Method for monitoring cell disruption and method for producing polyhydroxyalkanoate 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 lysis and a method for producing polyhydroxyalkanoates (PHA) using the same. [Background technology]

[0002] Polyhydroxyalkanoates (PHAs) are biodegradable polymers composed of several kinds of hydroxyl carboxylic acids produced by many microorganisms and are used as intracellular storage substances. Polyhydroxyalkanoates have similar physical properties to conventional petroleum-derived synthetic polymers such as polybutylene adipate terephthalate (PBAT), polybutylene succinate (PBS), polybutylene succinate terephthalate (PBST), and polybutylene succinate adipate (PBSA), and are completely biodegradable and have excellent biocompatibility.

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

[0004] The methods for extracting the PHA granules can be mainly classified into three types: organic solvent extraction, chemical extraction, and physical crushing.

[0005] First, the organic solvent extraction method is a method of dissolving and extracting PHA granules using an organic solvent. This method can obtain high-purity PHA, but has the disadvantage of requiring dedicated equipment because of the use of organic solvents. On the other hand, the chemical extraction method extracts PHA granules by destroying cell walls at high temperatures under acidic or basic conditions. It is easy to recover PHA granules, but problems such as a decrease in the molecular weight of the extracted PHA may occur due to harsh acidic or basic conditions and temperature conditions.

[0006] On the other hand, the physical disruption method is a method of extracting PHA granules after breaking down cell walls with physical energy such as high pressure. Compared to the above methods, this method has the disadvantage that the purity of the extracted PHA granules is low because the ability to remove impurities attached to the PHA granules is low. However, it has the advantage that the molecular weight reduction and decomposition of PHA can be minimized because the extraction is performed under mild conditions. This physical disruption method includes ultrasonic disruption, high-pressure disruption, and crushing. High-pressure disruption includes cell disruption using a high-pressure homogenizer (HPH), and crushing includes colloidal crushing, bead crushing, and ball crushing.

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

[0008] Specific examples of conventional disruption indicators used in physical disruption methods include optical microscopy, measurement of protein content, measurement of changes in target substance concentration, dielectric spectroscopy, flow cytometry, confirmation of particle size distribution, SDS-page, etc. These methods have the disadvantages of being complicated to analyze, taking a long time to analyze, and requiring sample pretreatment.

[0009] The present inventors have invented the present invention for the purpose of solving the problem of the prior art in which process conditions cannot be immediately changed according to the degree of cell disruption when physically disrupting cells using a high-pressure homogenizer, and of providing an effective method for indicating cell disruption. [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 polyhydroxyalkanoates (PHAs) using the same, which set optimal cell disruption conditions and enhance the stability of the impurity removal step following the disruption step. [How to solve 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 ascertained or the conditions for disruption are determined by analyzing 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 for monitoring cell disruption is provided, wherein the three-dimensional image analysis is three-dimensional image analysis using holotomography analysis 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, there is provided a method for producing polyhydroxyalkanoate (PHA), comprising the steps of: preparing a culture medium containing polyhydroxyalkanoate (PHA); preparing a feed solution containing the culture medium (or a supernatant obtained by centrifuging the culture medium); disrupting cells or PHA contained in the feed solution; and analyzing morphological parameters of the cells or PHA before, during, or after the disruption step.

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

[0016] In another embodiment, analysis of morphological parameters of cells or PHA contained in the feed solution is performed before the disruption step, and the method includes a step of quantifying the analyzed morphological parameters to detect the feed condition; and a step of determining the disruption pressure of the high-pressure homogenizer or the number of disruption treatments depending on the feed condition as a second disruption condition pre-determination 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 a step of quantifying the analyzed morphological parameters to detect the degree of disruption; and a step of determining the disruption pressure of the high-pressure homogenizer or the number of disruption treatments depending on 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 a supernatant obtained by centrifuging the culture medium); a first analysis step of analyzing morphological parameters of cells or PHA contained in the feed solution; a second pre-determination step of determining the disruption conditions of the disruption pressure or the number of disruption treatments of the high-pressure homogenizer based on the results of the first analysis; disrupting the cells or PHA contained in the feed solution; a second analysis step of analyzing morphological parameters of the cells or PHA contained in the disruption product solution; and a first re-determination step of determining the disruption conditions of the disruption pressure or the number of disruption treatments of the high-pressure homogenizer based on the results of the second analysis.

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

[0021] In the formula, ABS0 is the absorbance at 280 nm measured after five repeated disruption of the culture medium, feed solution, or disruption product solution at room temperature and 800 bar; ABS1 is the absorbance at 280 nm measured after one disruption of the culture medium, feed solution, or disruption product solution at room temperature and 300-1,500 bar.

[0022] In another embodiment, if the average sphericity of the cells or PHA contained in the feed solution is 0.7 or more, or the protein solubilization degree according to Equation 1 is 80% or more, in the second pre-determination step of 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 feed 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 the protein solubilization degree according to Equation 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 feed solution.

[0024] In another embodiment, the method further includes a step of performing an agglomeration test to measure the degree of agglomeration of the disruption product solution after performing the second analysis step; and a step of determining the disruption pressure of the high-pressure homogenizer or the number of disruption treatments depending on the degree of agglomeration as a second disruption condition redetermining step.

[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 ascertained or the conditions for disruption are determined by analyzing 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] The monitoring method according to one embodiment of the present invention allows accurate and real-time determination of the state of cells or PHA even with a simple measurement method and a small amount of analytical sample, and allows visualization of cell rupture by performing 3D stereoscopic photography, which allows instant modification of rupture process conditions according to the state of cells or PHA.

[0027] Furthermore, since the method for producing polyhydroxyalkanoate (PHA) according to one embodiment of the present invention utilizes the above-mentioned monitoring method, the state of cells or PHA can be determined immediately, and the disruption process conditions can be changed immediately according to the state of cells or PHA. Specifically, the state of cells or PHA contained in the culture medium is measured to set the disruption conditions in advance, and the supply state or degree of disruption of cells or PHA is measured to change the disruption conditions in real time.

[0028] Furthermore, in the method for producing polyhydroxyalkanoate (PHA) according to one embodiment of the present invention, the optimal crushing pressure or number of times of crushing treatment is determined by the above-mentioned monitoring method without further manipulating the temperature to a high temperature, and therefore the crushing conditions can be efficiently changed in real time.

[0029] In one embodiment of the present invention, a method for producing polyhydroxyalkanoate (PHA) measures the state of cells or PHA before, during, or after disruption and changes the process conditions for disruption, separation, and impurity removal, thereby making it possible to prevent aggregation of PHA in the separation process and impurity removal process after the disruption process and ensure process stability. [Brief description of the drawings]

[0030] [Figure 1] 1 shows a simplified method for producing polyhydroxyalkanoate (PHA) according to one embodiment of the present invention. [Diagram 2] 1 shows an algorithm for a method for producing polyhydroxyalkanoate (PHA) according to one embodiment of the present invention. [Diagram 3] 1 shows an example of an algorithm for controlling the fracturing pressure in the fracturing condition pre-determination step. [Figure 4] 13 shows an example of an algorithm for controlling the fracturing pressure in the fracturing condition redetermining step. [Diagram 5] 13 shows another example of an algorithm for controlling the fracturing pressure in the fracturing condition redetermining step. [Figure 6] 1 is a measurement result of an HTA image according to an embodiment. [Figure 7] FIG. 1 shows an example of a method for producing the polyhydroxyalkanoate of the present invention, illustrating risk areas in the separation process and impurity removal process depending on cell length and sphericity when the culture medium of the example is used. BEST MODE FOR CARRYING OUT THEINVENTION

[0031] The present invention is described in detail below. The present invention is not limited to the disclosed contents shown below, and can be modified in various forms as long as the gist of the present invention is not changed.

[0032] Throughout this specification, when a part is referred to as "comprising" certain elements, it is understood that other elements may be comprised, rather than excluding other elements, unless otherwise specified.

[0033] All numbers and expressions used herein relating to quantities of ingredients, reaction conditions, and the like, are understood to be modified by the term "about" unless otherwise specified.

[0034] In this specification, when an element is referred to as being formed "on" or "under" another element, it means not only that the element is directly formed "on" or "under" the other element, but also that the element is indirectly formed on or under the other element with another element(s) between them.

[0035] Throughout this specification, terms such as first and second are used to describe various components. However, the components should not be limited by the terms. The terms are used only to distinguish one component from another.

[0036] Method for monitoring cell lysis and system for monitoring cell lysis In a method for monitoring cell lysis according to one embodiment of the present invention, the degree of cell lysis can be confirmed or the lysis 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 another embodiment of the system for monitoring cell lysis according to the present invention, the degree of cell lysis can be confirmed or the lysis 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 holotomographic analysis (HTA), and the morphological parameters are one or more types selected from the group consisting of length, sphericity, volume, and surface area.

[0039] Holotomography analysis is a laser technique that measures the three-dimensional refractive index of a micro-sample such as a cell, and is a type of three-dimensional microscope. Through the measured refractive index, quantitative image information on the morphological parameters of the cell can be provided. As a result, the length, sphericity, volume, surface area, density, mass, etc. of the cell can be calculated.

[0040] Furthermore, holotomography analysis can clearly image cell membranes or intracellular organelles without the use of exogenous labels, and is therefore free of phototoxicity, photobleaching, or photodamage issues. Holotomography analysis can achieve three-dimensional stereoscopic photographs, allowing visualization of the extent of cell disruption.

[0041] Furthermore, holotomography analysis provides a spatial resolution of about 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, allowing accurate measurements even with small sample volumes of 10 μl of dilution. Rapid measurements are possible, as the total measurement time is less than 10 minutes. Thus, cell status can be checked in real time using holotomography analysis. Therefore, the disruption process conditions can be instantly changed or preset.

[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, but is not limited to, 1.35 to 1.50 or 1.35 to 1.45.

[0043] Three-dimensional image analysis allows the calculation of the length, sphericity, volume, surface area, density, or mass of cells or PHA.

[0044] Method for producing polyhydroxyalkanoate (PHA) 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 a supernatant obtained by centrifuging the culture medium) (feed solution preparation step); disrupting cells or PHA contained in the feed solution (disruption step); and analyzing morphological parameters of the cells or PHA before, during, or after the disruption step (analysis step).

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

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

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

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

[0049] Furthermore, in the method for producing polyhydroxyalkanoate (PHA) of the present invention, analysis of the morphological parameters of cells or PHA contained in the disruption product solution is carried out 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 depending on the degree of disruption as a first disruption condition redetermining step.

[0050] Specifically, the method for producing polyhydroxyalkanoate (PHA) of the present invention includes the steps of preparing a culture medium containing polyhydroxyalkanoate (PHA) (culture medium preparation step); analyzing morphological parameters of cells or PHA contained in the culture medium (preliminary analysis step); determining initial setting conditions for the culture medium according to the results of the preliminary analysis (first pre-determining step of disruption conditions); preparing a feed solution containing the culture medium (or a supernatant obtained by centrifuging the culture medium) (feed solution preparation step); a first analysis step of analyzing morphological parameters of cells or PHA contained in the feed solution (first analysis step); determining the disruption pressure or disruption condition of the high-pressure homogenizer; The method may include a step of determining the number of times of the crushing process depending on the first analysis result (second crushing condition pre-determination step); a step of crushing the cells or PHA contained in the supply solution (crushing step); a second analysis step of analyzing the morphological parameters of the cells or PHA contained in the crushing product solution (second analysis step); a step of determining the crushing pressure of the high-pressure homogenizer or the number of times of the crushing process depending on the second analysis result (first crushing condition redetermining step); a step of conducting an agglutination test to measure the degree of agglutination in the crushing product solution (aggregation test step); and / or a step of determining the crushing pressure of the high-pressure homogenizer or the number of times of the crushing process depending on the degree of agglutination (second crushing condition redetermining 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 redetermination step, the aggregation test step, and the second crushing condition redetermination step are each independent and can be selectively performed.

[0052] A method for producing a polyhydroxyalkanoate (PHA) according to one embodiment of the present invention can be carried out according to the following exemplary method. (1) A culture medium preparation step; a preliminary analysis step; and a disruption step. (2) a culture medium preparation step; a disruption step; and a second analysis step. (3) a culture medium preparation step; a first analysis step; a disruption step; and a second analysis step. (4) a culture medium preparation step, a first analysis step, a disruption step, a second analysis step, and a first disruption condition redetermining step. (5) a culture medium preparation step; a feed solution preparation step; a first analysis step; and a disruption step. (6) a culture medium preparation step; a preliminary analysis step; a feed solution preparation step; a first analysis step; and a disruption step. (7) a culture medium preparation step; a feed solution preparation step; a first analysis step; a second predetermination step of disruption conditions; and a disruption step. (8) a culture medium preparation step; a first analysis step; a second predetermination step of disruption conditions; a disruption step; a second analysis step; and a first re-determination step of disruption conditions. (9) a culture medium preparation step; a feed solution preparation step; a first analysis step; a second predetermination step of disruption conditions; a disruption step; a second analysis step; and a first re-determination step of disruption conditions. (10) a culture medium preparation step; a preliminary analysis step; a feed solution preparation step; a first analysis step; a second predetermination step of disruption conditions; a disruption step; a second analysis step; and a first re-determination step of disruption conditions.

[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 pre-determining step of the crushing conditions, the first or second re-determining step of the crushing conditions, and the crushing step may be repeatedly carried out.

[0054] Each step will now be described in detail.

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

[0056] Generally, polyhydroxyalkanoates can be produced by a microbial fermentation process. A crude product of polyhydroxyalkanoates can also be obtained by fermentation.

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

[0058] The culture medium is cultured with various microorganisms capable of producing polyhydroxyalkanoates (PHAs). The culture medium can be prepared by a conventional method in which the microorganism is cultured in a normal medium appropriately containing a carbon source, a nitrogen source, a phosphorus source, an inorganic compound, an amino acid, and / or a vitamin using a bioreactor at 15 to 50°C or 30 to 40°C for 1 to 160 hours. In order 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 the culture of the microorganism, but are not limited thereto.

[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. In addition, natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steep liquor may be used. Other carbon sources in appropriate amounts may be used in various ways without limitation. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

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

[0061] Examples of repeating units constituting 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). PHA may contain one or more repeating units selected from the above. For example, the PHA may be poly-3-hydroxybutyrate-co-4-hydroxybutyrate (hereinafter referred to as 3HB-co-4HB).

[0062] The polyhydroxyalkanoates (PHAs) may be homopolymers or copolymers. According to one embodiment, the polyhydroxyalkanoates may include copolymers, specifically copolymers that include two or more different repeat units with the different repeat units randomly distributed in the polymer chain.

[0063] Further, the polyhydroxyalkanoate may include isomers. For example, the polyhydroxyalkanoate may include structural isomers, enantiomers, or geometric isomers. Specifically, the polyhydroxyalkanoate may include structural isomers.

[0064] As an example, a polyhydroxyalkanoate (PHA) may contain 4-hydroxybutyrate (4-HB) repeat units. For example, the content of 4-HB repeat units in the polyhydroxyalkanoate can be 0.1% or more, 1% or more, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, or 60% or more by weight, based on the total weight of the polyhydroxyalkanoate; 100% or less, 99% or less, 90% or less, 80% or less, 70% or less, or 60% or less by weight; 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 content of 4-HB repeating units satisfies the above range, the PHA finally prepared has excellent biodegradability in soil and sea, and can further improve thermal properties, productivity, processability, etc., without reducing mechanical properties.

[0065] Furthermore, the polyhydroxyalkanoate (PHA) may have a weight average molecular weight of 10,000 to 1,200,000 g / mol. For example, the weight average molecular weight of the 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 normal cells, or may be autolytic cells in which the cell wall is weakened or the cells are ruptured due to overculture or autolysis. In the method according to one embodiment of the present invention, even if the cells are normal or autolytic, the disruption step can be effectively performed, and the productivity of the final PHA can be improved.

[0067] Feed solution preparation steps A method for producing polyhydroxyalkanoates (PHAs) according to one embodiment of the present invention includes preparing a feed solution comprising culture medium (or the supernatant obtained by centrifugation of the culture medium).

[0068] The feeding solution is a dilution of cells and PHA in distilled water and may further contain a pH adjuster and / or a surfactant.

[0069] When the supply solution contains a supernatant obtained by centrifuging a culture medium, the centrifugation can be carried out at a speed of 3,000 to 8,000 rpm, 4,000 to 6,000 rpm, or 4,000 to 5,000 rpm for 10 to 50 minutes, 20 to 40 minutes, or 25 to 35 minutes, but is not limited thereto.

[0070] The cells and PHA of the feed solution can be mixed with distilled water to a total solid content of 5-20% by weight, preferably 10-15% by weight. When the total solid content is within the above range, the disruption step can be carried out efficiently.

[0071] The feed solution may include a pH adjuster, which may be one or more basic solutions selected from the group consisting of sodium hydroxide, potassium hydroxide, lithium hydroxide, and sodium carbonate. Further, 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 feed 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 feed solution is adjusted to the above range, the cell disruption step and subsequent steps can be carried out more easily.

[0073] The feed solution may include a surfactant, which 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 employed in an amount of 0.05-0.5 wt%, 0.1-0.2 wt%, preferably 0.13-0.15 wt%, based on the total weight of the feed 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 polyhydroxyalkanoates (PHA) according to one embodiment of the present invention includes disrupting cells or PHA contained in a feed 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 the cells. The disruption pressure can be 300 to 1,500 bar, 300 to 600 bar, 300 to 500 bar, 400 to 1,000 bar, 400 to 700 bar, 400 to 600 bar, 400 to 500 bar, 600 to 1,000 bar, or 600 to 800 bar.

[0077] When the disruption pressure is within the above range, the target degree of cell disruption can be achieved and an appropriate viscosity range can be maintained, making it easier to carry out subsequent steps and increasing PHA productivity.

[0078] When the lower limit of the crushing pressure is reached, a crude polyhydroxyalkanoate solution is obtained immediately after the crushing step without carrying out the steps of analyzing the crushed product solution and testing for flocculation.

[0079] When the upper limit of the crushing pressure is reached, a crude polyhydroxyalkanoate solution is obtained after carrying out an analysis step of the crushing product solution without carrying out the flocculation test step.

[0080] Furthermore, the crushing temperature may be 0 to 40° C., 5 to 35° C., 10 to 30° C., 20 to 40° C., or 23 to 35° C. In order to prevent a rise in temperature due to high-pressure crushing, the temperature is kept constant by flowing cooling water.

[0081] The milling time may vary depending on the flow rate entering and exiting the high pressure homogenizer or the throughput per hour.

[0082] The feed solution that has passed through the high-pressure homogenizer is subjected to a single disruption step. When the disruption step is performed by changing the disruption conditions once or multiple times through the disruption condition pre-determination step and / or the disruption condition redetermination step, the determined disruption conditions (pressure, etc.) may be the same or different.

[0083] The disruption conditions may be determined according to the analysis results of the morphological parameters. Specifically, the morphological parameters of the cells or PHAs may be analyzed before, during, or after the disruption step, and the number and conditions of disruption may be determined according to the analysis results. Details of this will be described later in the pre-determination step of disruption conditions and the re-determination step of disruption conditions.

[0084] A step of centrifuging the cell disruption product solution 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 lysate product solution to obtain a crude polyhydroxyalkanoate solution.

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

[0086] Centrifugation can be carried out at a speed of 10,000 to 20,000 rpm, 12,000 to 18,000 rpm, or 15,000 to 16,000 rpm for 10 to 30 minutes or 10 to 20 minutes, and at a temperature of 0 to 40°C, 5 to 35°C, 10 to 30°C, 20 to 40°C, or 23 to 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 additional step of removing impurities by chemical or enzymatic treatment, and is formulated.

[0088] Analyzing the morphological parameters of the cells or PHA (analysis step) A method for producing polyhydroxyalkanoates (PHA) according to one embodiment of the present invention includes analyzing morphological parameters of cells or PHA.

[0089] The step of analyzing the morphological parameters may be, but is not limited to, three-dimensional image analysis using holotomography analysis.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 the holotomography analysis (HTA) are described above. In one embodiment of the invention, the step of analyzing morphological parameters of cells or PHAs may comprise 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 step may include a preliminary analysis step of taking multiple analytical samples from the culture medium, the feed solution, and / or the disruption product solution and analyzing morphological parameters of the cells or PHA contained in the culture medium depending on the analytical sample collection points; a first analysis step of analyzing morphological parameters of the cells or PHA contained in the feed solution; and / or a second analysis step of analyzing morphological parameters of the cells or PHA contained in the disruption product solution.

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

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

[0094] The average sphericity of the cells contained in the culture medium may be 0.4 or more, 0.5 or more, 0.6 or more, or 0.7 or more. Furthermore, the average length of the cells may be 3.0 μm or more, 3.5 μm or more, 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 can be 0.5 or more, 0.6 or more, 0.7 or more, or 0.8 or more.

[0096] A higher degree of autolysis results in a shorter average length of the cells in the culture medium and a higher average sphericity of the cells or PHA.

[0097] 2) Analysis of Cells or PHA in the Feed Solution (First Analysis Step) In one embodiment of the present invention, the step of analyzing the morphological parameters of the cells or PHA can include a first analysis step carried out before the disruption step, which is a step of analyzing the morphological parameters of the cells or PHA contained in the feeding solution and quantifying the analyzed morphological parameters to detect the feeding state.

[0098] The average sphericity of the cells in the feed solution can be 0.6 or more, 0.65 or more, 0.7 or more, 0.75 or more, 0.8 or more, 0.85 or more, or 0.9 or more, and the average length of the cells can be 3.28 μm or more, 3.4 μm or more, 3.6 μm or more, 3.8 μm or more, 4.0 μm or more, or 4.2 μm or more, or 6.0 μm or less, 5.5 μm or less, 5.0 μm or less, or 4.8 μm or less.

[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 contained in the supply solution may contain 40-90%, 40-80%, 45-70%, 50-70%, 55-65%, or 60-65% of cells having a sphericity of 0-0.8, or 10-60%, 20-55%, 30-55%, 30-50%, or 35-45% of cells having a sphericity of 0.8-1.0.

[0101] Additionally, the average sphericity of the PHA in the feed solution may be 0.6 or greater, 0.65 or greater, 0.7 or greater, 0.75 or greater, 0.8 or greater, 0.85 or greater, or 0.9 or greater.

[0102] Furthermore, the PHA may contain 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 of PHA having a sphericity of 0 to 0.8, or 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 of PHA having a sphericity of 0.8 to 1.0.

[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 the cells or PHA may comprise a second analysis step carried out during or after the disruption step, which is a step of analyzing the morphological parameters of the cells or PHA contained in the disruption product solution and quantifying the analyzed morphological parameters to detect the degree of disruption.

[0104] The final cells and PHA contained in the disruption product solution thus prepared 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 disruption product solution may be 0.65 or more, 0.7 or more, 0.8 or more, or 0.9 or more, and the average length of the cells may be 3.0 μm or more, or 3.5 μm or more, or 4.5 μm or less, or 4.0 μm or less.

[0106] Furthermore, the average sphericity of the PHA contained in the disruption product solution may be 0.6 or more, 0.65 or more, 0.7 or more, 0.75 or more, 0.8 or more, 0.85 or more, or 0.9 or more. 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, or 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] Three-dimensional images taken before and after cell disruption are shown in Figure 6. The bright areas (green) indicate the cell wall or cytoplasm, and the dark areas (red) indicate PHA. From the three-dimensional images, it can be visually confirmed that the length of the cells or PHA is shortened and the sphericity of the cells or PHA is increased upon disruption.

[0108] Analyzing the degree of protein solubilization or aggregation (auxiliary analytical step) 1) Analysis of protein solubility Protein solubility analysis or aggregation test may be performed as an auxiliary indicator to determine whether the cells and PHA contained in the disruption product solution are sufficiently disrupted and can proceed to subsequent steps and to ensure process stability.

[0109] Specifically, the degree of protein solubilization is an auxiliary indicator for determining cell rupture and cell destruction, and can be calculated according to the following formula 1 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 280 nm measured after the culture medium, the feed solution, or the disruption product solution is repeatedly disrupted five times at room temperature at 800 bar; ABS1 is the absorbance at 280 nm measured after the culture medium, the feed solution, or the disruption product solution is disrupted once at room temperature at 300 to 1,500 bar. More specifically, ABS0 and ABS1 are the absorbances measured after the culture medium, the feed solution, or the disruption product solution is disrupted under the above conditions, centrifuged at 15,000 rpm for 5 minutes, and the supernatant is diluted 100 times with distilled water.

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

[0113] The protein solubility of the disruption product solution according to one embodiment of the present invention according to the above formula 1 is 75 to 110%. Specifically, the protein solubility of the disruption product solution may be 85% or more, 90% or more, or 95% or more. If the protein solubility is less than 85%, the cell disruption efficiency decreases, and therefore the efficiency of obtaining PHA granules in the PHA production method may decrease.

[0114] 2) Agglutination test The flocculation test can be used as an auxiliary index 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 determine whether cells or PHA are flocculated and the degree of flocculation, and from this, the stability of the subsequent washing step and impurity removal step can be confirmed.

[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 with washing water. In such an event, the degree of agglomeration can be confirmed from the residue that is not dissolved in the washing water and does not pass through the sieve. If the degree of agglomeration is high, it is highly likely that agglomeration will occur in the subsequent washing and impurity removal steps. Therefore, the production of polyhydroxyalkanoate (PHA) is carried out by adjusting the disruption pressure or changing the process conditions of the washing and impurity removal steps.

[0116] Predetermining the crushing conditions step In one embodiment of the present invention, analysis of morphological parameters of cells or PHA contained in the culture medium is carried out before the disruption step, and the method includes a step of quantifying the analyzed morphological parameters to detect the degree of autolysis (pre-analysis step); and a step of determining the initial pressure of the high-pressure homogenizer or the number of disruption treatments depending on the degree of autolysis as a first disruption condition pre-determination step.

[0117] In another embodiment of the present invention, the analysis of the morphological parameters of the cells or PHA contained in the feed solution is carried out before the disruption step, and the method includes a step of quantifying the analyzed morphological parameters to detect the feed state (first analysis step); and a step of determining the disruption pressure of the high-pressure homogenizer or the number of disruption treatments for the feed solution according to the feed state as a second disruption condition predetermination step. Each disruption condition predetermination step is an individual step and is selected individually as necessary. When the first predetermination step and the second predetermination step are adopted simultaneously in one process, the second predetermination step is carried out after the first predetermination step.

[0118] 1) First, the predetermination of the crushing conditions according to the analysis of the morphological parameters As an example, when the average 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 more, the initial pressure may be determined to be 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 autolysis. Therefore, in the steps following the disruption step, morphological parameters can be analyzed and the disruption conditions adjusted to prevent clogging of pipes, etc.

[0120] 2) A second step of predetermining the fracture conditions according to the analysis of morphological parameters. As an example, when the average sphericity of the cells or PHA contained in the supply solution is 0.7 or more, the second pre-determining step of 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.

[0121] As another example, if the average sphericity of the cells or PHA contained in the supply solution is less than 0.7, the second pre-determination step of 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 setting pressure.

[0122] 3) A second step of predetermining the disruption conditions according to the analysis of protein solubilization degree As an example, when the protein solubilization degree of the supply solution according to the above formula 1 is 80% or more, the second pre-determining step of 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, when the protein solubilization degree of the supply solution according to the above formula 1 is less than 80%, the second pre-determining disruption conditions step 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 setting pressure.

[0124] A method for controlling the fracturing pressure in the second fracturing condition pre-determination step is specifically shown in FIG.

[0125] FIG. 3 relates to a step of determining the disruption pressure (P1) in the step of disrupting cells or PHA contained in a supply solution relative to the initial set pressure (P0) of a high-pressure homogenizer (second disruption condition pre-determination step).

[0126] When the average sphericity of the cells or PHA contained in the supply solution is less than 0.7 or the protein solubilization degree is less than 80%, the disruption pressure (P1) in the step of disrupting the cells or PHA contained in the supply solution is maintained at the same as the initial setting pressure (P0).

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

[0128] Crushing condition redetermining step In another embodiment of the present invention, analysis of morphological parameters of cells or PHA contained in the disruption product solution is carried out 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; and a step of determining the disruption pressure of the high-pressure homogenizer or the number of disruption treatments depending on the degree of disruption as a first disruption condition redetermining step.

[0129] In another embodiment of the present invention, the step of analyzing the morphological parameters of the cells or PHA contained in the disruption product solution is carried out during or after the disruption step, and the method may further include a step of performing an aggregation test to measure the degree of aggregation of the disruption product solution after the 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 times of the disruption treatment according to the degree of aggregation. Each disruption condition redetermining step is an individual step and is selected individually as necessary. When the first redetermining step and the second redetermining step are adopted simultaneously in one process, the second redetermining step is carried out after the first redetermining step.

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

[0131] As an 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 increase 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 disruption condition redetermining step by analyzing protein solubility As an example, when the protein solubilization degree of the disruption product solution according to the above formula 1 is less than 90%, the first disruption condition re-determination step may increase 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 the protein solubilization degree thereof. The disruption conditions may be adjusted based on the measurement results; if the degree of disruption is judged to be insufficient, the disruption pressure may be increased; or if the degree of disruption is judged to be excessive, the disruption pressure may be decreased.

[0134] 3) The second step of re-determining the crushing conditions according to the degree of aggregation In another embodiment of the present invention, the step of analyzing the morphological parameters of the 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 performing an aggregation test to measure the degree of aggregation 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 the continuously supplied feed solution depending on the degree of aggregation.

[0135] For example, when the average sphericity of PHA contained in the disruption product solution is 0.9 or more, or when the protein solubilization degree is 90% or more, an aggregation test is performed. If the degree of aggregation is measured to be equal to or greater than a predetermined value, the second disruption condition re-determination step may reduce the pressure by 10 to 100 bar from the disruption pressure in the step of disrupting 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 disruption condition re-determination step may maintain the disruption pressure in the step of disrupting the cells or PHA contained in the supply solution, or may increase the pressure by 10 to 100 bar.

[0137] Excessive physical disruption in the disruption step can lead to high levels of cell disruption, which can lead to PHA aggregation. Therefore, by determining the disruption conditions in real time using morphological parameter analysis, clogging of piping in subsequent steps can be prevented.

[0138] A method for controlling the fracturing pressure in the fracturing condition redetermining step is specifically shown in FIG. 4 and FIG.

[0139] FIG. 4 relates to a step of re-determining the disruption pressure (P2) of a high-pressure homogenizer for a continuously supplied feed solution relative to the disruption pressure (P1) in the step of disrupting cells or PHA contained in the feed solution.

[0140] A step (P1) of disrupting the cells or PHA contained in the feed solution; and an analysis step of analyzing the morphological parameters of the cells or PHA contained in the disruption product solution are carried out.

[0141] When the average sphericity of the cells or PHA contained in the disruption product solution is less than 0.9, or when the protein solubilization degree is less than 90%, the pressure increased by 50 bar from the disruption pressure (P1) in the step of disrupting the cells or PHA contained in the feed solution is determined as the disruption pressure (P2) of the high-pressure homogenizer for the continuously supplied feed solution.

[0142] When the average sphericity of the cells or PHA contained in the disruption product solution is 0.9 or more, or when the protein solubilization degree is 90% or more, an aggregation test is carried out to measure the degree of aggregation of the disruption product solution.

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

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

[0145] 5 shows a step of re-determining the disruption conditions after adjusting the pressure by an aggregation test in another embodiment of the present invention. This relates to a step of preparing a disruption product solution by increasing or decreasing the disruption pressure (Pn) in the step of disrupting cells or PHA contained in the supply solution by 10 to 100 bar from the preset disruption pressure (Pn-1), and then re-determining the disruption pressure (Pn+1) of the high-pressure homogenizer for the continuously supplied supply solution.

[0146] Specifically, a step of disrupting cells or PHA contained in a feed solution at a disruption pressure (Pn) reduced by 50 bar from a preset disruption pressure (Pn-1) is carried out; and an analysis step of analyzing the morphological parameters of cells or PHA contained in the disruption product solution is carried out.

[0147] Next, if the average sphericity of the cells or PHA contained in the disruption product solution is less than 0.9 or the protein solubilization degree is less than 90%, the first disruption condition re-determination step determines a pressure determined to be between the preset 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] When the average sphericity of the cells or PHA contained in the disruption product solution is 0.9 or more, or the protein solubilization degree is 90% or more, an aggregation test is performed to measure the degree of aggregation of the disruption 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 disruption pressure (Pn+1) of the high-pressure homogenizer for the continuously supplied supply solution to be the same as the disruption pressure (Pn) in the step of disrupting the cells or PHA contained in the supply solution.

[0150] If the degree of aggregation is measured to exceed a predetermined value (N), the second disruption condition re-determination step determines a pressure 50 bar lower than 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.

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

[0152] The present invention will now be described in more detail with reference to the following examples. However, the following examples are for the purpose of illustrating the present invention, and the scope of the examples is not limited thereto. EXAMPLES

[0153] 1. Preparation of Culture Medium A PHA culture medium was prepared using a genetically engineered E. coli strain. First, a certain amount of the PHA-producing strain was seed cultured, and then the main fermentation was carried out. Sucrose was used as the carbon source for the fermentation of the PHA-producing strain, 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 strain was carried out at 30-40°C and pH 6-9.

[0154] In Examples 1 and 2, genetically engineered E. coli strains and separate culture media using sucrose as the carbon source were used. Although the culture media in Examples 1 and 2 were prepared under the same culture conditions, the morphological parameters of the cells or PHA measured in Examples 1 and 2 may differ due to the viable cell nature of the strains.

[0155] 2. Simulation of autolysis of culture medium The degree of autolysis refers to the degree to which the cell wall weakens or the cells burst due to autolysis. To experimentally simulate this, an ultrasonic disperser (SONICS, Vibra cell) was used to simulate autolysis conditions. A normally fermented culture was treated with an ultrasonic disperser at 40% amplitude and 10 / 10 pulse for 1 or 2 minutes to rupture the cells and simulate the degree of autolysis, and then the cell disruption process was carried out.

[0156] In Examples 1-1 to 1-3, culture media that had been ultrasonically treated according to Table 1 below were used. Specifically, Example 1-1 is the culture medium of Example 1 that was not treated with an ultrasonic disperser, and Examples 1-2 and 1-3 are the culture media of Example 1 that were treated with an ultrasonic disperser for 1 minute or 2 minutes, respectively. Examples 2-1 to 2-3 are culture media that had been ultrasonically treated according to Table 2 below.

[0157] 3. Cell disruption process Each culture medium was subjected to separation of impurities from the biomass containing cells and / or PHA contained in the culture medium using a centrifuge (Hanil, Combi-514 R). The separated biomass was diluted with distilled water to a total solid content of 10-15 wt%. The diluted solution was adjusted to pH 10-11 using sodium hydroxide (40 w / w%, Deoksan Science), and then 0.13-0.15 wt% of anionic surfactant sodium laureth sulfate (30 w / w%, Miwon) was added to the liquid phase to prepare a feed solution. This feed solution for disruption 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 times.

[0158] [Test example] 1. Preparation of Samples for 3D 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 with distilled water (DIW) in 1.5 ml microtubes. 20 μl of the diluted samples were dispensed into TomoDish (Tomo Cube Co., Ltd.) and covered with a cover slide. Three-dimensional images of the prepared samples were taken using holotomography analysis (Tomo Cube Co., Ltd., HT-1H), and the length and sphericity of the cells were measured.

[0159] The refractive index (RI) used for performing the three-dimensional images and measuring the data was 1.3513-1.3745 (cells) and 1.3748-1.4081 (PHA). For each sample, 10 three-dimensional images were taken. The three-dimensional images were created using TomoStudio™ (Tomo Cube Co., Ltd.) and the sphericity of the images was analyzed. The average mass, volume, length, and sphericity of the cells and PHA were calculated.

[0160] 2. Measurement of protein content before and after cell lysis The analytical samples (feed solution or disruption product solution) of the examples and comparative examples were disrupted under conditions of 300 to 1,500 bar and room temperature, and then centrifuged at 15,000 rpm for 5 minutes using a microcentrifuge (Eppendorf, 5414R). The centrifuged supernatant was diluted 100 times 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). When measuring the absorbance, calibration was performed with distilled water.

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

[0162] In the formula, ABS0 is the absorbance at 280 nm measured after repeatedly crushing the analytical sample five times at room temperature and 800 bar; ABS1 is the absorbance at 280 nm measured after crushing the analytical sample once at room temperature and 300-1,500 bar.

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

[0164] 1) Results and analysis of autolysis simulation The analysis results of the length and sphericity of the cells 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 performed twice using different culture media. As a result, the average length of the cells in the culture media became shorter and the average sphericity of the cells became higher as the ultrasonic dispersion treatment time became longer.

[0165] [Table 1]

[0166] [Table 2]

[0167] 2) Analysis of the degree of cell disruption The feeding solutions were prepared using the culture media of Examples 1 and 2, respectively. Morphological parameters, protein solubilization degree, and degree of aggregation were measured for cells and PHA in the feed solution at the feed point of the high-pressure homogenizer; and for cells and PHA in the cell disruption product solution after one 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] As a result of the analysis, it was found that the tendency of the degree of aggregation varied depending on the degree of autolysis of the culture medium and the crushing pressure in the crushing step.

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

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

[0173] When the average cell length of the feed solution or the disruption product solution is 3.92 μm or less, and when 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 the process stability in the downstream process and is likely to cause problems such as pipe clogging.

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

[0175] In Examples 1 and 2, the degree of protein solubilization was calculated based on ABS0 of Example 1-1 or Example 2-1, which is a culture medium not subjected to ultrasonic treatment. When cells were disrupted 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 showed a high degree of autolysis. This is because a relatively large amount of protein is separated from the culture medium subjected to ultrasonic treatment in the process of preparing a feed solution containing the supernatant obtained by centrifuging the culture medium; this is understood to be because the residual protein content was lower in the culture medium not subjected to ultrasonic treatment.

[0176] In Examples 2-1 to 2-3, the degree of protein solubilization exceeding 100% is an error due to the absorbance measurement method, and it should be understood that this means that the degree of protein solubilization is 90% or more.

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

[0178] However, since Fig. 7 is an example prepared based on the culture medium of Examples 1 and 2 during fermentation, and the culture medium tends to aggregate relatively well; the ranges of the caution zone and safety zone of length and sphericity appear relatively narrow in Fig. 7. The value of each zone may vary when the type of strain or carbon source in the culture medium is changed.

Claims

1. A method for monitoring cell lysis, in which the degree of cell lysis is confirmed or the conditions for lysis are determined by analyzing morphological parameters of cells or PHA contained in a culture medium containing polyhydroxyalkanoate (PHA) by three-dimensional image analysis.

2. 2. The method of monitoring cell disruption as described in claim 1, wherein the three-dimensional image analysis is a three-dimensional image analysis using holotomography analysis, and the morphological parameters are one or more selected from the group consisting of length, sphericity, volume, and surface area.

3. A method for producing polyhydroxyalkanoate (PHA), comprising the steps of: Preparing a culture medium containing polyhydroxyalkanoate (PHA); preparing a feed solution comprising said culture medium; Disrupting the cells or PHA contained in the feeding solution; and The method comprises the step of analyzing morphological parameters of the cells or the PHA before, during or after the disruption step.

4. A step of analyzing morphological parameters of the cells or the PHA contained in the culture medium is carried out before the disruption step; The method for producing polyhydroxyalkanoate (PHA) according to claim 3, further comprising 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 times of the crushing treatment as a first crushing condition pre-determination step according to the degree of autolysis.

5. An analysis of morphological parameters of the cells or the PHA contained in the feed solution is carried out prior to the disruption step; The method for producing polyhydroxyalkanoate (PHA) according to claim 3, further comprising the steps of: quantifying the analyzed morphological parameters to detect the feeding condition; and determining the crushing pressure of the high-pressure homogenizer or the number of times of the crushing process according to the feeding condition as a second crushing condition pre-determining step.

6. An analysis of morphological parameters of the cells or the PHA contained in the disruption product solution is carried out during or after the disruption step; The method for producing polyhydroxyalkanoate (PHA) according to claim 3, comprising the steps of: quantifying the analyzed morphological parameters to detect the degree of crushing; and determining the crushing pressure of the high-pressure homogenizer or the number of times of crushing treatment according to the degree of crushing as a first crushing condition re-determination step.

7. The method for producing polyhydroxyalkanoates (PHA) according to claim 3, wherein the crushing pressure in the crushing step is 300 to 1,500 bar.

8. a first analysis step of analyzing morphological parameters of the cells or the PHA contained in the feed solution; a second pre-determining step of determining the crushing pressure of the high-pressure homogenizer or the number of times of the crushing treatment in accordance with the first analysis result; disrupting the cells or the PHA contained in the feed solution; A second analysis step of analyzing morphological parameters of the cells or the PHA contained in the disruption product solution; and The method for producing polyhydroxyalkanoate (PHA) according to claim 3, further comprising a first crushing condition redetermining step of determining the crushing pressure of the high-pressure homogenizer or the number of times of crushing treatment according to the second analysis result.

9. The following formula 1: [Formula 1] Protein solubilization degree (%) = ABS1 / ABS0 x 100 (%) (wherein ABS0 is the absorbance at 280 nm measured after five repeated disruption cycles of the culture medium, the feed solution or the disruption product solution at 800 bar and room temperature; ABS1 is the absorbance at 280 nm measured after one disruption cycle of the culture medium, the feed solution or the disruption product solution at 300-1,500 bar and room temperature). The method for producing a polyhydroxyalkanoate (PHA) according to any one of claims 3 to 8, wherein the degree of protein solubilization by the method is 75 to 110%.

10. When the average sphericity of the cells or the PHA contained in the supply solution is 0.7 or more, or when the average sphericity of the cells or the PHA contained in the supply solution is 0.7 or more, the average sphericity satisfies the following formula 1: [Formula 1] Protein solubilization degree (%) = ABS1 / ABS0 x 100 (%) (wherein ABS0 is the absorbance at 280 nm measured after five repeated disruption cycles of the culture medium, the feed solution or the disruption product solution at 800 bar and room temperature; ABS1 is the absorbance at 280 nm measured after one disruption cycle of the culture medium, the feed solution or the disruption product solution at 300-1,500 bar and room temperature).

6. The method for producing polyhydroxyalkanoate (PHA) according to claim 5, wherein in the second predetermining step, 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 when the protein solubilization degree by the predetermining step is 80% or more.

11. When the average sphericity of the cells or the PHA contained in the disruption product solution is less than 0.9, or when the average sphericity of the cells or the PHA contained in the disruption product solution is less than 0.9, the average sphericity of the cells or the PHA is less than 0.9, or when the average sphericity of the cells or the PHA contained in the disruption product solution is less than 0.9, the average sphericity of the cells or the P [Formula 1] Protein solubilization degree (%) = ABS1 / ABS0 x 100 (%) (wherein ABS0 is the absorbance at 280 nm measured after five repeated disruption cycles of the culture medium, the feed solution or the disruption product solution at 800 bar and room temperature; ABS1 is the absorbance at 280 nm measured after one disruption cycle of the culture medium, the feed solution or the disruption product solution at 300-1,500 bar and room temperature).

7. The method for producing polyhydroxyalkanoate (PHA) according to claim 6, wherein in the first re-determining step, the pressure is increased by 10 to 100 bar from the disruption pressure in the step of disrupting the cells or the PHA contained in the supply solution if the protein solubilization degree by the re-determining step is less than 90%.

12. The method for producing polyhydroxyalkanoate (PHA) according to claim 8, further comprising: a step of performing an agglomeration test to measure the degree of agglomeration of the disruption product solution after performing the second analysis step; and a step of determining the disruption pressure of the high-pressure homogenizer or the number of times of disruption treatment according to the degree of agglomeration as a second disruption condition re-determination step.

13. A system for monitoring cell lysis, in which the degree of cell lysis is confirmed or the conditions for lysis are determined by analyzing morphological parameters of cells or PHA contained in a culture medium containing polyhydroxyalkanoate (PHA) by three-dimensional image analysis.

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