WO2017195873A1 - Cellule de levure contenant de la l-hydroxyproline de yarrowia lipolytica ou produit de culture cellulaire ou son extrait, son utilisation, et procédé de production de l-hydroxyproline - Google Patents

Cellule de levure contenant de la l-hydroxyproline de yarrowia lipolytica ou produit de culture cellulaire ou son extrait, son utilisation, et procédé de production de 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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English (en)
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/ko
Priority to CN201780029019.0A priority patent/CN109072171A/zh
Priority to JP2018517082A priority patent/JP6869972B2/ja
Publication of WO2017195873A1 publication Critical patent/WO2017195873A1/fr

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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.

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Abstract

La présente invention concerne une cellule de levure de Yarrowia lipolytica, un produit de culture cellulaire de celle-ci ou un extrait de celle-ci. Ladite cellule de levure, ledit produit de culture cellulaire et ledit extrait de celle-ci sont caractérisés en ce qu'ils contiennent de la L-hydroxyproline, et le rapport [100 × Hyp/ (Pro + Hyp)] de la teneur (μg/mL) de L-hydroxyproline (Hyp) sur la teneur totale (μg/mL) de L-proline (Pro) et de L-hydroxyproline étant de 35 à 100.
PCT/JP2017/017937 2016-05-12 2017-05-11 Cellule de levure contenant de la l-hydroxyproline de yarrowia lipolytica ou produit de culture cellulaire ou son extrait, son utilisation, et procédé de production de l-hydroxyproline WO2017195873A1 (fr)

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WO2020113289A1 (fr) * 2018-12-07 2020-06-11 Atp Institute Pty Ltd Formulations comprenant de l'hydroxyproline non-dérivée d'animal et leur utilisation

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CN110760451B (zh) * 2019-10-21 2022-08-30 广东轻工职业技术学院 一株解脂耶式酵母及其在制备低糖低脂椰蓉营养粉中的用途
CN112198252B (zh) * 2020-09-27 2022-09-27 上海市质量监督检验技术研究院 一种检测纺织品中胶原蛋白含量的测定方法
KR102510688B1 (ko) * 2022-06-15 2023-03-17 아스티스(주) 야로위아 리폴리티카 변이균주 및 이를 포함하는 기능성 화장료 조성물

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