A method for extracting polyhydroxy fatty acid esters

Abstract

This invention discloses a method for extracting polyhydroxyalkanoates (PHAs). The method includes the following steps: S1: adding a chelating agent and lysozyme to a bacterial suspension containing PHA-producing bacteria for reaction; S2: adding a surfactant to the reaction system of step S1 for reaction, and collecting the crude PHA product. This extraction method is carried out at room temperature, with mild reaction conditions, short reaction time, and no difficult-to-treat wastewater is generated after subsequent washing, greatly saving time and subsequent wastewater treatment costs. It is a highly efficient and green extraction process.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a method for extracting polyhydroxy fatty acid esters. Background Technology

[0002] In recent years, the continuous increase in carbon dioxide (CO2) emissions has triggered serious problems such as climate change, impacting the sustainability of the global economy and environment. Driven by stringent environmental regulations and plastic restriction policies related to "dual carbon" targets, energy transition, and environmental protection, biodegradable materials have become a preferred alternative to traditional plastics. Among them, polyhydroxyalkanoate (PHA), a 100% biodegradable polymer synthesized by microorganisms, has attracted widespread attention due to its superior biocompatibility, biodegradability, and thermal processing properties, showing broad prospects in chemical, daily consumer goods, and pharmaceutical fields.

[0003] The two main extraction methods for PHA are organic solvent extraction and enzymatic hydrolysis. However, the main drawback of organic solvent extraction is the high cost of production due to the large amount of organic reagents required. Furthermore, the subsequent recovery of these reagents and the discharge of wastewater during production can damage the environment, and the addition of explosion-proof facilities during factory production increases costs. Another method involves cell digestion using ionic surfactants, but in actual production, this process results in poor purity of the extracted polyhydroxyalkali esters, along with other substandard indicators. The subsequent treatment of high-alkalinity, high-SDS wastewater also increases costs. Enzymatic hydrolysis can reduce the use of organic reagents, but in practice, incomplete enzymatic hydrolysis and low cell wall disruption efficiency can lead to cell membrane residues on the polyhydroxyalkali esters. Even with large amounts of water, these impurities cannot be removed, ultimately resulting in reduced product purity, excessive total nitrogen levels, and a strong odor. Therefore, researchers have developed a method combining lysozyme and SDS to disrupt cell walls. However, this method requires maintaining a very high pH and high temperature environment with an alkaline solution. Therefore, the amount of alkaline solution added needs to be strictly controlled. Excessive alkaline solution can lead to significant molecular weight loss in the material, resulting in a decrease in the purity of extracted PHA. In addition, a common extraction method is mechanical disruption, which uses physical methods such as ultrasonic disruption and high-pressure homogenization to break down cell structures and release PHA particles. However, mechanical disruption still suffers from high energy consumption and low efficiency in large-scale production, leading to extremely high PHA production costs. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes a method for extracting polyhydroxy fatty acid esters. This extraction method is carried out at room temperature, with mild reaction conditions, short processing time, and does not generate difficult-to-treat wastewater after subsequent washing, greatly saving time and subsequent wastewater treatment costs. It is a highly efficient and green extraction process.

[0005] According to a first aspect of the present invention, a method for extracting polyhydroxyalkanoates is provided, the method comprising the following steps: S1: A chelating agent and lysozyme are added to a bacterial suspension for reaction, wherein the bacterial suspension contains bacteria that produce polyhydroxy fatty acid esters; S2: Add a surfactant to the reaction system of step S1 to carry out the reaction, and collect the crude product of polyhydroxy fatty acid ester.

[0006] In reaction systems combining EDTA and lysozyme with Triton or Tween, lysozyme hydrolyzes the peptidoglycan backbone of microbial cell walls, catalyzing the cleavage of the β-1,4 glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycan, thus disintegrating the network structure of the cell wall. EDTA is a strong chelating agent that can bind to divalent cations (such as Ca2+) on the cell surface and in the cell wall. 2+ Mg 2+ Zn 2+ These cations are key bridges maintaining the connection between lipopolysaccharide (LPS) and peptidoglycan in the cell wall. After chelation, the LPS layer detaches, exposing the peptidoglycan, allowing lysozyme to directly contact its target site and enhancing the hydrolytic effect of lysozyme. Triton or Tween 20 are nonionic surfactants with a hydrophobic group at one end and a hydrophilic group at the other. They do not disrupt the hydrophobic structure of PHA. Their functions are as follows: First, dissolving cell membranes: The hydrophobic group inserts into the lipid bilayer of the cell membrane, disrupting the lipid structure, causing the cell membrane to rupture and releasing PHA particles, proteins, nucleic acids, etc. from the cell. Second, dispersing impurities: The hydrophilic group binds to the released water-soluble impurities such as proteins and nucleic acids, forming micelles dispersed in the aqueous phase, preventing impurities from agglomerating with PHA particles through hydrophobic interactions. Third, reducing interfacial tension: It reduces the adsorption force between PHA particles and the aqueous phase and cell debris, making PHA particles easier to separate in subsequent centrifugation and filtration.

[0007] In some embodiments of the present invention, step S1 involves adjusting the pH of the reaction system to 6-8 before the reaction.

[0008] In some embodiments of the present invention, the chelating agent in step S1 includes ethylenediaminetetraacetic acid.

[0009] In some embodiments of the present invention, the concentration of the chelating agent in step S1 in the reaction system is 0.5-2 g / L.

[0010] In some embodiments of the present invention, the concentration of the chelating agent in step S1 in the reaction system is 1-2 g / L.

[0011] In some embodiments of the present invention, the concentration of lysozyme in step S1 in the reaction system is 0.1-1.5 g / L.

[0012] In some embodiments of the present invention, the concentration of lysozyme in step S1 in the reaction system is 0.5-1 g / L.

[0013] In some embodiments of the present invention, the reaction time in step S1 is 15-45 min.

[0014] In some embodiments of the present invention, the reaction time in step S1 is 20-40 min.

[0015] In some embodiments of the present invention, the reaction described in step S1 is carried out under stirring conditions at a stirring speed of 400-800 rpm.

[0016] In some embodiments of the present invention, the surfactant in step S2 includes at least one of Triton X-100, Tween-20, and Tween-80.

[0017] In some embodiments of the present invention, the volume percentage of the surfactant in step S2 in the reaction system is 0.5%-1.25%.

[0018] In some embodiments of the present invention, the surfactant in step S2 has a volume percentage of 0.75%-1.25% in the reaction system.

[0019] In some embodiments of the present invention, the reaction time in step S2 is 1-2.5 h.

[0020] In some embodiments of the present invention, the reaction time in step S2 is 1-2 h.

[0021] In some embodiments of the present invention, the reaction described in step S2 is carried out under stirring conditions at a stirring speed of 400-800 rpm.

[0022] In some embodiments of the present invention, the bacteria that produce polyhydroxy fatty acid esters in step S1 include Halomonas and / or Pseudomonas.

[0023] In some embodiments of the present invention, the halomonas includes Halomonas LY03.

[0024] In some embodiments of the present invention, steps S1 and S2 are both performed at room temperature.

[0025] In some embodiments of the present invention, the crude product of polyhydroxyalkanoate collected in step S2 is collected by centrifugation.

[0026] In some embodiments of the present invention, the centrifugation conditions include centrifugation at 7000-9000 rpm for 20-40 minutes.

[0027] In some embodiments of the present invention, step S2 further includes washing the precipitate after centrifugation.

[0028] In some embodiments of the present invention, the washing is performed using deionized water.

[0029] The present invention has at least the following beneficial effects: The extraction method for polyhydroxyalkanoates provided by this invention involves adding a specific concentration of EDTA, lysozyme, and surfactant to the reaction system. The entire reaction process is carried out at room temperature, which has the advantages of mild conditions, economy, short reaction cycle, and high PHA quality. It is a mild, time-saving, and efficient PHA extraction method that does not require complex steps such as controlling heating, heat preservation, and cooling. At the same time, the neutral conditions reduce the number of water washings, reduce wastewater, simplify the process, reduce costs, and lower the difficulty of operation, making it suitable for industrial-scale production. Detailed Implementation

[0030] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0031] All instances of "w / v" in the following examples refer to g / 100 mL, i.e., 0.1% (w / v) corresponds to 0.1 g / 100 mL.

[0032] Example 1: Synergistic cell-wall disruption effect of different types of surfactants and EDTA Surfactants can not only alter cell membrane permeability and disrupt the structural properties of bacterial cells to break down cells and release intracellular substances, but also denature some proteins, thus reducing protein impurities after PHA extraction. Commonly used surfactants in this field include SDS, CAS, CTAB, Triton X-100, Tween-20, and Tween-80; EDTA is a strong chelating agent that can bind divalent cations (such as Ca2+) on the cell surface and in the cell wall. 2+ Mg 2+ Zn 2+These cations are key bridges for maintaining the connection between lipopolysaccharide and peptidoglycan in the cell wall. After chelation, the lipopolysaccharide layer is detached, exposing the peptidoglycan, allowing lysozyme to directly contact the target site and enhance the hydrolytic effect of lysozyme. At the same time, EDTA chelates metal ions in water, preventing them from reacting with surfactants to form precipitates, maintaining the cleanliness of the product, and preventing the oxidation and deterioration of PHA materials caused by metal ions during the cell wall breaking process.

[0033] Based on this, this embodiment tested the cell disruption effect of different surfactants combined with EDTA. The specific experimental methods and results are as follows: 1. Collection and resuspension of bacterial cells Collecting Halomonas Halomonas LY03 (deposited at Guangdong Provincial Center for Microbial Culture Collection, deposited on April 23, 2023, accession number GDMCC NO:63382) 3000 mL of bacterial culture from a 5 L fermenter after fermentation was centrifuged at room temperature for 30 min at 8000 rpm. The supernatant was discarded, and deionized water was added to restore the volume to 3000 mL. The suspension was resuspended, and centrifuged again under the same conditions, discarding the supernatant once more. This process was repeated twice. For the third resuspension, deionized water was added to restore the volume, and the suspension was resuspended to 3000 mL. The bacterial concentration of the resuspension at this point was OD0.05. 600 =600-800, this resuspended solution awaits cell wall disruption.

[0034] 2. Cell wall disruption Measure 100 mL of the resuspension collected in step 1 into each beaker, adjust the initial pH of the resuspension to 7.0, and adjust the acidity and alkaliness using 10% HCl and 2 M NaOH, respectively. First, add 0.1% (w / v) EDTA to the resuspension, then set up groups with no addition or with 0.1% (w / v) lysozyme, and stir the reaction for 0.5 h. Then, add 0.5% (v / v) surfactants SDS, CAS, CTAB, Triton X-100, Tween-20, and Tween-80, respectively, and stir the reaction for 2 h to obtain the cell wall-breaking solution. The entire reaction process was carried out at room temperature (25℃) and the stirring speed was maintained at 600 rpm.

[0035] 3. Collection of crude PHA products Collect the cell-wall-breaking liquid obtained after cell-wall breaking in step 2 into a centrifuge tube of appropriate volume and centrifuge at room temperature for 30 min at 8000 rpm. After discarding the supernatant, add an appropriate amount of deionized water to restore the volume, resuspend, and centrifuge under the same conditions, then discard the supernatant again. Repeat the above operation twice, and then place the bottom PHA solid material in an 80℃ oven to dry for 12 h to obtain crude PHA product.

[0036] 4. Determination of PHA content Take 30-40 mg of the crude PHA product obtained in step 3 and place it in a clean 10 mL digestion tube. Add 2 mL of esterification solution (ethanol containing 3% (v / v) 98% concentrated sulfuric acid and 1 g / L benzoic acid) and 2 mL of chloroform. Incubate at 100 °C for 4 h for esterification. Simultaneously, weigh 20 mg of PHA standard and treat it in the same way as a standard reference. Then, determine the PHA content using a GC-2014Pro gas chromatograph (Shimadzu, Japan).

[0037] The testing method was as follows: the initial temperature was maintained at 80℃ for 1.5 min; in the first stage, the temperature was increased to 140℃ at a rate of 30℃ / min; in the second stage, the temperature was increased to 240℃ at a rate of 40℃ / min, which took 2 min; the total analysis time was 8 min; the injection temperature was 240℃ and the detector temperature was 250℃. The results are shown in Table 1 (the contents in the table are all mean plus variance).

[0038] Table 1. PHA content in crude products obtained after cell wall disruption by combining different surfactants with EDTA.

[0039] As shown in Table 1, EDTA and the surfactant require the participation of lysozyme (0.1%) to lyse part of the cell membrane. With the addition of surfactant, the cells are further lysed, achieving the effect of cell wall disruption and releasing PHA material. Compared with ionic surfactants (SDS, CAS and CTAB), nonionic surfactants (Triton X-100, Tween-20 and Tween-80) are more effective and more suitable for cell wall disruption under mild conditions such as room temperature. Therefore, the subsequent optimization will mainly focus on nonionic surfactants.

[0040] Example 2: Effect of different concentrations of lysozyme on cell wall disruption Lysozyme is a natural enzyme that specifically breaks down bacterial cell walls. It primarily works by disrupting the peptidoglycan structure, leading to bacterial lysis. It also possesses antibacterial and anti-inflammatory functions and is widely used in pharmaceuticals, food, and daily chemical industries. The results of Example 1 confirm that lysing the cell membrane with lysozyme first improves the cell wall disruption effect. Therefore, the optimal concentration of lysozyme is crucial for cell wall disruption. This example tested the effect of different concentrations of lysozyme on the cell wall disruption effect. The specific experimental methods and results are as follows: 1. Collection and resuspension of bacterial cells: Same as step 1 in Example 1.

[0041] 2. Cell wall disruption Measure 100 mL of the resuspension collected in step 1 into each beaker, adjust the initial pH of the resuspension to 7.0, and adjust the acidity and alkaliness using 10% HCl and 2 M NaOH, respectively. First, add 0.1% (w / v) EDTA and different concentrations (0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3% w / v) of lysozyme to the system and stir for 0.5 h. Then, add 0.5% (v / v) of surfactants Triton X-100, Tween-20, and Tween-80, respectively, and stir for 2 h. The entire reaction process is carried out at room temperature (25℃) and the stirring speed is maintained at 600 rpm.

[0042] 3. Collection of crude PHA product: Same as step 1 in Example 1.

[0043] 4. Determination of PHA content: The result is consistent with step 1 of Example 1 and is shown in Table 2 (the contents in the table are the mean plus variance).

[0044] Table 2. Effects of different concentrations of lysozyme on PHA content

[0045] As shown in Table 2, among the nonionic surfactants (Triton X-100, Tween-20, and Tween-80), Triton X-100 and Tween-20 showed better performance. Furthermore, as the lysozyme concentration increased, the PHA content showed a trend of first increasing and then decreasing. The best effect was achieved when the lysozyme concentration was 0.1%, but the difference from 0.05% was not significant. Given the high price of lysozyme, a ratio of 0.05% was selected as the subsequent optimization scheme.

[0046] Example 3: Effect of different concentrations of EDTA on cell wall disruption EDTA can chelate metal ions during the cell wall disruption process, preventing them from reacting with surfactants to form precipitates, maintaining the product's cleanliness, and preventing the oxidation and deterioration of PHA materials caused by metal ions during disruption. This example tested the effect of different concentrations of EDTA on the cell wall disruption effect; the specific experimental methods and results are as follows: 1. Collection and resuspension of bacterial cells: Same as step 1 in Example 1.

[0047] 2. Cell wall disruption Measure 100 mL of the resuspension collected in step 1 into each beaker, adjust the initial pH of the resuspension to 7.0, and adjust the acidity and alkaliness using 10% HCl and 2 M NaOH, respectively. First, add different concentrations of EDTA (0, 0.05%, 0.1%, 0.15%, 0.2% w / v) and 0.05% (w / v) of lysozyme to the system and stir for 0.5 h. Then, add 0.5% (v / v) of surfactants Triton X-100, Tween-20, and Tween-80, respectively, and stir for 2 h. The entire reaction process is carried out at room temperature (25℃) and the stirring speed is maintained at 600 rpm.

[0048] 3. Collection of crude PHA product: Same as step 1 in Example 1.

[0049] 4. Determination of PHA content: The result is consistent with step 1 of Example 1 and is shown in Table 3 (the contents in the table are the mean plus variance).

[0050] Table 3. Effects of different concentrations of EDTA on PHA content

[0051] As shown in Table 3, the cell disruption effect is particularly poor without EDTA. However, with the increase of EDTA concentration, the PHA content after cell disruption first increases and then decreases, with the best effect observed at an EDTA concentration of 0.15%. Therefore, a concentration of 0.15% was selected as the subsequent optimization scheme. Furthermore, the above three examples illustrate that the PHA purification process requires the synergistic effect of specific concentrations of EDTA, lysozyme, and surfactant.

[0052] Example 4: Effect of different pH conditions on cell wall disruption pH plays a crucial regulatory role in the extraction of PHA, directly affecting the efficiency of lysozyme in disrupting cell walls, and profoundly influencing the subsequent PHA content, purity, and separation. This example tested the effect of different pH values ​​on the cell wall disruption effect; the specific experimental methods and results are as follows: 1. Collection and resuspension of bacterial cells: Same as step 1 in Example 1.

[0053] 2. Cell wall disruption Measure 100 mL of the resuspension collected in step 1 into various beakers, and adjust the initial pH of the resuspension to 6.0, 7.0, 8.0, and 9.0 respectively. Adjust the pH using 10% HCl and 2 M NaOH respectively. First, add 0.15% (w / v) EDTA and 0.05% (w / v) lysozyme to the system and stir for 0.5 h. Then, add 0.5% (v / v) of surfactants Triton X-100, Tween-20, and Tween-80 respectively, and stir for 2 h. The entire reaction process is carried out at room temperature (25℃) and the stirring speed is maintained at 600 rpm.

[0054] 3. Collection of crude PHA product: Same as step 1 in Example 1.

[0055] 4. Determination of PHA content: The procedure is the same as step 1 in Example 1, and the results are shown in Table 4 (the contents in the table are the mean plus variance).

[0056] Table 4. Effect of different pH values ​​on PHA content

[0057] As shown in Table 4, the PHA content after cell disruption remains high when the pH is within a wide range of 6-8. However, the PHA content drops sharply as the pH increases to 9.0. Therefore, this process has a good cell disruption effect in neutral and near-neutral pH ranges, and no additional acid or alkali adjustment is required for cell disruption.

[0058] Example 5: Effect of different processing times on cell wall disruption Processing time is a key parameter affecting efficiency, cost, and product quality in PHA cell wall disruption extraction, directly related to the degree of cell wall disruption, PHA release rate, and residual impurities. This example tested the effect of different processing times on the cell wall disruption effect; the specific experimental methods and results are as follows: 1. Collection and resuspension of bacterial cells: Same as step 1 in Example 1.

[0059] 2. Cell wall disruption Measure 100 mL of the resuspension collected in step 1 into each beaker, and adjust the initial pH of the resuspension to 7.0. Adjust the acidity and alkaliness using 10% HCl and 2 M NaOH, respectively. First, add 0.15% (w / v) EDTA and 0.05% (w / v) lysozyme to the system and stir for 0.5 h. Then, add 0.5% (v / v) of surfactants Triton X-100, Tween-20, and Tween-80, respectively, and maintain the pH at 7.0 throughout the process. The stirring time for this stage is 0.5, 1, 1.5, 2, and 2.5 h, respectively. The entire reaction process is carried out at room temperature (25℃) and the stirring speed is maintained at 600 rpm.

[0060] 3. Collection of crude PHA product: Same as step 1 in Example 1.

[0061] 4. Determination of PHA content: The result is consistent with step 1 of Example 1 and is shown in Table 5 (the contents in the table are the mean plus variance).

[0062] Table 5. Effect of different treatment times on PHA content

[0063] As shown in Table 5, the PHA content after cell disruption remains at a high level when the processing time is 1 h or more. Therefore, this process has a good cell disruption effect under the condition of short processing time of 1.5 h.

[0064] Example 6: Effect of different concentrations of surfactant on cell wall disruption Different concentrations of surfactants exhibit significantly different effects in PHA cell disruption extraction. The core difference lies in their ability to influence cell disruption efficiency, PHA release rate, and product purity by regulating the adsorption amount of molecules on the cell surface, micelle formation state, and interactions with cell wall components. This example tested the effect of different concentrations of surfactants on cell disruption; the specific experimental methods and results are as follows: 1. Collection and resuspension of bacterial cells: Same as step 1 in Example 1.

[0065] 2. Cell wall disruption Measure 100 mL of the resuspension collected in step 1 into each beaker, adjust the initial pH of the resuspension to 7.0, and adjust the acidity and alkaliness using 10% HCl and 2 M NaOH, respectively. First, add 0.15% (w / v) EDTA and 0.05% (w / v) lysozyme to the system and stir for 0.5 h. Then, add different concentrations (0.5%, 0.75%, 1%, 1.25% v / v) of surfactants Triton X-100, Tween-20, and Tween-80, respectively, and stir for 1.5 h. The entire reaction process is carried out at room temperature (25℃) and the stirring speed is maintained at 600 rpm.

[0066] 3. Collection of crude PHA product: Same as step 1 in Example 1.

[0067] 4. Determination of PHA content: The procedure is the same as step 1 in Example 1, and the results are shown in Table 6 (the contents in the table are the mean plus variance).

[0068] Table 6. Effect of different concentrations of surfactant on PHA content

[0069] As shown in Table 6, with the increase of surfactant concentration, the content of PHA after cell disruption first increases and then decreases, with the cell disruption effect being the best at a concentration of 1%.

[0070] Example 7: Effect of different extraction temperatures on cell wall disruption PHA is a thermosensitive biopolymer. High temperatures (typically exceeding 60°C) can lead to a decrease in its molecular weight and degradation, affecting the mechanical properties (such as strength and flexibility) and application value of the product. Therefore, temperature control during the PHA extraction process can effectively ensure the stability of the PHA material. This example tested the effect of different extraction temperatures on cell wall disruption. The specific experimental methods and results are as follows: 1. Collection and resuspension of bacterial cells: Same as step 1 in Example 1.

[0071] 2. Cell wall disruption Measure 100 mL of the resuspension collected in step 1 into each beaker, adjust the initial pH of the resuspension to 7.0, and adjust the acidity and alkaliness using 10% HCl and 2 M NaOH, respectively. First, add 0.15% (w / v) EDTA and 0.05% (w / v) lysozyme to the system and stir for 0.5 h. Then, add 1% (v / v) of surfactants Triton X-100, Tween-20, and Tween-80, respectively, and stir for 1.5 h. The reactions were carried out at room temperature (25℃), 37℃, 60℃, and 75℃, respectively, and the stirring speed was maintained at 600 rpm.

[0072] 3. Collection of crude PHA product: Same as step 1 in Example 1.

[0073] 4. Determination of PHA content: The result is consistent with step 1 of Example 1 and is shown in Table 7 (the contents in the table are the mean plus variance).

[0074] Table 7 Effect of different extraction temperatures on PHA content

[0075] As shown in Table 7, the PHA content extracted at room temperature is higher than that extracted under other high-temperature conditions. This allows for efficient PHA extraction without energy consumption, directly eliminating the need for electricity or fuel consumption in the heating process and significantly reducing production energy costs.

[0076] Example 8: The effect of different extraction mechanisms on PHA quality PHA material was extracted from bacterial cells using different extraction mechanisms, and its extraction rate, PHA content, whiteness, molecular weight, moisture content, and nitrogen content were evaluated. The specific experimental methods and results are as follows: 1. Collection and resuspension of bacterial cells: The specific operation is the same as in Example 1.

[0077] 2. Cell wall disruption: 1) Method 1: Place 100 mL of the resuspension collected in step 1 in a beaker and adjust the initial pH of the resuspension to 7.0. Adjust the acidity and alkaliness using 10% HCl and 2 M NaOH, respectively. First, add 0.15% (w / v) EDTA and 0.05% (w / v) lysozyme to the system and stir for 0.5 h. Then, add 1% (v / v) of the surfactant Triton X-100. Stir for 1.5 h during this stage. The entire reaction process is carried out at room temperature, and the stirring speed is maintained at 600 rpm throughout the process.

[0078] 2) Method 2: Place 100 mL of the resuspension collected in step 1 into a beaker. First, add 0.3% (w / v) lysozyme to the system and stir at 37°C for 2.0 h. Then add 0.5% (w / v) SDS, adjust the pH to 10.5, and stir at 75°C for 2 h. Maintain the stirring speed at 600 rpm throughout the process.

[0079] 3) Method 3: Place the collected resuspension from step 1 on a high-pressure homogenizer (1200 bar, 40 Hz) for crushing.

[0080] 3. Collection of crude PHA product: The specific operation is the same as in Example 1.

[0081] 4. PHA Quality Assessment: 1) Extraction rate: The ratio of the mass of crude PHA obtained from 100 mL of cell wall disruption (m0) to the mass of 100 mL of untreated dried bacterial cells (m1), the extraction rate (%) = m0 / m1 × 100%; 2) Whiteness: Using barium sulfate as a standard white plate of 100%, the reflectance factor of PHA dry powder was measured under blue light irradiation at a wavelength of 457 nm. The higher the value, the more blue light is reflected, and the whiter the sample is. 3) Nitrogen content: 1 g of PHA dry powder was digested with 0.5 g of copper sulfate and 5.5 g of potassium sulfate and 10 mL of concentrated sulfuric acid, and then the inorganic nitrogen was titrated by blowing out the Kjeldahl nitrogen. 4) Moisture content: Weigh PHA powder (M0) into a glass dish, dry in a 90℃ oven for 8 hours, and then measure the mass of PHA powder again (M1). The difference between the two is the moisture content. Divide the difference by the initial PHA mass to get the moisture content. Moisture content (%) = (M0-M1) / M0*100%; Molecular weight, notched impact strength, and flexural modulus were measured by sending samples to Zhuhai Medtronic Biomaterials Co., Ltd., and the results are shown in Table 8 (the contents in the table are all mean plus variance).

[0082] Table 8. Quality assessment of PHA obtained by different extraction methods

[0083] As shown in Table 8, the extraction of PHA material from bacteria using EDTA combined with the Triton process yielded comparable results to physical cell disruption method 3 (high-pressure homogenization) in terms of extraction rate, PHA content, whiteness, molecular weight, moisture content, and nitrogen content. The results were significantly better than the conventional SDS process (method 2). Furthermore, this process can largely preserve the original physical properties of PHA, making it particularly suitable for scenarios with high PHA requirements (such as medical materials and high-end packaging).

[0084] Example 9: A method for extracting PHA This embodiment, based on the optimization results of process parameters in Examples 1-8, provides a method for extracting PHA, with the specific steps as follows: 1. Collection and resuspension of bacterial cells Collecting Halomonas Halomonas After the fermentation of the LY03 5 L fermenter was completed, 3000 mL of bacterial culture was centrifuged at room temperature for 30 min at 8000 rpm. After discarding the supernatant, deionized water was added to restore the volume to 3000 mL, and the suspension was resuspended. After centrifugation under the same conditions, the supernatant was discarded again. The above operation was repeated twice. For the third time, deionized water was added to restore the volume, and the suspension was resuspended to 3000 mL. This resuspended solution was then left to break the cell wall.

[0085] 2. Cell wall disruption Measure 100 mL of the resuspension collected in step 1 into each beaker, adjust the initial pH of the resuspension to 7.0, and adjust the acidity and alkaliness using 10% HCl and 2 M NaOH, respectively. First, add 0.15% (w / v) EDTA and 0.05% (w / v) lysozyme to the resuspension and stir for 0.5 h. Then, add 1% (v / v) of the surfactant Triton X-100 and stir for 1.5 h to obtain the cell-wall-broken solution. The entire reaction process was carried out at room temperature (25℃) and the stirring speed was maintained at 600 rpm.

[0086] 3. Collection of crude PHA products Collect the cell-wall-breaking liquid obtained after cell-wall breaking in step 2 into a centrifuge tube of appropriate volume and centrifuge at room temperature for 30 min at 8000 rpm. After discarding the supernatant, add an appropriate amount of deionized water to restore the volume, resuspend, and centrifuge under the same conditions, then discard the supernatant again. Repeat the above operation twice, and then place the bottom PHA solid material in an 80℃ oven to dry for 12 h to obtain crude PHA product.

[0087] Currently, the extraction temperature of HA commonly used in industry is around 75℃. The energy consumption during the heating and cooling process of large-scale reaction vessels now accounts for nearly 5% of the total cost of PHA production. In the cell-wall breaking process, the pH is above 10, which consumes a large amount of alkali and degrades some PHA. At the same time, the high-alkalinity environment is highly corrosive to the equipment, and the alkaline environment increases the number of subsequent water washings, resulting in significant wastewater costs (accounting for 3% of the total cost). Physical extraction methods are extremely costly and inefficient. While conventional chemical-enzymatic methods can be mass-produced at a lower cost, the reaction conditions are harsh, and even slight changes in conditions can significantly affect material properties. The extraction method provided by this invention combines the advantages of the above two methods, obtaining materials under mild conditions, preserving the basic properties of the materials, and is low-cost and suitable for industrial production.

[0088] The novel extraction process proposed in this invention is carried out under neutral and room temperature conditions, which can significantly reduce the cost issues caused by the aforementioned two factors, while also reducing extraction time. The inventors have provided this method, commonly used industrial cell-wall breaking methods, and a physical cell-wall breaking control method to characterize the extracted PHA material. The physical extraction method can accurately reflect the true properties of the raw material, and the PHA material extracted by this method is close to that extracted using the physical extraction method in terms of PHA content, whiteness, molecular weight, moisture content, and nitrogen content, significantly better than currently used industrial cell-wall breaking methods. Reduced energy consumption and water consumption mean reduced carbon emissions, meeting green production requirements and synergizing with the biodegradable properties of PHA itself, further enhancing the environmental value of the product.

[0089] In summary, the PHA extraction method provided by this invention has the advantages of mild conditions, economy, short reaction cycle, and high PHA quality. It is a mild, time-saving, and efficient PHA extraction method that does not require complex steps such as controlling heating, heat preservation, and cooling. At the same time, the neutral conditions reduce the number of water washings, reduce wastewater, simplify the process flow, reduce costs and reduce operation difficulty, making it suitable for industrial-scale production.

[0090] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

Claims

1. A method for extracting polyhydroxyalkanoates, characterized in that, The extraction method for the polyhydroxy fatty acid ester includes the following steps: S1: A chelating agent and lysozyme are added to a bacterial suspension for reaction, wherein the bacterial suspension contains bacteria that produce polyhydroxy fatty acid esters; S2: Add a surfactant to the reaction system of step S1 to carry out the reaction, and collect the crude product of polyhydroxy fatty acid ester.

2. The method for extracting polyhydroxy fatty acid esters according to claim 1, characterized in that, The chelating agent mentioned in step S1 includes ethylenediaminetetraacetic acid.

3. The method for extracting polyhydroxy fatty acid esters according to claim 1, characterized in that, The concentration of the chelating agent in step S1 in the reaction system is 0.5-2 g / L.

4. The method for extracting polyhydroxy fatty acid esters according to claim 1, characterized in that, The concentration of lysozyme in the reaction system in step S1 is 0.1-1.5 g / L.

5. The method for extracting polyhydroxy fatty acid esters according to claim 1, characterized in that, The reaction time in step S1 is 15-45 min.

6. The method for extracting polyhydroxy fatty acid esters according to claim 1, characterized in that, The surfactant mentioned in step S2 includes at least one of Triton X-100, Tween-20, and Tween-80.

7. The method for extracting polyhydroxy fatty acid esters according to claim 1, characterized in that, The surfactant in step S2 has a volume percentage of 0.5%-1.25% in the reaction system.

8. The method for extracting polyhydroxy fatty acid esters according to claim 1, characterized in that, The reaction time in step S2 is 1-2.5 h.

9. The method for extracting polyhydroxy fatty acid esters according to claim 1, characterized in that, The pH conditions for the reaction described in step S2 are 6-8.

10. The method for extracting polyhydroxy fatty acid esters according to claim 1, characterized in that, Both steps S1 and S2 are performed at room temperature.

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