Method for preparing polyhydroxyalkanoate nanobeads

Abstract

The present invention relates to a method for preparing polyhydroxyalkanoate nanobeads. The present invention optimizes a process in which physical cell disruption is performed in order to directly recover polyhydroxyalkanoates, produced in the cells of polyhydroxyalkanoate-producing bacteria, in the original form thereof, and the recovered polyhydroxyalkanoates are washed. Using the optimized method for preparing polyhydroxyalkanoates according the present invention has the effect that polyhydroxyalkanoates can be prepared with high purity and high yield without an organic solvent, and polyhydroxyalkanoate nanobeads can be directly obtained without a separate forming process.

Description

Method for manufacturing polyhydroxyalkanoate in the form of nanobeads

[0001] The present invention relates to a method for manufacturing polyhydroxyalkanoate in the form of nanobeads.

[0002]

[0003] Plastics are polymer materials that have dramatically advanced the convenience of human life. Due to their lightweight nature, moldability, processability, cost-effectiveness, and excellent durability, they are used for a wide range of purposes, from industrial materials to disposable consumables. However, most plastics used universally—such as those for industrial packaging, food packaging, household use, and agriculture / horticulture—exist semi-permanently without decomposing in the natural environment. Consequently, environmental pollution issues arising from the disposal of discarded plastics have persisted. As an alternative to address this, research on biodegradable plastics is actively underway. Biodegradable plastics are plastics that can be broken down by bacteria or living organisms. Because they decompose without leaving behind pollutants, they are receiving significant attention in the field of biotechnology, as they are expected to potentially contribute to reducing solid waste, such as marine microplastics and carbon dioxide emissions from petroleum-based plastics. Bio-based, biodegradable plastics are referred to as biodegradable bioplastics, with representative examples including polylactic acid (PLA) and polyhydroxyalkanoates (PHA). When landfilled, biodegradable bioplastics can decompose into water and carbon dioxide within 6 months to 5 years. Currently, bioplastics account for approximately 1% (2 million tons) of the over 360 million tons of plastic produced annually, with about 1.2 million tons of this being biodegradable plastics. However, this figure is expected to increase to 1.8 million tons by 2025, with the market growth rate for polyhydroxyalkanoates projected to increase tenfold. As demand rises and the availability of high-performance biopolymers and products increases, the bioplastics market is expected to continue growing and diversifying.

[0004] Polyhydroxyalkanoates (PHAs) are intracellular energy storage compounds produced by various microorganisms under limited nutrient conditions and are biodegradable polymers composed of various types of hydroxycarboxylic acids. Polyhydroxyalkanoates have properties similar to existing synthetic polymers derived from petroleum, such as polybutylene adipate terephthalate (PBAT), polybutylene succinate (PBS), polybutylene succinate terephthalate (PBST), and polybutylene succinate adipate (PBSA), while exhibiting complete biodegradability and excellent biocompatibility. In addition, since mechanical properties and melting points can be controlled by adjusting the type and ratio of polyhydroxyalkanoate monomers, it is being used as an alternative to petroleum plastics in various industrial fields such as medicine, food, and energy.

[0005] [Prior Art Literature]

[0006] [Non-patent literature]

[0007] Yang, Yung-Hun, et al. “Optimization of growth media components for polyhydroxyalkanoate (PHA) production from organic acids by Ralstonia eutropha.” Applied microbiology and biotechnology 87.6 (2010): 2037-2045.

[0008]

[0009] The technical problem to be solved by the present invention is to provide a method for manufacturing polyhydroxyalkanoate and a polyhydroxyalkanoate manufactured therefrom.

[0010]

[0011] The technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned technical problems will be clearly understood by those skilled in the art to which the present invention belongs from the description below.

[0012]

[0013] To achieve the above technical objective, one embodiment of the present invention provides a method for producing polyhydroxyalkanoates, comprising: 1) a step of culturing bacteria capable of producing polyhydroxyalkanoates (PHA); 2) a step of physically lysing the bacterial cells to separate the polyhydroxyalkanoates; and 3) a step of washing the separated polyhydroxyalkanoates.

[0014] In an embodiment of the present invention, the polyhydroxyalkanoate is poly(3-hydroxypropionate) (PHP or P3HP), poly(3-hydroxybutyrate) (PHB or P3HB), poly(4-hydroxybutyrate) (P4HB), poly(3-hydroxyvalerate) (PHV or P3HV), poly(4-hydroxyvalerate) (P4HV), poly(5-hydroxyvalerate) (P5HV), poly(3-hydroxyhexanoate) (PHHx or P3HHx), poly(3-hydroxyoctanoate) (PHO or P3HO), poly(3-hydroxydecanoate) (PHD or P3HD), poly(3-hydroxyundecanoate) (PHU, P3HU), saturated or unsaturated, having a short-chain or medium-chain length. It may include PHA, or any copolymer thereof or any combination thereof.

[0015] In an embodiment of the present invention, the bacteria having the ability to produce polyhydroxyalkanoates are Cobetia, Ralstonia, Vibrio, Halomonas, Pseudomonas, Rhodopseudomonas, Bacillus, Methylobacterium, Methylosinus, Methylocella, Azotobacter, Alcaligenes, Aeromonas, Nocardia, Methylocapsa, Methyllocaldum, Methylocystis, Methyloversatilis, Rubrivax, Xanthobacter, It may belong to the genus Cupriavidus or Hydrogenophaga.

[0016] In an embodiment of the present invention, the bacteria may be Halomonas cyanhensis HN-1-3-2 (accession number KCTC 19195P).

[0017] In an embodiment of the present invention, the physical crushing may be performed by physically crushing by applying ultrasound or pressure.

[0018] In an embodiment of the present invention, physical crushing may be performed by applying pressure of 1000 to 1300 bar using a high-pressure homogenizer.

[0019] In an embodiment of the present invention, the cells may be physically ruptured by applying ultrasound for 5 to 20 minutes under conditions of an amplification intensity of 25 to 35%.

[0020] In an embodiment of the present invention, the washing may be performed by treating with sodium hydrogen sulfate (NaHSO3).

[0021] In an embodiment of the present invention, the sodium hydrogen sulfate may be treated at a concentration of 200 ppm to 300 ppm.

[0022] In an embodiment of the present invention, the polyhydroxyalkanoate washed in step 3) may have an average particle size of 800 nm to 900 nm.

[0023] In an embodiment of the present invention, a step of sterilization after step 3) may be further included.

[0024] To achieve the above technical problem, another embodiment of the present invention provides a polyhydroxyalkanoate prepared by the above method.

[0025] In an embodiment of the present invention, the polyhydroxyalkanoate may be dispersed in the form of nanobeads on a colloid.

[0026] In an embodiment of the present invention, the polyhydroxyalkanoate dispersed in the form of nanobeads on the colloid may have an average particle size of 600 nm to 800 nm.

[0027]

[0028] The present invention relates to a method for manufacturing polyhydroxyalkanoates in the form of nanobeads. In this invention, a process of physical cell disruption and washing is optimized to directly recover polyhydroxyalkanoates produced within the cells of bacteria that produce polyhydroxyalkanoates in their original form. By utilizing the optimized method for manufacturing polyhydroxyalkanoates according to the present invention, polyhydroxyalkanoates can be manufactured with high purity and high yield without organic solvents, and polyhydroxyalkanoates in the form of nanobeads can be obtained directly without separate molding processing.

[0029] The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the composition of the invention described in the description or claims of the present invention.

[0030]

[0031] Figure 1 shows the particle size according to washing at different concentrations of NaHSO3.

[0032] Figures 2 and 3 show the particle size of polyhydroxyalkanoates according to sterilization conditions. Figure 2 shows the analysis results according to temperature, and Figure 3 shows the analysis results according to time conditions.

[0033] Figure 4 shows the change in particle size according to temperature of a sample with added sucrose and glycerol in a finished product process according to an exemplary embodiment of the present invention.

[0034] Figures 5 and 6 show the DLS immediately after adding sucrose and glycerol 10% (v / v), 5% (v / v), and 1% (v / v) in the finished product process according to an exemplary embodiment of the present invention (Day 1, Fig. 4), and the DLS 7 days after adding sucrose and glycerol 10% (v / v), 5% (v / v), and 1% (v / v) (Day 8, Fig. 5).

[0035]

[0036] The present invention will be described in detail below.

[0037]

[0038] The present invention relates to a method for manufacturing polyhydroxyalkanoates.

[0039] The method of the present invention comprises: 1) a step of culturing bacteria capable of producing polyhydroxyalkanoates (PHA); 2) a step of physically lysing the bacterial cells to separate the polyhydroxyalkanoates; and 3) a step of washing the separated polyhydroxyalkanoates.

[0040] The above polyhydroxyalkanoates are, for example, poly(3-hydroxypropionate) (PHP or P3HP), poly(3-hydroxybutyrate) (PHB or P3HB), poly(4-hydroxybutyrate) (P4HB), poly(3-hydroxyvalerate) (PHV or P3HV), poly(4-hydroxyvalerate) (P4HV), poly(5-hydroxyvalerate) (P5HV), poly(3-hydroxyhexanoate) (PHHx or P3HHx), poly(3-hydroxyoctanoate) (PHO or P3HO), poly(3-hydroxydecanoate) (PHD or P3HD), poly(3-hydroxyundecanoate) (PHU, P3HU), saturated or unsaturated PHAs of short-chain or medium-chain length, or may include any copolymer of these or any combination thereof.

[0041] The bacteria having the ability to produce the above polyhydroxyalkanoates are, for example, Cobetia, Ralstonia, Vibrio, Halomonas, Pseudomonas, Rhodopseudomonas, Bacillus, Methylobacterium, Methylosinus, Methylocella, Azotobacter, Alcaligenes, Aeromonas, Nocardia, Methylocapsa, Methyllocaldum, Methylocystis, Methyloversatilis, Rubrivax, Xanthobacter, It may belong to the genus Cupriavidus or Hydrogenophaga, and more specifically, it may be Halomonas cyanhensis HN-1-3-2 (accession number KCTC 19195P).

[0042] The above physical crushing may be performed using means known in the art under known methods and conditions, for example, by physically crushing by applying ultrasound or pressure.

[0043] More specifically, the pressure can be applied at 1000 to 1300 bar using a high-pressure homogenizer, and the ultrasound can be performed for 5 to 20 minutes at 25 to 35% intensity (Amplication).

[0044] The above washing may be performed by treating with sodium bisulfate (NaHSO3). When washing with sodium bisulfate, the effect of preventing the separated polyhydroxyalkanoate nano beads from aggregating can be achieved.

[0045] The sodium hydrogen sulfate mentioned above may be treated at a concentration of, for example, 200 ppm to 300 ppm.

[0046] The polyhydroxyalkanoate obtained through the washing process above may have an average particle size of 800 nm to 900 nm. The polyhydroxyalkanoate biosynthesized within the cell may have an average particle size of less than about 1000 nm, and the polyhydroxyalkanoate separated through the physical disruption above may have an average particle size of 400 nm to 900 nm. Through the washing process above, polyhydroxyalkanoate having a uniform average particle size of 800 nm to 900 nm can be obtained.

[0047] The present invention may further include a step of sterilization as needed.

[0048] The above sterilization may be performed using means known in the art, such as methods and conditions. For example, in the present invention, sterilization may be performed by a high-pressure steam sterilization method. As a more specific example, sterilization may be performed at 70°C to 90°C for 50 to 70 minutes, and preferably at 80°C for 60 minutes.

[0049] Additionally, the present invention may further include a step of dispersing sterilized polyhydroxyalkanoates on a colloid as needed. This can be carried out using means known in the art under known methods and conditions, for example, by adding sugars (glycerol or sucrose). More specifically, it can be carried out by adding sucrose at a concentration of 1% (v / v) or more, or 1 to 10% (v / v). Additionally, it can be carried out by adding glycerol at a concentration of 5% (v / v) or more, or 5 to 10% (v / v). Through this step, the aggregation of sterilized PHA nanobeads can be suppressed to have an average particle size of 600 nm to 800 nm, and stability on the colloid can be ensured.

[0050] The present invention relates to a polyhydroxyalkanoate prepared by the above method.

[0051] The above polyhydroxyalkanoate may be dispersed in the form of nanobeads on a colloid.

[0052] The average particle size of the polyhydroxyalkanoate dispersed in the form of nanobeads on the above collide may be, for example, 600 nm to 800 nm.

[0053]

[0054] Hereinafter, in order to specifically explain the present invention, it will be described in detail with reference to examples.

[0055]

[0056] 1. Cell culture

[0057] Halomonas xianhensisHN-1-3-2 was used as the PHA-producing strain. The culture medium used for the strain growth was ASS Media, a recombinant medium modeled after MB medium. ASS medium consists of 5 g / L soy peptone, 1 g / L yeast extract, and 30 g / L artificial sea salt.

[0058] Strain culture for PHA production on a 5 L scale was carried out in the stages of Pre-culture, Transfer-culture, and Production-culture. Strain culture for the seed was performed overnight in a medium containing 5 g / L soy peptone, 1 g / L yeast extract, and 30 g / L artificial sea salt. The media for PHA production are divided into Transfer-culture and Production-culture. Since the purpose of Pre-culture is strain proliferation, glucose is not added; however, for Transfer-culture and Production-culture, 20 g / L glucose is added for PHA production, and the medium consists of 5 g / L soy peptone, 1 g / L yeast extract, and 30 g / L artificial sea salt, in accordance with the same basic medium composition as the seed.

[0059] Bacteria were inoculated at a 2% (v / v) level at each stage. The strains cultured in the flask were incubated at 37°C and 180 rpm at 1 / 5 of the flask's volume. For 5 L fermenter culture for PHA production, the maximum working volume was set to 3 L. Culture in the fermenter was conducted at 0.5 L / min (DO sat point 15%), 200–800 RPM, pH 7.6 (20–25% Ammonia solution), and 40°C.

[0060]

[0061] 2. Cell disruption

[0062] In this experiment, a high-pressure homogenizer (HPH) was used as the primary device for disintegration. The culture medium was recovered using a centrifuge at 5000 xg for 20 minutes at 4°C. The supernatant was completely removed from the recovered cells, and the moisture content was measured using a moisture analyzer (Kern, DAB 100-3 Moisture Analyser).

[0063] When using an ultrasonicator, cell lysis was performed according to the equipment and probe diameter. Cell lysis was carried out using the UltrasonicatorHD 4100 (BANDELIN) with the TS103 probe. Recovered cells with a moisture content of 50%–60% were prepared into a homogeneous suspension of 10% (w / w) using purified water. 5 mL of the suspension was placed into a 15 mL conical tube, and the tube was placed on ice to perform the lysis. The lysis was performed on ice with a reaction time of 10 minutes, a pulse duration of 3 sec on and 3 sec off, and an amplification of 30%.

[0064] When lysing cells using a high-pressure homogenizer (HPH), the cells were lysed by passing them twice in the range of 1000-1300 bar.

[0065]

[0066] 3. PHA Separation and Purification

[0067] After lysis, the cells were separated from PHA using a centrifuge at 3000 ×g for 20 minutes at 4°C. Subsequently, the supernatant was discarded, and the remaining pellet underwent a washing process. NaHSO3 aqueous solution and tertiary purified water were used as buffers for the washing process. For the first wash, NaHSO3(aq) at a concentration of 200–300 ppm or higher was added to an equal bed volume (5 mL for this experiment) to achieve a PHA concentration of 10% (w / v). The precipitated pellet was uniformly suspended (to maximize the surface area for PHA particles to react with NaHSO3(aq)) and reacted at room temperature for 5 minutes. Afterward, the NaHSO3 was removed by centrifugation (3000 ×g, 20 minutes, 4°C), and the remaining pellet was washed approximately twice more with tertiary purified water. All centrifugation conditions used for PHA separation and washing were performed identically at 3000 ×g, 20 min, and 4 ℃.

[0068] 3-1. Selection of PHA Washing Buffer

[0069] The PHA washing buffer was prepared using tertiary purified water (DW in Table 1 below) as a control group. 0.05 / 0.1 M NaOH, 0.05 / 0.1 M NH4OH, and 100 / 200 ppm NaHSO3 were used as comparison groups. Buffers used for PHA washing were selected through a literature review (Table 1), and buffers that prevented the aggregation of PHA nano-beads during washing were selected through a rapid test (data not shown), and basic experiments were conducted at different concentrations (Table 2). After cell lysis by passing the sample twice through a high-pressure homogenizer (HPH) at 1000 bar, the same bed volume (5 mL in this experiment) was added to the PHA separated from the lysate using a centrifuge (4 ℃, 3000 ×g, 20 min), and each candidate PHA washing buffer was added to achieve a PHA concentration of 10% (w / v). The mixture was homogeneously mixed using a loop and needle and reacted at room temperature for 5 minutes. After the reaction, the sample was washed twice using triple-distilled water as a buffer, and centrifugation was performed at 4°C and 3000 ×g for 20 minutes after each washing step. For the control sample (purified water), the same procedure was followed using purified water during the reaction step with the NaHSO3 solution.

[0070] MethodConcentrationConditionDW--NH3(aq)0.05-0.1 M aqueous NH3Heating block, 30 minutes, 45 / 75 / 140℃NaHSO3100-200 ppmVoltexing with 30 min reaction timeNaOH0.05-0.1 MVoltexing with 30 min reaction timeEthanolPHA was mixed with 1% (v / v) ethanol (96% v / v) stirred at 200 rpm for 3 hMethanolPHA:DCM:MeOH (w / v / v) = 1:10-25:10-25RT contact and washNaClO0.5-1% NaClO-

[0071] Washing bufferRecovery (%)Purity (%)Residual concentration after washing (ppm)DW59.06±1.8585.21±2.38-0.05 M NaOH52.44±0.4783.31±0.092.00.1 M NaOH55.71±2.3488.76±2.044.00.05 M NH4OH55.97±0.9280.81±2.232.00.1 M NH4OH55.54±0.8184.16±1.244.0100 ppm NaHSO359.36±0.1984.13±0.270.0001200 ppm NaHSO365.69±0.0897.35±2.070.0002

[0072] The washing efficiency of PHA after cell lysis was evaluated by recovery (%), purity (%), and residual concentration after washing (ppm). Residual concentration after washing (ppm) refers to the concentration of the washing buffer that ultimately remains after the washing process and was calculated using the following formula: C n =C0×r nr represents the centrifugal washing efficiency, n represents the number of washes, and C0 represents the initial concentration. The calculation process was performed by referring to Jue, E. et al., Sci. Rep. 10, 1940 (2020).

[0073] 3-2. Evaluation of Efficiency of Selected NaHSO3 at Different Concentrations

[0074] Through the above experiment, sodium bisulfite (NaHSO3) was selected as the main washing buffer. Experiments were conducted at different concentrations of NaHSO3 in increments of 50 ppm, ranging from 50 to 250 ppm.

[0075] Triple-distilled water (DW in Table 3 below) was used as the control group, and NaHSO₃ at concentrations of 50 / 100 / 150 / 200 / 250 ppm was set as the comparison group to evaluate efficiency. Cells were lysed by passing them twice through a high-pressure homogenizer (HPH) at 1000 bar. Subsequently, NaHSO₃ solutions of different concentrations with the same bed volume (5 mL in this experiment) were added to the PHA separated from the cell lysate using a centrifuge (4 ℃, 3000 ×g, 20 min). The mixture was homogeneously mixed using a loop and needle and reacted at room temperature for 5 minutes. After the reaction, two washes were performed using triple-distilled water as a buffer, and centrifugation was performed at 4 ℃, 3000 ×g for 20 minutes after each wash step. For the control group, the purified water sample, the same method was used with purified water in the reaction step of the NaHSO₃ solution (Table 3).

[0076] SampleRecovery (%)Purity (%)Residual concentration after washing (ppm)DW43.81±0.1275.01±1.00050 ppm45.34±0.5876.53±1.250.00005100 ppm47.10±0.8285.54±1.410.0001150 ppm48.42±1.2991.14±0.990.00015200 ppm50.15±0.4795.74±1.660.0002250 ppm50.51±1.1896.78±1.870.00025

[0077] DLS measurements were used to confirm changes in particles following washing at different concentrations of NaHSO3. After washing, the PHA was centrifuged at 4 ℃ and 3000 ×g for 20 minutes to form a pellet, and then dissolved in the same bed volume (5 mL in this experiment) of tertiary purified water to prepare a homogeneous suspension. 100 μL of the suspension was taken and 10 was added using tertiary purified water. 2 After diluting the sample, it was homogenized using a bath sonicator to prepare a sample for DLS. The results are shown in Figure 1.

[0078]

[0079] 4. Sterilization Process

[0080] Sterilization methods such as gamma ray sterilization, high-pressure steam sterilization, dry heat sterilization, ultraviolet sterilization, filtration sterilization, ethylene oxide gas sterilization, chemical sterilization, and ethanol sterilization are used in various ways depending on the purpose. In particular, gamma ray sterilization is most commonly used for raw material sterilization; however, this method is not preferred for liquid raw materials because there is a risk of microbial contamination if incomplete sealing or leakage occurs. Therefore, in the present invention, the raw materials were sterilized using a high-pressure steam sterilization method at 80°C, which is lower than Tm.

[0081] In addition, maintaining the morphology of PHA nano-beads throughout the entire manufacturing process is a crucial factor that must be controlled. To achieve this, common sterilization processes such as gamma ray sterilization can be used, but in this invention, high-pressure steam sterilization was chosen to improve the efficiency of the manufacturing process.

[0082] To confirm whether PHA nano-beads aggregated during the sterilization process, particle size was measured after sterilization using Dynamic Lighting Scattering (DLS, AntonPaar). Immediately after sterilization, 100 μL of the suspension was taken and 10 was added with tertiary purified water. 2 After diluting, the sample for DLS was prepared by homogenizing it using a Bath sonicator and measured.

[0083] To optimize autoclave conditions, experiments were conducted by gradually lowering the temperature in increments of approximately 20°C, based on the standard sterilization condition of 121°C for 15 minutes, and correspondingly doubling the sterilization time. For this purpose, 5 mL of PHA suspension was dispensed into each 15 mL conical tube, and sterilization was performed inside an autoclave. Thus, sterilization was carried out under a total of three conditions (121°C - 15 minutes, 100°C - 30 minutes, and 80°C - 60 minutes), and changes in particle size after treatment under each condition were compared and evaluated.

[0084] To verify the sterilization effect, 100 μL of each sample sterilized under the three conditions above was taken and LB (Luria-Bertani, BD Difco TM , USA), NB (Nutrient Broth, BD Difc TM , USA), MB (Marine Broth, MBcell, Korea), and TSB (Tryptic Soy Broth, BD Difco)TM The samples were spread onto solid media (USA). After incubating each medium at 37°C for 24 hours, the completeness of sterilization and the presence of contamination were evaluated by observing the presence or absence of colonies formed.

[0085] As a result, as the sterilization temperature and time increased, the average particle size of PHA particles showed a tendency to increase slightly, and it was confirmed that the growth of microorganisms was inhibited stepwise.

[0086] In particular, the sample treated at 80°C for 1 hour had a particle size of 700-900 nm before and after sterilization, which did not cause significant clumping while reducing microbial growth by more than 99%, and was evaluated as the condition with the best balance between morphological stability and sterilization effect.

[0087] On the other hand, although a complete sterilization effect was secured at higher temperatures (100 ℃ or higher), particle aggregation and an increase in size were observed, so in the present invention, conditions of 80 ℃ and 1 hour were adopted as the optimal sterilization conditions (Table 4 and Fig. 2).

[0088] ConditionCFU / mLPre-sterilization2.7 ×10 10 80℃, 60 min10100℃, 30 minND121℃, 15 minND

[0089] ND: Not Detected

[0090] (below detection limit < 10 CFU / mL; occasional single colony observed in 1 of 3 replicates)

[0091]

[0092] In addition, experimental results based on sterilization temperature confirmed that there was no significant change in particle size before and after treatment under 80 ℃ conditions.

[0093] Accordingly, in order to improve the sterilization effect while maintaining the morphological stability of the particles, the present invention conducted experiments by gradually increasing the sterilization time under the same 80°C condition to 1 hour, 1.5 hours, and 2 hours.

[0094] As a result, under sterilization conditions treated at 80°C for 1 hour, microbial growth was reduced by more than 99%, just as before, and it was confirmed that there was no significant change in particle size.

[0095] On the other hand, when the sterilization time at 80 ℃ was increased to 1.5 hours and 2 hours, almost no microbial growth was observed, but an increase in particle size was confirmed, indicating that some particle aggregation occurred during long-term treatment (Table 5 and Fig. 3).

[0096]

[0097] From the above results, it was confirmed that sterilization treatment at 80°C for 1 hour is the optimal condition for securing high sterilization efficiency while maintaining morphological stability without large clumping of particles.

[0098] ConditionCFU / mLPre-sterilization2.7 ×10 10 80℃, 60 min1080℃, 90 minND80℃, 120 minND

[0099] ND: Not Detected

[0100] (below detection limit < 10 CFU / mL; occasional single colony observed in 1 of 3 replicates)

[0101]

[0102] 5. Finished product process for storage

[0103] Sterilized PHA was used to ensure the colloidal stability of PHA nano-beads.

[0104] Since nanoparticles constantly undergo Brownian motion on the colloid, Van der Waals forces generated depending on the interparticle distance can lead to phenomena such as aggregation, potentially reducing stability. Typically, to suppress this, finishing processes such as adding sugars or silica, or lowering the pH to acidic levels, are performed. In this experiment, a sugar addition process was carried out on sterilized PHA to ensure the colloidal stability of PHA nano-beads. Sugars such as sucrose and glycerol readily adsorb to the interface between nanoparticles and the solvent, forming hydrogen bonds that create a dense hydration film around the nanoparticles. This hydration film maintains interparticle distance and provides hydration repulsion, thereby preventing nanoparticle aggregation caused by Van der Waals forces. By increasing the viscosity of the solvent, the Brownian motion of the nanoparticles is reduced, thereby suppressing nanoparticle aggregation and enhancing surface stability. The following experiment was conducted to verify whether sucrose and glycerol help improve PHA colloid stability.

[0105]

[0106] 5-1. Thermal Stability Evaluation

[0107] As a control group, a PHA sample dispersed at 10% (w / v) in triple purified water was set, and as a comparison group, 10% (v / v) of glycerol or sucrose was set in the PHA sample dispersed at 10% (w / v) in triple purified water.

[0108] 10 samples in tertiary purified water 2After dilution, the particles were placed in quartz cuvettes, and particle sizes at different temperatures were measured using Dynamic lighting scatter (DLS, AntonPaar). Measurements were taken while increasing the temperature from 25 ℃ to 80 ℃ in 5 ℃ increments.

[0109] Upon examining the results, it appears that the particle size decreases as the temperature rises, but above a certain temperature, it can be observed that the particle size increases. This is not because the actual particle size changes, but rather because the Brownian motion accelerates as the temperature increases, causing the intensity fluctuation rate measured by DLS to accelerate, which corresponds to the Stokes-Einstein equation D=k B T / 6 πηr The temperature (T) term directly affects the diffusion coefficient (D), causing the particle size calculated on the DLS to appear smaller. Therefore, the plotting showing a decrease in particle size as the temperature rises is a natural change associated with temperature in continuous DLS measurements. Along with the acceleration of Brownian motion due to rising temperature, particles move closer together, and aggregation occurs due to attractive forces such as Van der Waals forces. Consequently, the signal fluctuation pattern changes along with the intensity of the light scattering signal, and an increase in intensity values ​​is observed. This is interpreted as the particle motion accelerating and becoming too close to one another, disrupting the balance that maintained the stability of the colloid phase. Based on this, interpreting the results, it can be confirmed that the increase in particle size due to thermal fluctuations is most suppressed in the 75-80 ℃ range for Glycerol 10% (v / v) and sucrose 10% (v / v), compared to the 65-70 ℃ range for the control group with no additives. This means that both sucrose and glycerol contribute to improving the thermal stability of PHA colloids as buffers (Fig. 4).

[0110]

[0111] 5-2. Evaluation of Stability Maintenance Over Time

[0112] As in Experiment 5-1, a PHA sample dispersed at 10% (w / v) in triple-purified water was set as the control group. As comparison groups, samples containing 10% (v / v), 5% (v / v), or 1% (v / v) of glycerol or sucrose, respectively, were set to the PHA sample dispersed at 10% (w / v) in triple-purified water. The samples were dispersed 10 in triple-purified water 2 After dilution, the particles were placed in a quartz cuvette, and their particle sizes at different temperatures were measured using Dynamic lighting scatter (DLS, AntonPaar).

[0113] To confirm the effect of maintaining stability, storage experiments were conducted for 8 days after adding sucrose and glycerol at concentrations of 10% (v / v), 5% (v / v), and 1% (v / v), respectively, to samples with nothing added. As a result, the particle size of the control group without addition increased from 712 nm to 1182 nm, and aggregation occurred. The group with 1% (v / v) glycerol added aggregated and increased from 674 nm to the 1066 nm range, whereas the sucrose-added group (1–10% (v / v)) and the glycerol-added group (5% (v / v) or higher) maintained a stable particle size in the 600–800 nm range. This demonstrates that the addition of sucrose or glycerol at concentrations above a certain level is also effective in improving the stability of PHA nanoparticles over time (Figs. 5 and 6).

[0114] From the above results, the addition of sucrose and glycerol is considered an effective stabilization process that can simultaneously improve the thermal stability and long-term storage stability of PHA colloids. Therefore, regarding the addition of sucrose and glycerol as additives in the finished product process, we propose a composition in which sucrose is added at least 1% (v / v) and glycerol at least 5% (v / v).

[0115]

[0116] The present invention relates to a method for directly producing polyhydroxyalkanoates (PHAs) generated from bacteria in the form of nanobeads (average approximately 600–800 nm) through a process of physical crushing, washing with sodium bisulfate (NaHSO3), sterilization, and colloidal dispersion. Since high-purity and high-yield PHAs can be reproducibly obtained without organic solvents, they can be easily applied to mass production processes. The process of the present invention can be implemented using standard equipment such as high-pressure homogenization and ultrasound. Furthermore, since long-term storage and suitability for formulation are secured through sterilization at 80°C / 60 min and stabilization based on sucrose / glycerol, the PHA nanobeads can be directly integrated into continuous or batch downstream lines. Therefore, the PHA nanobeads according to the present invention can be commercially utilized as raw materials for PHA-based products such as eco-friendly packaging materials, agricultural films, ink / coating binders, cosmetic ingredients, and medical polymer materials, and their industrial applicability across the entire bioplastics industry is clear.

[0117] [Consignment Number]

[0118] Depository Name: Korea Research Institute of Biotechnology and Bioengineering Biological Resource Center (KCTC)

[0119] Trustee Number: KCTC19195P

[0120] Date of Trust: 20240603

[0121] [Correction pursuant to Rule 91 25.11.2025]

Claims

1. 1) A step of culturing bacteria capable of producing polyhydroxyalkanoates (PHA); 2) a step of physically lysing bacterial cells to isolate polyhydroxyalkanoates; and 3) A method for manufacturing polyhydroxyalkanoate comprising the step of washing the separated polyhydroxyalkanoate.

2. In claim 1, the polyhydroxyalkanoate is poly(3-hydroxypropionate) (PHP or P3HP), poly(3-hydroxybutyrate) (PHB or P3HB), poly(4-hydroxybutyrate) (P4HB), poly(3-hydroxyvalerate) (PHV or P3HV), poly(4-hydroxyvalerate) (P4HV), poly(5-hydroxyvalerate) (P5HV), poly(3-hydroxyhexanoate) (PHHx or P3HHx), poly(3-hydroxyoctanoate) (PHO or P3HO), poly(3-hydroxydecanoate) (PHD or P3HD), poly(3-hydroxyundecanoate) (PHU, P3HU), having a short-chain or medium-chain length. A method for preparing a polyhydroxyalkanoate comprising a saturated or unsaturated PHA, or any copolymer thereof or any combination thereof.

3. In claim 1, the bacteria having the ability to produce polyhydroxyalkanoates are Cobetia, Ralstonia, Vibrio, Halomonas, Pseudomonas, Rhodopseudomonas, Bacillus, Methylobacterium, Methylosinus, Methylocella, Azotobacter, Alcaligenes, Aeromonas, Nocardia, Methylocapsa, Methyllocaldum, Methylocystis, Methyloversatilis, Rubrivax, A method for preparing a polyhydroxyalkanoate belonging to the genus Xanthobacter, Cupriavidus, or Hydrogenophaga.

4. A method for preparing a polyhydroxyalkanoate according to claim 1, wherein the bacteria is Halomonas cyanhensis HN-1-3-2 (accession number KCTC 19195P).

5. A method for manufacturing polyhydroxyalkanoate according to claim 1, wherein the physical crushing is performed by physically crushing by applying ultrasound or pressure.

6. A method for manufacturing polyhydroxyalkanoate according to claim 5, wherein the polyhydroxyalkanoate is physically crushed by applying pressure of 1000 to 1300 bar using a high-pressure homogenizer.

7. A method for producing polyhydroxyalkanoate according to claim 5, wherein the cells are physically ruptured by applying ultrasound for 5 to 20 minutes under conditions of an amplification intensity of 25 to 35%.

8. A method for manufacturing a polyhydroxyalkanoate according to claim 1, wherein the washing is performed by treating with sodium hydrogen sulfate (NaHSO3).

9. A method for producing polyhydroxyalkanoate according to claim 8, wherein the sodium hydrogen sulfate is treated at a concentration of 200 ppm to 300 ppm.

10. A method for preparing a polyhydroxyalkanoate according to Claim 1, wherein the polyhydroxyalkanoate washed in step 3) has an average particle size of 800 nm to 900 nm.

11. A method for manufacturing a polyhydroxyalkanoate according to claim 1, further comprising a step of sterilization after step 3).

12. Polyhydroxyalkanoate prepared by the method of Claim 1.

13. The polyhydroxyalkanoate of claim 12, dispersed in the form of nanobeads on a colloid.

14. The polyhydroxyalkanoate of claim 13, wherein the polyhydroxyalkanoate has an average particle size of 600 nm to 800 nm.

Images