WO2021106462A1 - 液中での脂質類粒子の製造方法及び微生物の培養方法 - Google Patents

液中での脂質類粒子の製造方法及び微生物の培養方法 Download PDF

Info

Publication number
WO2021106462A1
WO2021106462A1 PCT/JP2020/040062 JP2020040062W WO2021106462A1 WO 2021106462 A1 WO2021106462 A1 WO 2021106462A1 JP 2020040062 W JP2020040062 W JP 2020040062W WO 2021106462 A1 WO2021106462 A1 WO 2021106462A1
Authority
WO
WIPO (PCT)
Prior art keywords
lipids
lipid particles
liquid
gas
melting point
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2020/040062
Other languages
English (en)
French (fr)
Japanese (ja)
Inventor
猪狩尊史
西森塩穂美
平野優
一力啓晃
神田彰久
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Kaneka Corp
Original Assignee
Kaneka Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kaneka Corp filed Critical Kaneka Corp
Priority to JP2021561232A priority Critical patent/JPWO2021106462A1/ja
Priority to CN202080081915.3A priority patent/CN114761573A/zh
Publication of WO2021106462A1 publication Critical patent/WO2021106462A1/ja
Priority to US17/742,035 priority patent/US20220266210A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/12—Bioreactors or fermenters specially adapted for specific uses for producing fuels or solvents
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C3/00—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
    • C12M27/02—Stirrer or mobile mixing elements
    • 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; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2/00—Processes or devices for granulating materials, e.g. fertilisers in general; Rendering particulate materials free flowing in general, e.g. making them hydrophobic
    • B01J2/02—Processes or devices for granulating materials, e.g. fertilisers in general; Rendering particulate materials free flowing in general, e.g. making them hydrophobic by dividing the liquid material into drops, e.g. by spraying, and solidifying the drops
    • B01J2/06—Processes or devices for granulating materials, e.g. fertilisers in general; Rendering particulate materials free flowing in general, e.g. making them hydrophobic by dividing the liquid material into drops, e.g. by spraying, and solidifying the drops in a liquid medium
    • 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
    • C12N2330/00—Production

Definitions

  • the present invention relates to a method for producing fine particles of lipids in a liquid, specifically, a method for producing fine particles of lipid particles which can be assimilated by a microorganism but has low solubility in water, and a method for producing fine particles of microorganisms. Regarding the culture method.
  • microbial production and substance production by microorganisms are increasing against the background of increasing awareness of environmental issues, food issues, health and safety, and increasing natural or natural orientation.
  • a carbon source carbon source for culturing, fermentation, etc.
  • Typical examples of the carbon source include sugars, lipids (for example, animal and vegetable fats and oils containing fatty acids) and the like.
  • lipids have a melting point higher than the culture temperature of microorganisms and have extremely low solubility in water. Since such lipids coagulate in the culture medium, the assimilation property of microorganisms is low, and they cannot be suitably used.
  • solubility of fatty acids such as lauric acid, myristic acid, palmitic acid, and stearic acid having a high melting point is 0.1 g / L or less at a water temperature of 25 ° C. even for lauric acid having the highest solubility in water.
  • Non-Patent Document 1 describes that the fact that lauric acid is solid at the culture temperature increases the difficulty of culturing.
  • Patent Document 1 It has also been reported that fatty acids are adjusted to the form of an oil-in-water emulsion so as to have a large specific surface area and supplied to the culture solution as a carbon source to improve bacterial cell growth.
  • Patent Document 2 There is also a report that fat or oil is heated above its melting point and added to a medium in a dispersed state and used for the production of bioproducts by microorganisms.
  • the present invention efficiently finely disperses lipids having low solubility in water at room temperature and solid at room temperature in a liquid having a temperature lower than the melting point of the lipids, and the lipids in the liquid.
  • a method for producing lipid particles and a method for producing microorganisms for producing similar particles.
  • the present invention is, in one or more embodiments, a method for producing lipid particles in which molten lipids are coagulated into particles in a liquid to obtain lipid particles, wherein the lipids are water at 25 ° C.
  • a method for producing lipid particles in which molten lipids are coagulated into particles in a liquid to obtain lipid particles, wherein the lipids are water at 25 ° C.
  • a step of obtaining lipid particles by directly injecting a gas into the liquid from a gas supply port of a fluid nozzle to disperse the molten lipids in the liquid and coagulate them while forming particles.
  • the bifluid nozzle is heated to a temperature higher than the melting point of the lipids by 10 ° C. or more, and the ratio D50 of the volume median diameter D50 of the lipid particles to the orifice diameter Nd of the liquid supply port of the bifluid nozzle is D50.
  • the present invention relates to a method for producing a lipid particle, wherein / Nd is 0.0017 or more and 0.17 or less.
  • the present invention is also characterized in that, in one or more embodiments, the lipid particles are prepared in a culture solution by the method for producing lipid particles, and the microorganism is cultured in the culture solution containing the lipid fine particles.
  • the present invention relates to a method for culturing microorganisms.
  • lipids having low solubility in water at room temperature (25 ° C.) and solid at room temperature are efficiently finely dispersed in a liquid having a temperature lower than the melting point of the lipids.
  • Lipid particles can be produced in a liquid.
  • lipids having low solubility in water at room temperature and solid at room temperature are efficiently finely dispersed in a culture solution having a temperature lower than the melting point of the lipids to form particles.
  • the lipid particles can be suitably assimilated by microorganisms, and therefore useful substances such as microbial metabolites such as PHA can be industrially and efficiently produced.
  • FIG. 1 is a schematic view of a device for producing lipid particles used in one or more embodiments of the present invention.
  • FIG. 2 is a graph plotting data of volume median diameter of Vg / Vf and PFAD particles / orifice diameter of the liquid supply port in Examples 6 to 14.
  • the present inventors have conducted diligent studies in order to solve the above-mentioned problems.
  • the molten lipids are directly injected into the liquid having a temperature lower than the melting point of the lipids from the liquid supply port of the two-fluid nozzle heated to a temperature higher than the melting point of the lipids by 10 ° C. or more, and the two fluids.
  • the ratio D50 / Nd of the volume median diameter D50 of the target lipid particles to the orifice diameter Nd of the liquid supply port is 0.0017 or more and 0.17 or less.
  • in liquid means an aqueous solution or an aqueous dispersion containing water as the main solvent.
  • using water as the main solvent means that the solvent contains 90% by mass or more of water, preferably 95% by mass or more of water, and more preferably 100% by mass or more of water. Means to be.
  • the "melted lipids” means that the temperature of the lipids is equal to or higher than the melting point.
  • the lipids are not particularly limited as long as they have a solubility in water at 25 ° C. of 10 g / L or less and are solid at 25 ° C.
  • any lipid can be used as long as it can be assimilated by a microorganism.
  • the lipids include fatty acids, hydrocarbons, sterols, and mixtures thereof. Among them, lipids in which the target microorganism has a metabolic pathway can be preferably used.
  • fatty acids include fatty acids, fatty acid salts, fatty acid esters and the like.
  • fatty acid salts include fatty acid sodium, fatty acid potassium, fatty acid calcium, fatty acid magnesium and the like.
  • fatty acid ester include fatty acid glycerin ester and the like.
  • fatty acid glycerin ester include triglyceride, diglyceride, monoglyceride and the like.
  • hydrocarbons examples include paraffin (paraffin wax), wax, polyethylene wax, petrolatum, ceresin and the like.
  • sterols examples include cholesterol, 24-methylene sterol and the like.
  • the lipids are preferably animal and vegetable fats and oils, or fatty acid glycerin esters or fatty acids derived from animal and vegetable fats and oils. Further, considering the influence on food problems, it is more preferable to use fats and oils, fatty acids, waste oils and the like for non-edible uses.
  • animal and vegetable oils and fats examples include beef oil, pig fat, milk fat, fish oil, soybean oil, rapeseed oil, sunflower oil, olive oil, sesame oil, canola oil, peanut oil, tung oil, rice oil, cottonseed oil, rice oil, and saflower oil.
  • palm kernel oil such as palm olein and palm double olein, which is a low melting point fraction obtained by separating palm oil
  • palm kernel oil such as palm kernel oil olein, which is a low melting point fraction obtained by separating palm kernel oil, and the like.
  • fatty acids derived from animal and vegetable fats and oils include fatty acids, fatty acid salts, fatty acid esters and the like, which are constituents of the above-mentioned animal and vegetable fats and oils. These can be used alone or in combination of two or more.
  • lipids derived from palm oil include crude palm oil, crude palm kernel oil, palm oil, palm kernel oil, palm olein, palm double olein, palm kernel oil olein, and PFAD (palm oil fatty acid). Distillate), PKFAD (palm kernel oil fatty acid distillate), POME (Palm Oil Mill Effluent, waste liquid discharged in the process of obtaining crude palm oil from abra palm fruit), and EFB juice (Empty Fruit Bunch Juice, abra palm) (By-products obtained in the process of obtaining palm oil pellets from palm oil), and crude refined oils, fractionated oils, hardened oils, and ester-exchanged oils of these fats and oils can be mentioned.
  • a component produced as a by-product in the process of refining the animal and vegetable fats and oils in order to avoid competition with food.
  • by-products in the purification treatment include fatty acid salts produced in the alkaline deoxidizing step, distillation residues produced in the distillation deoxidizing step, and distillation residues produced in the deodorizing step.
  • the distillation residue which is a by-product of the purification treatment, may contain monoglyceride, diglyceride, etc. in addition to the fatty acid as the main component.
  • the lipids preferably have, for example, a melting point of 30 ° C. or higher and 120 ° C. or lower.
  • a melting point of 30 ° C. or higher and 120 ° C. or lower.
  • the melting point of lipids is more preferably 35 ° C. or higher, further preferably 38 ° C. or higher, and particularly preferably 40 ° C. or higher.
  • the melting point is 120 ° C. or lower, the coagulation of lipids in the nozzle is likely to be suppressed, and the flow path is less likely to be blocked.
  • the melting point of the lipids is more preferably 100 ° C. or lower, and even more preferably 80 ° C. or lower.
  • lipids having a solubility in water at 25 ° C. of 10 g / L or less and solid at 25 ° C. are optionally combined with other components such as other carbon sources. It may be used in combination.
  • the two-fluid nozzle (also referred to as an pneumatic spray nozzle or an air spray nozzle) is not particularly limited, and is a ratio D50 / of the volume median diameter D50 of the target lipid particles to the orifice diameter Nd of the liquid supply port.
  • a known bifluid nozzle having Nd of 0.0017 or more and 0.17 or less and capable of atomizing the liquid can be appropriately used.
  • D50 / Nd is 0.0017 or more and 0.17 or less, finely divided lipid particles having, for example, a volume median diameter of less than 1 mm can be suitably produced.
  • D50 / Nd is more preferably 0.005 or more and 0.14 or less, further preferably 0.01 or more and 0.10 or less, and 0.02 or more and 0. It is particularly preferably 08 or less.
  • the two-fluid nozzle is not particularly limited, but for example, from the viewpoint of suppressing clogging of the nozzle, it is preferable that the two-fluid nozzle is an external mixture type in which liquid and air are mixed outside the nozzle.
  • the molten lipids are directly injected (supplied) from the liquid supply port of the two-fluid nozzle into the liquid at a temperature lower than the melting point of the lipids, and the gas is injected into the liquid from the gas supply port of the two-fluid nozzle.
  • the injected gas makes it easier to obtain lipid particles by dispersing the molten lipids in the liquid and coagulating them while forming particles.
  • the two-fluid nozzle may be attached so that the liquid injection hole and the gas injection hole are located lower than the liquid level and in the liquid so that the molten lipids and gas can be directly injected into the liquid.
  • the material of the two-fluid nozzle is not particularly limited, but is preferably stainless steel, ceramic, titanium or the like from the viewpoint of high stability in a liquid such as a culture solution.
  • the two-fluid nozzle is heated to a temperature higher than the melting point of the lipids by 10 ° C. or more. As a result, it is possible to suppress the coagulation of lipids in the nozzle.
  • the heating temperature of the two-fluid nozzle is preferably 15 ° C. or higher, more preferably 20 ° C. or higher, higher than the melting point of the lipids.
  • the heating temperature of the two-fluid nozzle is not particularly limited, but for example, from the viewpoint of suppressing the denaturation of the medium, the denaturation of the secretions of the microorganism into the medium, the denaturation of the cells of the microorganism, and the adverse effect on the growth. Therefore, it is preferably 200 ° C. or lower, more preferably 180 ° C. or lower, and further preferably 160 ° C. or lower.
  • the heating method is not particularly limited.
  • the spray pattern of the liquid ejected from the nozzle is not particularly limited, but a circular pattern is preferable from the viewpoint that the spray flow rate is large and the particle size distribution tends to be uniform.
  • the two-fluid nozzle is not particularly limited, but for example, from the viewpoint of easily atomizing lipids in a liquid, the ratio Vg / Vf of the injection linear velocity Vg of the gas and the injection linear velocity Vf of the lipids in the molten state is It is preferably 10 or more and 2000 or less, more preferably 12 or more and 1900 or less, further preferably 15 or more and 1800 or less, and particularly preferably 15 or more and 1200 or less.
  • the lipids may be directly supplied to the liquid in a molten state, and are not particularly limited, but the temperature of the lipids in the molten state from the viewpoint of suppressing coagulation of the lipids in the nozzle and easily forming fine particles. Is preferably 5 ° C. or higher, more preferably 10 ° C. or higher, and even more preferably 15 ° C. or higher than the melting point of lipids.
  • the temperature of the lipids is not particularly limited, but for example, from the viewpoint of suppressing the denaturation of the medium, the denaturation of the secretions of the microorganism into the medium, the denaturation of the cells of the microorganism, and the adverse effect on the growth.
  • the temperature is preferably 150 ° C. or lower, more preferably 120 ° C. or lower, still more preferably 95 ° C. or lower.
  • the heating method is not particularly limited.
  • the gas is not particularly limited, but for example, in the case of aerobic culture, air, oxygen, or a mixture thereof is used because the gas ejected from the nozzle can be used to supply oxygen to microorganisms. It is preferable to do so.
  • air, oxygen, or a mixture thereof is used because the gas ejected from the nozzle can be used to supply oxygen to microorganisms. It is preferable to do so.
  • nitrogen or a mixture of air and nitrogen because the dissolved oxygen concentration in the culture solution can be kept low.
  • hydrogen or a mixture of air and hydrogen because the gas ejected from the nozzle can be used to supply hydrogen to microorganisms.
  • the temperature of the gas is not particularly limited, but is preferably equal to or higher than the melting point of the lipids from the viewpoint of suppressing the coagulation of the lipids in the nozzle and easily atomizing the lipids in the liquid. It is more preferably 5 ° C. or higher than the melting point, and even more preferably 10 ° C. or higher.
  • the temperature of the gas is not particularly limited, but is 200, for example, from the viewpoint of suppressing the denaturation of the medium, the denaturation of the secretions of the microorganism into the medium, the denaturation of the cells of the microorganism, and the adverse effect on the growth.
  • the temperature is preferably 1 ° C. or lower, more preferably 180 ° C. or lower, still more preferably 160 ° C. or lower.
  • the heating method is not particularly limited.
  • the liquid that directly supplies the molten lipid particles and the gas.
  • Stirring can be performed using, for example, a stirrer using a propeller or the like.
  • the temperature of the solution may be lower than the melting point of the lipid particles, for example, 20 ° C. or higher, and in the case of the culture solution, for example, 25 ° C. or higher and 37 ° C. or lower.
  • the lipid particles preferably have a volume median diameter D50 of 150 ⁇ m or less, more preferably 80 ⁇ m or less, still more preferably 60 ⁇ m or less, from the viewpoint that microorganisms can easily assimilate as a carbon source. It is particularly preferably 40 ⁇ m or less. Further, for example, from the viewpoint of increasing the particleization efficiency, the lipid particles preferably have a volume median diameter of 1 ⁇ m or more, more preferably 5 ⁇ m or more, and further preferably 10 ⁇ m or more.
  • the span represented by the following mathematical formula (1) is 0.5 or more and 3.0 or less in the particle size distribution. It is preferable that the range of 0.8 or more and 2.8 or less is satisfied, and 1.0 or more and 2.5 or less is more preferable.
  • Span (D90-D10) / D50 (1)
  • the particle size distribution of lipid particles can be measured by a laser diffraction / scattering type measuring method.
  • a particle size distribution measuring device "MT3300EXII" manufactured by Microtrac.
  • the molten lipids and gases may be supplied to the culture solution all at once, or continuously or intermittently. Further, other carbon sources may be directly supplied into the culture solution at the same time as the molten lipids.
  • Lipid particles can be prepared in a culture solution by the above-mentioned method for producing lipid particles, and microorganisms can be cultured in a culture solution containing the lipid particles.
  • the microorganism is not particularly limited, and examples thereof include microorganisms capable of producing an environment-friendly biodegradable plastic having almost no adverse effect on the ecosystem. Among them, microorganisms that produce polyhydroxyalkanoates (hereinafter, also referred to as PHA), which are produced by using natural organic acids and fats and oils derived from plants as a carbon source and accumulated as an energy storage substance in cells, are preferable.
  • PHA polyhydroxyalkanoates
  • the PHA is a general term for polymers having 3-hydroxyalkanoic acid as a monomer unit.
  • the 3-hydroxyalkanoic acid is not particularly limited, and is, for example, 3-hydroxypropionate, 3-hydroxybutyrate, 3-hydroxyvalerate, 3-hydroxyhexanoate, 3-hydroxyheptanoate, and 3. -Hydroxyoctanoate and the like.
  • the PHA may be a homopolymer having one kind of 3-hydroxyalkanoic acid as a monomer unit, or may be a copolymer having two or more kinds of 3-hydroxyalkanoic acid as a monomer unit.
  • the copolymer includes a copolymer of 3-hydroxybutyrate (3HB) and another 3-hydroxyalkanoic acid, or 3-hydroxyalkanoate containing at least 3-hydroxyhexanoate (3HH) as a monomer unit.
  • Examples thereof include copolymers.
  • Specific examples of the PHA include poly (3-hydroxybutyrate) (PHB), poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBH), and poly (3-hydroxybutyrate-).
  • Co-3-hydroxyvalerate-co-3-hydroxyhexanoate poly (3-hydroxybutyrate-co-3-hydroxyhexanoate), poly (3-hydroxybutyrate-co-4-hydroxybutyate) Rate), poly (3-hydroxybutyrate-co-3-hydroxyoctanoate), poly (3-hydroxybutyrate-co-3-hydroxyoctanoate), etc. are industrially easy to produce. From the point of view, it is preferable.
  • the microorganism used for the production of PHA is not particularly limited as long as it is a microorganism capable of producing PHA.
  • Microorganisms isolated from nature, microorganisms deposited in a strain depository (for example, IFO, ATCC, etc.), or genetically engineered microorganisms such as mutants and transformants that can be prepared from them can be used.
  • the genus Capiliavidus such as Cupriavidus necator
  • the genus Alcaligenes such as Alcaligenes latas
  • Pseudomonas putida Pseudomonas fluorescens, Pseudomonas fluorescens, Pseudomonas fluorescens. aeruginosa
  • Pseudomonas genus Bacillus megaterium, etc.
  • modified biological cells that artificially produce PHA by introducing a PHA synthase gene or the like using a genetic engineering technique.
  • a genetic engineering technique for example, in addition to the above-mentioned microorganisms belonging to the genus Capiliavidas, Alkalinegenes, Pseudomonas, Bacillus, Azotobacta, Nocardia, Aeromonas, Larstonia, Wautersia, Comamonas, etc., Escherichia ( Glam-negative bacteria such as Escherichia, gram-positive bacteria such as Bacillus, yeasts such as Saccharomyces, Yarrowia, and Candida are preferably used. It is possible to obtain a modified biological cell that artificially produces PHA.
  • PHBH among PHA for example, a method using a microorganism that originally produces PHBH such as Aeromonas caviae or Aeromonas hydrophila, or a genetic engineering method for a microorganism that does not originally produce PHBH, a PHA synthase gene, etc.
  • a modified biological cell that artificially produces PHBH As the host microorganism into which the gene is introduced, for example, Cupriavidus necator can be preferably used.
  • a PHA synthase gene a PHA synthase gene derived from Aeromonas caviae, Aeromonas hydrophila or Chromobacterium sp., A variant thereof, or the like can be used.
  • a base sequence encoding a PHA synthase in which an amino acid group is deleted, added, inserted, or substituted can be used.
  • culturing microorganisms it is possible to cultivate by the same method as the usual culturing method of each microorganism except that a culture solution containing lipid particles prepared by the above-mentioned method for producing lipid particles is used. Specifically, a two-fluid nozzle is attached to the culture tank so that the liquid supply port and the gas supply port are located below the liquid level of the culture solution in the culture solution and in the culture solution, and the lipid fine particles in the culture solution.
  • the microorganism can be cultivated in the same manner as before, except that the mixture is prepared and supplied as a carbon source.
  • PHA can be recovered from the microorganisms cultured by the above culture method using a well-known method. For example, it can be performed by the following method. After completion of the culture, the cells are separated from the culture solution by a centrifuge or the like, and the cells are washed with distilled water, methanol or the like and dried. PHA is extracted from the dried cells using an organic solvent such as chloroform. The bacterial cell component is removed from the solution containing PHA by filtration or the like, and a poor solvent such as methanol or hexane is added to the filtrate to precipitate PHA. Further, the supernatant is removed by filtration or centrifugation and dried to recover PHA.
  • a well-known method For example, it can be performed by the following method. After completion of the culture, the cells are separated from the culture solution by a centrifuge or the like, and the cells are washed with distilled water, methanol or the like and dried. PHA is extracted from the dried cells using an organic solvent such as
  • FIG. 1 is a schematic view of a device for producing lipid particles used in one or more embodiments of the present invention.
  • the manufacturing apparatus used in the manufacturing method of the present invention is not limited to that shown in FIG.
  • the manufacturing apparatus 20 includes a container 1 such as a culture tank and a two-fluid nozzle 3.
  • the bifluid nozzle 3 is attached to the wall surface of the container 1 so that the liquid supply port 12 and the gas supply port 13 of the bifluid nozzle 3 are lower than the liquid level 2 and located in the liquid.
  • the temperature of the two-fluid nozzle 3 can be adjusted by the heater 4.
  • the molten lipids 10 supplied from the tank 5 with the temperature control function to the liquid flow path of the bifluid nozzle 3 through the line 7 with the temperature control function using the tube pump 6 are liquid supply ports (liquid injection holes). It is directly supplied (injected) into the liquid from No. 12.
  • the gas 11 supplied to the gas flow path of the two-fluid nozzle 3 through the flow rate adjusting valve 8 and the temperature adjusting heater 9 is directly supplied (injected) into the liquid from the gas supply port (gas injection hole) 13.
  • the molten lipids 10 are directly supplied from the liquid supply port 12 of the two-fluid nozzle 3 into a liquid having a temperature lower than the melting point of the lipids, and the gas 11 is supplied into the liquid from the gas supply port 13 of the two-fluid nozzle 3.
  • the lipid particles 14 can be obtained by directly injecting and coagulating the molten lipids 10 injected with the injected gas 11 while dispersing them in the liquid and forming them into particles.
  • the ratio D50 / Nd of the volume median diameter D50 of the target lipid particles to the orifice diameter Nd of the liquid supply port of the two-fluid nozzle is adjusted to be 0.0017 or more and 0.17 or less.
  • the manufacturing apparatus 20 includes a stirrer 16 with a propeller, whereby the lipid particles 14 can be uniformly dispersed in the culture solution.
  • Reference numeral 15 is an exhaust line.
  • the bifluid nozzle 3 has a structure in which a gas flow path surrounds the liquid flow path, and has a function of a heat insulating material that prevents the gas from cooling the liquid and the liquid flow path by the culture solution. In particular, in a preferred embodiment in which the temperature of the gas is higher than the melting point of the lipids, the function of the heat insulating material is further enhanced.
  • Example 1 Lipid particles were produced using the production apparatus shown in FIG. Put 150 L of water (25 ° C) in a 200 L container, and in the two-fluid nozzle, the nozzle injection port is located on the wall surface 300 mm above the lower end of the container, and the liquid supply port and gas supply port are located horizontally and in the liquid. I installed it so that it would.
  • the two-fluid nozzle has a circular spray pattern, and the nozzle material is an external mixing type two-fluid spray nozzle (1/4 JAUCO made by Spraying Systems, the orifice diameter of the liquid feed part (liquid supply port) is 0.6 mm, the gas feed part.
  • the ring area of was 1.6 ⁇ 10 -6 m 2 ).
  • the two-fluid nozzle was preheated to 75-80 ° C.
  • the air supplied to the two-fluid nozzle was heated to 60 ° C. and adjusted so that the air flow rate was 10 L / min (line speed 104.2 m / sec).
  • the temperature inside the container was 34 ° C., and 0.2 to 0.86 mL of molten fatty acid distillation (PFAD, melting point 50 ° C., density 0.87 g / mL, 25 ° C. solubility 10 g / L or less) heated to 60 ° C. It was added at a rate (flow rate) of / min and solidified in water.
  • PFAD molten fatty acid distillation
  • the ratio Vg / Vf of the jet velocity Vg of air to the jet velocity Vf of the fatty acid distillation in the molten state was 71. After long-term operation for 6 hours, nozzle clogging did not occur and lipid particles could be produced.
  • the particle size distribution of the obtained PFAD particles was measured with a particle size distribution measuring device (MT3300EXII manufactured by Microtrac) in Example 1, the volume median diameter (D50) was 20.1 ⁇ m, and the span [(D90-D10) / D50] was 2.51, and the volume median diameter of the PFAD particles / the orifice diameter of the liquid supply port was 0.033.
  • Example 1 The operation was performed under the same method and conditions as in Example 1 except that the nozzle was not heated. Within 5 minutes after the start of supply (spraying) of the molten fatty acid distillate and air, the nozzle was blocked and the operation became inoperable.
  • Examples 2 to 5 1L of water (34 ° C) is put into a 5L jar fermenter (BMS-5L manufactured by Biot), and the nozzle injection port of the two-fluid nozzle is located on the wall surface 50 mm above the lower end of the culture tank, and the air supplied to the two-fluid nozzle is Adjust the air volume to 6 L / min (line speed 62.5 m / sec), add 5 g of PFAD at a rate of 1 mL / min (line speed 0.06 m / sec), and set the nozzle temperature and PFAD temperature as follows. Fatty acid particles were obtained under the same method and conditions as in Example 1 except as shown in Table 1.
  • Example 2 the ratio Vg / Vf of the jet linear velocity Vg of air and the jet linear velocity Vf of the fatty acid distilled in the molten state was 1060.
  • the particle size distribution of the obtained PFAD particles was measured with a particle size distribution measuring device (MT3300EXII manufactured by Microtrac), and the results are shown in Table 1.
  • Example 6 Put 3.5 L of water in a 10 L jar fermenter (BMS-10L manufactured by Biot), and add 52.5 g of fatty acid distillate (PFAD, melting point 50 ° C., density 0.87 g / mL, 25 ° C. solubility 10 g / L or less).
  • PFAD fatty acid distillate
  • the air flow rate of the air supplied to the two-fluid nozzle was adjusted to 10 L / min (linear speed 104.2 m / sec), and the addition rate of PFAD (flow rate of PFAD) was as shown in Table 2 below.
  • Fatty acid particles were obtained under the same method and conditions as in Example 1 except.
  • the particle size distribution of the obtained PFAD particles was measured with a particle size distribution measuring device (MT3300EXII manufactured by Microtrac), and the results are shown in Table 2.
  • Example 14 Put 3.5 L of water (34 ° C.) in a 10 L jar fermenter (BMS-10L manufactured by Biot), and fatty acid distillate (PFAD, melting point 50 ° C., density 0.87 g / mL, 25 ° C. solubility 10 g / L or less) 52 Add .5 g, adjust the air flow rate of the air supplied to the two-fluid nozzle to 6 L / min (line speed 62.5 m / sec), and adjust the addition rate of PFAD to a rate of 20.2 mL / min (line speed). Fatty acid particles were obtained under the same method and conditions as in Example 1 except that the mixture was added at a speed of 1.19 m / sec.
  • BMS-10L manufactured by Biot fatty acid distillate
  • the ratio Vg / Vf of the jet velocity Vg of air to the jet velocity Vf of the fatty acid distillation in the molten state was 52.
  • the volume median diameter (D50) was 31.5 ⁇ m, and the span [(D90-D10) / D50] was 1. It was 29, and the volume median diameter of the PFAD particles / the orifice diameter of the liquid supply port was 0.053.
  • FIG. 2 shows a graph plotting data of the volume median diameter of Vg / Vf and PFAD particles / orifice diameter of the liquid supply port in Examples 6 to 14. From FIG. 2, it can be seen that in the PFAD liquid spraying by the two-fluid nozzle, Vg / Vf and the volume median diameter of the PFAD particles / the orifice diameter of the liquid supply port are correlated.
  • Example 2 (Comparative Example 2) Except that 1 L of water (34 ° C) was put in a cylindrical container with a diameter of 10 cm, an injection hole of an external mixed two-fluid spray nozzle was installed at a height of 5 cm from the water surface, and PFAD was injected from the nozzle toward the water surface. Spraying was carried out under the same method and conditions as in Example 14. The PFAD solidified and aggregated on the water surface, and coarse particles having a diameter of 3 mm or more were obtained.
  • Example 15 (Culturing cells using spray in lipid solution using an external mixed two-fluid spray nozzle)
  • KNK-631 strain (see Japanese Patent Application Laid-Open No. 2013-09627 and International Publication No. 2016/114128) was used, and seed mother culture, pre-culture, and main culture were carried out, and the cells were recovered.
  • Tanehaha medium 1 w / v% of Meat-extract, 1w / v% of Bacto-Tryptone, 0.2w / v% of Yeast-extract, 0.9w / v% of Na 2 HPO 4 ⁇ 12H 2 O and 0.15 w / v% KH 2 PO 4 were used, and the pH was set to 6.8.
  • composition of the preculture medium 1.1w / v% of Na 2 HPO 4 ⁇ 12H 2 O , 0.19w / v% of KH 2 PO 4, 1.29w / v % of (NH 4) 2 SO 4, 0.1 w / v% of MgSO 4 ⁇ 7H 2 O, 0.5v / v% trace metal salt solution (1.6 w in 0.1N HCl / v% of FeCl 3 ⁇ 6H 2 O, of 1 w / v% dissolve CaCl 2 ⁇ 2H 2 O, 0.02w / v% of CoCl 2 ⁇ 6H 2 O, 0.016w / v% of CuSO 4 ⁇ 5H 2 O, the NiCl 2 ⁇ 6H 2 O of 0.012w / v% It was said.
  • palm oil was added all at once at a concentration of 10 g / L.
  • composition of the main culture medium 0.385w / v% of Na 2 HPO 4 ⁇ 12H 2 O , 0.067w / v% of KH 2 PO 4, 0.291w / v % of (NH 4) 2 SO 4, 0.1 w / v% of MgSO 4 ⁇ 7H 2 O, 0.5v / v% trace metal salt solution (1.6 w of 0.1N hydrochloric acid / v% of FeCl 3 ⁇ 6H 2 O, 1w / v% of CaCl 2 ⁇ 2H 2 O, 0.02w / v% of CoCl 2 ⁇ 6H 2 O, 0.016w / v% of CuSO 4 ⁇ 5H 2 O, the NiCl 2 ⁇ 6H 2 O of 0.012w / v% (Melted) and 0.05 w / v% BIOSPUREX 200K (antifoaming agent: manufactured by Cognis Japan Co., Ltd.).
  • the glycerol stock (50 ⁇ L) of the KNK-631 strain was inoculated into the seed medium (10 mL) and cultured at 30 ° C. for 24 hours to perform seed mother culture.
  • the obtained seed mother culture solution was inoculated at 1.0 v / v% into a 3 L jar fermenter (MDL-300 type manufactured by Maruhishi Bioengineer) containing 1.8 L of preculture medium.
  • the operating conditions were a culture temperature of 30 ° C., a stirring speed of 600 rpm, an aeration rate of 1.8 L / min, and the culture was performed for 24 hours while controlling the pH at 6.5, and pre-culture was performed.
  • a 14% aqueous solution of ammonium hydroxide was used for pH control.
  • the obtained preculture solution was inoculated at 1.0 v / v% into a 10 L jar fermenter (BMS-10L manufactured by Biot) containing 6 L of the production medium.
  • the operating conditions were a culture temperature of 34 ° C., a stirring speed of 600 rpm, an aeration rate of 6.0 L / min, and a pH controlled to 6.5.
  • a 14% aqueous solution of ammonium hydroxide was used for pH control.
  • PFAD in a molten state heated to 60 ° C. PFAD obtained from FELDA via SUS piping so as not to come into contact with iron piping was used. Melting point 50 ° C., density 0.87 g / mL, 25 ° C.
  • solubility 10 g / L The following) was sprayed into the liquid with an external mixing type two-fluid spray nozzle heated to 70 ° C., and the mixture was added while controlling the concentration in the culture liquid.
  • the air supplied to the two-fluid nozzle was heated to 60 ° C. and adjusted so that the air flow rate was 6 L / min (line speed 62.5 m / sec).
  • PFAD was added at a rate of 0.2 to 0.86 mL / min, and a phosphoric acid solution was added at a constant rate during the culture.
  • the ratio Vg / Vf of the jet velocity Vg of air to the jet velocity Vf of the fatty acid distillation in the molten state was 1250.
  • the main culture was carried out for 48 hours, and after the completion of the culture, the cells were collected by centrifugation, washed with methanol, freeze-dried, and the weight of the dried cells was measured. 100 mL of chloroform was added to 1 g of the obtained dried cells, and the mixture was stirred at room temperature for 24 hours to extract PHBH in the cells. The cell residue was concentrated with an evaporator until the total volume reached 30 mL, 90 mL of hexane was gradually added, and the mixture was left for 1 hour with gentle stirring.
  • the precipitated PHBH was vacuum dried at 50 ° C. for 3 hours to obtain PHBH.
  • the productivity of PHBH and the yield of carbon source are shown in Table 3 below.
  • the productivity of PHBH is the yield of PHBH per volume of the culture solution (g / L), and the yield of carbon source is the yield of PHBH per supply weight of the carbon source (g / g).
  • Example 3 The culture was carried out under the same conditions and methods as in Example 15 except that the PFAD spray in the main culture was sprayed from the gas phase portion toward the liquid. The sprayed PFAD solidified on the water surface, and after agglomeration, adhered to the electrodes, stirring blades, and baffles inside the jar, and it was difficult to culture.
  • the melted lipids are sprayed into the culture solution with an external mixed two-fluid spray nozzle, so that the lipids have a temperature lower than the melting point of the lipids. It was found that the lipids were efficiently finely dispersed and granulated, and therefore the lipids were suitably assimilated by the microorganisms, and it was possible to efficiently produce microbial metabolites.
  • a method for producing lipid particles which obtains lipid particles by coagulating molten lipids into particles in a liquid.
  • the lipids have a solubility in water at 25 ° C. of 10 g / L or less and are solid at 25 ° C.
  • the two-fluid nozzle is heated to a temperature higher than the melting point of the lipids by 10 ° C. or more.
  • a method for producing lipid particles wherein the ratio D50 / Nd of the volume median diameter D50 of the lipid particles to the orifice diameter Nd of the liquid supply port of the two-fluid nozzle is 0.0017 or more and 0.17 or less. .. [2] The method for producing lipid particles according to [1], wherein the ratio Vg / Vf of the jet velocity Vg of the gas to the jet velocity Vf of the lipid in the molten state is 10 or more and 2000 or less.
  • [6] The method for producing lipid particles according to any one of [1] to [5], wherein the lipids are lipids derived from palm oil.
  • the method for producing lipid particles according to any one of [1] to [8], wherein the temperature of the molten lipid is 5 ° C. or higher higher than the melting point of the lipid.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biomedical Technology (AREA)
  • Sustainable Development (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Molecular Biology (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
PCT/JP2020/040062 2019-11-25 2020-10-26 液中での脂質類粒子の製造方法及び微生物の培養方法 Ceased WO2021106462A1 (ja)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP2021561232A JPWO2021106462A1 (https=) 2019-11-25 2020-10-26
CN202080081915.3A CN114761573A (zh) 2019-11-25 2020-10-26 液体中的脂质类粒子的制造方法及微生物的培养方法
US17/742,035 US20220266210A1 (en) 2019-11-25 2022-05-11 Production method for lipid particles in liquid and method for culturing microorganisms

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2019-212566 2019-11-25
JP2019212566 2019-11-25

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US17/742,035 Continuation US20220266210A1 (en) 2019-11-25 2022-05-11 Production method for lipid particles in liquid and method for culturing microorganisms

Publications (1)

Publication Number Publication Date
WO2021106462A1 true WO2021106462A1 (ja) 2021-06-03

Family

ID=76129333

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2020/040062 Ceased WO2021106462A1 (ja) 2019-11-25 2020-10-26 液中での脂質類粒子の製造方法及び微生物の培養方法

Country Status (4)

Country Link
US (1) US20220266210A1 (https=)
JP (1) JPWO2021106462A1 (https=)
CN (1) CN114761573A (https=)
WO (1) WO2021106462A1 (https=)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20230135375A (ko) * 2022-03-16 2023-09-25 한국생산기술연구원 랄스토니아 유트로파 균주를 이용한 폴리하이드록시알카노에이트 생산 방법
WO2025047344A1 (ja) * 2023-08-29 2025-03-06 株式会社カネカ エマルションの製造方法およびその利用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116510358B (zh) * 2023-05-25 2025-11-25 山东浩然特塑股份有限公司 一种聚砜类树脂的分散沉淀装置和方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS4943150B1 (https=) * 1970-12-28 1974-11-19
JPH06245700A (ja) * 1993-02-23 1994-09-06 Snow Brand Milk Prod Co Ltd 消化吸収性を低減した油脂組成物
JP2013009627A (ja) * 2011-06-29 2013-01-17 Kaneka Corp 微生物の培養方法
WO2019230644A1 (ja) * 2018-05-30 2019-12-05 株式会社カネカ ポリヒドロキシアルカン酸の製造方法

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2003225895A1 (en) * 2002-03-19 2003-10-08 Purdue Research Foundation Microencapsulation using ultrasonic atomizers
WO2014045962A1 (ja) * 2012-09-20 2014-03-27 株式会社カネカ 微生物培養用乳化物、これを用いた微生物の培養方法、及び、微生物代謝産物の製造方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS4943150B1 (https=) * 1970-12-28 1974-11-19
JPH06245700A (ja) * 1993-02-23 1994-09-06 Snow Brand Milk Prod Co Ltd 消化吸収性を低減した油脂組成物
JP2013009627A (ja) * 2011-06-29 2013-01-17 Kaneka Corp 微生物の培養方法
WO2019230644A1 (ja) * 2018-05-30 2019-12-05 株式会社カネカ ポリヒドロキシアルカン酸の製造方法

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20230135375A (ko) * 2022-03-16 2023-09-25 한국생산기술연구원 랄스토니아 유트로파 균주를 이용한 폴리하이드록시알카노에이트 생산 방법
KR102728692B1 (ko) * 2022-03-16 2024-11-13 한국생산기술연구원 랄스토니아 유트로파 균주를 이용한 폴리하이드록시알카노에이트 생산 방법
WO2025047344A1 (ja) * 2023-08-29 2025-03-06 株式会社カネカ エマルションの製造方法およびその利用

Also Published As

Publication number Publication date
CN114761573A (zh) 2022-07-15
JPWO2021106462A1 (https=) 2021-06-03
US20220266210A1 (en) 2022-08-25

Similar Documents

Publication Publication Date Title
US20220266210A1 (en) Production method for lipid particles in liquid and method for culturing microorganisms
JP6994057B2 (ja) Ｐｈａ合成酵素をコードする遺伝子を複数有する微生物、およびそれを用いたｐｈａの製造方法
Możejko et al. Saponified waste palm oil as an attractive renewable resource for mcl-polyhydroxyalkanoate synthesis
EP2418277B1 (en) METHOD FOR CULTURING MICROORGANISM, AND PROCESS FOR PRODUCING PHAs WITH MICROORGANISM
Diniz et al. High-cell-density cultivation of Pseudomonas putida IPT 046 and medium-chain-length polyhydroxyalkanoate production from sugarcane carbohydrates
JP6313708B2 (ja) 高分子量ｐｈａ生産微生物とそれを用いた高分子量ｐｈａの製造方法
EP2818561A1 (en) Utilization of the novel, environmental isolates Pseudomonas sp. IPB-B26 and N-128 for the efficient high yield production of mcl/lcl-PHAs
EP2899264B1 (en) Emulsion for microbial growth, method for growing microorganisms by using said emulsion, and method for producing microbial metabolite
US12600993B2 (en) Method for producing poly(3-hydroxyalkanoate)
JP5839854B2 (ja) 微生物の培養方法
WO2025047344A1 (ja) エマルションの製造方法およびその利用
JP2015002678A (ja) 微生物の培養方法、ならびに微生物によるｐｈａの製造方法
WO2014042076A1 (ja) 改質油脂組成物を用いたポリヒドロキシアルカノエートの製造方法
JP2007228894A (ja) 遺伝子組み換え微生物及びそれを用いたポリエステルの製造方法
CN1703502A (zh) 控制共聚聚酯组成的培养方法
JP2014521338A (ja) 油含有供給原料のバイオ発酵のための組成物及び方法
WO2024075597A1 (ja) 共重合ポリヒドロキシアルカン酸混合物の製造方法、及び形質転換微生物
JP2024118674A (ja) ポリヒドロキシアルカノエートの製造方法
Freches Bio-valorization of crude glycerol through polyhydroxyalkanoates production
Ye 32 Enzyme-Catalyzed Production of Lipid-Based Esters for the Food Industry
Fava et al. Production of vanillin from wheat bran hydrolyzates via microbial bioconversion
Ucha et al. Enrichment of a mixed microbial culture for PHA production with raw used cooking oil as substrate
Gumel Biosynthesis of Chiral Poly-Hydroxyacyl Esters and their Enzymatic Functionalization as Glycopolymers
Piacentini et al. New developments for the controlled fabrication of microstructured multiphase bioreactor using membrane emulsification technology
Wongsirichot et al. 8 Stage IV results, publication: An integrated biorefining approach for the production of polyhydroxyalkanoates from enzymatically hydrolyzed rapeseed meal under nitrogen-limited conditions

Legal Events

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

Ref document number: 20892349

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2021561232

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20892349

Country of ref document: EP

Kind code of ref document: A1