WO2017195873A1 - L-hydroxyproline-containing yeast cell of yarrowia lipolytica or cell culture product or extract of same, use thereof, and method for producing l-hydroxyproline - Google Patents

L-hydroxyproline-containing yeast cell of yarrowia lipolytica or cell culture product or extract of same, use thereof, and method for producing l-hydroxyproline Download PDF

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WO2017195873A1
WO2017195873A1 PCT/JP2017/017937 JP2017017937W WO2017195873A1 WO 2017195873 A1 WO2017195873 A1 WO 2017195873A1 JP 2017017937 W JP2017017937 W JP 2017017937W WO 2017195873 A1 WO2017195873 A1 WO 2017195873A1
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hydroxyproline
yeast
cell
culture
hyp
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PCT/JP2017/017937
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French (fr)
Japanese (ja)
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幸彦 成田
祐子 福井
龍雄 松岡
順 小川
慎 日比
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サントリーホールディングス株式会社
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Priority to KR1020187032008A priority Critical patent/KR102330633B1/en
Priority to CN201780029019.0A priority patent/CN109072171A/en
Priority to JP2018517082A priority patent/JP6869972B2/en
Publication of WO2017195873A1 publication Critical patent/WO2017195873A1/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
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • 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
    • A23L31/00—Edible extracts or preparations of fungi; Preparation or treatment thereof
    • A23L31/10—Yeasts or derivatives thereof
    • 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
    • A23L33/14—Yeasts or derivatives thereof
    • 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
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/175—Amino acids
    • 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
    • A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
    • A61K8/44—Aminocarboxylic acids or derivatives thereof, e.g. aminocarboxylic acids containing sulfur; Salts; Esters or N-acylated derivatives 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
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products 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
    • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
    • 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
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D207/10—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D207/16—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • 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
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • 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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/24—Proline; Hydroxyproline; Histidine
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

Definitions

  • the present invention relates to a yeast cell or a cell culture or an extract thereof containing L-hydroxyproline, its use, and a method for producing L-hydroxyproline.
  • the invention also relates to the use of yeast for producing L-hydroxyproline.
  • the present invention also relates to a food / beverage product, a cosmetic, a cosmetic raw material, a composition for reinforcing L-hydroxyproline, and the like containing a yeast cell or cell culture or an extract thereof.
  • L-hydroxyproline (hydroxy-L-proline) is an amino acid having a structure in which a hydroxyl group is bonded to the 4-position carbon atom of L-proline.
  • L-hydroxyproline has the following effects: promotion of collagen production in fibroblasts, promotion of epidermal cell proliferation, moisturizing effect equivalent to or better than collagen, prevention of skin aging, transdermal absorbability higher than tripeptide, wrinkle improvement effect, The improvement effect of atopic dermatitis etc. are mentioned. Since L-hydroxyproline is safe for the human body, it can be used by being contained in foods and drinks, cosmetics, pharmaceuticals, etc., and its usefulness is very high.
  • L-hydroxyproline can be produced by an organic synthesis method, but a production method using a microorganism is also being studied.
  • Patent Document 1 discloses that a transformant obtained by introducing a polynucleotide encoding L-proline cis-4-hydroxylase derived from Rhizobium rhizobia into a host cell is cultured in a medium, and the cis- A method for producing cis-4-hydroxy-L-proline is described in which 4-hydroxy-L-proline is produced and accumulated, and cis-4-hydroxy-L-proline is collected from the culture.
  • yeast is a microorganism which has been tried for various industrial uses from the old days in the food and drink field, etc., and yeast cells or cell cultures or extracts thereof containing L-hydroxyproline are L -It is useful as a raw material for cosmetics, foods and drinks where the effect of hydroxyproline is expected.
  • yeast cells or cell cultures or extracts thereof containing L-hydroxyproline are L -It is useful as a raw material for cosmetics, foods and drinks where the effect of hydroxyproline is expected.
  • a yeast that accumulates L-hydroxyproline in bacterial cells or bacterial cell cultures has not yet been reported.
  • the main object of the present invention is to provide a yeast cell or cell culture or yeast extract containing L-hydroxyproline, its use, and a method for producing L-hydroxyproline.
  • the present inventors have found that when Yarrowia lipolytica (yeast of yeast) is aerobically cultured, L-hydroxyproline is added to the cells or cell cultures. Found to accumulate.
  • the obtained Yarrowia lipolytica cell or cell culture containing L-hydroxyproline or an extract thereof is the total weight of L-proline (Pro) and L-hydroxyproline (Hyp). It has been found that the ratio of the weight content of L-hydroxyproline to the content (100 ⁇ Hyp / (Pro + Hyp)) can be in a specific range.
  • the present inventors have further studied based on these findings and have completed the present invention.
  • a yeast cell or a cell culture of the yeast of the present invention or an extract thereof is a cell or a cell culture of yeast Yarrowia lipolytica or an extract thereof, wherein the yeast cell or The cell culture or these extracts contain L-hydroxyproline, and the content of L-hydroxyproline ( ⁇ g / mL) relative to the total content ( ⁇ g / mL) of L-proline (Pro) and L-hydroxyproline (Hyp). / ML) (100 ⁇ Hyp / (Pro + Hyp)) is 35 to 100.
  • the yeast cells or cell cultures or extracts thereof of the present invention preferably have an L-hydroxyproline content of 10 ⁇ g / mL or more.
  • the yeast cell or cell culture or an extract thereof is a yeast cell or cell culture of yeast Yarrowia lipolytica or an extract thereof.
  • the hydroxyproline content is 10 ⁇ g / mL or more.
  • the yeast cell or cell culture is obtained by aerobically culturing yeast Yarrowia lipolytica in a liquid medium containing a carbon source and a nitrogen source.
  • the L-hydroxyproline-containing peptide is preferably a collagen peptide.
  • the average molecular weight of the collagen peptide is preferably 1000 to 10,000.
  • the aerobic culture is preferably performed for 10 to 100 hours.
  • the invention also encompasses the use of the yeast Yarrowia lipolytica to produce L-hydroxyproline.
  • the use of the present invention can be carried out by aerobically cultivating the yeast Yarrowia lipolytica in a liquid medium containing a carbon source and a nitrogen source, whereby L-hydroxyl in the yeast cell or cell culture. It is preferable that the nitrogen source is a nitrogen source containing an L-hydroxyproline-containing peptide.
  • the L-hydroxyproline-containing peptide is preferably a collagen peptide.
  • the average molecular weight of the collagen peptide is preferably 1000 to 10,000.
  • the aerobic culture is preferably performed for 10 to 100 hours.
  • composition of the present invention is characterized in that it comprises the yeast or cell culture of the yeast of the present invention or an extract thereof.
  • the food / beverage products of this invention are characterized by including the microbial cell or microbial cell culture of this invention, or these extracts.
  • the cosmetic or cosmetic raw material of the present invention is characterized by containing the yeast or bacterial culture of the yeast of the present invention or an extract thereof.
  • the cosmetic or cosmetic raw material of the present invention comprises collagen production promotion, epidermal cell growth promotion, skin moisturization, skin aging prevention, skin sagging prevention or improvement, skin firmness improvement, wrinkle prevention or improvement and It is preferably used for applications selected from the improvement of atopic dermatitis.
  • the cosmetic or cosmetic raw material of the present invention is a cosmetic raw material, and preferably has an L-hydroxyproline content of 5 to 300 ppm.
  • the cosmetic or cosmetic raw material of the present invention is a cosmetic and preferably has an L-hydroxyproline content of 0.01 to 20 ppm. In this specification, ppm means weight ppm.
  • the composition for reinforcing L-hydroxyproline of the present invention is characterized by comprising a cell or a cell culture of yeast Yarrowia lipolytica containing L-hydroxyproline or an extract thereof.
  • the yeast cell or cell culture or extract thereof contains the total content ( ⁇ g of L-proline (Pro) and L-hydroxyproline (Hyp)).
  • the ratio (100 ⁇ Hyp / (Pro + Hyp)) of L-hydroxyproline content ( ⁇ g / mL) to (mL / mL) is preferably 35 to 100.
  • the yeast cells or cell cultures or extracts thereof preferably have an L-hydroxyproline content of 10 ⁇ g / mL or more.
  • yeast cell or bacterial cell culture or an extract thereof and its use containing L-hydroxyproline, a method for producing L-hydroxyproline, and the like.
  • the yeast cell or cell culture of the present invention or an extract thereof is suitably used as a raw material for foods and beverages, cosmetics and the like.
  • FIG. 1 is a graph showing the amount of L-hydroxyproline (Hyp) accumulated in yeast.
  • FIG. 2 is a graph showing the amount of Yyp accumulation of Yarrowia lipolytica cultured in each medium ((a): Condition 1, (b): Condition 2).
  • FIG. 3 is a graph showing the Hyp accumulation amount of Yarrowia lipolytica cultured for 5 days in PD medium or PD medium supplemented with 0.125% gelatin (white: PD medium containing 0.125% gelatin, black: PD medium) ).
  • FIG. 1 is a graph showing the amount of L-hydroxyproline (Hyp) accumulated in yeast.
  • FIG. 2 is a graph showing the amount of Yyp accumulation of Yarrowia lipolytica cultured in each medium ((a): Condition 1, (b): Condition 2).
  • FIG. 3 is a graph showing the Hyp accumulation amount of Yarrowia lipolytica cultured for 5 days in PD medium or PD
  • FIG. 4 is a graph showing the Hyp accumulation amount of Yarrowia lipolytica cultured in YPD media having different compositions ((a): Hyp accumulation amount ( ⁇ g / mL), (b): Hyp amount per cell ( ⁇ g / mL). / OD600)).
  • FIG. 5 is a graph showing the Hyp accumulation amount, the Hyp amount per cell, and OD600 of Yarrowia lipolytica cultured in a medium containing peptone or collagen peptide ((a): Hyp accumulation amount ( ⁇ g / mL), (b) : Hyp amount per cell ( ⁇ g / mL / OD600), (c): OD600)).
  • FIG. 5 is a graph showing the Hyp accumulation amount, the Hyp amount per cell, and OD600 of Yarrowia lipolytica cultured in a medium containing peptone or collagen peptide ((a): Hyp accumulation amount ( ⁇ g / mL), (b
  • FIG. 6 is a diagram showing the amount of Hyp accumulated in Yarrowia lipolytica cultured by shaking culture or stationary culture ((a): Hyp equivalent per culture medium in culture sample (intracellular), (b): in culture supernatant. Hyp amount)). It is a graph which shows the ethanol concentration and glucose concentration of the culture supernatant of Yarrowia lipolytica which carried out shaking culture or stationary culture ((a): ethanol concentration (v / v%), (b): glucose concentration (weight%)).
  • FIG. 8 is an HPLC chart obtained by analyzing a 0.1N hydrochloric acid solution containing amino acid mixed standard solution H and L-hydroxyproline (each amino acid concentration 20 ⁇ mol / L) ((a): Ch1 excitation wavelength 350 nm, fluorescence wavelength. Detection at 450 nm, (b): Ch2 excitation wavelength 266 nm, fluorescence wavelength 305 nm detection).
  • a yeast cell or cell culture or an extract thereof according to the first aspect of the present invention is a cell or cell culture of yeast Yarrowia lipolytica or an extract thereof, Yeast cells or cell cultures or extracts thereof contain L-hydroxyproline, and L-hydroxy with respect to the total content ( ⁇ g / mL) of L-proline (Pro) and L-hydroxyproline (Hyp).
  • the ratio of proline content ( ⁇ g / mL) (100 ⁇ Hyp / (Pro + Hyp)) is 35 to 100.
  • the yeast cell or cell culture or the extract thereof according to the second aspect of the present invention is a yeast cell or a cell culture of yeast Yarrowia lipolytica or an extract thereof.
  • the content of hydroxyproline is 10 ⁇ g / mL or more.
  • the yeast cells or cell cultures of the yeast according to the first aspect and the second aspect of the present invention, or extracts thereof are collectively referred to as the yeast cells or cell cultures of the yeast of the present invention or extracts thereof. Also called.
  • the yeast cells or cell cultures or extracts thereof of the present invention are yeast cells or cell cultures of yeast Yarrowia lipolytica or extracts thereof.
  • the yeast in this invention should just be a yeast which belongs to Yarrowia lipolytica ( Yarrowia lipolytica ), and may use only 1 type and may use 2 or more types.
  • Yeast Yarrowia lipolytica is available from various depository institutions. Examples of depository organizations include the National Institute of Technology and Evaluation (2-5-8, Kazusa Kamashichi, Kisarazu City, Chiba Prefecture, Japan). It can also be separated from nature.
  • L-hydroxyproline in the present invention is 4-hydroxy-L-proline.
  • L-hydroxyproline contained in yeast cells or cell cultures or extracts thereof refers to free L-hydroxyproline.
  • the L-hydroxyproline content or accumulated amount in yeast cells or cell cultures or extracts thereof refers to the amount of free L-hydroxyproline. No yeast has been reported that accumulates free L-hydroxyproline in its cells or cell cultures.
  • the yeast or cell culture of yeast according to the first aspect of the present invention, or an extract thereof contains L-hydroxy with respect to the total content ( ⁇ g / mL) of L-proline (Pro) and L-hydroxyproline (Hyp).
  • the ratio of proline content ( ⁇ g / mL) (100 ⁇ Hyp / (Pro + Hyp)) is 35 to 100.
  • Such yeast cells or cell cultures or extracts thereof are suitably used for foods and beverages and cosmetic raw materials for which L-hydroxyproline is expected to be effective.
  • “Ratio of L-hydroxyproline content ( ⁇ g / mL) to total content ( ⁇ g / mL) of L-proline (Pro) and L-hydroxyproline (Hyp)” (100 ⁇ Hyp / (Pro + Hyp)) Then, it is also referred to as “(Hyp / (Pro + Hyp)) ratio”.
  • the L-proline content in the above ratio (Hyp / (Pro + Hyp)) refers to the free L-proline content contained in yeast cells or cell cultures or extracts thereof.
  • the yeast cell or cell culture of the second embodiment of the present invention or an extract thereof has a (Hyp / (Pro + Hyp)) ratio of 35 to 100.
  • the (Hyp / (Pro + Hyp)) ratio is preferably 40 to 100, more preferably 50 to 100, more preferably 60 to 100, still more preferably 70 to 100, and still more preferably. 80 to 100, particularly preferably 90 to 100.
  • the above yeast (Hyp / (Pro + Hyp)) ratio of such yeast cells or cell cultures or extracts thereof have a high Hyp content ratio and are expected to be effective for L-hydroxyproline. It is particularly suitable as a raw material for the material.
  • the yeast cell or cell culture or the extract thereof according to the second aspect of the present invention has an L-hydroxyproline content of 10 ⁇ g / mL or more.
  • the yeast cell or cell culture or extract thereof according to the first aspect of the present invention preferably has an L-hydroxyproline content of 10 ⁇ g / mL or more.
  • a yeast cell or cell culture or an extract thereof having a content of L-hydroxyproline within the above range is suitable as a raw material for foods and beverages and cosmetics for which the effect of L-hydroxyproline is expected.
  • the content of L-hydroxyproline in the yeast or cell culture of the yeast of the present invention or these extracts is more preferably 15 ⁇ g / mL or more, more preferably 17 ⁇ g / mL or more, and more preferably 20 ⁇ g / mL or more. More preferably, 30 ⁇ g / mL or more is more preferable, 40 ⁇ g / mL or more is further preferable, 50 ⁇ g / mL or more is further preferable, 70 ⁇ g / mL or more is further preferable, 100 ⁇ g / mL or more is further preferable, and 150 ⁇ g / mL or more is more preferable.
  • the upper limit of the content of L-hydroxyproline in yeast cells or cell cultures or extracts thereof is not particularly limited and is preferably large, but is usually 6000 ⁇ g / mL or less, 3000 ⁇ g / mL or less, or 2000 ⁇ g. / ML or less may be sufficient. According to the present invention, it is possible to provide a yeast cell or cell culture, or an extract thereof, wherein the content of L-hydroxyproline derived from the yeast cell or cell culture is in the above range. .
  • the L-proline content ( ⁇ g / mL) and the L-hydroxyproline content ( ⁇ g / mL) in yeast cells or cell cultures or extracts thereof are measured by high performance liquid chromatography (HPLC). be able to.
  • HPLC high performance liquid chromatography
  • yeast cells or cell cultures or extracts of these contain cells, use the one that crushes cells by heating, etc. by self-digestion or enzymatic degradation, and elutes the cell contents. Then, the contents of L-proline and L-hydroxyproline are measured.
  • HPLC measurement conditions, and the like the methods and conditions described in the Examples may be employed.
  • amino acids are sequentially derivatized with o-phthalaldehyde (OPA) and 4-chloro-7-nitrobenzofurazan (NBD-Cl) and analyzed by HPLC (excitation wavelength 503 nm / fluorescence wavelength 541 nm).
  • OPA o-phthalaldehyde
  • NBD-Cl 4-chloro-7-nitrobenzofurazan
  • the yeast cells or cell culture of the yeast of the present invention or extracts thereof are obtained by aerobically culturing yeast Yarrowia lipolytica in a liquid medium, and crushing the cells as necessary.
  • the yeast cell culture preferably contains yeast cells and / or culture supernatant, and may contain yeast cell contents.
  • the yeast cells may be live or dead.
  • yeast cells cultured cells obtained by aerobic culture of the yeast and cell culture liquid containing the culture supernatant, the bacteria Examples include yeast cells collected from a body culture solution (bacteria) or culture supernatants obtained by removing the cells from the cell culture solution.
  • the culture supernatant of the cell culture medium is simply referred to as the culture supernatant.
  • the cell culture is preferably a cell culture solution or culture supernatant containing yeast cells and culture supernatant.
  • the extract of a microbial cell or a microbial cell culture contains a microbial cell content normally, and it is preferable that a microbial cell content and a culture supernatant are included.
  • the microbial cell culture or a microbial cell culture containing the microbial cell is subjected to microbial cell disruption treatment such as self-digestion treatment or enzymatic degradation treatment, What eluted yeast cell contents in the culture solution (broken cell), removed the cell residue from the broken cell or cell culture (broken cell)
  • microbial cell disruption treatment such as self-digestion treatment or enzymatic degradation treatment, What eluted yeast cell contents in the culture solution (broken cell), removed the cell residue from the broken cell or cell culture (broken cell)
  • the microbial cell culture solution bacterial cell crushed material
  • the culture supernatant obtained by removing the cell residue from the cell crushed material It is a thing.
  • the yeast cells or cell cultures of the present invention or extracts thereof are usually yeast cells obtained by aerobic culture of yeast Yarrowia lipolytica in a liquid medium.
  • the bacterial cell culture medium containing the culture supernatant is prepared by subjecting the bacterial cell culture solution to treatment such as collection and disruption of the bacterial cells as necessary.
  • the L-hydroxyproline contained in the yeast cell or cell culture of the present invention or the extract thereof is preferably derived from the yeast cell or cell culture obtained by the above aerobic culture. It is preferable that the L-hydroxyproline contained in the yeast cell or cell culture of the present invention or the extract thereof is substantially absent before the aerobic culture.
  • the yeast cells or cell cultures or extracts thereof of the present invention can be suitably used as raw materials for foods and drinks including cosmetics and liquors, for example.
  • the yeast cell or cell culture of the present invention or an extract thereof has a value ( ⁇ g / mL / OD600) obtained by dividing the L-hydroxyproline content ( ⁇ g / mL) by OD600 (0.5 or more). It is preferable that A value obtained by dividing the content of L-hydroxyproline ( ⁇ g / mL) by OD600 ( ⁇ g / mL / OD600) is hereinafter also referred to as a Hyp / OD600 value. A higher Hyp / OD600 value is preferred because the L-hydroxyproline content per cell is higher.
  • the upper limit of the Hyp / OD600 value of yeast cells or cell cultures or extracts thereof is not particularly limited and is preferably as large as possible, but is usually 300 or less.
  • the Hyp / OD600 value is more preferably 1 or more, for example, 1 to 150 is preferable.
  • the Hyp / OD600 value is more preferably 25 or more, and even more preferably 50 or more.
  • OD is an abbreviation for optical density and refers to optical density. OD represents a cell concentration or the like. In general, absorbance OD600 or OD660 with respect to visible light having a wavelength of 600 nm or 660 nm is measured (Bio Experiment Illustrated (7) Yeast that can be used Two Hybrid, Shujunsha, published in 2003).
  • the OD600 used for the calculation of the Hyp / OD600 value is the cell culture solution containing the cells and culture supernatant (cells and culture supernatant used for the preparation of the cells or cell cultures or extracts thereof)
  • OD600 is: It is the light absorbency OD600 of the microbial cell culture solution (the microbial cell culture solution containing the microbial cell of the yeast before microbial cell disruption, and a culture supernatant) used for the preparation. OD600 can be measured with a spectrophotometer, for example.
  • the yeast cells or cell cultures or extracts thereof of the present invention preferably have an ethanol content of 1 v / v% or less.
  • the ethanol content is 1 v / v% or less, it can be particularly preferably used as a raw material for various foods and cosmetics. If ethanol exceeds 1 v / v%, there may be adverse effects on yeast growth and the like.
  • the ethanol content of the yeast or the cell culture of the yeast of the present invention or the extract thereof is more preferably 0.8 v / v% or less, and further preferably 0.5 v / v% or less.
  • the ethanol content can be measured by a known method.
  • the form of the yeast cells or cell cultures or extracts thereof of the present invention is not particularly limited, and examples thereof include pastes, suspensions, extracts, and liquids.
  • the yeast cells or cell cultures of the present invention or extracts thereof can be suitably used as a raw material for cosmetics, foods and drinks, etc. as described later.
  • the yeast cells or cell cultures of the yeast of the present invention or extracts thereof can be used after being powdered by drying or the like.
  • the yeast cell or cell culture of the present invention or an extract thereof is obtained by aerobically cultivating the yeast Yarrowia lipolytica in a liquid medium containing a carbon source and a nitrogen source. It can be obtained by disrupting the cells. More specifically, the nitrogen source includes an L-hydroxyproline-containing peptide.
  • Yeastia lipolytica ( Yarrowia lipolytica ) is aerobically cultured in a liquid medium containing a carbon source and a nitrogen source containing an L-hydroxyproline-containing peptide, whereby the yeast cell or cell culture is obtained. L-hydroxyproline accumulates, and yeast cells or cell cultures containing L-hydroxyproline are obtained.
  • Yeastia lipolytica ( Yarrowia lipolytica ) is aerobically cultured in a liquid medium containing a carbon source and a nitrogen source containing an L-hydroxyproline-containing peptide, whereby the yeast cell or cell culture is obtained.
  • a method including the step of accumulating L-hydroxyproline is preferable as a method for producing the above-described yeast cell or cell culture of the present invention or an extract thereof, or a method for producing L-hydroxyproline.
  • the yeast cell or cell culture is obtained by aerobically culturing yeast Yarrowia lipolytica in a liquid medium containing a carbon source and a nitrogen source.
  • a step of accumulating L-hydroxyproline (hereinafter also referred to as a Hyp accumulation step).
  • the nitrogen source is a nitrogen source containing an L-hydroxyproline-containing peptide.
  • the production method of the present invention may have steps other than the Hyp accumulation step as desired. For example, you may have 1 or 2 or more processes, such as the preculture process mentioned later, a microbe collection process, and a microbial cell crushing process.
  • the above-described yeast cell or cell culture of the present invention or an extract thereof can be obtained.
  • a yeast cell or cell culture containing L-hydroxyproline is obtained.
  • the obtained yeast cells or cell cultures usually have the above (Hyp / (Pro + Hyp)) ratio of 35 to 100.
  • the yeast cell or cell culture obtained by the Hyp accumulation step usually has an L-hydroxyproline content of 10 ⁇ g / mL or more.
  • Such yeast cells or cell cultures can be used as the yeast cells or cell cultures of the present invention described above.
  • the obtained bacterial cells or bacterial cell culture is further subjected to treatment such as cell disruption as required to prepare a yeast bacterial cell or bacterial cell culture extract containing L-hydroxyproline.
  • the method for producing L-hydroxyproline including the Hyp accumulation step is also preferable as a method for producing the above-described yeast cells or cell cultures of the present invention or extracts thereof.
  • the Hyp accumulation step and preferred embodiments thereof are the same as the Hyp accumulation step and preferred embodiments thereof in the method for producing L-hydroxyproline.
  • yeast Yarrowia lipolytica can be added to a liquid medium by inoculating a small amount of cells directly into the liquid medium containing a carbon source and a nitrogen source.
  • the medium used for the pre-culture is not particularly limited, and may be the same medium as the liquid medium used in the Hyp accumulation step (usually main culture), or a known medium that can be used for yeast.
  • the preculture time is usually 10 to 72 hours, preferably 12 to 48 hours.
  • the preculture temperature is preferably 15 to 40 ° C.
  • the amount inoculated with the pre-cultured bacterial solution is usually 1/10000 to 1/2 of the amount of medium used in the Hyp accumulation step, preferably 1/1000 to 1/10, and preferably 1/200 to 1/10. Is more preferable, and 1/200 to 1/20 is even more preferable. If the inoculation amount is in the above range, the yeast Yarrowia lipolytica grows rapidly in the Hyp accumulation step, and L-hydroxyproline can be efficiently accumulated.
  • the nitrogen source of the liquid medium used in the Hyp accumulation step is a nitrogen source containing an L-hydroxyproline-containing peptide.
  • L-hydroxyproline When the yeast Yarrowia lipolytica is aerobically cultured using such a nitrogen source, L-hydroxyproline accumulates in the cells or cell cultures.
  • Such nitrogen sources may be used alone or in combination of two or more.
  • the number of L-hydroxyproline-containing peptides may be one, or two or more.
  • the L-hydroxyproline-containing peptide may be any peptide that contains L-hydroxyproline as a constituent amino acid, but is preferably a peptide in which 10% by weight or more of the constituent amino acid is L-hydroxyproline.
  • the nitrogen source containing the L-hydroxyproline-containing peptide is also preferably an L-hydroxyproline-containing peptide.
  • a nitrogen source containing an L-hydroxyproline-containing peptide can be obtained, for example, by hydrolyzing an L-hydroxyproline-containing protein.
  • the L-hydroxyproline-containing protein may be a protein containing L-hydroxyproline as a constituent amino acid, but is preferably a protein in which 10% by weight or more of the constituent amino acid is L-hydroxyproline.
  • collagenous protein and the like are preferable.
  • the collagenous protein include proteins prepared from tissues containing collagen such as viscera, skin, fish scales, and bones; collagen and gelatin.
  • the origin of the collagen protein is not particularly limited.
  • collagen-derived proteins derived from animals such as cow-derived, pig-derived and fish-derived can be preferably used.
  • As the collagenous protein a commercially available product can be used. Hydrolysis of the L-hydroxyproline-containing protein can be performed by a known method using an enzyme or the like.
  • animal-derived peptone can be preferably used as a nitrogen source containing an L-hydroxyproline-containing peptide.
  • Peptone derived from cow, pig or fish is preferred, and peptone derived from cow or fish is more preferred.
  • animal peptone, myocardial peptone, and gelatin peptone are also preferable as peptone.
  • An example of a commercial product of a nitrogen source containing an L-hydroxyproline-containing peptide that can be used in the present invention is the product name Pepton (# 211677) (Bacto).
  • the L-hydroxyproline-containing peptide is preferably a collagen peptide.
  • Collagen peptide means hydrolyzed collagen, which can be either gelatin modified by heat treatment of natural collagen, collagen peptide hydrolyzed from natural collagen, or those chemically or enzymatically modified. Good. Hydrolysis can be performed with an enzyme, acid, alkali, or the like, and preferably with an enzyme.
  • gelatin modified by heat treatment of natural collagen or collagen peptide hydrolyzed from natural collagen is used.
  • the origin of the collagen peptide is not particularly limited.
  • animal-derived collagen peptides such as cow-derived, pig-derived and fish-derived can be preferably used.
  • it is a collagen peptide derived from fish.
  • a commercially available collagen peptide can be used.
  • Examples of commercially available collagen peptides that can be used in the present invention include, for example, “Collagen Peptide Iquos HDL-50SP” (product name) (average molecular weight 5000), “Collagen Peptide Type S” (product) manufactured by Nitta Gelatin Co., Ltd. Name) (average molecular weight 1200), “super collagen peptide SCP-2000” (product name) (average molecular weight 2000), “collagen peptide P-5000” (product name) (average molecular weight 5000) manufactured by Yasu Chemical Co., Ltd.
  • Collagen Peptide F-5000 product name
  • Marine Collagen Oligo CF product name
  • Marine Collagen Oligo MF product name
  • Product name (average molecular weight 900-1500) and the like.
  • “collagen peptide Type S” average molecular weight 1200
  • “collagen peptide Iquos HDL-50SP” average molecular weight 5000) and the like are preferable.
  • “collagen peptide Iquos HDL-50SP”, “collagen peptide Type S”, “collagen peptide F-5000”, “marine collagen CF”, “marine collagen oligo MF” are derived from fish.
  • “Collagen Peptide P-5000” and “Super Collagen Peptide SCP-2000” are derived from pigs.
  • the nitrogen source comprising the L-hydroxyproline-containing peptide is preferably a collagen peptide (more preferably a collagen peptide derived from fish) and / or a peptone (more preferably derived from cow, pig or fish, More preferred is peptone derived from cattle or fish, and particularly preferred is a collagen peptide.
  • a nitrogen source containing such an L-hydroxyproline-containing peptide is used, the amount of L-hydroxyproline accumulated in yeast cells or cell cultures increases.
  • the peptone may be animal meat peptone, heart muscle peptone, gelatin peptone.
  • An example of a preferred embodiment of the method for producing L-hydroxyproline of the present invention is that the yeast Yarrowia lipolytica is aerobically cultured in a liquid medium containing a carbon source and a collagen peptide and / or peptone.
  • the nitrogen source containing the L-hydroxyproline-containing peptide preferably has an average molecular weight of 10,000 or less, and preferably has an average molecular weight of 100 to 10,000, for example.
  • the L-hydroxyproline-containing peptide preferably has an average molecular weight of 10,000 or less, and preferably has an average molecular weight of 100 to 10,000, for example.
  • the L-hydroxyproline-containing peptide preferably has a molecular weight of 10,000 or less.
  • the collagen peptide preferably has an average molecular weight of 1000 to 10,000.
  • the average molecular weight of the L-hydroxyproline-containing peptide is calculated by gel filtration or the like.
  • the average molecular weight of the collagen peptide is usually a value calculated by the method described in “20-2 Average Molecular Weight” of the 10th edition of Photographic Gelatin Test Method (PAGI Method).
  • PAGI Method Photographic Gelatin Test Method
  • the average molecular weight of a peptide refers to a weight average molecular weight.
  • the average molecular weight of the collagen peptide is more preferably 1000 to 6000, still more preferably 1000 to 5500, and particularly preferably 1000 to 5000.
  • the average molecular weight of the collagen peptide is more preferably 1000 to 3000, and even more preferably 1000 to 1500.
  • the average molecular weight of the collagen peptide is more preferably 2000 to 5500, further preferably 3000 to 5000.
  • the concentration of the nitrogen source containing the L-hydroxyproline-containing peptide in the liquid medium is preferably 0.1 to 10% by weight, more preferably 0.25 to 5% by weight, and further preferably 1 to 5% by weight. preferable.
  • concentration of the nitrogen source is within the above range, L-hydroxyproline accumulates in the bacterial cells or bacterial cell culture. Therefore, for example, a yeast cell or cell culture or an extract thereof having an L-hydroxyproline content of 10 ⁇ g / mL or more can be obtained.
  • cells or cell cultures or extracts thereof having a (Hyp / (Pro + Hyp)) ratio of 35 to 100 can be obtained.
  • the concentration of the nitrogen source in the liquid medium is still more preferably 1.5 to 4.5% by weight, particularly preferably 2 to 4% by weight.
  • the concentration of the nitrogen source may be the above concentration at the start of culture.
  • the concentration of the collagen peptide or peptone in the liquid medium is preferably in the above range.
  • the carbon source is not particularly limited. Sugars or sugar alcohols; organic acids such as acetic acid, citric acid, or gluconic acid.
  • a carbon source may be used individually by 1 type, and may mix and use 2 or more types. Among them, the carbon source is preferably a sugar such as glucose, fructose, or sucrose, and glucose is particularly preferable.
  • the concentration of the carbon source in the liquid medium is preferably 0.1 to 20% by weight, preferably 0.5 to 15% by weight, more preferably 1 to 10% by weight, more preferably 1 to 5% by weight, more preferably 2-5% by weight. It is preferable that the concentration of the carbon source in the liquid medium is 1% by weight or more because the growth rate of the bacterial cells is high. In addition, the density
  • the weight ratio (C / N) of the carbon source (C) and the nitrogen source (N) containing the L-hydroxyproline-containing peptide is preferably 0.25 to 20.
  • a weight ratio of C / N within the above range is preferable because the amount of L-hydroxyproline accumulated in the bacterial cells or bacterial cell culture increases.
  • the C / N weight ratio is more preferably 0.25 to 5, more preferably 0.3 to 3, and further preferably 0.4 to 1.5.
  • the weight ratio of C / N is within the above range, the amount of L-hydroxyproline accumulated in the microbial cells or microbial cell culture is increased.
  • the C / N weight ratio is 0. 25 to 5 is more preferable, 0.3 to 3 is more preferable, 0.4 to 1.5 is further preferable, and 0.5 to 1.3 is particularly preferable.
  • the C / N weight ratio is preferably 0.5 to 20. The weight ratio of C / N may be in the above range at the start of culture.
  • the liquid medium may contain components other than the above-described carbon source and a nitrogen source containing an L-hydroxyproline-containing peptide, and preferably contains, for example, a yeast extract.
  • Yeast extracts usually do not contain L-hydroxyproline-containing peptides and are not included in nitrogen sources that contain L-hydroxyproline-containing peptides.
  • the yeast extract is not particularly limited as long as it can be used for yeast culture, and a commercially available product can be used.
  • the product name Bacto yeast Extract (# 212750) (Bacto) can be preferably used.
  • the concentration of the yeast extract is preferably 0.1 to 3% by weight, more preferably 0.5 to 3% by weight, based on the liquid medium.
  • the concentration of the yeast extract may be the above concentration at the start of culture.
  • the pH of the liquid medium is preferably 3 to 9, more preferably 4 to 9, more preferably more than 4 and 9 or less, still more preferably 4.5 to 8.8, even more preferably 5 to 8.7. Is particularly preferred.
  • the pH of the liquid medium may be adjusted as appropriate.
  • a known acid or alkali agent can be used for pH adjustment, such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, glutamic acid, acetic acid, butyric acid, lactic acid, formic acid, succinic acid, maleic acid, malic acid, oxalic acid, citric acid, Examples thereof include sodium hydroxide, potassium hydroxide, calcium hydroxide, aqueous ammonia, and sodium glutamate.
  • the culture temperature is preferably 15 to 45 ° C, more preferably 20 to 40 ° C, and further preferably 25 to 35 ° C.
  • the culture temperature is within this temperature range, the yeast Yarrowia lipolytica grows rapidly, and the amount of L-hydroxyproline accumulated in the cells or cell cultures increases.
  • the method for aerobic culture is not particularly limited, and a liquid medium inoculated with yeast Yarrowia lipolytica may be subjected to, for example, shaking culture or stirring culture.
  • the speed of shaking or stirring is not particularly limited, but is preferably 30 to 600 rpm, and in one aspect, more preferably 50 to 600 rpm, and still more preferably 100 to 600 rpm. In another preferred embodiment, the speed of shaking or stirring is more preferably 30 to 500 rpm, further preferably 50 to 300 rpm. Shaking culture or stirring culture at such a rate is preferable because the amount of accumulated L-hydroxyproline increases. More preferably, the shaking culture is performed at the above speed. In addition, bubbling may be performed with sterilized air or oxygen if desired.
  • the culture format may be batch culture, fed-batch culture, or continuous culture, but batch culture is preferred. In the production method of the present invention, static culture may be performed.
  • the culture time is not particularly limited and may be set as appropriate. For example, it is preferable to perform aerobic culture for 10 to 100 hours. When the culture time is within the above range, L-hydroxyproline accumulates in the yeast cells or cell culture. In addition, usually, yeast cells or cell cultures or extracts thereof having a (Hyp / (Pro + Hyp)) ratio of 35 to 100, yeast cells having an L-hydroxyproline content of 10 ⁇ g / mL or more. Alternatively, a bacterial cell culture or an extract thereof can be obtained. In addition, yeast cells or cell cultures or extracts thereof having a low ethanol content (for example, 1 v / v% or less) can be obtained.
  • the culture time is less than 10 hours, the amount of accumulated L-hydroxyproline is small, or the yeast cell or cell culture obtained or the extract (Hyp / (Pro + Hyp)) ratio is less than 35. It may become.
  • the culture time exceeds 100 hours, the ethanol concentration of the obtained yeast or bacterial cell culture or the extract thereof may exceed 1 v / v%. In addition, contamination may easily occur, and coloring due to self-digestion may occur after the death of the yeast.
  • the culture time is more preferably 10 to 80 hours, still more preferably 12 to 72 hours, still more preferably 20 to 60 hours, still more preferably 24 to 55 hours, and particularly preferably 24 hours. ⁇ 50 hours.
  • aerobic culture it is preferable to perform aerobic culture until the L-hydroxyproline content in the microbial cells or the microbial cell culture becomes 10 ⁇ g / mL or more.
  • aerobic culture is usually performed as main culture, but it may be preculture or aerobic culture may be performed in preculture and main culture.
  • L-hydroxyproline accumulates in the cells of the yeast Yarrowia lipolytica or the cell culture.
  • the cell culture may be a cell culture solution containing yeast cells and culture supernatant, may be yeast cells, or may be a culture supernatant of a cell culture solution. Good.
  • a cell or a cell culture of yeast Yarrowia lipolytica containing L-hydroxyproline and having a (Hyp / (Pro + Hyp)) ratio of 35 to 100 is obtained.
  • a bacterial cell or a bacterial cell culture of yeast Yarrowia lipolytica having an L-hydroxyproline content of 10 ⁇ g / mL or more can be obtained.
  • An extract of yeast cells or cell cultures can be obtained by subjecting yeast cells or cell cultures to a cell disruption treatment, for example.
  • a cell culture comprising a yeast cell obtained in the Hyp accumulation step and a culture supernatant.
  • the solution can be used as it is as a yeast cell culture containing L-hydroxyproline.
  • yeast cells may be collected from the cell culture medium, and the obtained cell bodies may be used as yeast cells or cell cultures. It can also be a body culture. Further, the cells or the cell culture medium is subjected to a treatment for crushing the cells as necessary, and the cell contents are eluted in the culture solution to prepare a cell or cell culture extract.
  • a step of removing bacterial cell residues may be performed after disrupting the bacterial cells. Moreover, you may perform processes, such as disinfection and a heating, to a microbial cell or a microbial cell culture, or these extracts as needed.
  • the production method of the present invention may include one or more steps such as such a collection process, a microbial cell disruption process, a microbial cell residue removal process, and a sterilization process.
  • a method for collecting the cells from the cell culture solution is not particularly limited, and a commonly used method can be employed, and examples thereof include centrifugation.
  • the method for disrupting the cells is not particularly limited, and a commonly used method can be employed, and examples thereof include an autolysis method, an enzymatic decomposition method, and an alkali extraction method. Of these, the autolysis method is preferred.
  • the self-digestion method for example, the cells or the cell cultures may be heated at 40 to 60 ° C. for 60 to 180 minutes, or at 95 to 100 ° C. for 5 to 15 minutes.
  • the method for removing the cell residue is not particularly limited. For example, what is necessary is just to remove a microbial cell residue by well-known methods, such as filtration and centrifugation.
  • yeast cells or cell cultures or extracts thereof it is preferable to heat yeast cells or cell cultures or extracts thereof at 75 to 90 ° C. (more preferably 80 ° C.) and 45 to 90 minutes (more preferably 60 minutes). .
  • removing the microbial cell residue and sterilizing either may be performed first.
  • the yeast cells or cell cultures or extracts thereof obtained in the present invention are the yeast cells or cell cultures or extracts thereof of the present invention described above, and the extracts thereof. This is the same as the preferred embodiment.
  • the yeast Yarrowia lipolytica having a (Hyp / (Pro + Hyp)) ratio of 35 to 100 and an L-hydroxyproline content of 10 ⁇ g / mL.
  • Body or cell cultures or extracts thereof can be produced.
  • L-hydroxyproline in the yeast or yeast culture of yeast Yarrowia lipolytica or an extract thereof is usually derived from the yeast or bacterial culture of the yeast.
  • L-hydroxyproline derived or synthesized from natural products may be further added to yeast cells or cell cultures or extracts thereof obtained by the above method.
  • yeast cells are used.
  • the L-hydroxyproline contained in the cell culture or the extract thereof is L-hydroxyproline derived from the yeast Yarrowia lipolytica obtained by the above Hyp accumulation step or the cell culture. Consists of.
  • the yeast cells or cell cultures or extracts thereof containing L-hydroxyproline obtained in the present invention can be used as raw materials for foods and beverages, cosmetics and the like described later. Further, in the method for producing L-hydroxyproline of the present invention, a step of purifying L-hydroxyproline from the obtained yeast cell or cell culture or an extract thereof may be performed. Purification of L-hydroxyproline may be performed by a known method such as column chromatography.
  • the invention also encompasses the use of the yeast Yarrowia lipolytica to produce L-hydroxyproline.
  • the above Yarrowia lipolytica is also suitably used for producing yeast cells or cell cultures containing L-hydroxyproline, or extracts thereof.
  • the yeast cells or cell cultures or extracts thereof containing L-hydroxyproline preferably have a (Hyp / (Pro + Hyp)) ratio of 35 to 100. It is also preferable that the yeast cells or cell cultures or extracts thereof containing L-hydroxyproline have an L-hydroxyproline content of 10 ⁇ g / mL or more.
  • Preferred embodiments of the yeast cells or cell cultures or extracts thereof containing L-hydroxyproline are the same as the preferred embodiments of the yeast cells or cell cultures or extracts thereof of the present invention described above. It is.
  • the use of the present invention can be accomplished by aerobically cultivating yeast Yarrowia lipolytica in a liquid medium containing a carbon source and a nitrogen source, thereby producing L-hydroxyproline in the yeast cell or cell culture.
  • a nitrogen source is preferably a nitrogen source containing an L-hydroxyproline-containing peptide.
  • the L-hydroxyproline-containing peptide is preferably a collagen peptide.
  • the average molecular weight of the collagen peptide is preferably 1000 to 10,000.
  • the concentration of the nitrogen source containing the L-hydroxyproline-containing peptide in the liquid medium is preferably 0.25 to 5% by weight.
  • the weight ratio (C / N) of the carbon source (C) and the nitrogen source (N) containing the L-hydroxyproline-containing peptide is preferably 0.25 to 20. In one embodiment, the C / N ratio is preferably 0.5 to 20.
  • the aerobic culture is preferably performed for 10 to 100 hours.
  • the liquid medium, the carbon source and the nitrogen source containing the L-hydroxyproline-containing peptide and preferred embodiments thereof are the same as those in the above-described method for producing L-hydroxyproline.
  • the aerobic culture conditions and preferred embodiments thereof are also the same as those in the above-described method for producing L-hydroxyproline.
  • the use of the present invention may include one or more processes such as the above-described collection process, microbial cell disruption process, microbial cell removal process, and sterilization process.
  • the yeast cell or cell culture of the present invention described above or an extract thereof can be blended in various compositions such as cosmetics, foods and drinks, and pharmaceuticals.
  • the yeast cell or cell culture of the present invention or a composition containing an extract thereof is also encompassed in the present invention.
  • the composition of the present invention may include any one of the yeast cell or cell culture of the yeast of the first aspect and the second aspect of the present invention described above, or an extract thereof, and may include both.
  • the yeast cell or cell culture of the present invention or a composition containing these extracts contains L-hydroxyproline derived from the yeast cell or cell culture or these extracts.
  • compositions of the present invention include cosmetics (cosmetic compositions), foods and drinks (food and beverage compositions), pharmaceuticals (pharmaceutical compositions), quasi drugs (quasi drugs) and the like.
  • the composition may be these raw materials.
  • the composition is preferably a cosmetic or a food or drink, or a raw material thereof.
  • the content of the yeast cell culture or cell culture or the extract thereof in the composition of the present invention is not particularly limited, and can be appropriately set depending on the type and use of the composition.
  • the solid content of the yeast cell or cell culture or extract thereof is preferably 0.00001 to 50% by weight, preferably 0.00005 to 20% by weight, based on the composition. Is more preferable, and 0.0001 to 10% by weight is more preferable.
  • the dosage form is not particularly limited, and any dosage form such as a solution, paste, gel, solid, or powder can be used.
  • Cosmetics are not particularly limited. , Perfume, powder, eau de cologne, body soap, soap, bath salt, sunscreen cream and the like.
  • the cosmetics or cosmetic raw materials containing the above-described yeast cells or cell cultures of the present invention or extracts thereof are one of the preferred embodiments of the present invention.
  • the cosmetics and cosmetic raw materials may contain components other than the yeast cells or cell cultures or extracts thereof.
  • Various ingredients that are usually blended in cosmetics can be blended in cosmetics and cosmetic raw materials.
  • oils, fragrances, surfactants, humectants, antioxidants, ultraviolet absorbers, preservatives, pigments, dyes and the like can be appropriately blended. What is necessary is just to select these compounding ratios suitably.
  • the cosmetic raw material of the present invention is suitably used for producing the cosmetic of the present invention.
  • the usage and dosage of the cosmetic can be appropriately determined according to the type of cosmetic.
  • the cosmetic or cosmetic raw material of the present invention contains L-hydroxyproline, for example, promotion of collagen production, promotion of epidermal cell proliferation, skin moisturization, prevention of skin aging, prevention or improvement of skin sagging, It is preferably used for applications selected from the improvement of skin firmness, prevention or improvement of wrinkles and the improvement of atopic dermatitis, and is preferably used for applications selected from improvement of skin firmness and prevention or improvement of wrinkles. .
  • the content of the yeast cells or cell culture or the extract thereof in the cosmetic is preferably 0.00001 to 10% by weight, preferably 0.0001 to 10% by weight in terms of solid content with respect to the cosmetic. It is preferably 0.0001 to 5% by weight, more preferably 0.001 to 5% by weight, still more preferably 0.01 to 3% by weight, and particularly preferably 0.05 to 2% by weight.
  • the content of the yeast cell culture or cell culture or extract thereof in the cosmetic is 0.00005 to 1% by weight in terms of solid content relative to the cosmetic. Preferably, 0.0001 to 0.5% by weight is more preferable.
  • the content of the yeast cell or cell culture or extract thereof in the cosmetic raw material is preferably 0.001 to 20% by weight, for example, in terms of solid content, based on the cosmetic raw material, Is more preferably from 10 to 10% by weight, further preferably from 0.05 to 5% by weight, particularly preferably from 0.1 to 2% by weight.
  • the L-hydroxyproline content in the cosmetic raw material is, for example, preferably from 5 to 300 ppm, more preferably from 10 to 200 ppm, still more preferably from 50 to 100 ppm.
  • the L-hydroxyproline content in the cosmetic can be, for example, 0.01 to 20 ppm, preferably 0.03 to 15 ppm, and more preferably 0.05 to 10 ppm. It is preferable to blend the yeast cells or cell cultures or extracts thereof so that the L-hydroxyproline content falls within the above range.
  • the food or drink is not particularly limited.
  • the form of the food or drink may be any of liquid, semi-liquid or solid, and paste, and may be any of general food and drink, health food, functional food, and the like.
  • General food and drink is not particularly limited, and includes alcoholic beverages.
  • Healthy food means food that is considered healthy or healthy, and includes nutritional supplements, natural foods, and the like.
  • Nutritional supplements refer to foods that are enriched with specific nutritional components.
  • Functional foods refer to foods for supplementing nutritional components that fulfill the body's regulatory functions, and include foods for specified health use and functional nutritional foods.
  • dietary supplements include beauty drinks and supplements.
  • the food and drink of the present invention may be in the form of a pharmaceutical preparation such as a capsule, a drink or the like.
  • Various components that are permitted to be blended in food and drink can be blended in the food and drink. Examples of such components include binders, thickeners, colorants, stabilizers, emulsifiers, dispersants, disintegrants, suspending agents, surfactants, preservatives, sweeteners, and sour agents.
  • the above-described yeast cell or cell culture of the present invention or a food or drink containing these extracts is one of the preferred embodiments of the present invention.
  • the content of the yeast cells or cell culture or the extract thereof in the food or drink is preferably 0.0001 to 10% by weight in terms of solid content with respect to the food or drink. 001 to 5% by weight is more preferable, and 0.01 to 1% by weight is more preferable. Further, the L-hydroxyproline content in the food and drink is preferably 0.0001 to 0.01% by weight, and the yeast cell or cell culture or the culture of the yeast so that the L-hydroxyproline content falls within the above range. It is preferable to blend these extracts.
  • the L-hydroxyproline content in the cosmetics, cosmetic raw materials, foods and beverages, etc. is a free L-hydroxyproline content.
  • L-hydroxyproline is preferably derived from the above-mentioned yeast cells or cell cultures or extracts thereof.
  • compositions such as cosmetics, foods and drinks, and these raw materials containing yeast cells or bacterial cell cultures or extracts thereof according to the present invention are the types of raw materials, additives, etc. that are usually used in these.
  • the yeast cells or cell cultures of the yeast of the present invention or extracts thereof can be blended and produced by a known technique.
  • the present invention accumulates L-hydroxyproline in the yeast cells or cell cultures by aerobic culture of yeast Yarrowia lipolytica in a liquid medium containing a carbon source and a nitrogen source.
  • a method for producing an L-hydroxyproline-containing cosmetic raw material including a step (Hyp accumulation step).
  • the nitrogen source is a nitrogen source containing an L-hydroxyproline-containing peptide.
  • the preferred embodiment of the method for producing an L-hydroxyproline-containing cosmetic raw material of the present invention is the same as the preferred embodiment of the method for producing L-hydroxyproline described above.
  • the method for producing the L-hydroxyproline-containing cosmetic raw material is preferable as the method for producing the cosmetic raw material of the present invention.
  • yeast cell or cell culture containing L-hydroxyproline By performing the Hyp accumulation step, a yeast cell or cell culture containing L-hydroxyproline is obtained.
  • an extract of the cells or cell cultures of yeast Yarrowia lipolytica containing L-hydroxyproline can be obtained. can get.
  • yeast cells or cell cultures or extracts thereof, and preferred embodiments thereof are the yeast cells or cell cultures of the present invention described above or extracts thereof, and The preferred embodiment is the same.
  • L-hydroxyproline-containing cosmetic raw material containing L-hydroxyproline-containing yeast cells or cell cultures or extracts thereof, if desired, containing additives or the like commonly used in cosmetics Can be used as In addition, L-hydroxyproline purified from yeast cells or cell cultures or extracts thereof containing L-hydroxyproline may be blended with additives ordinarily used in cosmetics, if desired.
  • a hydroxyproline-containing cosmetic raw material can also be produced.
  • the cosmetic raw material containing L-hydroxyproline obtained by the present invention has the following effects: collagen production promotion, epidermal cell growth promotion, skin moisturization, skin aging prevention, skin sagging prevention or improvement, skin firmness improvement, wrinkle It is suitably used for cosmetics for uses selected from prevention or improvement and improvement of atopic dermatitis.
  • the present invention also includes a composition for reinforcing L-hydroxyproline comprising a cell or a cell culture of yeast Yarrowia lipolytica containing L-hydroxyproline or an extract thereof.
  • the yeast cell or cell culture or extract thereof contains L-hydroxyproline, but the L relative to the total content ( ⁇ g / mL) of L-proline (Pro) and L-hydroxyproline (Hyp).
  • the ratio (100 ⁇ Hyp / (Pro + Hyp)) of the hydroxyproline content ( ⁇ g / mL) is preferably 35-100.
  • the yeast cells or cell cultures or extracts thereof preferably have an L-hydroxyproline content of 10 ⁇ g / mL or more.
  • a composition for reinforcing L-hydroxyproline containing such yeast cells or cell cultures or extracts thereof is an additive for reinforcing, supplementing or strengthening L-hydroxyproline in cosmetics, foods and drinks, etc.
  • Preferred embodiments of yeast cells or cell cultures or extracts thereof are the same as the yeast cells or cell cultures or extracts thereof of the present invention described above and preferred embodiments thereof.
  • the L-hydroxyproline reinforcing composition can be suitably used as an additive composition for reinforcing L-hydroxyproline in foods and drinks, cosmetics and the like.
  • the composition for reinforcing L-hydroxyproline can also be referred to as a composition for supplementing L-hydroxyproline or a composition for reinforcing L-hydroxyproline.
  • the L-hydroxyproline reinforcing composition of the present invention only needs to contain a cell or a cell culture of yeast Yarrowia lipolytica containing L-hydroxyproline, or an extract thereof.
  • the content of the body or cell culture or these extracts may be 100% by weight, but may contain other components as desired.
  • the L-hydroxyproline reinforcing composition when used as a food additive, it may contain one or more known additives used in foods.
  • the L-hydroxyproline reinforcing composition of the present invention is also suitably used as a cosmetic additive, for example.
  • the L-hydroxyproline reinforcing composition when used as a cosmetic additive, it contains a yeast or a yeast culture of yeast Yarrowia lipolytica or an extract thereof.
  • the content of the cells or cell cultures or their extracts may be 100% by weight, but if desired, it contains one or more known additives used in cosmetics. May be.
  • the method for producing a composition for reinforcing L-hydroxyproline comprises the yeast yeast or Yarrowia lipolytica by aerobic culture in a liquid medium containing a carbon source and a nitrogen source. It is preferable to include a step of accumulating L-hydroxyproline in the cell culture. A method for producing an L-hydroxyproline reinforcing composition including such steps is also encompassed by the present invention.
  • the nitrogen source is a nitrogen source containing an L-hydroxyproline-containing peptide.
  • the production method of L-hydroxyproline reinforcing composition of the present invention and preferred embodiments thereof are the same as the above-described production method of L-hydroxyproline and preferred embodiments thereof.
  • the method for producing an L-hydroxyproline reinforcing composition of the present invention comprises a step of adding a known food additive, cosmetic additive, etc. to yeast cells or cell cultures or extracts thereof, if desired. May be included.
  • a method for producing L-hydroxyproline by aerobically cultivating yeast Yarrowia lipolytica in a liquid medium containing a carbon source and a nitrogen source.
  • the nitrogen source preferably contains an L-hydroxyproline-containing protein or an L-hydroxyproline-containing peptide.
  • the L-hydroxyproline-containing protein is preferably a collagenous protein, and the L-hydroxyproline-containing peptide is preferably a collagen peptide.
  • the collagen protein and collagen peptide preferably have an average molecular weight of 1,000 to 100,000.
  • the concentration of the nitrogen source in the liquid medium is 0.25 to 5% by weight, and the weight ratio (C / C) of the carbon source (C) and the nitrogen source (N).
  • N) is preferably from 0.5 to 20.
  • the aerobic culture is preferably performed for 10 to 100 hours.
  • Another use of the present invention is the use of the yeast Yarrowia lipolytica for producing L-hydroxyproline, wherein the yeast is preferably used in a liquid medium containing a carbon source and a nitrogen source.
  • L-hydroxyproline is accumulated in the yeast or cell culture of the yeast by air culture, and the nitrogen source contains an L-hydroxyproline-containing protein or an L-hydroxyproline-containing peptide.
  • the L-hydroxyproline-containing protein is preferably a collagenous protein
  • the L-hydroxyproline-containing peptide is preferably a collagen peptide.
  • the collagen protein and collagen peptide preferably have an average molecular weight of 1,000 to 100,000.
  • the concentration of the nitrogen source in the liquid medium is 0.25 to 5% by weight, and the weight ratio (C / N) of the carbon source (C) and the nitrogen source (N). ) Is preferably 0.5-20.
  • the aerobic culture is preferably performed for 10 to 100 hours.
  • L-hydroxyproline-containing protein examples include the aforementioned collagenous protein.
  • the average molecular weight of the L-hydroxyproline-containing protein such as collagenous protein is preferably more than 10,000 and not more than 100,000.
  • the average molecular weight of protein refers to the weight average molecular weight.
  • the average molecular weight of the L-hydroxyproline-containing protein such as collagenous protein is calculated by gel filtration or the like.
  • test examples for more specifically explaining the present invention will be shown.
  • the present invention is not limited only to these test examples.
  • “%” means “% by weight” unless otherwise specified.
  • L-4-hydroxyproline manufactured by Nacalai Tesque Co., Ltd. was used to prepare an L-hydroxyproline (Hyp) standard solution used for preparing a calibration curve.
  • L-proline manufactured by Nacalai Tesque Co., Ltd. was used for the preparation of L-proline (Pro) standard solution and the like.
  • L-hydroxyproline and L-proline measured in the test examples are both free L-hydroxyproline and L-proline.
  • All of the media used in the test examples are liquid media.
  • the YPD medium used in the test examples unless otherwise specified, Y: P: D is 1: 2: 2 (weight ratio) (Y: yeast extract 1.0%, P: peptone 2.0%, D: glucose 2.0%).
  • the yeast extract used for medium preparation is the product name Yeast Extract (# 212750) manufactured by Bacto.
  • Peptone used was a product name Pepton (# 211677) manufactured by Bacto, which was obtained by degrading bovine cells with an enzyme derived from porcine pancreas.
  • the pH of the medium before the start of culture was about 6.5 to 8.5.
  • Hyp L-Hydroxyproline
  • Hyp was quantified by HPLC.
  • culture 1 mL of each strain was inoculated into 1 mL of YPD medium supplemented with 0.5% L-proline (hereinafter referred to as “Pro”), cultured at 28 ° C. for 24 hours with shaking (300 rpm), and then allowed to stand at room temperature for 4 to 7 days. Culturing was performed to obtain a yeast cell culture solution (cell culture).
  • Pro 0.5% L-proline
  • Example preparation The yeast cell culture solution was subjected to the following treatment to prepare a culture sample, and the Hyp content was measured. After the above culture, the cells were collected and the cells were washed with 1 mL of physiological saline. The cells were suspended in 0.2 mL of 50 mM potassium phosphate buffer (hereinafter referred to as KPB) (pH 6.0), boiled for 10 minutes, and the cell contents were eluted by autolysis (boiling method). The cell residue was removed from the obtained cell extract by centrifugation (14000 rpm, 5 min, 4 ° C.). This prepared the culture sample for measuring the amount of Hyp accumulation.
  • KPB potassium phosphate buffer
  • the culture sample was derivatized by the AccQTag method using Waters (AccQ-Fluor Reagent Kit), and Hyp was quantified by HPLC under the following conditions.
  • FIG. 1 is a graph showing the amount of Hyp accumulated in yeast.
  • the vertical axis (Hyp ( ⁇ g / mL)) in FIG. 1 indicates Hyp ( ⁇ g) per 1 mL of the culture sample. From the above tests, yeasts that accumulate Hyp were found. In particular, Yarrowia lipolytica had a large amount of Hyp accumulation.
  • Yarrowia lipolytica NBRC0717 was used and the following liquid medium was used.
  • Yarrowia lipolytica NBRC0717 was obtained from the National Institute of Technology and Evaluation (2-5-8, Kazusa Kama feet, Kisarazu City, Chiba Prefecture, Japan).
  • YPD 1% yeast extract, 2% peptone, 2% glucose
  • YTD 1% yeast extract, 2% tryptone, 2% glucose
  • YM 1% yeast extract, 0.3% malt extract, 0.5% peptone, 2% glucose
  • PD 0.4% potato extract, 2% glucose
  • SD synthetic medium, 1% glucose
  • Condition 2 Pre-culture was performed under the same conditions as Condition 1 except that SD medium supplemented with 0.5% Pro was used instead of YPD medium supplemented with 0.5% Pro.
  • Each medium supplemented with 0.5% Pro (the above YPD, YTD, PD or SD) 2 mL, was inoculated preculture 0.2 mL, 28 ° C. 45 hours by shaking culture at (300 rpm) and bacteria Yarrowia lipolytica by A body culture solution was obtained.
  • Example preparation After culturing under condition 1 or 2, the yeast cell culture solution was subjected to the following treatment to prepare a culture sample, and the Hyp content was measured. After the above culture, the cells were collected and the cells were washed with 1 mL of physiological saline. The cells were suspended in 0.2 mL of 50 mM KPB (pH 6.0), boiled for 10 minutes, and the cell contents were eluted by autolysis (boiling method). The cell residue was removed from the obtained cell extract by centrifugation (14000 rpm, 5 min, 4 ° C.). This prepared the culture sample for measuring the amount of Hyp accumulation.
  • a calibration curve was prepared by preparing 50 mM KPB (pH 6.0) solutions of 5 ⁇ mol / L, 10 ⁇ mol / L, 20 ⁇ mol / L, 50 ⁇ mol / L, 100 ⁇ mol / L, 250 ⁇ mol / L of Hyp.
  • FIG. 2 is a graph showing the amount of Yyp accumulation of Yarrowia lipolytica cultured in each medium ((a): Condition 1, (b): Condition 2).
  • the Hyp ( ⁇ g / mL) on the vertical axis in FIG. 2 is Hyp ( ⁇ g) per 1 mL of the culture sample.
  • a large amount of hydroxyproline accumulated in the cells cultured in YPD and YM media (medium containing peptone).
  • Hyp accumulated in the cells cultured in the YPD medium.
  • ⁇ Test Example 3> The variation of the accumulation amount with the number of culture days was examined.
  • Yarrowia lipolytica strains NBRC1551, NBRC0717, NBRC0746 and NBRC1195 were cultured in YPD medium for 1, 2, or 3 days, respectively, and Hyp eluted from yeast cells (cells) was quantified using HPLC.
  • yeasts identified by the NBRC number were obtained from the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamashitsu, Kisarazu City, Chiba Prefecture, Japan).
  • Pre-culture One platinum loop of each strain was inoculated into 1 mL of YPD medium, followed by shaking culture (300 rpm) at 28 ° C. for 24 hours to obtain a preculture solution.
  • Example preparation and amino acid determination Culture samples were prepared in the same manner as in Test Example 2, and derivatized with OPA and NBD-Cl. Analysis by HPLC was performed under the same conditions as in Test Example 2, and Hyp and Pro were quantified. Pro calibration curves were prepared by preparing Pro 5 ⁇ mol / L, 10 ⁇ mol / L, 20 ⁇ mol / L, 50 ⁇ mol / L, 100 ⁇ mol / L, 250 ⁇ mol / L 50 mM KPB (pH 6.0) solutions.
  • Table 2 shows the amount of Pro and Hyp of each culture sample. Further, from the measured values of Pro and Hyp, the ratio (100 ⁇ Hyp / (Pro + Hyp)) of the content of Hyp ( ⁇ g / mL) to the total content of Pro and Hyp ( ⁇ g / mL) was calculated. The results are also shown in Table 2.
  • Yarrowia lipolytica accumulated L-hydroxyproline by culture.
  • the obtained cell culture contained L-hydroxyproline. Further, the (Hyp / (Pro + Hyp)) ratio was 35 or more.
  • Yarrowia lipolytica NBRC1551 strain, NBRC0717 strain and NBRC0746 strain are used to culture for 5 days in PD medium or PD medium supplemented with 0.125% gelatin, and the cell contents are eluted from the cells and Hyp is analyzed by HPLC. Quantified. The same medium as used in Test Example 2 was used as the PD medium.
  • FIG. 3 is a graph showing the amount of Hyp accumulated in Yarrowia lipolytica cultured in PD medium or PD medium supplemented with 0.125% gelatin for 5 days (white: PD medium containing 0.125% gelatin (PD + gel), black : PD medium).
  • Hyp ( ⁇ g / mL) on the vertical axis is Hyp ( ⁇ g) per 1 mL of culture sample. From FIG. 3, when high molecular gelatin was added to the medium as it was, the accumulation of Hyp was less than that of Test Example 3.
  • Table 3 shows the YPD composition of each medium # 1 to # 12.
  • Y is yeast extract
  • P is peptone
  • D glucose.
  • the yeast extract (Y) was kept constant, and the peptone (P) and glucose (D) were changed (the values in the table are final concentrations (% by weight)).
  • the yeast Yarrowia lipolytica cultured in the above YPD medium accumulated Hyp.
  • the amount of Hyp accumulated was large.
  • FIG. 4 shows the results when cultured in # 1 and # 7-11 media.
  • FIG. 4 is a graph showing the Hyp accumulation amount of Yarrowia lipolytica cultured in YPD media having different compositions ((a): Hyp accumulation amount ( ⁇ g / mL), (b): Hyp amount per cell ( ⁇ g / mL). / OD600)).
  • shaft of (a) of FIG. 4 is Hyp (microgram) per 1 mL of culture samples.
  • (Hyp / OD) on the vertical axis of FIG. 4B is the amount of Hyp per cell, and the value obtained by dividing the amount of Hyp ( ⁇ g) in 1 mL of the culture sample by the measured value of OD600 ( ⁇ g / mL / OD600).
  • OD600 was obtained by diluting 60 ⁇ L of the cell culture solution used for preparation of the culture sample with 1150 ⁇ L of ultrapure water using a nucleic acid protein spectrophotometer (device name Bio spec mini, Shimadzu Corporation), and measuring the absorbance (600 nm ) Measured
  • YPD media # 1 and # 10 used in Test Example 5 the peptone used as P was replaced with a collagen peptide (product name: Super Collagen Peptide SCP-2000, manufactured by Nitta Gelatin Co., Ltd., average molecular weight 2000). The amount of Hyp accumulated was examined. Yarrowia lipolytica NBRC0717 was used as the yeast.
  • a YPD medium a medium in which part or all of peptone is replaced with a collagen peptide can be said to be a YPD-modified medium.
  • YPD # 1-Normal (YPD # 1)
  • YPD # 1-CP50 (CP50 is used instead of peptone in YPD # 1)
  • YPD # 1-CP100 (CP100 is used instead of peptone in YPD # 1)
  • YPD # 10-normal (YPD # 10)
  • YPD # 10-CP50 (CP50 is used instead of peptone in YPD # 10)
  • YPD # 10-CP100 (CP100 is used instead of peptone in YPD # 10)
  • FIG. 5 is a graph showing Hyp accumulation amount, Hyp amount per cell, and OD600 of Yarrowia lipolytica cultured in a medium containing peptone or collagen peptide.
  • N means P is a normal medium
  • 50 means P is a CP50 medium
  • 100 means P is a CP100 medium.
  • Yarrowia lipolytica was able to grow even in a medium in which peptone was completely replaced with collagen peptide, and Hyp was accumulated.
  • Yarrowia lipolytica NBRC0717 was used to examine the influence of aeration during culture.
  • the YPD media # 1 and # 10 of Test Example 5 were used as the media.
  • FIG. 6 is a diagram showing the amount of Hyp accumulated in Yarrowia lipolytica cultured by shaking culture or stationary culture ((a): Hyp equivalent per culture medium in culture sample (intracellular), (b): in culture supernatant. Hyp amount). Yarrowia lipolytica had a large amount of Hyp accumulation when aerobically cultured.
  • Test Example 8 The ethanol concentration and glucose concentration in the culture supernatant obtained in Test Example 7 were quantified by HPLC (fermentation column (manufactured by Bio-Rad, product name: Aminex fermentation monitor column)). The results are shown in FIG.
  • FIG. 7 is a graph showing the ethanol concentration and glucose concentration of the culture supernatant of Yarrowia lipolytica subjected to shaking culture or stationary culture ((a): ethanol concentration (v / v%), (b): glucose concentration (weight). %)).
  • Yarrowia lipolytica NBRC0717 the main culture was performed while changing the composition of YPD medium (ratio of Y: P: D and the type of P) and the culture time, and the Hyp content and Pro content in the cell culture were measured.
  • Pre-culture One platinum loop of Yarrowia lipolytica NBRC0717 was inoculated into 3 mL of YPD medium, and statically cultured at 30 ° C. for 1 day to obtain a preculture solution.
  • peptone or collagen peptide (CP) was used as P in the YPD medium.
  • Collagen peptides include collagen peptide Iquos HDL-50SP (product name, Nitta Gelatin Co., Ltd., average molecular weight 5000) (hereinafter referred to as collagen peptide (CP1)) or collagen peptide Type S (product name, Nitta).
  • collagen peptide (CP2) collagen peptide
  • Table 4 shows the culture conditions 1 to 12 used in the main culture.
  • the Yarrowia lipolytica cell culture solution obtained in the main culture was subjected to the following autolysis and sterilization steps to prepare a culture sample, and the Hyp content was measured.
  • the bacterial cell culture solution of Yarrowia lipolytica was incubated at 50 ° C. for 2 hours, and the bacterial cell contents were eluted into the culture solution by autolysis.
  • the culture solution self-digested above was incubated at 80 ° C. for 1 hour.
  • the cell residue was removed from the obtained extract by centrifugation (3000 rpm, 5 min, 1 ° C.). This prepared the culture sample for measuring the amount of Hyp accumulation.
  • sample preparation The obtained culture sample was diluted with 0.1N hydrochloric acid solution to prepare a sample for HPLC.
  • the culture sample prepared from the bacterial cell culture medium with a culture time of 1 day is diluted 10 times, and the culture sample prepared from the bacterial cell culture medium with a culture time of 2 days is diluted 40 times except for the condition 12, Diluted 20 times.
  • HPLC HPLC analysis
  • primary amino acids primary amino groups
  • secondary amino acids secondary amino groups
  • OPA o-phthalaldehyde
  • FMOC chloroformate-9-fluorenylmethyl
  • the HPLC sample prepared above was filtered through a 0.45 ⁇ m filter in a sample vial and set in an autosampler. An empty vial was charged with 30 ⁇ L of MPA (mercaptopropionic acid) reagent, 15 ⁇ L of OPA reagent, and 5 ⁇ L of the above sample and allowed to stand for 1 minute, and then 5 ⁇ L of FMOC reagent was added and 1 ⁇ L of the reacted solution was injected into HPLC. OPA reacts with primary amino acid, and Hyp and Pro having the remaining secondary amino group react with FMOC. By detecting each fluorescence wavelength with two channels, it is possible to detect all amino acids simultaneously.
  • MPA mercaptopropionic acid
  • Each of these solutions was reacted with each reagent of MPA, OPA and FMOC by the above method, and analyzed by HPLC to draw a calibration curve.
  • the amino acid mixed standard solution H type solution and 0.1N hydrochloric acid solution containing L-hydroxyproline were similarly reacted with each reagent and analyzed by HPLC.
  • the amino acid mixed standard solution H type used is manufactured by Wako Pure Chemical Industries, Ltd.
  • FIG. 8 is an HPLC chart obtained by analyzing a 0.1N hydrochloric acid solution containing amino acid mixed standard solution H and L-hydroxyproline (each amino acid concentration 20 ⁇ mol / L) ((a): Ch1 excitation wavelength 350 nm, fluorescence wavelength. Detection at 450 nm, (b): Ch2 excitation wavelength 266 nm, fluorescence wavelength 305 nm detection).
  • Table 5 shows the quantification results of Hyp and Pro of the culture sample.
  • CP1 is the above-described collagen peptide (CP1) (product name collagen peptide Iquos HDL-50SP), and CP2 is collagen peptide (CP2) (product name collagen peptide Type S).
  • CP1 is the above-described collagen peptide
  • CP2 is collagen peptide (CP2) (product name collagen peptide Type S).
  • CP1 collagen peptide
  • CP2 collagen peptide
  • the culture conditions in the table are the conditions for main culture. Each medium before the start of culture contained almost no free Hyp.
  • YPD modified medium yeast extract 1.0%, collagen peptide (CP1) 2.0%, glucose 1.0%) as the medium and shaking culture (200-500 rpm) at 28 ° C for 2 days.
  • the main culture was performed in the same manner as in Test Example 9.
  • Collagen peptide (CP1) is the same as in Test Example 9.
  • a culture sample was prepared by subjecting the bacterial culture of Yarrowia lipolytica obtained in the main culture to a self-digestion step and a sterilization step in the same manner as in Test Example 9.
  • the Hyp content and the Pro content were measured by the same method as in Test Example 9.
  • ⁇ Test Example 11> Using Yarrowia lipolytica used in Test Examples 1 to 10, the cells were cultured in the same manner as in Test Examples 9 to 10 to obtain a cell culture solution. This was incubated at 50 ° C. for 2 hours to elute the cell contents into the culture solution by autolysis, and then incubated at 80 ° C. for 1 hour. Thereafter, the mixture was centrifuged at 1 ° C. (3000 rpm, 5 min) to obtain an L-hydroxyproline-containing cell culture extract (extract of Hyp-containing cell culture) excluding cell residues. The extract of the Hyp-containing cell culture can be used after appropriately diluted.
  • Table 8 shows the blending amounts of shampoo raw materials.
  • a preservative dissolved in 1,3-butylene glycol was added to purified water. After uniformly stirring, sodium laureth sulfate and coconut oil fatty acid monoethanolamide were added, and then a pigment, a fragrance and the remaining 1,3-butylene glycol were added, and each Hyp-containing cell culture extract was added, The mixture was uniformly mixed and stirred.
  • Table 9 shows the blending amounts of the conditioner raw materials.
  • Stearyldimethylbenzylammonium chloride and sodium chloride were added to purified water and heated to 80 ° C. to dissolve.
  • Cetostearyl alcohol, hydrogenated polyisobutene and glycerin monostearate were heated to 80 ° C. and dissolved.
  • the mixture was cooled to 50 ° C. with stirring, each Hyp-containing cell culture extract was added, and the mixture was further cooled to 35 ° C. with stirring.
  • Table 10 shows the blending amounts of the hair tonic raw materials. Vitamin E and L-menthol dissolved in salicylic acid, glycerin, and ethanol are added to purified water, and dipotassium glycyrrhizinate dissolved in a portion of purified water is added, and then each Hyp-containing cell culture extract is added. And mixed uniformly to prepare.
  • Table 11 shows the amounts of the mist raw materials. Citric acid and sodium citrate were added to purified water and dissolved. Thereafter, an antiseptic and polysorbate 80 dissolved in ethanol were added. Thereafter, each Hyp-containing cell culture extract was added and stirred uniformly.
  • Table 12 shows the amounts of the lotion ingredients. Citric acid and sodium citrate were added to purified water and dissolved. Next, glycerin, 1,3-butylene glycol and trisodium ethylenediaminetetraacetate were sequentially added, and polyoxyethylene (18) oleyl alcohol ether dissolved in ethanol, vitamin E and methylparaben were added and stirred until uniform. Thereafter, each Hyp-containing cell culture extract was added and stirred uniformly.
  • Table 13 shows the amounts of the emulsion raw materials.
  • Stearic acid, cetyl alcohol, octyldodecyl myristate and liquid paraffin were heated to 80 ° C. and dissolved.
  • Triethanolamine, sodium hyaluronate, glycerin, 1,3-butylene glycol, polyoxyethylene (10) monooleate and sodium ethylenediaminehydroxytriacetate were added to purified water and heated to 80 ° C.
  • the mixture was cooled to 50 ° C., an extract of each Hyp-containing cell culture was added, and further cooled to 35 ° C. for preparation.
  • Table 14 shows the blending amounts of the cream raw materials.
  • Stearic acid, glyceryl monostearate, sorbitan sesquistearate, polyoxyethylene sorbitan monostearate, cetostearyl alcohol, squalane, hexa (hydroxystearic acid / stearic acid / rosinic acid) dipentaerythlit, olive oil, myristic Octyldodecyl acid and methylpolysiloxane were dissolved by heating to 80 ° C.
  • Glycerin, 1,3-butylene glycol, sodium hydroxide and methylparaben were added to purified water and heated to 80 ° C. to dissolve.
  • the yeast, Yarrowia lipolytica microbial cells, bacterial cell cultures and extracts thereof containing L-hydroxyproline of the present invention are useful as raw materials for cosmetics, foods and drinks and the like.

Abstract

The present invention relates to a yeast cell of Yarrowia lipolytica, a cell culture product thereof or an extract of the same. The aforesaid yeast cell, cell culture product and extract of the same are characterized by containing L-hydroxyproline, and the ratio [100 × Hyp/(Pro + Hyp)] of the content (μg/mL) of L-hydroxyproline (Hyp) to the total content (μg/mL) of L-proline (Pro) and L-hydroxyproline being 35-100.

Description

Ｌ－ヒドロキシプロリンを含有する酵母ヤロウィア・リポリティカの菌体もしくは菌体培養物又はこれらの抽出物及びその用途並びにＬ－ヒドロキシプロリンの製造方法Yeast Yarrowia lipolytica cells containing L-hydroxyproline or cell cultures or extracts thereof and uses thereof, and method for producing L-hydroxyproline
本発明は、Ｌ－ヒドロキシプロリンを含有する酵母の菌体もしくは菌体培養物又はこれらの抽出物及びその用途、並びに、Ｌ－ヒドロキシプロリンの製造方法に関する。本発明はまた、Ｌ－ヒドロキシプロリンを製造するための酵母の使用に関する。本発明はまた、酵母の菌体もしくは菌体培養物又はこれらの抽出物を含有する飲食品、化粧料、化粧料原料及びＬ－ヒドロキシプロリン補強用組成物等に関する。 The present invention relates to a yeast cell or a cell culture or an extract thereof containing L-hydroxyproline, its use, and a method for producing L-hydroxyproline. The invention also relates to the use of yeast for producing L-hydroxyproline. The present invention also relates to a food / beverage product, a cosmetic, a cosmetic raw material, a composition for reinforcing L-hydroxyproline, and the like containing a yeast cell or cell culture or an extract thereof.
Ｌ－ヒドロキシプロリン（ヒドロキシ－Ｌ－プロリン）は、Ｌ－プロリンの４位の炭素原子に水酸基が結合した構造を有するアミノ酸である。Ｌ－ヒドロキシプロリンの効能として、線維芽細胞におけるコラーゲン産生促進、表皮細胞の増殖促進、コラーゲンと同等以上の保湿効果、皮膚の老化防止、トリペプチドよりも高い経皮吸収性、しわの改善効果、アトピー性皮膚炎の改善効果等が挙げられる。Ｌ－ヒドロキシプロリンは人体に対して安全であることから、飲食品、化粧料、医薬品等に含有させて使用することができ、その有益性は非常に高い。 L-hydroxyproline (hydroxy-L-proline) is an amino acid having a structure in which a hydroxyl group is bonded to the 4-position carbon atom of L-proline. L-hydroxyproline has the following effects: promotion of collagen production in fibroblasts, promotion of epidermal cell proliferation, moisturizing effect equivalent to or better than collagen, prevention of skin aging, transdermal absorbability higher than tripeptide, wrinkle improvement effect, The improvement effect of atopic dermatitis etc. are mentioned. Since L-hydroxyproline is safe for the human body, it can be used by being contained in foods and drinks, cosmetics, pharmaceuticals, etc., and its usefulness is very high.
Ｌ－ヒドロキシプロリンは、有機合成法によって製造することができるが、微生物を利用する製造方法も検討されている。例えば特許文献１には、ミヤコグサ根粒菌由来のＬ－プロリン　シス－４－ヒドロキシラーゼをコードするポリヌクレオチドを宿主細胞に導入して得られる形質転換体を培地に培養し、培養物中にシス－４－ヒドロキシ－Ｌ－プロリンを生成、蓄積させ、該培養物中からシス－４－ヒドロキシ－Ｌ－プロリンを採取するシス－４－ヒドロキシ－Ｌ－プロリンの製造方法が記載されている。 L-hydroxyproline can be produced by an organic synthesis method, but a production method using a microorganism is also being studied. For example, Patent Document 1 discloses that a transformant obtained by introducing a polynucleotide encoding L-proline cis-4-hydroxylase derived from Rhizobium rhizobia into a host cell is cultured in a medium, and the cis- A method for producing cis-4-hydroxy-L-proline is described in which 4-hydroxy-L-proline is produced and accumulated, and cis-4-hydroxy-L-proline is collected from the culture.
特許第５５０６６６８号公報Japanese Patent No. 5506668
ところで、酵母は、飲食品分野等で古くから様々な工業的利用が試みられている微生物であり、Ｌ－ヒドロキシプロリンを含有する酵母の菌体もしくは菌体培養物又はこれらの抽出物は、Ｌ－ヒドロキシプロリンの効能が期待される化粧料、飲食品等の原料として有用である。しかしながら、菌体又は菌体培養物中にＬ－ヒドロキシプロリンを蓄積する酵母は、未だ報告されていない。 By the way, yeast is a microorganism which has been tried for various industrial uses from the old days in the food and drink field, etc., and yeast cells or cell cultures or extracts thereof containing L-hydroxyproline are L -It is useful as a raw material for cosmetics, foods and drinks where the effect of hydroxyproline is expected. However, a yeast that accumulates L-hydroxyproline in bacterial cells or bacterial cell cultures has not yet been reported.
本発明は、Ｌ－ヒドロキシプロリンを含有する酵母の菌体もしくは菌体培養物又はこれらの抽出物及びその用途、並びに、Ｌ－ヒドロキシプロリンの製造方法を提供することを主な目的とする。 The main object of the present invention is to provide a yeast cell or cell culture or yeast extract containing L-hydroxyproline, its use, and a method for producing L-hydroxyproline.
本発明者らは、上記課題を解決するために鋭意研究を行った結果、酵母のヤロウィア・リポリティカ(Yarrowia lipolytica)を好気培養すると、その菌体又は菌体培養物中にＬ－ヒドロキシプロリンを蓄積することを見出した。また、得られるＬ－ヒドロキシプロリンを含有するヤロウィア・リポリティカ(Yarrowia lipolytica)の菌体もしくは菌体培養物又はこれらの抽出物は、Ｌ－プロリン（Ｐｒｏ）及びＬ－ヒドロキシプロリン（Ｈｙｐ）の合計重量含量に対するＬ－ヒドロキシプロリンの重量含量の割合（１００×Ｈｙｐ／（Ｐｒｏ＋Ｈｙｐ））が特定範囲になり得ることを見出した。本発明者らは、これらの知見に基づきさらに研究を行い、本発明を完成するに至った。 As a result of intensive studies to solve the above problems, the present inventors have found that when Yarrowia lipolytica (yeast of yeast) is aerobically cultured, L-hydroxyproline is added to the cells or cell cultures. Found to accumulate. In addition, the obtained Yarrowia lipolytica cell or cell culture containing L-hydroxyproline or an extract thereof is the total weight of L-proline (Pro) and L-hydroxyproline (Hyp). It has been found that the ratio of the weight content of L-hydroxyproline to the content (100 × Hyp / (Pro + Hyp)) can be in a specific range. The present inventors have further studied based on these findings and have completed the present invention.
本発明の酵母の菌体もしくは菌体培養物又はこれらの抽出物は、酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)の菌体もしくは菌体培養物又はこれらの抽出物であって、上記酵母の菌体もしくは菌体培養物又はこれらの抽出物は、Ｌ－ヒドロキシプロリンを含有し、Ｌ－プロリン（Ｐｒｏ）及びＬ－ヒドロキシプロリン（Ｈｙｐ）の合計含量（μｇ／ｍＬ）に対するＬ－ヒドロキシプロリンの含量（μｇ／ｍＬ）の割合（１００×Ｈｙｐ／（Ｐｒｏ＋Ｈｙｐ））が、３５～１００であることを特徴とする。
本発明の酵母の菌体もしくは菌体培養物又はこれらの抽出物は、Ｌ－ヒドロキシプロリンの含量が１０μｇ／ｍＬ以上であることが好ましい。 A yeast cell or a cell culture of the yeast of the present invention or an extract thereof is a cell or a cell culture of yeast Yarrowia lipolytica or an extract thereof, wherein the yeast cell or The cell culture or these extracts contain L-hydroxyproline, and the content of L-hydroxyproline (μg / mL) relative to the total content (μg / mL) of L-proline (Pro) and L-hydroxyproline (Hyp). / ML) (100 × Hyp / (Pro + Hyp)) is 35 to 100.
The yeast cells or cell cultures or extracts thereof of the present invention preferably have an L-hydroxyproline content of 10 μg / mL or more.
本発明の別の態様の酵母の菌体もしくは菌体培養物又はこれらの抽出物は、酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)の菌体もしくは菌体培養物又はこれらの抽出物であって、Ｌ－ヒドロキシプロリンの含量が１０μｇ／ｍＬ以上であることを特徴とする。 In another aspect of the present invention, the yeast cell or cell culture or an extract thereof is a yeast cell or cell culture of yeast Yarrowia lipolytica or an extract thereof. The hydroxyproline content is 10 μg / mL or more.
本発明のＬ－ヒドロキシプロリンの製造方法は、酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)を、炭素源及び窒素源を含む液体培地中で好気培養することにより、上記酵母の菌体又は菌体培養物中にＬ－ヒドロキシプロリンを蓄積させる工程を含み、上記窒素源がＬ－ヒドロキシプロリン含有ペプチドを含む窒素源であることを特徴とする。 In the method for producing L-hydroxyproline of the present invention, the yeast cell or cell culture is obtained by aerobically culturing yeast Yarrowia lipolytica in a liquid medium containing a carbon source and a nitrogen source. A step of accumulating L-hydroxyproline therein, wherein the nitrogen source is a nitrogen source containing an L-hydroxyproline-containing peptide.
本発明の製造方法においては、上記Ｌ－ヒドロキシプロリン含有ペプチドがコラーゲンペプチドであることが好ましい。上記コラーゲンペプチドの平均分子量は１０００～１００００であることが好ましい。
本発明の製造方法においては、上記好気培養を１０～１００時間行うことが好ましい。 In the production method of the present invention, the L-hydroxyproline-containing peptide is preferably a collagen peptide. The average molecular weight of the collagen peptide is preferably 1000 to 10,000.
In the production method of the present invention, the aerobic culture is preferably performed for 10 to 100 hours.
本発明は、酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)の、Ｌ－ヒドロキシプロリンを製造するための使用も包含する。
本発明の使用は、上記酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)を、炭素源及び窒素源を含む液体培地中で好気培養することにより、上記酵母の菌体又は菌体培養物中にＬ－ヒドロキシプロリンを蓄積させることを含み、上記窒素源がＬ－ヒドロキシプロリン含有ペプチドを含む窒素源であることが好ましい。 The invention also encompasses the use of the yeast Yarrowia lipolytica to produce L-hydroxyproline.
The use of the present invention can be carried out by aerobically cultivating the yeast Yarrowia lipolytica in a liquid medium containing a carbon source and a nitrogen source, whereby L-hydroxyl in the yeast cell or cell culture. It is preferable that the nitrogen source is a nitrogen source containing an L-hydroxyproline-containing peptide.
本発明の使用においては、上記Ｌ－ヒドロキシプロリン含有ペプチドがコラーゲンペプチドであることが好ましい。上記コラーゲンペプチドの平均分子量は１０００～１００００であることが好ましい。
本発明の使用においては、上記好気培養を１０～１００時間行うことが好ましい。 In the use of the present invention, the L-hydroxyproline-containing peptide is preferably a collagen peptide. The average molecular weight of the collagen peptide is preferably 1000 to 10,000.
In the use of the present invention, the aerobic culture is preferably performed for 10 to 100 hours.
本発明の組成物は、本発明の酵母の菌体もしくは菌体培養物又はこれらの抽出物を含むことを特徴とする。
本発明の飲食品は、本発明の酵母の菌体もしくは菌体培養物又はこれらの抽出物を含むことを特徴とする。 The composition of the present invention is characterized in that it comprises the yeast or cell culture of the yeast of the present invention or an extract thereof.
The food / beverage products of this invention are characterized by including the microbial cell or microbial cell culture of this invention, or these extracts.
本発明の化粧料又は化粧料原料は、本発明の酵母の菌体もしくは菌体培養物又はこれらの抽出物を含むことを特徴とする。
本発明の化粧料又は化粧料原料は、コラーゲン産生促進、表皮細胞の増殖促進、皮膚の保湿、皮膚の老化防止、皮膚のたるみの予防又は改善、皮膚のハリの改善、しわの予防又は改善及びアトピー性皮膚炎の改善から選ばれる用途に用いられることが好ましい。
一態様において、本発明の化粧料又は化粧料原料は、化粧料原料であり、Ｌ－ヒドロキシプロリン含量が５～３００ｐｐｍであることが好ましい。
本発明の化粧料又は化粧料原料は、化粧料であり、Ｌ－ヒドロキシプロリン含量が０．０１～２０ｐｐｍであることも好ましい。本明細書中、ｐｐｍは、重量ｐｐｍを意味する。 The cosmetic or cosmetic raw material of the present invention is characterized by containing the yeast or bacterial culture of the yeast of the present invention or an extract thereof.
The cosmetic or cosmetic raw material of the present invention comprises collagen production promotion, epidermal cell growth promotion, skin moisturization, skin aging prevention, skin sagging prevention or improvement, skin firmness improvement, wrinkle prevention or improvement and It is preferably used for applications selected from the improvement of atopic dermatitis.
In one embodiment, the cosmetic or cosmetic raw material of the present invention is a cosmetic raw material, and preferably has an L-hydroxyproline content of 5 to 300 ppm.
The cosmetic or cosmetic raw material of the present invention is a cosmetic and preferably has an L-hydroxyproline content of 0.01 to 20 ppm. In this specification, ppm means weight ppm.
本発明のＬ－ヒドロキシプロリン補強用組成物は、Ｌ－ヒドロキシプロリンを含有する酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)の菌体もしくは菌体培養物又はこれらの抽出物を含むことを特徴とする。
本発明のＬ－ヒドロキシプロリン補強用組成物においては、上記酵母の菌体もしくは菌体培養物又はこれらの抽出物は、Ｌ－プロリン（Ｐｒｏ）及びＬ－ヒドロキシプロリン（Ｈｙｐ）の合計含量（μｇ／ｍＬ）に対するＬ－ヒドロキシプロリンの含量（μｇ／ｍＬ）の割合（１００×Ｈｙｐ／（Ｐｒｏ＋Ｈｙｐ））が、３５～１００であることが好ましい。また、上記酵母の菌体もしくは菌体培養物又はこれらの抽出物は、Ｌ－ヒドロキシプロリンの含量が１０μｇ／ｍＬ以上であることが好ましい。 The composition for reinforcing L-hydroxyproline of the present invention is characterized by comprising a cell or a cell culture of yeast Yarrowia lipolytica containing L-hydroxyproline or an extract thereof.
In the L-hydroxyproline reinforcing composition of the present invention, the yeast cell or cell culture or extract thereof contains the total content (μg of L-proline (Pro) and L-hydroxyproline (Hyp)). The ratio (100 × Hyp / (Pro + Hyp)) of L-hydroxyproline content (μg / mL) to (mL / mL) is preferably 35 to 100. Further, the yeast cells or cell cultures or extracts thereof preferably have an L-hydroxyproline content of 10 μg / mL or more.
本発明によれば、Ｌ－ヒドロキシプロリンを含有する酵母の菌体もしくは菌体培養物又はこれらの抽出物及びその用途、並びに、Ｌ－ヒドロキシプロリンの製造方法等を提供することができる。本発明の酵母の菌体もしくは菌体培養物又はこれらの抽出物は、飲食品、化粧料等の原料等として好適に使用される。 INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a yeast cell or bacterial cell culture or an extract thereof and its use containing L-hydroxyproline, a method for producing L-hydroxyproline, and the like. The yeast cell or cell culture of the present invention or an extract thereof is suitably used as a raw material for foods and beverages, cosmetics and the like.
図１は、酵母のＬ－ヒドロキシプロリン（Ｈｙｐ）蓄積量を示すグラフである。FIG. 1 is a graph showing the amount of L-hydroxyproline (Hyp) accumulated in yeast. 図２は、各培地で培養したYarrowia lipolyticaのＨｙｐ蓄積量を示すグラフである（（ａ）：条件１、（ｂ）：条件２）。FIG. 2 is a graph showing the amount of Yyp accumulation of Yarrowia lipolytica cultured in each medium ((a): Condition 1, (b): Condition 2). 図３は、ＰＤ培地又は０．１２５％ゼラチンを添加したＰＤ培地で５日間培養したYarrowia lipolyticaのＨｙｐ蓄積量を示すグラフである（白：０．１２５％ゼラチンを含むＰＤ培地、黒：ＰＤ培地）。FIG. 3 is a graph showing the Hyp accumulation amount of Yarrowia lipolytica cultured for 5 days in PD medium or PD medium supplemented with 0.125% gelatin (white: PD medium containing 0.125% gelatin, black: PD medium) ). 図４は、組成が異なるＹＰＤ培地で培養したYarrowia lipolyticaのＨｙｐ蓄積量を示すグラフである（（ａ）：Ｈｙｐ蓄積量（μｇ／ｍＬ）、（ｂ）：細胞あたりのＨｙｐ量（μｇ／ｍＬ／ＯＤ６００））。FIG. 4 is a graph showing the Hyp accumulation amount of Yarrowia lipolytica cultured in YPD media having different compositions ((a): Hyp accumulation amount (μg / mL), (b): Hyp amount per cell (μg / mL). / OD600)). 図５は、ペプトン又はコラーゲンペプチドを含む培地で培養したYarrowia lipolyticaのＨｙｐ蓄積量、細胞あたりのＨｙｐ量及びＯＤ６００を示すグラフである（（ａ）：Ｈｙｐ蓄積量（μｇ／ｍＬ）、（ｂ）：細胞あたりのＨｙｐ量（μｇ／ｍＬ／ＯＤ６００）、（ｃ）：ＯＤ６００））。FIG. 5 is a graph showing the Hyp accumulation amount, the Hyp amount per cell, and OD600 of Yarrowia lipolytica cultured in a medium containing peptone or collagen peptide ((a): Hyp accumulation amount (μg / mL), (b) : Hyp amount per cell (μg / mL / OD600), (c): OD600)). 図６は、振盪培養又は静置培養したYarrowia lipolyticaのＨｙｐ蓄積量を示す図である（（ａ）：培養物サンプル（細胞内）の培地あたり換算Ｈｙｐ量、（ｂ）：培養上清中のＨｙｐ量））。FIG. 6 is a diagram showing the amount of Hyp accumulated in Yarrowia lipolytica cultured by shaking culture or stationary culture ((a): Hyp equivalent per culture medium in culture sample (intracellular), (b): in culture supernatant. Hyp amount)). 振盪培養又は静置培養したYarrowia lipolyticaの培養上清のエタノール濃度及びグルコース濃度を示すグラフである（（ａ）：エタノール濃度（ｖ／ｖ％）、（ｂ）：グルコース濃度（重量％））。It is a graph which shows the ethanol concentration and glucose concentration of the culture supernatant of Yarrowia lipolytica which carried out shaking culture or stationary culture ((a): ethanol concentration (v / v%), (b): glucose concentration (weight%)). 図８は、アミノ酸混合標準液Ｈ型及びＬ－ヒドロキシプロリンを含む０．１Ｎ塩酸溶液（各アミノ酸濃度２０μｍｏｌ／Ｌ）を分析したＨＰＬＣチャートである（（ａ）：Ｃｈ１の励起波長３５０ｎｍ、蛍光波長４５０ｎｍで検出、（ｂ）：Ｃｈ２の励起波長２６６ｎｍ、蛍光波長３０５ｎｍで検出）。FIG. 8 is an HPLC chart obtained by analyzing a 0.1N hydrochloric acid solution containing amino acid mixed standard solution H and L-hydroxyproline (each amino acid concentration 20 μmol / L) ((a): Ch1 excitation wavelength 350 nm, fluorescence wavelength. Detection at 450 nm, (b): Ch2 excitation wavelength 266 nm, fluorescence wavelength 305 nm detection).
以下、本発明の実施形態について具体的に説明する。しかしながら、本発明は、以下の実施形態に限定されるものではなく、本発明の要旨を変更しない範囲において適宜変更して適用することができる。
本明細書中、酵母の属種は、The Yeasts, a Taxonomic Study Fifth Edition（Elsevier発行、2011年）に記載の属種名で記載した。 Hereinafter, embodiments of the present invention will be specifically described. However, the present invention is not limited to the following embodiments, and can be applied with appropriate modifications without departing from the scope of the present invention.
In this specification, the genus species of yeast were described by the genus name described in The Yeasts, a Taxonomic Study Fifth Edition (issued by Elsevier, 2011).
本発明の第一の態様の酵母の菌体もしくは菌体培養物又はこれらの抽出物は、酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)の菌体もしくは菌体培養物又はこれらの抽出物であって、上記酵母の菌体もしくは菌体培養物又はこれらの抽出物は、Ｌ－ヒドロキシプロリンを含有し、Ｌ－プロリン（Ｐｒｏ）及びＬ－ヒドロキシプロリン（Ｈｙｐ）の合計含量（μｇ／ｍＬ）に対するＬ－ヒドロキシプロリンの含量（μｇ／ｍＬ）の割合（１００×Ｈｙｐ／（Ｐｒｏ＋Ｈｙｐ））が、３５～１００である。 A yeast cell or cell culture or an extract thereof according to the first aspect of the present invention is a cell or cell culture of yeast Yarrowia lipolytica or an extract thereof, Yeast cells or cell cultures or extracts thereof contain L-hydroxyproline, and L-hydroxy with respect to the total content (μg / mL) of L-proline (Pro) and L-hydroxyproline (Hyp). The ratio of proline content (μg / mL) (100 × Hyp / (Pro + Hyp)) is 35 to 100.
本発明の第二の態様の酵母の菌体もしくは菌体培養物又はこれらの抽出物は、酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)の菌体もしくは菌体培養物又はこれらの抽出物であって、Ｌ－ヒドロキシプロリンの含量が１０μｇ／ｍＬ以上である。
以下、本発明の第一の態様及び第二の態様の酵母の菌体もしくは菌体培養物又はこれらの抽出物を、まとめて本発明の酵母の菌体もしくは菌体培養物又はこれらの抽出物ともいう。 The yeast cell or cell culture or the extract thereof according to the second aspect of the present invention is a yeast cell or a cell culture of yeast Yarrowia lipolytica or an extract thereof. -The content of hydroxyproline is 10 μg / mL or more.
Hereinafter, the yeast cells or cell cultures of the yeast according to the first aspect and the second aspect of the present invention, or extracts thereof are collectively referred to as the yeast cells or cell cultures of the yeast of the present invention or extracts thereof. Also called.
本発明の酵母の菌体もしくは菌体培養物又はこれらの抽出物は、酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)の菌体もしくは菌体培養物又はこれらの抽出物である。
本発明における酵母は、ヤロウィア・リポリティカ(Yarrowia lipolytica)に属する酵母であればよく、１種のみ使用してもよく、２種以上を使用してもよい。
酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)は、さまざまな寄託機関等より入手可能である。寄託機関としては、例えば、独立行政法人製品評価技術基盤機構（日本国千葉県木更津市かずさ鎌足２－５－８）等が挙げられる。また、自然界から分離することもできる。 The yeast cells or cell cultures or extracts thereof of the present invention are yeast cells or cell cultures of yeast Yarrowia lipolytica or extracts thereof.
The yeast in this invention should just be a yeast which belongs to Yarrowia lipolytica ( Yarrowia lipolytica ), and may use only 1 type and may use 2 or more types.
Yeast Yarrowia lipolytica is available from various depository institutions. Examples of depository organizations include the National Institute of Technology and Evaluation (2-5-8, Kazusa Kamashichi, Kisarazu City, Chiba Prefecture, Japan). It can also be separated from nature.
本発明の酵母の菌体もしくは菌体培養物又はこれらの抽出物は、Ｌ－ヒドロキシプロリン（Ｈｙｐ）を含有する。本発明におけるＬ－ヒドロキシプロリンは、４－ヒドロキシ－Ｌ－プロリンである。本明細書中、酵母の菌体もしくは菌体培養物又はこれらの抽出物に含まれるＬ－ヒドロキシプロリンは、遊離のＬ－ヒドロキシプロリンを指す。酵母の菌体もしくは菌体培養物又はこれらの抽出物におけるＬ－ヒドロキシプロリン含量又は蓄積量は、遊離のＬ－ヒドロキシプロリン量を指す。その菌体又は菌体培養物中に遊離のＬ－ヒドロキシプロリンを蓄積する酵母は報告されていなかった。 The yeast or cell culture of the yeast of the present invention or an extract thereof contains L-hydroxyproline (Hyp). L-hydroxyproline in the present invention is 4-hydroxy-L-proline. In the present specification, L-hydroxyproline contained in yeast cells or cell cultures or extracts thereof refers to free L-hydroxyproline. The L-hydroxyproline content or accumulated amount in yeast cells or cell cultures or extracts thereof refers to the amount of free L-hydroxyproline. No yeast has been reported that accumulates free L-hydroxyproline in its cells or cell cultures.
本発明の第一の態様の酵母の菌体もしくは菌体培養物又はこれらの抽出物は、Ｌ－プロリン（Ｐｒｏ）及びＬ－ヒドロキシプロリン（Ｈｙｐ）の合計含量（μｇ／ｍＬ）に対するＬ－ヒドロキシプロリンの含量（μｇ／ｍＬ）の割合（１００×Ｈｙｐ／（Ｐｒｏ＋Ｈｙｐ））が、３５～１００である。このような酵母の菌体もしくは菌体培養物又はこれらの抽出物は、Ｌ－ヒドロキシプロリンの効能が期待される飲食品や化粧料の原料等に好適に使用される。「Ｌ－プロリン（Ｐｒｏ）及びＬ－ヒドロキシプロリン（Ｈｙｐ）の合計含量（μｇ／ｍＬ）に対するＬ－ヒドロキシプロリンの含量（μｇ／ｍＬ）の割合」（１００×Ｈｙｐ／（Ｐｒｏ＋Ｈｙｐ））を、以下では「（Ｈｙｐ／（Ｐｒｏ＋Ｈｙｐ））割合」ともいう。本明細書中、上記（Ｈｙｐ／（Ｐｒｏ＋Ｈｙｐ））割合におけるＬ－プロリン含量は、酵母の菌体もしくは菌体培養物又はこれらの抽出物に含まれる遊離のＬ－プロリン含量を指す。
好ましい態様において、本発明の第二の態様の酵母の菌体もしくは菌体培養物又はこれらの抽出物は、（Ｈｙｐ／（Ｐｒｏ＋Ｈｙｐ））割合が、３５～１００である。
上記（Ｈｙｐ／（Ｐｒｏ＋Ｈｙｐ））割合は、好ましくは４０～１００であり、より好ましくは５０～１００であり、より好ましくは６０～１００であり、さらに好ましくは７０～１００であり、さらにより好ましくは８０～１００であり、特に好ましくは９０～１００である。上記（Ｈｙｐ／（Ｐｒｏ＋Ｈｙｐ））割合がこのような酵母の菌体もしくは菌体培養物又はこれらの抽出物は、Ｈｙｐの含量割合が高く、Ｌ－ヒドロキシプロリンの効能が期待される飲食品や化粧料の原料等として特に好適である。 The yeast or cell culture of yeast according to the first aspect of the present invention, or an extract thereof, contains L-hydroxy with respect to the total content (μg / mL) of L-proline (Pro) and L-hydroxyproline (Hyp). The ratio of proline content (μg / mL) (100 × Hyp / (Pro + Hyp)) is 35 to 100. Such yeast cells or cell cultures or extracts thereof are suitably used for foods and beverages and cosmetic raw materials for which L-hydroxyproline is expected to be effective. “Ratio of L-hydroxyproline content (μg / mL) to total content (μg / mL) of L-proline (Pro) and L-hydroxyproline (Hyp)” (100 × Hyp / (Pro + Hyp)) Then, it is also referred to as “(Hyp / (Pro + Hyp)) ratio”. In the present specification, the L-proline content in the above ratio (Hyp / (Pro + Hyp)) refers to the free L-proline content contained in yeast cells or cell cultures or extracts thereof.
In a preferred embodiment, the yeast cell or cell culture of the second embodiment of the present invention or an extract thereof has a (Hyp / (Pro + Hyp)) ratio of 35 to 100.
The (Hyp / (Pro + Hyp)) ratio is preferably 40 to 100, more preferably 50 to 100, more preferably 60 to 100, still more preferably 70 to 100, and still more preferably. 80 to 100, particularly preferably 90 to 100. The above yeast (Hyp / (Pro + Hyp)) ratio of such yeast cells or cell cultures or extracts thereof have a high Hyp content ratio and are expected to be effective for L-hydroxyproline. It is particularly suitable as a raw material for the material.
本発明の第二の態様の酵母の菌体もしくは菌体培養物又はこれらの抽出物は、Ｌ－ヒドロキシプロリンの含量が１０μｇ／ｍＬ以上である。
本発明の第一の態様の酵母の菌体もしくは菌体培養物又はこれらの抽出物は、Ｌ－ヒドロキシプロリンの含量が１０μｇ／ｍＬ以上であることが好ましい。Ｌ－ヒドロキシプロリンの含量が上記範囲である酵母の菌体もしくは菌体培養物又はこれらの抽出物は、Ｌ－ヒドロキシプロリンの効能が期待される飲食品や化粧料の原料等として好適である。
本発明の酵母の菌体もしくは菌体培養物又はこれらの抽出物のＬ－ヒドロキシプロリンの含量は、１５μｇ／ｍＬ以上であることがより好ましく、１７μｇ／ｍＬ以上がより好ましく、２０μｇ／ｍＬ以上がより好ましく、３０μｇ／ｍＬ以上がさらに好ましく、４０μｇ／ｍＬ以上がさらに好ましく、５０μｇ／ｍＬ以上がさらに好ましく、７０μｇ／ｍＬ以上がさらに好ましく、１００μｇ／ｍＬ以上がさらに好ましく、１５０μｇ／ｍＬ以上がさらに好ましく、２００μｇ／ｍＬ以上が特に好ましく、２５０μｇ／ｍＬ以上が特に好ましく、３００μｇ／ｍＬ以上が特に好ましく、４００μｇ／ｍＬ以上が特に好ましく、４５０μｇ／ｍＬ以上が特に好ましく、４７５μｇ／ｍＬ以上が特に好ましく、５００μｇ／ｍＬ以上が最も好ましい。
酵母の菌体もしくは菌体培養物又はこれらの抽出物のＬ－ヒドロキシプロリンの含量の上限は特に限定されず、多い方が好ましいが、通常、６０００μｇ／ｍＬ以下であり、３０００μｇ／ｍＬ以下又は２０００μｇ／ｍＬ以下であってもよい。
本発明によれば、酵母の菌体又は菌体培養物に由来するＬ－ヒドロキシプロリンの含量が、上記範囲である酵母の菌体もしくは菌体培養物又はこれらの抽出物を提供することができる。 The yeast cell or cell culture or the extract thereof according to the second aspect of the present invention has an L-hydroxyproline content of 10 μg / mL or more.
The yeast cell or cell culture or extract thereof according to the first aspect of the present invention preferably has an L-hydroxyproline content of 10 μg / mL or more. A yeast cell or cell culture or an extract thereof having a content of L-hydroxyproline within the above range is suitable as a raw material for foods and beverages and cosmetics for which the effect of L-hydroxyproline is expected.
The content of L-hydroxyproline in the yeast or cell culture of the yeast of the present invention or these extracts is more preferably 15 μg / mL or more, more preferably 17 μg / mL or more, and more preferably 20 μg / mL or more. More preferably, 30 μg / mL or more is more preferable, 40 μg / mL or more is further preferable, 50 μg / mL or more is further preferable, 70 μg / mL or more is further preferable, 100 μg / mL or more is further preferable, and 150 μg / mL or more is more preferable. 200 μg / mL or more is particularly preferable, 250 μg / mL or more is particularly preferable, 300 μg / mL or more is particularly preferable, 400 μg / mL or more is particularly preferable, 450 μg / mL or more is particularly preferable, 475 μg / mL or more is particularly preferable, 500 μg / ML or more is most preferable.
The upper limit of the content of L-hydroxyproline in yeast cells or cell cultures or extracts thereof is not particularly limited and is preferably large, but is usually 6000 μg / mL or less, 3000 μg / mL or less, or 2000 μg. / ML or less may be sufficient.
According to the present invention, it is possible to provide a yeast cell or cell culture, or an extract thereof, wherein the content of L-hydroxyproline derived from the yeast cell or cell culture is in the above range. .
酵母の菌体もしくは菌体培養物又はこれらの抽出物中のＬ－プロリンの含量（μｇ／ｍＬ）及びＬ－ヒドロキシプロリンの含量（μｇ／ｍＬ）は、高速液体クロマトグラフィー（ＨＰＬＣ）により測定することができる。酵母の菌体もしくは菌体培養物又はこれらの抽出物が菌体を含む場合には、加熱等による自己消化や酵素分解処理等により菌体を破砕し、菌体内容物を溶出したものを用いて、Ｌ－プロリン及びＬ－ヒドロキシプロリンの含量を測定する。Ｌ－プロリン及びＬ－ヒドロキシプロリン含量の測定方法及びＨＰＬＣの測定条件等は、実施例に記載の方法及び条件等を採用すればよい。好ましくは、（１）ｏ－フタルアルデヒド（ＯＰＡ）及び４－クロロ－７－ニトロベンゾフラザン（ＮＢＤ－Ｃｌ）により順次アミノ酸を誘導体化して、ＨＰＬＣにより分析する方法（励起波長５０３ｎｍ／蛍光波長５４１ｎｍで検出）、又は、（２）メルカプトプロピオン酸、ＯＰＡで１級アミノ基を誘導体化後クロロ蟻酸－９－フルオレニルメチル（ＦＭＯＣ）により２級アミノ酸を誘導体化して、ＨＰＬＣにより分析する方法（励起波長２６６ｎｍ、蛍光波長３０５ｎｍで検出）により、より好ましくは上記（２）の方法により、Ｌ－プロリン及びＬ－ヒドロキシプロリンを定量する。 The L-proline content (μg / mL) and the L-hydroxyproline content (μg / mL) in yeast cells or cell cultures or extracts thereof are measured by high performance liquid chromatography (HPLC). be able to. When yeast cells or cell cultures or extracts of these contain cells, use the one that crushes cells by heating, etc. by self-digestion or enzymatic degradation, and elutes the cell contents. Then, the contents of L-proline and L-hydroxyproline are measured. As a method for measuring L-proline and L-hydroxyproline content, HPLC measurement conditions, and the like, the methods and conditions described in the Examples may be employed. Preferably, (1) a method in which amino acids are sequentially derivatized with o-phthalaldehyde (OPA) and 4-chloro-7-nitrobenzofurazan (NBD-Cl) and analyzed by HPLC (excitation wavelength 503 nm / fluorescence wavelength 541 nm). (2) A method in which a primary amino group is derivatized with mercaptopropionic acid or OPA, and then a secondary amino acid is derivatized with chloroformic acid-9-fluorenylmethyl (FMOC) and analyzed by HPLC ( L-proline and L-hydroxyproline are quantified by detection at an excitation wavelength of 266 nm and a fluorescence wavelength of 305 nm, more preferably by the method of (2) above.
本発明の酵母の菌体もしくは菌体培養物又はこれらの抽出物は、酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)を液体培地中で好気培養し、必要に応じて菌体破砕等を行って得られるものである。
酵母の菌体培養物は、酵母の菌体及び／又は培養上清を含むことが好ましく、酵母の菌体内容物を含んでいてもよい。酵母の菌体は、生菌であってもよく、死菌であってもよい。酵母の菌体及び／又は培養上清を含む菌体培養物として、上記酵母を好気培養して得られる酵母の菌体（培養菌体）及び培養上清を含む菌体培養液、該菌体培養液から酵母の菌体を集菌したもの（菌体）、又は該菌体培養液から菌体を除去した培養上清が挙げられる。菌体培養液の培養上清を、単に培養上清という。菌体培養物は、好ましくは、酵母の菌体及び培養上清を含む菌体培養液又は培養上清である。
また、菌体又は菌体培養物の抽出物は、通常、菌体内容物を含み、菌体内容物及び培養上清を含むことが好ましい。菌体又は菌体培養物の抽出物として、菌体又は菌体を含む菌体培養物（好ましくは菌体培養液）に、自己消化処理や酵素分解処理等の菌体破砕処理を行って、酵母菌体内容物を培養液中等に溶出したもの（菌体破砕物）、菌体破砕処理を行った菌体又は菌体培養物（菌体破砕物）から菌体残渣を除去したものが挙げられ、好ましくは、菌体破砕処理を行った菌体培養液（菌体破砕物）又は該菌体破砕物から菌体残渣を除去して得られる、菌体内容物及び培養上清を含む抽出物である。 The yeast cells or cell culture of the yeast of the present invention or extracts thereof are obtained by aerobically culturing yeast Yarrowia lipolytica in a liquid medium, and crushing the cells as necessary. Is.
The yeast cell culture preferably contains yeast cells and / or culture supernatant, and may contain yeast cell contents. The yeast cells may be live or dead. As a bacterial cell culture containing yeast cells and / or culture supernatant, yeast cells (cultured cells) obtained by aerobic culture of the yeast and cell culture liquid containing the culture supernatant, the bacteria Examples include yeast cells collected from a body culture solution (bacteria) or culture supernatants obtained by removing the cells from the cell culture solution. The culture supernatant of the cell culture medium is simply referred to as the culture supernatant. The cell culture is preferably a cell culture solution or culture supernatant containing yeast cells and culture supernatant.
Moreover, the extract of a microbial cell or a microbial cell culture contains a microbial cell content normally, and it is preferable that a microbial cell content and a culture supernatant are included. As an extract of the microbial cell or the microbial cell culture, the microbial cell culture or a microbial cell culture containing the microbial cell (preferably microbial cell culture solution) is subjected to microbial cell disruption treatment such as self-digestion treatment or enzymatic degradation treatment, What eluted yeast cell contents in the culture solution (broken cell), removed the cell residue from the broken cell or cell culture (broken cell) Preferably, the microbial cell culture solution (bacterial cell crushed material) that has been subjected to the cell disruption treatment or an extract containing the cell contents and the culture supernatant obtained by removing the cell residue from the cell crushed material It is a thing.
上記のように、本発明の酵母の菌体もしくは菌体培養物又はこれらの抽出物は、通常、酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)を液体培地中で好気培養して得られる酵母の菌体及び培養上清を含む菌体培養液に、必要に応じて集菌、菌体破砕等の処理を行って調製される。
本発明の酵母の菌体もしくは菌体培養物又はこれらの抽出物に含まれるＬ－ヒドロキシプロリンは、上記の好気培養により得られる酵母の菌体又は菌体培養物に由来することが好ましい。本発明の酵母の菌体もしくは菌体培養物又はこれらの抽出物に含まれるＬ－ヒドロキシプロリンは、上記の好気培養前には実質的に存在しないことが好ましい。
本発明の酵母の菌体もしくは菌体培養物又はこれらの抽出物は、例えば、化粧料、酒類を含む飲食品等の原料として好適に使用することができるものである。 As described above, the yeast cells or cell cultures of the present invention or extracts thereof are usually yeast cells obtained by aerobic culture of yeast Yarrowia lipolytica in a liquid medium. In addition, the bacterial cell culture medium containing the culture supernatant is prepared by subjecting the bacterial cell culture solution to treatment such as collection and disruption of the bacterial cells as necessary.
The L-hydroxyproline contained in the yeast cell or cell culture of the present invention or the extract thereof is preferably derived from the yeast cell or cell culture obtained by the above aerobic culture. It is preferable that the L-hydroxyproline contained in the yeast cell or cell culture of the present invention or the extract thereof is substantially absent before the aerobic culture.
The yeast cells or cell cultures or extracts thereof of the present invention can be suitably used as raw materials for foods and drinks including cosmetics and liquors, for example.
本発明の酵母の菌体もしくは菌体培養物又はこれらの抽出物は、Ｌ－ヒドロキシプロリンの含量（μｇ／ｍＬ）を、ＯＤ６００で除した値（μｇ／ｍＬ／ＯＤ６００）が、０．５以上であることが好ましい。Ｌ－ヒドロキシプロリンの含量（μｇ／ｍＬ）を、ＯＤ６００で除した値（μｇ／ｍＬ／ＯＤ６００）を、以下、Ｈｙｐ／ＯＤ６００値ともいう。Ｈｙｐ／ＯＤ６００値が高いほど、細胞あたりのＬ－ヒドロキシプロリンの含量が多いため好ましい。酵母の菌体もしくは菌体培養物又はこれらの抽出物のＨｙｐ／ＯＤ６００値の上限は特に限定されず、多いほど好ましいが、通常、３００以下である。Ｈｙｐ／ＯＤ６００値は、１以上であることがより好ましく、例えば、１～１５０が好ましい。Ｈｙｐ／ＯＤ６００値は、２５以上がさらに好ましく、５０以上がさらにより好ましい。 The yeast cell or cell culture of the present invention or an extract thereof has a value (μg / mL / OD600) obtained by dividing the L-hydroxyproline content (μg / mL) by OD600 (0.5 or more). It is preferable that A value obtained by dividing the content of L-hydroxyproline (μg / mL) by OD600 (μg / mL / OD600) is hereinafter also referred to as a Hyp / OD600 value. A higher Hyp / OD600 value is preferred because the L-hydroxyproline content per cell is higher. The upper limit of the Hyp / OD600 value of yeast cells or cell cultures or extracts thereof is not particularly limited and is preferably as large as possible, but is usually 300 or less. The Hyp / OD600 value is more preferably 1 or more, for example, 1 to 150 is preferable. The Hyp / OD600 value is more preferably 25 or more, and even more preferably 50 or more.
ＯＤとはｏｐｔｉｃａｌ　ｄｅｎｓｉｔｙの略で、光学密度を指す。ＯＤは、細胞の濃度などを表す。一般的には６００ｎｍ又は６６０ｎｍの波長の可視光に対する吸光度ＯＤ６００又はＯＤ６６０を測定する（バイオ実験イラストレイテッド▲７▼使おう酵母　できるＴｗｏ　Ｈｙｂｒｉｄ、秀潤社、２００３年発行）。
Ｈｙｐ／ＯＤ６００値の計算に使用されるＯＤ６００は、菌体もしくは菌体培養物又はこれらの抽出物の調製に用いた、菌体及び培養上清を含む菌体培養液（菌体及び培養上清を含む菌体培養物）の６００ｎｍの吸光度である。
より具体的には、上記の酵母を液体培地中で好気培養して得られる酵母の菌体培養液をそのまま菌体培養物とする場合には、ＯＤ６００は、該菌体培養液（菌体培養物）の吸光度ＯＤ６００である。酵母の菌体もしくは菌体培養物又はこれらの抽出物が、酵母の菌体を破砕して菌体内容物を溶出させた菌体又は菌体培養物の抽出物の場合には、ＯＤ６００は、その調製に使用した菌体培養液（菌体破砕前の酵母の菌体及び培養上清を含む菌体培養液）の吸光度ＯＤ６００である。ＯＤ６００は、例えば、分光光度計により測定することができる。 OD is an abbreviation for optical density and refers to optical density. OD represents a cell concentration or the like. In general, absorbance OD600 or OD660 with respect to visible light having a wavelength of 600 nm or 660 nm is measured (Bio Experiment Illustrated (7) Yeast that can be used Two Hybrid, Shujunsha, published in 2003).
The OD600 used for the calculation of the Hyp / OD600 value is the cell culture solution containing the cells and culture supernatant (cells and culture supernatant used for the preparation of the cells or cell cultures or extracts thereof) The absorbance at 600 nm of the bacterial cell culture containing
More specifically, when the yeast cell culture solution obtained by aerobic culture of the above yeast in a liquid medium is used as it is as a cell culture, OD600 is the cell culture solution (cells). Absorbance OD600 of the culture). In the case of yeast cells or cell cultures or extracts thereof, the microbial cells or cell culture extract obtained by crushing yeast cells and eluting the cell contents, OD600 is: It is the light absorbency OD600 of the microbial cell culture solution (the microbial cell culture solution containing the microbial cell of the yeast before microbial cell disruption, and a culture supernatant) used for the preparation. OD600 can be measured with a spectrophotometer, for example.
本発明の酵母の菌体もしくは菌体培養物又はこれらの抽出物は、エタノール含量が１ｖ／ｖ％以下であることが好ましい。エタノール含量が１ｖ／ｖ％以下であると、各種飲食品や化粧料等の原料として特に好適に使用することができる。エタノールが１ｖ／ｖ％を超えると、酵母の増殖などに悪い影響がある場合がある。本発明の酵母の菌体もしくは菌体培養物又はこれらの抽出物のエタノール含量は、より好ましくは０．８ｖ／ｖ％以下であり、さらに好ましくは０．５ｖ／ｖ％以下である。エタノール含量は、公知の方法により測定することができる。 The yeast cells or cell cultures or extracts thereof of the present invention preferably have an ethanol content of 1 v / v% or less. When the ethanol content is 1 v / v% or less, it can be particularly preferably used as a raw material for various foods and cosmetics. If ethanol exceeds 1 v / v%, there may be adverse effects on yeast growth and the like. The ethanol content of the yeast or the cell culture of the yeast of the present invention or the extract thereof is more preferably 0.8 v / v% or less, and further preferably 0.5 v / v% or less. The ethanol content can be measured by a known method.
本発明の酵母の菌体もしくは菌体培養物又はこれらの抽出物の形態は特に限定されないが、例えば、ペースト状、懸濁状、エキス状、液状等が挙げられる。
本発明の酵母の菌体もしくは菌体培養物又はこれらの抽出物は、後述するように化粧料、飲食品等の原料として好適に使用することができるものである。本発明の酵母の菌体もしくは菌体培養物又はこれらの抽出物を乾燥等により粉末化して使用することもできる。 The form of the yeast cells or cell cultures or extracts thereof of the present invention is not particularly limited, and examples thereof include pastes, suspensions, extracts, and liquids.
The yeast cells or cell cultures of the present invention or extracts thereof can be suitably used as a raw material for cosmetics, foods and drinks, etc. as described later. The yeast cells or cell cultures of the yeast of the present invention or extracts thereof can be used after being powdered by drying or the like.
本発明の酵母の菌体もしくは菌体培養物又はこれらの抽出物は、酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)を、炭素源及び窒素源を含有する液体培地中で好気培養し、必要に応じて菌体破砕等することにより、得ることができる。より具体的には、窒素源は、Ｌ－ヒドロキシプロリン含有ペプチドを含む。
酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)を、炭素源、及び、Ｌ－ヒドロキシプロリン含有ペプチドを含む窒素源を含む液体培地中で好気培養することにより、該酵母の菌体又は菌体培養物中にＬ－ヒドロキシプロリンが蓄積し、Ｌ－ヒドロキシプロリンを含有する酵母の菌体又は菌体培養物が得られる。酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)を、炭素源、及び、Ｌ－ヒドロキシプロリン含有ペプチドを含む窒素源を含む液体培地中で好気培養することにより、該酵母の菌体又は菌体培養物中にＬ－ヒドロキシプロリンを蓄積させる工程を含む方法は、上述した本発明の酵母の菌体もしくは菌体培養物又はこれらの抽出物の製造方法、又は、Ｌ－ヒドロキシプロリンの製造方法として好ましい。 The yeast cell or cell culture of the present invention or an extract thereof is obtained by aerobically cultivating the yeast Yarrowia lipolytica in a liquid medium containing a carbon source and a nitrogen source. It can be obtained by disrupting the cells. More specifically, the nitrogen source includes an L-hydroxyproline-containing peptide.
Yeastia lipolytica ( Yarrowia lipolytica ) is aerobically cultured in a liquid medium containing a carbon source and a nitrogen source containing an L-hydroxyproline-containing peptide, whereby the yeast cell or cell culture is obtained. L-hydroxyproline accumulates, and yeast cells or cell cultures containing L-hydroxyproline are obtained. Yeastia lipolytica ( Yarrowia lipolytica ) is aerobically cultured in a liquid medium containing a carbon source and a nitrogen source containing an L-hydroxyproline-containing peptide, whereby the yeast cell or cell culture is obtained. A method including the step of accumulating L-hydroxyproline is preferable as a method for producing the above-described yeast cell or cell culture of the present invention or an extract thereof, or a method for producing L-hydroxyproline.
本発明のＬ－ヒドロキシプロリンの製造方法は、酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)を、炭素源及び窒素源を含む液体培地中で好気培養することにより、上記酵母の菌体又は菌体培養物中にＬ－ヒドロキシプロリンを蓄積させる工程（以下、Ｈｙｐ蓄積工程ともいう）を含む。上記窒素源は、Ｌ－ヒドロキシプロリン含有ペプチドを含む窒素源である。
本発明の製造方法は、所望によりＨｙｐ蓄積工程以外の工程を有してもよい。例えば、後述する前培養工程、集菌工程、菌体破砕工程等の１又は２以上の工程を有していてもよい。 In the method for producing L-hydroxyproline of the present invention, the yeast cell or cell culture is obtained by aerobically culturing yeast Yarrowia lipolytica in a liquid medium containing a carbon source and a nitrogen source. A step of accumulating L-hydroxyproline (hereinafter also referred to as a Hyp accumulation step). The nitrogen source is a nitrogen source containing an L-hydroxyproline-containing peptide.
The production method of the present invention may have steps other than the Hyp accumulation step as desired. For example, you may have 1 or 2 or more processes, such as the preculture process mentioned later, a microbe collection process, and a microbial cell crushing process.
本発明のＬ－ヒドロキシプロリンの製造方法においては、上述した本発明の酵母の菌体もしくは菌体培養物又はこれらの抽出物を得ることができる。
上記Ｈｙｐ蓄積工程を行うことにより、Ｌ－ヒドロキシプロリンを含有する酵母の菌体又は菌体培養物が得られる。得られる酵母の菌体又は菌体培養物は、通常、上記（Ｈｙｐ／（Ｐｒｏ＋Ｈｙｐ））割合が３５～１００である。また、Ｈｙｐ蓄積工程により得られる酵母の菌体又は菌体培養物は、通常、Ｌ－ヒドロキシプロリンの含量が１０μｇ／ｍＬ以上である。このような酵母の菌体又は菌体培養物は、上述した本発明の酵母の菌体又は菌体培養物として使用できる。また、得られた菌体又は菌体培養物に、所望によりさらに菌体破砕処理等の処理を行って、Ｌ－ヒドロキシプロリンを含有する酵母の菌体又は菌体培養物の抽出物を調製することもできる。
Ｈｙｐ蓄積工程を含むＬ－ヒドロキシプロリンの製造方法は、上述した本発明の酵母の菌体もしくは菌体培養物又はこれらの抽出物の製造方法としても好ましい。本発明の酵母の菌体もしくは菌体培養物又はこれらの抽出物を得る場合、Ｈｙｐ蓄積工程及びその好ましい態様は、Ｌ－ヒドロキシプロリンの製造方法におけるＨｙｐ蓄積工程及びその好ましい態様と同じである。 In the method for producing L-hydroxyproline of the present invention, the above-described yeast cell or cell culture of the present invention or an extract thereof can be obtained.
By performing the Hyp accumulation step, a yeast cell or cell culture containing L-hydroxyproline is obtained. The obtained yeast cells or cell cultures usually have the above (Hyp / (Pro + Hyp)) ratio of 35 to 100. In addition, the yeast cell or cell culture obtained by the Hyp accumulation step usually has an L-hydroxyproline content of 10 μg / mL or more. Such yeast cells or cell cultures can be used as the yeast cells or cell cultures of the present invention described above. Further, the obtained bacterial cells or bacterial cell culture is further subjected to treatment such as cell disruption as required to prepare a yeast bacterial cell or bacterial cell culture extract containing L-hydroxyproline. You can also
The method for producing L-hydroxyproline including the Hyp accumulation step is also preferable as a method for producing the above-described yeast cells or cell cultures of the present invention or extracts thereof. When obtaining the yeast cells or cell cultures or extracts thereof of the present invention, the Hyp accumulation step and preferred embodiments thereof are the same as the Hyp accumulation step and preferred embodiments thereof in the method for producing L-hydroxyproline.
Ｈｙｐ蓄積工程において、酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)の液体培地ヘの添加方法は、炭素源及び窒素源を含有する液体培地中に直接少量の菌体を接種することで増殖させることができるが、短期間で菌体濃度を上昇させるためには、前培養した菌液を接種することが好ましい。前培養に用いる培地は特に限定されず、Ｈｙｐ蓄積工程（通常、本培養）で使用される液体培地と同じ培地でもよく、酵母に使用することができる公知の培地を使用してもよい。前培養の時間は、通常１０～７２時間であり、好ましくは１２～４８時間である。前培養温度は、１５～４０℃とすることが好ましい。前培養した菌液を接種する量としては、通常、Ｈｙｐ蓄積工程で使用する培地量の１／１０００００～１／２であり、１／１０００～１／１０が好ましく、１／２００～１／１０がより好ましく、１／２００～１／２０がさらに好ましい。接種量が上記範囲であれば、Ｈｙｐ蓄積工程において酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)の増殖が速く、Ｌ－ヒドロキシプロリンを効率よく蓄積させることができる。 In the Hyp accumulation step, yeast Yarrowia lipolytica can be added to a liquid medium by inoculating a small amount of cells directly into the liquid medium containing a carbon source and a nitrogen source. In order to increase the bacterial cell concentration in a short period, it is preferable to inoculate a pre-cultured bacterial solution. The medium used for the pre-culture is not particularly limited, and may be the same medium as the liquid medium used in the Hyp accumulation step (usually main culture), or a known medium that can be used for yeast. The preculture time is usually 10 to 72 hours, preferably 12 to 48 hours. The preculture temperature is preferably 15 to 40 ° C. The amount inoculated with the pre-cultured bacterial solution is usually 1/10000 to 1/2 of the amount of medium used in the Hyp accumulation step, preferably 1/1000 to 1/10, and preferably 1/200 to 1/10. Is more preferable, and 1/200 to 1/20 is even more preferable. If the inoculation amount is in the above range, the yeast Yarrowia lipolytica grows rapidly in the Hyp accumulation step, and L-hydroxyproline can be efficiently accumulated.
Ｈｙｐ蓄積工程で使用される液体培地の窒素源は、Ｌ－ヒドロキシプロリン含有ペプチドを含む窒素源である。このような窒素源を用いて酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)を好気培養すると、菌体又は菌体培養物中にＬ－ヒドロキシプロリンが蓄積する。このような窒素源は１種のみ使用してもよく、２種以上を使用してもよい。Ｌ－ヒドロキシプロリン含有ペプチドは１種であってもよく、２種以上であってもよい。
Ｌ－ヒドロキシプロリン含有ペプチドとは、Ｌ－ヒドロキシプロリンを構成アミノ酸に含むペプチドであればよいが、好ましくは、構成アミノ酸の１０重量％以上がＬ－ヒドロキシプロリンであるペプチドである。一態様において、Ｌ－ヒドロキシプロリン含有ペプチドを含む窒素源は、Ｌ－ヒドロキシプロリン含有ペプチドであることも好ましい。 The nitrogen source of the liquid medium used in the Hyp accumulation step is a nitrogen source containing an L-hydroxyproline-containing peptide. When the yeast Yarrowia lipolytica is aerobically cultured using such a nitrogen source, L-hydroxyproline accumulates in the cells or cell cultures. Such nitrogen sources may be used alone or in combination of two or more. The number of L-hydroxyproline-containing peptides may be one, or two or more.
The L-hydroxyproline-containing peptide may be any peptide that contains L-hydroxyproline as a constituent amino acid, but is preferably a peptide in which 10% by weight or more of the constituent amino acid is L-hydroxyproline. In one embodiment, the nitrogen source containing the L-hydroxyproline-containing peptide is also preferably an L-hydroxyproline-containing peptide.
Ｌ－ヒドロキシプロリン含有ペプチドを含む窒素源は、例えば、Ｌ－ヒドロキシプロリン含有タンパク質を加水分解することにより得ることができる。Ｌ－ヒドロキシプロリン含有タンパク質とは、構成アミノ酸にＬ－ヒドロキシプロリンを含むタンパク質であればよいが、好ましくは、構成アミノ酸の１０重量％以上がＬ－ヒドロキシプロリンであるタンパク質である。上記Ｌ－ヒドロキシプロリン含有タンパク質として、コラーゲン性タンパク質等が好ましい。コラーゲン性タンパク質として、例えば、内臓、皮、魚鱗、骨等のコラーゲンを含む組織から調製されるタンパク質；コラーゲン、ゼラチンが挙げられる。コラーゲン性タンパク質の原料由来は特に限定されない。例えば牛由来、豚由来、魚由来等の動物由来のコラーゲン性タンパク質を好適に使用することができる。コラーゲン性タンパク質は、市販品を使用することができる。Ｌ－ヒドロキシプロリン含有タンパク質の加水分解は、酵素等により公知の方法で行うことができる。 A nitrogen source containing an L-hydroxyproline-containing peptide can be obtained, for example, by hydrolyzing an L-hydroxyproline-containing protein. The L-hydroxyproline-containing protein may be a protein containing L-hydroxyproline as a constituent amino acid, but is preferably a protein in which 10% by weight or more of the constituent amino acid is L-hydroxyproline. As the L-hydroxyproline-containing protein, collagenous protein and the like are preferable. Examples of the collagenous protein include proteins prepared from tissues containing collagen such as viscera, skin, fish scales, and bones; collagen and gelatin. The origin of the collagen protein is not particularly limited. For example, collagen-derived proteins derived from animals such as cow-derived, pig-derived and fish-derived can be preferably used. As the collagenous protein, a commercially available product can be used. Hydrolysis of the L-hydroxyproline-containing protein can be performed by a known method using an enzyme or the like.
Ｌ－ヒドロキシプロリン含有ペプチドを含む窒素源として、例えば、動物由来のペプトンを好適に使用することができる。好ましくは、牛、豚又は魚由来のペプトンであり、より好ましくは牛又は魚由来のペプトンである。本発明の一態様において、ペプトンとして、獣肉ペプトン、心筋ペプトン、ゼラチンペプトンも好ましい。
本発明において使用できるＬ－ヒドロキシプロリン含有ペプチドを含む窒素源の市販品の一例として、例えば、製品名Pepton（#211677）（Bacto社）等が挙げられる。 As a nitrogen source containing an L-hydroxyproline-containing peptide, for example, animal-derived peptone can be preferably used. Peptone derived from cow, pig or fish is preferred, and peptone derived from cow or fish is more preferred. In one embodiment of the present invention, animal peptone, myocardial peptone, and gelatin peptone are also preferable as peptone.
An example of a commercial product of a nitrogen source containing an L-hydroxyproline-containing peptide that can be used in the present invention is the product name Pepton (# 211677) (Bacto).
一態様において、上記Ｌ－ヒドロキシプロリン含有ペプチドは、好ましくはコラーゲンペプチドである。コラーゲンペプチドとは、加水分解コラーゲンを意味し、天然コラーゲンを熱処理して変性させたゼラチン又は天然コラーゲンを加水分解したコラーゲンペプチド、又は、これらを化学的、酵素的に修飾したものの何れであってもよい。加水分解は、酵素、酸、アルカリ等により行うことができ、好ましくは酵素により行われる。好ましくは、天然コラーゲンを熱処理して変性させたゼラチン又は天然コラーゲンを加水分解したコラーゲンペプチドを用いる。コラーゲンペプチドの原料由来は特に限定されない。例えば牛由来、豚由来、魚由来等の動物由来のコラーゲンペプチドを好適に使用することができる。好ましくは、魚由来のコラーゲンペプチドである。コラーゲンペプチドは、市販品を使用することができる。 In one embodiment, the L-hydroxyproline-containing peptide is preferably a collagen peptide. Collagen peptide means hydrolyzed collagen, which can be either gelatin modified by heat treatment of natural collagen, collagen peptide hydrolyzed from natural collagen, or those chemically or enzymatically modified. Good. Hydrolysis can be performed with an enzyme, acid, alkali, or the like, and preferably with an enzyme. Preferably, gelatin modified by heat treatment of natural collagen or collagen peptide hydrolyzed from natural collagen is used. The origin of the collagen peptide is not particularly limited. For example, animal-derived collagen peptides such as cow-derived, pig-derived and fish-derived can be preferably used. Preferably, it is a collagen peptide derived from fish. A commercially available collagen peptide can be used.
本発明において使用できるコラーゲンペプチドの市販品の一例として、例えば、新田ゼラチン（株）製の「コラーゲンペプチド　イクオスＨＤＬ－５０ＳＰ」（製品名）（平均分子量５０００）、「コラーゲンペプチドＴｙｐｅ　Ｓ」（製品名）（平均分子量１２００）、「スーパーコラーゲンペプチド　ＳＣＰ－２０００」（製品名）（平均分子量２０００）、野洲化学工業（株）製の「コラーゲンペプチドＰ－５０００」（製品名）（平均分子量５０００）、「コラーゲンペプチドＦ－５０００」（製品名）（平均分子量５０００）、日祥（株）製の「マリンコラーゲンオリゴＣＦ」（製品名）（平均分子量９００～１１００）、「マリンコラーゲンオリゴＭＦ」（製品名）（平均分子量９００～１５００）等が挙げられる。中でも、「コラーゲンペプチドＴｙｐｅ　Ｓ」（平均分子量１２００）、「コラーゲンペプチド　イクオスＨＤＬ－５０ＳＰ」（平均分子量５０００）等が好ましい。
これらの中で、例えば「コラーゲンペプチド　イクオスＨＤＬ－５０ＳＰ」、「コラーゲンペプチドＴｙｐｅ　Ｓ」、「コラーゲンペプチドＦ－５０００」、「マリンコラーゲンＣＦ」、「マリンコラーゲンオリゴＭＦ」は魚に由来するものであり、「コラーゲンペプチドＰ－５０００」、「スーパーコラーゲンペプチド　ＳＣＰ－２０００」は豚に由来するものである。 Examples of commercially available collagen peptides that can be used in the present invention include, for example, “Collagen Peptide Iquos HDL-50SP” (product name) (average molecular weight 5000), “Collagen Peptide Type S” (product) manufactured by Nitta Gelatin Co., Ltd. Name) (average molecular weight 1200), “super collagen peptide SCP-2000” (product name) (average molecular weight 2000), “collagen peptide P-5000” (product name) (average molecular weight 5000) manufactured by Yasu Chemical Co., Ltd. "Collagen Peptide F-5000" (product name) (average molecular weight 5000), "Marine Collagen Oligo CF" (product name) (average molecular weight 900-1100) manufactured by Nissho Co., Ltd., "Marine Collagen Oligo MF" ( Product name) (average molecular weight 900-1500) and the like. Among these, “collagen peptide Type S” (average molecular weight 1200), “collagen peptide Iquos HDL-50SP” (average molecular weight 5000) and the like are preferable.
Among these, for example, “collagen peptide Iquos HDL-50SP”, “collagen peptide Type S”, “collagen peptide F-5000”, “marine collagen CF”, “marine collagen oligo MF” are derived from fish. “Collagen Peptide P-5000” and “Super Collagen Peptide SCP-2000” are derived from pigs.
本発明の好ましい実施態様においては、Ｌ－ヒドロキシプロリン含有ペプチドを含む窒素源は、好ましくはコラーゲンペプチド（より好ましくは魚由来のコラーゲンペプチド）及び／又はペプトン（より好ましくは牛、豚又は魚由来、さらに好ましくは牛又は魚由来のペプトン）であり、特に好ましくはコラーゲンペプチドである。このようなＬ－ヒドロキシプロリン含有ペプチドを含む窒素源を使用すると、酵母の菌体又は菌体培養物中のＬ－ヒドロキシプロリンの蓄積量が多くなる。ペプトンは、獣肉ペプトン、心筋ペプトン、ゼラチンペプトンであってよい。本発明のＬ－ヒドロキシプロリンの製造方法の好ましい実施態様の一例は、酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)を、炭素源、並びに、コラーゲンペプチド及び／又はペプトンを含む液体培地中で好気培養することにより、上記酵母の菌体又は菌体培養物中にＬ－ヒドロキシプロリンを蓄積させる工程を含む。 In a preferred embodiment of the invention, the nitrogen source comprising the L-hydroxyproline-containing peptide is preferably a collagen peptide (more preferably a collagen peptide derived from fish) and / or a peptone (more preferably derived from cow, pig or fish, More preferred is peptone derived from cattle or fish, and particularly preferred is a collagen peptide. When a nitrogen source containing such an L-hydroxyproline-containing peptide is used, the amount of L-hydroxyproline accumulated in yeast cells or cell cultures increases. The peptone may be animal meat peptone, heart muscle peptone, gelatin peptone. An example of a preferred embodiment of the method for producing L-hydroxyproline of the present invention is that the yeast Yarrowia lipolytica is aerobically cultured in a liquid medium containing a carbon source and a collagen peptide and / or peptone. The step of accumulating L-hydroxyproline in the yeast or bacterial cell culture of the yeast.
一態様において、Ｌ－ヒドロキシプロリン含有ペプチドを含む窒素源は、平均分子量が１００００以下であることが好ましく、例えば、平均分子量が１００～１００００が好ましい。Ｌ－ヒドロキシプロリン含有ペプチドは、平均分子量が１００００以下であることが好ましく、例えば、平均分子量が１００～１００００が好ましい。また、Ｌ－ヒドロキシプロリン含有ペプチドは、分子量が１００００以下であることも好ましい。
コラーゲンペプチドは、平均分子量が１０００～１００００であることが好ましい。平均分子量が上記範囲のコラーゲンペプチドを窒素源に使用すると、菌体又は菌体培養物中のＬ－ヒドロキシプロリンの蓄積量が多くなる。
Ｌ－ヒドロキシプロリン含有ペプチドの平均分子量は、ゲル濾過などにより算出される。コラーゲンペプチドの平均分子量は、通常、写真用ゼラチン試験法（ＰＡＧＩ法）第１０版「２０－２　平均分子量」に記載されている方法により算出される値である。ペプチドの平均分子量は、重量平均分子量を指す。 In one embodiment, the nitrogen source containing the L-hydroxyproline-containing peptide preferably has an average molecular weight of 10,000 or less, and preferably has an average molecular weight of 100 to 10,000, for example. The L-hydroxyproline-containing peptide preferably has an average molecular weight of 10,000 or less, and preferably has an average molecular weight of 100 to 10,000, for example. In addition, the L-hydroxyproline-containing peptide preferably has a molecular weight of 10,000 or less.
The collagen peptide preferably has an average molecular weight of 1000 to 10,000. When a collagen peptide having an average molecular weight in the above range is used as a nitrogen source, the amount of L-hydroxyproline accumulated in the bacterial cells or bacterial cell culture increases.
The average molecular weight of the L-hydroxyproline-containing peptide is calculated by gel filtration or the like. The average molecular weight of the collagen peptide is usually a value calculated by the method described in “20-2 Average Molecular Weight” of the 10th edition of Photographic Gelatin Test Method (PAGI Method). The average molecular weight of a peptide refers to a weight average molecular weight.
コラーゲンペプチドの平均分子量は、より好ましくは１０００～６０００であり、さらに好ましくは１０００～５５００であり、特に好ましくは１０００～５０００である。このようなコラーゲンペプチドを窒素源に使用すると、菌体又は菌体培養物中のＬ－ヒドロキシプロリンの蓄積量がより多くなる。また、一態様において、コラーゲンペプチドの平均分子量として、１０００～３０００がより好ましく、１０００～１５００がさらに好ましい。本発明の別の好ましい態様においては、コラーゲンペプチドの平均分子量は、２０００～５５００がより好ましく、３０００～５０００がさらに好ましい。 The average molecular weight of the collagen peptide is more preferably 1000 to 6000, still more preferably 1000 to 5500, and particularly preferably 1000 to 5000. When such a collagen peptide is used as a nitrogen source, the amount of L-hydroxyproline accumulated in the microbial cells or microbial cell culture is increased. In one embodiment, the average molecular weight of the collagen peptide is more preferably 1000 to 3000, and even more preferably 1000 to 1500. In another preferred embodiment of the present invention, the average molecular weight of the collagen peptide is more preferably 2000 to 5500, further preferably 3000 to 5000.
上記液体培地中のＬ－ヒドロキシプロリン含有ペプチドを含む窒素源の濃度は、０．１～１０重量％が好ましく、０．２５～５重量％がより好ましく、１～５重量％であることがさらに好ましい。上記窒素源の濃度が上記範囲であると、菌体又は菌体培養物中にＬ－ヒドロキシプロリンが蓄積する。このため、例えば、Ｌ－ヒドロキシプロリン含量が１０μｇ／ｍＬ以上である酵母の菌体もしくは菌体培養物又はこれらの抽出物を得ることができる。また、（Ｈｙｐ／（Ｐｒｏ＋Ｈｙｐ））割合が３５～１００である菌体もしくは菌体培養物又はこれらの抽出物を得ることができる。液体培地中の上記窒素源の濃度は、１．５～４．５重量％がさらにより好ましく、２～４重量％が特に好ましい。上記窒素源の濃度は、培養開始時に上記濃度であればよい。
一態様において、上記液体培地中のコラーゲンペプチド又はペプトンの濃度が、上記範囲であることが好ましい。 The concentration of the nitrogen source containing the L-hydroxyproline-containing peptide in the liquid medium is preferably 0.1 to 10% by weight, more preferably 0.25 to 5% by weight, and further preferably 1 to 5% by weight. preferable. When the concentration of the nitrogen source is within the above range, L-hydroxyproline accumulates in the bacterial cells or bacterial cell culture. Therefore, for example, a yeast cell or cell culture or an extract thereof having an L-hydroxyproline content of 10 μg / mL or more can be obtained. In addition, cells or cell cultures or extracts thereof having a (Hyp / (Pro + Hyp)) ratio of 35 to 100 can be obtained. The concentration of the nitrogen source in the liquid medium is still more preferably 1.5 to 4.5% by weight, particularly preferably 2 to 4% by weight. The concentration of the nitrogen source may be the above concentration at the start of culture.
In one embodiment, the concentration of the collagen peptide or peptone in the liquid medium is preferably in the above range.
上記炭素源は特に限定されないが、例えば、グルコース、フルクトース、スクロース、ラフィノース、マンノース、マルトース、ガラクトース、マンニトール、トレハロース、メレビトース、セロビオース、澱粉、糖蜜、ソルビトール、Ｌ－ソルボース、グリセロール、エタノール、グルシトール等の糖質又は糖アルコール；酢酸、クエン酸、又はグルコン酸等の有機酸等が挙げられる。炭素源は、１種単独で使用してもよく、また２種以上を混合して使用してもよい。中でも、炭素源はグルコース、フルクトース、スクロース等の糖が好ましく、グルコースが特に好ましい。 The carbon source is not particularly limited. Sugars or sugar alcohols; organic acids such as acetic acid, citric acid, or gluconic acid. A carbon source may be used individually by 1 type, and may mix and use 2 or more types. Among them, the carbon source is preferably a sugar such as glucose, fructose, or sucrose, and glucose is particularly preferable.
液体培地中の炭素源の濃度は、好ましくは０．１～２０重量％であり、好ましくは０．５～１５重量％であり、より好ましくは１～１０重量％であり、より好ましくは１～５重量％であり、さらに好ましくは２～５重量％である。液体培地中の炭素源の濃度が１重量％以上であると、菌体の増殖速度が速いため好ましい。なお、炭素源の濃度は、培養開始時に上記濃度であればよい。 The concentration of the carbon source in the liquid medium is preferably 0.1 to 20% by weight, preferably 0.5 to 15% by weight, more preferably 1 to 10% by weight, more preferably 1 to 5% by weight, more preferably 2-5% by weight. It is preferable that the concentration of the carbon source in the liquid medium is 1% by weight or more because the growth rate of the bacterial cells is high. In addition, the density | concentration of a carbon source should just be the said density | concentration at the time of a culture | cultivation start.
本発明の製造方法においては、炭素源（Ｃ）とＬ－ヒドロキシプロリン含有ペプチドを含む窒素源（Ｎ）との重量比（Ｃ／Ｎ）が０．２５～２０であることが好ましい。Ｃ／Ｎの重量比が上記範囲であると、菌体又は菌体培養物中のＬ－ヒドロキシプロリンの蓄積量が多くなるため好ましい。一態様において、上記Ｃ／Ｎの重量比は、０．２５～５がより好ましく、０．３～３がより好ましく、０．４～１．５がさらに好ましい。Ｃ／Ｎの重量比が上記範囲であると、菌体又は菌体培養物中のＬ－ヒドロキシプロリンの蓄積量がより多くなる。一態様において、例えばＬ－ヒドロキシプロリン含有ペプチドを含む窒素源（Ｎ）にコラーゲンペプチド（好ましくは平均分子量が１０００～５０００）又はペプトンを使用する場合には、上記Ｃ／Ｎの重量比は０．２５～５がより好ましく、０．３～３がより好ましく、０．４～１．５がさらに好ましく、０．５～１．３が特に好ましい。
別の好ましい態様において、上記Ｃ／Ｎの重量比は、０．５～２０も好ましい。
上記Ｃ／Ｎの重量比は、培養開始時に上記範囲であればよい。 In the production method of the present invention, the weight ratio (C / N) of the carbon source (C) and the nitrogen source (N) containing the L-hydroxyproline-containing peptide is preferably 0.25 to 20. A weight ratio of C / N within the above range is preferable because the amount of L-hydroxyproline accumulated in the bacterial cells or bacterial cell culture increases. In one embodiment, the C / N weight ratio is more preferably 0.25 to 5, more preferably 0.3 to 3, and further preferably 0.4 to 1.5. When the weight ratio of C / N is within the above range, the amount of L-hydroxyproline accumulated in the microbial cells or microbial cell culture is increased. In one embodiment, for example, when a collagen peptide (preferably having an average molecular weight of 1000 to 5000) or peptone is used as the nitrogen source (N) containing the L-hydroxyproline-containing peptide, the C / N weight ratio is 0. 25 to 5 is more preferable, 0.3 to 3 is more preferable, 0.4 to 1.5 is further preferable, and 0.5 to 1.3 is particularly preferable.
In another preferred embodiment, the C / N weight ratio is preferably 0.5 to 20.
The weight ratio of C / N may be in the above range at the start of culture.
液体培地は、上述した炭素源及びＬ－ヒドロキシプロリン含有ペプチドを含む窒素源以外の成分を含んでもよく、例えば、酵母抽出物（Ｙｅａｓｔ　ｅｘｔｒａｃｔ）を含むことが好ましい。酵母抽出物は、通常Ｌ－ヒドロキシプロリン含有ペプチドを含まず、Ｌ－ヒドロキシプロリン含有ペプチドを含む窒素源には含まれない。酵母抽出物は、酵母の培養に使用できるものであればよく、特に限定されず、市販品を使用することができる。例えば、製品名Bacto yeast Extract(#212750)（Bacto社）等を好適に使用することができる。
酵母抽出物を使用する場合、酵母抽出物の濃度は、液体培地に対して０．１～３重量％が好ましく、０．５～３重量％がより好ましい。酵母抽出物の濃度は、培養開始時に上記濃度であればよい。 The liquid medium may contain components other than the above-described carbon source and a nitrogen source containing an L-hydroxyproline-containing peptide, and preferably contains, for example, a yeast extract. Yeast extracts usually do not contain L-hydroxyproline-containing peptides and are not included in nitrogen sources that contain L-hydroxyproline-containing peptides. The yeast extract is not particularly limited as long as it can be used for yeast culture, and a commercially available product can be used. For example, the product name Bacto yeast Extract (# 212750) (Bacto) can be preferably used.
When using a yeast extract, the concentration of the yeast extract is preferably 0.1 to 3% by weight, more preferably 0.5 to 3% by weight, based on the liquid medium. The concentration of the yeast extract may be the above concentration at the start of culture.
一態様において、液体培地のｐＨは、３～９が好ましく、４～９がより好ましく、４を超えて９以下がさらに好ましく、４．５～８．８がさらにより好ましく、５～８．７が特に好ましい。液体培地のｐＨは、適宜調整すればよい。ｐＨ調整には、公知の酸又はアルカリ剤を使用でき、例えば塩酸、硫酸、リン酸、硝酸、グルタミン酸、酢酸、酪酸、乳酸、蟻酸、コハク酸、マレイン酸、リンゴ酸、シュウ酸、クエン酸、水酸化ナトリウム、水酸化カリウム、水酸化カルシウム、アンモニア水、グルタミン酸ナトリウム等が挙げられる。 In one embodiment, the pH of the liquid medium is preferably 3 to 9, more preferably 4 to 9, more preferably more than 4 and 9 or less, still more preferably 4.5 to 8.8, even more preferably 5 to 8.7. Is particularly preferred. The pH of the liquid medium may be adjusted as appropriate. A known acid or alkali agent can be used for pH adjustment, such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, glutamic acid, acetic acid, butyric acid, lactic acid, formic acid, succinic acid, maleic acid, malic acid, oxalic acid, citric acid, Examples thereof include sodium hydroxide, potassium hydroxide, calcium hydroxide, aqueous ammonia, and sodium glutamate.
培養温度は、１５～４５℃が好ましく、２０～４０℃がより好ましく、２５～３５℃がさらに好ましい。培養温度がこの温度範囲であると、酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)の増殖が速く、菌体又は菌体培養物中のＬ－ヒドロキシプロリンの蓄積量が多くなる。 The culture temperature is preferably 15 to 45 ° C, more preferably 20 to 40 ° C, and further preferably 25 to 35 ° C. When the culture temperature is within this temperature range, the yeast Yarrowia lipolytica grows rapidly, and the amount of L-hydroxyproline accumulated in the cells or cell cultures increases.
好気培養を行う方法は特に限定されず、酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)を接種した液体培地を、例えば、振盪培養又は攪拌培養すればよい。振盪又は攪拌の速度は特に限定されないが、好ましくは３０～６００ｒｐｍであり、一態様において、より好ましくは５０～６００ｒｐｍであり、さらに好ましくは、１００～６００ｒｐｍである。また別の好ましい態様において、振盪又は攪拌の速度は、より好ましくは３０～５００ｒｐｍであり、さらに好ましくは５０～３００ｒｐｍである。このような速度で振盪培養又は攪拌培養すると、Ｌ－ヒドロキシプロリンの蓄積量が多くなるため好ましい。より好ましくは、上記速度で振盪培養する。また、所望により滅菌された空気又は酸素でバブリングを行ってもよい。また、培養形式は、回分培養、流加培養、連続培養のいずれでもよいが、回分培養が好ましい。本発明の製造方法においては、静置培養を行ってもよい。 The method for aerobic culture is not particularly limited, and a liquid medium inoculated with yeast Yarrowia lipolytica may be subjected to, for example, shaking culture or stirring culture. The speed of shaking or stirring is not particularly limited, but is preferably 30 to 600 rpm, and in one aspect, more preferably 50 to 600 rpm, and still more preferably 100 to 600 rpm. In another preferred embodiment, the speed of shaking or stirring is more preferably 30 to 500 rpm, further preferably 50 to 300 rpm. Shaking culture or stirring culture at such a rate is preferable because the amount of accumulated L-hydroxyproline increases. More preferably, the shaking culture is performed at the above speed. In addition, bubbling may be performed with sterilized air or oxygen if desired. The culture format may be batch culture, fed-batch culture, or continuous culture, but batch culture is preferred. In the production method of the present invention, static culture may be performed.
培養時間は特に限定されず、適宜設定すればよいが、例えば、好気培養を１０～１００時間行うことが好ましい。培養時間が上記範囲であると、上記酵母の菌体又は菌体培養物中にＬ－ヒドロキシプロリンが蓄積する。また、通常、（Ｈｙｐ／（Ｐｒｏ＋Ｈｙｐ））割合が３５～１００である、酵母の菌体もしくは菌体培養物又はこれらの抽出物、Ｌ－ヒドロキシプロリン含量が１０μｇ／ｍＬ以上である酵母の菌体もしくは菌体培養物又はこれらの抽出物を得ることができる。また、エタノール含量が少ない（例えば、１ｖ／ｖ％以下）酵母の菌体もしくは菌体培養物又はこれらの抽出物が得られる。培養時間が１０時間未満であると、Ｌ－ヒドロキシプロリンの蓄積量が少ない場合や、得られる酵母の菌体もしくは菌体培養物又はこれらの抽出物の（Ｈｙｐ／（Ｐｒｏ＋Ｈｙｐ））割合が３５未満となる場合がある。培養時間が１００時間を超えると、得られる酵母の菌体もしくは菌体培養物又はこれらの抽出物のエタノール濃度が１ｖ／ｖ％を超える場合がある。また、コンタミネーションが起こりやすくなったり、酵母の死滅後に自己消化による着色等が生じたりする場合がある。培養時間は、より好ましくは１０～８０時間であり、さらに好ましくは１２～７２時間であり、さらにより好ましくは２０～６０時間であり、さらにより好ましくは２４～５５時間であり、特に好ましくは２４～５０時間である。また、本発明の製造方法においては、菌体又は菌体培養物中のＬ－ヒドロキシプロリン含量が１０μｇ／ｍＬ以上となるまで、好気培養を行うことが好ましい。
本発明の製造方法において、好気培養は、通常、本培養として行われるが、前培養であってもよく、前培養及び本培養において好気培養を行ってもよい。 The culture time is not particularly limited and may be set as appropriate. For example, it is preferable to perform aerobic culture for 10 to 100 hours. When the culture time is within the above range, L-hydroxyproline accumulates in the yeast cells or cell culture. In addition, usually, yeast cells or cell cultures or extracts thereof having a (Hyp / (Pro + Hyp)) ratio of 35 to 100, yeast cells having an L-hydroxyproline content of 10 μg / mL or more. Alternatively, a bacterial cell culture or an extract thereof can be obtained. In addition, yeast cells or cell cultures or extracts thereof having a low ethanol content (for example, 1 v / v% or less) can be obtained. When the culture time is less than 10 hours, the amount of accumulated L-hydroxyproline is small, or the yeast cell or cell culture obtained or the extract (Hyp / (Pro + Hyp)) ratio is less than 35. It may become. When the culture time exceeds 100 hours, the ethanol concentration of the obtained yeast or bacterial cell culture or the extract thereof may exceed 1 v / v%. In addition, contamination may easily occur, and coloring due to self-digestion may occur after the death of the yeast. The culture time is more preferably 10 to 80 hours, still more preferably 12 to 72 hours, still more preferably 20 to 60 hours, still more preferably 24 to 55 hours, and particularly preferably 24 hours. ~ 50 hours. In the production method of the present invention, it is preferable to perform aerobic culture until the L-hydroxyproline content in the microbial cells or the microbial cell culture becomes 10 μg / mL or more.
In the production method of the present invention, aerobic culture is usually performed as main culture, but it may be preculture or aerobic culture may be performed in preculture and main culture.
上記の好気培養を行うことにより、酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)の菌体又は菌体培養物中にＬ－ヒドロキシプロリンが蓄積する。菌体培養物は、酵母の菌体及び培養上清を含む菌体培養液であってもよく、酵母の菌体であってもよく、又は、菌体培養液の培養上清であってもよい。Ｈｙｐ蓄積工程を行うことにより、Ｌ－ヒドロキシプロリンを含有し、（Ｈｙｐ／（Ｐｒｏ＋Ｈｙｐ））割合が、３５～１００である酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)の菌体又は菌体培養物を得ることができる。また、Ｌ－ヒドロキシプロリンの含量が１０μｇ／ｍＬ以上である酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)の菌体又は菌体培養物を得ることができる。酵母の菌体又は菌体培養物に、例えば菌体破砕処理を行うことにより、酵母の菌体又は菌体培養物の抽出物を得ることができる。 By carrying out the above aerobic culture, L-hydroxyproline accumulates in the cells of the yeast Yarrowia lipolytica or the cell culture. The cell culture may be a cell culture solution containing yeast cells and culture supernatant, may be yeast cells, or may be a culture supernatant of a cell culture solution. Good. By performing the Hyp accumulation step, a cell or a cell culture of yeast Yarrowia lipolytica containing L-hydroxyproline and having a (Hyp / (Pro + Hyp)) ratio of 35 to 100 is obtained. Can do. In addition, a bacterial cell or a bacterial cell culture of yeast Yarrowia lipolytica having an L-hydroxyproline content of 10 μg / mL or more can be obtained. An extract of yeast cells or cell cultures can be obtained by subjecting yeast cells or cell cultures to a cell disruption treatment, for example.
本発明においてＬ－ヒドロキシプロリンを含有する酵母の菌体もしくは菌体培養物又はその抽出物を調製する場合は、例えば、Ｈｙｐ蓄積工程で得られる酵母の菌体及び培養上清を含む菌体培養液をそのままＬ－ヒドロキシプロリンを含有する酵母の菌体培養物とすることができる。また、菌体培養液から酵母の菌体を集菌し、得られた菌体を酵母の菌体又は菌体培養物としてもよく、菌体培養液から菌体を除去した培養上清を菌体培養物とすることもできる。さらに、上記菌体又は菌体培養液に、必要に応じて菌体を破砕する処理を行って、菌体内容物を培養液中等に溶出させて菌体又は菌体培養物の抽出物を調製することもできる。菌体又は菌体培養物の抽出物の調製においては、菌体を破砕した後、菌体残渣を除去する工程を行ってもよい。また、菌体もしくは菌体培養物又はこれらの抽出物には、必要に応じて殺菌、加熱等の処理を行ってもよい。本発明の製造方法は、このような集菌工程、菌体破砕工程、菌体残渣除去工程、殺菌工程等の１又は２以上の工程を含んでもよい。 In the present invention, when preparing a yeast cell or cell culture or an extract thereof containing L-hydroxyproline in the present invention, for example, a cell culture comprising a yeast cell obtained in the Hyp accumulation step and a culture supernatant. The solution can be used as it is as a yeast cell culture containing L-hydroxyproline. Alternatively, yeast cells may be collected from the cell culture medium, and the obtained cell bodies may be used as yeast cells or cell cultures. It can also be a body culture. Further, the cells or the cell culture medium is subjected to a treatment for crushing the cells as necessary, and the cell contents are eluted in the culture solution to prepare a cell or cell culture extract. You can also In the preparation of an extract of bacterial cells or bacterial cell cultures, a step of removing bacterial cell residues may be performed after disrupting the bacterial cells. Moreover, you may perform processes, such as disinfection and a heating, to a microbial cell or a microbial cell culture, or these extracts as needed. The production method of the present invention may include one or more steps such as such a collection process, a microbial cell disruption process, a microbial cell residue removal process, and a sterilization process.
菌体培養液から菌体を集菌する方法は特に限定されず、通常行われている方法を採用することができ、例えば、遠心分離等が挙げられる。
上記菌体を破砕する方法は特に限定されず、通常行われている方法を採用することができ、例えば、自己消化法、酵素分解法、アルカリ抽出法等が挙げられる。中でも、自己消化法が好ましい。自己消化法においては、例えば菌体又は菌体培養物を４０～６０℃で６０～１８０分間、又は、９５～１００℃で５～１５分間加熱すればよい。
菌体残渣を除去する方法は特に限定されない。例えば、濾過、遠心分離等の公知の方法により菌体残渣を除去すればよい。
殺菌を行う場合には、酵母の菌体もしくは菌体培養物又はこれらの抽出物を７５～９０℃（より好ましくは８０℃）、４５～９０分（より好ましくは６０分）加熱することが好ましい。菌体残渣除去及び殺菌を行う場合、いずれを先に行ってもよい。 A method for collecting the cells from the cell culture solution is not particularly limited, and a commonly used method can be employed, and examples thereof include centrifugation.
The method for disrupting the cells is not particularly limited, and a commonly used method can be employed, and examples thereof include an autolysis method, an enzymatic decomposition method, and an alkali extraction method. Of these, the autolysis method is preferred. In the self-digestion method, for example, the cells or the cell cultures may be heated at 40 to 60 ° C. for 60 to 180 minutes, or at 95 to 100 ° C. for 5 to 15 minutes.
The method for removing the cell residue is not particularly limited. For example, what is necessary is just to remove a microbial cell residue by well-known methods, such as filtration and centrifugation.
In the case of sterilization, it is preferable to heat yeast cells or cell cultures or extracts thereof at 75 to 90 ° C. (more preferably 80 ° C.) and 45 to 90 minutes (more preferably 60 minutes). . When removing the microbial cell residue and sterilizing, either may be performed first.
本発明において得られる酵母の菌体もしくは菌体培養物又はこれらの抽出物、及び、その好ましい態様は、上述した本発明の酵母の菌体もしくは菌体培養物又はこれらの抽出物、及び、その好ましい態様と同じである。本発明の製造方法によれば、例えば、（Ｈｙｐ／（Ｐｒｏ＋Ｈｙｐ））割合が、３５～１００であり、かつ、Ｌ－ヒドロキシプロリン含量が１０μｇ／ｍＬである酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)の菌体もしくは菌体培養物又はこれらの抽出物を製造することができる。酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)の菌体もしくは菌体培養物又はこれらの抽出物中のＬ－ヒドロキシプロリンは、通常、上記酵母の菌体又は菌体培養物に由来するものである。
上記方法により得られる酵母の菌体もしくは菌体培養物又はこれらの抽出物に、さらに天然物由来又は合成されたＬ－ヒドロキシプロリンを添加してもよいが、好ましい態様においては、酵母の菌体もしくは菌体培養物又はこれらの抽出物に含まれるＬ－ヒドロキシプロリンは、上記のＨｙｐ蓄積工程により得られる酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)の菌体又は菌体培養物に由来するＬ－ヒドロキシプロリンからなる。 The yeast cells or cell cultures or extracts thereof obtained in the present invention, and preferred embodiments thereof are the yeast cells or cell cultures or extracts thereof of the present invention described above, and the extracts thereof. This is the same as the preferred embodiment. According to the production method of the present invention, for example, the yeast Yarrowia lipolytica having a (Hyp / (Pro + Hyp)) ratio of 35 to 100 and an L-hydroxyproline content of 10 μg / mL. Body or cell cultures or extracts thereof can be produced. L-hydroxyproline in the yeast or yeast culture of yeast Yarrowia lipolytica or an extract thereof is usually derived from the yeast or bacterial culture of the yeast.
L-hydroxyproline derived or synthesized from natural products may be further added to yeast cells or cell cultures or extracts thereof obtained by the above method. In a preferred embodiment, yeast cells are used. Alternatively, the L-hydroxyproline contained in the cell culture or the extract thereof is L-hydroxyproline derived from the yeast Yarrowia lipolytica obtained by the above Hyp accumulation step or the cell culture. Consists of.
本発明で得られるＬ－ヒドロキシプロリンを含有する酵母の菌体もしくは菌体培養物又はこれらの抽出物は、後述する飲食品、化粧料等の原料等として使用することができる。また、本発明のＬ－ヒドロキシプロリンの製造方法においては、得られる酵母の菌体もしくは菌体培養物又はこれらの抽出物から、Ｌ－ヒドロキシプロリンを精製する工程を行ってもよい。Ｌ－ヒドロキシプロリンの精製は、例えば、カラムクロマトグラフィー等の公知の方法により行えばよい。 The yeast cells or cell cultures or extracts thereof containing L-hydroxyproline obtained in the present invention can be used as raw materials for foods and beverages, cosmetics and the like described later. Further, in the method for producing L-hydroxyproline of the present invention, a step of purifying L-hydroxyproline from the obtained yeast cell or cell culture or an extract thereof may be performed. Purification of L-hydroxyproline may be performed by a known method such as column chromatography.
本発明は、酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)の、Ｌ－ヒドロキシプロリンを製造するための使用も包含する。
上記ヤロウィア・リポリティカ(Yarrowia lipolytica)は、Ｌ－ヒドロキシプロリンを含有する酵母の菌体もしくは菌体培養物又はこれらの抽出物を製造するためにも好適に使用される。Ｌ－ヒドロキシプロリンを含有する酵母の菌体もしくは菌体培養物又はこれらの抽出物は、（Ｈｙｐ／（Ｐｒｏ＋Ｈｙｐ））割合が、３５～１００であることが好ましい。Ｌ－ヒドロキシプロリンを含有する酵母の菌体もしくは菌体培養物又はこれらの抽出物は、Ｌ－ヒドロキシプロリンの含量が１０μｇ／ｍＬ以上であることも好ましい。Ｌ－ヒドロキシプロリンを含有する酵母の菌体もしくは菌体培養物又はこれらの抽出物の好ましい態様は、上述した本発明の酵母の菌体もしくは菌体培養物又はこれらの抽出物の好ましい態様と同じである。 The invention also encompasses the use of the yeast Yarrowia lipolytica to produce L-hydroxyproline.
The above Yarrowia lipolytica is also suitably used for producing yeast cells or cell cultures containing L-hydroxyproline, or extracts thereof. The yeast cells or cell cultures or extracts thereof containing L-hydroxyproline preferably have a (Hyp / (Pro + Hyp)) ratio of 35 to 100. It is also preferable that the yeast cells or cell cultures or extracts thereof containing L-hydroxyproline have an L-hydroxyproline content of 10 μg / mL or more. Preferred embodiments of the yeast cells or cell cultures or extracts thereof containing L-hydroxyproline are the same as the preferred embodiments of the yeast cells or cell cultures or extracts thereof of the present invention described above. It is.
本発明の使用は、酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)を、炭素源及び窒素源を含む液体培地中で好気培養することにより、上記酵母の菌体又は菌体培養物中にＬ－ヒドロキシプロリンを蓄積させることを含むことが好ましい。
上記窒素源は、Ｌ－ヒドロキシプロリン含有ペプチドを含む窒素源であることが好ましい。 The use of the present invention can be accomplished by aerobically cultivating yeast Yarrowia lipolytica in a liquid medium containing a carbon source and a nitrogen source, thereby producing L-hydroxyproline in the yeast cell or cell culture. Preferably comprising accumulating.
The nitrogen source is preferably a nitrogen source containing an L-hydroxyproline-containing peptide.
本発明の使用においては、上記Ｌ－ヒドロキシプロリン含有ペプチドがコラーゲンペプチドであることが好ましい。また、上記コラーゲンペプチドの平均分子量は１０００～１００００であることが好ましい。 In the use of the present invention, the L-hydroxyproline-containing peptide is preferably a collagen peptide. The average molecular weight of the collagen peptide is preferably 1000 to 10,000.
本発明の使用においては、上記液体培地中のＬ－ヒドロキシプロリン含有ペプチドを含む窒素源の濃度が０．２５～５重量％であることが好ましい。また、炭素源（Ｃ）とＬ－ヒドロキシプロリン含有ペプチドを含む窒素源（Ｎ）との重量比（Ｃ／Ｎ）が０．２５～２０であることが好ましい。一態様において、上記Ｃ／Ｎ比は０．５～２０であることも好ましい。本発明の使用においては、上記好気培養を１０～１００時間行うことが好ましい。 In the use of the present invention, the concentration of the nitrogen source containing the L-hydroxyproline-containing peptide in the liquid medium is preferably 0.25 to 5% by weight. The weight ratio (C / N) of the carbon source (C) and the nitrogen source (N) containing the L-hydroxyproline-containing peptide is preferably 0.25 to 20. In one embodiment, the C / N ratio is preferably 0.5 to 20. In the use of the present invention, the aerobic culture is preferably performed for 10 to 100 hours.
本発明の使用における液体培地、炭素源及びＬ－ヒドロキシプロリン含有ペプチドを含む窒素源並びにこれらの好ましい態様は、上述したＬ－ヒドロキシプロリンの製造方法におけるものと同じである。また、好気培養の条件及びその好ましい態様も、上述したＬ－ヒドロキシプロリンの製造方法におけるものと同じである。本発明の使用は、上述した集菌工程、菌体破砕工程、菌体除去工程、殺菌工程等の１又は２以上の工程を含んでもよい。 In the use of the present invention, the liquid medium, the carbon source and the nitrogen source containing the L-hydroxyproline-containing peptide and preferred embodiments thereof are the same as those in the above-described method for producing L-hydroxyproline. The aerobic culture conditions and preferred embodiments thereof are also the same as those in the above-described method for producing L-hydroxyproline. The use of the present invention may include one or more processes such as the above-described collection process, microbial cell disruption process, microbial cell removal process, and sterilization process.
上述した本発明の酵母の菌体もしくは菌体培養物又はこれらの抽出物は、化粧料、飲食品、医薬品等の各種組成物に配合することができる。本発明の酵母の菌体もしくは菌体培養物又はこれらの抽出物を含む組成物も、本発明に包含される。本発明の組成物は、上述した本発明の第一の態様及び第二の態様の酵母の菌体もしくは菌体培養物又はこれらの抽出物のいずれかを含めばよく、両方を含んでよい。本発明の酵母の菌体もしくは菌体培養物又はこれらの抽出物を含む組成物は、該酵母の菌体もしくは菌体培養物又はこれらの抽出物に由来するＬ－ヒドロキシプロリンを含む。
本発明の組成物として、例えば、化粧料（化粧料組成物）、飲食品（飲食品組成物）、医薬品（医薬品組成物）、医薬部外品（医薬部外品組成物）等が挙げられる。組成物は、これらの原料であってもよい。一態様において、組成物は、化粧料又は飲食品、これらの原料であることが好ましい。 The yeast cell or cell culture of the present invention described above or an extract thereof can be blended in various compositions such as cosmetics, foods and drinks, and pharmaceuticals. The yeast cell or cell culture of the present invention or a composition containing an extract thereof is also encompassed in the present invention. The composition of the present invention may include any one of the yeast cell or cell culture of the yeast of the first aspect and the second aspect of the present invention described above, or an extract thereof, and may include both. The yeast cell or cell culture of the present invention or a composition containing these extracts contains L-hydroxyproline derived from the yeast cell or cell culture or these extracts.
Examples of the composition of the present invention include cosmetics (cosmetic compositions), foods and drinks (food and beverage compositions), pharmaceuticals (pharmaceutical compositions), quasi drugs (quasi drugs) and the like. . The composition may be these raw materials. In one embodiment, the composition is preferably a cosmetic or a food or drink, or a raw material thereof.
本発明の組成物中の上記酵母の菌体もしくは菌体培養物又はこれらの抽出物の含量は特に限定されず、該組成物の種類、用途に応じて適宜設定することができる。例えば、組成物に対して、上記酵母の菌体もしくは菌体培養物又はこれらの抽出物の固形分換算の含量を０．００００１～５０重量％とすることが好ましく、０．００００５～２０重量％がより好ましく、０．０００１～１０重量％がさらに好ましい。 The content of the yeast cell culture or cell culture or the extract thereof in the composition of the present invention is not particularly limited, and can be appropriately set depending on the type and use of the composition. For example, the solid content of the yeast cell or cell culture or extract thereof is preferably 0.00001 to 50% by weight, preferably 0.00005 to 20% by weight, based on the composition. Is more preferable, and 0.0001 to 10% by weight is more preferable.
本発明の組成物が化粧料又は医薬品である場合、その剤型は特に限定されず、溶液状、ペースト状、ゲル状、固体状、粉末状等任意の剤型をとることができる。
化粧料は特に限定されず、例えば、クレンジング剤、洗顔料、化粧水、乳液、クリーム、美容液、育毛剤、オイル、ゲル、シャンプー、ヘアリンス、ヘアコンディショナー、エナメル、ファンデーション、リップスティック、おしろい、パック、香水、パウダー、オーデコロン、ボディソープ、石鹸、入浴剤、日焼け止めクリーム等とすることができる。 When the composition of the present invention is a cosmetic or a pharmaceutical, the dosage form is not particularly limited, and any dosage form such as a solution, paste, gel, solid, or powder can be used.
Cosmetics are not particularly limited. , Perfume, powder, eau de cologne, body soap, soap, bath salt, sunscreen cream and the like.
上述した本発明の酵母の菌体もしくは菌体培養物又はこれらの抽出物を含む化粧料又は化粧料原料は、本発明における好ましい態様の１つである。化粧料及び化粧料原料は、上記酵母の菌体もしくは菌体培養物又はこれらの抽出物以外の成分を含んでいてもよい。化粧料及び化粧料原料には、化粧料に通常配合される種々の成分を配合することができる。例えば、油分、香料、界面活性剤、保湿剤、酸化防止剤、紫外線吸収剤、防腐剤、顔料、色素等を適宜配合することができる。これらの配合比率は適宜選択すればよい。本発明の化粧料原料は、本発明の化粧料を製造するために好適に使用される。
化粧料の用法及び用量は、化粧料の種類等に応じて、適宜決定することができる。
本発明の化粧料又は化粧料原料は、Ｌ－ヒドロキシプロリンを含有することから、例えば、コラーゲン産生促進、表皮細胞の増殖促進、皮膚の保湿、皮膚の老化防止、皮膚のたるみの予防又は改善、皮膚のハリの改善、しわの予防又は改善及びアトピー性皮膚炎の改善から選ばれる用途に好適に用いられ、皮膚のハリの改善、及び、しわの予防又は改善から選ばれる用途により好適に用いられる。 The cosmetics or cosmetic raw materials containing the above-described yeast cells or cell cultures of the present invention or extracts thereof are one of the preferred embodiments of the present invention. The cosmetics and cosmetic raw materials may contain components other than the yeast cells or cell cultures or extracts thereof. Various ingredients that are usually blended in cosmetics can be blended in cosmetics and cosmetic raw materials. For example, oils, fragrances, surfactants, humectants, antioxidants, ultraviolet absorbers, preservatives, pigments, dyes and the like can be appropriately blended. What is necessary is just to select these compounding ratios suitably. The cosmetic raw material of the present invention is suitably used for producing the cosmetic of the present invention.
The usage and dosage of the cosmetic can be appropriately determined according to the type of cosmetic.
Since the cosmetic or cosmetic raw material of the present invention contains L-hydroxyproline, for example, promotion of collagen production, promotion of epidermal cell proliferation, skin moisturization, prevention of skin aging, prevention or improvement of skin sagging, It is preferably used for applications selected from the improvement of skin firmness, prevention or improvement of wrinkles and the improvement of atopic dermatitis, and is preferably used for applications selected from improvement of skin firmness and prevention or improvement of wrinkles. .
化粧料中の上記酵母の菌体もしくは菌体培養物又はこれらの抽出物の含量は、化粧料に対して、固形分換算で０．００００１～１０重量％が好ましく、０．０００１～１０重量％とすることが好ましく、０．０００１～５重量％がより好ましく、０．００１～５重量％がより好ましく、０．０１～３重量％がさらに好ましく、０．０５～２重量％が特に好ましい。また、別の好ましい態様において、化粧料中の上記酵母の菌体もしくは菌体培養物又はこれらの抽出物の含量は、化粧料に対して、固形分換算で０．００００５～１重量％がより好ましく、０．０００１～０．５重量％がさらに好ましい。
化粧料原料中の上記酵母の菌体もしくは菌体培養物又はこれらの抽出物の含量は、化粧料原料に対して、例えば、固形分換算で０．００１～２０重量％が好ましく、０．０１～１０重量％がより好ましく、０．０５～５重量％がさらに好ましく、０．１～２重量％が特に好ましい。化粧料原料中のＬ－ヒドロキシプロリン含量は、例えば、５～３００ｐｐｍが好ましく、１０～２００ｐｐｍがより好ましく、５０～１００ｐｐｍがさらに好ましい。一態様において、化粧料中のＬ－ヒドロキシプロリン含量は、例えば、０．０１～２０ｐｐｍとすることができ、０．０３～１５ｐｐｍが好ましく、０．０５～１０ｐｐｍがより好ましい。Ｌ－ヒドロキシプロリン含量が上記の範囲となるように、上記酵母の菌体もしくは菌体培養物又はこれらの抽出物を配合することが好ましい。 The content of the yeast cells or cell culture or the extract thereof in the cosmetic is preferably 0.00001 to 10% by weight, preferably 0.0001 to 10% by weight in terms of solid content with respect to the cosmetic. It is preferably 0.0001 to 5% by weight, more preferably 0.001 to 5% by weight, still more preferably 0.01 to 3% by weight, and particularly preferably 0.05 to 2% by weight. In another preferred embodiment, the content of the yeast cell culture or cell culture or extract thereof in the cosmetic is 0.00005 to 1% by weight in terms of solid content relative to the cosmetic. Preferably, 0.0001 to 0.5% by weight is more preferable.
The content of the yeast cell or cell culture or extract thereof in the cosmetic raw material is preferably 0.001 to 20% by weight, for example, in terms of solid content, based on the cosmetic raw material, Is more preferably from 10 to 10% by weight, further preferably from 0.05 to 5% by weight, particularly preferably from 0.1 to 2% by weight. The L-hydroxyproline content in the cosmetic raw material is, for example, preferably from 5 to 300 ppm, more preferably from 10 to 200 ppm, still more preferably from 50 to 100 ppm. In one embodiment, the L-hydroxyproline content in the cosmetic can be, for example, 0.01 to 20 ppm, preferably 0.03 to 15 ppm, and more preferably 0.05 to 10 ppm. It is preferable to blend the yeast cells or cell cultures or extracts thereof so that the L-hydroxyproline content falls within the above range.
本発明の組成物が飲食品（飲食品組成物）である場合、飲食品は特に限定されない。飲食品の形態は、液状、半液体状又は固体状、ペースト状のいずれであってもよく、例えば、一般的な飲食品、健康食品、機能性食品等のいずれでもよい。
一般的な飲食品は特に限定されず、酒類も含まれる。健康食品とは、健康的な又は健康によいとされる食品をいい、栄養補助食品、自然食品等を含む。栄養補助食品とは、特定の栄養成分が強化されている食品をいう。機能性食品とは、体の調節機能を果たす栄養成分を補給するための食品をいい、特定保健用食品、栄養機能食品を含む。 When the composition of the present invention is a food or drink (food or drink composition), the food or drink is not particularly limited. The form of the food or drink may be any of liquid, semi-liquid or solid, and paste, and may be any of general food and drink, health food, functional food, and the like.
General food and drink is not particularly limited, and includes alcoholic beverages. Healthy food means food that is considered healthy or healthy, and includes nutritional supplements, natural foods, and the like. Nutritional supplements refer to foods that are enriched with specific nutritional components. Functional foods refer to foods for supplementing nutritional components that fulfill the body's regulatory functions, and include foods for specified health use and functional nutritional foods.
栄養補助食品として、美容ドリンク、サプリメント等が挙げられる。本発明の飲食品は、カプセル等の医薬製剤の形態、ドリンク剤等であってもよい。
飲食品には、飲食品に配合することが認められている種々の成分を配合することができる。このような成分としては例えば、結合剤、増粘剤、着色剤、安定剤、乳化剤、分散剤、崩壊剤、懸濁化剤、界面活性剤、防腐剤、甘味料、酸味料等が挙げられる。
上述した本発明の酵母の菌体もしくは菌体培養物又はこれらの抽出物を含む飲食品は、本発明における好ましい態様の１つである。 Examples of dietary supplements include beauty drinks and supplements. The food and drink of the present invention may be in the form of a pharmaceutical preparation such as a capsule, a drink or the like.
Various components that are permitted to be blended in food and drink can be blended in the food and drink. Examples of such components include binders, thickeners, colorants, stabilizers, emulsifiers, dispersants, disintegrants, suspending agents, surfactants, preservatives, sweeteners, and sour agents. .
The above-described yeast cell or cell culture of the present invention or a food or drink containing these extracts is one of the preferred embodiments of the present invention.
飲食品中の上記酵母の菌体もしくは菌体培養物又はこれらの抽出物の含量は、例えば、飲食品に対して、固形分換算で０．０００１～１０重量％とすることが好ましく、０．００１～５重量％がより好ましく、０．０１～１重量％がさらに好ましい。また、飲食品中のＬ－ヒドロキシプロリン含量は、０．０００１～０．０１重量％が好ましく、Ｌ－ヒドロキシプロリン含量が上記の範囲となるように、上記酵母の菌体もしくは菌体培養物又はこれらの抽出物を配合することが好ましい。上記化粧料、化粧料原料、飲食品等の組成物中のＬ－ヒドロキシプロリン含量は、遊離のＬ－ヒドロキシプロリン含量である。Ｌ－ヒドロキシプロリンは、好ましくは、上記酵母の菌体もしくは菌体培養物又はこれらの抽出物に由来する。 The content of the yeast cells or cell culture or the extract thereof in the food or drink is preferably 0.0001 to 10% by weight in terms of solid content with respect to the food or drink. 001 to 5% by weight is more preferable, and 0.01 to 1% by weight is more preferable. Further, the L-hydroxyproline content in the food and drink is preferably 0.0001 to 0.01% by weight, and the yeast cell or cell culture or the culture of the yeast so that the L-hydroxyproline content falls within the above range. It is preferable to blend these extracts. The L-hydroxyproline content in the cosmetics, cosmetic raw materials, foods and beverages, etc. is a free L-hydroxyproline content. L-hydroxyproline is preferably derived from the above-mentioned yeast cells or cell cultures or extracts thereof.
本発明の酵母の菌体もしくは菌体培養物又はこれらの抽出物を含有する化粧料、飲食品、これらの原料等の組成物は、これらに通常使用されている原料、添加剤等をその種類に応じて選択して配合し、これに本発明の酵母の菌体もしくは菌体培養物又はこれらの抽出物を配合し、公知の手法によって製造することができる。 Compositions such as cosmetics, foods and drinks, and these raw materials containing yeast cells or bacterial cell cultures or extracts thereof according to the present invention are the types of raw materials, additives, etc. that are usually used in these. The yeast cells or cell cultures of the yeast of the present invention or extracts thereof can be blended and produced by a known technique.
本発明は、酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)を、炭素源及び窒素源を含む液体培地中で好気培養することにより、上記酵母の菌体又は菌体培養物中にＬ－ヒドロキシプロリンを蓄積させる工程（Ｈｙｐ蓄積工程）を含む、Ｌ－ヒドロキシプロリン含有化粧料原料の製造方法も包含する。上記窒素源は、Ｌ－ヒドロキシプロリン含有ペプチドを含む窒素源である。本発明のＬ－ヒドロキシプロリン含有化粧料原料の製造方法の好ましい態様は、上述したＬ－ヒドロキシプロリンの製造方法の好ましい態様と同じである。上記Ｌ－ヒドロキシプロリン含有化粧料原料の製造方法は、本発明の化粧料原料の製造方法として好ましい。
Ｈｙｐ蓄積工程を行うことにより、Ｌ－ヒドロキシプロリンを含有する酵母の菌体又は菌体培養物が得られる。また、酵母の菌体又は菌体培養物に上述した菌体破砕処理を行うことにより、Ｌ－ヒドロキシプロリンを含有する酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)の菌体又は菌体培養物の抽出物が得られる。このようにして得られる酵母の菌体もしくは菌体培養物又はこれらの抽出物、及び、その好ましい態様は、上述した本発明の酵母の菌体もしくは菌体培養物又はこれらの抽出物、及び、その好ましい態様と同じである。得られるＬ－ヒドロキシプロリンを含有する酵母の菌体もしくは菌体培養物又はこれらの抽出物は、所望により化粧料に通常使用される添加剤等を配合して、Ｌ－ヒドロキシプロリン含有化粧料原料として使用することができる。また、Ｌ－ヒドロキシプロリンを含有する酵母の菌体もしくは菌体培養物又はこれらの抽出物から精製したＬ－ヒドロキシプロリンに、所望により化粧料に通常使用される添加剤等を配合して、Ｌ－ヒドロキシプロリン含有化粧料原料を製造することもできる。本発明により得られるＬ－ヒドロキシプロリン含有化粧料原料は、コラーゲン産生促進、表皮細胞の増殖促進、皮膚の保湿、皮膚の老化防止、皮膚のたるみの予防又は改善、皮膚のハリの改善、しわの予防又は改善及びアトピー性皮膚炎の改善から選ばれる用途の化粧料に好適に使用される。 The present invention accumulates L-hydroxyproline in the yeast cells or cell cultures by aerobic culture of yeast Yarrowia lipolytica in a liquid medium containing a carbon source and a nitrogen source. And a method for producing an L-hydroxyproline-containing cosmetic raw material, including a step (Hyp accumulation step). The nitrogen source is a nitrogen source containing an L-hydroxyproline-containing peptide. The preferred embodiment of the method for producing an L-hydroxyproline-containing cosmetic raw material of the present invention is the same as the preferred embodiment of the method for producing L-hydroxyproline described above. The method for producing the L-hydroxyproline-containing cosmetic raw material is preferable as the method for producing the cosmetic raw material of the present invention.
By performing the Hyp accumulation step, a yeast cell or cell culture containing L-hydroxyproline is obtained. In addition, by performing the above-described cell disruption treatment on yeast cells or cell cultures, an extract of the cells or cell cultures of yeast Yarrowia lipolytica containing L-hydroxyproline can be obtained. can get. Thus obtained yeast cells or cell cultures or extracts thereof, and preferred embodiments thereof are the yeast cells or cell cultures of the present invention described above or extracts thereof, and The preferred embodiment is the same. L-hydroxyproline-containing cosmetic raw material containing L-hydroxyproline-containing yeast cells or cell cultures or extracts thereof, if desired, containing additives or the like commonly used in cosmetics Can be used as In addition, L-hydroxyproline purified from yeast cells or cell cultures or extracts thereof containing L-hydroxyproline may be blended with additives ordinarily used in cosmetics, if desired. A hydroxyproline-containing cosmetic raw material can also be produced. The cosmetic raw material containing L-hydroxyproline obtained by the present invention has the following effects: collagen production promotion, epidermal cell growth promotion, skin moisturization, skin aging prevention, skin sagging prevention or improvement, skin firmness improvement, wrinkle It is suitably used for cosmetics for uses selected from prevention or improvement and improvement of atopic dermatitis.
本発明は、Ｌ－ヒドロキシプロリンを含有する酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)の菌体もしくは菌体培養物又はこれらの抽出物を含む、Ｌ－ヒドロキシプロリン補強用組成物も包含する。
上記酵母の菌体もしくは菌体培養物又はこれらの抽出物は、Ｌ－ヒドロキシプロリンを含有するが、Ｌ－プロリン（Ｐｒｏ）及びＬ－ヒドロキシプロリン（Ｈｙｐ）の合計含量（μｇ／ｍＬ）に対するＬ－ヒドロキシプロリンの含量（μｇ／ｍＬ）の割合（１００×Ｈｙｐ／（Ｐｒｏ＋Ｈｙｐ））が、３５～１００であることが好ましい。上記酵母の菌体もしくは菌体培養物又はこれらの抽出物は、Ｌ－ヒドロキシプロリンの含量が１０μｇ／ｍＬ以上が好ましい。このような酵母の菌体もしくは菌体培養物又はこれらの抽出物を含むＬ－ヒドロキシプロリン補強用組成物は、化粧品、飲食品等のＬ－ヒドロキシプロリンを補強、補充又は強化するための添加剤として特に好適に使用することができる。酵母の菌体もしくは菌体培養物又はこれらの抽出物の好ましい態様は、上述した本発明の酵母の菌体もしくは菌体培養物又はこれらの抽出物及びその好ましい態様と同じである。Ｌ－ヒドロキシプロリン補強用組成物は、Ｌ－ヒドロキシプロリン補強用の添加剤組成物として、飲食品、化粧料等に好適に使用することができる。Ｌ－ヒドロキシプロリン補強用組成物は、Ｌ－ヒドロキシプロリン補充用組成物又はＬ－ヒドロキシプロリン強化用組成物と言い換えることもできる。 The present invention also includes a composition for reinforcing L-hydroxyproline comprising a cell or a cell culture of yeast Yarrowia lipolytica containing L-hydroxyproline or an extract thereof.
The yeast cell or cell culture or extract thereof contains L-hydroxyproline, but the L relative to the total content (μg / mL) of L-proline (Pro) and L-hydroxyproline (Hyp). The ratio (100 × Hyp / (Pro + Hyp)) of the hydroxyproline content (μg / mL) is preferably 35-100. The yeast cells or cell cultures or extracts thereof preferably have an L-hydroxyproline content of 10 μg / mL or more. A composition for reinforcing L-hydroxyproline containing such yeast cells or cell cultures or extracts thereof is an additive for reinforcing, supplementing or strengthening L-hydroxyproline in cosmetics, foods and drinks, etc. Can be used particularly preferably. Preferred embodiments of yeast cells or cell cultures or extracts thereof are the same as the yeast cells or cell cultures or extracts thereof of the present invention described above and preferred embodiments thereof. The L-hydroxyproline reinforcing composition can be suitably used as an additive composition for reinforcing L-hydroxyproline in foods and drinks, cosmetics and the like. The composition for reinforcing L-hydroxyproline can also be referred to as a composition for supplementing L-hydroxyproline or a composition for reinforcing L-hydroxyproline.
本発明のＬ－ヒドロキシプロリン補強用組成物は、Ｌ－ヒドロキシプロリンを含有する酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)の菌体もしくは菌体培養物又はこれらの抽出物を含んでいればよく、該菌体もしくは菌体培養物又はこれらの抽出物の含量が１００重量％であってもよいが、所望により、他の成分を含んでいてもよい。例えばＬ－ヒドロキシプロリン補強用組成物を食品添加剤として使用する場合には、食品に使用される公知の添加剤１種又は２種以上を含んでいてもよい。 The L-hydroxyproline reinforcing composition of the present invention only needs to contain a cell or a cell culture of yeast Yarrowia lipolytica containing L-hydroxyproline, or an extract thereof. The content of the body or cell culture or these extracts may be 100% by weight, but may contain other components as desired. For example, when the L-hydroxyproline reinforcing composition is used as a food additive, it may contain one or more known additives used in foods.
本発明のＬ－ヒドロキシプロリン補強用組成物は、例えば、化粧料添加剤としても好適に使用される。Ｌ－ヒドロキシプロリン補強用組成物を化粧料添加剤として使用する場合は、該組成物中、酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)の菌体もしくは菌体培養物又はこれらの抽出物を含んでいればよく、該菌体もしくは菌体培養物又はこれらの抽出物の含量が１００重量％であってもよいが、所望により、化粧料に使用される公知の添加剤１種又は２種以上を含んでいてもよい。 The L-hydroxyproline reinforcing composition of the present invention is also suitably used as a cosmetic additive, for example. When the L-hydroxyproline reinforcing composition is used as a cosmetic additive, it contains a yeast or a yeast culture of yeast Yarrowia lipolytica or an extract thereof. Well, the content of the cells or cell cultures or their extracts may be 100% by weight, but if desired, it contains one or more known additives used in cosmetics. May be.
本発明のＬ－ヒドロキシプロリン補強用組成物の製造方法は、酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)を、炭素源及び窒素源を含む液体培地中で好気培養することにより、上記酵母の菌体又は菌体培養物中にＬ－ヒドロキシプロリンを蓄積させる工程を含むことが好ましい。このような工程を含むＬ－ヒドロキシプロリン補強用組成物の製造方法も本発明に包含される。上記窒素源は、Ｌ－ヒドロキシプロリン含有ペプチドを含む窒素源である。本発明のＬ－ヒドロキシプロリン補強用組成物の製造方法及びその好ましい態様は、上述したＬ－ヒドロキシプロリンの製造方法及びその好ましい態様と同じである。本発明のＬ－ヒドロキシプロリン補強用組成物の製造方法は、所望により、酵母の菌体もしくは菌体培養物又はこれらの抽出物に公知の食品添加剤、化粧料添加剤等を添加する工程を含んでいてもよい。 The method for producing a composition for reinforcing L-hydroxyproline according to the present invention comprises the yeast yeast or Yarrowia lipolytica by aerobic culture in a liquid medium containing a carbon source and a nitrogen source. It is preferable to include a step of accumulating L-hydroxyproline in the cell culture. A method for producing an L-hydroxyproline reinforcing composition including such steps is also encompassed by the present invention. The nitrogen source is a nitrogen source containing an L-hydroxyproline-containing peptide. The production method of L-hydroxyproline reinforcing composition of the present invention and preferred embodiments thereof are the same as the above-described production method of L-hydroxyproline and preferred embodiments thereof. The method for producing an L-hydroxyproline reinforcing composition of the present invention comprises a step of adding a known food additive, cosmetic additive, etc. to yeast cells or cell cultures or extracts thereof, if desired. May be included.
本発明の別の態様のＬ－ヒドロキシプロリンの製造方法は、酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)を、炭素源及び窒素源を含む液体培地中で好気培養することにより、上記酵母の菌体又は菌体培養物中にＬ－ヒドロキシプロリンを蓄積させる工程を含む。
本発明の別の態様の製造方法においては、上記窒素源が、Ｌ－ヒドロキシプロリン含有タンパク質又はＬ－ヒドロキシプロリン含有ペプチドを含むことが好ましい。
本発明の別の態様の製造方法においては、上記Ｌ－ヒドロキシプロリン含有タンパク質がコラーゲン性タンパク質であり、上記Ｌ－ヒドロキシプロリン含有ペプチドがコラーゲンペプチドであることが好ましい。本発明の別の態様の製造方法においては、上記コラーゲン性タンパク質及びコラーゲンペプチドの平均分子量が１０００～１０００００であることが好ましい。
本発明の別の態様の製造方法においては、上記液体培地中の窒素源の濃度が０．２５～５重量％であり、炭素源（Ｃ）と窒素源（Ｎ）との重量比（Ｃ／Ｎ）が０．５～２０であることが好ましい。また、上記好気培養を１０～１００時間行うことが好ましい。 According to another aspect of the present invention, there is provided a method for producing L-hydroxyproline by aerobically cultivating yeast Yarrowia lipolytica in a liquid medium containing a carbon source and a nitrogen source. A step of accumulating L-hydroxyproline in the cell culture.
In the production method of another aspect of the present invention, the nitrogen source preferably contains an L-hydroxyproline-containing protein or an L-hydroxyproline-containing peptide.
In the production method of another aspect of the present invention, the L-hydroxyproline-containing protein is preferably a collagenous protein, and the L-hydroxyproline-containing peptide is preferably a collagen peptide. In the production method of another aspect of the present invention, the collagen protein and collagen peptide preferably have an average molecular weight of 1,000 to 100,000.
In the production method of another aspect of the present invention, the concentration of the nitrogen source in the liquid medium is 0.25 to 5% by weight, and the weight ratio (C / C) of the carbon source (C) and the nitrogen source (N). N) is preferably from 0.5 to 20. The aerobic culture is preferably performed for 10 to 100 hours.
本発明の別の態様の使用は、酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)の、Ｌ－ヒドロキシプロリンを製造するための使用であって、上記酵母を、炭素源及び窒素源を含む液体培地中で好気培養することにより、上記酵母の菌体又は菌体培養物中にＬ－ヒドロキシプロリンを蓄積させることを含み、上記窒素源がＬ－ヒドロキシプロリン含有タンパク質又はＬ－ヒドロキシプロリン含有ペプチドを含むことが好ましい。
本発明の別の態様の使用においては、上記Ｌ－ヒドロキシプロリン含有タンパク質がコラーゲン性タンパク質であり、上記Ｌ－ヒドロキシプロリン含有ペプチドがコラーゲンペプチドであることが好ましい。本発明の別の態様の使用においては、上記コラーゲン性タンパク質及びコラーゲンペプチドの平均分子量が１０００～１０００００であることが好ましい。
本発明の別の態様の使用においては、上記液体培地中の窒素源の濃度が０．２５～５重量％であり、炭素源（Ｃ）と窒素源（Ｎ）との重量比（Ｃ／Ｎ）が０．５～２０であることが好ましい。また、上記好気培養を１０～１００時間行うことが好ましい。 Another use of the present invention is the use of the yeast Yarrowia lipolytica for producing L-hydroxyproline, wherein the yeast is preferably used in a liquid medium containing a carbon source and a nitrogen source. L-hydroxyproline is accumulated in the yeast or cell culture of the yeast by air culture, and the nitrogen source contains an L-hydroxyproline-containing protein or an L-hydroxyproline-containing peptide. preferable.
In the use of another aspect of the present invention, the L-hydroxyproline-containing protein is preferably a collagenous protein, and the L-hydroxyproline-containing peptide is preferably a collagen peptide. In the use of another aspect of the present invention, the collagen protein and collagen peptide preferably have an average molecular weight of 1,000 to 100,000.
In another embodiment of the present invention, the concentration of the nitrogen source in the liquid medium is 0.25 to 5% by weight, and the weight ratio (C / N) of the carbon source (C) and the nitrogen source (N). ) Is preferably 0.5-20. The aerobic culture is preferably performed for 10 to 100 hours.
上記Ｌ－ヒドロキシプロリン含有タンパク質としては、例えば、上述したコラーゲン性タンパク質が挙げられる。コラーゲン性タンパク質等のＬ－ヒドロキシプロリン含有タンパク質の平均分子量は、好ましくは１００００を超えて１０００００以下である。タンパク質の平均分子量は、重量平均分子量を指す。コラーゲン性タンパク質等のＬ－ヒドロキシプロリン含有タンパク質の平均分子量は、ゲル濾過などにより算出される。 Examples of the L-hydroxyproline-containing protein include the aforementioned collagenous protein. The average molecular weight of the L-hydroxyproline-containing protein such as collagenous protein is preferably more than 10,000 and not more than 100,000. The average molecular weight of protein refers to the weight average molecular weight. The average molecular weight of the L-hydroxyproline-containing protein such as collagenous protein is calculated by gel filtration or the like.
以下、本発明をより具体的に説明する試験例等を示す。なお、本発明はこれらの試験例等のみに限定されるものではない。試験例中、特に断らない場合には、「％」は「重量％」を意味する。 Hereinafter, test examples for more specifically explaining the present invention will be shown. The present invention is not limited only to these test examples. In the test examples, “%” means “% by weight” unless otherwise specified.
試験例中、検量線の作成に使用するＬ－ヒドロキシプロリン（Ｈｙｐ）標準溶液等の調製には、ナカライテスク（株）製のＬ－４－ヒドロキシプロリンを使用した。Ｌ－プロリン（Ｐｒｏ）標準溶液等の調製等には、ナカライテスク（株）製のＬ－プロリンを使用した。試験例で測定したＬ－ヒドロキシプロリン及びＬ－プロリンは、いずれも遊離のＬ－ヒドロキシプロリン及びＬ－プロリンである。 In the test examples, L-4-hydroxyproline manufactured by Nacalai Tesque Co., Ltd. was used to prepare an L-hydroxyproline (Hyp) standard solution used for preparing a calibration curve. For the preparation of L-proline (Pro) standard solution and the like, L-proline manufactured by Nacalai Tesque Co., Ltd. was used. L-hydroxyproline and L-proline measured in the test examples are both free L-hydroxyproline and L-proline.
試験例中で使用した培地は、全て液体培地である。試験例中で使用したＹＰＤ培地は、特に断らない場合は、Ｙ：Ｐ：Ｄが１：２：２（重量比）（Ｙ：酵母抽出物１．０％、Ｐ：ペプトン２．０％、Ｄ：グルコース２．０％）とした。
培地調製に使用した酵母抽出物は、Bacto社製の製品名Yeast Extract(#212750)である。ペプトンは、Bacto社製の製品名Pepton(#211677)、ウシの細胞をブタの膵臓由来の酵素で分解したものを使用した。
試験例において、培養開始前の培地のｐＨは、約６．５～８．５であった。 All of the media used in the test examples are liquid media. The YPD medium used in the test examples, unless otherwise specified, Y: P: D is 1: 2: 2 (weight ratio) (Y: yeast extract 1.0%, P: peptone 2.0%, D: glucose 2.0%).
The yeast extract used for medium preparation is the product name Yeast Extract (# 212750) manufactured by Bacto. Peptone used was a product name Pepton (# 211677) manufactured by Bacto, which was obtained by degrading bovine cells with an enzyme derived from porcine pancreas.
In the test examples, the pH of the medium before the start of culture was about 6.5 to 8.5.
＜試験例１＞
Ｌ－ヒドロキシプロリン（以下、Ｈｙｐ）を蓄積する酵母のスクリーニング
表１に示す６０株の酵母を使用し、Ｈｙｐを培養物中に蓄積する酵母のスクリーニングを行った。表１に各菌株の属種名を示す。 <Test Example 1>
Screening of Yeast Accumulating L-Hydroxyproline (hereinafter Hyp) 60 yeast strains shown in Table 1 were used to screen for yeast that accumulates Hyp in the culture. Table 1 shows the genus name of each strain.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
以下の条件で、ＹＰＤ培地で酵母を培養した後、ＨＰＬＣでＨｙｐを定量した。 After culturing the yeast in the YPD medium under the following conditions, Hyp was quantified by HPLC.
（培養）
０．５％Ｌ－プロリン（以下、Ｐｒｏ）を添加したＹＰＤ培地１ｍＬに、各菌株を１白金耳接種し、２８℃で２４時間振盪培養（３００ｒｐｍ）した後、室温で４～７日間静置培養して酵母の菌体培養液（菌体培養物）を得た。 (culture)
1 mL of each strain was inoculated into 1 mL of YPD medium supplemented with 0.5% L-proline (hereinafter referred to as “Pro”), cultured at 28 ° C. for 24 hours with shaking (300 rpm), and then allowed to stand at room temperature for 4 to 7 days. Culturing was performed to obtain a yeast cell culture solution (cell culture).
（サンプル調製）
酵母の菌体培養液に以下の処理を行って、培養物サンプルを調製し、Ｈｙｐ含量を測定した。
上記の培養後、集菌し、菌体を１ｍＬの生理食塩水で洗浄した。菌体を０．２ｍＬの５０ｍＭ　カリウムリン酸緩衝液（以下、ＫＰＢ）（ｐＨ６．０）に懸濁し、１０分間煮沸して、自己消化により菌体内容物を溶出させた（煮沸法）。得られた菌体抽出物から遠心分離（１４０００ｒｐｍ、５ｍｉｎ、４℃）により菌体残渣を除いた。これにより、Ｈｙｐ蓄積量を測定するための培養物サンプルを調製した。 (Sample preparation)
The yeast cell culture solution was subjected to the following treatment to prepare a culture sample, and the Hyp content was measured.
After the above culture, the cells were collected and the cells were washed with 1 mL of physiological saline. The cells were suspended in 0.2 mL of 50 mM potassium phosphate buffer (hereinafter referred to as KPB) (pH 6.0), boiled for 10 minutes, and the cell contents were eluted by autolysis (boiling method). The cell residue was removed from the obtained cell extract by centrifugation (14000 rpm, 5 min, 4 ° C.). This prepared the culture sample for measuring the amount of Hyp accumulation.
培養物サンプルをウォーターズ社の（AccQ-Fluor Reagent Kit）を使用して、ＡｃｃＱＴａｇ法により誘導体化し、以下の条件でＨＰＬＣでＨｙｐを定量した。 The culture sample was derivatized by the AccQTag method using Waters (AccQ-Fluor Reagent Kit), and Hyp was quantified by HPLC under the following conditions.
ＨＰＬＣ分析に使用した装置及び条件を以下に示す。
（装置）
高速液体クロマトグラフ： Prominence（（株）島津製作所）
カラム： XBridge C18 5μm（2.1 x 150 mm、ウォーターズ社製）
（測定条件）
溶離液Ａ：酢酸アンモニウム（１０ｍＭ、ｐＨ５）
溶離液Ｂ：メタノール（０～０．５ｍｉｎ．（０％→１％），０．５～１８ｍｉｎ．（１％→５％），１８～１９ｍｉｎ．（５％→９％），１９～２９．５ｍｉｎ．（９％→１７％），２９．５～４０ｍｉｎ．（１７％→６０％），４０～４３ｍｉｎ．（６０％））
流速：０．３ｍＬ／ｍｉｎ
温度：４０℃
検出：蛍光検出器（励起波長：２５０ｎｍ、検出波長：３９５ｎｍ）
溶離液Ｂの濃度（％）は、ｖ／ｖ％である。
結果を図１に示す。 The apparatus and conditions used for the HPLC analysis are shown below.
(apparatus)
High-performance liquid chromatograph: Prominence (Shimadzu Corporation)
Column: XBridge C18 5μm (2.1 x 150 mm, Waters)
(Measurement condition)
Eluent A: ammonium acetate (10 mM, pH 5)
Eluent B: Methanol (0-0.5 min. (0% → 1%), 0.5-18 min. (1% → 5%), 18-19 min. (5% → 9%), 19-29. 5 min. (9% → 17%), 29.5-40 min. (17% → 60%), 40-43 min. (60%))
Flow rate: 0.3 mL / min
Temperature: 40 ° C
Detection: fluorescence detector (excitation wavelength: 250 nm, detection wavelength: 395 nm)
The concentration (%) of the eluent B is v / v%.
The results are shown in FIG.
図１は、酵母のＨｙｐ蓄積量を示すグラフである。図１の縦軸（Ｈｙｐ（μｇ／ｍＬ））は、培養物サンプル１ｍＬあたりのＨｙｐ（μｇ）を示す。
上記の試験から、Ｈｙｐを蓄積する酵母が見出された。特にYarrowia lipolyticaが、Ｈｙｐ蓄積量が多かった。 FIG. 1 is a graph showing the amount of Hyp accumulated in yeast. The vertical axis (Hyp (μg / mL)) in FIG. 1 indicates Hyp (μg) per 1 mL of the culture sample.
From the above tests, yeasts that accumulate Hyp were found. In particular, Yarrowia lipolytica had a large amount of Hyp accumulation.
＜試験例２＞
酵母Yarrowia lipolyticaについて、培地によってＨｙｐ蓄積量が変化するかを調べた。Yarrowia lipolytica NBRC0717を使用し、以下の液体培地を使用した。Yarrowia lipolytica NBRC0717は、独立行政法人製品評価技術基盤機構（日本国千葉県木更津市かずさ鎌足２－５－８）から入手した。 <Test Example 2>
About yeast Yarrowia lipolytica , it was investigated whether the amount of Hyp accumulation changed with culture media. Yarrowia lipolytica NBRC0717 was used and the following liquid medium was used. Yarrowia lipolytica NBRC0717 was obtained from the National Institute of Technology and Evaluation (2-5-8, Kazusa Kama feet, Kisarazu City, Chiba Prefecture, Japan).
ＹＰＤ（１％酵母抽出物、２％ペプトン、２％グルコース）
ＹＴＤ（１％酵母抽出物、２％トリプトン、２％グルコース）
ＹＭ（０．３％酵母抽出物、０．３％麦芽エキス、０．５％ペプトン、２％グルコース）
ＰＤ（０．４％ポテト抽出物、２％グルコース）
ＳＤ（合成培地、１％グルコース） YPD (1% yeast extract, 2% peptone, 2% glucose)
YTD (1% yeast extract, 2% tryptone, 2% glucose)
YM (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 2% glucose)
PD (0.4% potato extract, 2% glucose)
SD (synthetic medium, 1% glucose)
（前培養及び本培養）
（１）条件１
０．５％Ｐｒｏを添加したＹＰＤ培地１ｍＬに、Yarrowia lipolytica NBRC0717を１白金耳接種し、２８℃で２４時間振盪培養（３００ｒｐｍ）して前培養液を得た。
０．５％Ｐｒｏを添加した各培地（上記ＹＰＤ、ＹＴＤ、ＹＭ、ＰＤ又はＳＤ）２ｍＬに、前培養液０．２ｍＬを接種して、２８℃で３６時間振盪培養（３００ｒｐｍ）してYarrowia lipolyticaの菌体培養液を得た。
（２）条件２
０．５％Ｐｒｏを添加したＹＰＤ培地の代わりに、０．５％Ｐｒｏを添加したＳＤ培地を使用した以外は、条件１と同じ条件で前培養を行った。０．５％Ｐｒｏを添加した各培地（上記ＹＰＤ、ＹＴＤ、ＰＤ又はＳＤ）２ｍＬに、前培養液０．２ｍＬを接種して、２８℃で４５時間振盪培養（３００ｒｐｍ）してYarrowia lipolyticaの菌体培養液を得た。 (Pre-culture and main culture)
(1) Condition 1
One platinum loop of Yarrowia lipolytica NBRC0717 was inoculated into 1 mL of YPD medium supplemented with 0.5% Pro, and precultured at 28 ° C. for 24 hours with shaking (300 rpm).
Inoculate 2 mL of each medium (YPD, YTD, YM, PD or SD) to which 0.5% Pro has been added with 0.2 mL of the pre-culture solution, and shake culture (300 rpm) at 28 ° C. for 36 hours to give Yarrowia lipolytica The bacterial cell culture solution was obtained.
(2) Condition 2
Pre-culture was performed under the same conditions as Condition 1 except that SD medium supplemented with 0.5% Pro was used instead of YPD medium supplemented with 0.5% Pro. Each medium supplemented with 0.5% Pro (the above YPD, YTD, PD or SD) 2 mL, was inoculated preculture 0.2 mL, 28 ° C. 45 hours by shaking culture at (300 rpm) and bacteria Yarrowia lipolytica by A body culture solution was obtained.
（サンプル調製）
条件１又は２の培養後、酵母の菌体培養液に以下の処理を行って培養物サンプルを調製し、Ｈｙｐ含量を測定した。
上記の培養後、集菌し、菌体を１ｍＬの生理食塩水で洗浄した。菌体を０．２ｍＬの５０ｍＭ　ＫＰＢ（ｐＨ６．０）に懸濁し、１０分間煮沸して、自己消化により菌体内容物を溶出させた（煮沸法）。得られた菌体抽出物から遠心分離（１４０００ｒｐｍ、５ｍｉｎ、４℃）により菌体残渣を除いた。これにより、Ｈｙｐ蓄積量を測定するための培養物サンプルを調製した。 (Sample preparation)
After culturing under condition 1 or 2, the yeast cell culture solution was subjected to the following treatment to prepare a culture sample, and the Hyp content was measured.
After the above culture, the cells were collected and the cells were washed with 1 mL of physiological saline. The cells were suspended in 0.2 mL of 50 mM KPB (pH 6.0), boiled for 10 minutes, and the cell contents were eluted by autolysis (boiling method). The cell residue was removed from the obtained cell extract by centrifugation (14000 rpm, 5 min, 4 ° C.). This prepared the culture sample for measuring the amount of Hyp accumulation.
（Ｈｙｐ定量）
培養物サンプルをｏ－フタルアルデヒド（ＯＰＡ）及び４－クロロ－７－ニトロベンゾフラザン（ＮＢＤ－Ｃｌ）により誘導体化し、ＨＰＬＣでＨｙｐを定量した。
（誘導体化方法）
4-Chloro-7-nitrobenzofurazan（ＮＢＤ－Ｃｌ）による誘導体化
０．４Ｍ　ホウ酸カリウム緩衝液（ｐＨ９．５）を５０μＬ分注し、培養物サンプルを２μＬ加えた。３００ｍＭ　ＯＰＡ（Ｗａｋｏ　１６７－０９２６３）（メタノールに溶解）を２μＬ加えて混合し、３７℃で２０分間反応後、２ｍＭ　ＮＢＤ－Ｃｌ（Ｓｉｇｍａ２５４５５）（メタノールに溶解）を５０μＬ加えて混合し、３７℃で２０分間反応した液に、１Ｎ　ＨＣｌを２５μＬ、５０％メタノールを７５μＬ加え、１４０００ｒｐｍで５分間遠心した上清をＨＰＬＣ測定用のサンプルとし、ＨＰＬＣ用のバイアルへ移した。 (Hyp quantification)
Culture samples were derivatized with o-phthalaldehyde (OPA) and 4-chloro-7-nitrobenzofurazan (NBD-Cl) and Hyp was quantified by HPLC.
(Derivatization method)
Derivatization with 4-Chloro-7-nitrobenzofurazan (NBD-Cl) 50 μL of 0.4 M potassium borate buffer (pH 9.5) was dispensed, and 2 μL of the culture sample was added. 2 μL of 300 mM OPA (Wako 167-09263) (dissolved in methanol) was added and mixed. After 20 minutes of reaction at 37 ° C., 50 μL of 2 mM NBD-Cl (Sigma 25455) (dissolved in methanol) was added and mixed at 37 ° C. The supernatant obtained by adding 25 μL of 1N HCl and 75 μL of 50% methanol to the reaction solution for 20 minutes and centrifuging at 14000 rpm for 5 minutes was used as a sample for HPLC measurement, and transferred to a vial for HPLC.
ＨＰＬＣ分析に使用した装置及び条件を以下に示す。
カラム：ＸＢｒｉｄｇｅ　Ｃ１８　ｃｏｌｕｍｎ（５μｍ；２．１ｍｍ×１５０ｍｍ；Ｗａｔｅｒｓ）
Ａ液：１０ｍＭ　酢酸アンモニウム（ｐＨ５．０）
Ｂ液：メタノール
グラジエント：
０～０．５分：Ｂ．Ｃｏｎｃ　０ｖ／ｖ％→１ｖ／ｖ％
０．５～１８分：Ｂ．Ｃｏｎｃ　１ｖ／ｖ％→５ｖ／ｖ％
１８～１９分：Ｂ．Ｃｏｎｃ　５ｖ／ｖ％→９ｖ／ｖ％
１９～２９．５分：Ｂ．Ｃｏｎｃ　９ｖ／ｖ％→１７ｖ／ｖ％
２９．５～４０分：Ｂ．Ｃｏｎｃ　１７ｖ／ｖ％→６０ｖ／ｖ％
４０～４３分：Ｂ．Ｃｏｎｃ　６０ｖ／ｖ％
流速：０．３ｍＬ／分
カラム温度：４０℃
検出器：蛍光検出器、励起波長５０３ｎｍ／蛍光波長５４１ｎｍ
注入量：１０μＬ The apparatus and conditions used for the HPLC analysis are shown below.
Column: XBridge C18 column (5 μm; 2.1 mm × 150 mm; Waters)
Liquid A: 10 mM ammonium acetate (pH 5.0)
B liquid: Methanol gradient:
0-0.5 min. Conc 0v / v% → 1v / v%
0.5-18 minutes: B.I. Conc 1v / v% → 5v / v%
18-19 min. Conc 5v / v% → 9v / v%
19-29.5 min. Conc 9v / v% → 17v / v%
29.5-40 minutes: B.I. Con 17v / v% → 60v / v%
40-43 min. Conc 60v / v%
Flow rate: 0.3 mL / min Column temperature: 40 ° C
Detector: Fluorescence detector, excitation wavelength 503 nm / fluorescence wavelength 541 nm
Injection volume: 10 μL
検量線は、Ｈｙｐの、５μｍｏｌ／Ｌ、１０μｍｏｌ／Ｌ、２０μｍｏｌ／Ｌ、５０μｍｏｌ／Ｌ、１００μｍｏｌ／Ｌ、２５０μｍｏｌ／Ｌの５０ｍＭ　ＫＰＢ（ｐＨ６．０）溶液を調製して作成した。 A calibration curve was prepared by preparing 50 mM KPB (pH 6.0) solutions of 5 μmol / L, 10 μmol / L, 20 μmol / L, 50 μmol / L, 100 μmol / L, 250 μmol / L of Hyp.
結果を図２に示す。図２は、各培地で培養したYarrowia lipolyticaのＨｙｐ蓄積量を示すグラフである（（ａ）：条件１、（ｂ）：条件２）。図２の縦軸のＨｙｐ（μｇ／ｍＬ）は、培養物サンプル１ｍＬあたりのＨｙｐ（μｇ）である。条件１の培養では、ＹＰＤ及びＹＭ培地で培養した菌体にヒドロキシプロリンが多く蓄積した（ペプトンを含む培地）。条件２の培養では、ＹＰＤ培地で培養した菌体にＨｙｐが蓄積した。 The results are shown in FIG. FIG. 2 is a graph showing the amount of Yyp accumulation of Yarrowia lipolytica cultured in each medium ((a): Condition 1, (b): Condition 2). The Hyp (μg / mL) on the vertical axis in FIG. 2 is Hyp (μg) per 1 mL of the culture sample. In the culture of Condition 1, a large amount of hydroxyproline accumulated in the cells cultured in YPD and YM media (medium containing peptone). In the culture of Condition 2, Hyp accumulated in the cells cultured in the YPD medium.
＜試験例３＞
培養日数による蓄積量の変動を調べた。
Yarrowia lipolyticaのNBRC1551株、NBRC0717株、NBRC0746株及びNBRC1195株をそれぞれＹＰＤ培地で１日、２日又は３日間培養し、酵母の菌体（細胞）から溶出させたＨｙｐをＨＰＬＣを用いて定量した。ＮＢＲＣ番号で特定されるこれらの酵母は、独立行政法人製品評価技術基盤機構（日本国千葉県木更津市かずさ鎌足２－５－８）から入手した。 <Test Example 3>
The variation of the accumulation amount with the number of culture days was examined.
Yarrowia lipolytica strains NBRC1551, NBRC0717, NBRC0746 and NBRC1195 were cultured in YPD medium for 1, 2, or 3 days, respectively, and Hyp eluted from yeast cells (cells) was quantified using HPLC. These yeasts identified by the NBRC number were obtained from the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamashitsu, Kisarazu City, Chiba Prefecture, Japan).
（前培養）
ＹＰＤ培地１ｍＬに、各菌株を１白金耳接種し、２８℃で２４時間振盪培養（３００ｒｐｍ）して前培養液を得た。 (Pre-culture)
One platinum loop of each strain was inoculated into 1 mL of YPD medium, followed by shaking culture (300 rpm) at 28 ° C. for 24 hours to obtain a preculture solution.
（本培養）
ＹＰＤ培地２ｍＬに、前培養液０．２ｍＬを接種して、２８℃で１～３日間振盪培養（３００ｒｐｍ）して菌体培養液を得た。 (Main culture)
2 mL of YPD medium was inoculated with 0.2 mL of the preculture solution, and cultured at 28 ° C. for 1 to 3 days with shaking (300 rpm) to obtain a cell culture solution.
（サンプル調製及びアミノ酸定量）
試験例２と同じ方法で培養物サンプルを調製し、ＯＰＡ及びＮＢＤ－Ｃｌにより誘導体化した。
試験例２と同じ条件でＨＰＬＣによる分析を行い、Ｈｙｐ及びＰｒｏを定量した。Ｐｒｏの検量線は、Ｐｒｏの５μｍｏｌ／Ｌ、１０μｍｏｌ／Ｌ、２０μｍｏｌ／Ｌ、５０μｍｏｌ／Ｌ、１００μｍｏｌ／Ｌ、２５０μｍｏｌ／Ｌの５０ｍＭ　ＫＰＢ（ｐＨ６．０）溶液を調製して作成した。 (Sample preparation and amino acid determination)
Culture samples were prepared in the same manner as in Test Example 2, and derivatized with OPA and NBD-Cl.
Analysis by HPLC was performed under the same conditions as in Test Example 2, and Hyp and Pro were quantified. Pro calibration curves were prepared by preparing Pro 5 μmol / L, 10 μmol / L, 20 μmol / L, 50 μmol / L, 100 μmol / L, 250 μmol / L 50 mM KPB (pH 6.0) solutions.
各培養物サンプルのＰｒｏ及びＨｙｐ量を表２に示す。また、Ｐｒｏ及びＨｙｐの測定値から、Ｐｒｏ及びＨｙｐの合計含量（μｇ／ｍＬ）に対するＨｙｐの含量（μｇ／ｍＬ）の割合（１００×Ｈｙｐ／（Ｐｒｏ＋Ｈｙｐ））を計算した。その結果も表２に示す。 Table 2 shows the amount of Pro and Hyp of each culture sample. Further, from the measured values of Pro and Hyp, the ratio (100 × Hyp / (Pro + Hyp)) of the content of Hyp (μg / mL) to the total content of Pro and Hyp (μg / mL) was calculated. The results are also shown in Table 2.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
Yarrowia lipolyticaは、培養によってＬ－ヒドロキシプロリンを蓄積した。得られた菌体培養物は、Ｌ－ヒドロキシプロリンを含有した。また、（Ｈｙｐ／（Ｐｒｏ＋Ｈｙｐ））割合が３５以上であった。 Yarrowia lipolytica accumulated L-hydroxyproline by culture. The obtained cell culture contained L-hydroxyproline. Further, the (Hyp / (Pro + Hyp)) ratio was 35 or more.
＜試験例４＞
Yarrowia lipolyticaのゼラチン資化性を検討した。Yarrowia lipolyticaのNBRC1551株、NBRC0717株及びNBRC0746株を使用して、ＰＤ培地又は０．１２５％ゼラチンを添加したＰＤ培地で５日間培養し、菌体から菌体内容物を溶出させてＨｙｐをＨＰＬＣにより定量した。ＰＤ培地は、試験例２で使用したものと同じ培地を使用した。 <Test Example 4>
The gelatin utilization of Yarrowia lipolytica was examined. Yarrowia lipolytica NBRC1551 strain, NBRC0717 strain and NBRC0746 strain are used to culture for 5 days in PD medium or PD medium supplemented with 0.125% gelatin, and the cell contents are eluted from the cells and Hyp is analyzed by HPLC. Quantified. The same medium as used in Test Example 2 was used as the PD medium.
（前培養）
ＰＤ培地１ｍＬに、各菌株を１白金耳接種し、２８℃で２４時間振盪培養（３００ｒｐｍ）して前培養液を得た。
（本培養）
ＰＤ培地又は０．１２５％ゼラチン（和光純薬工業（株）製、製品名ゼラチン）を添加したＰＤ培地２ｍＬに、前培養液０．２ｍＬを接種して、２８℃で５日間振盪培養（３００ｒｐｍ）して菌体培養液を得た。
（サンプル調製及びＨｙｐ定量）
培養物サンプルの調製及びＨｙｐ定量は、試験例２と同じ方法で行った。結果を図３に示す。 (Pre-culture)
1 mL of each strain was inoculated into 1 mL of PD medium, and cultured with shaking (300 rpm) at 28 ° C. for 24 hours to obtain a preculture solution.
(Main culture)
PD culture medium or 0.125% gelatin (manufactured by Wako Pure Chemical Industries, Ltd., product name gelatin) was added to 2 mL of PD culture medium, and 0.2 mL of the preculture was inoculated, followed by shaking culture (300 rpm) at 28 ° C. for 5 days. ) To obtain a bacterial cell culture solution.
(Sample preparation and Hyp quantification)
Preparation of culture samples and Hyp quantification were performed in the same manner as in Test Example 2. The results are shown in FIG.
図３は、ＰＤ培地又は０．１２５％ゼラチンを添加したＰＤ培地で５日間培養したYarrowia lipolyticaのＨｙｐ蓄積量を示すグラフである（白：０．１２５％ゼラチンを含むＰＤ培地（ＰＤ＋ｇｅｌ）、黒：ＰＤ培地）。縦軸のＨｙｐ（μｇ／ｍＬ）は、培養物サンプル１ｍＬあたりのＨｙｐ（μｇ）である。図３から、高分子ゼラチンのまま培地に添加すると、Ｈｙｐの蓄積は試験例３と比較して少なかった。 FIG. 3 is a graph showing the amount of Hyp accumulated in Yarrowia lipolytica cultured in PD medium or PD medium supplemented with 0.125% gelatin for 5 days (white: PD medium containing 0.125% gelatin (PD + gel), black : PD medium). Hyp (μg / mL) on the vertical axis is Hyp (μg) per 1 mL of culture sample. From FIG. 3, when high molecular gelatin was added to the medium as it was, the accumulation of Hyp was less than that of Test Example 3.
＜試験例５＞
Yarrowia lipolytica NBRC0717を用いて、ＹＰＤ培地組成によりＨｙｐ蓄積量が変化するか検討した。表３に＃１～＃１２の各培地のＹＰＤ組成を示す。表３中、Ｙは、酵母抽出物、Ｐはペプトン、Ｄはグルコースである。酵母抽出物（Ｙ）を一定にし、ペプトン（Ｐ）及びグルコース（Ｄ）を変化させた（表中の数値は終濃度（重量％））。 <Test Example 5>
Using Yarrowia ipolytica NBRC0717, it was examined whether the amount of Hyp accumulation changes depending on the YPD medium composition. Table 3 shows the YPD composition of each medium # 1 to # 12. In Table 3, Y is yeast extract, P is peptone, and D is glucose. The yeast extract (Y) was kept constant, and the peptone (P) and glucose (D) were changed (the values in the table are final concentrations (% by weight)).
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
（前培養）
各ＹＰＤ培地１ｍＬに、Yarrowia lipolytica NBRC0717を１白金耳接種し、２８℃で２４時間振盪培養（３００ｒｐｍ）して前培養液を得た。
（本培養）
各ＹＰＤ培地２ｍＬに、前培養液０．２ｍＬを接種して、２８℃で４日間振盪培養（３００ｒｐｍ）して菌体培養液を得た。
（サンプル調製及びＨｙｐ定量）
培養物サンプルの調製及びＨｙｐ定量は、試験例２と同じ方法で行った。 (Pre-culture)
One platinum loop of Yarrowia lipolytica NBRC0717 was inoculated into 1 mL of each YPD medium, followed by shaking culture (300 rpm) at 28 ° C. for 24 hours to obtain a preculture solution.
(Main culture)
Each YPD medium (2 mL) was inoculated with 0.2 mL of a preculture solution, and cultured at 28 ° C. for 4 days with shaking (300 rpm) to obtain a cell culture solution.
(Sample preparation and Hyp quantification)
Preparation of culture samples and Hyp quantification were performed in the same manner as in Test Example 2.
上記のＹＰＤ培地で培養した酵母Yarrowia lipolyticaは、Ｈｙｐを蓄積した。＃１及び＃７～１１の培地で培養した場合にＨｙｐ蓄積量が多かった。＃１及び＃７～１１の培地で培養した場合の結果を図４に示す。 The yeast Yarrowia lipolytica cultured in the above YPD medium accumulated Hyp. When cultured in media # 1 and # 7-11, the amount of Hyp accumulated was large. FIG. 4 shows the results when cultured in # 1 and # 7-11 media.
図４は、組成が異なるＹＰＤ培地で培養したYarrowia lipolyticaのＨｙｐ蓄積量を示すグラフである（（ａ）：Ｈｙｐ蓄積量（μｇ／ｍＬ）、（ｂ）：細胞あたりのＨｙｐ量（μｇ／ｍＬ／ＯＤ６００））。図４の（ａ）の縦軸は、培養物サンプル１ｍＬあたりのＨｙｐ（μｇ）である。図４の（ｂ）の縦軸の（Ｈｙｐ／ＯＤ）は、細胞あたりのＨｙｐ量であり、培養物サンプル１ｍＬ中のＨｙｐ量（μｇ）をＯＤ６００の測定値で除した値（μｇ／ｍＬ／ＯＤ６００）である。
ＯＤ６００は、核酸蛋白質分光光度計（装置名Bio spec mini、（株）島津製作所）を用いて、培養物サンプルの調製に使用した菌体培養液６０μＬを超純水１１５０μＬで希釈し、吸光度（６００ｎｍ）を測定した FIG. 4 is a graph showing the Hyp accumulation amount of Yarrowia lipolytica cultured in YPD media having different compositions ((a): Hyp accumulation amount (μg / mL), (b): Hyp amount per cell (μg / mL). / OD600)). The vertical axis | shaft of (a) of FIG. 4 is Hyp (microgram) per 1 mL of culture samples. (Hyp / OD) on the vertical axis of FIG. 4B is the amount of Hyp per cell, and the value obtained by dividing the amount of Hyp (μg) in 1 mL of the culture sample by the measured value of OD600 (μg / mL / OD600).
OD600 was obtained by diluting 60 μL of the cell culture solution used for preparation of the culture sample with 1150 μL of ultrapure water using a nucleic acid protein spectrophotometer (device name Bio spec mini, Shimadzu Corporation), and measuring the absorbance (600 nm ) Measured
＜試験例６＞
試験例５で使用したＹＰＤ培地＃１及び＃１０において、Ｐとして使用したペプトンをコラーゲンペプチド（新田ゼラチン社製の製品名スーパーコラーゲンペプチド　ＳＣＰ－２０００、ブタ由来、平均分子量２０００）に置換して、Ｈｙｐの蓄積量を調べた。酵母は、Yarrowia lipolytica NBRC0717を使用した。ＹＰＤ培地において、ペプトンの一部又は全部をコラーゲンペプチドに置換した培地は、ＹＰＤ改変培地ともいえる。
ＹＰＤ培地のＰの組成
ノーマル：ペプトン１００％
ＣＰ５０：ペプトン５０％　コラーゲンペプチド５０％
ＣＰ１００：コラーゲンペプチド１００％ <Test Example 6>
In YPD media # 1 and # 10 used in Test Example 5, the peptone used as P was replaced with a collagen peptide (product name: Super Collagen Peptide SCP-2000, manufactured by Nitta Gelatin Co., Ltd., average molecular weight 2000). The amount of Hyp accumulated was examined. Yarrowia lipolytica NBRC0717 was used as the yeast. In a YPD medium, a medium in which part or all of peptone is replaced with a collagen peptide can be said to be a YPD-modified medium.
Composition of P in YPD medium Normal: 100% peptone
CP50: Peptone 50% Collagen peptide 50%
CP100: Collagen peptide 100%
培地組成
（１）ＹＰＤ＃１－ノーマル（ＹＰＤ＃１）
（２）ＹＰＤ＃１－ＣＰ５０（ＹＰＤ＃１において、ペプトンの代わりにＣＰ５０を使用）
（３）ＹＰＤ＃１－ＣＰ１００（ＹＰＤ＃１において、ペプトンの代わりにＣＰ１００を使用）
（４）ＹＰＤ＃１０－ノーマル（ＹＰＤ＃１０）
（５）ＹＰＤ＃１０－ＣＰ５０（ＹＰＤ＃１０において、ペプトンの代わりにＣＰ５０を使用）
（６）ＹＰＤ＃１０－ＣＰ１００（ＹＰＤ＃１０において、ペプトンの代わりにＣＰ１００を使用） Medium composition (1) YPD # 1-Normal (YPD # 1)
(2) YPD # 1-CP50 (CP50 is used instead of peptone in YPD # 1)
(3) YPD # 1-CP100 (CP100 is used instead of peptone in YPD # 1)
(4) YPD # 10-normal (YPD # 10)
(5) YPD # 10-CP50 (CP50 is used instead of peptone in YPD # 10)
(6) YPD # 10-CP100 (CP100 is used instead of peptone in YPD # 10)
（前培養）
ＹＰＤ培地１ｍＬに、Yarrowia lipolytica NBRC0717を１白金耳接種し、２８℃で２４時間振盪培養（３００ｒｐｍ）して前培養液を得た。
（本培養）
上記（１）～（６）の各培地２ｍＬに、前培養液０．１ｍＬを接種して、２８℃で４日間振盪培養（３００ｒｐｍ）して菌体培養液を得た。
（サンプル調製及びＨｙｐ定量）
培養物サンプルの調製及びＨｙｐ定量は、試験例２と同じ方法で行った。
結果を図５示す。 (Pre-culture)
One platinum loop of Yarrowia lipolytica NBRC0717 was inoculated into 1 mL of YPD medium, and a preculture was obtained by shaking culture (300 rpm) at 28 ° C. for 24 hours.
(Main culture)
To 2 mL of each of the above media (1) to (6), 0.1 mL of the preculture solution was inoculated and cultured at 28 ° C. for 4 days with shaking (300 rpm) to obtain a cell culture solution.
(Sample preparation and Hyp quantification)
Preparation of culture samples and Hyp quantification were performed in the same manner as in Test Example 2.
The results are shown in FIG.
図５は、ペプトン又はコラーゲンペプチドを含む培地で培養したYarrowia lipolyticaのＨｙｐ蓄積量、細胞あたりのＨｙｐ量及びＯＤ６００を示すグラフである。（（ａ）：Ｈｙｐ蓄積量（μｇ／ｍＬ）、（ｂ）：細胞あたりのＨｙｐ量（μｇ／ｍＬ／ＯＤ６００）、（ｃ）：ＯＤ６００）。図５の（ａ）～（ｃ）中、Ｎは、Ｐがノーマルの培地、５０は、ＰがＣＰ５０の培地、１００はＰがＣＰ１００の培地であることを意味する。ペプトンを完全にコラーゲンペプチドに置換した培地でもYarrowia lipolyticaは増殖でき、Ｈｙｐを蓄積した。 FIG. 5 is a graph showing Hyp accumulation amount, Hyp amount per cell, and OD600 of Yarrowia lipolytica cultured in a medium containing peptone or collagen peptide. ((A): Hyp accumulation amount (μg / mL), (b): Hyp amount per cell (μg / mL / OD600), (c): OD600). In FIGS. 5A to 5C, N means P is a normal medium, 50 means P is a CP50 medium, and 100 means P is a CP100 medium. Yarrowia lipolytica was able to grow even in a medium in which peptone was completely replaced with collagen peptide, and Hyp was accumulated.
＜試験例７＞
Yarrowia lipolytica NBRC0717を用いて、培養時のエアレーションの影響を検討した。培地は、試験例５のＹＰＤ培地＃１及び＃１０を使用した。 <Test Example 7>
Yarrowia lipolytica NBRC0717 was used to examine the influence of aeration during culture. The YPD media # 1 and # 10 of Test Example 5 were used as the media.
（前培養）
ＹＰＤ培地１ｍＬに、Yarrowia lipolytica NBRC0717を１白金耳接種し、２８℃で２４時間振盪培養（３００ｒｐｍ）して前培養液を得た。
（本培養）
各ＹＰＤ培地（＃１又は＃１０）２ｍＬに、前培養液０．１ｍＬを接種して、２８℃で３日間振盪培養（３００ｒｐｍ）又は静置培養して菌体培養液を得た。
（サンプル調製及びＨｙｐ定量）
培養物サンプルの調製及びＨｙｐ定量は、試験例２と同じ方法で行った。また、培養上清についても、培養物サンプルと同じ方法で誘導体化し、ＨＰＬＣでＨｙｐを定量した。
結果を図６に示す。 (Pre-culture)
One platinum loop of Yarrowia lipolytica NBRC0717 was inoculated into 1 mL of YPD medium, and a preculture was obtained by shaking culture (300 rpm) at 28 ° C. for 24 hours.
(Main culture)
To 2 mL of each YPD medium (# 1 or # 10), 0.1 mL of the preculture solution was inoculated, and shake culture (300 rpm) or static culture at 28 ° C. for 3 days to obtain a cell culture solution.
(Sample preparation and Hyp quantification)
Preparation of culture samples and Hyp quantification were performed in the same manner as in Test Example 2. The culture supernatant was also derivatized by the same method as the culture sample, and Hyp was quantified by HPLC.
The results are shown in FIG.
図６は、振盪培養又は静置培養したYarrowia lipolyticaのＨｙｐ蓄積量を示す図である（（ａ）：培養物サンプル（細胞内）の培地あたり換算Ｈｙｐ量、（ｂ）：培養上清中のＨｙｐ量）。
Yarrowia lipolyticaは、好気培養するとＨｙｐ蓄積量が多かった。 FIG. 6 is a diagram showing the amount of Hyp accumulated in Yarrowia lipolytica cultured by shaking culture or stationary culture ((a): Hyp equivalent per culture medium in culture sample (intracellular), (b): in culture supernatant. Hyp amount).
Yarrowia lipolytica had a large amount of Hyp accumulation when aerobically cultured.
＜試験例８＞
試験例７で得られた培養上清中のエタノール濃度及びグルコース濃度をＨＰＬＣ（発酵カラム（バイオ・ラッド社製、製品名Aminex発酵モニター用カラム）を使用）で定量した。
結果を図７に示す。 <Test Example 8>
The ethanol concentration and glucose concentration in the culture supernatant obtained in Test Example 7 were quantified by HPLC (fermentation column (manufactured by Bio-Rad, product name: Aminex fermentation monitor column)).
The results are shown in FIG.
図７は、振盪培養又は静置培養したYarrowia lipolyticaの培養上清のエタノール濃度及びグルコース濃度を示すグラフである（（ａ）：エタノール濃度（ｖ／ｖ％）、（ｂ）：グルコース濃度（重量％））。 FIG. 7 is a graph showing the ethanol concentration and glucose concentration of the culture supernatant of Yarrowia lipolytica subjected to shaking culture or stationary culture ((a): ethanol concentration (v / v%), (b): glucose concentration (weight). %)).
＜試験例９＞
Yarrowia lipolytica NBRC0717について、ＹＰＤ培地の組成（Ｙ：Ｐ：Ｄの比率及びＰの種類）及び培養時間を変化させて本培養を行い、菌体培養物中のＨｙｐ含量及びＰｒｏ含量を測定した。 <Test Example 9>
For Yarrowia lipolytica NBRC0717, the main culture was performed while changing the composition of YPD medium (ratio of Y: P: D and the type of P) and the culture time, and the Hyp content and Pro content in the cell culture were measured.
（前培養）
ＹＰＤ培地３ｍＬに、Yarrowia lipolytica NBRC0717を１白金耳接種し、３０℃で１日静置培養して前培養液を得た。 (Pre-culture)
One platinum loop of Yarrowia lipolytica NBRC0717 was inoculated into 3 mL of YPD medium, and statically cultured at 30 ° C. for 1 day to obtain a preculture solution.
（本培養）
上記で得られた前培養液１００μＬを、下記のＹＰＤ培地５ｍＬに接種して３０℃で振盪培養（６０ｒｐｍ）した。培養時間は、１日又は２日として菌体培養液（菌体培養物）を得た。
本培養では、ＹＰＤ培地のＰとして、ペプトン又はコラーゲンペプチド（ＣＰ）を使用した。コラーゲンペプチドには、コラーゲンペプチド　イクオス　ＨＤＬ－５０ＳＰ（製品名、新田ゼラチン（株）製、平均分子量５０００）（以下、コラーゲンペプチド（ＣＰ１）と記載する）又はコラーゲンペプチドＴｙｐｅ　Ｓ（製品名、新田ゼラチン（株）製、平均分子量１２００）（以下、コラーゲンペプチド（ＣＰ２）と記載する）を使用した。
また、本培養におけるＹＰＤ培地のＹ：Ｐ：Ｄの比率は、（１）Ｙ：Ｐ：Ｄ＝１：２：２（重量比）（Ｙ：酵母抽出物１．０％、Ｐ：ペプトン又はコラーゲンペプチド２．０％、Ｄ：グルコース２．０％）、又は、（２）Ｙ：Ｐ：Ｄ＝１：４：５（重量比）（Ｙ：酵母抽出物１．０％、Ｐ：ペプトン又はコラーゲンペプチド４．０％、Ｄ：グルコース５．０％）とした。表４に、本培養で使用した培養条件１～１２を示す。 (Main culture)
100 μL of the preculture solution obtained above was inoculated into 5 mL of the following YPD medium, followed by shaking culture (60 rpm) at 30 ° C. The culture time was 1 day or 2 days to obtain a cell culture solution (cell culture).
In the main culture, peptone or collagen peptide (CP) was used as P in the YPD medium. Collagen peptides include collagen peptide Iquos HDL-50SP (product name, Nitta Gelatin Co., Ltd., average molecular weight 5000) (hereinafter referred to as collagen peptide (CP1)) or collagen peptide Type S (product name, Nitta). Gelatin Co., Ltd., average molecular weight 1200) (hereinafter referred to as collagen peptide (CP2)) was used.
Moreover, the ratio of Y: P: D of the YPD medium in the main culture is (1) Y: P: D = 1: 2: 2 (weight ratio) (Y: yeast extract 1.0%, P: peptone or Collagen peptide 2.0%, D: glucose 2.0%), or (2) Y: P: D = 1: 4: 5 (weight ratio) (Y: yeast extract 1.0%, P: peptone Or collagen peptide 4.0%, D: glucose 5.0%). Table 4 shows the culture conditions 1 to 12 used in the main culture.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
本培養で得られたYarrowia lipolyticaの菌体培養液に、以下の自己消化工程及び殺菌工程を行って培養物サンプルを調製し、Ｈｙｐ含量を測定した。
（自己消化工程）
Yarrowia lipolyticaの菌体培養液を５０℃で２時間インキュベートして、自己消化により菌体内容物を培養液中に溶出した。
（殺菌工程）
上記で自己消化した培養液を８０℃で１時間インキュベートした。
得られた抽出物から遠心分離（３０００ｒｐｍ、５ｍｉｎ、１℃）により菌体残渣を除いた。これにより、Ｈｙｐ蓄積量を測定するための培養物サンプルを調製した。 The Yarrowia lipolytica cell culture solution obtained in the main culture was subjected to the following autolysis and sterilization steps to prepare a culture sample, and the Hyp content was measured.
(Self digestion process)
The bacterial cell culture solution of Yarrowia lipolytica was incubated at 50 ° C. for 2 hours, and the bacterial cell contents were eluted into the culture solution by autolysis.
(Sterilization process)
The culture solution self-digested above was incubated at 80 ° C. for 1 hour.
The cell residue was removed from the obtained extract by centrifugation (3000 rpm, 5 min, 1 ° C.). This prepared the culture sample for measuring the amount of Hyp accumulation.
（サンプル調製）
得られた培養物サンプルを０．１Ｎ塩酸溶液で希釈して、ＨＰＬＣのサンプルを調製した。培養時間１日の菌体培養液から調製した培養物サンプルは、１０倍希釈、培養時間２日の菌体培養液から調製した培養物サンプルは、条件１２以外は４０倍希釈し、条件１２は２０倍希釈した。 (Sample preparation)
The obtained culture sample was diluted with 0.1N hydrochloric acid solution to prepare a sample for HPLC. The culture sample prepared from the bacterial cell culture medium with a culture time of 1 day is diluted 10 times, and the culture sample prepared from the bacterial cell culture medium with a culture time of 2 days is diluted 40 times except for the condition 12, Diluted 20 times.
（ＨＰＬＣ分析）
ＨＰＬＣでは、オートサンプラーで１級アミノ酸（１級アミノ基）、２級アミノ酸（２級アミノ基）の蛍光標識化を行い、逆相のＨＰＬＣで分析を行うシステムを用いた。１級アミノ基の蛍光標識には、ｏ－フタルアルデヒド（ＯＰＡ）を、２級アミノ基の蛍光標識には、クロロ蟻酸－９－フルオレニルメチル（ＦＭＯＣ）を、それぞれ使用した。 (HPLC analysis)
In HPLC, a system in which primary amino acids (primary amino groups) and secondary amino acids (secondary amino groups) were fluorescently labeled with an autosampler and analyzed by reversed-phase HPLC was used. For primary amino group fluorescent labeling, o-phthalaldehyde (OPA) was used, and for secondary amino group fluorescent labeling, chloroformate-9-fluorenylmethyl (FMOC) was used.
上記で調製したＨＰＬＣのサンプルを、サンプルバイアルに０．４５μｍのフィルターでろ過してオートサンプラーにセットした。空バイアルにＭＰＡ（メルカプトプロピオン酸）試薬３０μＬ、ＯＰＡ試薬１５μＬ、上記サンプル５μＬを入れて１分静置後、ＦＭＯＣ試薬を５μＬ加え２分間反応した溶液１μＬをＨＰＬＣに注入した。ＯＰＡと１級アミノ酸が反応し、残った２級アミノ基を持つＨｙｐ及びＰｒｏがＦＭＯＣと反応する。それぞれの蛍光波長を２チャンネルで検出することにより、すべてのアミノ酸を同時に検出することが可能である。 The HPLC sample prepared above was filtered through a 0.45 μm filter in a sample vial and set in an autosampler. An empty vial was charged with 30 μL of MPA (mercaptopropionic acid) reagent, 15 μL of OPA reagent, and 5 μL of the above sample and allowed to stand for 1 minute, and then 5 μL of FMOC reagent was added and 1 μL of the reacted solution was injected into HPLC. OPA reacts with primary amino acid, and Hyp and Pro having the remaining secondary amino group react with FMOC. By detecting each fluorescence wavelength with two channels, it is possible to detect all amino acids simultaneously.
ＨＰＬＣ分析に使用した装置及び条件を以下に示す。
（装置）
（株）島津製作所製の高速液体クロマトグラフ　Ｎｅｘｅｒａ　Ｘ２　システム（製品名）
システムコントローラ：ＣＢＭ－２０Ａ、送液ユニット：ＬＣ－３０ＡＤ（２台）、脱気ユニット：ＤＧＵ－２０Ａ５Ｒ、ミキサ：ＭＲ１８０μＬ　ＩＩ、オートサンプラー：ＳＩＬ－３０ＡＣ、カラムオーブン：ＣＴＯ－２０ＡＣ、蛍光検出器：ＲＦ－２０ＡＸＳ、ワークステーション：ＬａｂＳｏｌｕｔｉｏｎｓ　ＬＣ／ＧＣ The apparatus and conditions used for the HPLC analysis are shown below.
(apparatus)
High-performance liquid chromatograph Nexera X2 system (product name) manufactured by Shimadzu Corporation
System controller: CBM-20A, liquid feeding unit: LC-30AD (2 units), deaeration unit: DGU-20A5R, mixer: MR180 μL II, autosampler: SIL-30AC, column oven: CTO-20AC, fluorescence detector: RF-20AXS, workstation: LabSolutions LC / GC
（測定条件）
カラム：Ｉｎｅｒｔｓｉｌ　ＯＤＳ－４　１００ｍｍ×３．０ｍｍφ　Ｓ－２μｍ　（ジーエルサイエンス（株））
ガードカラム：ＵＨＰＬＣ　Ｆｉｔｔｉｎｇ（製品名、ジーエルサイエンス（株））（Ｍａｘ．　Ｐｒｅｓｓｕｒｅ：１３０ＭＰａ）
移動相
Ａ液：１５ｍｍｏｌ／Ｌ　ＫＨ２ＰＯ４及び５ｍｍｏｌ／Ｌ　Ｋ２ＨＰＯ４（ｐＨ６．５）
Ｂ液：１５／４５／４０（ｖ／ｖ／ｖ）＝水／アセトニトリル／メタノール
Ｒ０（リンス液）：水／メタノール＝２０／８０（ｖ／ｖ）
Ｒ３（リンス液）：水／アセトニトリル＝８０／２０（ｖ／ｖ）
初期Ｂ液濃度：１０ｖ／ｖ％
流量：０．８ｍＬ／ｍｉｎ
カラムオーブン温度：３５℃
注入量：１μＬ
検出：
Ｃｈ１：励起波長３５０ｎｍ、蛍光波長４５０ｎｍ
Ｃｈ２：励起波長２６６ｎｍ、蛍光波長３０５ｎｍ
セル温度:２５℃、Gain: ×4、Sensitivity: Midium (Measurement condition)
Column: Inertsil ODS-4 100 mm × 3.0 mmφ S-2 μm (GL Sciences Inc.)
Guard column: UHPLC Fitting (product name, GL Sciences Inc.) (Max. Pressure: 130 MPa)
Mobile phase A solution: 15 mmol / L KH 2 PO 4 and 5 mmol / L K 2 HPO 4 (pH 6.5)
Liquid B: 15/45/40 (v / v / v) = water / acetonitrile / methanol R0 (rinse liquid): water / methanol = 20/80 (v / v)
R3 (rinse solution): water / acetonitrile = 80/20 (v / v)
Initial solution B concentration: 10 v / v%
Flow rate: 0.8mL / min
Column oven temperature: 35 ° C
Injection volume: 1 μL
detection:
Ch1: excitation wavelength 350 nm, fluorescence wavelength 450 nm
Ch2: excitation wavelength 266 nm, fluorescence wavelength 305 nm
Cell temperature: 25 ° C, Gain: × 4, Sensitivity: Midium
グラジエントプログラム
０～１．５分：Ｂ．Ｃｏｎｃ　１０ｖ／ｖ％
１．５～６分：Ｂ．Ｃｏｎｃ　１０ｖ／ｖ％→３０ｖ／ｖ％のグラジエント
６～１１分：Ｂ．Ｃｏｎｃ　３０ｖ／ｖ％→４０ｖ／ｖ％のグラジエント
１１～１５分：Ｂ．Ｃｏｎｃ　１００ｖ／ｖ％
２０～２１．５分：Ｂ．Ｃｏｎｃ　１００ｖ／ｖ％→１０ｖ／ｖ％
２５分：コントローラ停止
検量線：Ｈｙｐ及びＰｒｏそれぞれについて、６．２５μｍｏｌ／Ｌ、２５μｍｏｌ／Ｌ、５０μｍｏｌ／Ｌ、１００μｍｏｌ／Ｌの０．１Ｎ　ＨＣｌ溶液を調製した。これらの各溶液を、上記の方法でＭＰＡ、ＯＰＡ及びＦＭＯＣの各試薬と反応させ、ＨＰＬＣで分析して検量線を引いた。
アミノ酸混合標準液Ｈ型溶液及びＬ－ヒドロキシプロリンを含む０．１Ｎ塩酸溶液についても、同様に各試薬と反応させてＨＰＬＣで分析した。使用したアミノ酸混合標準液Ｈ型は、和光純薬工業（株）製である。 Gradient program 0-1.5 minutes: B. Conc 10v / v%
1.5-6 minutes: B.I. Conc 10 v / v% → 30 v / v% gradient 6-11 minutes: Conc 30v / v% → 40v / v% gradient 11-15 min: Conc 100v / v%
20-21.5 min. Conc 100v / v% → 10v / v%
25 min: Controller stop calibration curve: For each of Hyp and Pro, 6.25 μmol / L, 25 μmol / L, 50 μmol / L, and 100 μmol / L 0.1N HCl solutions were prepared. Each of these solutions was reacted with each reagent of MPA, OPA and FMOC by the above method, and analyzed by HPLC to draw a calibration curve.
The amino acid mixed standard solution H type solution and 0.1N hydrochloric acid solution containing L-hydroxyproline were similarly reacted with each reagent and analyzed by HPLC. The amino acid mixed standard solution H type used is manufactured by Wako Pure Chemical Industries, Ltd.
図８は、アミノ酸混合標準液Ｈ型及びＬ－ヒドロキシプロリンを含む０．１Ｎ塩酸溶液（各アミノ酸濃度２０μｍｏｌ／Ｌ）を分析したＨＰＬＣチャートである（（ａ）：Ｃｈ１の励起波長３５０ｎｍ、蛍光波長４５０ｎｍで検出、（ｂ）：Ｃｈ２の励起波長２６６ｎｍ、蛍光波長３０５ｎｍで検出）。 FIG. 8 is an HPLC chart obtained by analyzing a 0.1N hydrochloric acid solution containing amino acid mixed standard solution H and L-hydroxyproline (each amino acid concentration 20 μmol / L) ((a): Ch1 excitation wavelength 350 nm, fluorescence wavelength. Detection at 450 nm, (b): Ch2 excitation wavelength 266 nm, fluorescence wavelength 305 nm detection).
培養物サンプルのＨｙｐ及びＰｒｏの定量結果を表５に示す。表中、ＣＰ１は、上記のコラーゲンペプチド（ＣＰ１）（製品名コラーゲンペプチド　イクオス　ＨＤＬ－５０ＳＰ）であり、ＣＰ２は、コラーゲンペプチド（ＣＰ２）（製品名コラーゲンペプチド　Ｔｙｐｅ　Ｓ）である。Ｈｙｐ及びＰｒｏの定量結果から、各培養物サンプル中のＰｒｏ及びＨｙｐの合計含量に対するＨｙｐの含量の割合（１００×Ｈｙｐ／（Ｐｒｏ＋Ｈｙｐ））を計算した。その結果も表５に示す。表の培養条件は、本培養の条件である。なお、培養開始前の各培地には、遊離のＨｙｐはほとんど含まれなかった。 Table 5 shows the quantification results of Hyp and Pro of the culture sample. In the table, CP1 is the above-described collagen peptide (CP1) (product name collagen peptide Iquos HDL-50SP), and CP2 is collagen peptide (CP2) (product name collagen peptide Type S). From the quantification results of Hyp and Pro, the ratio of the content of Hyp to the total content of Pro and Hyp in each culture sample (100 × Hyp / (Pro + Hyp)) was calculated. The results are also shown in Table 5. The culture conditions in the table are the conditions for main culture. Each medium before the start of culture contained almost no free Hyp.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
＜試験例１０＞
（前培養）
ＹＰＤ培地３ｍＬに、Yarrowia lipolyticaを１白金耳接種し、２８℃で１日振盪培養（１００ｒｐｍ）して前培養液を得た。 <Test Example 10>
(Pre-culture)
One platinum loop of Yarrowia lipolytica was inoculated into 3 mL of YPD medium, and precultured at 28 ° C. for 1 day with shaking (100 rpm).
（本培養）
培地にＹＰＤ改変培地（酵母抽出物１．０％、コラーゲンペプチド（ＣＰ１）２．０％、グルコース１．０％）を使用し、２８℃で２日間、振盪培養（２００～５００ｒｐｍ）した以外は、試験例９と同じ方法で本培養を行った。コラーゲンペプチド（ＣＰ１）は、試験例９と同じものである。
本培養で得られたYarrowia lipolyticaの菌体培養液に、試験例９と同じ方法で自己消化工程及び殺菌工程等を行って培養物サンプルを調製した。試験例９と同じ方法で、Ｈｙｐ含量及びＰｒｏ含量を測定した。試験はｎ＝４で行った。結果を表６に示す (Main culture)
Except for using YPD modified medium (yeast extract 1.0%, collagen peptide (CP1) 2.0%, glucose 1.0%) as the medium and shaking culture (200-500 rpm) at 28 ° C for 2 days. The main culture was performed in the same manner as in Test Example 9. Collagen peptide (CP1) is the same as in Test Example 9.
A culture sample was prepared by subjecting the bacterial culture of Yarrowia lipolytica obtained in the main culture to a self-digestion step and a sterilization step in the same manner as in Test Example 9. The Hyp content and the Pro content were measured by the same method as in Test Example 9. The test was performed with n = 4. The results are shown in Table 6.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
＜試験例１１＞
試験例１～１０で使用したYarrowia lipolyticaを用いて、試験例９～１０と同じ方法で培養し、菌体培養液を得た。これを５０℃で２時間インキューベートすることにより、自己消化により菌体内容物を培養液中に溶出した後、８０℃で１時間インキュベートした。その後、１℃で遠心分離（３０００ｒｐｍ、５ｍｉｎ）し、菌体残渣を除いたＬ－ヒドロキシプロリン含有菌体培養物の抽出物（Ｈｙｐ含有菌体培養物の抽出物）を得た。Ｈｙｐ含有菌体培養物の抽出物は、適宜希釈して使用することもできる。 <Test Example 11>
Using Yarrowia lipolytica used in Test Examples 1 to 10, the cells were cultured in the same manner as in Test Examples 9 to 10 to obtain a cell culture solution. This was incubated at 50 ° C. for 2 hours to elute the cell contents into the culture solution by autolysis, and then incubated at 80 ° C. for 1 hour. Thereafter, the mixture was centrifuged at 1 ° C. (3000 rpm, 5 min) to obtain an L-hydroxyproline-containing cell culture extract (extract of Hyp-containing cell culture) excluding cell residues. The extract of the Hyp-containing cell culture can be used after appropriately diluted.
以下、試験例１１で得た各Ｈｙｐ含有菌体培養物の抽出物を配合した皮膚外用剤組成物の製造例の一例を示す。
＜製造例１＞石鹸
原料の配合量を表７に示す。
石鹸素地を混合攪拌し、その後各Ｈｙｐ含有菌体培養物の抽出物を投入し均一に混合後、成型した。 Hereinafter, an example of the manufacture example of the skin external preparation composition which mix | blended the extract of each Hyp containing microbial cell culture obtained in Test Example 11 is shown.
<Production Example 1> Table 7 shows blending amounts of soap raw materials.
The soap base was mixed and stirred, and then an extract of each Hyp-containing cell culture was added and uniformly mixed, followed by molding.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
＜製造例２＞シャンプー
原料の配合量を表８に示す。
精製水に１，３－ブチレングリコールに溶解した防腐剤を投入した。均一に攪拌後、ラウレス硫酸ナトリウム、ヤシ油脂肪酸モノエタノールアミドを投入し、その後色素、香料及び残りの１，３－ブチレングリコールを投入し、各Ｈｙｐ含有菌体培養物の抽出物を投入後、均一に混合攪拌した。 <Production Example 2> Table 8 shows the blending amounts of shampoo raw materials.
A preservative dissolved in 1,3-butylene glycol was added to purified water. After uniformly stirring, sodium laureth sulfate and coconut oil fatty acid monoethanolamide were added, and then a pigment, a fragrance and the remaining 1,3-butylene glycol were added, and each Hyp-containing cell culture extract was added, The mixture was uniformly mixed and stirred.
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
＜製造例３＞コンディショナー
原料の配合量を表９に示す。
（１）塩化ステアリルジメチルベンジルアンモニウム及び食塩を精製水に投入し、８０℃まで加温し溶解した。
（２）セトステアリルアルコール、水素添加ポリイソブテン及びグリセリンモノステアレートを８０℃まで加温し溶解した。
（３）（１）をホモミキサーで攪拌しながら（２）を添加し、添加後５分間予備攪拌を行った。
（４）予備攪拌終了後、５０℃まで攪拌しながら冷却し、各Ｈｙｐ含有菌体培養物の抽出物を添加しさらに３５℃まで攪拌冷却し調製した。 <Production Example 3> Table 9 shows the blending amounts of the conditioner raw materials.
(1) Stearyldimethylbenzylammonium chloride and sodium chloride were added to purified water and heated to 80 ° C. to dissolve.
(2) Cetostearyl alcohol, hydrogenated polyisobutene and glycerin monostearate were heated to 80 ° C. and dissolved.
(3) (2) was added while stirring (1) with a homomixer, and preliminary stirring was performed for 5 minutes after the addition.
(4) After completion of the pre-stirring, the mixture was cooled to 50 ° C. with stirring, each Hyp-containing cell culture extract was added, and the mixture was further cooled to 35 ° C. with stirring.
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
＜製造例４＞ヘアトニック
原料の配合量を表１０に示す。
精製水にサリチル酸、グリセリン、エタノールに溶解したビタミンＥ、Ｌ－メントールを投入し、さらに精製水の一部に溶解したグリチルリチン酸ジカリウムを投入し、その後各Ｈｙｐ含有菌体培養物の抽出物を投入し、均一に混合し調製した。 <Production Example 4> Table 10 shows the blending amounts of the hair tonic raw materials.
Vitamin E and L-menthol dissolved in salicylic acid, glycerin, and ethanol are added to purified water, and dipotassium glycyrrhizinate dissolved in a portion of purified water is added, and then each Hyp-containing cell culture extract is added. And mixed uniformly to prepare.
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000010
＜製造例５＞ミスト
原料の配合量を表１１に示す。
精製水にクエン酸及びクエン酸ナトリウムを投入し溶解した。その後エタノールに溶解した防腐剤及びポリソルベート８０を投入した。その後各Ｈｙｐ含有菌体培養物の抽出物を投入し均一に攪拌し調製した。 <Production Example 5> Table 11 shows the amounts of the mist raw materials.
Citric acid and sodium citrate were added to purified water and dissolved. Thereafter, an antiseptic and polysorbate 80 dissolved in ethanol were added. Thereafter, each Hyp-containing cell culture extract was added and stirred uniformly.
Figure JPOXMLDOC01-appb-T000011
Figure JPOXMLDOC01-appb-T000011
＜製造例６＞化粧水
原料の配合量を表１２に示す。
精製水にクエン酸及びクエン酸ナトリウムを投入し溶解した。次にグリセリン、１，３－ブチレングリコール及びエチレンジアミン四酢酸三ナトリウムを順次投入し、さらにエタノールに溶解したポリオキシエチレン（１８）オレイルアルコールエーテル、ビタミンＥ及びメチルパラベンを投入し均一になるまで攪拌した。その後各Ｈｙｐ含有菌体培養物の抽出物を投入し均一に攪拌し調製した。 <Production Example 6> Table 12 shows the amounts of the lotion ingredients.
Citric acid and sodium citrate were added to purified water and dissolved. Next, glycerin, 1,3-butylene glycol and trisodium ethylenediaminetetraacetate were sequentially added, and polyoxyethylene (18) oleyl alcohol ether dissolved in ethanol, vitamin E and methylparaben were added and stirred until uniform. Thereafter, each Hyp-containing cell culture extract was added and stirred uniformly.
Figure JPOXMLDOC01-appb-T000012
Figure JPOXMLDOC01-appb-T000012
＜製造例７＞乳液
原料の配合量を表１３に示す。
（１）ステアリン酸、セチルアルコール、ミリスチン酸オクチルドデシル及び流動パラフィンを８０℃まで加温し溶解した。
（２）トリエタノールアミン、ヒアルロン酸ナトリウム、グリセリン、１，３－ブチレングリコール、ポリオキシエチレン（１０）モノオレイン酸エステル及びエチレンジアミンヒドロキシ三酢酸ナトリウムを精製水に投入し８０℃まで加温した。
（３）（１）をホモミキサーで攪拌しながら（２）を投入し、投入後５分間予備攪拌した。
（４）予備攪拌終了後、５０℃まで冷却し、各Ｈｙｐ含有菌体培養物の抽出物を加え、さらに３５℃まで冷却し調製した。 <Production Example 7> Table 13 shows the amounts of the emulsion raw materials.
(1) Stearic acid, cetyl alcohol, octyldodecyl myristate and liquid paraffin were heated to 80 ° C. and dissolved.
(2) Triethanolamine, sodium hyaluronate, glycerin, 1,3-butylene glycol, polyoxyethylene (10) monooleate and sodium ethylenediaminehydroxytriacetate were added to purified water and heated to 80 ° C.
(3) While stirring (1) with a homomixer, (2) was added and pre-stirred for 5 minutes after the addition.
(4) After completion of preliminary stirring, the mixture was cooled to 50 ° C., an extract of each Hyp-containing cell culture was added, and further cooled to 35 ° C. for preparation.
Figure JPOXMLDOC01-appb-T000013
Figure JPOXMLDOC01-appb-T000013
＜製造例８＞クリーム
原料の配合量を表１４に示す。
（１）ステアリン酸、モノステアリン酸グリセリル、セスキステアリン酸ソルビタン、モノステアリン酸ポリオキシエチレンソルビタン、セトステアリルアルコール、スクワラン、ヘキサ（ヒドロキシステアリン酸／ステアリン酸／ロジン酸）ジペンタエリスリット、オリーブ油、ミリスチン酸オクチルドデシル及びメチルポリシロキサンを８０℃まで加温し溶解した。
（２）精製水にグリセリン、１，３－ブチレングリコール、水酸化ナトリウム及びメチルパラベンを投入し、８０℃まで加温し溶解した。
（３）（１）をホモミキサーで攪拌しながら（２）を投入し、投入後５分間予備攪拌した。
（４）予備攪拌終了後、５０℃まで冷却し、各Ｈｙｐ含有菌体培養物の抽出物を加え、さらに３５℃まで冷却し調製した。 <Production Example 8> Table 14 shows the blending amounts of the cream raw materials.
(1) Stearic acid, glyceryl monostearate, sorbitan sesquistearate, polyoxyethylene sorbitan monostearate, cetostearyl alcohol, squalane, hexa (hydroxystearic acid / stearic acid / rosinic acid) dipentaerythlit, olive oil, myristic Octyldodecyl acid and methylpolysiloxane were dissolved by heating to 80 ° C.
(2) Glycerin, 1,3-butylene glycol, sodium hydroxide and methylparaben were added to purified water and heated to 80 ° C. to dissolve.
(3) While stirring (1) with a homomixer, (2) was added and pre-stirred for 5 minutes after the addition.
(4) After completion of preliminary stirring, the mixture was cooled to 50 ° C., an extract of each Hyp-containing cell culture was added, and further cooled to 35 ° C. for preparation.
Figure JPOXMLDOC01-appb-T000014
Figure JPOXMLDOC01-appb-T000014
本発明のＬ－ヒドロキシプロリンを含有する酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)の菌体、菌体培養物及びこれらの抽出物は、化粧料、飲食品等の原料として有用である。 The yeast, Yarrowia lipolytica microbial cells, bacterial cell cultures and extracts thereof containing L-hydroxyproline of the present invention are useful as raw materials for cosmetics, foods and drinks and the like.

Claims (21)

  1. 酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)の菌体もしくは菌体培養物又はこれらの抽出物であって、
    前記酵母の菌体もしくは菌体培養物又はこれらの抽出物は、Ｌ－ヒドロキシプロリンを含有し、Ｌ－プロリン（Ｐｒｏ）及びＬ－ヒドロキシプロリン（Ｈｙｐ）の合計含量（μｇ／ｍＬ）に対するＬ－ヒドロキシプロリンの含量（μｇ／ｍＬ）の割合（１００×Ｈｙｐ／（Ｐｒｏ＋Ｈｙｐ））が、３５～１００であることを特徴とする酵母の菌体もしくは菌体培養物又はこれらの抽出物。     A yeast or yeast culture of yeast Yarrowia lipolytica or an extract thereof,
    The yeast cells or cell cultures or extracts thereof contain L-hydroxyproline, and L-hydroxy to the total content (μg / mL) of L-proline (Pro) and L-hydroxyproline (Hyp). A yeast cell or a cell culture or an extract thereof, wherein the content of hydroxyproline (μg / mL) (100 × Hyp / (Pro + Hyp)) is 35 to 100.
  2. Ｌ－ヒドロキシプロリンの含量が１０μｇ／ｍＬ以上である請求項１に記載の酵母の菌体もしくは菌体培養物又はこれらの抽出物。 The yeast cell or cell culture or extract thereof according to claim 1, wherein the content of L-hydroxyproline is 10 µg / mL or more.
  3. 酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)の菌体もしくは菌体培養物又はこれらの抽出物であって、Ｌ－ヒドロキシプロリンの含量が１０μｇ／ｍＬ以上であることを特徴とする酵母の菌体もしくは菌体培養物又はこれらの抽出物。 A yeast cell or cell culture of yeast Yarrowia lipolytica or an extract thereof, wherein the content of L-hydroxyproline is 10 μg / mL or more Cultures or extracts thereof.
  4. 酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)を、炭素源及び窒素源を含む液体培地中で好気培養することにより、前記酵母の菌体又は菌体培養物中にＬ－ヒドロキシプロリンを蓄積させる工程を含み、前記窒素源がＬ－ヒドロキシプロリン含有ペプチドを含む窒素源であることを特徴とするＬ－ヒドロキシプロリンの製造方法。 A process of accumulating L-hydroxyproline in a yeast cell or a cell culture of the yeast by aerobic culture of yeast Yarrowia lipolytica in a liquid medium containing a carbon source and a nitrogen source. The method for producing L-hydroxyproline, wherein the nitrogen source is a nitrogen source containing an L-hydroxyproline-containing peptide.
  5. 前記Ｌ－ヒドロキシプロリン含有ペプチドがコラーゲンペプチドである請求項４に記載の製造方法。 The production method according to claim 4, wherein the L-hydroxyproline-containing peptide is a collagen peptide.
  6. 前記コラーゲンペプチドの平均分子量が１０００～１００００である請求項５に記載の製造方法。 The production method according to claim 5, wherein the collagen peptide has an average molecular weight of 1,000 to 10,000.
  7. 前記好気培養を１０～１００時間行う請求項４～６のいずれかに記載の製造方法。 The production method according to any one of claims 4 to 6, wherein the aerobic culture is performed for 10 to 100 hours.
  8. 酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)の、Ｌ－ヒドロキシプロリンを製造するための使用。 Use of the yeast Yarrowia lipolytica for the production of L-hydroxyproline.
  9. 前記酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)を、炭素源及び窒素源を含む液体培地中で好気培養することにより、前記酵母の菌体又は菌体培養物中にＬ－ヒドロキシプロリンを蓄積させることを含み、前記窒素源がＬ－ヒドロキシプロリン含有ペプチドを含む窒素源である請求項８に記載の使用。 The yeast Yarrowia lipolytica is aerobically cultured in a liquid medium containing a carbon source and a nitrogen source, thereby accumulating L-hydroxyproline in the yeast cell or cell culture. The use according to claim 8, wherein the nitrogen source is a nitrogen source comprising an L-hydroxyproline-containing peptide.
  10. 前記Ｌ－ヒドロキシプロリン含有ペプチドがコラーゲンペプチドである請求項９に記載の使用。 The use according to claim 9, wherein the L-hydroxyproline-containing peptide is a collagen peptide.
  11. 前記コラーゲンペプチドの平均分子量が１０００～１００００である請求項１０に記載の使用。 The use according to claim 10, wherein the collagen peptide has an average molecular weight of 1,000 to 10,000.
  12. 前記好気培養を１０～１００時間行う請求項９～１１のいずれかに記載の使用。 The use according to any one of claims 9 to 11, wherein the aerobic culture is performed for 10 to 100 hours.
  13. 請求項１～３のいずれかに記載の酵母の菌体もしくは菌体培養物又はこれらの抽出物を含むことを特徴とする組成物。 A composition comprising the yeast cell or cell culture or extract thereof according to any one of claims 1 to 3.
  14. 請求項１～３のいずれかに記載の酵母の菌体もしくは菌体培養物又はこれらの抽出物を含むことを特徴とする飲食品。 A food or drink comprising the yeast cell or bacterial cell culture according to any one of claims 1 to 3 or an extract thereof.
  15. 請求項１～３のいずれかに記載の酵母の菌体もしくは菌体培養物又はこれらの抽出物を含むことを特徴とする化粧料又は化粧料原料。 A cosmetic or a cosmetic raw material comprising the yeast cell culture or cell culture or extract thereof according to any one of claims 1 to 3.
  16. コラーゲン産生促進、表皮細胞の増殖促進、皮膚の保湿、皮膚の老化防止、皮膚のたるみの予防又は改善、皮膚のハリの改善、しわの予防又は改善及びアトピー性皮膚炎の改善から選ばれる用途に用いられる請求項１５に記載の化粧料又は化粧料原料。 For applications selected from promotion of collagen production, promotion of epidermal cell growth, skin moisturization, prevention of skin aging, prevention or improvement of skin sagging, improvement of skin firmness, prevention or improvement of wrinkles and improvement of atopic dermatitis The cosmetic or cosmetic raw material according to claim 15, which is used.
  17. 前記化粧料又は化粧料原料は、化粧料原料であり、Ｌ－ヒドロキシプロリン含量が５～３００ｐｐｍである請求項１５又は１６に記載の化粧料又は化粧料原料。 The cosmetic or cosmetic raw material according to claim 15 or 16, wherein the cosmetic or cosmetic raw material is a cosmetic raw material and has an L-hydroxyproline content of 5 to 300 ppm.
  18. 前記化粧料又は化粧料原料は、化粧料であり、Ｌ－ヒドロキシプロリン含量が０．０１～２０ｐｐｍである請求項１５又は１６に記載の化粧料又は化粧料原料。 The cosmetic or cosmetic raw material according to claim 15 or 16, wherein the cosmetic or cosmetic raw material is a cosmetic and has an L-hydroxyproline content of 0.01 to 20 ppm.
  19. Ｌ－ヒドロキシプロリンを含有する酵母ヤロウィア・リポリティカ(Yarrowia lipolytica)の菌体もしくは菌体培養物又はこれらの抽出物を含むことを特徴とするＬ－ヒドロキシプロリン補強用組成物。 A composition for reinforcing L-hydroxyproline, comprising a cell or a cell culture of yeast Yarrowia lipolytica containing L-hydroxyproline or an extract thereof.
  20. 前記酵母の菌体もしくは菌体培養物又はこれらの抽出物は、Ｌ－プロリン（Ｐｒｏ）及びＬ－ヒドロキシプロリン（Ｈｙｐ）の合計含量（μｇ／ｍＬ）に対するＬ－ヒドロキシプロリンの含量（μｇ／ｍＬ）の割合（１００×Ｈｙｐ／（Ｐｒｏ＋Ｈｙｐ））が、３５～１００である請求項１９に記載のＬ－ヒドロキシプロリン補強用組成物。 The yeast cells or cell cultures or extracts thereof have an L-hydroxyproline content (μg / mL) relative to the total content (μg / mL) of L-proline (Pro) and L-hydroxyproline (Hyp). The composition for reinforcing L-hydroxyproline according to claim 19, wherein the ratio (100 × Hyp / (Pro + Hyp)) is 35 to 100.
  21. 前記酵母の菌体もしくは菌体培養物又はこれらの抽出物は、Ｌ－ヒドロキシプロリンの含量が１０μｇ／ｍＬ以上である請求項１９又は２０に記載のＬ－ヒドロキシプロリン補強用組成物。 21. The composition for reinforcing L-hydroxyproline according to claim 19 or 20, wherein the yeast cell or cell culture or an extract thereof has an L-hydroxyproline content of 10 μg / mL or more.
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