・培養日数：14日間
　・炭素源の種類：グルコース
　・炭素源濃度（重量％）：（比較例１）１％、（比較例２）３％、（実施例１）５％、（実施例２）７％、（実施例３）９％、（実施例４）１８％、（実施例５）２５％、（実施例６）３０％
　４×ＭＡ培地に上記濃度となるようにグルコースを加えた培地５ｍＬに、Galdieria sulphuraria CCCryo 127-00（1倍体）を加え、試験管培養を行った。４×ＭＡ培地の鉄イオン濃度は、０．２３７ｍＭである。 Culture conditions (1) Experiment A (examination of carbon source concentration, Comparative Examples 1-2, Examples 1-6)
・Temperature: 40℃
・Shaking: 330 rpm
・Culture days: 14 days ・Type of carbon source: glucose ・Concentration of carbon source (% by weight): (Comparative Example 1) 1%, (Comparative Example 2) 3%, (Example 1) 5%, (Example 2 ) 7%, (Example 3) 9%, (Example 4) 18%, (Example 5) 25%, (Example 6) 30%
Galdieria sulphuraria CCCryo 127-00 (haploid) was added to 5 mL of a 4×MA medium with glucose added to the above concentration, and cultured in a test tube. The iron ion concentration of 4xMA medium is 0.237 mM.
（２）実験Ｂ（炭素源の検討、実施例７～８）
　・温度：40 ℃
　・振盪：330 rpm
　・培養日数：12日間
　・炭素源の種類：（実施例７）グルコース、（実施例８）スクロース
　・炭素源濃度（重量％）：（実施例７）９％、（実施例８）９％
　１×ＭＡ培地に上記濃度となるように炭素源を加えた培地５ｍＬに、Galdieria sulphuraria CCCryo 127-00（1倍体）を加え、試験管培養を行った。１×ＭＡ培地の鉄イオン濃度は、０．０５９２ｍＭである。 (2) Experiment B (Study of carbon source, Examples 7-8)
・Temperature: 40℃
・Shaking: 330 rpm
・Culture days: 12 days ・Types of carbon source: (Example 7) glucose, (Example 8) sucrose ・Carbon source concentration (% by weight): (Example 7) 9%, (Example 8) 9%
Galdieria sulphuraria CCCryo 127-00 (haploid) was added to 5 mL of a 1×MA medium containing a carbon source at the above concentration, and cultured in a test tube. The iron ion concentration of 1×MA medium is 0.0592 mM.
（３）実験Ｃ（鉄イオンの効果確認、実施例９～１６）
　・温度：４０ ℃
　・振盪：330 rpm
　・培養日数：14日間
　・炭素源の種類：グルコース
　・炭素源濃度（重量％）：１８％
　・鉄イオン濃度：（実施例９）０．０５９２ｍＭ、（実施例１０）０．１１８４ｍＭ、（実施例１１）０．２９６０ｍＭ、（実施例１２）０．５９１９ｍＭ、（実施例１３）０．８８７９ｍＭ、（実施例１４）１．１８３９ｍＭ、（実施例１５）２．９５９７ｍＭ、（実施例１６）５．９１９３ｍＭ
　１０×ＭＡ（－）培地に、上記炭素源濃度及び鉄イオン濃度となるようにグルコース及びFeを加えた培地５ｍＬに、Galdieria sulphuraria CCCryo 127-00（1倍体）を加え、試験管培養を行った。 (3) Experiment C (confirmation of the effect of iron ions, Examples 9 to 16)
・Temperature: 40℃
・Shaking: 330 rpm
・Culture days: 14 days ・Type of carbon source: glucose ・Concentration of carbon source (% by weight): 18%
- Iron ion concentration: (Example 9) 0.0592 mM, (Example 10) 0.1184 mM, (Example 11) 0.2960 mM, (Example 12) 0.5919 mM, (Example 13) 0.8879 mM, (Example 14) 1.1839 mM, (Example 15) 2.9597 mM, (Example 16) 5.9193 mM
Galdieria sulphuraria CCCryo 127-00 (haploid) was added to 5 mL of a medium obtained by adding glucose and Fe to 10×MA (-) medium so that the carbon source concentration and iron ion concentration were as described above, and cultured in test tubes. Ta.
［評価］
（１）ガルデリアの増殖確認
　ガルデリアの増殖確認は、培養液200μLをFALCON（登録商標）96 wellプレートに加え、室温における750 nmの光学密度（以下、「OD750nm」とする）を、分光光度計（Thermo Fisher Scientific社製、Thermo Scientific Multiskan Go）を用いて測定することにより行った。 [evaluation]
(1) Confirmation of growth of Galderia To confirm the growth of Galderia, add 200 μL of the culture solution to a FALCON (registered trademark) 96-well plate and measure the optical density at 750 nm at room temperature (hereinafter referred to as “OD750 nm”) with a spectrophotometer ( Measurement was performed using Thermo Scientific Multiskan Go (manufactured by Thermo Fisher Scientific).
（２）培養液中の藻体量（ｇ／Ｌ）（藻体量／培養液）
　培養液１ｍＬを1.5mL容量のマイクロチューブに分注して、遠心分離（TOMY社製、微量高速冷却遠心機MX-307）を行った（15000×g、10分）。上清を取り除いてから１×ＭＡ培地１ｍＬをマイクロチューブに添加し、ダイレクトミキサー（アズワン社製、ダイレクトミキサーDM-301）で５分間懸濁した。再び遠心分離を行い、上清を除いた（洗浄）。洗浄操作をもう一度行い、得られた藻体を－20℃で凍結させた。
　凍結乾燥機（EYELA社製、凍結乾燥機FDU-1200型）で数時間乾燥させて、重さを測定した（ｇ／Ｌ）。 (2) Amount of algae in culture medium (g/L) (Amount of algae/culture medium)
1 mL of the culture solution was dispensed into a 1.5 mL microtube and centrifuged (TOMY, high-speed microcentrifuge MX-307) (15000×g, 10 minutes). After removing the supernatant, 1 mL of 1×MA medium was added to the microtube and suspended for 5 minutes with a direct mixer (manufactured by AS ONE, Direct Mixer DM-301). Centrifugation was performed again and the supernatant was removed (washing). The washing operation was performed once more, and the obtained alga bodies were frozen at -20°C.
It was dried for several hours with a freeze dryer (manufactured by EYELA, freeze dryer FDU-1200 model) and weighed (g/L).
（３）藻体中のグリコーゲン量（ｍｇ／Ｌ）（グリコーゲン量／培養液）
　（２）培養液中の藻体量(ｇ／Ｌ)と同様に藻体を凍結したサンプルに、グリコーゲン抽出操作を行った。凍結藻体にイオン交換水400μLを加えてダイレクトミキサーで混合し、95℃のアルミブロック（アズワン社製、アルミブロック恒温槽CB-100A）で10分間の加温を行った。15000×gで10分間遠心を行って、得られた上清をグリコーゲン抽出液とした。
　グリコーゲン定量には、市販のキットを使用した（BioVision社製、Glycogen Assay Kit）。抽出液は10～1000倍に段階的に希釈し、適切な濃度（mg/L）の希釈液を測定に使用した。なお、グリコーゲン量に関するデータは、実験系列間の比較に適さない。 (3) Amount of glycogen in alga (mg/L) (amount of glycogen/culture solution)
(2) Glycogen extraction was performed on a sample obtained by freezing alga bodies in the same manner as the amount (g/L) of alga bodies in the culture medium. 400 μL of ion-exchanged water was added to the frozen algal body, mixed with a direct mixer, and heated for 10 minutes in a 95° C. aluminum block (Aluminum block constant temperature bath CB-100A, manufactured by AS ONE). After centrifugation at 15000×g for 10 minutes, the resulting supernatant was used as a glycogen extract.
A commercially available kit was used for glycogen quantification (Glycogen Assay Kit manufactured by BioVision). The extract was serially diluted 10- to 1000-fold, and the diluted solution with an appropriate concentration (mg/L) was used for the measurement. Data on glycogen content are not suitable for comparison between experimental series.
（４）グリコーゲン濃度／OD750nm（ｍｇ／Ｌ）
　（３）で得られた藻体中のグリコーゲン量（ｍｇ／Ｌ）を（１）で得られたOD750nmの値で割って算出した（データ示さず）。 (4) Glycogen concentration/OD750nm (mg/L)
It was calculated by dividing the amount of glycogen (mg/L) in the algal body obtained in (3) by the value of OD750nm obtained in (1) (data not shown).
（５）藻体中のグリコーゲン量／藻体量（ｍｇ／ｇ）＝藻体中のグリコーゲン濃度
　（３）で得られた藻体中のグリコーゲン量（ｍｇ／Ｌ）（グリコーゲン量／培養液）を（２）で得られた培養液中の藻体量（ｇ／Ｌ）（藻体量／培養液）で割って算出した。得られた値は藻体中のグリコーゲン濃度を意味する。 (5) Amount of glycogen in alga body/Amount of alga body (mg/g) = Glycogen concentration in alga body Amount of glycogen in alga body (mg/L) obtained in (3) (amount of glycogen/culture solution) was divided by the algal mass (g/L) in the culture solution obtained in (2) (alga mass/culture solution). The obtained value means the glycogen concentration in the algal body.
［結果］
（１）実験Ａ（グルコース濃度の検討、比較例１～２、実施例１～６）
　実験Ａの結果を表２に示す。 [result]
(1) Experiment A (examination of glucose concentration, Comparative Examples 1-2, Examples 1-6)
The results of Experiment A are shown in Table 2.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　表２に示されるとおり、炭素源であるグルコースの濃度を５．０％以上で培養した場合に、培養液中の藻体量が増加した。９～３０％のグルコース濃度で培養した場合（実施例３～６）、培養液中の藻体量が２０ｇ／Ｌを超え、培養液中の藻体量の増加が著しかった。特に、１８～２５％のグルコース濃度で培養した場合（実施例４、５）に培養液中の藻体量が多かった。また、炭素源であるグルコースの濃度を５．０％以上で培養した場合に、藻体量当たりのグリコーゲン量も増加した。 As shown in Table 2, the amount of algal bodies in the culture solution increased when the culture was performed at a concentration of glucose, which is a carbon source, of 5.0% or higher. When cultured at a glucose concentration of 9 to 30% (Examples 3 to 6), the amount of algae in the culture solution exceeded 20 g/L, and the increase in the amount of algae in the culture solution was remarkable. In particular, when cultured at a glucose concentration of 18 to 25% (Examples 4 and 5), the amount of algal bodies in the culture solution was large. In addition, the amount of glycogen per algal mass increased when the algae were cultured at a glucose concentration of 5.0% or more, which is a carbon source.
　実験Ａの結果から、炭素源を５％以上含む培地で培養することにより、イデユコゴメ綱に属する藻類の培養効率を改善できることが示された。また、炭素源を５％以上含む培地で培養することにより、イデユコゴメ綱に属する藻類からグリコーゲンの生産効率を改善できることが示された。 From the results of experiment A, it was shown that culturing in a medium containing 5% or more of the carbon source can improve the efficiency of culturing algae belonging to the class Idycogome. In addition, it was shown that culturing in a medium containing 5% or more of a carbon source can improve the production efficiency of glycogen from algae belonging to the class Idycogome.
（２）実験Ｂ（炭素源の検討、実施例７～８）
　実験Ｂの結果を表３に示す。 (2) Experiment B (Study of carbon source, Examples 7-8)
The results of Experiment B are shown in Table 3.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　表３に示されるとおり、実験Ｂでは、炭素源としてグルコース（実施例７）又はスクロース（実施例８）を用いて培養した場合に、同程度のＯＤ７５０値であった。この結果は、実施例７と実施例８では、イデユコゴメ綱に属する藻類が同程度増殖したことを示す。また、炭素源としてグリセロール（９％）を使用した場合も、イデユコゴメ綱に属する藻類の増殖が維持されることが示された。
　実験Ｂの結果から、イデユコゴメ綱に属する藻類の増殖効率に対する炭素源による向上効果に関して、炭素源の違いによる影響はないか、あったとしても小さいと考えられることが示された。 As shown in Table 3, Experiment B had similar OD750 values when cultured using glucose (Example 7) or sucrose (Example 8) as the carbon source. This result indicates that algae belonging to the class Idycogome grew to the same extent in Examples 7 and 8. It was also shown that the use of glycerol (9%) as a carbon source maintained the growth of algae belonging to the class Idycogome.
The results of Experiment B indicated that the effect of the carbon source on the growth efficiency of algae belonging to the class Idyucogome is considered to be small, if at all, due to differences in the carbon source.
（３）実験Ｃ（鉄イオンの効果確認、実施例９～１６）
　実験Ｃの結果を表４に示す。 (3) Experiment C (confirmation of the effect of iron ions, Examples 9 to 16)
The results of Experiment C are shown in Table 4.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　表４に示されるとおり、培地中の鉄イオン濃度を０．０５９２ｍＭ以上で培養した場合に、藻体量あたりの藻体中のグリコーゲン量が増加した。０．１１８４～５．９１９３ｍＭの鉄イオン濃度で培養した場合に（実施例１０～１６）、グリコーゲン量の増加が著しかった。
　実験Ｃの結果から、鉄イオン濃度を０．０５９２ｍＭ以上含む培地で培養することにより、イデユコゴメ綱に属する藻類のグリコーゲンの生産効率を向上できることが示された。 As shown in Table 4, the amount of glycogen in the alga per unit amount of alga increased when the culture was carried out at an iron ion concentration of 0.0592 mM or higher in the medium. When cultured at iron ion concentrations of 0.1184 to 5.9193 mM (Examples 10 to 16), the amount of glycogen increased significantly.
The results of Experiment C showed that culturing in a medium containing an iron ion concentration of 0.0592 mM or more can improve the glycogen production efficiency of algae belonging to the class Idycogome.
　本発明により、イデユコゴメ綱に属する藻類の培養方法が提供される。本発明の培養方法により、イデユコゴメ綱に属する藻類の藻体量が増加し、イデユコゴメ綱に属する藻類の培養効率が改善する。 The present invention provides a method for culturing algae belonging to the class Idycogome. The culturing method of the present invention increases the amount of algal bodies of algae belonging to the class Idyunicogome and improves the efficiency of culturing the algae belonging to the class Idyuteicogome.
　本発明の別の態様において、本発明の培養方法により、グリコーゲンの生産量が増加する。また、本発明により、グリコーゲンの生産方法が提供される。したがって、本発明によれば、イデユコゴメ綱に属する藻類によるグリコーゲンの生産効率が改善する。 In another aspect of the present invention, the culture method of the present invention increases glycogen production. The present invention also provides a method for producing glycogen. Therefore, according to the present invention, the efficiency of glycogen production by algae belonging to the class Idycogome is improved.
　本発明の別の態様において、イデユコゴメ綱に属する藻類の培養効率を改善させつつ、かつ、イデユコゴメ綱に属する藻類によるグリコーゲンの生産効率を改善させることができる。 In another aspect of the present invention, it is possible to improve the efficiency of culturing algae belonging to the class Idyucogome, and at the same time improve the efficiency of glycogen production by algae belonging to the class Idyucogome.
　本発明の別の態様において、イデユコゴメ綱に属する藻類の培養物を乾燥した乾燥体が提供される。乾燥体はグリコーゲンを多く含むことから、本発明により、藻類由来のバイオマス燃料の原料になり得るグリコーゲンの効率の良い製造が可能になる。 In another aspect of the present invention, there is provided a dried culture of algae belonging to the class Idycogome. Since the dried matter contains a large amount of glycogen, the present invention enables efficient production of glycogen that can be used as a raw material for biomass fuel derived from algae.

Claims (14)

  1. 　イデユコゴメ綱に属する藻類を、５～４０重量％の炭素源を含む培地で培養することを含む、イデユコゴメ綱に属する藻類を培養する方法。 A method for culturing algae belonging to the class Idyucogome, comprising culturing the algae belonging to the class Idyucogome in a medium containing 5 to 40% by weight of a carbon source.
  2. 　前記炭素源が、単糖類、二糖類又は糖アルコールである、請求項１に記載の方法。 The method according to claim 1, wherein the carbon source is a monosaccharide, disaccharide or sugar alcohol.
  3. 　培養が回分培養である、請求項１又は２に記載の方法。 The method according to claim 1 or 2, wherein the culture is batch culture.
  4. 　培地が０．０７～１０ｍＭの鉄イオンをさらに含む、請求項１～３のいずれか一項記載の方法。 The method according to any one of claims 1 to 3, wherein the medium further contains 0.07 to 10 mM iron ions.
  5. 　請求項１～３いずれか一項記載の培養方法によりグリコーゲンを生産する方法。 A method for producing glycogen by the culture method according to any one of claims 1 to 3.
  6. 　培養物からグリコーゲンを回収することをさらに含む、請求項５に記載の方法。 The method according to claim 5, further comprising recovering glycogen from the culture.
  7. 　グリコーゲンが、培養物中のイデユコゴメ綱に属する藻類に対し１０ｍｇ／ｇ以上含まれる、請求項５又は６に記載の方法。 The method according to claim 5 or 6, wherein glycogen is contained at 10 mg/g or more for algae belonging to the class Idycogome in the culture.
  8. 　請求項１～７のいずれか一項の方法で得られるイデユコゴメ綱に属する藻類の培養物を乾燥した、イデユコゴメ綱に属する藻類の乾燥物。 A dried product of algae belonging to the class Idycogome, which is obtained by drying the culture of algae belonging to the class Ideucogome obtained by the method according to any one of claims 1 to 7.
  9. 　イデユコゴメ綱に属する藻類を、５～４０重量％の炭素源を含む培地で培養することを含む、イデユコゴメ綱に属する藻類にグリコーゲンを生産させる方法。 A method for producing glycogen in algae belonging to the class Idyucogome, comprising culturing the algae belonging to the class Idyucogome in a medium containing 5 to 40% by weight of a carbon source.
  10. 　前記炭素源が、単糖類、二糖類又は糖アルコールである、請求項９に記載の方法。 The method according to claim 9, wherein the carbon source is a monosaccharide, disaccharide or sugar alcohol.
  11. 　培地が０．０７～１０ｍＭの鉄イオンをさらに含む、請求項９又は１０に記載の方法。 The method according to claim 9 or 10, wherein the medium further contains 0.07-10 mM iron ions.
  12. 　グリコーゲンが、培養物中のイデユコゴメ綱に属する藻類に対し１０ｍｇ／ｇ以上含まれる、請求項９～１１のいずれか一項に記載の方法。 The method according to any one of claims 9 to 11, wherein glycogen is contained at 10 mg/g or more for algae belonging to the class Idycogome in the culture.
  13. 　５～４０重量％の炭素源を含む、イデユコゴメ綱に属する藻類を培養するための培地。 A medium for culturing algae belonging to the class Idycogome, containing 5 to 40% by weight of carbon source.
  14. 　０．０７～１０ｍＭの鉄イオンをさらに含む、請求項１３に記載の培地。 The medium according to claim 13, further comprising 0.07-10 mM iron ions.
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Publication number Priority date Publication date Assignee Title
WO2017050917A1 (en) * 2015-09-25 2017-03-30 Fermentalg Novel method for the culture of unicellular red algae
WO2020161280A1 (en) * 2019-02-08 2020-08-13 Fermentalg Optimized method for industrial exploitation of unicellular red algae

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Publication number Priority date Publication date Assignee Title
WO2017050917A1 (en) * 2015-09-25 2017-03-30 Fermentalg Novel method for the culture of unicellular red algae
WO2020161280A1 (en) * 2019-02-08 2020-08-13 Fermentalg Optimized method for industrial exploitation of unicellular red algae

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