WO2023236718A1 - 一种聚羟基脂肪酸酯凝集体的制备方法 - Google Patents

一种聚羟基脂肪酸酯凝集体的制备方法 Download PDF

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Publication number
WO2023236718A1
WO2023236718A1 PCT/CN2023/093510 CN2023093510W WO2023236718A1 WO 2023236718 A1 WO2023236718 A1 WO 2023236718A1 CN 2023093510 W CN2023093510 W CN 2023093510W WO 2023236718 A1 WO2023236718 A1 WO 2023236718A1
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Prior art keywords
polyhydroxyalkanoate
suspension
pha
aggregate
preparation
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PCT/CN2023/093510
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English (en)
French (fr)
Inventor
汪东升
马文彦
吴雅琨
张进城
王杰鹏
孙强
曹钰
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Bluepha Co Ltd
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Beijing Bluepha Microbiology Technology Co Ltd
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Priority to US18/859,681 priority Critical patent/US20250277084A1/en
Priority to JP2024528548A priority patent/JP2024541371A/ja
Priority to EP23818883.3A priority patent/EP4484473A4/en
Publication of WO2023236718A1 publication Critical patent/WO2023236718A1/zh
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/12—Powdering or granulating
    • C08J3/16—Powdering or granulating by coagulating dispersions
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
    • C08G63/02—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds
    • C08G63/06—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds derived from hydroxycarboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
    • C08G63/88—Post-polymerisation treatment
    • C08G63/90—Purification; Drying
    • 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
    • C12P7/625—Polyesters of hydroxy carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2367/00—Characterised by the use of polyesters obtained by reactions forming a carboxylic ester link in the main chain; Derivatives of such polymers
    • C08J2367/04—Polyesters derived from hydroxy carboxylic acids, e.g. lactones

Definitions

  • the invention relates to the field of bioengineering technology, and specifically relates to a method for preparing polyhydroxyalkanoate aggregates.
  • PHA Polyhydroxyalkanoates
  • PHA produced by microorganisms usually accumulates in the cell bodies of microorganisms in the form of inclusion bodies, it is necessary to separate and recover the PHA particles inside the cells.
  • PHA particles When PHA particles are used in plastic preparation, they need to have good processing properties. Therefore, the purity of PHA particles is required to minimize interference from impurities such as inorganic salts or cell lysates.
  • the invention patent with publication number EP1609868B1 discloses a method for collecting high-purity polyhydroxyalkanoates from microbial cells.
  • the method includes physical disruption and low-temperature alkali addition in an aqueous suspension of microbial cells containing PHA. Efficiently disrupt the cells to recover PHA, then treat the PHA with enzymes and/or surfactants, followed by washing the PHA particles with a hydrophilic solvent for degreasing, deodorizing and decolorizing, causing the PHA particles to aggregate by heating and stirring, This is followed by suction filtration and drying.
  • this method has mild reaction conditions and a high recovery rate, it requires the use of a large amount of organic solvents to wash the PHA particles, which makes downstream wastewater treatment difficult. It also requires explosion-proof workshops and explosion-proof equipment, making industrial production more difficult.
  • the invention patent with publication number EP2357247B1 discloses a method for preparing poly3-hydroxyalkanoate, which method includes performing physical disruption treatment and low-temperature alkali addition in an aqueous suspension of microbial cells containing PHA to effectively destroy the cells for recovery. PHA, then treating the PHA with enzymes and/or surfactants, followed by washing the PHA particles with large amounts of water, by adjusting the content of organic nitrogen in the aqueous suspension containing poly-3-hydroxyalkanoic acid to less than 1500 ppm per weight of poly-3-hydroxyalkanoic acid, and then the PHA particles are aggregated by heating and stirring, followed by suction filtration and drying.
  • this method has mild reaction conditions and a high recovery rate, it requires the use of a large amount of water to wash the particles through centrifugation. The preparation process generates a large amount of wastewater, which increases the difficulty of downstream wastewater treatment.
  • the invention patent with publication number EP3027761B1 discloses a method for recovering and purifying polyhydroxyalkanoate from cell culture, and a method for recovering and purifying PHA from cell culture, including acidifying the cell culture and physically disrupting the cells to obtain PHA suspension. liquid. Subsequently, the PHA suspension base until the pH value is equal to or higher than 8, and then perform tangential filtration after dilution. The PHA suspension is then bleached, diluted and tangentially filtered again and then dried to obtain the finished product. This method can be carried out continuously without the use of organic solvents and obtain PHA in pure form without reducing the molecular weight.
  • the invention patent with publication number US9683076B2 obtains PHA particles by spray drying a PHA suspension.
  • spray drying consumes a lot of energy and mixes the inorganic salts and cell lysates remaining in the suspension into the PHA particles, which reduces the cost of the finished PHA.
  • the purity increases the difficulty for downstream material processing.
  • the methods disclosed in the prior art may require the use of a large amount of organic solvents or water to clean the PHA particles, or the use of flocculants, which reduces the purity of the finished product, or may have problems such as complex technological processes and high production costs.
  • the object of the present invention is to provide a method for preparing polyhydroxyalkanoate aggregates and the polyhydroxyalkanoate aggregates prepared by the method.
  • the present invention has conducted extensive research on the cohesion process of polyhydroxyalkanoate.
  • the present invention unexpectedly discovered that the turbulent state of the polyhydroxyalkanoate suspension is a key factor in promoting the agglomeration of polyhydroxyalkanoate particles: When the particles are cleaned, it is difficult to achieve the agglomeration of polyhydroxyalkanoate with low-speed stirring or even in a vortex state.
  • the present invention provides a method for preparing a polyhydroxyalkanoate agglomerate, which method includes the step of bringing the polyhydroxyalkanoate suspension to and maintaining a turbulent flow state to aggregate the polyhydroxyalkanoate.
  • the above-mentioned turbulent flow state is a state in which the Reynolds coefficient of the polyhydroxyalkanoate suspension is greater than the critical Reynolds coefficient of the polyhydroxyalkanoate suspension reaching the critical Reynolds coefficient of turbulence in the equipment in which it is located.
  • the above turbulent flow state can be carried out in the reactor or in the pipeline.
  • D turbine diameter (m); N: stirrer speed (r/s); ⁇ : material liquid density (kg/m 3 ); ⁇ : material liquid viscosity (Ns/m 2 ).
  • v fluid flow rate (m/s)
  • ⁇ material liquid density (kg/m 3 )
  • ⁇ material liquid viscosity (Ns/m 2 )
  • d characteristic length.
  • the turbulent flow state is the state of the suspension when the Reynolds coefficient of the polyhydroxyalkanoate suspension is greater than 1000.
  • the turbulent flow state is the state of the suspension when the Reynolds coefficient of the polyhydroxyalkanoate suspension is greater than 4000.
  • the coagulation of polyhydroxyalkanoate is carried out in a reactor, and the turbulent flow state has a Reynolds coefficient greater than 1000, preferably greater than 1200, and more preferably greater than 4000.
  • the present invention has no special limitation on the reactor, and it can be any equipment capable of placing the feed liquid in a turbulent flow state.
  • the polyhydroxyalkanoate suspension is subjected to continuous Stir the suspension to achieve and maintain turbulence.
  • the nitrogen content of the polyhydroxyalkanoate particles is less than 7000ppm (relative to the polyhydroxyalkanoate per unit mass), and the nitrogen content of the suspension clear liquid is less than 3000ppm (relative to the polyhydroxyalkanoate per unit mass). relative to clear liquid per unit mass of suspension).
  • the method for detecting the nitrogen content of the above-mentioned polyhydroxyalkanoate particles and suspension clear liquid is as follows: centrifuge the polyhydroxyalkanoate suspension at 10,000 rpm for 5 minutes, the supernatant is the suspension clear liquid, and the precipitate is After washing with water twice, centrifuging and drying, polyhydroxyalkanoate particles were obtained, and the nitrogen content of the suspension liquid and polyhydroxyalkanoate particles were measured respectively.
  • the polyhydroxyalkanoate suspension described above is obtained by using the biological fermentation liquid of polyhydroxyalkanoate as raw material, collecting the polyhydroxyalkanoate precipitate through cell crushing and solid-liquid separation, and then suspending it in water.
  • the above-mentioned biological fermentation liquid can be a fermentation liquid containing organisms used for fermentation prepared by fermentation culture using any organism capable of fermenting to produce polyhydroxyalkanoate.
  • microorganisms include, for example, Aeromonas, Alcaligenes, Azotobacter, Bacillus, Clostridium, and Salobacterium Halobacterium, Nocardia, Rhodospirillum, Pseudomonas, Ralstonia, Zoogloea, etc. microorganism.
  • examples of the genus Aeromonas include, for example, Aeromonas caviae and the like; examples of the genus Alcaligenes include, for example, Alcaligenes lipolytica, Alcaligenes Bacteria (Alcaligenes latus), etc.; examples of the genus Ralstonia (Ralstonia) include, for example, Ralstonia eutropha (Ralstonia eutropha), etc.
  • the agglomerate preparation method of the present invention has relatively simple requirements for the preparation process of the suspension.
  • the fermentation liquid can be adjusted to alkalinity and then the cells are mechanically broken to release the polyhydroxyalkanoate particles in the cells.
  • the pH of the fermentation broth to alkaline (preferably 10.5-11.5), leave it for 1-3 hours, and then use mechanical methods to disrupt the cells.
  • Solid-liquid separation method Any method that can separate polyhydroxyalkanoates and non-polyhydroxyalkanoates can be used. Centrifugation, filtration, etc. can be used. Among them, non-polyhydroxyalkanoates can be used.
  • Fatty acid ester substances include, but are not limited to, cell debris, proteins, nucleic acids and other impurities.
  • the present invention is not particularly limited to the concentration of polyhydroxyalkanoate in the polyhydroxyalkanoate suspension. If you want to increase the yield of polyhydroxyalkanoate aggregates, it is preferred to use a higher concentration of polyhydroxyalkanoate suspension. .
  • the content of polyhydroxyalkanoate in the polyhydroxyalkanoate suspension is not less than 20 g/L. More preferably, it is 50 g/L or more.
  • the present invention has no special restrictions on the pH of the polyhydroxyalkanoate suspension used for coagulation.
  • the polyhydroxyalkanoate suspension can be acidic, neutral or alkaline.
  • the fermentation broth is adjusted to alkaline, then the cells are disrupted to release the polyhydroxyalkanoate particles in the cells, the polyhydroxyalkanoate precipitate is collected through solid-liquid separation, and then the polyhydroxyalkanoate is prepared by a water suspension method. Suspension, there is no need to adjust the pH of the rehydrated suspension. At this time, the pH of the suspension is 8.5-10.5.
  • the suspension can be directly used for the agglomeration of polyhydroxyalkanoate. During the agglomeration process of polyhydroxyalkanoate, keeping the temperature not lower than 20°C is helpful to promote agglomeration.
  • the temperature of the polyhydroxyalkanoate suspension is maintained at not lower than 20°C.
  • the temperature of the hydroxy fatty acid ester suspension is not lower than 30°C.
  • the temperature of the polyhydroxyalkanoate suspension is maintained at not less than 40°C.
  • the temperature of the polyhydroxyalkanoate suspension is maintained at 20-70°C, more preferably at 20-40°C.
  • the time for maintaining the turbulent flow state can be based on the preference.
  • the temperature of the polyhydroxyalkanoate suspension is maintained at not less than 20°C.
  • the particle size of the agglomerates is monitored, and when the desired particle size is reached, the agglomerates are collected. Particle size monitoring can be carried out through visual inspection or online real-time detection.
  • the particle size of the polyhydroxyalkanoate agglomerate is not particularly limited. Any particle size suitable for industrial separation can be used. Generally, it is preferably 5 ⁇ m or more, and more preferably 50 ⁇ m or more, so as to be better suitable for industrial solid-liquid separation.
  • the polyhydroxyalkanoate agglomerates are aggregated until polyhydroxyalkanoate agglomerates of suitable particle size are obtained, and then the polyhydroxyalkanoate agglomerates are collected through solid-liquid separation.
  • the aggregation of polyhydroxyalkanoate particles usually occurs within 0.1-3 hours, and the particle size of the polyhydroxyalkanoate agglomerate increases significantly.
  • the preferred time to maintain the turbulent flow state is 40. -180min.
  • the method of the present invention it is possible to obtain aggregates with an average particle size of polyhydroxyalkanoate aggregates of 10 ⁇ m or more, 50 ⁇ m or more, or even 100 ⁇ m or more.
  • the upper limit of the particle diameter is not particularly limited, but the average diameter is preferably 5000 ⁇ m or less, and preferably 3000 ⁇ m or less.
  • the preparation method of the aggregate includes: using a polyhydroxyalkanoate suspension as a raw material, stirring the polyhydroxyalkanoate suspension so that the polyhydroxyalkanoate suspension
  • the fatty acid ester suspension reaches and maintains a turbulent flow state while maintaining the temperature not lower than 20°C to obtain a polyhydroxyalkanoate agglomerate with an enlarged particle size.
  • the preparation method of the aggregates further includes the step of using enzymes to remove impurities in the polyhydroxyalkanoate suspension.
  • the enzyme is used to process peptidoglycan, lipids, polysaccharides, and nucleic acid impurities attached to the surface of polyhydroxyalkanoate particles, including but not limited to proteolytic enzymes, lipid-decomposing enzymes, and cell wall-decomposing enzymes. , one or more of DNA-degrading enzymes.
  • the above-mentioned enzymes can be used alone, or two or more types can be used in combination. There is no special restriction on the source of the enzyme, and it can be a commercially available enzyme preparation or a commercially available enzyme detergent for washing.
  • the amount of enzyme added depends on the type and activity of the enzyme. Although there is no particular limit, in order to reduce costs, it is preferable to use an enzyme activity of 100,000-2 million U/mL and an enzyme added amount of 0.001mL/L-10mL. /L (relative to polyhydroxyalkanoate suspension).
  • the temperature and pH can be adjusted according to the optimal temperature and optimal pH of the enzyme used, for example, the temperature is controlled at 20-70°C and the pH is 5-10. Since the optimal temperature and optimal pH of enzymes are common knowledge among those skilled in the art, they will not be described in detail in the present invention.
  • the polyhydroxyalkanoate suspension can also be removed with a surfactant while adding enzymes. impurities in the liquid to further remove impurities attached to the surface of PHA particles.
  • the surfactant mentioned above is one or more selected from anionic surfactants, cationic surfactants, amphoteric surfactants, and nonionic surfactants, and is preferably an anionic surfactant.
  • the amount of surfactant added is not particularly limited, a preferred amount is 1 g/L to 20 g/L (relative to the polyhydroxyalkanoate suspension).
  • the surfactant is SDS
  • the dosage of SDS is preferably Choose 5-20g/L.
  • the surfactant is sodium dodecylbenzene sulfonate, and the dosage of sodium dodecylbenzene sulfonate is preferably 2-10g/L.
  • the preparation method of the aggregate of the present invention Coagulation and impurity removal (impurity removal using enzymes and/or surfactants) can be carried out in the same reaction system, which greatly simplifies the preparation process and reduces waste liquid production.
  • the step of removing impurities in the polyhydroxyalkanoate suspension and the aggregation of the polyhydroxyalkanoate are performed in the same reaction system.
  • Methods disclosed in the prior art mostly involve washing polyhydroxyalkanoate particles with organic solvents and then performing agglomeration reactions, or performing agglomeration reactions in a system containing organic solvents.
  • the preparation method of the agglomerates of the present invention can be achieved without using organic solvents. Clean and effectively agglomerate polyhydroxyalkanoates in a system that does not contain organic solvents.
  • the coagulated system of polyhydroxyalkanoate does not contain organic solvents.
  • the preparation method of the polyhydroxyalkanoate aggregate includes: using a polyhydroxyalkanoate suspension as a raw material, stirring the polyhydroxyalkanoate suspension so that the polyhydroxyalkanoate When the ester suspension reaches a turbulent flow state, add enzyme to maintain the turbulent flow state while maintaining the temperature at no less than 20°C to obtain polyhydroxyalkanoate agglomerates with increased particle size. When the target particle size is reached, collect the polyhydroxyalkanoate agglomerates. collective.
  • the preparation method of the polyhydroxyalkanoate aggregate includes: using a polyhydroxyalkanoate suspension as a raw material, stirring the polyhydroxyalkanoate suspension so that the polyhydroxyalkanoate When the ester suspension reaches a turbulent flow state, add enzymes and surfactants to maintain the turbulent flow state while maintaining the temperature at no less than 20°C to obtain polyhydroxyalkanoate agglomerates with increased particle size. When the target particle size is reached, collect the polyhydroxyalkanoate Fatty acid ester aggregates.
  • the polyhydroxyalkanoate aggregates can be collected by solid-liquid separation.
  • the difficulty of solid-liquid separation of polyhydroxyalkanoate agglomerates is significantly reduced, which can reduce equipment costs in industrial production. Therefore, there are no special requirements for solid-liquid separation methods and equipment.
  • methods such as filtration (plate and frame filter, basket bag filter, etc.) or centrifugation (decanter centrifuge, etc.) can be used.
  • the polyhydroxyalkanoate aggregates are collected through solid-liquid separation.
  • the above-mentioned polyhydroxyalkanoate agglomerate can also be dried to obtain a dry agglomerate.
  • the drying method is not particularly limited and can be ventilation drying at normal temperature, ventilation drying with heating, etc.
  • the present invention provides polyhydroxyalkanoate aggregates prepared by the above-mentioned preparation method of polyhydroxyalkanoate aggregates.
  • the particle size of the polyhydroxyalkanoate aggregate is at least 5 ⁇ m, more preferably at least 50 ⁇ m.
  • the particle size of the polyhydroxyalkanoate aggregate is 50 to 100 ⁇ m.
  • the particle size of the polyhydroxyalkanoate aggregate is 100 to 300 ⁇ m.
  • the polyhydroxyalkanoate (PHA) can be various polyhydroxyalkanoate products, including but not limited to polyhydroxybutyric acid (PHB), poly3-hydroxybutyric acid and 3-hydroxyvaleric acid.
  • Polyester (PHBV) poly 3-hydroxybutyric acid and 3-hydroxycaproic acid copolyester (PHBH), poly 3-hydroxybutyric acid and 4-hydroxybutyric acid copolyester (P3HB4HB), etc.
  • the method for preparing polyhydroxyalkanoate aggregates provided by the present invention can produce polyhydroxyalkanoate aggregates with a particle size of 50 ⁇ m or more, so as to be suitable for industrial solid-liquid separation equipment, and the prepared
  • the polyhydroxyalkanoate agglomerate has high purity and yield; the method has mild process conditions, simple steps, simple wastewater treatment, low cost, low equipment requirements, and can achieve large-scale industrial production.
  • the present invention Compared with existing methods of increasing the particle size of polyhydroxyalkanoate particles, the present invention
  • the obvious method has at least the following advantages:
  • the method for preparing the polyhydroxyalkanoate agglomerate provided by the present invention does not use any organic solvent during the preparation process, the production environment is simple and safe, and there is no organic solvent residue in the prepared polyhydroxyalkanoate agglomerate. .
  • the method for preparing polyhydroxyalkanoate agglomerates provided by the present invention does not use inorganic salts or flocculants during the preparation process, which effectively reduces the cost of preparation reagents.
  • the method for preparing polyhydroxyalkanoate agglomerates provided by the present invention can achieve efficient agglomeration of polyhydroxyalkanoate under lower temperature conditions, does not rely on high-temperature operation, effectively reduces energy consumption, and has simple extraction equipment. , saving costs.
  • the preparation method of polyhydroxyalkanoate agglomerates does not require cleaning of polyhydroxyalkanoate particles before agglomeration.
  • the enzymatic decontamination process can be carried out in the same reaction system as the agglomeration process, without the need for enzymatic decontamination first. Coagulation is performed after removing impurities, which effectively simplifies the preparation process and reduces the amount of waste water.
  • the detection method of PHA purity is as follows:
  • the preparation method of the esterification liquid is as follows: weigh 0.5g of benzoic acid and add it to a methanol reagent bottle containing 485mL, slowly add 15mL of concentrated sulfuric acid into the methanol reagent bottle, and mix well to complete the preparation of the esterification liquid.
  • the method for measuring nitrogen content is as follows: According to the method in 4.3 of GB/T-32019-2013, use a Kjeldahl nitrogen analyzer to measure nitrogen content.
  • the detection method of PHA aggregate particle size is as follows: use the Truth Optics LT2100 laser particle size analyzer to measure the particle size.
  • the PHA suspension used in the following examples was prepared as follows: Utilizing PHA production strains After fermentation, obtain a fermentation broth containing 80% to 85% PHA with a biomass of 200-220g/L; adjust the pH of the fermentation broth to 11, maintain it for 2 hours, and mechanically disrupt the cells. Use a cup centrifuge to centrifuge at 10,000 rpm for 5 minutes. Pour off the supernatant and redissolve it with water to the original volume to obtain a PHA suspension.
  • the PHA content is 163g/L.
  • the nitrogen content of the PHA particles is 6530ppm.
  • the clear liquid contains The nitrogen content is 2459ppm.
  • This embodiment provides a method for preparing PHA aggregates.
  • the steps of the method are as follows: Pour the PHA suspension into a stirring reactor, start stirring at 600r/min, raise the temperature to 40°C, and add SDS with a final concentration of 8g/L. , neutral protease with a final concentration of 1mL/L was used to start the purification reaction, during which the particle size of the PHA aggregate was measured every 10 minutes until the particle size no longer continued to increase.
  • the diameter of the turbine is 6cm
  • the viscosity of the material liquid is 0.0075Ns/m 2
  • the density of the material liquid is 1030kg/m 3 . It is obtained by using the Reynolds coefficient calculation formula. After starting the stirring, the Reynolds coefficient of the material liquid in the reactor is 4944, achieving sufficient turbulence. state.
  • This embodiment provides a method for preparing PHA aggregates.
  • the steps of the method are as follows: Pour the PHA suspension into a stirring reactor, start stirring at 150r/min, raise the temperature to 40°C, and add SDS with a final concentration of 8g/L. , neutral protease with a final concentration of 1mL/L was used to start the purification reaction, during which the particle size of the PHA aggregate was measured every 10 minutes until the particle size no longer continued to increase.
  • the diameter of the turbine is 6cm
  • the viscosity of the material liquid is 0.0075Ns/m 2
  • the density of the material liquid is 1030kg/m 3 . It is obtained by using the Reynolds coefficient calculation formula. After starting the stirring, the Reynolds coefficient of the material liquid in the reactor is 1236, a turbulent flow state was reached.
  • This embodiment provides a method for preparing PHA aggregates.
  • the steps of the method are as follows: Pour the PHA suspension into a stirring reactor, start stirring at 150r/min, raise the temperature to 40°C, and add SDS with a final concentration of 8g/L. , papain with a final concentration of 1mL/L was used to start the purification reaction, during which the PHA aggregate particle size was measured every 10 minutes until the particle size no longer continued to increase.
  • the diameter of the turbine is 6cm
  • the viscosity of the feed liquid is 0.0075Ns/m 2
  • the density of the feed liquid is 1030kg/m 3 , which is obtained by using the Reynolds coefficient calculation formula.
  • the Reynolds coefficient of the feed liquid in the reactor is 1236, reaching a turbulent flow state.
  • This embodiment provides a method for preparing PHA aggregates.
  • the steps of the method are as follows: PHA suspension was poured into the stirring reactor, stirred at 150r/min, raised to 40°C, added SDS with a final concentration of 10g/L, papain with a final concentration of 0.5mL/L, and started the purification reaction every 10min. Detect the particle size of the PHA aggregates until the particle size no longer increases.
  • the diameter of the turbine is 6cm
  • the viscosity of the feed liquid is 0.0075Ns/m 2
  • the density of the feed liquid is 1030kg/m 3 , which is obtained by using the Reynolds coefficient calculation formula.
  • the Reynolds coefficient of the feed liquid in the reactor is 1236, reaching a turbulent flow state.
  • This embodiment provides a method for preparing PHA aggregates.
  • the steps of the method are as follows: Pour the PHA suspension into the reactor, start stirring at 150r/min, raise the temperature to 40°C, and add 12 mg/L with a final concentration of 4g/L. Sodium alkyl benzene sulfonate and neutral protease with a final concentration of 0.8 mL/L were used to start the purification reaction. During this period, the particle size of the PHA aggregate was measured every 10 minutes until the particle size no longer continued to increase.
  • the diameter of the turbine is 6cm
  • the viscosity of the feed liquid is 0.0075Ns/m 2
  • the density of the feed liquid is 1030kg/m 3 , which is obtained by using the Reynolds coefficient calculation formula.
  • the Reynolds coefficient of the feed liquid in the reactor is 1236, reaching a turbulent flow state.
  • This embodiment provides a method for preparing PHA agglomerates.
  • the steps of the method are as follows: Pour the PHA suspension into the reactor, start stirring at 150r/min, the temperature is 20°C, and start the purification reaction. During this period, the PHA agglomerates are detected every 10 minutes. collective particle size until the particle size no longer continues to increase.
  • the diameter of the turbine is 6cm
  • the viscosity of the feed liquid is 0.0075Ns/m 2
  • the density of the feed liquid is 1030kg/m 3 , which is obtained by using the Reynolds coefficient calculation formula.
  • the Reynolds coefficient of the feed liquid in the reactor is 1236, reaching a turbulent flow state.
  • This embodiment provides a method for preparing PHA aggregates.
  • the steps of the method are as follows: Pour the PHA suspension into a stirring reactor, start stirring at 600r/min, raise the temperature to 40°C, and add medium with a final concentration of 1mL/L. protease, start the purification reaction, and detect the particle size of the PHA aggregates every 10 minutes until the particle size no longer continues to increase.
  • the diameter of the turbine is 6cm
  • the viscosity of the material liquid is 0.0075Ns/m 2
  • the density of the material liquid is 1030kg/m 3 . It is obtained by using the Reynolds coefficient calculation formula. After starting the stirring, the Reynolds coefficient of the material liquid in the reactor is 4944, achieving sufficient turbulence. state.
  • This embodiment provides a method for preparing PHA aggregates.
  • the steps of the method are as follows: add 2 times the volume of water to the PHA suspension used in Example 1 to dilute it so that the content of PHA in the suspension is about 50g/L. , pour into the stirred reactor, start stirring at 600r/min, raise the temperature to 40°C, add SDS with a final concentration of 8g/L, and neutral protease with a final concentration of 1mL/L, start the purification reaction, and detect PHA every 10 minutes. Aggregate particle size until the particle size no longer continues to increase.
  • the diameter of the turbine is 6cm
  • the viscosity of the material liquid is 0.0075Ns/m 2
  • the density of the material liquid is 1030kg/m 3 . It is obtained by using the Reynolds coefficient calculation formula. After starting the stirring, the Reynolds coefficient of the material liquid in the reactor is 4944, achieving sufficient turbulence. state.
  • the PHA suspension (the PHA suspension is the same as in Example 1) into the stirring reactor, start stirring at 100r/min, raise the temperature to 40°C, add SDS with a final concentration of 8g/L, and a final concentration of 1mL/L.
  • Neutral protease is used to start the purification reaction, during which the PHA particle size is measured every 10 minutes until the particle size no longer continues to increase.
  • the diameter of the turbine is 6cm
  • the viscosity of the material liquid is 0.0075Ns/m 2
  • the density of the material liquid is 1030kg/m 3 . It is obtained by using the Reynolds coefficient calculation formula. After starting the stirring, the Reynolds coefficient of the material liquid in the reactor is 824, which does not reach the turbulent flow state.
  • the PHA suspension (the PHA suspension is the same as in Example 1) into the stirring reactor, start stirring at 100r/min, the temperature is 20°C, and start the purification reaction. During this period, the PHA particle size is detected every 10 minutes until the particle size is no longer Continue to increase. Among them, the diameter of the turbine is 6cm, the viscosity of the material liquid is 0.0075Ns/m 2 , and the density of the material liquid is 1030kg/m 3 . It is obtained by using the Reynolds coefficient calculation formula. After starting the stirring, the Reynolds coefficient of the material liquid in the reactor is 824, which does not reach turbulent flow.
  • the invention provides a method for preparing polyhydroxyalkanoate aggregates.
  • the method for preparing the polyhydroxyalkanoate agglomerate provided by the present invention includes the step of bringing the polyhydroxyalkanoate suspension to and maintaining a turbulent flow state to aggregate the polyhydroxyalkanoate.
  • the preparation method of the present invention can produce polyhydroxyalkanoate agglomerates with a particle size of 50 ⁇ m or more, and the produced polyhydroxyalkanoate agglomerates have high purity and yield; the method has mild process conditions and simple steps. , Wastewater treatment is simple, low cost, low equipment requirements, can achieve large-scale industrial production, and has good economic value and application prospects.

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Abstract

本发明涉及生物工程技术领域，具体涉及一种聚羟基脂肪酸酯凝集体的制备方法。本发明提供的聚羟基脂肪酸酯凝集体的制备方法包括使聚羟基脂肪酸酯悬浮液达到并维持湍流状态，以使得聚羟基脂肪酸酯凝聚的步骤。本发明的制备方法能够制得粒径50μm以上的聚羟基脂肪酸酯凝集体，且制得的聚羟基脂肪酸酯凝集体具有较高的纯度和收率；该方法的工艺条件温和、步骤简单、废水处理简单，成本低廉、对设备要求低，可实现大规模工业化生产。

Description

一种聚羟基脂肪酸酯凝集体的制备方法
交叉引用
本申请要求2022年6月6日提交的专利名称为“一种聚羟基脂肪酸酯凝集体的制备方法”的第202210635146.8号中国专利申请的优先权，其全部公开内容通过引用整体并入本文。
技术领域
本发明涉及生物工程技术领域，具体涉及一种聚羟基脂肪酸酯凝集体的制备方法。
背景技术
聚羟基脂肪酸酯(polyhydroxyalkanoates，简称PHA)是微生物在其他营养限制而碳源过剩条件下合成的一种储藏性颗粒物，是一种生物高分子聚酯化合物。PHA具有普通热塑性塑料的特性，但有别于传统的石油化工塑料，具备良好的生物相容性、可降解性、压电性和光学活性，使其在工业、农业、医药卫生、食品等领域有着广阔的应用前景。在可降解农用地膜领域和可降解塑料用品和医学领域，PHA作为目前最佳的生物可降解材料已经显现出巨大的潜力。
由于微生物产生的PHA通常以包涵体的形式积累在微生物的细胞体中，因此需要将细胞内部的PHA颗粒进行分离回收。PHA颗粒用于塑料制备时，需要具有良好的加工性能，因此对PHA颗粒的纯度要求较高，以尽量减少无机盐或细胞裂解物等杂质的干扰。
然而，由于微生物细胞内成分相当复杂，PHA的提取难度相当大。在PHA发展的漫长历程中，研究者们对其提取方法进行了大量的研究。已有的提取工艺包括有机溶剂抽提法、机械破碎法、次氯酸钠-表面活性剂法、酶法等。其中，有机溶剂法的缺点是溶剂回收困难，生产环境危险，只适合实验室提取；机械破碎法主要包括超声波破壁或者高压匀浆破壁，能耗大，放大生产难度大；次氯酸钠-表面活性剂法的缺点是次氯酸钠刺激性强， 工作环境差，对PHA分子量破坏严重，且含有表面活性剂的废水处理难度大；酶法的缺点是要用到多种昂贵的酶类，成本很高。上述方法存在的各种问题阻碍PHA的工业化生产。
将含有PHA的微生物细胞破碎得到PHA悬浮液，在从PHA悬浮液中回收PHA颗粒时均会遇到由于PHA颗粒太小，无法进行过滤或者离心分离得到PHA颗粒的问题。现有技术虽然公开了一些使PHA颗粒粒径增大以便于分离的方法，但是均有不同的缺点，具体如下：
公开号EP1609868B1的发明专利公开了一种从微生物细胞中收集高纯度聚羟基链烷酸酯的方法，该方法包括通过在含有PHA的微生物细胞的含水悬浮液中进行物理破碎处理和低温碱添加以有效地破坏细胞以回收PHA，然后用酶和/或表面活性剂处理PHA，随后用一种亲水性溶剂洗涤PHA颗粒以进行脱脂，除臭和脱色后，通过加热和搅拌使PHA颗粒聚集，随后进行抽滤和干燥。该方法虽然反应条件温和，回收率较高，但是需要使用大量的有机溶剂进行PHA颗粒的洗涤，导致下游废水处理困难，且需要防爆车间和防爆设备，使得工业化生产较为困难。
公开号EP2357247B1的发明专利公开了聚3-羟基链烷酸酯的制备方法，该方法包括通过在含有PHA的微生物细胞的含水悬浮液中进行物理破碎处理和低温碱添加以有效地破坏细胞以回收PHA，然后用酶和/或表面活性剂处理PHA，随后用大量水清洗PHA颗粒，通过将含有聚-3-羟基链烷酸的水性悬浮液中的有机氮的含量调节至低于1500ppm每重量的聚-3-羟基链烷酸，再通过加热和搅拌使PHA颗粒聚集，随后进行抽滤和干燥。该方法虽然反应条件温和，回收率较高，但是需要使用大量的水通过离心的方式进行颗粒的洗涤，制备过程产生大量废水，增加了下游废水处理的难度。
公开号EP3027761B1的发明专利公开了从细胞培养物中回收和纯化聚羟基链烷酸酯的方法，从细胞培养物中回收和纯化PHA的方法，包括酸化细胞培养物并物理破碎细胞以获得PHA悬浮液。随后将PHA悬浮液碱 化至pH值等于或高于8，稀释后进行切向过滤。然后将PHA悬浮液漂白，稀释并再次进行切向过滤，然后干燥得到成品。该方法可以在不使用有机溶剂的情况下连续进行，并以纯净形式获得PHA而不会导致分子量降低，但是由于PHA颗粒较小，在切向过滤时容易发生设备堵塞，需要加入氧化钙、硫酸铝、磷酸及其混合物作为絮凝剂，使PHA颗粒发生聚集，增大PHA颗粒粒径。由于絮凝剂得到的颗粒强度并不总是很高，而且金属盐以及残留的细胞碎片会污染PHA聚合物，降低了成品PHA的纯度，为下游材料加工增加了困难。
公开号US9683076B2的发明专利将PHA悬浮液通过喷雾干燥的方式得到PHA颗粒，但是喷雾干燥能耗大，且会将悬浮液中残留的无机盐和细胞裂解物混合在PHA颗粒中，降低了成品PHA的纯度，为下游材料加工增加了困难。
综上所述，现有技术公开的方法或需要使用大量有机溶剂或者水进行PHA颗粒的清洗，或需要使用絮凝剂进而降低了成品的纯度，或存在工艺流程复杂、生产成本高等问题。
发明内容
本发明的目的是提供一种聚羟基脂肪酸酯凝集体的制备方法以及由该方法制备得到的聚羟基脂肪酸酯凝集体。
为解决现有技术公开的使聚羟基脂肪酸酯凝聚以增大粒径的方法中需要使用大量有机溶剂或水清洗聚羟基脂肪酸酯颗粒才能使其凝聚、以及能耗高、工艺复杂等问题，本发明对聚羟基脂肪酸酯的凝聚过程进行了大量研究。在研发过程中，本发明意外地发现，聚羟基脂肪酸酯悬浮液处于湍流状态是促进聚羟基脂肪酸酯颗粒凝聚的关键因素：在不对聚羟基脂肪酸酯悬浮液中的聚羟基脂肪酸酯颗粒进行清洗的情况下，低转速的搅拌、甚至达到涡流状态均很难实现聚羟基脂肪酸酯的凝聚，而一旦达到湍流状态，聚羟基脂肪酸酯悬浮液中的聚羟基脂肪酸酯开始快速自发发生聚集，粒径明显增大，在较短时间内即可获得大粒径的聚羟基脂肪酸酯凝集体。 虽然现有技术公开的凝聚方法中也涉及了搅拌，但是从其凝聚工艺和结果来看，这些方法使用的搅拌并没有使得聚羟基脂肪酸酯悬浮液达到湍流状态，从未有现有技术发现湍流状态对聚羟基脂肪酸酯凝聚的重要性。
基于上述发现，本发明提供以下技术方案：
本发明提供一种聚羟基脂肪酸酯凝集体的制备方法，所述方法包括使聚羟基脂肪酸酯悬浮液达到并维持湍流状态，以使得聚羟基脂肪酸酯凝聚的步骤。
以上所述的湍流状态为聚羟基脂肪酸酯悬浮液的雷诺系数大于聚羟基脂肪酸酯悬浮液在其所处设备中达到湍流的临界雷诺系数时所处状态。
上述湍流状态可在反应器中进行，也可在管道中进行。
以反应器为例，反应器中的雷诺系数的计算公式如下：
其中，D：涡轮直径(m)；N：搅拌器转速(r/s)；ρ：料液密度(kg/m3)；μ：料液粘度(N.s/m2)。
当在管道中进行湍流时，雷诺系数的计算公式为：Re＝ρvd/μ，
其中，v：流体流速(m/s)、ρ：料液密度(kg/m3)、μ：料液粘度(N.s/m2)，d：特征长度。
优选地，当聚羟基脂肪酸酯悬浮液所处设备为反应器时，湍流状态为聚羟基脂肪酸酯悬浮液的雷诺系数大于1000时，悬浮液所处状态。
当聚羟基脂肪酸酯悬浮液所处设备为管道时，湍流状态为聚羟基脂肪酸酯悬浮液的雷诺系数大于4000时，悬浮液所处状态。
在本发明的一些实施方式中，聚羟基脂肪酸酯的凝聚在反应器中进行，所述湍流状态为雷诺系数大于1000，优选大于1200，更优选大于4000。
对于反应器，本发明没有特殊限制，可以是任何能够使料液处于湍流状态的设备。
在本发明的一些实施方式中，通过对聚羟基脂肪酸酯悬浮液进行不断 的搅拌以使得悬浮液达到并维持湍流状态。
对于聚羟基脂肪酸酯悬浮液，优选其中的聚羟基脂肪酸酯颗粒的含氮量低于7000ppm(相对于每单位质量聚羟基脂肪酸酯)，悬浮液清液的含氮量低于3000ppm(相对于每单位质量悬浮液清液)。
以上所述的聚羟基脂肪酸酯颗粒和悬浮液清液的含氮量的检测方法如下：将聚羟基脂肪酸酯悬浮液在10000rpm条件下离心5min，上清即为悬浮液清液，将沉淀水洗两次离心烘干后，得到聚羟基脂肪酸酯颗粒，分别检测悬浮液清液和聚羟基脂肪酸酯颗粒的含氮量。
若聚羟基脂肪酸酯颗粒、悬浮液清液的含氮量高于上述目标值，会明显抑制聚羟基脂肪酸酯凝集体的产生，甚至导致不会出现凝集体，或者成品杂质含量过高，聚羟基脂肪酸酯凝集体的纯度降低。
以上所述的聚羟基脂肪酸酯悬浮液以聚羟基脂肪酸酯的生物发酵液为原料，经细胞破碎、固液分离收集聚羟基脂肪酸酯沉淀，再以水悬浮得到。
上述生物发酵液可为采用任意能够发酵产生聚羟基脂肪酸酯的生物体经发酵培养制得的含有用于发酵的生物体的发酵液。
对于生物体，只要是能够在细胞内积累聚羟基脂肪酸酯的生物体均可使用，没有特别的限定。优选为微生物，可以举出，例如，气单胞菌属(Aeromonas)、产碱菌属(Alcaligenes)、固氮菌属(Azotobacter)、芽孢杆菌属(Bacillus)、梭菌属(Clostridium)、盐杆菌属(Halobacterium)、诺卡氏菌属(Nocardia)、红螺菌属(Rhodospirillum)、假单胞菌属(Pseudomonas)、罗尔斯通氏菌属(Ralstonia)、动胶菌属(Zoogloea)等微生物。
具体地，作为气单胞菌属，可以举出，例如豚鼠气单胞菌(Aeromonas caviae)等；作为产碱菌属，可以举出，例如解脂产碱菌(Alcaligenes lipolytica)、广泛产碱菌(Alcaligenes latus)等；作为罗尔斯通氏菌属(Ralstonia)，可以举出，例如罗氏真养菌(Ralstonia eutropha)等。
上述聚羟基脂肪酸酯悬浮液的制备过程中，对于细胞破碎的方法没有 特别的限定，只要能将细胞内的聚羟基脂肪酸酯释放出来的方法均可使用，可采用化学法、机械法、生物法等。
本发明的凝集体制备方法对于悬浮液的制备工艺要求较为简单，可采用将发酵液调节至碱性后，再进行机械法破碎细胞的方法释放细胞内的聚羟基脂肪酸酯颗粒。
在本发明的一些实施方式中，将发酵液的pH调节至碱性(优选为10.5-11.5)后，放置1-3h，再采用机械法破碎细胞。
对于固液分离的方法也没有特别的限定，只要能将聚羟基脂肪酸酯和非聚羟基脂肪酸酯分离开来的方法均可以使用，可采用离心方式、过滤方式等，其中，非聚羟基脂肪酸酯物质包括但不仅限于细胞碎片、蛋白质、核酸等杂质。
本发明对于聚羟基脂肪酸酯悬浮液中聚羟基脂肪酸酯的浓度并不特别限定，若想要提高聚羟基脂肪酸酯凝集体的产量，优选使用较高浓度的聚羟基脂肪酸酯悬浮液。
在本发明的一些实施方式中，优选聚羟基脂肪酸酯悬浮液中聚羟基脂肪酸酯的含量不低于20g/L。更优选为50g/L以上。
与现有技术公开的聚羟基脂肪酸酯凝聚的方法不同，本发明对于用于凝聚的聚羟基脂肪酸酯悬浮液的pH没有特别限制，聚羟基脂肪酸酯悬浮液可为酸性、中性或碱性。
若采用将发酵液调节至碱性后，再进行细胞破碎释放细胞内的聚羟基脂肪酸酯颗粒，经固液分离收集聚羟基脂肪酸酯沉淀，再以水悬浮的方法制备聚羟基脂肪酸酯悬浮液，无需对复水后的悬浮液进行pH调节，此时悬浮液的pH为8.5-10.5，该悬浮液可直接用于聚羟基脂肪酸酯的凝聚。在聚羟基脂肪酸酯凝聚过程中，保持温度不低于20℃有利于促进凝集的进行。
优选地，在维持湍流状态的过程中，同时维持聚羟基脂肪酸酯悬浮液的温度不低于20℃。
在本发明的一些实施方式中，在维持湍流状态的过程中，同时维持聚 羟基脂肪酸酯悬浮液的温度不低于30℃。
在本发明的一些实施方式中，在维持湍流状态的过程中，同时维持聚羟基脂肪酸酯悬浮液的温度不低于40℃。
优选地，在维持湍流状态的过程中，同时维持聚羟基脂肪酸酯悬浮液的温度为20-70℃，更优选为20-40℃。
以上所述的凝集体制备方法中，维持湍流状态的时间可根据优选地，在维持湍流状态的过程中，同时维持聚羟基脂肪酸酯悬浮液的温度不低于20℃。
想要得到的凝集体的粒径大小而定，所需聚羟基脂肪酸酯凝集体粒径越大，所需维持时间越长。在凝集过程中，通过对凝集体进行粒径监测，在达到所需粒径时，收集凝集体。粒径监测可通过目测或在线实时检测等方式进行。
对于聚羟基脂肪酸酯凝集体的粒径并没有特别限定，适用于工业分离的粒径均可，一般优选为5μm以上，更优选为50μm以上，以更好地适用于工业化固液分离。为了得到适用于固液分离的聚羟基脂肪酸酯凝集体，凝聚直至得到合适粒径的聚羟基脂肪酸酯凝集体，再经固液分离收集聚羟基脂肪酸凝集体。
对于固液分离的方法没有特殊限制，包括但不限于离心、过滤等。
以上所述的凝集体制备方法中，通常在0.1-3h的时间内出现聚羟基脂肪酸酯颗粒的聚集，聚羟基脂肪酸酯凝集体的粒径明显增大，优选维持湍流状态的时间为40-180min。
通过本发明的方法，可以得到聚羟基脂肪酸酯凝集体的平均粒径为10μm或10μm以上，50μm或50μm以上，甚至100μm或100μm以上的凝集体。对于粒径的上限没有特别的限定，但优选平均直径为5000μm或5000μm以下，优选3000μm或3000μm以下。
在本发明的一些实施方式中，所述凝集体的制备方法包括：以聚羟基脂肪酸酯悬浮液为原料，对聚羟基脂肪酸酯悬浮液进行搅拌以使得聚羟基 脂肪酸酯悬浮液达到并维持湍流状态，同时维持温度不低于20℃，获得粒径增大的聚羟基脂肪酸酯凝集体。
进一步地，为更好地去除悬浮液中的杂质、促进凝集体的形成，所述凝集体的制备方法还包括以酶去除聚羟基脂肪酸酯悬浮液中的杂质的步骤。
所述酶用于对聚羟基脂肪酸酯颗粒表面附着的肽聚糖、脂质类、多糖类、核酸类杂质进行处理，包括但不限于蛋白质分解酶、脂质类分解酶、细胞壁分解酶、DNA分解酶中的一种或多种。在具体应用时，上述酶可以单独使用，也可以两种或两种以上并用。对于酶的来源没有特殊限制，可为市售的酶制剂，或市售的洗涤用酶洗涤剂等。
所述酶的添加量依赖于酶的种类以及活性，虽然没有特别的限定，但是为了降低成本，优选采用酶活力为10万-200万U/mL，酶的添加量为0.001mL/L-10mL/L(相对于聚羟基脂肪酸酯悬浮液)。
当向凝聚体系中添加酶进行除杂处理时，温度和pH可根据所用酶的最适温度和最适pH进行调整，例如控制温度在20-70℃，pH为5-10。由于酶的最适温度和最适pH属于本领域技术人员的公知常识，本发明不再赘述。
进一步优选地，为了保证最终凝集体成品的质量(主要指产品的纯度)，以使其更好地适用于下游材料加工，还可在加入酶的同时以表面活性剂去除聚羟基脂肪酸酯悬浮液中的杂质，以更进一步地去除附着在PHA颗粒表面的杂质。
以上所述的表面活性剂为选自阴离子表面活性剂、阳离子表面活性剂、两性表面活性剂、非离子表面活性剂中的一种或多种，优选为阴离子表面活性剂。
对于表面活性剂的添加量虽无需特别限定，优选的添加量为1g/L～20g/L(相对于聚羟基脂肪酸酯悬浮液)。
在本发明的一些实施方式中，所述表面活性剂为SDS，SDS的用量优 选为5-20g/L。
在本发明的一些实施方式中，所述表面活性剂为十二烷基苯磺酸钠，十二烷基苯磺酸钠的用量优选为2-10g/L。
与现有技术公开的先以酶和/或表面活性剂除杂，再清洗、分离聚羟基脂肪酸酯后，再进行聚羟基脂肪酸酯的凝聚的方法不同，本发明的凝集体的制备方法可实现凝聚和除杂(利用酶和/或表面活性剂除杂)在同一反应体系中进行，极大地简化了制备工艺，同时减少了废液产生。
优选地，所述凝集体的制备方法中，上述去除聚羟基脂肪酸酯悬浮液中的杂质的步骤与聚羟基脂肪酸酯的凝聚在同一反应体系中进行。
现有技术公开的方法多涉及采用有机溶剂洗涤聚羟基脂肪酸酯颗粒后再进行凝聚反应，或在含有有机溶剂的体系中进行凝集反应，本发明的凝集体的制备方法可实现不使用有机溶剂清洗、且在不含有有机溶剂的体系中进行聚羟基脂肪酸酯的有效凝聚。
优选地，所述聚羟基脂肪酸酯的凝聚体系中不包含有机溶剂。
在本发明的一些实施方式中，所述聚羟基脂肪酸酯凝集体的制备方法包括：以聚羟基脂肪酸酯悬浮液为原料，对聚羟基脂肪酸酯悬浮液进行搅拌以使得聚羟基脂肪酸酯悬浮液达到湍流状态，加入酶，维持湍流状态，同时维持温度不低于20℃，获得粒径增大的聚羟基脂肪酸酯凝集体，达到目标粒径时，收集聚羟基脂肪酸酯凝集体。
在本发明的一些实施方式中，所述聚羟基脂肪酸酯凝集体的制备方法包括：以聚羟基脂肪酸酯悬浮液为原料，对聚羟基脂肪酸酯悬浮液进行搅拌以使得聚羟基脂肪酸酯悬浮液达到湍流状态，加入酶和表面活性剂，维持湍流状态，同时维持温度不低于20℃，获得粒径增大的聚羟基脂肪酸酯凝集体，达到目标粒径时，收集聚羟基脂肪酸酯凝集体。
收集聚羟基脂肪酸酯凝集体可通过固液分离进行。伴随着聚羟基脂肪酸酯凝集体粒径的增大，聚羟基脂肪酸酯凝集体固液分离的难度明显降低，在工业生产上可以减少设备费用。因此对固液分离方法和设备没有特别的 限定，可以使用过滤(板框过滤机、篮袋式过滤机等)或离心(卧螺离心机等)等方法。
为了使聚羟基脂肪酸酯凝集体中的杂质去除更为彻底，优选对收集的聚羟基脂肪酸酯凝集体进行清洗后再进行固液分离，收集清洗后的聚羟基脂肪酸酯凝集体。
在本发明的一些实施方式中，以水对收集的聚羟基脂肪酸酯凝集体进行清洗后，经固液分离收集聚羟基脂肪酸酯凝集体。
对于上述聚羟基脂肪酸酯凝集体，还可进行干燥以制得干燥的凝集体，干燥方法没有特别限定，可以为常温通风干燥、加热通风干燥等。
进一步地，本发明提供采用以上所述的聚羟基脂肪酸酯凝集体的制备方法制备得到的聚羟基脂肪酸酯凝集体。
优选地，所述聚羟基脂肪酸酯凝集体的粒径为至少5μm，更优选为至少50μm。
在本发明的一些实施方式中，聚羟基脂肪酸酯凝集体的粒径为50～100μm。
在本发明的一些实施方式中，聚羟基脂肪酸酯凝集体的粒径为100～300μm。
本发明中，所述聚羟基脂肪酸酯(PHA)可为各种聚羟基脂肪酸酯产品，包括但不限于聚羟基丁酸(PHB)、聚3-羟基丁酸和3-羟基戊酸共聚酯(PHBV)、聚3-羟基丁酸和3-羟基己酸共聚酯(PHBH)、聚3-羟基丁酸和4-羟基丁酸共聚酯(P3HB4HB)等。
本发明的有益效果至少在于：本发明提供的聚羟基脂肪酸酯凝集体的制备方法能够制得粒径50μm以上的聚羟基脂肪酸酯凝集体，以适合工业化固液分离设备，且制得的聚羟基脂肪酸酯凝集体具有较高的纯度和收率；该方法的工艺条件温和、步骤简单、废水处理简单，成本低廉、对设备要求低，可实现大规模工业化生产。
具体而言，与现有的增大聚羟基脂肪酸酯颗粒粒径的方法相比，本发 明的方法至少具有以下优势：
1、本发明提供的聚羟基脂肪酸酯凝集体的制备方法，在制备过程中不使用任何有机溶剂，生产环境简单、安全，且制得的聚羟基脂肪酸酯凝集体中不存在有机溶剂残留。
2、本发明提供的聚羟基脂肪酸酯凝集体的制备方法，在制备过程中不使用无机盐、无需使用絮凝剂，有效减少了制备的试剂成本。
3、本发明提供的聚羟基脂肪酸酯凝集体的制备方法，在较低温度条件下即可实现聚羟基脂肪酸酯的高效凝聚，不依赖高温操作，有效降低了能耗，且提取设备简单，节约了成本。
4、本发明提供的聚羟基脂肪酸酯凝集体的制备方法，无需先对聚羟基脂肪酸酯颗粒进行清洗再进行凝聚，酶解除杂过程可与凝聚过程在同一反应体系中进行，无需先酶解除杂后再进行凝聚，有效简化了制备工艺，减少了废水量。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。
以下实施例中，PHA纯度的检测方法如下：
取30mg干燥后的待测样品，加入2mL酯化液和2mL三氯甲烷，100℃加热4h，反应结束后，待样品冷却至室温后，加入1mL去离子水，涡旋振荡，静置30-60min，使其分层，上层为水相，下层为有机相；取1mL下层有机相，使用岛津气相色谱仪进行气相色谱(GC)检测，并计算PHA纯度。其中，酯化液的配制方法为：称取0.5g苯甲酸加入装有485mL的甲醇试剂瓶中，取15mL浓硫酸缓慢加入至甲醇试剂瓶中，混匀后完成酯化液的配制。
含氮量的测定方法如下：按照GB/T-32019-2013中4.3的方法，使用凯式定氮仪进行含氮量的测定。
PHA凝集体粒径的检测方法如下：使用真理光学LT2100激光粒度分析仪测定粒度大小。
以下实施例中使用的PHA悬浮液的制备方法如下：利用PHA生产菌株 发酵后，获得生物量为200～220g/L的含80％～85％PHA的发酵液；将发酵液pH调节至11后，维持2h，机械法破碎细胞。使用杯式离心机离心，10000rpm离心5min后，倒掉上清液，加水复溶至原体积，得到PHA悬浮液，其中PHA含量为163g/L，PHA颗粒含氮量为6530ppm，清液中含氮量为2459ppm。
实施例1
本实施例提供一种PHA凝集体的制备方法，该方法的步骤如下：将PHA悬浮液倒入搅拌反应器中，开启搅拌600r/min，升温至40℃，加入终浓度为8g/L的SDS，终浓度为1mL/L的中性蛋白酶，开始纯化反应，期间每隔10min检测PHA凝集体粒径，直至粒径不再继续增加。
其中，涡轮直径6cm，料液粘度0.0075N.s/m2，料液密度1030kg/m3，利用雷诺系数计算公式得到，启动搅拌后，反应器中的料液雷诺系数为4944，达到了充分的湍流状态。
上述方法中，纯化阶段PHA颗粒聚集时粒径变化情况如表1所示。
表1
当PHA凝集体粒径增加至100μm以上时，使用布氏漏斗抽滤，将得到的滤饼复水水洗两次后，过滤，使用鼓风干燥箱80℃烘干，检测PHA纯度为99.1％，回收率98.5％。
实施例2
本实施例提供一种PHA凝集体的制备方法，该方法的步骤如下：将PHA悬浮液倒入搅拌反应器中，开启搅拌150r/min，升温至40℃，加入终浓度为8g/L的SDS，终浓度为1mL/L的中性蛋白酶，开始纯化反应，期间每隔10min检测PHA凝集体粒径，直至粒径不再继续增加。
其中，涡轮直径6cm，料液粘度0.0075N.s/m2，料液密度1030kg/m3，利用雷诺系数计算公式得到，启动搅拌后，反应器中的料液雷诺系数为 1236，达到了湍流状态。
上述方法中，纯化阶段PHA颗粒聚集时粒径变化情况如表2所示。
表2
当PHA凝集体粒径增加至100μm以上时，使用布氏漏斗抽滤，将得到的滤饼复水水洗两次后，过滤，使用鼓风干燥箱80℃烘干，检测PHA纯度为99.2％，回收率98.4％。
实施例3
本实施例提供一种PHA凝集体的制备方法，该方法的步骤如下：将PHA悬浮液倒入搅拌反应器中，开启搅拌150r/min，升温至40℃，加入终浓度为8g/L的SDS，终浓度为1mL/L的木瓜蛋白酶，开始纯化反应，期间每隔10min检测PHA凝集体粒径，直至粒径不再继续增加。
其中，涡轮直径6cm，料液粘度0.0075N.s/m2，料液密度1030kg/m3，利用雷诺系数计算公式得到，启动搅拌后，反应器中的料液雷诺系数为1236，达到了湍流状态。
上述方法中，纯化阶段PHA颗粒聚集时粒径变化情况如表3所示。
表3
当PHA凝集体粒径增加至100μm以上时，使用布氏漏斗抽滤，将得到的滤饼复水水洗两次后，过滤，使用鼓风干燥箱80℃烘干，检测PHA纯度为98.4％，回收率98.3％。
实施例4
本实施例提供一种PHA凝集体的制备方法，该方法的步骤如下：将 PHA悬浮液倒入搅拌反应器中，开启搅拌150r/min，升温至40℃，加入终浓度为10g/L的SDS，终浓度为0.5mL/L的木瓜蛋白酶，开始纯化反应，期间每隔10min检测PHA凝集体粒径，直至粒径不再继续增加。
其中，涡轮直径6cm，料液粘度0.0075N.s/m2，料液密度1030kg/m3，利用雷诺系数计算公式得到，启动搅拌后，反应器中的料液雷诺系数为1236，达到了湍流状态。
上述方法中，纯化阶段PHA颗粒聚集时粒径变化情况如表4所示。
表4
当PHA凝集体粒径增加至100μm以上时，使用布氏漏斗抽滤，将得到的滤饼复水水洗两次后，过滤，使用鼓风干燥箱80℃烘干，检测PHA纯度为98.7％，回收率98.4％。
实施例5
本实施例提供一种PHA凝集体的制备方法，该方法的步骤如下：将PHA悬浮液倒入反应器中，开启搅拌150r/min，升温至40℃，加入终浓度为4g/L的十二烷基苯磺酸钠，终浓度为0.8mL/L的中性蛋白酶，开始纯化反应，期间每隔10min检测PHA凝集体粒径，直至粒径不再继续增加。
其中，涡轮直径6cm，料液粘度0.0075N.s/m2，料液密度1030kg/m3，利用雷诺系数计算公式得到，启动搅拌后，反应器中的料液雷诺系数为1236，达到了湍流状态。
上述方法中，纯化阶段PHA颗粒聚集时粒径变化情况如表5所示。
表5
当PHA凝集体粒径增加至100μm以上时，使用布氏漏斗抽滤，将得到的滤饼复水水洗两次后，过滤，使用鼓风干燥箱80℃烘干，检测PHA纯度为98.6％，回收率98.5％。
实施例6
本实施例提供一种PHA凝集体的制备方法，该方法的步骤如下：将PHA悬浮液倒入反应器中，开启搅拌150r/min，温度20℃，开始纯化反应，期间每隔10min检测PHA凝集体粒径，直至粒径不再继续增加。
其中，涡轮直径6cm，料液粘度0.0075N.s/m2，料液密度1030kg/m3，利用雷诺系数计算公式得到，启动搅拌后，反应器中的料液雷诺系数为1236，达到了湍流状态。
上述方法中，纯化阶段PHA颗粒聚集时粒径变化情况如表6所示。
表6
当PHA凝集体粒径增加至50μm以上时，使用布氏漏斗抽滤，将得到的滤饼复水水洗两次后，过滤，使用鼓风干燥箱80℃烘干，检测PHA纯度为94.6％，回收率87.3％。
实施例7
本实施例提供一种PHA凝集体的制备方法，该方法的步骤如下：将PHA悬浮液倒入搅拌反应器中，开启搅拌600r/min，升温至40℃，加入终浓度为1mL/L的中性蛋白酶，开始纯化反应，期间每隔10min检测PHA凝集体粒径，直至粒径不再继续增加。
其中，涡轮直径6cm，料液粘度0.0075N.s/m2，料液密度1030kg/m3，利用雷诺系数计算公式得到，启动搅拌后，反应器中的料液雷诺系数为4944，达到了充分的湍流状态。
上述方法中，纯化阶段PHA颗粒聚集时粒径变化情况如表7所示。
表7
当PHA凝集体粒径增加至100μm以上时，使用布氏漏斗抽滤，将得到的滤饼复水水洗两次后，过滤，使用鼓风干燥箱80℃烘干，检测PHA纯度为97.1％，回收率98.3％。
实施例8
本实施例提供一种PHA凝集体的制备方法，该方法的步骤如下：将实施例1中使用的PHA悬浮液，加入2倍体积的水稀释，使得悬浮液中PHA的含量约为50g/L，倒入搅拌反应器中，开启搅拌600r/min，升温至40℃，加入终浓度为8g/L的SDS，终浓度为1mL/L的中性蛋白酶，开始纯化反应，期间每隔10min检测PHA凝集体粒径，直至粒径不再继续增加。
其中，涡轮直径6cm，料液粘度0.0075N.s/m2，料液密度1030kg/m3，利用雷诺系数计算公式得到，启动搅拌后，反应器中的料液雷诺系数为4944，达到了充分的湍流状态。
上述方法中，纯化阶段PHA颗粒聚集时粒径变化情况如表8所示。
表8
当PHA凝集体粒径增加至50μm以上时，使用布氏漏斗抽滤，将得到的滤饼复水水洗两次后，过滤，使用鼓风干燥箱80℃烘干，检测PHA纯度为99.1％，回收率97.4％。
对比例1
将PHA悬浮液(PHA悬浮液与实施例1相同)倒入搅拌反应器中，开启搅拌100r/min，升温至40℃，加入终浓度为8g/L的SDS，终浓度为1mL/L的 中性蛋白酶，开始纯化反应，期间每隔10min检测PHA颗粒粒径，直至颗粒粒径不再继续增加。
其中，涡轮直径6cm，料液粘度0.0075N.s/m2，料液密度1030kg/m3，利用雷诺系数计算公式得到，启动搅拌后，反应器中的料液雷诺系数为824，未达到湍流状态。
纯化阶段PHA颗粒的粒径变化情况如表9所示。结果显示，纯化维持3h以上，PHA颗粒粒径未出现明显增大。
表9
对比例2
将PHA悬浮液(PHA悬浮液与实施例1相同)倒入搅拌反应器中，开启搅拌100r/min，温度20℃，开始纯化反应，期间每隔10min检测PHA颗粒粒径，直至颗粒粒径不再继续增加。其中，涡轮直径6cm，料液粘度0.0075N.s/m2，料液密度1030kg/m3，利用雷诺系数计算公式得到，启动搅拌后，反应器中的料液雷诺系数为824，未达到湍流。
纯化阶段PHA颗粒的粒径变化情况如表10所示。结果显示，纯化3h以上PHA颗粒未出现粒径增加。
表10
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
工业实用性
本发明提供一种聚羟基脂肪酸酯凝集体的制备方法。本发明提供的聚羟基脂肪酸酯凝集体的制备方法包括使聚羟基脂肪酸酯悬浮液达到并维持湍流状态，以使得聚羟基脂肪酸酯凝聚的步骤。本发明的制备方法能够制得粒径50μm以上的聚羟基脂肪酸酯凝集体，且制得的聚羟基脂肪酸酯凝集体具有较高的纯度和收率；该方法的工艺条件温和、步骤简单、废水处理简单，成本低廉、对设备要求低，可实现大规模工业化生产，具有较好的经济价值和应用前景。

Claims (10)

  1. 一种聚羟基脂肪酸酯凝集体的制备方法，其特征在于，所述方法包括使聚羟基脂肪酸酯悬浮液达到并维持湍流状态，以使得聚羟基脂肪酸酯凝聚的步骤。
  2. 根据权利要求1所述的聚羟基脂肪酸酯凝集体的制备方法，其特征在于，所述湍流状态为所述聚羟基脂肪酸酯悬浮液的雷诺系数大于所述聚羟基脂肪酸酯悬浮液在其所处设备中达到湍流的临界雷诺系数时所处状态；
    优选地，所述设备为反应器，所述湍流状态为所述聚羟基脂肪酸酯悬浮液的雷诺系数大于1000时所处状态；
    或者，所述设备为管道，所述湍流状态为所述聚羟基脂肪酸酯悬浮液的雷诺系数大于4000时所处状态。
  3. 根据权利要求1或2所述的聚羟基脂肪酸酯凝集体的制备方法，其特征在于，所述聚羟基脂肪酸酯悬浮液中，聚羟基脂肪酸酯颗粒的含氮量低于7000ppm，悬浮液清液的含氮量低于3000ppm。
  4. 根据权利要求3所述的聚羟基脂肪酸酯凝集体的制备方法，其特征在于，所述聚羟基脂肪酸酯悬浮液以聚羟基脂肪酸酯的生物发酵液为原料，经细胞破碎、固液分离收集聚羟基脂肪酸酯沉淀，再以水悬浮得到；
    优选地，所述聚羟基脂肪酸酯悬浮液中，聚羟基脂肪酸酯的含量不低于20g/L。
  5. 根据权利要求1～4任一项所述的聚羟基脂肪酸酯凝集体的制备方法，其特征在于，在维持湍流状态的过程中，同时维持聚羟基脂肪酸酯悬浮液的温度不低于20℃。
  6. 根据权利要求1～5任一项所述的聚羟基脂肪酸酯凝集体的制备方法，其特征在于，所述方法还包括以酶去除聚羟基脂肪酸酯悬浮液中的杂质的步骤；
    优选地，所述酶为选自蛋白质分解酶、脂质类分解酶、细胞壁分解酶、 DNA分解酶中的一种或多种。
  7. 根据权利要求6所述的聚羟基脂肪酸酯凝集体的制备方法，其特征在于，所述方法还包括以表面活性剂去除聚羟基脂肪酸酯悬浮液中的杂质的步骤。
  8. 根据权利要求6或7所述的聚羟基脂肪酸酯凝集体的制备方法，其特征在于，所述去除聚羟基脂肪酸酯悬浮液中的杂质的步骤与聚羟基脂肪酸酯的凝聚在同一反应体系中进行。
  9. 根据权利要求1～8任一项所述的制备方法，其特征在于，聚羟基脂肪酸酯凝聚的反应体系中不包含有机溶剂。
  10. 权利要求1～9任一项所述的聚羟基脂肪酸酯凝集体的制备方法制备得到的聚羟基脂肪酸酯凝集体；
    优选地，所述聚羟基脂肪酸酯凝集体的粒径为至少5μm，更优选为至少50μm。
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Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1694963A (zh) * 2002-09-30 2005-11-09 株式会社钟化 聚－3－羟基链烷酸的凝集方法
WO2010116681A1 (ja) * 2009-03-30 2010-10-14 株式会社カネカ ポリヒドロキシアルカノエートの回収方法
EP1609868B1 (en) 2003-01-20 2015-11-11 Kaneka Corporation Method of collecting highly pure polyhydroxyalkanoate from microbial cells
EP2357247B1 (en) 2008-12-09 2016-03-09 Kaneka Corporation Method for producing poly-3-hydroxyalkanoate
US9683076B2 (en) 2013-07-30 2017-06-20 Bio-On S.P.A. Processes for recovering and purifying polyhydroxyalkanoates from cell cultures
JP2019041606A (ja) * 2017-08-30 2019-03-22 株式会社カネカ ポリヒドロキシアルカノエートの製造方法
CN111349218A (zh) * 2020-04-29 2020-06-30 吉林中粮生化有限公司 聚羟基脂肪酸酯的分离方法及其制备的聚羟基脂肪酸酯
CN111393625A (zh) * 2020-06-03 2020-07-10 中粮营养健康研究院有限公司 溶菌酶结合sds下利用超声提取聚羟基脂肪酸酯的方法和系统
CN116144046A (zh) * 2022-06-06 2023-05-23 北京蓝晶微生物科技有限公司 一种聚羟基脂肪酸酯凝集体的制备方法

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9215791D0 (en) * 1992-07-24 1992-09-09 Ici Plc Increasing the particle size of polymers
US7435567B2 (en) * 2004-03-04 2008-10-14 Kaneka Corporation Method for degradation of nucleic acids and use thereof
TW200617171A (en) * 2004-06-29 2006-06-01 Procter & Gamble Improved process for the solvent-based extraction of polyhydroxyalkanoates from biomass
US20070038295A1 (en) * 2005-08-12 2007-02-15 Cook Incorporated Artificial valve prosthesis having a ring frame
DE102012014536B4 (de) * 2012-07-21 2016-11-24 Perkinelmer Chemagen Technologie Gmbh Sphärische, magnetisierbare Polyvinylalkohol-Mikropartikel, Verfahren für deren Herstellung sowie deren Verwendung
CN109939617A (zh) * 2019-03-14 2019-06-28 开滦能源化工股份有限公司 一种共聚甲醛催化剂加入装置及方法
CN214167689U (zh) * 2020-08-31 2021-09-10 南京中电环保水务有限公司 一种氨氮废水高效处理装置

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1694963A (zh) * 2002-09-30 2005-11-09 株式会社钟化 聚－3－羟基链烷酸的凝集方法
EP1609868B1 (en) 2003-01-20 2015-11-11 Kaneka Corporation Method of collecting highly pure polyhydroxyalkanoate from microbial cells
EP2357247B1 (en) 2008-12-09 2016-03-09 Kaneka Corporation Method for producing poly-3-hydroxyalkanoate
WO2010116681A1 (ja) * 2009-03-30 2010-10-14 株式会社カネカ ポリヒドロキシアルカノエートの回収方法
US9683076B2 (en) 2013-07-30 2017-06-20 Bio-On S.P.A. Processes for recovering and purifying polyhydroxyalkanoates from cell cultures
EP3027761B1 (en) 2013-07-30 2019-12-11 BIO-ON S.p.A. Process for recovering and purifying polyhydroxyalkanoates from a cell culture
JP2019041606A (ja) * 2017-08-30 2019-03-22 株式会社カネカ ポリヒドロキシアルカノエートの製造方法
CN111349218A (zh) * 2020-04-29 2020-06-30 吉林中粮生化有限公司 聚羟基脂肪酸酯的分离方法及其制备的聚羟基脂肪酸酯
CN111393625A (zh) * 2020-06-03 2020-07-10 中粮营养健康研究院有限公司 溶菌酶结合sds下利用超声提取聚羟基脂肪酸酯的方法和系统
CN116144046A (zh) * 2022-06-06 2023-05-23 北京蓝晶微生物科技有限公司 一种聚羟基脂肪酸酯凝集体的制备方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP4484473A4

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