WO2022255477A1 - Shewanella bacteria and use thereof - Google Patents

Shewanella bacteria and use thereof Download PDF

Info

Publication number
WO2022255477A1
WO2022255477A1 PCT/JP2022/022593 JP2022022593W WO2022255477A1 WO 2022255477 A1 WO2022255477 A1 WO 2022255477A1 JP 2022022593 W JP2022022593 W JP 2022022593W WO 2022255477 A1 WO2022255477 A1 WO 2022255477A1
Authority
WO
WIPO (PCT)
Prior art keywords
strain
shewanella
mutant
medium
epa
Prior art date
Application number
PCT/JP2022/022593
Other languages
French (fr)
Japanese (ja)
Inventor
史明 祖父江
広顕 山崎
遼太郎 光増
Original Assignee
Dic株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dic株式会社 filed Critical Dic株式会社
Priority to JP2023525927A priority Critical patent/JP7468788B2/en
Publication of WO2022255477A1 publication Critical patent/WO2022255477A1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/36—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9728—Fungi, e.g. yeasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q1/00—Make-up preparations; Body powders; Preparations for removing make-up
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q1/00—Make-up preparations; Body powders; Preparations for removing make-up
    • A61Q1/02—Preparations containing skin colorants, e.g. pigments
    • A61Q1/04—Preparations containing skin colorants, e.g. pigments for lips
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q1/00—Make-up preparations; Body powders; Preparations for removing make-up
    • A61Q1/02—Preparations containing skin colorants, e.g. pigments
    • A61Q1/08—Preparations containing skin colorants, e.g. pigments for cheeks, e.g. rouge
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q1/00—Make-up preparations; Body powders; Preparations for removing make-up
    • A61Q1/02—Preparations containing skin colorants, e.g. pigments
    • A61Q1/10—Preparations containing skin colorants, e.g. pigments for eyes, e.g. eyeliner, mascara
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q5/00—Preparations for care of the hair
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q5/00—Preparations for care of the hair
    • A61Q5/02—Preparations for cleaning the hair
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q5/00—Preparations for care of the hair
    • A61Q5/12—Preparations containing hair conditioners
    • 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
    • C12N1/20—Bacteria; Culture media therefor
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • the present invention relates to Shewanella bacteria and uses thereof.
  • it relates to new strains of Shewanella udii.
  • It also relates to compositions, dry powders and extracts comprising said novel strains.
  • Eicosapentaenoic acid is a type of omega-3 fatty acid that has anti-obesity and neutral lipid-lowering effects.
  • EPA is known to be abundant in fish.
  • the fish may contain bioaccumulated heavy metal components, and there is concern about health hazards due to heavy metals. It may also lead to overexploitation of fish, a limited marine resource.
  • Non-Patent Documents 1 to 3 Marine bacteria such as the genus Shewanella are known to produce fatty acids such as EPA (eg, Patent Document 1, Non-Patent Documents 1 to 3).
  • Shewanella bacteria often do not grow well unless a medium containing animal-derived components is used (Non-Patent Documents 3 and 4). Therefore, industrial production of EPA using Shewanella bacteria has not been realized.
  • Shewanella bacteria with high EPA-producing ability can grow satisfactorily in a completely synthetic medium containing glucose and the like as the sole organic carbon source.
  • the present invention provides a Shewanella bacterium that grows well on glucose as the sole organic carbon source and has a high EPA-producing ability, a composition containing the Shewanella bacterium, and a dry powder and extract of the Shewanella bacterium.
  • the task is to provide
  • Shewanella udii strain GS001 (accession number NITE BP-03460) or a mutant strain thereof.
  • a composition comprising the Shewanella udii GS001 strain or a mutant strain thereof.
  • the composition according to [2] which is a food, feed, feed, or cosmetic.
  • a Shewanella bacterium that grows well on glucose as the sole organic carbon source and has a high EPA-producing ability
  • a composition comprising the Shewanella bacterium, and a dry powder and extract of the Shewanella bacterium is provided.
  • FIG. 5 shows the results of culturing an isolate of Shewanella woodyi (strain GS001) using 50 mL of modified M9+glucose (Glc) liquid medium placed in a 100 mL Erlenmeyer flask. Shown are the results of phylogenetic analysis of Shewanella udii strain GS001 based on the 16S rDNA sequence. Shown are the results of a culture test of Shewanella udii strain GS001 using a jar fermenter. Shown are the results of measuring the EPA content per dry cell weight in a culture test of Shewanella udii strain GS001 using a jar fermenter.
  • Fig. 2 shows the results of examining the effect of NaCl concentration on the growth of strain GS001.
  • Fig. 3 shows the results of a growth test in modified M9 + Glc liquid medium using Shewanella udii strain GS001 or Shewanella electrodiphila ATCC BAA-2408.
  • the term “comprise” means that it may include components other than the target components.
  • the term “consist of” means containing no elements other than the subject element.
  • the term “consistently of” means that it does not include constituent elements other than the subject constituent elements in a mode that exhibits a special function (such as a mode that completely loses the effects of the invention). means.
  • the term “comprise” includes aspects that "consist of” and aspects that "consist essentially of.”
  • Bacteria can be isolated. "Isolated” means separated from the natural state or other components. “Isolated” can be substantially free of other components. “Substantially free of other components” means that the content of other components contained in the isolated component is negligible. The content of other components contained in the isolated component is, for example, 10% by mass or less, 5% by mass or less, 4% by mass or less, 3% by mass or less, 2% by mass or less, 1% by mass or less, 0.5% by mass or less. It may be 5% by mass or less, or 0.1% by mass or less.
  • a bacterium described herein can be an isolated bacterium.
  • “Growing using glucose as the sole organic carbon source” means growing in a medium containing only glucose as the organic carbon source.
  • a “synthetic medium” refers to a medium in which the chemical components contained in the medium are defined. Synthetic media are prepared by mixing chemical components with well-defined chemical names.
  • a “semi-synthetic medium” refers to a medium obtained by adding a biological substance, which is a mixture of substances whose chemical composition is unclear, to a synthetic medium. Biological substances include yeast extract, peptone, casamino acid, meat extract and the like.
  • eicosapentaenoic acid or "EPA” includes eicosapentaenoic acid (free form) and its derivatives.
  • Derivatives of eicosapentaenoic acid include esterified type, phospholipid-linked type, phosphatidylglycerol-linked type, monoglyceride type, diglyceride type, triglyceride type and salts thereof.
  • the invention provides Shewanella woodyi strain GS001 (Accession No. NITE BP-03460) or a variant thereof.
  • the Shewanella udii strain GS001 (hereinafter referred to as "GS001 strain") is a strain isolated from the intestinal tract of Nigisu, a deep-sea fish.
  • the GS001 strain was identified as Shewanella udii by phylogenetic analysis based on 16S rDNA sequences (see Figure 2).
  • the 16S rDNA sequence of strain GS001 is shown in SEQ ID NO:1.
  • the GS001 strain is characterized by being able to grow well on glucose as the sole organic carbon source.
  • the GS001 strain was cultured with shaking (150 rpm) at 15°C using a 100 mL flask containing 50 mL of a modified M9 liquid medium containing 10% (w/v) glucose (modified M9 + Glc liquid medium) for 48 hours. It can grow to a cell density of 5 or more at OD600nm .
  • the GS001 strain is characterized by being able to grow well in a semi-synthetic medium containing glucose and yeast extract. For example, the GS001 strain is aerated at 15° C.
  • the GS001 strain is characterized by a high EPA content. For example, the GS001 strain is aerated at 15 ° C.
  • the GS001 strain is characterized by being able to grow in sodium chloride concentrations as low as 0.5% (w/v). For example, the GS001 strain was cultured with shaking (150 rpm) at 15°C in a 100 mL flask containing 50 mL of modified M9 + Glc medium with a sodium chloride concentration of 0.5% (w/v). 600 nm can grow to a cell density of 3 or more. Since the GS001 strain has the characteristics described above, it is suitable for industrial production of EPA.
  • the GS001 strain was designated as accession number NITE P-03460 on April 15, 2021 by the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan Room 122). and has been transferred to the International Deposit on April 4, 2022 under accession number NITE BP-03460.
  • Name of Depositor Gaku Ebara Address of Depositor: 631 Sakado, Sakura City, Chiba Prefecture, Japan
  • the Depositor authorizes Applicant to refer to the deposited organism in this application. The depositor has given consent to the applicant that the deposited organism will be made available to the public.
  • a “mutant strain” refers to a strain in which a mutation has occurred in the genome of the original strain. Mutations may be naturally occurring or artificially occurring. The method of artificially mutating the genome is not particularly limited. Techniques for artificially generating mutations include, for example, irradiation with high-energy electromagnetic waves such as ultraviolet irradiation and radiation irradiation; chemical treatment with nitrous acid; genetic engineering techniques such as gene transfer and genome editing.
  • the mutant strain has a ratio of mutation to the entire genome of the original strain, for example, 10% or less, 5% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.3% or less, Alternatively, it is preferably 0.1% or less.
  • the mutant 16S rDNA sequence may have the nucleotide sequence set forth in SEQ ID NO:1.
  • the mutant strain of the GS001 strain (hereinafter referred to as "GS001 mutant strain") has a specific growth rate when cultured with glucose as the sole organic carbon source that is equal to or greater than that of the GS001 strain cultured under the same conditions. is preferred.
  • the specific growth rate of the GS001 mutant is 0.7 (or 0.75, 0.8, 0.85, 0.9.0) to that of the GS001 strain. It is preferably 0.95, 0.97, 0.98, 0.99, or a value multiplied by 1).
  • the GS001 mutant has an OD 600 nm of 5 or more in 48 hours when cultured with shaking (150 rpm) at 15 ° C. using a 100 mL flask containing 30 mL of a modified M9 liquid medium containing 10% glucose (modified M9 + Glc medium). It is preferable to grow to a bacterial density of
  • the GS001 mutant has a specific growth rate when cultured in a semi-synthetic medium containing glucose and yeast extract that is equal to or greater than that of the GS001 strain cultured under the same conditions.
  • the specific growth rate of the GS001 mutant is 0.7 (or 0.75, 0.8, 0.85, 0.9.0) to that of the GS001 strain. It is preferably 0.95, 0.97, 0.98, 0.99, or a value multiplied by 1).
  • the GS001 mutant strain was cultured at 15° C.
  • the GS001 mutant strain has an EPA content per dry cell weight when cultured using glucose as the sole organic carbon source, which is about the same as the GS001 strain cultured under the same conditions.
  • the EPA content per dry cell weight of the GS001 mutant strain is 0.7 (or 0.75, 0.8, 0.85, It is preferably about 0.9, 0.95, 0.97, 0.98, 0.99, or a value obtained by multiplying 1).
  • the GS001 mutant strain preferably has an EPA content per dry cell weight when cultured in a semi-synthetic medium containing glucose and yeast extract, which is about the same as the GS001 strain cultured under the same conditions.
  • the EPA content per dry cell weight of the GS001 mutant strain is 0.7 (or 0.75, 0.8, 0.85, It is preferably about 0.9, 0.95, 0.97, 0.98, 0.99, or a value obtained by multiplying 1).
  • the GS001 mutant strain was cultured under aerobic conditions at 15° C.
  • the GS001 mutant can grow in media containing 0.5% (w/v) sodium chloride.
  • the specific growth rate when cultured in a medium containing glucose as the sole organic carbon source and containing 0.5% (w/v) sodium chloride is equal to or higher than that of the GS001 strain cultured under the same conditions. is preferred.
  • the specific growth rate of the GS001 mutant is 0.7 (or 0.75, 0.8, 0.85, 0.9.0) to that of the GS001 strain. It is preferably 0.95, 0.97, 0.98, 0.99, or a value multiplied by 1).
  • the GS001 mutant strain was cultured with shaking (150 rpm) at 15 ° C.
  • the GS001 strain and the GS001 mutant strain can be cultured using a medium commonly used for culturing heterotrophic bacteria.
  • the medium include those containing organic carbon sources, nitrogen sources, phosphorus sources, trace elements (zinc, boron, cobalt, copper, manganese, molybdenum, iron, nickel, etc.) and the like.
  • Organic carbon sources include, for example, glucose, dextran, starch, yeast extract and the like.
  • Nitrogen sources include, for example, ammonium salts, nitrates, nitrites, amino acids, peptones, and the like.
  • Phosphorus sources include, for example, phosphates and the like.
  • the medium may be, for example, a minimal medium such as M9 medium or a modified M9 medium (modified M9 medium, etc.) added with an organic carbon source.
  • a preferred organic carbon source is glucose.
  • preferred organic carbon sources include yeast extract.
  • the semi-synthetic medium may contain glucose and yeast extract as organic carbon sources.
  • the medium preferably contains 0.5-5% (w/v) sodium chloride. More preferably, the sodium chloride concentration of the medium is 0.5-3% (w/v).
  • the medium may be a solid medium or a liquid medium.
  • a solid medium For maintenance, it is preferred to use a solid medium.
  • a liquid medium For growth, it is preferable to use a liquid medium.
  • the pH of the medium includes, for example, pH 6-9.
  • the pH of the medium is, for example, preferably pH 7-8.5, more preferably pH 7.5-8.5.
  • the culture temperature is 0 to 30°C.
  • the culture temperature is preferably 10 to 30°C, more preferably 12 to 20°C, even more preferably 15 to 18°C.
  • the culture may be static culture, aerobic culture, or shaking culture. From the viewpoint of preventing oxygen deficiency, aerobic culture or shaking culture is preferred.
  • the GS001 strain or GS001 mutant may be passaged as appropriate during the culture period.
  • the subculture interval is, for example, half a month to one month.
  • passage intervals include, for example, 2-10 days, or 2-5 days.
  • the GS001 strain and the GS001 mutant strain contain a high content of EPA in the cells. Therefore, the GS001 strain or GS001 mutant strain can be used for the production of EPA.
  • EPA can be extracted from the GS001 strain or the GS001 mutant strain by a known method. For example, cells are collected from the culture medium of the GS001 strain and the GS001 mutant strain by centrifugation, filtration, or the like, and all lipids are extracted from the cells with an organic solvent such as chloroform, methanol, or toluene.
  • the amount of extraction solvent used for cells is not particularly limited.
  • the amount of extraction solvent can be, for example, about 1 to 1000 times (preferably about 5 to 200 times) the amount of cells.
  • the extraction operation can usually be carried out under normal pressure in the range from room temperature to the boiling point of the solvent.
  • the extraction operation may be performed only once or may be performed multiple times. For example, a fresh extraction solvent may be added again to the cell residue that has been subjected to the extraction operation once, or the extraction operation may be performed again.
  • cell debris may be removed by centrifugation, filtration, ultrafiltration, or the like.
  • the extraction solvent may be removed by heating or distillation under reduced pressure using an evaporator or the like.
  • various purification treatments may be performed to purify EPA.
  • Purification treatments include, for example, salting out, dialysis, recrystallization, reprecipitation, solvent extraction, adsorption, concentration, filtration, gel filtration, ultrafiltration, various chromatography (thin layer chromatography, column chromatography, ion exchange chromatography, high-performance liquid chromatography, adsorption chromatography, etc.), but are not limited thereto.
  • a specific example of the method for extracting EPA from the GS001 strain or the GS001 mutant strain is the method described in Examples below.
  • EPA can be used as nutritional supplements, food additives, etc.
  • composition comprising Shewanella udii strain GS001 or a variant thereof.
  • the GS001 strain and GS001 mutant contain useful ingredients such as EPA. Therefore, the GS001 strain or the GS001 mutant strain can be used in various compositions.
  • compositions include, for example, foods, feeds, feeds, cosmetics, and the like.
  • Feeds include, for example, various pet foods.
  • the feed includes, for example, feed for ornamental fish, feed for cultured fish, and the like.
  • Cosmetics include, for example, skin cosmetics (lotions, milky lotions, serums, creams, etc.), hair cosmetics (hair styling, shampoos, rinses, conditioners, etc.), makeup cosmetics (foundations, cheeks, eye shadows, lipstick, etc.).
  • the GS001 strain or GS001 mutant strain can be added as an additive to the various compositions described above. In addition, it may be mixed with other ingredients and prepared as foods, feeds, feeds, cosmetics, and the like. Other components can be appropriately selected depending on the use of the composition.
  • ingredients that can be used for food, feed, or feed can be used without particular limitation.
  • ingredients that can be used for foods, feeds, or feeds include fish meat, vegetables, grains, dairy products, fermented products, spices, proteins, amino acids, sugars, various seasonings, sweeteners, corrigents, flavors, Oils and fats, vitamins, thickeners, gelling agents, antioxidants, preservatives, chelating agents, pH adjusters, coloring agents, etc., but not limited to these.
  • any ingredient that can be used in cosmetics can be used without any particular restrictions.
  • ingredients that can be used in cosmetics include hydrocarbons, lipids (oils such as animal and vegetable oils and mineral oils, waxes, fatty acid esters, fatty acids, ceramides, etc.), alcohols (higher alcohols, lower alcohols and polyhydric alcohols).
  • proteins collagen, etc.
  • polysaccharides hyaluronic acid, chitosan, cellulose, xanthan gum, etc.
  • various high-molecular compounds polyethylene glycol, silicone, etc.
  • surfactants UV absorbers, whitening agents, anti-inflammatory agents, blood circulation promoters, anti-aging agents, moisturizers, vitamins, thickeners, gelling agents, antioxidants, preservatives, antibacterial agents, chelates agents, pH adjusters, powders, fragrances, colorants, etc., but are not limited to these.
  • the invention provides a dry powder of strain GS001 or GS001 variant.
  • GS001 strain dry powder refers to GS001 strain cells dried and powdered.
  • dry powder of GS001 mutant refers to a powder obtained by drying GS001 mutant cells.
  • a dry powder of the GS001 strain or GS001 mutant can be produced by a known method. For example, GS001 strain or GS001 mutant strain is cultured, and cells are collected by centrifugation or the like. A dry powder can then be obtained by drying the cells and pulverizing them.
  • a method for drying the cells is not particularly limited, and examples thereof include natural drying, heat drying, reduced pressure drying, freeze drying and the like. Dried cells may be physically pulverized into a powder.
  • the cells may be subjected to washing treatment, sterilization treatment, etc. before drying.
  • a washing solution such as water or a buffer solution can be used.
  • the sterilization method include sterilization with hypochlorous acid, ultraviolet treatment, ozone treatment, heat treatment, and the like.
  • the GS001 strain or GS001 mutant dry powder can be used as a food, feed, or fodder.
  • the dry powder of the GS001 strain or the GS001 mutant can be used as an additive in foods, feeds, feeds, or cosmetics.
  • the invention provides an extract of the GS001 strain or GS001 mutant strain.
  • GS001 strain extract refers to a cell component extracted from GS001 strain cells.
  • GS001 mutant extract refers to the extraction of cell components from GS001 mutant cells.
  • the extract of the GS001 strain or the GS001 mutant strain may be a cell disruption product obtained by destroying the cells of the GS001 strain or the GS001 mutant strain. Methods for disrupting cells include, for example, mechanical disruption using a homogenizer or the like, ultrasonic treatment, freeze-thaw treatment, and the like.
  • the extract of the GS001 strain or the GS001 mutant strain may be a cell lysate obtained by lysing the cells of the GS001 strain or the GS001 mutant strain.
  • Methods for lysing cells include, for example, enzymatic treatment using enzymes such as protease and lysozyme, surfactant treatment, and the like.
  • the extract of the GS001 strain or the GS001 mutant strain may be obtained by adding an extraction solvent to the cells, cell disruptions, or cell lysates of the GS001 strain or the GS001 mutant strain, and performing an extraction treatment. .
  • the same extraction solvent as mentioned above can be used.
  • the GS001 strain or the GS001 mutant strain extract can be used as food, feed, or fodder. Moreover, the GS001 strain or the GS001 mutant strain extract can be used as an additive in foods, feeds, feeds, or cosmetics.
  • the present disclosure provides a method of producing EPA comprising culturing the GS001 strain or GS001 mutant strain. In one embodiment, the present disclosure provides a method for producing EPA, comprising culturing strain GS001 or a mutant strain GS001 in a medium containing glucose as an organic carbon source. In one embodiment, the present disclosure comprises culturing the GS001 strain or the GS001 mutant strain in a medium containing glucose as an organic carbon source, recovering cells of the GS001 strain or the GS001 mutant strain, and removing EPA from the cells. and extracting the EPA.
  • the present disclosure provides a method for culturing strain GS001 or GS001 mutant comprising culturing strain GS001 or GS001 mutant in a medium containing glucose as an organic carbon source. In one embodiment, the present disclosure provides a method for producing a GS001 strain or a GS001 mutant comprising culturing the GS001 strain or the GS001 mutant in a medium containing glucose as an organic carbon source.
  • the above-mentioned subculture was carried out for about 6 months, and strains with good growth in modified M9+Glc liquid medium and high EPA content were selected.
  • the GS001 strain was obtained as a strain that grew well in a modified M9+Glc liquid medium and had a high EPA content.
  • Tables 1-3 show the composition of the modified M9+Glc liquid medium.
  • the modified M9+Glc medium is M9 minimal medium supplemented with Fe solution (Table 2), Trace Metal solution (Table 3), and glucose.
  • FIG. 1 shows the results of growing the GS001 strain under the same culture conditions as above using 50 mL of modified M9+Glc liquid medium placed in a 100 mL Erlenmeyer flask.
  • the GS001 strain reached an OD 600 of about 5 to 6 about 48 hours after the start of culture, showing good growth.
  • FIG. 2 shows the results of phylogenetic analysis of the GS001 strain based on the 16S rDNA sequence. Phylogenetic analysis was performed by Satoi et al. (Masataka Satoi et al., International Journal of Systematic and Evolutionary Microbiology (2003), 53, 491-499.) and Zhang et al.
  • ⁇ Culture of GS001 strain in semi-synthetic medium A modified M9+Glc liquid medium containing 0.5% (w/v) yeast extract (M9+Glc+YE liquid medium) was used as a semi-synthetic medium. 2 L of modified M9+Glc+Y. E.
  • the GS001 strain was inoculated into the liquid medium and cultured. The culture temperature was set to 15° C., and aeration culture (1 vvm air, 150 to 750 rpm) was performed. Cultivation was performed using two jar fermenters.
  • the cap was tightly tightened, and the test tube was heated at 100° C. for 5 minutes using a heat block or the like. After that, the test tube was sufficiently cooled again. 1 mL of hexane and saturated saline were added to the test tube and thoroughly mixed. The mixed sample solution with the saturated saline solution was centrifuged at 1450 ⁇ g for 5 minutes at room temperature, and an appropriate amount of the obtained upper layer (organic layer) was placed in a vial and subjected to gas chromatography (GC) analysis.
  • GC gas chromatography
  • Figure 3A shows the change in OD600 and dry cell weight.
  • Figure 3B shows the EPA content per dry cell weight.
  • OD J1 and DCW J1 indicate OD 600 and dry cell weight in the first jar fermenter, respectively.
  • OD J2 and DCW J2 indicate the OD 600 and dry cell weight in the second jar fermenter, respectively.
  • the GS001 strain reached a dry cell weight of 35 g/L or more in any jar fermenter and showed good growth. It should be noted that the dry cell weight reached by Shewanella bacteria reported in the past is about 10.3 to 10.5 g/L.
  • J1 indicates the EPA content per dry cell weight in the first jar fermenter.
  • J2 indicates the EPA content per dry cell weight in the second jar fermenter.
  • the GS001 strain showed a high EPA content.
  • the results are shown in Figure 4.
  • the GS001 strain was able to grow in a NaCl concentration range of 0.5-5% (w/v).
  • the GS001 strain grew well, especially in the NaCl concentration range of 0.5-3% (w/v).
  • Tables 4 and 5 summarize the effect of NaCl concentration on the growth of previously reported strains of Shewanella udii (Esra Ersoy Omeroglu et al., Folia Microbiol (2014) 59:79-92) and the GS001 strain.
  • data other than GS001 strain are quoted from Table 2 in Esra Ersoy Omeroglu et al., Folia Microbiol (2014) 59:79-92.
  • "+" indicates growth
  • "-" indicates non-growth
  • N.D.” indicates non-implementation.
  • the GS001 strain can grow even at a NaCl concentration of 0.5% (w/v).
  • no other Shewanella udii strains have been reported that can grow at a NaCl concentration of 0.5% (w/v). From this result, it was confirmed that the GS001 strain can grow at a lower salt concentration than the previously reported Shewanella udii strains.
  • GS001 strain or Shewanella electrodiphila ATCC BAA-2408 (hereinafter, "ATCC BAA-2408") was precultured in a modified M9 + Glc liquid medium for 48 hours (culture temperature 15 ° C., shaking culture (rotation speed: 150 rpm).
  • the precultured strain GS001 or ATCC BAA-2408 was inoculated into 50 mL modified M9+Glc liquid medium in a 100 mL flask and cultured.
  • the culture temperature was set to 15° C., and shaking culture was performed using a bioshaker (rotation speed: 150 rpm).
  • Shewanella electrodyphila is a bacterium of the genus Shewanella that has been reported to grow well using glucose as the sole organic carbon source (Jinwei Zhang, and J. Grant Burgess. PLoS One. 2017 Nov 27;12(11 ):).
  • the growth rate of Shewanella electrodyphila when glucose is the sole organic carbon source is an index of glucose utilization.
  • the results are shown in Figure 5.
  • the GS001 strain had a shorter induction period and a faster growth rate than ATCC BAA-2408. From this result, it was confirmed that the GS001 strain has a higher ability to assimilate glucose compared to ATCC BAA-2408.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Mycology (AREA)
  • Dermatology (AREA)
  • Biochemistry (AREA)
  • Birds (AREA)
  • Epidemiology (AREA)
  • Food Science & Technology (AREA)
  • Botany (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Emergency Medicine (AREA)
  • Polymers & Plastics (AREA)
  • Medicinal Chemistry (AREA)
  • Virology (AREA)
  • Nutrition Science (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

Shewanella woodyi strain GS001 (accession number NITE BP-03460) or mutant strains thereof. A composition containing shewanella woodyi strain GS001 or mutant strains thereof. A dry powder or extract of shewanella woodyi strain GS001 or mutant strains thereof.

Description

シェワネラ属細菌、及びその使用Shewanella bacteria and uses thereof
　本発明は、シェワネラ属細菌、及びその使用に関する。特に、シェワネラ・ウーディイの新規菌株に関する。また、前記新規菌株を含む組成物、乾燥粉末、及び抽出物に関する。
　本願は、２０２１年６月４日に、日本に出願された特願２０２１－０９４３８２号に基づき優先権を主張し、その内容をここに援用する。 The present invention relates to Shewanella bacteria and uses thereof. In particular, it relates to new strains of Shewanella udii. It also relates to compositions, dry powders and extracts comprising said novel strains.
This application claims priority based on Japanese Patent Application No. 2021-094382 filed in Japan on June 4, 2021, the contents of which are incorporated herein.
　エイコサペンタエン酸（ＥＰＡ）は、抗肥満効果及び中性脂質低下効果等を有するオメガ３脂肪酸の一種である。ＥＰＡは、魚に多く含まれることが知られている。しかしながら、魚からＥＰＡを製造する場合、魚に生物濃縮された重金属成分が含まれている可能性があり、重金属による健康被害が懸念される。また、限りある海洋資源である魚の乱獲にもつながる可能性がある。 Eicosapentaenoic acid (EPA) is a type of omega-3 fatty acid that has anti-obesity and neutral lipid-lowering effects. EPA is known to be abundant in fish. However, when EPA is produced from fish, there is a possibility that the fish may contain bioaccumulated heavy metal components, and there is concern about health hazards due to heavy metals. It may also lead to overexploitation of fish, a limited marine resource.
　シェワネラ（Ｓｈｅｗａｎｅｌｌａ）属等の海洋細菌は、ＥＰＡ等の脂肪酸を生産することが知られている（例えば、特許文献１、非特許文献１～３）。しかしながら、シェワネラ属細菌は、動物由来成分を含む培地を用いないと、良好に増殖しないことが多い（非特許文献３、４）。そのため、シェワネラ属細菌を用いたＥＰＡの工業生産は実現していない。 Marine bacteria such as the genus Shewanella are known to produce fatty acids such as EPA (eg, Patent Document 1, Non-Patent Documents 1 to 3). However, Shewanella bacteria often do not grow well unless a medium containing animal-derived components is used (Non-Patent Documents 3 and 4). Therefore, industrial production of EPA using Shewanella bacteria has not been realized.
特公平７－６１２７２号公報Japanese Patent Publication No. 7-61272
　シェワネラ属細菌を用いてＥＰＡを工業生産するためには、高いＥＰＡ生産能を有するシェワネラ属細菌が、グルコース等を唯一の有機炭素源として含む完全合成培地で、良好に増殖できることが好ましい。 In order to industrially produce EPA using Shewanella bacteria, it is preferable that Shewanella bacteria with high EPA-producing ability can grow satisfactorily in a completely synthetic medium containing glucose and the like as the sole organic carbon source.
　そこで、本発明は、グルコースを唯一の有機炭素源として良好に増殖し、且つ高いＥＰＡ生産能を有するシェワネラ属細菌、前記シェワネラ属細菌を含む組成物、並びに前記シェワネラ属細菌の乾燥粉末及び抽出物を提供することを課題とする。 Accordingly, the present invention provides a Shewanella bacterium that grows well on glucose as the sole organic carbon source and has a high EPA-producing ability, a composition containing the Shewanella bacterium, and a dry powder and extract of the Shewanella bacterium. The task is to provide
　本開示は、以下の態様を含む。
［１］シェワネラ・ウーディイ　ＧＳ００１株（受託番号ＮＩＴＥ　ＢＰ－０３４６０）又はその変異株。
［２］シェワネラ・ウーディイ　ＧＳ００１株又はその変異株を含む、組成物。
［３］食品、飼料、又は餌料、又は化粧料である、［２］に記載の組成物。
［４］シェワネラ・ウーディイ　ＧＳ００１株又はその変異株の乾燥粉末。
［５］シェワネラ・ウーディイ　ＧＳ００１株又はその変異株の抽出物。 The present disclosure includes the following aspects.
[1] Shewanella udii strain GS001 (accession number NITE BP-03460) or a mutant strain thereof.
[2] A composition comprising the Shewanella udii GS001 strain or a mutant strain thereof.
[3] The composition according to [2], which is a food, feed, feed, or cosmetic.
[4] Dry powder of Shewanella udii strain GS001 or a mutant strain thereof.
[5] An extract of Shewanella udii strain GS001 or a mutant strain thereof.
　本開示によれば、グルコースを唯一の有機炭素源として良好に増殖し、且つ高いＥＰＡ生産能を有するシェワネラ属細菌、前記シェワネラ属細菌を含む組成物、並びに前記シェワネラ属細菌の乾燥粉末及び抽出物が提供される。 According to the present disclosure, a Shewanella bacterium that grows well on glucose as the sole organic carbon source and has a high EPA-producing ability, a composition comprising the Shewanella bacterium, and a dry powder and extract of the Shewanella bacterium is provided.
１００ｍＬ三角フラスコに入れた５０ｍＬの改変Ｍ９＋グルコース（Ｇｌｃ）液体培地を用いて、シェワネラ・ウーディイ（Ｓｈｅｗａｎｅｌｌａ　ｗｏｏｄｙｉ）の単離株（ＧＳ００１株）を培養した結果を示す。5 shows the results of culturing an isolate of Shewanella woodyi (strain GS001) using 50 mL of modified M9+glucose (Glc) liquid medium placed in a 100 mL Erlenmeyer flask. １６ＳｒＤＮＡ配列に基づく、シェワネラ・ウーディイ　ＧＳ００１株の系統解析の結果を示す。Shown are the results of phylogenetic analysis of Shewanella udii strain GS001 based on the 16S rDNA sequence. ジャーファーメンターを用いた、シェワネラ・ウーディイ　ＧＳ００１株の培養試験の結果を示す。Shown are the results of a culture test of Shewanella udii strain GS001 using a jar fermenter. ジャーファーメンターを用いた、シェワネラ・ウーディイ　ＧＳ００１株の培養試験において、乾燥細胞重量当たりのＥＰＡ含有量を測定した結果を示す。Shown are the results of measuring the EPA content per dry cell weight in a culture test of Shewanella udii strain GS001 using a jar fermenter. ＧＳ００１株の増殖に対するＮａＣｌ濃度の影響を調べた結果を示す。Fig. 2 shows the results of examining the effect of NaCl concentration on the growth of strain GS001. シェワネラ・ウーディイ　ＧＳ００１株又はシェワネラ・エレクトロディフィラ（Ｓｈｅｗａｎｅｌｌａ　ｅｌｅｃｔｒｏｄｉｐｈｉｌａ）ＡＴＣＣ　ＢＡＡ－２４０８を用いた改変Ｍ９＋Ｇｌｃ液体培地での増殖試験の結果を示す。Fig. 3 shows the results of a growth test in modified M9 + Glc liquid medium using Shewanella udii strain GS001 or Shewanella electrodiphila ATCC BAA-2408.
　「を含む」（ｃｏｍｐｒｉｓｅ）という用語は、対象となる構成要素以外の構成要素を含んでいてもよいことを意味する。「からなる」（ｃｏｎｓｉｓｔ　ｏｆ）という用語は、対象となる構成要素以外の構成要素を含まないことを意味する。「から本質的になる」（ｃｏｎｓｉｓｔ　ｅｓｓｅｎｔｉａｌｌｙ　ｏｆ）という用語は、対象となる構成要素以外の構成要素を特別な機能を発揮する態様（発明の効果を完全に喪失させる態様など）では含まないことを意味する。本明細書において、「を含む」（ｃｏｍｐｒｉｓｅ）と記載する場合、「からなる」（ｃｏｎｓｉｓｔ　ｏｆ）態様、及び「から本質的になる」（ｃｏｎｓｉｓｔ　ｅｓｓｅｎｔｉａｌｌｙ　ｏｆ）態様を包含する。 The term "comprise" means that it may include components other than the target components. The term "consist of" means containing no elements other than the subject element. The term "consistently of" means that it does not include constituent elements other than the subject constituent elements in a mode that exhibits a special function (such as a mode that completely loses the effects of the invention). means. As used herein, the term "comprise" includes aspects that "consist of" and aspects that "consist essentially of."
　細菌は、単離されたものであり得る。「単離された」とは、天然状態又は他の成分から分離された状態を意味する。「単離された」ものは、他の成分を実質的に含まないものであり得る。「他の成分を実質的に含まない」とは、単離された成分に含まれる他の成分の含有量が無視できる程度であることを意味する。単離された成分に含まれる他の成分の含有量は、例えば、１０質量％以下、５質量％以下、４質量％以下、３質量％以下、２質量％以下、１質量％以下、０．５質量％以下、又は０．１質量％以下であり得る。本明細書に記載される細菌は、単離された細菌であり得る。 Bacteria can be isolated. "Isolated" means separated from the natural state or other components. "Isolated" can be substantially free of other components. "Substantially free of other components" means that the content of other components contained in the isolated component is negligible. The content of other components contained in the isolated component is, for example, 10% by mass or less, 5% by mass or less, 4% by mass or less, 3% by mass or less, 2% by mass or less, 1% by mass or less, 0.5% by mass or less. It may be 5% by mass or less, or 0.1% by mass or less. A bacterium described herein can be an isolated bacterium.
　「グルコースを唯一の有機炭素源として増殖する」とは、有機炭素源としてグルコースのみを含有する培地で、増殖することをいう。
　「合成培地」とは、培地に含まれる化学成分が明確である培地をいう。合成培地は、化学物質名が明確である化学成分を混合して調製される。
　「半合成培地」とは、合成培地に、化学的組成が不明確な物質の混合物である生物由来物質を添加した培地をいう。生物由来物質としては、酵母エキス、ペプトン、カザミノ酸、肉エキス等が挙げられる。 "Growing using glucose as the sole organic carbon source" means growing in a medium containing only glucose as the organic carbon source.
A “synthetic medium” refers to a medium in which the chemical components contained in the medium are defined. Synthetic media are prepared by mixing chemical components with well-defined chemical names.
A “semi-synthetic medium” refers to a medium obtained by adding a biological substance, which is a mixture of substances whose chemical composition is unclear, to a synthetic medium. Biological substances include yeast extract, peptone, casamino acid, meat extract and the like.
　本明細書において、「エイコサペンタエン酸」又は「ＥＰＡ」という用語は、エイコサペンタエン酸（遊離型）及びその誘導体を包含する。エイコサペンタエン酸の誘導体としては、エステル化型、リン脂質結合型、ホスファチジルグリセロール結合型、モノグリセリド型、ジグリセリド型、トリグリセリド型、これらの塩等が挙げられる。 As used herein, the term "eicosapentaenoic acid" or "EPA" includes eicosapentaenoic acid (free form) and its derivatives. Derivatives of eicosapentaenoic acid include esterified type, phospholipid-linked type, phosphatidylglycerol-linked type, monoglyceride type, diglyceride type, triglyceride type and salts thereof.
＜シェワネラ・ウーディイの単離株＞
　一態様において、本発明は、シェワネラ・ウーディイ（Ｓｈｅｗａｎｅｌｌａ　ｗｏｏｄｙｉ）　ＧＳ００１株（受託番号ＮＩＴＥ　ＢＰ－０３４６０）又はその変異株を提供する。 <Isolate strain of Shewanella udii>
In one aspect, the invention provides Shewanella woodyi strain GS001 (Accession No. NITE BP-03460) or a variant thereof.
　シェワネラ・ウーディイ　ＧＳ００１株（以下、「ＧＳ００１株」という）は、深海魚であるニギスの腸管から単離された菌株である。ＧＳ００１株は、１６ＳｒＤＮＡ配列に基づく系統解析により、シェワネラ・ウーディイと同定された（図２参照）。ＧＳ００１株の１６ＳｒＤＮＡ配列を配列番号１に示す。 The Shewanella udii strain GS001 (hereinafter referred to as "GS001 strain") is a strain isolated from the intestinal tract of Nigisu, a deep-sea fish. The GS001 strain was identified as Shewanella udii by phylogenetic analysis based on 16S rDNA sequences (see Figure 2). The 16S rDNA sequence of strain GS001 is shown in SEQ ID NO:1.
　ＧＳ００１株は、グルコースを唯一の有機炭素源として良好に増殖することができるという特徴を有する。例えば、ＧＳ００１株は、１０％（ｗ／ｖ）グルコースを含む改変Ｍ９液体培地（改変Ｍ９＋Ｇｌｃ液体培地）を５０ｍＬ入れた１００ｍＬフラスコ用いて、１５℃で振盪培養（１５０ｒｐｍ）したとき、４８時間で、ＯＤ６００ｎｍが５以上の菌密度に増殖することができる。
　ＧＳ００１株は、グルコース及び酵母エキスを含む半合成培地で良好に増殖することができるという特徴を有する。例えば、ＧＳ００１株は、０．５％（ｗ／ｖ）の酵母エキスを含む改変Ｍ９＋Ｇｌｃ液体培地を２Ｌ入れた３Ｌジャーファーメンター用いて、１５℃で通気培養（１．０ｖｖｍ　ａｉｒ、１５０～７５０ｒｐｍ）し、有機炭素源としてのグルコース溶液、窒素源としてのアンモニウム水溶液をそれぞれ適切な速度で流加したとき、７５時間で、乾燥細胞重量が３５ｇ／Ｌ以上に増殖することができる。
　ＧＳ００１株は、ＥＰＡ含有量が高いという特徴を有する。例えば、ＧＳ００１株を、０．５％（ｗ／ｖ）の酵母エキスを含む改変Ｍ９＋Ｇｌｃ液体培地を２Ｌ入れた３Ｌジャーファーメンター用いて、１５℃で通気培養（１ｖｖｍ　ａｉｒ、１５０～７５０ｒｐｍ）したとき、乾燥細胞重量当たりのＥＰＡ含有量は、０．５ｇ／１００ｇ乾燥細胞重量以上に維持され得る。
　ＧＳ００１株は、０．５％（ｗ／ｖ）という低い塩化ナトリウム濃度で増殖可能であるという特徴を有する。例えば、ＧＳ００１株は、塩化ナトリウム濃度を０．５％（ｗ／ｖ）に変更した改変Ｍ９＋Ｇｌｃ培地を５０ｍＬ入れた１００ｍＬフラスコ用いて、１５℃で振盪培養（１５０ｒｐｍ）したとき、４８時間で、ＯＤ６００ｎｍが３以上の菌密度に増殖することができる。
　ＧＳ００１株は、上記のような特徴を有するため、ＥＰＡの工業生産に適している。 The GS001 strain is characterized by being able to grow well on glucose as the sole organic carbon source. For example, the GS001 strain was cultured with shaking (150 rpm) at 15°C using a 100 mL flask containing 50 mL of a modified M9 liquid medium containing 10% (w/v) glucose (modified M9 + Glc liquid medium) for 48 hours. It can grow to a cell density of 5 or more at OD600nm .
The GS001 strain is characterized by being able to grow well in a semi-synthetic medium containing glucose and yeast extract. For example, the GS001 strain is aerated at 15° C. (1.0 vvm air, 150-750 rpm) using a 3 L jar fermenter containing 2 L of modified M9+Glc liquid medium containing 0.5% (w/v) yeast extract. Then, when a glucose solution as an organic carbon source and an ammonium aqueous solution as a nitrogen source are fed at appropriate rates, respectively, the dry cell weight can grow to 35 g/L or more in 75 hours.
The GS001 strain is characterized by a high EPA content. For example, the GS001 strain is aerated at 15 ° C. (1 vvm air, 150 to 750 rpm) using a 3 L jar fermenter containing 2 L of a modified M9 + Glc liquid medium containing 0.5% (w / v) yeast extract. , the EPA content per dry cell weight can be maintained above 0.5 g/100 g dry cell weight.
The GS001 strain is characterized by being able to grow in sodium chloride concentrations as low as 0.5% (w/v). For example, the GS001 strain was cultured with shaking (150 rpm) at 15°C in a 100 mL flask containing 50 mL of modified M9 + Glc medium with a sodium chloride concentration of 0.5% (w/v). 600 nm can grow to a cell density of 3 or more.
Since the GS001 strain has the characteristics described above, it is suitable for industrial production of EPA.
　ＧＳ００１株は、２０２１年４月１５日付で、受託番号ＮＩＴＥ　Ｐ－０３４６０として、独立行政法人製品評価技術基盤機構特許微生物寄託センター（日本国千葉県木更津市かずさ鎌足２－５－８　１２２号室）に寄託され、受託番号ＮＩＴＥ　ＢＰ－０３４６０として、２０２２年４月４日付で国際寄託に移管されている。
　寄託者氏名：江原　岳
　寄託者住所：日本国千葉県佐倉市坂戸６３１
　寄託者は、本出願において寄託生物について言及する権限を出願人に与えている。寄託者は、寄託生物が公衆に利用可能となる旨の同意を出願人に与えている。　 The GS001 strain was designated as accession number NITE P-03460 on April 15, 2021 by the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan Room 122). and has been transferred to the International Deposit on April 4, 2022 under accession number NITE BP-03460.
Name of Depositor: Gaku Ebara Address of Depositor: 631 Sakado, Sakura City, Chiba Prefecture, Japan
The Depositor authorizes Applicant to refer to the deposited organism in this application. The depositor has given consent to the applicant that the deposited organism will be made available to the public.
　「変異株」とは、元の菌株のゲノムに変異が生じた菌株をいう。変異は、自然発生的に生じたものであってもよく、人為的に生じたものであってもよい。人為的にゲノムに変異を生じさせる手法は、特に限定されない。人為的に変異を生じさせる手法としては、例えば、紫外線照射、放射線照射等の高エネルギー電磁波の照射；亜硝酸などによる化学的処理；遺伝子導入、ゲノム編集などの遺伝子工学的手法等が挙げられる。
　変異株は、元の菌株の全ゲノムに対する変異の割合が、例えば、１０％以下、５％以下、３％以下、２％以下、１％以下、０．５％以下、０．３％以下、又は０．１％以下であることが好ましい。変異株の１６ＳｒＤＮＡ配列は、配列番号１に記載のヌクレオチド配列を有してもよい。 A "mutant strain" refers to a strain in which a mutation has occurred in the genome of the original strain. Mutations may be naturally occurring or artificially occurring. The method of artificially mutating the genome is not particularly limited. Techniques for artificially generating mutations include, for example, irradiation with high-energy electromagnetic waves such as ultraviolet irradiation and radiation irradiation; chemical treatment with nitrous acid; genetic engineering techniques such as gene transfer and genome editing.
The mutant strain has a ratio of mutation to the entire genome of the original strain, for example, 10% or less, 5% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.3% or less, Alternatively, it is preferably 0.1% or less. The mutant 16S rDNA sequence may have the nucleotide sequence set forth in SEQ ID NO:1.
　ＧＳ００１株の変異株（以下、「ＧＳ００１変異株」という）は、グルコースを唯一の有機炭素源として培養したときの比増殖速度が、同じ条件で培養したＧＳ００１株と同等程度又は同等以上であることが好ましい。例えば、同じ培養条件下で培養したとき、ＧＳ００１変異株の比増殖速度は、ＧＳ００１株の比増殖速度に０．７（又は、０．７５、０．８、０．８５、０．９．０．９５、０．９７、０．９８、０．９９、若しくは１）を乗じた値以上であることが好ましい。例えば、ＧＳ００１変異株は、１０％グルコースを含む改変Ｍ９液体培地（改変Ｍ９＋Ｇｌｃ培地）を３０ｍＬ入れた１００ｍＬフラスコ用いて、１５℃で振盪培養（１５０ｒｐｍ）したとき、４８時間で、ＯＤ６００ｎｍが５以上の菌密度に増殖することが好ましい。 The mutant strain of the GS001 strain (hereinafter referred to as "GS001 mutant strain") has a specific growth rate when cultured with glucose as the sole organic carbon source that is equal to or greater than that of the GS001 strain cultured under the same conditions. is preferred. For example, when cultured under the same culture conditions, the specific growth rate of the GS001 mutant is 0.7 (or 0.75, 0.8, 0.85, 0.9.0) to that of the GS001 strain. It is preferably 0.95, 0.97, 0.98, 0.99, or a value multiplied by 1). For example, the GS001 mutant has an OD 600 nm of 5 or more in 48 hours when cultured with shaking (150 rpm) at 15 ° C. using a 100 mL flask containing 30 mL of a modified M9 liquid medium containing 10% glucose (modified M9 + Glc medium). It is preferable to grow to a bacterial density of
　ＧＳ００１変異株は、グルコース及び酵母エキスを含む半合成培地で培養したときの比増殖速度が、同じ条件で培養したＧＳ００１株と同等程度又は同等以上であることが好ましい。例えば、同じ培養条件下で培養したとき、ＧＳ００１変異株の比増殖速度は、ＧＳ００１株の比増殖速度に０．７（又は、０．７５、０．８、０．８５、０．９．０．９５、０．９７、０．９８、０．９９、若しくは１）を乗じた値以上であることが好ましい。例えば、ＧＳ００１変異株は、０．５％（ｗ／ｖ）の酵母エキスを含む改変Ｍ９＋Ｇｌｃ液体培地を２Ｌ入れた３Ｌジャーファーメンター用いて、１５℃で通気培養（１ｖｖｍ　ａｉｒ、１５０～７５０ｒｐｍ）したとき、７５時間で、乾燥細胞重量が３０ｇ／Ｌ以上に増殖することが好ましい。 It is preferable that the GS001 mutant has a specific growth rate when cultured in a semi-synthetic medium containing glucose and yeast extract that is equal to or greater than that of the GS001 strain cultured under the same conditions. For example, when cultured under the same culture conditions, the specific growth rate of the GS001 mutant is 0.7 (or 0.75, 0.8, 0.85, 0.9.0) to that of the GS001 strain. It is preferably 0.95, 0.97, 0.98, 0.99, or a value multiplied by 1). For example, the GS001 mutant strain was cultured at 15° C. under aerobic conditions (1 vvm air, 150-750 rpm) using a 3 L jar fermenter containing 2 L of modified M9+Glc liquid medium containing 0.5% (w/v) yeast extract. At 75 hours, it is preferable to grow to a dry cell weight of 30 g/L or more.
　ＧＳ００１変異株は、グルコースを唯一の有機炭素源として培養したときの乾燥細胞重量当たりのＥＰＡ含有量が、同じ条件で培養したＧＳ００１株と同等程度であることが好ましい。例えば、同じ培養条件下で培養したとき、ＧＳ００１変異株の乾燥細胞重量当たりのＥＰＡ含有量が、ＧＳ００１株のＥＰＡ含有量に０．７（又は、０．７５、０．８、０．８５、０．９．０．９５、０．９７、０．９８、０．９９、若しくは１）を乗じた値程度であることが好ましい。 It is preferable that the GS001 mutant strain has an EPA content per dry cell weight when cultured using glucose as the sole organic carbon source, which is about the same as the GS001 strain cultured under the same conditions. For example, when cultured under the same culture conditions, the EPA content per dry cell weight of the GS001 mutant strain is 0.7 (or 0.75, 0.8, 0.85, It is preferably about 0.9, 0.95, 0.97, 0.98, 0.99, or a value obtained by multiplying 1).
　ＧＳ００１変異株は、グルコース及び酵母エキスを含む半合成培地で培養したときの乾燥細胞重量当たりのＥＰＡ含有量が、同じ条件で培養したＧＳ００１株と同等程度であることが好ましい。例えば、同じ培養条件下で培養したとき、ＧＳ００１変異株の乾燥細胞重量当たりのＥＰＡ含有量が、ＧＳ００１株のＥＰＡ含有量に０．７（又は、０．７５、０．８、０．８５、０．９．０．９５、０．９７、０．９８、０．９９、若しくは１）を乗じた値程度であることが好ましい。例えば、ＧＳ００１変異株を、０．５％（ｗ／ｖ）の酵母エキスを含む改変Ｍ９＋Ｇｌｃ液体培地を２Ｌ入れた３Ｌジャーファーメンター用いて、１５℃で通気培養（１ｖｖｍ　ａｉｒ、１５０～７５０ｒｐｍ）したとき、乾燥細胞重量当たりのＥＰＡ含有量は、０．５ｇ／１００ｇ乾燥細胞重量程度に維持されることが好ましい。 The GS001 mutant strain preferably has an EPA content per dry cell weight when cultured in a semi-synthetic medium containing glucose and yeast extract, which is about the same as the GS001 strain cultured under the same conditions. For example, when cultured under the same culture conditions, the EPA content per dry cell weight of the GS001 mutant strain is 0.7 (or 0.75, 0.8, 0.85, It is preferably about 0.9, 0.95, 0.97, 0.98, 0.99, or a value obtained by multiplying 1). For example, the GS001 mutant strain was cultured under aerobic conditions at 15° C. (1 vvm air, 150-750 rpm) using a 3 L jar fermenter containing 2 L of modified M9+Glc liquid medium containing 0.5% (w/v) yeast extract. When the EPA content per dry cell weight is preferably maintained at around 0.5 g/100 g dry cell weight.
　ＧＳ００１変異株は、０．５％（ｗ／ｖ）の塩化ナトリウムを含む培地で増殖できることが好ましい。例えば、グルコースを唯一の有機炭素源として含み、且つ０．５％（ｗ／ｖ）の塩化ナトリウムを含む培地で培養したときの比増殖速度が、同じ条件で培養したＧＳ００１株と同等以上であることが好ましい。例えば、同じ培養条件下で培養したとき、ＧＳ００１変異株の比増殖速度は、ＧＳ００１株の比増殖速度に０．７（又は、０．７５、０．８、０．８５、０．９．０．９５、０．９７、０．９８、０．９９、若しくは１）を乗じた値以上であることが好ましい。例えば、ＧＳ００１変異株は、塩化ナトリウム濃度を０．５％（ｗ／ｖ）に変更した改変Ｍ９＋Ｇｌｃ培地を５０ｍＬ入れた１００ｍＬフラスコ用いて、１５℃で振盪培養（１５０ｒｐｍ）したとき、４８時間で、ＯＤ６００ｎｍが３以上の菌密度に増殖することが好ましい。 Preferably, the GS001 mutant can grow in media containing 0.5% (w/v) sodium chloride. For example, the specific growth rate when cultured in a medium containing glucose as the sole organic carbon source and containing 0.5% (w/v) sodium chloride is equal to or higher than that of the GS001 strain cultured under the same conditions. is preferred. For example, when cultured under the same culture conditions, the specific growth rate of the GS001 mutant is 0.7 (or 0.75, 0.8, 0.85, 0.9.0) to that of the GS001 strain. It is preferably 0.95, 0.97, 0.98, 0.99, or a value multiplied by 1). For example, the GS001 mutant strain was cultured with shaking (150 rpm) at 15 ° C. using a 100 mL flask containing 50 mL of a modified M9 + Glc medium with a sodium chloride concentration of 0.5% (w / v). It is preferable to grow to a cell density of OD 600 nm of 3 or more.
　ＧＳ００１株及びＧＳ００１変異株は、従属栄養細菌の培養に通常用いられる培地を用いて、培養することができる。培地としては、例えば、有機炭素源、窒素源、リン源、微量元素（亜鉛、ホウ素、コバルト、銅、マンガン、モリブデン、鉄、ニッケルなど）等を含む培地が挙げられる。有機炭素源としては、例えば、グルコース、デキストラン、スターチ、酵母エキス等が挙げられる。窒素源としては、例えば、アンモニウム塩、硝酸塩、亜硝酸塩、アミノ酸、ペプトン等が挙げられる。リン源としては、例えば、リン酸塩等が挙げられる。培地は、例えば、Ｍ９培地、Ｍ９培地の改変培地（改変Ｍ９培地等）等の最小培地に有機炭素源を添加したものであってもよい。 The GS001 strain and the GS001 mutant strain can be cultured using a medium commonly used for culturing heterotrophic bacteria. Examples of the medium include those containing organic carbon sources, nitrogen sources, phosphorus sources, trace elements (zinc, boron, cobalt, copper, manganese, molybdenum, iron, nickel, etc.) and the like. Organic carbon sources include, for example, glucose, dextran, starch, yeast extract and the like. Nitrogen sources include, for example, ammonium salts, nitrates, nitrites, amino acids, peptones, and the like. Phosphorus sources include, for example, phosphates and the like. The medium may be, for example, a minimal medium such as M9 medium or a modified M9 medium (modified M9 medium, etc.) added with an organic carbon source.
　合成培地を用いる場合、好ましい有機炭素源としては、グルコースが挙げられる。半合成培地を用いる場合、好ましい有機炭素源としては、酵母エキスが挙げられる。半合成培地は、有機炭素源として、グルコース及び酵母エキスを含んでもよい。 When using a synthetic medium, a preferred organic carbon source is glucose. When semi-synthetic media are used, preferred organic carbon sources include yeast extract. The semi-synthetic medium may contain glucose and yeast extract as organic carbon sources.
　培地は、０．５～５％（ｗ／ｖ）の塩化ナトリウムを含むことが好ましい。培地の塩化ナトリウム濃度は、０．５～３％（ｗ／ｖ）がより好ましい。 The medium preferably contains 0.5-5% (w/v) sodium chloride. More preferably, the sodium chloride concentration of the medium is 0.5-3% (w/v).
　培地は、固体培地であってもよく、液体培地であってもよい。維持のためには、固体培地を用いることが好ましい。増殖させる場合には、液体培地を用いることが好ましい。 The medium may be a solid medium or a liquid medium. For maintenance, it is preferred to use a solid medium. For growth, it is preferable to use a liquid medium.
　培地のｐＨとしては、例えば、ｐＨ６～９が挙げられる。培地のｐＨは、例えば、ｐＨ７～８．５が好ましく、ｐＨ７．５～８．５がより好ましい。 The pH of the medium includes, for example, pH 6-9. The pH of the medium is, for example, preferably pH 7-8.5, more preferably pH 7.5-8.5.
　培養温度としては、０～３０℃が挙げられる。培養温度は、１０～３０℃が好ましく、１２～２０℃がより好ましく、１５～１８℃がさらに好ましい。 　The culture temperature is 0 to 30°C. The culture temperature is preferably 10 to 30°C, more preferably 12 to 20°C, even more preferably 15 to 18°C.
　培養は、静置培養であってもよく、通気培養であってもよく、振盪培養であってもよい。酸素不足を防ぐ観点から、通気培養又は振盪培養することが好ましい。 The culture may be static culture, aerobic culture, or shaking culture. From the viewpoint of preventing oxygen deficiency, aerobic culture or shaking culture is preferred.
　培養期間中、ＧＳ００１株又はＧＳ００１変異株は、適宜継代してもよい。固体培地で維持培養する場合、継代の間隔としては、例えば、半月～１カ月が挙げられる。液体培地で増殖させる場合、継代の間隔としては、例えば、２日～１０日、又は２～５日が挙げられる。 The GS001 strain or GS001 mutant may be passaged as appropriate during the culture period. In the case of maintenance culture on a solid medium, the subculture interval is, for example, half a month to one month. When grown in liquid medium, passage intervals include, for example, 2-10 days, or 2-5 days.
　ＧＳ００１株及びＧＳ００１変異株は、細胞内に、高い含有量でＥＰＡを含む。そのため、ＧＳ００１株又はＧＳ００１変異株は、ＥＰＡの製造に用いることができる。ＧＳ００１株又はＧＳ００１変異株からのＥＰＡの抽出は、公知の方法で行うことができる。例えば、ＧＳ００１株及びＧＳ００１変異株の培養液から遠心分離又はろ過等により細胞を回収し、クロロホルム、メタノール、トルエン等の有機溶媒により細胞から全脂質を抽出する。細胞に対して使用する抽出溶媒の量は、特に限定されない。抽出溶媒の量は、例えば、細胞に対して、１～１０００倍量程度（好ましくは５～２００倍量程度）とすることができる。抽出操作は、通常、常圧下、常温～溶媒の沸点の範囲で行うことができる。抽出操作は、１回のみ行ってもよく、複数回行ってもよい。例えば、１度抽出操作を行った細胞残渣に再度新鮮な抽出溶媒を添加しても再度抽出操作を行ってもよい。抽出操作後、必要に応じて、遠心分離、ろ過、限外ろ過等により細胞残渣を除去してもよい。また、加熱、エバポレーター等を用いた減圧蒸留により、抽出溶媒を除去してもよい。さらに、各種精製処理を行い、ＥＰＡを精製してもよい。精製処理としては、例えば、塩析、透析、再結晶、再沈殿、溶媒抽出、吸着、濃縮、ろ過、ゲルろ過、限外ろ過、各種クロマトグラフィ（薄層クロマトグラフィ、カラムクロマトグラフィ、イオン交換クロマトグラフィ、高速液体クロマトグラフィ、吸着クロマトグラフィなど）等が挙げられるが、これらに限定されない。
　ＧＳ００１株又はＧＳ００１変異株からのＥＰＡ抽出方法の具体例としては、後述の実施例に記載の方法が挙げられる。 The GS001 strain and the GS001 mutant strain contain a high content of EPA in the cells. Therefore, the GS001 strain or GS001 mutant strain can be used for the production of EPA. EPA can be extracted from the GS001 strain or the GS001 mutant strain by a known method. For example, cells are collected from the culture medium of the GS001 strain and the GS001 mutant strain by centrifugation, filtration, or the like, and all lipids are extracted from the cells with an organic solvent such as chloroform, methanol, or toluene. The amount of extraction solvent used for cells is not particularly limited. The amount of extraction solvent can be, for example, about 1 to 1000 times (preferably about 5 to 200 times) the amount of cells. The extraction operation can usually be carried out under normal pressure in the range from room temperature to the boiling point of the solvent. The extraction operation may be performed only once or may be performed multiple times. For example, a fresh extraction solvent may be added again to the cell residue that has been subjected to the extraction operation once, or the extraction operation may be performed again. After the extraction operation, if necessary, cell debris may be removed by centrifugation, filtration, ultrafiltration, or the like. Alternatively, the extraction solvent may be removed by heating or distillation under reduced pressure using an evaporator or the like. Furthermore, various purification treatments may be performed to purify EPA. Purification treatments include, for example, salting out, dialysis, recrystallization, reprecipitation, solvent extraction, adsorption, concentration, filtration, gel filtration, ultrafiltration, various chromatography (thin layer chromatography, column chromatography, ion exchange chromatography, high-performance liquid chromatography, adsorption chromatography, etc.), but are not limited thereto.
A specific example of the method for extracting EPA from the GS001 strain or the GS001 mutant strain is the method described in Examples below.
　ＥＰＡは、栄養補助食品、食品添加物等として、使用することができる。 EPA can be used as nutritional supplements, food additives, etc.
＜組成物＞
　一態様において、本発明は、シェワネラ・ウーディイ　ＧＳ００１株又はその変異株を含む、組成物を提供する。 <Composition>
In one aspect, the invention provides a composition comprising Shewanella udii strain GS001 or a variant thereof.
　ＧＳ００１株及びＧＳ００１変異株は、ＥＰＡ等の有用成分を含有する。そのため、ＧＳ００１株又はＧＳ００１変異株を、各種組成物に利用することができる。 The GS001 strain and GS001 mutant contain useful ingredients such as EPA. Therefore, the GS001 strain or the GS001 mutant strain can be used in various compositions.
　ＧＳ００１株又はＧＳ００１変異株を含む組成物の種類は、特に限定されない。組成物としては、例えば、食品、飼料、餌料、化粧料等が挙げられる。 The type of composition containing the GS001 strain or GS001 mutant strain is not particularly limited. Compositions include, for example, foods, feeds, feeds, cosmetics, and the like.
　食品としては、例えば、菓子類（キャラメル、キャンディー等）、氷菓類（アイスクリーム等）、乳製品（ヨーグルト等）、各種加工食品、各種調味料、各種飲料、機能性食品、栄養補助食品、サプリメント等が挙げられる。
　飼料としては、例えば、各種ペットフードが挙げられる。
　餌料としては、例えば、観賞魚用の餌料、養殖魚用の餌料等が挙げられる。
　化粧料としては、例えば、皮膚化粧料（化粧水、乳液、美容液、クリーム等）、頭髪化粧料（整髪料、シャンプー、リンス、コンディショナー等）、メーキャップ用化粧料（ファンデーション、チーク、アイシャドウ、口紅等）が挙げられる。 Examples of foods include confectionery (caramel, candy, etc.), frozen desserts (ice cream, etc.), dairy products (yogurt, etc.), various processed foods, various seasonings, various beverages, functional foods, dietary supplements, and supplements. etc.
Feeds include, for example, various pet foods.
The feed includes, for example, feed for ornamental fish, feed for cultured fish, and the like.
Cosmetics include, for example, skin cosmetics (lotions, milky lotions, serums, creams, etc.), hair cosmetics (hair styling, shampoos, rinses, conditioners, etc.), makeup cosmetics (foundations, cheeks, eye shadows, lipstick, etc.).
　ＧＳ００１株又はＧＳ００１変異株は、上記のような各種組成物に、添加剤として添加することができる。また、他の成分と混合し、食品、飼料、餌料、化粧料等として、調製してもよい。他の成分は、組成物の用途に応じて、適宜選択可能である。 The GS001 strain or GS001 mutant strain can be added as an additive to the various compositions described above. In addition, it may be mixed with other ingredients and prepared as foods, feeds, feeds, cosmetics, and the like. Other components can be appropriately selected depending on the use of the composition.
　例えば、組成物が食品、飼料、又は餌料である場合、食品、飼料、又は餌料に使用可能な成分を特に制限なく用いることができる。食品、飼料、又は餌料に使用可能な成分としては、例えば、魚肉類、野菜類、穀類、乳製品、発酵製品、香辛料、タンパク質、アミノ酸、糖類、各種調味料、甘味剤、矯味剤、香料、油脂類、ビタミン類、増粘剤、ゲル化剤、酸化防止剤、防腐剤、キレート剤、ｐＨ調整剤、着色剤等が挙げられるが、これらに限定されない。 For example, when the composition is food, feed, or feed, ingredients that can be used for food, feed, or feed can be used without particular limitation. Examples of ingredients that can be used for foods, feeds, or feeds include fish meat, vegetables, grains, dairy products, fermented products, spices, proteins, amino acids, sugars, various seasonings, sweeteners, corrigents, flavors, Oils and fats, vitamins, thickeners, gelling agents, antioxidants, preservatives, chelating agents, pH adjusters, coloring agents, etc., but not limited to these.
　例えば、組成物が化粧料である場合、化粧料に使用可能な成分を特に制限なく用いることができる。化粧料に使用可能な成分としては、例えば、炭化水素、脂質類（動植物性油脂や鉱物油等の油脂、ロウ、脂肪酸エステル、脂肪酸、セラミド等）、アルコール類（高級アルコール、低級アルコールや多価アルコール等）のアルコール類、タンパク質類（コラーゲン等）、多糖類（ヒアルロン酸、キトサン、セルロース、キサンタンガム等）、各種高分子化合物（ポリエチレングリコール、シリコーン等）、動植物や微生物由来成分、アミノ酸、無機鉱物、各種界面活性剤、紫外線吸収剤、美白剤、抗炎症剤、血行促進剤、抗老化剤、保湿剤、ビタミン類、増粘剤、ゲル化剤、酸化防止剤、防腐剤、抗菌剤、キレート剤、ｐＨ調整剤、粉体、香料、着色剤等が挙げられるが、これらに限定されない。 For example, when the composition is a cosmetic, any ingredient that can be used in cosmetics can be used without any particular restrictions. Examples of ingredients that can be used in cosmetics include hydrocarbons, lipids (oils such as animal and vegetable oils and mineral oils, waxes, fatty acid esters, fatty acids, ceramides, etc.), alcohols (higher alcohols, lower alcohols and polyhydric alcohols). alcohol, etc.), proteins (collagen, etc.), polysaccharides (hyaluronic acid, chitosan, cellulose, xanthan gum, etc.), various high-molecular compounds (polyethylene glycol, silicone, etc.), components derived from animals, plants and microorganisms, amino acids, inorganic minerals , Various surfactants, UV absorbers, whitening agents, anti-inflammatory agents, blood circulation promoters, anti-aging agents, moisturizers, vitamins, thickeners, gelling agents, antioxidants, preservatives, antibacterial agents, chelates agents, pH adjusters, powders, fragrances, colorants, etc., but are not limited to these.
＜乾燥粉末＞
　一態様において、本発明は、ＧＳ００１株又はＧＳ００１変異株の乾燥粉末を提供する。 <Dry powder>
In one aspect, the invention provides a dry powder of strain GS001 or GS001 variant.
　「ＧＳ００１株の乾燥粉末」とは、ＧＳ００１株の細胞を乾燥し、粉末状にしたものをいう。「ＧＳ００１変異株の乾燥粉末」とは、ＧＳ００１変異株の細胞を乾燥し、粉末状にしたものをいう。ＧＳ００１株又はＧＳ００１変異株の乾燥粉末は、公知の方法により製造することができる。例えば、ＧＳ００１株又はＧＳ００１変異株を培養し、遠心分離等により細胞を回収する。次いで、細胞を乾燥し、粉末状とすることにより、乾燥粉末を得ることができる。細胞の乾燥方法は、特に限定されないが、例えば、自然乾燥、加熱乾燥、減圧乾燥、凍結乾燥等が挙げられる。乾燥した細胞は、物理的に粉砕して、粉末状としてもよい。細胞は、乾燥する前に、洗浄処理、殺菌処理等を行ってもよい。洗浄処理には、例えば、水、緩衝液等の洗浄液を用いることができる。殺菌処理の方法としては、例えば、次亜塩素酸等の殺菌による処理、紫外線処理、オゾン処理、加熱処理等が挙げられる。 "GS001 strain dry powder" refers to GS001 strain cells dried and powdered. The term "dry powder of GS001 mutant" refers to a powder obtained by drying GS001 mutant cells. A dry powder of the GS001 strain or GS001 mutant can be produced by a known method. For example, GS001 strain or GS001 mutant strain is cultured, and cells are collected by centrifugation or the like. A dry powder can then be obtained by drying the cells and pulverizing them. A method for drying the cells is not particularly limited, and examples thereof include natural drying, heat drying, reduced pressure drying, freeze drying and the like. Dried cells may be physically pulverized into a powder. The cells may be subjected to washing treatment, sterilization treatment, etc. before drying. For the washing treatment, for example, a washing solution such as water or a buffer solution can be used. Examples of the sterilization method include sterilization with hypochlorous acid, ultraviolet treatment, ozone treatment, heat treatment, and the like.
　乾燥粉末とすることにより、ＧＳ００１株又はＧＳ００１変異株が含有するＥＰＡ等の有用成分が濃縮される。そのため、ＧＳ００１株又はＧＳ００１変異株の乾燥粉末は、食品、飼料、又は餌料として用いることができる。また、ＧＳ００１株又はＧＳ００１変異株の乾燥粉末は、添加剤として、食品、飼料、餌料、又は化粧料に使用することができる。 By making it into a dry powder, useful ingredients such as EPA contained in the GS001 strain or the GS001 mutant strain are concentrated. Therefore, the GS001 strain or GS001 mutant dry powder can be used as a food, feed, or fodder. Moreover, the dry powder of the GS001 strain or the GS001 mutant can be used as an additive in foods, feeds, feeds, or cosmetics.
＜抽出物＞
　一態様において、本発明は、ＧＳ００１株又はＧＳ００１変異株の抽出物を提供する。 <Extract>
In one aspect, the invention provides an extract of the GS001 strain or GS001 mutant strain.
　「ＧＳ００１株の抽出物」とは、ＧＳ００１株の細胞から細胞成分を抽出したものをいう。「ＧＳ００１変異株の抽出物」とは、ＧＳ００１変異株の細胞から細胞成分を抽出したものをいう。ＧＳ００１株又はＧＳ００１変異株の抽出物は、ＧＳ００１株又はＧＳ００１変異株の細胞を破壊した細胞破壊物であってもよい。細胞の破壊方法としては、例えば、ホモジナイザー等による機械的破砕処理、超音波処理、凍結融解処理等が挙げられる。また、ＧＳ００１株又はＧＳ００１変異株の抽出物は、ＧＳ００１株又はＧＳ００１変異株の細胞を溶解した細胞溶解物であってもよい。細胞の溶解方法としては、例えば、プロテアーゼ、リゾチーム等の酵素を用いた酵素処理、界面活性剤処理等が挙げられる。
　ＧＳ００１株又はＧＳ００１変異株の抽出物は、ＧＳ００１株又はＧＳ００１変異株の細胞、細胞破壊物、又は細胞溶解物に対して、抽出溶媒を添加して、抽出処理を行ったものであってもよい。抽出溶媒は、上記と同様のものを用いることができる。 The term “GS001 strain extract” refers to a cell component extracted from GS001 strain cells. The term “GS001 mutant extract” refers to the extraction of cell components from GS001 mutant cells. The extract of the GS001 strain or the GS001 mutant strain may be a cell disruption product obtained by destroying the cells of the GS001 strain or the GS001 mutant strain. Methods for disrupting cells include, for example, mechanical disruption using a homogenizer or the like, ultrasonic treatment, freeze-thaw treatment, and the like. Moreover, the extract of the GS001 strain or the GS001 mutant strain may be a cell lysate obtained by lysing the cells of the GS001 strain or the GS001 mutant strain. Methods for lysing cells include, for example, enzymatic treatment using enzymes such as protease and lysozyme, surfactant treatment, and the like.
The extract of the GS001 strain or the GS001 mutant strain may be obtained by adding an extraction solvent to the cells, cell disruptions, or cell lysates of the GS001 strain or the GS001 mutant strain, and performing an extraction treatment. . The same extraction solvent as mentioned above can be used.
　抽出物とすることにより、ＧＳ００１株又はＧＳ００１変異株が含有するＥＰＡ等の有用成分が濃縮される。そのため、ＧＳ００１株又はＧＳ００１変異株の抽出物は、食品、飼料、又は餌料として用いることができる。また、ＧＳ００１株又はＧＳ００１変異株の抽出物は、添加剤として、食品、飼料、餌料、又は化粧料に使用することができる。 By making it into an extract, useful ingredients such as EPA contained in the GS001 strain or the GS001 mutant strain are concentrated. Therefore, the GS001 strain or the GS001 mutant strain extract can be used as food, feed, or fodder. Moreover, the GS001 strain or the GS001 mutant strain extract can be used as an additive in foods, feeds, feeds, or cosmetics.
　一実施形態において、本開示は、ＧＳ００１株又はＧＳ００１変異株を培養することを含む、ＥＰＡの製造方法を提供する。
　一実施形態において、本開示は、ＧＳ００１株又はＧＳ００１変異株を、グルコースを有機炭素源として含む培地で培養することを含む、ＥＰＡの製造方法を提供する。
　一実施形態において、本開示は、ＧＳ００１株又はＧＳ００１変異株を、グルコースを有機炭素源として含む培地で培養すること、ＧＳ００１株又はＧＳ００１変異株の菌体を回収すること、及び前記菌体からＥＰＡを抽出すること、を含む、ＥＰＡの製造方法を提供する。
　一実施形態において、本開示は、ＧＳ００１株又はＧＳ００１変異株を、グルコースを有機炭素源として含む培地で培養することを含む、ＧＳ００１株又はＧＳ００１変異株の培養方法を提供する。
　一実施形態において、本開示は、ＧＳ００１株又はＧＳ００１変異株を、グルコースを有機炭素源として含む培地で培養することを含む、ＧＳ００１株又はＧＳ００１変異株の製造方法を提供する。 In one embodiment, the present disclosure provides a method of producing EPA comprising culturing the GS001 strain or GS001 mutant strain.
In one embodiment, the present disclosure provides a method for producing EPA, comprising culturing strain GS001 or a mutant strain GS001 in a medium containing glucose as an organic carbon source.
In one embodiment, the present disclosure comprises culturing the GS001 strain or the GS001 mutant strain in a medium containing glucose as an organic carbon source, recovering cells of the GS001 strain or the GS001 mutant strain, and removing EPA from the cells. and extracting the EPA.
In one embodiment, the present disclosure provides a method for culturing strain GS001 or GS001 mutant comprising culturing strain GS001 or GS001 mutant in a medium containing glucose as an organic carbon source.
In one embodiment, the present disclosure provides a method for producing a GS001 strain or a GS001 mutant comprising culturing the GS001 strain or the GS001 mutant in a medium containing glucose as an organic carbon source.
　以下、実施例により本発明を説明するが、本発明は以下の実施例に限定されるものではない。 The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.
＜グルコース資化能の高いシェワネラ属細菌の単離＞
　深海魚であるニギスの腸管から、Ｍａｒｉｎｅ　Ｂｒｏｔｈ培地（Ｄｉｆｃｏ社製）を用いて、細菌を単離した。Ｍａｒｉｎｅ　Ｂｒｏｔｈ寒天培地に出現したコロニーを、１００ｍＬ三角フラスコに入れた５０ｍＬの改変Ｍ９＋Ｇｌｃ液体培地に接種して培養した。培養温度１５℃とし、バイオシェーカーを用いて振盪培養（回転数：１５０ｒｐｍ）した。培地のＯＤ６００が４～５に達した時点で、５００μＬの培養液を採取し、新しい５０ｍＬの改変Ｍ９＋Ｇｌｃ液体培地に接種した。前記の継代培養を６カ月程度行い、改変Ｍ９＋Ｇｌｃ液体培地での増殖が良好で、ＥＰＡ含量が高い菌株を選抜した。その結果、改変Ｍ９＋Ｇｌｃ液体培地での増殖が良好で、ＥＰＡ含量が高い菌株として、ＧＳ００１株を取得した。表１～３に、改変Ｍ９＋Ｇｌｃ液体培地の組成を示す。改変Ｍ９＋Ｇｌｃ培地は、Ｍ９最小培地に、Ｆｅ　ｓｏｌｕｔｉｏｎ（表２）、Ｔｒａｃｅ　Ｍｅｔａｌ　ｓｏｌｕｔｉｏｎ（表３）、及びグルコースを添加したものである。 <Isolation of Shewanella bacteria with high glucose utilization>
Bacteria were isolated from the intestinal tract of nigisu, a deep-sea fish, using Marine Broth medium (manufactured by Difco). Colonies that appeared on Marine Broth agar medium were inoculated into 50 mL of modified M9+Glc liquid medium placed in a 100 mL Erlenmeyer flask and cultured. The culture temperature was set to 15° C., and shaking culture was performed using a bioshaker (rotation speed: 150 rpm). When the OD 600 of the medium reached 4-5, 500 μL of culture was harvested and inoculated into 50 mL of fresh modified M9+Glc liquid medium. The above-mentioned subculture was carried out for about 6 months, and strains with good growth in modified M9+Glc liquid medium and high EPA content were selected. As a result, the GS001 strain was obtained as a strain that grew well in a modified M9+Glc liquid medium and had a high EPA content. Tables 1-3 show the composition of the modified M9+Glc liquid medium. The modified M9+Glc medium is M9 minimal medium supplemented with Fe solution (Table 2), Trace Metal solution (Table 3), and glucose.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　１００ｍＬ三角フラスコに入れた５０ｍＬの改変Ｍ９＋Ｇｌｃ液体培地を用いて、上記と同様の培養条件で、ＧＳ００１株を増殖させた結果を図１に示す。ＧＳ００１株は、培養開始から４８時間程度で、ＯＤ６００が５～６程度に達し、良好な増殖を示した。 FIG. 1 shows the results of growing the GS001 strain under the same culture conditions as above using 50 mL of modified M9+Glc liquid medium placed in a 100 mL Erlenmeyer flask. The GS001 strain reached an OD 600 of about 5 to 6 about 48 hours after the start of culture, showing good growth.
＜ＧＳ００１株の同定＞
　ＧＳ００１株の懸濁液を鋳型として、以下のプライマーセットを用いてＰＣＲを行い、１６ＳｒＤＮＡを増幅した。
　フォワードプライマー：ＧＴＴＴＧＡＴＣＣＴＧＧＣＴＣＡ（配列番号２）
　リバースプライマー：ＴＡＣＣＡＧＧＧＴＡＴＣＴＡＡＴＣＣ（配列番号３） <Identification of GS001 strain>
Using the GS001 strain suspension as a template, PCR was performed using the following primer set to amplify 16S rDNA.
Forward primer: GTTTGATCCTGGCTCA (SEQ ID NO: 2)
Reverse primer: TACCAGGGTATCTAATCC (SEQ ID NO: 3)
　増幅断片の配列解析を行い、ＧＳ００１株の１６ＳｒＤＮＡ配列（配列番号１）を決定した。ＧＳ００１株の１６ＳｒＤＮＡ配列をクエリー配列としてＢＬＡＳＴ検索を行った。ＢＬＡＳＴ検索の結果から、ＧＳ００１株は、シェワネラ・ウーディイと同定された。図２に、１６ＳｒＤＮＡ配列に基づく、ＧＳ００１株の系統解析の結果を示す。
　系統解析は、Ｓａｔｏｉら（Masataka Satoi et al., International Journal of Systematicand Evolutionary Microbiology(2003),53,491-499.）及びＺｈａｎｇら（Jinwei Zhang, et al., International Journal of Systematicand Evolutionary Microbiology(2015),65,2882-2889.）に記載の方法を参考にして行った。シェワネラ・ウーディイを含む１９種類のシェワネラ属細菌と近縁種の１６ＳｒＲＮＡ配列をＤＤＢＪデータベースから収集し、それらの配列とＧＳ００１株の１６ｓｒＲＮＡ配列について、解析用ソフトウェア「ＣＬＣ　Ｇｅｎｏｍｉｃｓ　Ｗｏｒｋｂｅｎｃｈ」（ＣＬＣ　ｂｉｏ社製）を用いて系統樹を作成した（クラスタ解析法はＮＪメソッド、遺伝距離はＫｉｍｕｒａモデルに基づいて行った）。 Sequence analysis of the amplified fragment was performed to determine the 16S rDNA sequence (SEQ ID NO: 1) of strain GS001. A BLAST search was performed using the GS001 strain 16S rDNA sequence as a query sequence. From the results of the BLAST search, strain GS001 was identified as Shewanella udii. FIG. 2 shows the results of phylogenetic analysis of the GS001 strain based on the 16S rDNA sequence.
Phylogenetic analysis was performed by Satoi et al. (Masataka Satoi et al., International Journal of Systematic and Evolutionary Microbiology (2003), 53, 491-499.) and Zhang et al. (Jinwei Zhang, et al., International Journal of Systematic and Evolutionary Microbiology (2015), 65 , 2882-2889.). The 16S rRNA sequences of 19 types of Shewanella bacteria including Shewanella udii and related species were collected from the DDBJ database, and those sequences and the 16S rRNA sequence of the GS001 strain were analyzed using the analysis software "CLC Genomics Workbench" (manufactured by CLC bio). was used to create a phylogenetic tree (the cluster analysis method was the NJ method, and the genetic distance was based on the Kimura model).
＜半合成培地によるＧＳ００１株の培養＞
　半合成培地として、０．５％（ｗ／ｖ）の酵母エキスを含む改変Ｍ９＋Ｇｌｃ液体培地（Ｍ９＋Ｇｌｃ＋Ｙ．Ｅ．液体培地）を用いた。３Ｌのジャーファーメンターに入れた２Ｌの改変Ｍ９＋Ｇｌｃ＋Ｙ．Ｅ．液体培地に、ＧＳ００１株を接種して培養した。培養温度は１５℃とし、通気培養（１ｖｖｍ　ａｉｒ、１５０～７５０ｒｐｍ）した。培養は、２台のジャーファーメンターを使用して行った。 <Culture of GS001 strain in semi-synthetic medium>
A modified M9+Glc liquid medium containing 0.5% (w/v) yeast extract (M9+Glc+YE liquid medium) was used as a semi-synthetic medium. 2 L of modified M9+Glc+Y. E. The GS001 strain was inoculated into the liquid medium and cultured. The culture temperature was set to 15° C., and aeration culture (1 vvm air, 150 to 750 rpm) was performed. Cultivation was performed using two jar fermenters.
（乾燥細胞重量の測定）
　任意の容積の培養液を、１００００×ｇ、４℃で５～１０分間遠心分離し、沈殿を得た。これを１％（ｗ／ｖ）酢酸アンモニウム水溶液で洗浄し、再度遠心分離する工程を２度行った。沈殿を少量の１％（ｗ／ｖ）酢酸アンモニウム水溶液に再懸濁し、予め秤量した樹脂チューブに移した。－８０℃で凍結後、凍結乾燥を行い、乾燥細胞を得た。細胞を含む樹脂チューブを再度秤量し、容積当たりの乾燥細胞重量を得た。 (Measurement of dry cell weight)
An arbitrary volume of culture was centrifuged at 10000×g at 4° C. for 5-10 minutes to obtain a precipitate. This was washed with a 1% (w/v) ammonium acetate aqueous solution, and the step of centrifuging again was performed twice. The precipitate was resuspended in a small volume of 1% (w/v) aqueous ammonium acetate solution and transferred to a pre-weighed resin tube. After freezing at -80°C, freeze-drying was performed to obtain dry cells. The resin tube containing the cells was weighed again to give the dry cell weight per volume.
（ＥＰＡの測定）
　ねじ口試験管に乾燥細胞を３０ｍｇ程度とり、抽出溶媒を０．５ｍＬ添加した。抽出溶媒はトルエン：メタノール＝５２：４８（ｖ／ｖ）の割合で混合した混合溶剤を用いた。
　抽出溶媒と乾燥細胞を混合した溶液に０．５Ｍ水酸化ナトリウム－メタノール溶液を１ｍＬ添加した。キャップを固く締め、ヒートブロックなどで試験管を１００℃、５分間加熱した。その後、試験管を十分に放冷した。
　試験管内に三フッ化ホウ素－メタノール溶液（三フッ化ホウ素濃度１２～１５％（ｗ／ｖ））を１ｍＬ添加した。キャップを固く締め、ヒートブロックなどで試験管を１００℃、５分間加熱した。その後、再び試験管を十分に放冷した。
　試験管にヘキサン１ｍＬおよび飽和食塩水を加え、十分に混和した。前記飽和食塩水との混合試料溶液を１４５０×ｇ、室温で５分間遠心分離し、得られた上層（有機層）をバイアルに適量とり、ガスクロマトグラフィー（ＧＣ）分析を行った。 (Measurement of EPA)
About 30 mg of dried cells were placed in a screw cap test tube, and 0.5 mL of an extraction solvent was added. A mixed solvent of toluene:methanol=52:48 (v/v) was used as an extraction solvent.
1 mL of 0.5 M sodium hydroxide-methanol solution was added to the mixed solution of the extraction solvent and dried cells. The cap was tightly tightened, and the test tube was heated at 100° C. for 5 minutes using a heat block or the like. The test tube was then allowed to cool sufficiently.
1 mL of a boron trifluoride-methanol solution (boron trifluoride concentration 12 to 15% (w/v)) was added to the test tube. The cap was tightly tightened, and the test tube was heated at 100° C. for 5 minutes using a heat block or the like. After that, the test tube was sufficiently cooled again.
1 mL of hexane and saturated saline were added to the test tube and thoroughly mixed. The mixed sample solution with the saturated saline solution was centrifuged at 1450×g for 5 minutes at room temperature, and an appropriate amount of the obtained upper layer (organic layer) was placed in a vial and subjected to gas chromatography (GC) analysis.
　図３Ａに、ＯＤ６００及び乾燥細胞重量の変化を示す。図３Ｂに、乾燥細胞重量当たりのＥＰＡ含有量を示す。
　図３Ａ中、ＯＤ　Ｊ１及びＤＣＷ　Ｊ１は、１台目のジャーファーメンターにおけるＯＤ６００及び乾燥細胞重量をそれぞれ示す。図３Ａ中、ＯＤ　Ｊ２及びＤＣＷ　Ｊ２は、２台目のジャーファーメンターにおけるＯＤ６００及び乾燥細胞重量をそれぞれ示す。ＧＳ００１株は、いずれのジャーファーメンターにおいても、乾燥細胞重量が３５ｇ／Ｌ以上に達しており良好な増殖を示した。なお、従来報告されているシェワネラ属細菌が到達する乾燥細胞重量は、１０．３～１０．５ｇ／Ｌ程度である。（鈴木信雄ら、Nippon Suisan Gakkaishi 58(2),323-328(1992).）
　図３Ｂ中、Ｊ１は、１台目のジャーファーメンターにおける乾燥細胞重量当たりのＥＰＡ含有量を示す。図３Ｂ中、Ｊ２は、２台目のジャーファーメンターにおける乾燥細胞重量当たりのＥＰＡ含有量を示す。いずれのジャーファーメンターにおいても、ＧＳ００１株は、高いＥＰＡ含有量を示した。 Figure 3A shows the change in OD600 and dry cell weight. Figure 3B shows the EPA content per dry cell weight.
In FIG. 3A, OD J1 and DCW J1 indicate OD 600 and dry cell weight in the first jar fermenter, respectively. In FIG. 3A, OD J2 and DCW J2 indicate the OD 600 and dry cell weight in the second jar fermenter, respectively. The GS001 strain reached a dry cell weight of 35 g/L or more in any jar fermenter and showed good growth. It should be noted that the dry cell weight reached by Shewanella bacteria reported in the past is about 10.3 to 10.5 g/L. (Nobuo Suzuki et al., Nippon Suisan Gakkaishi 58(2), 323-328(1992).)
In FIG. 3B, J1 indicates the EPA content per dry cell weight in the first jar fermenter. In FIG. 3B, J2 indicates the EPA content per dry cell weight in the second jar fermenter. In both jar fermenters, the GS001 strain showed a high EPA content.
＜ＧＳ００１株の増殖に対するＮａＣｌ濃度の影響＞
　ＮａＣｌ濃度を０．５～１０％（ｗ／ｖ）に変更した改変Ｍ９＋Ｇｌｃ液体培地を用いて、ＧＳ００１株の増殖に対するＮａＣｌ濃度の影響を検討した。１００ｍＬ三角フラスコに入れた５０ｍＬの改変Ｍ９＋Ｇｌｃ液体培地を用いて、１５℃で、振盪培養（１５０ｒｐｍ）した。培養開始から４８時間後に、培養液を採取し、波長６００ｎｍのＯＤを測定した。 <Effect of NaCl concentration on growth of GS001 strain>
Using a modified M9+Glc liquid medium in which the NaCl concentration was changed from 0.5 to 10% (w/v), the effect of NaCl concentration on the growth of strain GS001 was examined. Shaking culture (150 rpm) was performed at 15° C. using 50 mL of modified M9+Glc liquid medium placed in a 100 mL Erlenmeyer flask. 48 hours after the start of culture, the culture solution was collected and OD was measured at a wavelength of 600 nm.
　結果を図４に示す。ＧＳ００１株は、０．５～５％（ｗ／ｖ）のＮａＣｌ濃度範囲で、増殖することができた。ＧＳ００１株は、特に、０．５～３％（ｗ／ｖ）のＮａＣｌ濃度範囲では、増殖が良好であった。 The results are shown in Figure 4. The GS001 strain was able to grow in a NaCl concentration range of 0.5-5% (w/v). The GS001 strain grew well, especially in the NaCl concentration range of 0.5-3% (w/v).
　表４及び表５に、以前に報告されたシェワネラ・ウーディイの菌株（Esra Ersoy Omeroglu et al., Folia Microbiol (2014) 59:79-92）及びＧＳ００１株の増殖に対するＮａＣｌ濃度の影響をまとめた。表４及び表５において、ＧＳ００１株以外のデータは、Esra Ersoy Omeroglu et al., Folia Microbiol (2014) 59:79-92にＴａｂｌｅ　２から引用した。「＋」は生育可、「－」は生育不可、「Ｎ．Ｄ．」は非実施を示す。 Tables 4 and 5 summarize the effect of NaCl concentration on the growth of previously reported strains of Shewanella udii (Esra Ersoy Omeroglu et al., Folia Microbiol (2014) 59:79-92) and the GS001 strain. In Tables 4 and 5, data other than GS001 strain are quoted from Table 2 in Esra Ersoy Omeroglu et al., Folia Microbiol (2014) 59:79-92. "+" indicates growth, "-" indicates non-growth, and "N.D." indicates non-implementation.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　ＧＳ００１株は、０．５％（ｗ／ｖ）のＮａＣｌ濃度でも生育可能である。一方、他のシェワネラ・ウーディイ菌株で０．５％（ｗ／ｖ）のＮａＣｌ濃度で生育可能なものは報告されていない。この結果から、ＧＳ００１株は、従来報告されていたシェワネラ・ウーディイ菌株よりも、低塩濃度で生育可能であることが確認された。 The GS001 strain can grow even at a NaCl concentration of 0.5% (w/v). On the other hand, no other Shewanella udii strains have been reported that can grow at a NaCl concentration of 0.5% (w/v). From this result, it was confirmed that the GS001 strain can grow at a lower salt concentration than the previously reported Shewanella udii strains.
＜グルコースを唯一の有機炭素源とする合成培地での増殖試験＞
　ＧＳ００１株又はシェワネラ・エレクトロディフィラ（Ｓｈｅｗａｎｅｌｌａ　ｅｌｅｃｔｒｏｄｉｐｈｉｌａ）ＡＴＣＣ　ＢＡＡ－２４０８（以下、「ＡＴＣＣ　ＢＡＡ－２４０８」）を、改変Ｍ９＋Ｇｌｃ液体培地で４８時間前培養した（培養温度１５℃、振盪培養（回転数：１５０ｒｐｍ）。
　前培養したＧＳ００１株又はＡＴＣＣ　ＢＡＡ－２４０８を、１００ｍＬフラスコに入れた５０ｍＬの改変Ｍ９＋Ｇｌｃ液体培地に接種して培養した。培養温度１５℃とし、バイオシェーカーを用いて振盪培養（回転数：１５０ｒｐｍ）した。
　シェワネラ・エレクトロディフィラは、グルコースを唯一の有機炭素源として良好に生育することが報告されているシェワネラ属細菌である（Jinwei Zhang, and J. Grant Burgess. PLoS One. 2017 Nov 27;12(11):）。グルコースを唯一の有機炭素源としたときのシェワネラ・エレクトロディフィラの増殖速度は、グルコース資化能の指標となる。 <Growth test in synthetic medium with glucose as sole organic carbon source>
GS001 strain or Shewanella electrodiphila ATCC BAA-2408 (hereinafter, "ATCC BAA-2408") was precultured in a modified M9 + Glc liquid medium for 48 hours (culture temperature 15 ° C., shaking culture (rotation speed: 150 rpm).
The precultured strain GS001 or ATCC BAA-2408 was inoculated into 50 mL modified M9+Glc liquid medium in a 100 mL flask and cultured. The culture temperature was set to 15° C., and shaking culture was performed using a bioshaker (rotation speed: 150 rpm).
Shewanella electrodyphila is a bacterium of the genus Shewanella that has been reported to grow well using glucose as the sole organic carbon source (Jinwei Zhang, and J. Grant Burgess. PLoS One. 2017 Nov 27;12(11 ):). The growth rate of Shewanella electrodyphila when glucose is the sole organic carbon source is an index of glucose utilization.
　結果を図５に示す。ＧＳ００１株は、ＡＴＣＣ　ＢＡＡ－２４０８と比較して、誘導期が短く、増殖速度が速かった。この結果から、ＧＳ００１株は、ＡＴＣＣ　ＢＡＡ－２４０８と比較して、グルコースの資化能力が高いことが確認された。 The results are shown in Figure 5. The GS001 strain had a shorter induction period and a faster growth rate than ATCC BAA-2408. From this result, it was confirmed that the GS001 strain has a higher ability to assimilate glucose compared to ATCC BAA-2408.
　以上、本発明の好ましい実施形態を説明および図示してきたが、これらは本発明を例示するものであり、限定的なものとみなされるべきではないことを理解すべきである。本発明の精神または範囲から逸脱することなく、追加、省略、置換、およびその他の変更を行うことができる。したがって、本発明は、前述の説明によって限定されるものとはみなされず、添付の請求項の範囲によってのみ限定される。 While the preferred embodiments of the invention have been described and illustrated above, it should be understood that they are intended to be illustrative of the invention and should not be taken as limiting. Additions, omissions, substitutions, and other changes can be made without departing from the spirit or scope of the invention. Accordingly, the present invention should not be viewed as limited by the foregoing description, but only by the scope of the appended claims.

Claims (5)

  1. 　シェワネラ・ウーディイ　ＧＳ００１株（受託番号ＮＩＴＥ　ＢＰ－０３４６０）又はその変異株。 Shewanella udii strain GS001 (acceptance number NITE BP-03460) or a variant thereof.
  2. 　シェワネラ・ウーディイ　ＧＳ００１株又はその変異株を含む、組成物。 A composition containing the Shewanella udii strain GS001 or a mutant strain thereof.
  3. 　食品、飼料、又は餌料、又は化粧料である、請求項２に記載の組成物。 The composition according to claim 2, which is food, feed, feed, or cosmetics.
  4. 　シェワネラ・ウーディイ　ＧＳ００１株又はその変異株の乾燥粉末。 　Shewanella udii GS001 strain or a dry powder thereof.
  5. 　シェワネラ・ウーディイ　ＧＳ００１株又はその変異株の抽出物。 Extract of Shewanella udii strain GS001 or its mutants.
PCT/JP2022/022593 2021-06-04 2022-06-03 Shewanella bacteria and use thereof WO2022255477A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2023525927A JP7468788B2 (en) 2021-06-04 2022-06-03 Shewanella bacteria and uses thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2021094382 2021-06-04
JP2021-094382 2021-06-04

Publications (1)

Publication Number Publication Date
WO2022255477A1 true WO2022255477A1 (en) 2022-12-08

Family

ID=84322598

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2022/022593 WO2022255477A1 (en) 2021-06-04 2022-06-03 Shewanella bacteria and use thereof

Country Status (3)

Country Link
JP (1) JP7468788B2 (en)
TW (1) TW202313961A (en)
WO (1) WO2022255477A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023157948A1 (en) * 2022-02-21 2023-08-24 Dic株式会社 Oral composition

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK 31 October 1997 (1997-10-31), ANONYMOUS: "Shewanella woodyi 16S ribosomal RNA gene, complete sequence", XP093011807, retrieved from NUCLEOTIDE Database accession no. AF003549 *
THEBERGE ALLISON L., ALSABIA SAHAR M., MORTENSEN CHRISTOPHER T., BLAIR ANNA G., WENDEL NINA M., BIFFINGER JUSTIN C.: "Soluble electron acceptors affect bioluminescence from Shewanella woodyi", LUMINESCENCE: THE JOURNAL OF BIOLOGICAL AND CHEMICAL LUMINESCENCE, vol. 35, no. 3, 1 May 2020 (2020-05-01), GB , pages 427 - 433, XP093011805, ISSN: 1522-7235, DOI: 10.1002/bio.3744 *
THEBERGE ALLISON LINDSEY: "An Investigation Correlating Bioluminescence and Metal Ruduction Utilizing Shewanella woodyI", OHIOLINK ELECTRONIC THESES AND DISSERTATIONS CENTER, UNIVERSITY OF DAYTON / OHIOLINK, 1 January 2019 (2019-01-01), pages 1 - 65, XP093011799, Retrieved from the Internet <URL:http://rave.ohiolink.edu/etdc/view?acc_num=dayton1555331326931868>> [retrieved on 20230105] *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023157948A1 (en) * 2022-02-21 2023-08-24 Dic株式会社 Oral composition

Also Published As

Publication number Publication date
JPWO2022255477A1 (en) 2022-12-08
TW202313961A (en) 2023-04-01
JP7468788B2 (en) 2024-04-16

Similar Documents

Publication Publication Date Title
Labrenz et al. Staleya guttiformis gen. nov., sp. nov. and Sulfitobacter brevis sp. nov., alpha-3-Proteobacteria from hypersaline, heliothermal and meromictic antarctic Ekho Lake.
Fuerst et al. Porphyrobacter neustonensis gen. nov., sp. nov., an aerobic bacteriochlorophyll-synthesizing budding bacterium from fresh water
WO2018225556A1 (en) Novel lactic acid bacteria and use thereof
Vorobjeva Propionibacteria
JP6523579B2 (en) Extracellular polysaccharide of lactic acid bacteria and use thereof
WO2022255477A1 (en) Shewanella bacteria and use thereof
Hamzehlou et al. Production of vitamins B3, B6 and B9 by Lactobacillus isolated from traditional yogurt samples from 3 cities in Iran, winter 2016
US20230380462A1 (en) Fermented composition
Chowdhury et al. Optimization, dynamics, and enhanced production of a free radical scavenging extracellular polysaccharide (EPS) from hydrodynamic sediment attached Bacillus megaterium RB-05
JP4799895B2 (en) Production method of carotenoid pigment, glycosphingolipid, ubiquinone Q-10
Oh et al. Gracilibacillus kimchii sp. nov., a halophilic bacterium isolated from kimchi
JP4104741B2 (en) Vitamin K high-producing strain and method for producing vitamin K using the same
Mohammed et al. Local culture medium from the legumes mixture as a novel media for the growth and stimulation of prodigiosin pigment which production from Serratia marcescens that isolated environmentally
WO2023112407A1 (en) Cyanidioschyzon merolae which can be heterotrophically proliferated in dark place, and use thereof
Abedi et al. Isolation and Identification of Lactobacillus strains from dairy products and evaluation of carbon sources effects on bacterial growth and phytase activity: supplement for fish feed
JP2009232716A (en) New lactic acid bacterium
Rattanaporn et al. Production of pyridoxal 5՛-phosphate and pyridoxineby Lactobacillus pentosus L47I-A
Khadija et al. Phenotypic and Genotypic Identification of the Most Acidifiers LAB Strains Isolated from Fermented Food
JP2019208457A (en) Novel microorganism, and method for producing urolithin using the microorganism
JP2019129721A (en) Method for producing seaweed-derived lactic acid fermentation solution
RU2816652C1 (en) Bacterial strain lacticaseibacillus paracasei subspecies paracasei 1338 vkm b-3753d for production of fermented milk products and as probiotic
KR102049970B1 (en) Composition for improving oxidative stress due to the fine dust containing Lactobacillus casei HY2782 as effective component
Pham et al. Cholesterol-lowering potential and exopolysaccharide biosynthesis of Lactobacillus spp. isolated from human milk
Makut et al. Molecular identification of lactic acid bacteria isolated from fermented rice
Ezemba et al. Microbacterium lacticum; a Lysine Producing Bacterium Isolated from Oil-contaminated Soil in South-East Nigeria

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22816222

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2023525927

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE