A method for monitoring cell disruption and a method for producing polyhydroxyalkanoates using the same.

Abstract

The present invention relates to a method for producing polyhydroxyalkanoate (PHA) and a method for monitoring cell disruption. The present invention provides a method for producing polyhydroxyalkanoate (PHA) that enables the length and sphericity of cells or PHA to be measured by three-dimensional image analysis utilizing the difference in refractive index of the cells or PHA, thereby identifying the degree of autolysis, supply state, or degree of disruption of PHA cells, thereby enabling optimal disruption conditions to be set and enhancing the stability of the impurity removal process, which is a process subsequent to the disruption process.

Description

Technical Field

[0001] The present invention relates to a method for monitoring cell disruption and a method for producing polyhydroxyalkanoate (PHA) using the same.

Background Art

[0002] Polyhydroxyalkanoate (PHA) is a biodegradable polymer composed of several types of hydroxyl carboxylic acids produced by many microorganisms and is used as an intracellular storage substance. Polyhydroxyalkanoate has physical properties similar to those of conventional petroleum-derived synthetic polymers such as polybutylene adipate terephthalate (PBAT), polybutylene succinate (PBS), polybutylene succinate terephthalate (PBST), and polybutylene succinate adipate (PBSA), shows complete biodegradability, and has excellent biocompatibility.

[0003] Since PHA has the property of forming and accumulating granules in microbial cells, the most important step in the preparation and utilization process of PHA is the step of extracting PHA granules from the cells by cell disruption.

[0004] This method for extracting PHA granules can be mainly classified into three types: an organic solvent extraction method, a chemical extraction method, and a physical disruption method.

[0005] First, the organic solvent extraction method is a method of dissolving and extracting PHA granules using an organic solvent. Although high-purity PHA can be obtained, there is a drawback that dedicated equipment is required because an organic solvent is used. On the other hand, the chemical extraction method extracts PHA granules by destroying the cell wall at a high temperature under acidic or basic conditions. Although the recovery of PHA granules is easy, problems such as a decrease in the molecular weight of the extracted PHA may occur due to severe acid or base conditions and temperature conditions.

[0006] On the other hand, physical disruption is a method of extracting PHA granules after destroying the cell wall with physical energy such as high pressure. Compared to the above method, it has the disadvantage of having a lower ability to remove impurities attached to the PHA granules, resulting in lower purity of the extracted PHA granules. However, it has the advantage that the reduction in molecular weight and degradation of PHA can be minimized because the extraction is performed under mild conditions. Physical disruption methods include ultrasonic disruption, high-pressure disruption, and pulverization. High-pressure disruption includes cell disruption using a high-pressure homogenizer (HPH), and pulverization includes colloidal pulverization, bead pulverization, and ball pulverization.

[0007] However, with physical disruption methods, even when the same physical energy is applied, the degree of cell disruption may differ depending on the cell state (size, PHA content, cell wall thickness and composition, etc.). Therefore, in order to optimize and stabilize the process, it is necessary to quantitatively and qualitatively monitor the cell state in real time before, during, or after the cell disruption step.

[0008] Conventional fragmentation indicators used in physical fragmentation methods include, specifically, optical microscopy, measurement of protein content, measurement of changes in target substance concentration, dielectric spectroscopy, flow cytometry, confirmation of particle size distribution, and SDS-page. These methods have drawbacks such as being complicated to analyze, requiring long analysis times, and necessitating sample pretreatment.

[0009] The inventors of this invention devised this invention with the aim of solving the problems of the prior art, which does not allow for immediate change of process conditions according to the degree of cell disruption during physical disruption using a high-pressure homogenizer, and to provide an effective disruption indicator method. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Britta, Eggenreich, "A combination of HPLC and automated data analysis for monitoring the efficiency of high-pressure homogenization," Microbial Cell Factories, Volume 16, Article No.:134 (2017) Detailed description of the invention

[0011] [Technical issues] The present invention provides a method for monitoring cell disruption, which determines the degree of cell disruption by measuring cell length and cell sphericity through three-dimensional image analysis utilizing the difference in refractive index of cells; and a method for producing polyhydroxyalkanoate (PHA) using this method to set optimal cell disruption conditions and enhance the stability of the impurity removal step following the disruption step. [Solutions to the problem]

[0012] According to one aspect of the present invention, a method for monitoring cell disruption is provided, in which the degree of cell disruption is confirmed or the disruption conditions are determined by analyzing the morphological parameters of cells or PHA contained in a culture medium containing polyhydroxyalkanoate (PHA) by three-dimensional image analysis.

[0013] In one embodiment, a method is provided for monitoring cell disruption, wherein the three-dimensional image analysis is a three-dimensional image analysis using holotomography, and the morphological parameters are one or more selected from the group consisting of length, sphericity, volume, and surface area.

[0014] According to another aspect of the present invention, a method for producing polyhydroxyalkanoate (PHA) is provided, comprising the steps of: preparing a culture medium containing polyhydroxyalkanoate (PHA); preparing a feed solution containing the culture medium (or the supernatant obtained by centrifugation of the culture medium); disrupting cells or PHA contained in the feed solution; and analyzing the morphological parameters of the cells or PHA before, during, or after the disruption step.

[0015] In one embodiment, the analysis of morphological parameters of cells or PHA contained in the culture medium is performed before the disruption step, and the method includes the steps of: quantifying the analyzed morphological parameters to detect the degree of autolysis; and determining the initial pressure of the high-pressure homogenizer or the number of disruption treatments according to the degree of autolysis as a first disruption condition predetermination step.

[0016] In another embodiment, the analysis of morphological parameters of cells or PHA contained in the supply solution is performed prior to the disruption step, and the method includes the steps of: detecting the supply condition by quantifying the analyzed morphological parameters; and determining the disruption pressure of the high-pressure homogenizer or the number of disruption treatments according to the supply condition as a second disruption condition predetermination step.

[0017] In another embodiment, analysis of morphological parameters of cells or PHA contained in the disruption product solution is performed during or after the disruption step, and the method includes the steps of: quantifying the analyzed morphological parameters to detect the degree of disruption; and determining the disruption pressure of the high-pressure homogenizer or the number of disruption treatments according to the degree of disruption as a first disruption condition re-determination step.

[0018] In another embodiment, the crushing pressure in the crushing step is 300 to 1,500 bar.

[0019] In another embodiment, the method includes the steps of: preparing a culture medium containing polyhydroxyalkanoate (PHA); preparing a feed solution containing the culture medium (or the supernatant obtained by centrifugation of the culture medium); a first analytical step of analyzing the morphological parameters of cells or PHA contained in the feed solution; a second pre-determination step of disruption conditions of determining the disruption pressure of a high-pressure homogenizer or the number of disruption treatments according to the results of the first analytical step; disrupting the cells or PHA contained in the feed solution; a second analytical step of analyzing the morphological parameters of cells or PHA contained in the disruption product solution; and a first re-determination step of disruption conditions of determining the disruption pressure of a high-pressure homogenizer or the number of disruption treatments according to the results of the second analytical step.

[0020] In another embodiment, the degree of protein solubility according to the following formula 1 is 75-110%. [Formula 1] Protein solubility (%) = ABS1 / ABS0 × 100 (%)

[0021] In the formula, ABS0 is the absorbance at a wavelength of 280 nm measured after crushing the culture medium, feed solution, or crushed product solution five times at 800 bar at room temperature; ABS1 is the absorbance at a wavelength of 280 nm measured after crushing the culture medium, feed solution, or crushed product solution once at 300 to 1,500 bar at room temperature.

[0022] In another embodiment, if the average sphericity of the cells or PHA contained in the supply solution is 0.7 or higher, or if the degree of protein solubilization according to Formula 1 is 80% or higher, in the second step of predetermining the disruption conditions, the pressure is reduced by 10 to 100 bar from the initial pressure of the step of disrupting the cells or PHA contained in the supply solution.

[0023] In another embodiment, if the average sphericity of the cells or PHA contained in the disruption product solution is less than 0.9, or if the degree of protein solubilization according to Formula 1 is less than 90%, in the first disruption condition re-determination step, the pressure is increased by 10 to 100 bar from the disruption pressure in the step of disrupting the cells or PHA contained in the supply solution.

[0024] In another embodiment, the method further comprises performing an agglutination test for measuring the degree of agglutination of the crushed product solution after performing the second analysis step; and determining the crushing pressure of the high-pressure homogenizer or the number of crushing treatments as a second crushing condition redetermination step according to the degree of agglutination.

[0025] According to another aspect of the present invention, there is provided a system for monitoring cell disruption, in which the degree of cell disruption is confirmed or the disruption conditions are determined by analyzing the morphological parameters of cells or PHA contained in a culture medium containing polyhydroxyalkanoate (PHA) by three-dimensional image analysis. [Advantageous Effects of the Invention]

[0026] According to the monitoring method according to an embodiment of the present invention, even when a simple measurement method and a small amount of analysis sample are used, it is possible to accurately and real-time determine the state of cells or PHA, and cell disruption can be visualized by performing 3D stereoscopic photography. Thereby, it is possible to immediately change the disruption process conditions according to the state of cells or PHA.

[0027] Furthermore, since the method for producing polyhydroxyalkanoate (PHA) according to an embodiment of the present invention utilizes the above monitoring method, it is possible to immediately determine the state of cells or PHA, and it is possible to immediately change the disruption process conditions according to the state of cells or PHA. Specifically, the disruption conditions are set in advance by measuring the state of cells or PHA contained in the culture medium, and the disruption conditions are changed in real time by measuring the supply state of cells or PHA or the degree of disruption.

[0028] Furthermore, since the method for producing polyhydroxyalkanoate (PHA) according to an embodiment of the present invention determines the optimal crushing pressure or the number of crushing treatments by the above monitoring method without operating the temperature to a higher temperature, the crushing conditions can be efficiently changed in real time.

[0029] A method for producing polyhydroxyalkanoate (PHA) according to one embodiment of the present invention involves measuring the state of cells or PHA before, during, or after crushing, and changing the process conditions for crushing, separation, and impurity removal. This makes it possible to prevent aggregation of PHA in the separation and impurity removal processes after the crushing process and to ensure process stability. [Brief explanation of the drawing]

[0030] [Figure 1] A simplified method for producing polyhydroxyalkanoate (PHA) according to one embodiment of the present invention is shown below. [Figure 2] An algorithm for a method of producing polyhydroxyalkanoate (PHA) according to one embodiment of the present invention is shown. [Figure 3] This document presents an example of an algorithm for controlling the crushing pressure in the step of pre-determining the crushing conditions. [Figure 4] An example of an algorithm for controlling the crushing pressure in the crushing condition re-determination step is shown. [Figure 5] Here is another example of an algorithm for controlling the crushing pressure in the crushing condition re-determination step. [Figure 6] This is the measurement result of an HTA image according to one embodiment. [Figure 7] An example of a method for producing the polyhydroxyalkanoate of the present invention is shown, illustrating the risk areas in the separation process and impurity removal process according to cell length and sphericity when using the culture medium of the example. [Best Mode for Carrying Out the Invention]

[0031] The present invention will now be described in detail. The present invention is not limited to the disclosures shown below and can be modified in various forms as long as the essence of the invention is not altered.

[0032] Throughout this specification, when a part is said to "comprising" an element, unless otherwise specified, it is understood that other elements may be included rather than excluded.

[0033] All numerical values ​​and expressions relating to the quantities of ingredients and reaction conditions used herein are understood to be modified by the term "approximately" unless otherwise specified.

[0034] In this specification, when it is said that one element is formed "on" or "under" another element, it means not only that the element is formed directly "on" or "under" another element, but also that the element is formed indirectly on or under another element with other elements interposed between them.

[0035] Throughout this specification, terms such as "1," "2," etc., are used to describe various components. However, components should not be limited by these terms. The terms are used solely for the purpose of distinguishing one component from another.

[0036] Method for monitoring cell disruption and system for monitoring cell disruption In a method for monitoring cell disruption according to one embodiment of the present invention, the degree of cell disruption can be confirmed or the disruption conditions can be determined by analyzing the morphological parameters of cells or PHA contained in a culture medium containing polyhydroxyalkanoate (PHA) by three-dimensional image analysis.

[0037] In a system for monitoring cell disruption according to another embodiment of the present invention, the degree of cell disruption can be confirmed or the disruption conditions can be determined by analyzing the morphological parameters of cells or PHA contained in a culture medium containing polyhydroxyalkanoate (PHA) by three-dimensional image analysis.

[0038] The three-dimensional image analysis according to one embodiment of the present invention may be a three-dimensional image analysis using holotomography (HTA), and the morphological parameters are one or more types selected from the group consisting of length, sphericity, volume, and surface area.

[0039] Holotomography is a laser technique that measures the three-dimensional refractive index of minute samples such as cells, and is a type of three-dimensional microscope. The measured refractive index provides quantitative image information regarding the morphological parameters of the cell. As a result, cell length, sphericity, volume, surface area, density, and mass can be calculated.

[0040] Furthermore, holotomography analysis can clearly image cell membranes or intracellular organelles without the use of exogenous labels, thus avoiding problems of phototoxicity, photobleaching, or photodamage. By achieving three-dimensional stereoscopic images through holotomography analysis, it is possible to visualize the degree of cell disruption.

[0041] Furthermore, holotomography analysis provides a spatial resolution of approximately 100 nm and a temporal resolution of several hundred frames per second, depending on the numerical aperture of the objective lens used and the resolution of the image sensor, enabling accurate measurements even with small sample sizes of 10 μl of diluent. Rapid measurement is possible, as the total measurement time is less than 10 minutes. Thus, cell conditions can be monitored in real time using holotomography analysis. Therefore, disruption process conditions can be changed immediately or pre-set.

[0042] Holotomography analysis can be performed by diluting the sample to be analyzed 10 to 100 times using distilled water (DIW) and taking a three-dimensional image. The refractive index (RI) used when taking the three-dimensional image may be 1.35 to 1.50 or 1.35 to 1.45, but is not limited to these values.

[0043] Three-dimensional image analysis can be used to calculate the length, sphericity, volume, surface area, density, or mass of cells or PHAs.

[0044] Method for producing polyhydroxyalkanoates (PHAs) A method for producing polyhydroxyalkanoate (PHA) according to one embodiment of the present invention includes the steps of: preparing a culture medium containing polyhydroxyalkanoate (PHA) (culture medium preparation step); preparing a feed solution containing the culture medium (or the supernatant obtained by centrifugation of the culture medium) (feed solution preparation step); disrupting cells or PHA contained in the feed solution (disruption step); and analyzing the morphological parameters of the cells or PHA before, during, or after the disruption step (analysis step).

[0045] A method for producing polyhydroxyalkanoate (PHA) is shown in Figure 1 or Figure 2, which includes a step of analyzing the morphological parameters of cells or PHA before, during, and / or after the disruption step (analysis step).

[0046] Cell disruption can be carried out as a batch reaction or as a continuous manufacturing process. In a continuous manufacturing process, the disruption step can be performed by analyzing the morphological parameters of the cells or PHA in real time and adjusting the disruption conditions of the continuously supplied supply solution in real time according to the analysis results.

[0047] The crushing conditions may be the same or different. The crushing step can be carried out by repeating the above procedure and re-determining the crushing conditions in real time.

[0048] Furthermore, the method for producing polyhydroxyalkanoate (PHA) according to the present invention may include analyzing the morphological parameters of cells or PHA contained in the supply solution (first analysis step); and disrupting the cells or PHA contained in the supply solution (disruption step).

[0049] Furthermore, in the method for producing polyhydroxyalkanoate (PHA) of the present invention, the analysis of morphological parameters of cells or PHA contained in the disruption product solution is performed during or after the disruption step, and the method may include a step of quantifying the analyzed morphological parameters to detect the degree of disruption (second analysis step); and a step of determining the disruption pressure of the high-pressure homogenizer or the number of disruption treatments according to the degree of disruption as a first disruption condition re-determination step.

[0050] Specifically, the method for producing polyhydroxyalkanoate (PHA) according to the present invention comprises the steps of: preparing a culture medium containing polyhydroxyalkanoate (PHA) (culture medium preparation step); analyzing the morphological parameters of cells or PHA contained in the culture medium (preliminary analysis step); determining the initial conditions of the culture medium according to the preliminary analysis results (first crushing condition pre-determination step); preparing a supply solution containing the culture medium (or supernatant obtained by centrifugation of the culture medium) (supply solution preparation step); a first analysis step in which the morphological parameters of cells or PHA contained in the supply solution are analyzed (first analysis step); and crushing pressure or crushing pressure of a high-pressure homogenizer. The process may include the steps of: determining the number of crushing operations according to the results of the first analysis (second crushing condition pre-determination step); crushing cells or PHA contained in the supply solution (crushing step); a second analysis step of analyzing the morphological parameters of cells or PHA contained in the crushed product solution (second analysis step); determining the crushing pressure of the high-pressure homogenizer or the number of crushing operations according to the results of the second analysis (first crushing condition re-determination step); conducting an agglutination test to measure the degree of agglutination of the crushed product solution (agglutination test step); and / or determining the crushing pressure of the high-pressure homogenizer or the number of crushing operations according to the degree of agglutination (second crushing condition re-determination step).

[0051] The preliminary analysis step, the first crushing condition pre-determination step, the first analysis step, the second crushing condition pre-determination step, the second analysis step, the first crushing condition re-determination step, the coagulation test step, and the second crushing condition re-determination step are all independent and can be performed selectively.

[0052] A method for producing polyhydroxyalkanoate (PHA) according to one embodiment of the present invention can be carried out by the following exemplary method. (1) Culture medium preparation step; preliminary analysis step; and crushing step (2) Culture medium preparation step; crushing step; and second analysis step (3) Culture medium preparation step; first analysis step; crushing step; and second analysis step (4) Culture medium preparation step; first analysis step; crushing step; second analysis step; and first crushing condition re-determination step (5) Culture medium preparation step; supply solution preparation step; first analysis step; and crushing step (6) Culture medium preparation step; preliminary analysis step; supply solution preparation step; first analysis step; and crushing step (7) Culture medium preparation step; supply solution preparation step; first analysis step; second crushing condition pre-determination step; and crushing step (8) Culture medium preparation step; first analysis step; second crushing condition pre-determination step; crushing step; second analysis step; and first crushing condition re-determination step (9) Culture medium preparation step; supply solution preparation step; first analysis step; second crushing condition pre-determination step; crushing step; second analysis step; and first crushing condition re-determination step (10) Culture medium preparation step; preliminary analysis step; supply solution preparation step; first analysis step; second crushing condition pre-determination step; crushing step; second analysis step; and first crushing condition re-determination step

[0053] Furthermore, the method for producing polyhydroxyalkanoate (PHA) according to one embodiment of the present invention can be carried out by various steps in addition to the steps exemplified above. If necessary, the first analysis step, the first or second crushing condition pre-determination step, the first or second crushing condition re-determination step, and the crushing step may be repeated.

[0054] The following explains each step in detail.

[0055] Culture medium preparation step A method for producing polyhydroxyalkanoate (PHA) according to one embodiment of the present invention comprises preparing a culture medium containing polyhydroxyalkanoate (PHA).

[0056] Generally, polyhydroxyalkanoates can be produced by microbial fermentation processes. Crude polyhydroxyalkanoates can also be obtained by fermentation.

[0057] Methods for producing polyhydroxyalkanoates (PHAs) by fermentation can be carried out by known methods. For example, polyhydroxyalkanoates can be produced by fermentation using wild-type or genetically modified microorganisms cultured on a specific substrate, and are not particularly limited. Examples of microorganisms include, but are not limited to, those of the genus Escherichia or Escherichia coli.

[0058] The culture medium is prepared by culturing various microorganisms capable of producing polyhydroxyalkanoates (PHAs). The culture medium can be prepared by conventional methods, in which microorganisms are cultured in a bioreactor at 15-50°C or 30-40°C for 1-160 hours in a standard medium containing a carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids, and / or vitamins as appropriate. To adjust the pH of the medium, compounds such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid may be added to the medium in an appropriate manner during microbial cultivation, but are not limited to these.

[0059] Examples of carbon sources include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, and maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, and citric acid; and amino acids such as glutamic acid, methionine, and lysine. Furthermore, natural organic nutrient sources such as starch hydrolysates, molasses, brown sugar molasses, rice bran, cassava, bagasse, and corn steep liquor may be used. Appropriate amounts of other carbon sources can be used in a variety of ways without limitation. These carbon sources may be used alone or in combination of two or more, but are not limited to these uses.

[0060] Polyhydroxyalkanoates (PHAs) may be formed in living cells by enzymatic polymerization of one or more monomer repeating units. Based on their molecular structure, they can be classified as crystalline, semi-crystalline, or amorphous polyhydroxyalkanoates.

[0061] Examples of repeating units that constitute polyhydroxyalkanoates (PHAs) include lactic acid, glycolic acid, 2-hydroxybutyrate (2-HB), 3-hydroxybutyrate (3-HB), 3-hydroxypropionate (3-HP), 3-hydroxyvalerate (3-HV), 3-hydroxyhexanoate (3-HH), 3-hydroxyheptanoate (3-HHep), 3-hydroxyoctanoate (3-HO), 3-hydroxynonanoate (3-HN), 3-hydroxydecanoate (3-HD), 3-hydroxydodecanoate (3-HDd), 4-hydroxybutyrate (4-HB), 4-hydroxyvalerate (4-HV), 5-hydroxyvalerate (5-HV), and 6-hydroxyhexanoate (6-HH). A PHA may contain one or more repeating units selected from the above. For example, PHA may be poly-3-hydroxybutyrate-co-4-hydroxybutyrate (hereinafter referred to as 3HB-co-4HB).

[0062] Polyhydroxyalkanoates (PHAs) may be homopolymers or copolymers. According to one embodiment, the polyhydroxyalkanoate may include copolymers, specifically copolymers comprising two or more different repeating units having different repeating units randomly distributed in the polymer chain.

[0063] Furthermore, polyhydroxyalkanoates may contain isomers. For example, polyhydroxyalkanoates may contain structural isomers, enantiomers, or geometric isomers. Specifically, polyhydroxyalkanoates may contain structural isomers.

[0064] For example, polyhydroxyalkanoates (PHAs) may contain 4-hydroxybutyrate (4-HB) repeating units. For example, the content of 4-HB repeating units in polyhydroxyalkanoates may be 0.1% by weight or more, 1% by weight or more, 5% by weight or more, 10% by weight or more, 20% by weight or more, 30% by weight or more, 40% by weight or more, 50% by weight or more, or 60% by weight or more, based on the total weight of the polyhydroxyalkanoate; 100% by weight or less, 99% by weight or less, 90% by weight or less, 80% by weight or less, 70% by weight or less, or 60% by weight or less; and 0.1-60% by weight, 0.1-55% by weight, 0.5-60% by weight, 0.5-55% by weight, 1-60% by weight, 1-55% by weight, 1-50% by weight, 2-55% by weight, 3-55% by weight, 3-50% by weight, 5-55% by weight, 5-50% by weight, 10-55% by weight, 10-50% by weight, or 1-40% by weight. By ensuring that the 4-HB repeating unit content meets the above range, the final PHA prepared exhibits excellent biodegradability in soil and seawater, and can further improve thermal properties, productivity, and processability without reducing its mechanical properties.

[0065] Furthermore, the weight-average molecular weight of the polyhydroxyalkanoate (PHA) may range from 10,000 to 1,200,000 g / mol. For example, the weight-average molecular weight of PHA may be 10,000 to 1,200,000 g / mol, 50,000 to 1,200,000 g / mol, 100,000 to 1,000,000 g / mol, 100,000 to 900,000 g / mol, 200,000 to 800,000 g / mol, 10,000 to 600,000 g / mol, or 600,000 to 1,200,000 g / mol.

[0066] Microbial cells for producing polyhydroxyalkanoate (PHA) may be in a normal state, or they may be in a state of autodegradation where the cell wall is weakened due to overculture or autodigestion, or the cells are ruptured. In the method according to one embodiment of the present invention, the disruption step can be effectively carried out even when the cells are in a normal state or an autodegradation state, and the productivity of the final PHA can be improved.

[0067] Supply solution preparation step A method for producing polyhydroxyalkanoate (PHA) according to one embodiment of the present invention includes preparing a supply solution containing a culture medium (or the supernatant obtained by centrifugation of the culture medium).

[0068] The supply solution is a mixture of cells and PHA diluted with distilled water. It may further contain pH adjusters and / or surfactants.

[0069] If the supply solution contains the supernatant obtained by centrifugation of the culture medium, centrifugation may be performed at speeds of 3,000–8,000 rpm, 4,000–6,000 rpm, or 4,000–5,000 rpm for 10–50 minutes, 20–40 minutes, or 25–35 minutes, but is not limited to these speeds.

[0070] The cells and PHA in the supply solution can be mixed with distilled water so that the total solids content is 5-20% by weight, preferably 10-15% by weight. When the total solids content meets the above content range, the crushing step can be carried out efficiently.

[0071] The supply solution may contain a pH adjuster. The pH adjuster may be one or more basic solutions selected from the group consisting of sodium hydroxide, potassium hydroxide, lithium hydroxide, and sodium carbonate. Furthermore, the concentration of the basic solution is such that the basic solute is 20-60 w / w%, 30-50 w / w%, or 35-45 w / w% based on the total weight of the basic solution.

[0072] The pH of the supply solution containing the pH adjuster may be pH 8 to pH 13, or pH 9 to pH 12, preferably pH 10 to pH 11. When the pH of the supply solution is adjusted to the above range, the cell disruption step and subsequent steps can be carried out more easily.

[0073] The supply solution may contain a surfactant. The surfactant may be one or more anionic surfactants selected from the group consisting of sodium laureth sulfate, sodium lauryl sulfate, sodium dodecyl sulfate, and sodium dodecylbenzenesulfonate.

[0074] The surfactant can be used in an amount of 0.05-0.5% by weight, 0.1-0.2% by weight, preferably 0.13-0.15% by weight, based on the total weight of the supply solution. When the surfactant content is adjusted to the above range, the cell disruption step and subsequent steps can be carried out more easily.

[0075] A step of disrupting cells or PHA (disruption step) A method for producing polyhydroxyalkanoate (PHA) according to one embodiment of the present invention comprises disrupting cells or PHA contained in a supply solution.

[0076] The disruption step is a physical cell disruption step using a high-pressure homogenizer. A high-pressure homogenizer is a device that disrupts cells by applying high pressure to them. The disruption pressure can be 300-1,500 bar, 300-600 bar, 300-500 bar, 400-1,000 bar, 400-700 bar, 400-600 bar, 400-500 bar, 600-1,000 bar, or 600-800 bar.

[0077] When the disruption pressure meets the above range, the target degree of cell disruption can be achieved, and an appropriate viscosity range can be maintained, thus facilitating subsequent steps and increasing the productivity of PHA.

[0078] When the lower limit of the crushing pressure is reached, a crude polyhydroxyalkanoate solution can be obtained immediately after the crushing step without performing the analysis step of the crushed product solution or the agglutination test step.

[0079] If the upper limit of the crushing pressure is reached, the crude polyhydroxyalkanoate solution is obtained after performing the analysis step of the crushing product solution without carrying out the agglomeration test step.

[0080] Furthermore, the crushing temperature can be 0-40°C, 5-35°C, 10-30°C, 20-40°C, or 23-35°C. To prevent temperature rise due to high-pressure crushing, cooling water is circulated to maintain a constant temperature.

[0081] The crushing time may vary depending on the flow rate entering and leaving the high-pressure homogenizer or the processing rate per hour.

[0082] A crushing step is performed once on the feed solution that has passed through the high-pressure homogenizer. If the crushing step is performed with the crushing conditions changed once or multiple times through the crushing condition pre-determination step and / or crushing condition re-determination step, the determined crushing conditions (pressure, etc.) may be the same or different.

[0083] The disruption conditions may be determined based on the results of morphological parameter analysis. Specifically, the morphological parameters of cells or PHA can be analyzed before, during, or after the disruption step, and the number and conditions of disruption can be determined based on the analysis results. Details will be explained later in the steps for pre-determining disruption conditions and re-determining disruption conditions.

[0084] The cell disruption product solution is centrifuged to obtain a crude polyhydroxyalkanoate solution (separation step). A method for producing polyhydroxyalkanoate (PHA) according to one embodiment of the present invention includes centrifuging a cell disruption product solution to obtain a crude polyhydroxyalkanoate solution.

[0085] The step of preparing the crude polyhydroxyalkanoate solution involves centrifuging the cell disruption product solution to separate impurities from the cells and PHA contained in the disruption product solution.

[0086] Centrifugation can be performed at speeds of 10,000–20,000 rpm, 12,000–18,000 rpm, or 15,000–16,000 rpm for 10–30 minutes or 10–20 minutes. Furthermore, centrifugation can be performed at temperatures of 0–40°C, 5–35°C, 10–30°C, 20–40°C, or 23–35°C.

[0087] A crude polyhydroxyalkanoate solution containing the distillate formed by centrifugation is obtained. The crude polyhydroxyalkanoate solution thus obtained is then subjected to a washing step and an impurity removal step by additional chemical or enzymatic treatment to be formulated.

[0088] Steps to analyze the morphological parameters of cells or PHA (analysis steps) A method for producing polyhydroxyalkanoate (PHA) according to one embodiment of the present invention includes analyzing the morphological parameters of cells or PHA.

[0089] The step of analyzing morphological parameters may, but is not limited to, three-dimensional image analysis using holotomography. Furthermore, the morphological parameters may be one or more selected from the group consisting of length, sphericity, mass, protein concentration, volume, and surface area.

[0090] Details of holotomography analysis (HTA) are as described above. In one embodiment of the present invention, the step of analyzing the morphological parameters of cells or PHA may include a preliminary analysis step and a first analysis step performed before the disruption step; and / or a second analysis step performed during or after the disruption step.

[0091] The analysis steps may include: a preliminary analysis step of taking multiple analytical samples from the culture medium, feed solution, and / or disruption product solution and analyzing the morphological parameters of cells or PHA contained in the culture medium according to the analytical sample collection points; a first analysis step of analyzing the morphological parameters of cells or PHA contained in the feed solution; and / or a second analysis step of analyzing the morphological parameters of cells or PHA contained in the disruption product solution.

[0092] Each analysis step is a separate step and is selected individually as needed. When multiple analysis steps are employed simultaneously in a single process, each analysis step can be performed in the following order: preliminary analysis step, first analysis step, second analysis step, and auxiliary analysis step.

[0093] 1) Analysis of cells or PHA contained in the culture medium (preliminary analysis step) The preliminary analysis step involves analyzing the morphological parameters of cells or PHA contained in the culture medium, quantifying the analyzed morphological parameters, and detecting the degree of autolysis.

[0094] The average sphericity of cells contained in the culture medium may be 0.4 or higher, 0.5 or higher, 0.6 or higher, or 0.7 or higher. Furthermore, the average length of cells may be 3.0 μm or higher, 3.5 μm or higher, or 6.0 μm or less, 5.0 μm or less, or 4.0 μm or less.

[0095] The average sphericity of the PHA contained in the culture medium may be 0.5 or higher, 0.6 or higher, 0.7 or higher, or 0.8 or higher.

[0096] As the degree of autodegradation increases, the average length of cells in the culture medium decreases, and the average sphericity of the cells or PHA increases.

[0097] 2) Analysis of cells or PHA in the supply solution (first analytical step) In one embodiment of the present invention, the step of analyzing the morphological parameters of cells or PHA may include a first analysis step performed before the disruption step. The first analysis step is to analyze the morphological parameters of cells or PHA contained in the supply solution and to quantify the analyzed morphological parameters to detect the supply state.

[0098] The average sphericity of the cells contained in the supply solution may be 0.6 or higher, 0.65 or higher, 0.7 or higher, 0.75 or higher, 0.8 or higher, 0.85 or higher, or 0.9 or higher. The average length of the cells may also be 3.28 μm or higher, 3.4 μm or higher, 3.6 μm or higher, 3.8 μm or higher, 4.0 μm or higher, or 4.2 μm or higher, and may be 6.0 μm or lower, 5.5 μm or lower, 5.0 μm or lower, or 4.8 μm or lower.

[0099] Specifically, the average length of the cells contained in the supply solution may be 3.28 μm to 4.8 μm, 3.28 μm to 4.2 μm, or 4.0 μm to 4.8 μm, and the average sphericity of the cells may be 0.6 to 0.99, 0.65 to 0.9, 0.7 to 0.9, 0.6 to 0.7, or 0.75 to 0.85.

[0100] More specifically, the cells in the supply solution may contain cells with a sphericity of 0-0.8 in proportions of 40-90%, 40-80%, 45-70%, 50-70%, 55-65%, or 60-65%. Furthermore, cells with a sphericity of 0.8-1.0 may contain cells in proportions of 10-60%, 20-55%, 30-55%, 30-50%, or 35-45%.

[0101] Furthermore, the average sphericity of the PHA contained in the supply solution may be 0.6 or higher, 0.65 or higher, 0.7 or higher, 0.75 or higher, 0.8 or higher, 0.85 or higher, or 0.9 or higher.

[0102] Furthermore, the PHA may contain PHA with a sphericity of 0 to 0.8 in proportions of 23% or more, 25% or more, 30% or more, 34% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, or 65% or more, and may contain PHA with a sphericity of 0.8 to 1.0 in proportions of 77% or less, 75% or less, 70% or less, 66% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, or 35% or less.

[0103] 3) Analysis of cells or PHA contained in the disruption product solution (second analysis step) In one embodiment of the present invention, the step of analyzing the morphological parameters of cells or PHA may include a second analysis step performed during or after the disruption step. The second analysis step is to analyze the morphological parameters of cells or PHA contained in the disruption product solution and to quantify the analyzed morphological parameters to detect the degree of disruption.

[0104] The final cells and PHA contained in the disruption product solution prepared in this manner can be obtained by a method for producing polyhydroxyalkanoate (PHA) according to one embodiment of the present invention.

[0105] The average sphericity of the cells contained in the lysation product solution may be 0.65 or higher, 0.7 or higher, 0.8 or higher, or 0.9 or higher. The average length of the cells may be 3.0 μm or higher, or 3.5 μm or higher, or 4.5 μm or lower, or 4.0 μm or lower.

[0106] Furthermore, the average sphericity of the PHA contained in the crushed product solution may be 0.6 or higher, 0.65 or higher, 0.7 or higher, 0.75 or higher, 0.8 or higher, 0.85 or higher, or 0.9 or higher. The PHA may contain 34% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, or 65% or more of PHA having a sphericity of 0.8 to 1.0, and may contain 66% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, or 35% or less of PHA having a sphericity of 0 to 0.8.

[0107] Figure 6 shows three-dimensional images measured before and after cell disruption. The bright areas (green) represent the cell wall or cytoplasm, and the dark areas (red) represent PHA. From the three-dimensional images, it can be visually confirmed that the length of the cells or PHA shortens and the sphericity of the cells or PHA increases during disruption.

[0108] Step to analyze the degree of protein solubility or aggregation (auxiliary analysis step) 1) Analysis of protein solubility To determine whether the cells and PHA in the disruption product solution are sufficiently disrupted and can proceed to the subsequent steps, and as an auxiliary indicator to ensure process stability, protein solubility analysis or agglutination testing may be performed.

[0109] Specifically, protein solubility is an auxiliary indicator for determining cell rupture and cell lysis, and can be calculated according to Equation 1 below by measuring the absorbance at a wavelength of 280 nm using a UV spectrometer.

[0110] [Formula 1] Protein solubility (%) = ABS1 / ABS0 × 100 (%)

[0111] In the formula, ABS0 is the absorbance at a wavelength of 280 nm measured after crushing the culture medium, feed solution, or crushed product solution five times at 800 bar at room temperature; ABS1 is the absorbance at a wavelength of 280 nm measured after crushing the culture medium, feed solution, or crushed product solution once at 300 to 1,500 bar at room temperature. More specifically, ABS0 and ABS1 are the absorbances measured after crushing the culture medium, feed solution, or crushed product solution under the above conditions, centrifuging at 15,000 rpm for 5 minutes, and diluting the supernatant 100-fold with distilled water.

[0112] In this invention, room temperature refers to a constant temperature, which may be 10-40°C, 20-35°C, 23-35°C, or approximately 25°C. In this invention, in order to prevent temperature rise due to high-pressure crushing, cooling water is circulated from the outside to perform heat exchange and maintain a constant temperature for equipment such as a high-pressure homogenizer.

[0113] The degree of protein solubilization of the disrupted product solution according to one embodiment of the present invention of Formula 1 is 75 to 110%. Specifically, the degree of protein solubilization of the disrupted product solution may be 85% or more, 90% or more, or 95% or more. If the degree of protein solubilization is less than 85%, the cell disruption efficiency decreases, which may reduce the efficiency of obtaining PHA granules in the PHA production method.

[0114] 2) Agglomeration test The agglutination test can be used as an auxiliary indicator to confirm the stability of solid-liquid separation after cell disruption. By applying shear force or centrifugal force to the cell disruption product solution after physical cell disruption using a high-pressure homogenizer, it is possible to measure whether cells or PHA are agglutinating and the degree of agglutination, thereby confirming the stability of the subsequent washing and impurity removal steps.

[0115] In this test, the cell disruption product solution is centrifuged at 3,800 rpm for 20 minutes, the supernatant is removed, and the crude polyhydroxyalkanoate solution is passed through a 150 μm sieve using washing water. In such an event, the degree of aggregation can be confirmed from the residue that does not dissolve in the washing water and does not pass through the sieve. If the degree of aggregation is high, aggregation is likely to occur in the subsequent washing and impurity removal steps. Therefore, the production of polyhydroxyalkanoates (PHAs) is carried out by adjusting the disruption pressure or by changing the process conditions of the washing and impurity removal steps.

[0116] Steps to determine crushing conditions in advance In one embodiment of the present invention, the analysis of morphological parameters of cells or PHA contained in the culture medium is performed before the disruption step, and the method includes the steps of: quantifying the analyzed morphological parameters to detect the degree of autolysis (preliminary analysis step); and determining the initial pressure of the high-pressure homogenizer or the number of disruption treatments according to the degree of autolysis as a first disruption condition predetermination step.

[0117] In another embodiment of the present invention, the analysis of morphological parameters of cells or PHA contained in the supply solution is performed before the disruption step, and the method includes the steps of: quantifying the analyzed morphological parameters to detect the supply state (first analysis step); and determining the disruption pressure of the high-pressure homogenizer or the number of disruption treatments for the supply solution as a second disruption condition predetermination step according to the supply state. Each disruption condition predetermination step is a separate step and is selected individually as needed. If the first and second predetermination steps are employed simultaneously in a single process, the second predetermination step is performed after the first predetermination step.

[0118] 1) First step of predetermining crushing conditions based on analysis of morphological parameters For example, if the mean sphericity of the cells or PHA analyzed in the step of analyzing the morphological parameters of the cells or PHA contained in the culture medium is 0.7 or higher, the initial pressure may be set to 700 bar or less or 600 bar or less.

[0119] During the centrifugation and impurity removal steps after cell disruption, PHA aggregation may occur depending on the degree of cell autodegradation. Therefore, in the steps following the disruption step, morphological parameters can be analyzed and the disruption conditions adjusted to prevent clogging of the piping, etc.

[0120] 2) A second step of pre-determining crushing conditions based on the analysis of morphological parameters. For example, if the average sphericity of the cells or PHA contained in the supply solution is 0.7 or higher, the second step of predetermining the disruption conditions may involve reducing the pressure by 10 to 100 bar from the initial pressure during the step of disrupting the cells or PHA contained in the supply solution.

[0121] As another example, if the mean sphericity of the cells or PHA contained in the supply solution is less than 0.7, the second step of predetermining the disruption conditions may determine the disruption pressure in the step of disrupting the cells or PHA contained in the supply solution to be the same as the initial pressure.

[0122] 3) Second step of pre-determining disruption conditions based on analysis of protein solubility. For example, if the protein solubility of the supply solution according to formula 1 above is 80% or higher, the second step of pre-determining the disruption conditions may involve reducing the pressure by 10 to 100 bar from the initial pressure in the step of disrupting the cells or PHA contained in the supply solution.

[0123] As another example, if the protein solubility of the supply solution according to Equation 1 above is less than 80%, the second step of predetermining the disruption conditions may determine the disruption pressure in the step of disrupting the cells or PHA contained in the supply solution to be the same as the initial pressure.

[0124] Figure 3 specifically shows the method for controlling the crushing pressure in the second step of pre-determining the crushing conditions.

[0125] Figure 3 relates to the step of determining the disruption pressure (P1) in the step of disrupting cells or PHA contained in the supply solution, relative to the initial pressure (P0) of the high-pressure homogenizer (the second step of predetermining the disruption conditions).

[0126] If the mean sphericity of the cells or PHA in the supply solution is less than 0.7, or the degree of protein solubility is less than 80%, the disruption pressure (P1) in the step of disrupting the cells or PHA in the supply solution is maintained at the same initial pressure (P0).

[0127] If the average sphericity of the cells or PHA contained in the supply solution is 0.7 or higher, or if the degree of protein solubilization is 80% or higher, the disruption pressure (P1) in the step of disrupting the cells or PHA contained in the supply solution is reduced by 10 to 100 bar, for example, 50 bar, from the initial pressure (P0).

[0128] Step to re-determine crushing conditions In another embodiment of the present invention, the analysis of morphological parameters of cells or PHA contained in the disruption product solution is performed during or after the disruption step, and the method may include the steps of: quantifying the analyzed morphological parameters to detect the degree of disruption; and determining the disruption pressure of the high-pressure homogenizer or the number of disruption treatments according to the degree of disruption as a first disruption condition re-determination step.

[0129] In another embodiment of the present invention, the step of analyzing the morphological parameters of cells or PHA contained in the disruption product solution is performed during or after the disruption step, and the method may further include a step of performing an agglutination test to measure the degree of aggregation of the disruption product solution after a step of quantifying the analyzed morphological parameters to detect the degree of disruption; and a second disruption condition pre-determination step of determining the disruption pressure of the high-pressure homogenizer or the number of disruption treatments according to the degree of aggregation. Each disruption condition re-determination step is a separate step and is selected individually as needed. If the first re-determination step and the second re-determination step are employed simultaneously in one process, the second re-determination step is performed after the first re-determination step.

[0130] 1) First step of re-determining the crushing conditions by analyzing morphological parameters. The step of analyzing the morphological parameters of cells or PHA contained in the disruption product solution is performed during or after the disruption step, and the method may include a first disruption condition re-determination step of quantifying the analyzed morphological parameters and redetermining the disruption pressure of the high-pressure homogenizer or the number of disruption treatments for a continuously supplied supply solution.

[0131] For example, if the average sphericity of the cells or PHA contained in the disruption product solution is less than 0.9, the first disruption condition re-determination step may involve increasing the pressure by 10 to 100 bar from the disruption pressure in the step of disrupting the cells or PHA contained in the supply solution.

[0132] 2) First step of re-determining the disruption conditions by analyzing the degree of protein solubility. For example, if the protein solubility of the disruption product solution according to Formula 1 above is less than 90%, the first step of re-determining the disruption conditions may involve increasing the pressure by 10 to 100 bar from the disruption pressure in the step of disrupting the cells or PHA contained in the supply solution.

[0133] The degree of disruption can be measured by the average sphericity of the cells or PHA contained in the disruption product solution, or by the degree of protein solubility thereof. The disruption conditions may be adjusted based on the measurement results; if the degree of disruption is deemed insufficient, the disruption pressure may be increased; or if the degree of disruption is deemed excessive, the disruption pressure may be decreased.

[0134] 3) Second step of re-determining crushing conditions according to the degree of aggregation In another embodiment of the present invention, the step of analyzing the morphological parameters of cells or PHA contained in the disruption product solution is performed during or after the disruption step, and the method may include the step of performing an agglutination test to measure the degree of agglutination of the disruption product solution; and a second disruption condition re-determination step of determining the disruption pressure of the high-pressure homogenizer or the number of disruption treatments for a continuously supplied supply solution according to the degree of agglutination.

[0135] As an example, if the average sphericity of the PHA contained in the disrupted product solution is 0.9 or higher, or if the degree of protein solubilization is 90% or higher, an agglutination test is performed. If the degree of agglutination is measured to be above a predetermined value, the second step of re-determining the disruption conditions may involve lowering the pressure by 10 to 100 bar from the disruption pressure used in the step of disrupting the cells or PHA contained in the supply solution.

[0136] As another example, if the degree of aggregation is measured to be below a predetermined value, the second step of re-determining the disruption conditions may involve maintaining the disruption pressure in the step of disrupting the cells or PHA contained in the supply solution, or increasing it by 10 to 100 bar.

[0137] Excessive physical disruption during the disruption step can lead to a high degree of cell disruption, potentially causing PHA aggregation. Therefore, determining the disruption conditions in real time through morphological parameter analysis can prevent clogging of piping in subsequent steps.

[0138] Figures 4 and 5 specifically illustrate the method for controlling the crushing pressure in the crushing condition re-determination step.

[0139] Figure 4 relates to a step of redetermining the disruption pressure (P2) of a high-pressure homogenizer relative to the disruption pressure (P1) in the step of disrupting cells or PHA contained in the supply solution, for a continuously supplied solution.

[0140] Steps are performed to disrupt the cells or PHA contained in the supply solution (P1); and to analyze the morphological parameters of the cells or PHA contained in the disruption product solution.

[0141] If the mean sphericity of the cells or PHA contained in the disrupted product solution is less than 0.9, or if the degree of protein solubilization is less than 90%, the disruption pressure (P2) of the high-pressure homogenizer for the continuously supplied supply solution is determined to be 50 bar higher than the disruption pressure (P1) in the step of disrupting the cells or PHA contained in the supply solution.

[0142] If the average sphericity of cells or PHA contained in the lysation product solution is 0.9 or higher, or if the degree of protein solubilization is 90% or higher, an agglutination test is performed to measure the degree of aggregation of the lysation product solution.

[0143] If the degree of aggregation is measured to be below a predetermined value (Y), the crushing pressure (P2) of the high-pressure homogenizer for the continuously supplied supply solution is determined to be 50 bar higher than the crushing pressure (P1) in the step of crushing the cells or PHA contained in the supply solution.

[0144] If the degree of aggregation is measured to exceed a predetermined value (N), the crushing pressure (P2) of the high-pressure homogenizer for the continuously supplied supply solution is determined to be 50 bar lower than the crushing pressure (P1) in the step of crushing the cells or PHA contained in the supply solution.

[0145] Figure 5 shows the step of readjusting the disruption conditions after adjusting the pressure by an agglutination test in another embodiment of the present invention. The step relates to readjusting the disruption pressure (Pn+1) of the high-pressure homogenizer for the continuously supplied supply solution after preparing the disruption product solution by increasing or decreasing the disruption pressure (Pn) of the disruption pressure (Pn-1) by 10 to 100 bar from the pre-set disruption pressure (Pn-1) in the step of disrupting cells or PHA contained in the supply solution.

[0146] Specifically, the process involves the steps of: disrupting cells or PHA contained in the supply solution with a disruption pressure (Pn) that is 50 bar lower than the pre-set disruption pressure (Pn-1); and analyzing the morphological parameters of the cells or PHA contained in the disruption product solution.

[0147] Next, if the mean sphericity of the cells or PHA contained in the disrupted product solution is less than 0.9, or if the degree of protein solubility is less than 90%, the first disruption condition re-determination step determines the pressure, which is determined to be between the pre-set disruption pressure (Pn-1) and the disruption pressure (Pn) in the step of disrupting the cells or PHA contained in the supply solution, as the disruption pressure (Pn+1) of the high-pressure homogenizer for the continuously supplied supply solution.

[0148] If the average sphericity of the cells or PHA contained in the lysation product solution is 0.9 or higher, or if the degree of protein solubilization is 90% or higher, an agglutination test is performed to measure the degree of aggregation of the lysation product solution.

[0149] If the degree of aggregation is measured to be below a predetermined value (Y), the second disruption condition re-determination step determines the same pressure (Pn) as the disruption pressure (Pn) in the step of disrupting the cells or PHA contained in the supply solution, as the disruption pressure (Pn+1) of the high-pressure homogenizer for the continuously supplied supply solution.

[0150] If the degree of aggregation is measured to exceed a predetermined value (N), the second step of re-determining the disruption conditions determines the disruption pressure (Pn+1) of the high-pressure homogenizer for the continuously supplied supply solution as a pressure obtained by reducing the disruption pressure (Pn) in the step of disrupting the cells or PHA contained in the supply solution by 50 bar.

[0151] In the step of pre-determining the disruption conditions and the step of re-determining the disruption conditions, the criteria for determining the disruption conditions according to the morphological parameters of the cells or PHA, the degree of protein solubility, or the degree of aggregation can be adjusted according to the culture medium supplied to the entire process. Embodiments for carrying out the invention

[0152] The present invention will be described in more detail below with reference to the following examples. However, the following examples are for illustrative purposes only, and the scope of the examples is not limited thereto. [Examples]

[0153] 1. Preparation of culture medium PHA culture media were prepared using genetically modified E. coli strains. First, a certain quantity of PHA-producing strains was obtained through seed culture, and then the main fermentation was carried out. Sucrose was used as the carbon source for the fermentation of the PHA-producing strains, and various mineral components were added as nutrients. Sodium hydroxide solution or ammonia gas was used to adjust the fermentation pH. The fermentation process using the PHA-producing strains was carried out at 30-40°C and pH 6-9.

[0154] Examples 1 and 2 used separate culture media containing genetically modified E. coli strains and sucrose as a carbon source. Although the culture media for Examples 1 and 2 were prepared under the same culture conditions, the morphological parameters of the cells or PHA measured in Example 1 or Example 2 may differ due to the viable cell properties of the bacterial strains.

[0155] 2. Simulation of the autodegradation of culture media The degree of autolysis refers to the extent to which the cell wall weakens or the cell ruptures due to autolysis. To experimentally simulate this, autolysis conditions were simulated using an ultrasonic disperser (SONICS, Vibra cell). Normally fermented cultures were treated with an ultrasonic disperser at an amplitude of 40% and a pulse of 10 / 10 for 1 or 2 minutes to rupture the cells and simulate the degree of autolysis, after which the cell disruption process was carried out.

[0156] Examples 1-1 to 1-3 used culture media treated with ultrasound according to Table 1 below. Specifically, Example 1-1 is the culture media of Example 1 without treatment with an ultrasonic disperser, while Examples 1-2 and 1-3 are the culture media of Example 1 treated with an ultrasonic disperser for 1 minute or 2 minutes, respectively. Examples 2-1 to 2-3 are culture media treated with ultrasound according to Table 2 below.

[0157] 3. Cell disruption process Each culture medium was subjected to centrifugation using a centrifuge (Hanil, Combi-514 R) to separate impurities from the cells and / or PHA-containing biomass contained in the culture medium. The separated biomass was diluted with distilled water to a total solids content of 10-15% by weight. The diluted solution was adjusted to a pH of 10-11 using sodium hydroxide (40 w / w%, Deoksan Science), and then 0.13-0.15% by weight of the anionic surfactant sodium laureth sulfate (30 w / w%, Miwon) was added to the liquid phase to prepare the feed solution. This lysation feed solution was thoroughly stirred and subjected to physical cell disruption using a high-pressure homogenizer (Bertoli, ATOMO 3.0) at 600-1,000 bar for 1-5 cycles.

[0158] [Example Test] 1. Preparation of samples for three-dimensional image analysis and measurement. The analytical samples (culture medium, feed solution, or disruption product solution) of the examples and comparative examples were diluted 10-fold or 100-fold, respectively, in 1.5 ml microcentrifuge tubes using distilled water (DIW). 20 μl of the diluted sample was dispensed into a TomoDish (Tomo Cube Co., Ltd.) and covered with a coverslide. Three-dimensional images of the prepared samples were taken using holotomography analysis (Tomo Cube Co., Ltd., HT-1H), and the cell length and sphericity were measured.

[0159] The refractive indices (RI) used for 3D imaging and data measurement were 1.3513–1.3745 (cells) and 1.3748–1.4081 (PHA). Ten 3D images were taken for each sample. 3D images were created using TomoStudio (trademark) (Tomo Cube Co., Ltd.), and the sphericity of the images was analyzed. The average mass, volume, length, and sphericity of cells and PHA were calculated.

[0160] 2. Measurement of protein content before and after cell disruption The analytical samples (supply solution or crushing product solution) of the examples and comparative examples were crushed at 300-1,500 bar at room temperature, and then centrifuged at 15,000 rpm for 5 minutes using a microcentrifuge (Eppendorf, 5414R). The supernatant obtained from centrifugation was diluted 100-fold with distilled water in a 2.0 ml microtube, and the absorbance at a wavelength of 280 nm was measured using a UV spectrometer (HACH, DR6000). Calibration was performed with distilled water when measuring absorbance.

[0161] The degree of protein solubility was calculated according to formula 1 below. [Formula 1] Protein solubility (%) = ABS1 / ABS0 × 100 (%)

[0162] In the formula, ABS0 is the absorbance at a wavelength of 280 nm measured after the analyte sample was crushed five times at 800 bar and room temperature; ABS1 is the absorbance at a wavelength of 280 nm measured after the analyte sample was crushed once at 300-1,500 bar and room temperature.

[0163] 3. Agglomeration test 50 ml of the analytical sample (solubil) was centrifuged at 3,800 rpm for 20 minutes (Hanil, Combi-514R), and the supernatant was removed. The distillate remaining at the bottom was passed through a 150 μm sieve with washing water. In such events, residues that did not dissolve in water and did not pass through the sieve were observed with the naked eye. The degree of aggregation was classified as ×, △, ○, and ◎ based on the degree of residue, and is shown in Tables 3 and 4 below. If the degree of aggregation was × or △, it was determined to be below the predetermined value (Y in Figures 4 and 5).

[0164] 1) Results and analysis of the self-decomposition simulation The analysis results of cell length and sphericity in the culture media of Examples 1-1 to 1-3 and Examples 2-1 to 2-3 are summarized in Tables 1 and 2 below. To confirm reproducibility, the experiment was repeated twice with different culture media. As a result, as the sonication treatment time increased, the average length of cells in the culture media decreased and the average sphericity of the cells increased.

[0165] [Table 1]

[0166] [Table 2]

[0167] 2) Analysis of the degree of cell disruption The supply solutions were prepared using the culture media from Examples 1 and 2, respectively. Morphological parameters, protein solubility, and degree of aggregation were measured for cells and PHA in the supply solution at the supply point of the high-pressure homogenizer, and for cells and PHA in the cell disruption product solution after a single cell disruption at a disruption pressure of 600-1,000 bar in the high-pressure homogenizer. The results are shown in Tables 3 and 4 below.

[0168] [Table 3]

[0169] [Table 4]

[0170] The analysis revealed that the degree of aggregation varied depending on the degree of autodegradation of the culture medium and the crushing pressure during the crushing step.

[0171] Specifically, when the average cell length of the feed solution or the disruption product solution is 4.00 μm or greater, and the average cell sphericity of the feed solution or the average PHA sphericity of the disruption product solution is 0.85 or less, the degree of aggregation is hardly observed, resulting in high process stability in downstream processes.

[0172] When the average cell length of the feed solution or the disrupted product solution is 3.28–4.20 μm, and the average cell sphericity of the feed solution or the average PHA sphericity of the disrupted product solution is 0.75–0.85, some degree of aggregation is observed, and process stability in downstream processes is slightly reduced, but normal process operation is still possible.

[0173] If the average cell length of the feed solution or the disruption product solution is 3.92 μm or less, and the average cell sphericity of the feed solution or the average PHA sphericity of the disruption product solution is 0.83 to 1.00, a significant or high level of aggregation is observed, which significantly reduces process stability in downstream processes and is likely to cause problems such as pipe clogging.

[0174] Furthermore, when the number of cells with a sphericity of 0-0.8 was 50.0% or more of the total, the degree of aggregation was observed at a low level or hardly at all. In particular, when the percentage was 70.0% or more, the degree of aggregation was hardly observed.

[0175] In Example 1 or 2, the degree of protein solubilization was calculated based on ABS0 of Example 1-1 or Example 2-1, respectively, which were culture media not subjected to sonication. When cells were lysed using a high-pressure homogenizer at a disruption pressure of 600 bar or more, protein solubilization was 90% or more in Examples 1-1, 1-2, and 2-1, whereas protein solubilization was less than 90% in Examples 1-3, 2-2, and 2-3, which exhibited a higher degree of autodegradation. This is understood to be because, in culture media subjected to sonication, a relatively large amount of protein is separated in the process of preparing the supply solution containing the supernatant obtained by centrifugation of the culture medium; therefore, the residual protein content was lower compared to culture media not subjected to sonication.

[0176] In Examples 2-1 to 2-3, the protein solubilization exceeding 100% is due to errors in the absorbance measurement method, and should be understood as meaning that the protein solubilization is 90% or higher.

[0177] Based on the above results, the range of cell or PHA length (average cell length or average PHA length) and sphericity (average cell sphericity or average PHA sphericity) contained in the supply solution for determining process stability was classified into three zones, as shown in Figure 7. 1. Safety zone: Length 4.00 μm or more, and sphericity 0.00 to 0.85 2. Caution zone: Length 3.28–4.20 μm and sphericity 0.75–0.85 3. High-risk zone: Length 3.92 μm or less, and sphericity 0.83-1.00.

[0178] However, Figure 7 shows an example prepared based on the culture media of Examples 1 and 2 during fermentation, and since the culture media tends to aggregate relatively well, the ranges of the caution zone and safety zone for length and sphericity appear relatively narrow in Figure 7. The values ​​for each zone may vary depending on the strain or the type of carbon source in the culture medium.

Claims

1. A method for monitoring cell disruption, wherein the degree of cell disruption is confirmed or the disruption conditions are determined by analyzing the morphological parameters of cells or PHA contained in a culture medium containing polyhydroxyalkanoate (PHA) using three-dimensional image analysis, The aforementioned three-dimensional image analysis is a three-dimensional image analysis using holotomography analysis. The morphological parameters are one or more selected from the group consisting of length, sphericity, volume, and surface area. A method for determining the number of crushing operations, or increasing or decreasing the crushing pressure by 10 to 100 bar, by comparing the average sphericity and / or average length among the morphological parameters with a preset threshold.

2. A method for producing polyhydroxyalkanoates (PHAs), A step of preparing a culture medium containing polyhydroxyalkanoate (PHA); A step of preparing a supply solution containing the culture medium; A disruption step of disrupting cells or PHA contained in the supply solution; and The process includes a step of analyzing the morphological parameters of the cells or PHA by three-dimensional image analysis before, during, or after the disruption step. The aforementioned three-dimensional image analysis is a three-dimensional image analysis using holotomography analysis. The morphological parameters are one or more selected from the group consisting of length, sphericity, volume, and surface area. A method for determining the number of crushing operations, or increasing or decreasing the crushing pressure by 10 to 100 bar, by comparing the average sphericity and / or average length among the morphological parameters with a preset threshold.

3. The step of analyzing the morphological parameters of the cells or PHA contained in the culture medium is performed before the disruption step. A method for producing a polyhydroxyalkanoate (PHA) according to claim 2, wherein the method includes the steps of: quantifying the analyzed morphological parameters to detect the degree of autodegradation; and determining the initial pressure of the high-pressure homogenizer or the number of crushing treatments according to the degree of autodegradation as a first crushing condition pre-determination step.

4. The morphological parameters of the cells or PHA contained in the supply solution are analyzed prior to the disruption step. A method for producing polyhydroxyalkanoate (PHA) according to claim 2, wherein the method includes the steps of: quantifying the analyzed morphological parameters to detect the supply state; and determining the crushing pressure of a high-pressure homogenizer or the number of crushing operations according to the supply state as a second crushing condition pre-determination step.

5. Analysis of the morphological parameters of the cells or PHA contained in the disruption product solution is performed during or after the disruption step. A method for producing a polyhydroxyalkanoate (PHA) according to claim 2, wherein the method includes the steps of: quantifying the analyzed morphological parameters to detect the degree of crushing; and determining the crushing pressure of a high-pressure homogenizer or the number of crushing treatments according to the degree of crushing as a first crushing condition re-determination step.

6. A method for producing polyhydroxyalkanoate (PHA) according to claim 2, wherein the crushing pressure in the crushing step is 300 to 1,500 bar.

7. A first analytical step involves analyzing the morphological parameters of the cells or PHA contained in the supply solution; A second step of predetermining crushing conditions, in which the crushing pressure of the high-pressure homogenizer or the number of crushing cycles is determined according to the results of the first analysis; A step of disrupting the cells or PHA contained in the supply solution; A second analytical step of analyzing the morphological parameters of the cells or PHA contained in the disruption product solution; and A method for producing polyhydroxyalkanoate (PHA) according to claim 2, comprising a first crushing condition re-determination step of determining the crushing pressure of the high-pressure homogenizer or the number of crushing treatments according to the second analysis result.

8. Formula 1 below: [Formula 1] Protein solubility (%) = ABS1 / ABS0 × 100 (%) (In the formula, ABS0 is the absorbance at a wavelength of 280 nm measured after repeatedly crushing the culture medium, the supply solution, or the crushed product solution at 800 bar at room temperature five times; ABS1 is the absorbance at a wavelength of 280 nm measured after crushing the culture medium, the supply solution, or the crushed product solution at 300 to 1,500 bar at room temperature once.) A method for producing a polyhydroxyalkanoate (PHA) according to any one of claims 2 to 7, wherein the degree of protein solubility is 75 to 110%.

9. If the average sphericity of the cells or PHA contained in the supply solution is 0.7 or higher, or if the following formula 1: [Formula 1] Protein solubility (%) = ABS1 / ABS0 × 100 (%) (In the formula, ABS0 is the absorbance at a wavelength of 280 nm measured after repeatedly crushing the culture medium, the supply solution, or the crushed product solution at 800 bar at room temperature five times; ABS1 is the absorbance at a wavelength of 280 nm measured after crushing the culture medium, the supply solution, or the crushed product solution at 300 to 1,500 bar at room temperature once.) A method for producing polyhydroxyalkanoate (PHA) according to claim 4, wherein, if the degree of protein solubilization is 80% or more, in the second step of predetermining the disruption conditions, the pressure is reduced by 10 to 100 bar from the initial pressure in the step of disrupting the cells or PHA contained in the supply solution.

10. If the average sphericity of the cells or PHA contained in the disruption product solution is less than 0.9, or if the following formula 1: [Formula 1] Protein solubility (%) = ABS1 / ABS0 × 100 (%) (In the formula, ABS0 is the absorbance at a wavelength of 280 nm measured after repeatedly crushing the culture medium, the supply solution, or the crushed product solution at 800 bar at room temperature five times; ABS1 is the absorbance at a wavelength of 280 nm measured after crushing the culture medium, the supply solution, or the crushed product solution at 300 to 1,500 bar at room temperature once.) A method for producing polyhydroxyalkanoate (PHA) according to claim 5, wherein, if the degree of protein solubility is less than 90%, in the first step of re-determining the disruption conditions, the pressure is increased by 10 to 100 bar from the disruption pressure in the step of disrupting the cells or PHA contained in the supply solution.

11. A method for producing a polyhydroxyalkanoate (PHA) according to claim 7, further comprising: a step of performing an agglutination test to measure the degree of agglutination of the crushed product solution after performing the second analysis step; and a step of determining the crushing pressure of the high-pressure homogenizer or the number of crushing treatments according to the degree of agglutination as a second crushing condition re-determination step.

12. A system for monitoring cell disruption in which the degree of cell disruption is confirmed or the disruption conditions are determined by analyzing the morphological parameters of cells or PHA contained in a culture medium containing polyhydroxyalkanoate (PHA) using three-dimensional image analysis, The aforementioned three-dimensional image analysis is a three-dimensional image analysis using holotomography analysis. The morphological parameters are one or more selected from the group consisting of length, sphericity, volume, and surface area. A system that, among the morphological parameters, compares the average sphericity and / or average length with a preset threshold to maintain or increase / decrease the crushing pressure by 10 to 100 bar, or to determine the number of crushing operations.

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