Method for formulating analysis sample of component derived from biopolymer-producing microorganism, analysis method, and method for identifying biopolymer-producing microorganism

Abstract

A method for formulating an analysis sample of a component derived from a biopolymer-producing microorganism according to the present invention comprises: a step for preparing a sample solution containing constituent components of the microorganism and the biopolymer; a step for centrifuging the sample solution; a step for performing ultrafiltration of a supernatant obtained through the centrifugation; and a step for formulating an analysis sample using a filtrate or a residue obtained through the ultrafiltration.

Description

Method for preparing a sample for analysis of components derived from microorganisms that produce biopolymers, method for analysis, and method for identifying microorganisms that produce biopolymers

[0001] The present invention relates to a method for preparing a sample for analysis of components derived from microorganisms that produce biopolymers, an analytical method, and a method for identifying microorganisms that produce biopolymers.

[0002] Some microorganisms produce polymeric compounds such as polysaccharides, polyamino acids, and polyesters. These polymeric compounds derived from microorganisms are called biopolymers. Some biopolymers have surface-active properties, and are also called biosurfactants. Biosurfactants are highly biodegradable and have low toxicity to living organisms, so they are expected to be used to purify petroleum-contaminated soil and water.

[0003] One method for analyzing biosurfactants is to use matrix-assisted laser desorption / ionization (MALDI) mass spectrometry. For example, Non-Patent Document 1 describes a method for analyzing lipopeptide biosurfactants produced by the Gram-positive bacterium Bacillus subtilis using a MALDI mass spectrometer. In this method, Bacillus subtilis is cultured in a medium that promotes biosurfactant production, and the medium containing the cultured cells is then mixed with an organic solvent consisting of 70% acetonitrile and 0.1% trifluoroacetic acid to extract the biosurfactant. Because the cell walls of Gram-positive bacteria are not easily destroyed by treatment with an organic solvent, the method described in Non-Patent Document 1 allows the biosurfactant produced extracellularly by Bacillus subtilis to be extracted and subjected to MALDI mass spectrometry.

[0004] In contrast, when Gram-negative bacteria are treated with an organic solvent, the cell walls are destroyed, and therefore, in the method of Non-Patent Document 1, microbial components present within the bacteria, such as proteins and lipids, are extracted with the organic solvent along with biopolymers produced extracellularly by the Gram-negative bacteria.

[0005] On the other hand, biopolymers can be produced not only extracellularly but also intracellularly by microorganisms. To analyze biopolymers produced intracellularly, the cell wall must be disrupted and then extracted with an organic solvent. However, this method extracts not only intracellular biosurfactants, but also extracellularly produced biosurfactants and intracellular microbial components with the organic solvent.

[0006] Joachim Vater, and 4 others, "Whole Cell" - Matrix-Assisted Laser Desorption Ionization-Time of Flight- Mass Spectrometry, an Emerging Technique for Efficient Screening of Biocombinatorial Libraries of Natural Compounds - Present State of Research", Combinatorial Chemistry & High throughput Screening, 2003, 6, pp. 557-567

[0007] When extracts are subjected to MALDI mass spectrometry directly, the peaks derived from the intracellular and extracellular biopolymers contained in the extract, or the peaks derived from the intracellular and extracellular biopolymers and the peaks derived from the microbial components, may have similar mass-to-charge ratios (m / z). Furthermore, the intensities of the peaks derived from the microbial components may be significantly greater than those of the biopolymer peaks. In such cases, it becomes difficult to observe the peaks derived from the intracellular and extracellular biopolymers. Therefore, it is necessary to separate the intracellular and extracellular biopolymers in the extract from other components before subjecting the extract to MALDI mass spectrometry.

[0008] Chromatography is a common separation method, but it requires appropriate setting of separation conditions, such as the type of column, mobile phase, and eluent, as well as the elution rate, which can be time-consuming and require a long time for separation.

[0009] The problem to be solved by the present invention is to separate biopolymers and the components of the microorganisms that produce the biopolymers without requiring much time and effort.

[0010] The present invention has been made to solve the above-mentioned problems by providing a method for preparing an analytical sample of a component derived from a microorganism that produces a biopolymer, the method comprising the steps of: preparing a sample solution containing a biopolymer and components of the microorganism that produces the biopolymer; centrifuging the sample solution; ultrafiltering the supernatant obtained by the centrifugation; and preparing an analytical sample using the filtrate obtained by the ultrafiltration.

[0011] Another aspect of the method for preparing an analytical sample of a component derived from a microorganism that produces a biopolymer according to the present invention, which has been made to solve the above-mentioned problems, comprises the steps of: preparing a sample solution containing a biopolymer and components of the microorganism that produces the biopolymer; centrifuging the sample solution; ultrafiltering the supernatant obtained by the centrifugation; and preparing an analytical sample using the residue obtained by the ultrafiltration.

[0012] According to the method of the present invention for preparing a sample for analysis of components derived from a microorganism that produces a biopolymer, the biopolymer and the components of the microorganism that produces the biopolymer can be separated without much effort or time.

[0013] 1 is a flowchart showing the procedure for the method for analyzing a component derived from a microorganism that produces a biopolymer according to a first embodiment. 2 is a flowchart showing the procedure for the method for analyzing a component derived from a microorganism that produces a biopolymer according to a second embodiment. 3 is a flowchart showing the procedure for the method for analyzing a component derived from a microorganism that produces a biopolymer according to a third embodiment. 4 is a mass spectrum of the component derived from a microorganism in Example 1. 5 is a mass spectrum of the component derived from a microorganism in Example 2. 6 is a mass spectrum of the component derived from a microorganism in Example 3.

[0014] Hereinafter, an embodiment of the present invention will be described with reference to the drawings. The microorganism in the present invention produces a biopolymer at least either extracellularly or intracellularly. A biopolymer refers to a polymer compound produced by a microorganism, and is, for example, a compound comprising polysaccharides, polyamino acids, polylactic acids, polyhydroxyalkanoates, 3-hydroxybutyric acid-3-hydroxyhexanoic acid copolymer polyesters, etc.

[0015] First Embodiment The first embodiment of the method for analyzing components derived from microorganisms that produce biopolymers is intended to analyze biopolymers produced intracellularly or extracellularly by microorganisms. FIG. 1 is a flowchart illustrating the steps of the first embodiment of the method for analyzing components derived from microorganisms that produce biopolymers. First, an operator prepares a sample containing microorganisms that produce biopolymers (step S101). Next, the sample is subjected to a process for disrupting the microbial cell walls, thereby preparing a sample solution containing the biopolymer and microbial components (step S102). Examples of processes for disrupting the microbial cell walls include chemical methods such as the addition of organic solvents, surfactants, acids, or alkalis; physical methods such as ultrasonic treatment, bead crushing, and freeze-fracturing; and biochemical methods such as enzyme treatment. When the microbial cell walls are disrupted, microbial components such as cytoplasm and nucleoids are eluted into the sample solution. Biopolymers produced extracellularly or intracellularly are also eluted into the sample solution.

[0016] Next, the obtained sample solution is centrifuged (step S103). The sample solution is separated into a supernatant and a precipitate by centrifugation. The supernatant contains biopolymers and cytoplasmic components, and the precipitate contains nucleoids, cell membranes, etc.

[0017] After centrifugation, the resulting supernatant is subjected to ultrafiltration (step S104). The ultrafiltration membrane is permeable to biopolymers but not to cytoplasmic components. Therefore, the cytoplasmic components do not permeate the ultrafiltration membrane and remain as residue on the membrane, while the biopolymers are transferred to the filtrate. Note that microfiltration may optionally be performed before ultrafiltration.

[0018] Next, an analytical sample is prepared using the filtrate obtained by ultrafiltration (step S105). Specifically, a matrix solution is added to the filtrate, and the mixture is dropped into a well of a sample plate of a MALDI mass spectrometer and then dried. This prepares an analytical sample. This analytical sample is then subjected to MALDI mass spectrometry (step S106).

[0019] According to the analytical method of the first embodiment, the biopolymer produced by the microorganism and the microbial constituents are separated by a simple operation, and the microbial constituents are removed from the analytical sample, thereby enabling the biopolymer to be analyzed with high accuracy.

[0020] (Regarding the sample) The sample is not particularly limited as long as it contains microorganisms that produce biopolymers, but it preferably contains bacteria belonging to the genera Sphingomonas, Gordonia, and Pseudarthrobacter. When analyzing microorganisms contained in a collected object, a portion containing the microorganisms is removed from the object and added to a liquid medium or spread on an agar medium to culture the microorganisms. For example, microorganisms recovered from a liquid medium by centrifugation or microorganisms contained in colonies on an agar medium can be used as samples. Alternatively, a suspension of these can be used as a sample.

[0021] (Regarding the treatment for disrupting the cell wall of microorganisms) The treatment for disrupting the cell wall of microorganisms is not particularly limited as long as it can elute intracellular components. When disrupting the cell wall by adding an organic solvent, examples of the organic solvent that can be used include methanol, ethanol, and acetonitrile. The organic solvent may be a mixture with an aqueous solvent (a solvent containing water as the main component (e.g., a solvent in which 70 to 100% by volume of water is present)). Gram-negative bacteria have a thin cell wall that covers the cells, so the cell wall is easily dissolved by organic solvent treatment, resulting in cell disruption. On the other hand, Gram-positive bacteria have a thick cell wall that makes the cells more difficult to disrupt.

[0022] When cell walls are disrupted by ultrasonic treatment, the method is not particularly limited, and known methods can be used. For example, ultrasonic treatment can be carried out by continuously or intermittently irradiating a solution containing a sample with ultrasonic waves using an ultrasonic cleaner or ultrasonic disrupter. The duration of ultrasonic treatment is not particularly limited, and can be appropriately set to 30 seconds to 5 minutes, etc.

[0023] When disrupting cell walls by bead crushing, the method is not particularly limited, and known methods can be used. For example, a bead cell crusher or a bead homogenizer can be used to vibrate a container containing a mixture of a sample solution and beads. The time required for the bead crushing process is not particularly limited, and can be set appropriately between 30 seconds and 5 minutes.

[0024] Physical disruption treatments such as ultrasonication or bead crushing disrupt cells regardless of whether they are gram-negative or gram-positive bacteria.

[0025] The above-mentioned cell wall disruption treatment is preferably carried out to an extent that microbial components, such as protein complexes, are not decomposed, in order to ensure that microbial components remain on the ultrafiltration membrane reliably and efficiently during ultrafiltration, which will be described later.

[0026] (Regarding Centrifugation) The method of centrifugation is not particularly limited, and can be performed by known methods using a centrifuge, etc. The conditions for centrifugation are not particularly limited, but from the viewpoint of separating biopolymers and cytoplasmic components from nucleoids and cell membranes, a rotation speed of 10,000 g or more is preferable, and 12,000 g or more is more preferable. The time for centrifugation is also not particularly limited, and can be appropriately set to 1 to 5 minutes, etc.

[0027] (Regarding Microfiltration) In order to remove relatively large molecules from a sample, it is preferable to microfilter the supernatant obtained by centrifugation. This not only removes impurities but also prevents clogging during ultrafiltration. The pore size of the microfiltration membrane used for microfiltration is preferably 0.5 μm or less, more preferably 0.45 μm or less. The smaller the pore size, the wider the range of molecules corresponding to molecular weights that can be removed from the sample. The method of microfiltration is not particularly limited, but for example, centrifugation to promote filtration is preferable for efficient microfiltration. A microfiltration membrane with a large pore size is preferable because it allows for efficient separation in a short time when microfiltration is performed using centrifugation or the like.

[0028] (About Ultrafiltration) Ultrafiltration separates macromolecules such as proteins and fine particles dissolved in water using the pores in a porous ultrafiltration membrane. Due to variations in pore size and the difficulty of measuring, the molecular weight cutoff, rather than pore size, is used as an indicator of the membrane's separation performance. Each ultrafiltration membrane manufacturer defines the nominal molecular weight limit (NMWL) using different standards. Molecules with molecular weights similar to the molecular weight cutoff may or may not pass through the ultrafiltration membrane. In the following, the molecular weight cutoff is defined as the molecular weight at which a rejection rate of 90% is achieved on a fractionation curve obtained by introducing multiple standard substances of different molecular weights into an ultrafiltration membrane.

[0029] The ultrafiltration membrane in this embodiment allows biopolymers to pass through but does not allow cytoplasmic components to pass through. Here, "permeable" to a substance refers to the molecular weight cutoff of the ultrafiltration membrane being greater than the molecular weight of the substance, while "impermeable" to a substance refers to the molecular weight cutoff of the ultrafiltration membrane being equal to or less than the molecular weight of the substance. Therefore, the molecular weight cutoff of the ultrafiltration membrane can be set based on the molecular weights of the biopolymer and microbial components. When the molecular weight of the biopolymer to be separated is between 500 and 20,000, the molecular weight cutoff of the ultrafiltration membrane is preferably greater than 3 kDa, more preferably greater than 10 kDa, and even more preferably greater than 30 kDa. The higher the molecular weight cutoff, the more efficient the separation in a shorter time. Furthermore, the molecular weight cutoff of the ultrafiltration membrane is preferably less than 100 kDa.

[0030] Although the method of ultrafiltration is not particularly limited, for example, centrifugation is preferred to promote filtration in order to more efficiently prepare the analytical sample. In this embodiment, the filtrate (filtered fraction) of the sample subjected to ultrafiltration that has permeated the ultrafiltration membrane is subjected to mass spectrometry.

[0031] (Mass Analysis) The mass analysis method is not particularly limited as long as it can detect the target peak. From the viewpoint of obtaining measurement data that is easy to analyze, ionization is preferably performed using the aforementioned MALDI, which easily generates singly charged ions. Furthermore, from the viewpoint of accurately analyzing high-mass ions, time-of-flight mass analysis is preferable. Therefore, mass analysis is preferably performed using a MALDI time-of-flight mass spectrometer capable of ionization by the MALDI method. The MALDI method is a type of soft ionization method. To analyze substances that do not easily absorb laser light or substances that are easily damaged by laser light, such as proteins, the analyte is mixed with a matrix that easily absorbs laser light and ionizes it, and the analyte is ionized by irradiating the mixture with laser light. Ionization in mass analysis can be performed in either positive ion mode or negative ion mode. Mass analysis can be performed using a single mass spectrometer, or fragment ions can be analyzed in multiple stages.

[0032] The matrix solution used when performing MALDI mass spectrometry is a solution in which a matrix is ​​added to a solvent for matrix preparation, and may contain an additive to improve the accuracy of mass spectrometry.

[0033] The solvent used to prepare the matrix is ​​preferably an organic solvent or a mixture of an organic solvent and an aqueous solvent. The type of organic solvent or aqueous solvent is not particularly limited. For example, the solvent may be an aqueous solution containing trifluoroacetic acid (TFA) prepared at a predetermined volume percent concentration of 0% to 1% and acetonitrile (ACN) at an arbitrary volume percent concentration. The concentration of acetonitrile can be appropriately set to several tens of percent (especially 50%).

[0034] The type of matrix is ​​not particularly limited as long as it can perform appropriate ionization, and examples that can be used include α-cyano-4-hydroxycinnamic acid (CHCA), sinapic acid (SA), 2,5-dihydroxybenzoic acid (DHB), 1,5-diaminonaphthalene (1,5-DAN), dithranol (DIT), and trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene]malononitrile (DCTB).

[0035] The matrix additive is not particularly limited as long as it is expected to have some effect in mass spectrometry, such as reducing noise or increasing the detection sensitivity of analyte ions. From the viewpoint of increasing detection sensitivity, the additive preferably contains at least one of a compound containing a phosphonic acid group and a surfactant. The compound containing a phosphonic acid group is preferably an alkylphosphonic acid. The surfactant is preferably a nonionic surfactant.

[0036] Second Embodiment The method for analyzing a component derived from a microorganism that produces a biopolymer according to the second embodiment is for analyzing the constituent components of the microorganism. The method for analyzing a component derived from a microorganism that produces a biopolymer according to the second embodiment differs from the analytical method of the first embodiment in that an analytical sample is prepared using a residue (capture fraction) instead of a filtrate obtained by ultrafiltration. Other than that, the method is the same as the first embodiment, and therefore a description of the overlapping parts will be omitted.

[0037] 2 is a flowchart showing the procedure of the method for analyzing microbial components according to the second embodiment. In the second embodiment, steps S201 to S204 are the same as steps S101 to S104 in the first embodiment.

[0038] After step S204, the residue obtained by ultrafiltration is used to prepare an analytical sample (step S205). Specifically, a matrix solution is added to the residue to redissolve it, and the redissolved solution is dropped into a well of a sample plate of a MALDI mass spectrometer and then dried. The prepared analytical sample is then subjected to MALDI mass spectrometry (step S206).

[0039] According to the analytical method of the second embodiment, biopolymers produced by microorganisms and microbial components are separated by simple operations, and the biopolymers are removed from the analytical sample. This allows for accurate analysis of microbial components such as proteins. <Third Embodiment> The analytical method of the third embodiment for a component derived from a microorganism that produces a biopolymer is intended for analyzing a biopolymer produced intracellularly by the microorganism. The analytical method of the third embodiment for a component derived from a microorganism that produces a biopolymer differs from the analytical method of the first embodiment in that the microorganisms contained in the sample are identified as Gram-positive bacteria, and that after the sample is prepared, biopolymers produced extracellularly by the microorganism are removed before the sample is subjected to a cell disruption process. Other aspects are the same as those of the first embodiment, and therefore, a description of the overlapping parts will be omitted.

[0040] 3 is a flowchart showing the procedure for the method for analyzing components derived from microorganisms that produce biopolymers according to the third embodiment. First, an operator prepares a sample containing Gram-positive bacteria as microorganisms (step S301). Next, the sample is mixed with an organic solvent and centrifuged (step S302). As described above, Gram-positive bacteria have thick cell walls, making them less susceptible to cell destruction even when in contact with an organic solvent. Therefore, in step S302, biopolymers produced outside the cells of the Gram-positive bacteria are extracted into the organic solvent, and the precipitate obtained by centrifugation contains microorganisms from which the biopolymers produced on the cell surface have been removed.

[0041] Next, the resulting precipitate is subjected to a process for disrupting the cell walls of the microorganisms to prepare a sample solution containing the biopolymers produced within the cells and the microbial components (step S303). Specifically, the precipitate is redissolved in an organic solvent or aqueous solvent, and the cell walls are disrupted by a physical method such as ultrasonic treatment, bead crushing, or freeze crushing.

[0042] Thereafter, steps S304 to S307 are carried out in the same manner as steps S103 to S106 in the first embodiment.

[0043] According to the analysis method of the third embodiment, it is possible to separate biopolymers produced extracellularly from biopolymers produced intracellularly by Gram-positive bacteria.

[0044] (Identification of Microorganisms) The types of components and the combinations of components of biopolymers produced by microorganisms vary from microorganism to microorganism. Therefore, microorganisms can be identified using the mass spectrum obtained by the method of analyzing components derived from microorganisms that produce biopolymers according to the present invention. The mass spectrum obtained by the analysis is compared with the mass spectra of multiple known microbial species in a database. Peaks derived from biopolymers can serve as marker peaks for identifying microorganisms. The data of known microbial species to be compared is not limited to mass spectra, and any data showing the m / z of peaks derived from biopolymers and microbial components can be used.

[0045] The analytical method for the components derived from microorganisms that produce biopolymers according to the present invention will be described below with reference to several examples, but these are merely illustrative and the present invention is not limited thereto. In the following, % indicates % by volume unless otherwise specified.

[0046] [Experimental Example 1] In Experimental Example 1, an analytical sample (Comparative Example 1) was prepared by mixing a sample with a matrix solution containing an organic solvent, without centrifuging or ultrafiltration, and an analytical sample (Example 1) was prepared by mixing a sample with an organic solvent, centrifuging and ultrafiltration, and using the filtrate obtained by ultrafiltration. Mass spectrometry was then performed on each of these samples to evaluate the separation effects of biopolymers and microbial components.

[0047] Comparative Example 1 1. Preparation of Analytical Samples The Gram-negative bacterium Sphingomonas adhaesiva (NBRC 15099T, a strain provided by the National Biotechnology Center, National Institute of Technology and Evaluation (NBRC)) was cultured on standard agar medium at 30°C for 2 days. The grown bacteria were mixed with an α-cyano-4-hydroxycinnamic acid (CHCA) solution (matrix solution) prepared at 10 mg / mL in a 50% acetonitrile (ACN) aqueous solution containing 1% trifluoroacetic acid (TFA). 1 μL of this solution was then dropped into a well of a MALDI-MS sample plate and dried, preparing a sample / matrix mixed crystal as the analytical sample.

[0048] 2. Mass spectrometry Mass spectrometry was performed using a MALDI time-of-flight mass spectrometer (MALDI-MS, product name: MALDI-8020, manufactured by Shimadzu Corporation). The sample plate was inserted into the MALDI-MS, and the analytical sample was irradiated with a laser to ionize it, followed by measurement in linear and positive modes.

[0049] 3. Results Figure 4(a) shows the MALDI mass spectrum obtained by mass spectrometry in Comparative Example 1 in the m / z range of 2500-7500. In Figure 4(a), the vertical axis indicates relative intensity (%). This is the same for the subsequent mass spectra. In Figure 4(a), peaks derived from microbial components were observed around m / z 5000, in the m / z ranges of 6000-6500 and 7000-7500, and peaks derived from biopolymers were observed in the m / z range of 2500-6000. Because Sphingomonas bacteria are Gram-negative bacteria with thin cell walls, simply mixing them with a matrix solution containing an organic solvent (acetonitrile) and an organic acid (trifluoroacetic acid) disrupted the cell walls, resulting in the observation of peaks derived from intracellular cytoplasmic components and peaks derived from biopolymers produced intracellularly or extracellularly.

[0050] Example 1 1. Preparation of Analytical Samples The Gram-negative bacterium Sphingomonas adhaesiva (NBRC 15099T) was cultured on standard agar medium at 30°C for 2 days. The grown bacteria were dispersed in 70% ethanol for lysis and centrifuged at 15,000 g for 5 minutes. The resulting supernatant was subjected to ultrafiltration using an Amicon Ultra (NMWL 30 KDa). The same matrix solution as in Comparative Example 1 was added to the filtrate obtained by ultrafiltration, and 1 μL of the solution was dropped into a well of a MALDI sample plate and dried. The sample / matrix mixed crystals were prepared as analytical samples.

[0051] 2. Mass spectrometry Mass spectrometry was performed in the same manner as in Comparative Example 1.

[0052] 3. Results Figure 4(b) shows the MALDI mass spectrum in the m / z range of 2500-7500 obtained by the mass spectrometry in Example 1. In Figure 4(b), the peaks derived from microbial components observed around m / z 5000 and in the m / z ranges of 6000-6500 and 7000-7500 in Figure 4(a) were not observed, and peaks derived from biopolymers were clearly observed in the m / z range of 2500-6500.

[0053] [Experimental Example 2] In Experimental Example 2, an analytical sample (Comparative Example 2) was prepared by subjecting a solution containing a sample to a bead-crushing treatment, without centrifuging or ultrafiltration, and an analytical sample (Example 2) was prepared by subjecting a solution containing a sample to a bead-crushing treatment, then centrifuging and ultrafiltration, and using the filtrate obtained by ultrafiltration. Mass spectrometry was then performed on each of these samples to evaluate the separation effect of biopolymers and microbial components.

[0054] Comparative Example 2 1. Preparation of Analytical Samples Gordonia sputi (NBRC 100414T) was cultured on an agar medium containing 0.4% yeast extract, 1% meat extract, and 0.4% glucose at 30°C for 3 days. Because Gordonia bacteria are Gram-positive and contain long-chain fatty acids called mycolic acids on their cell surface, lysis with organic solvents or organic acids does not disrupt the cells, preventing the extraction of cytoplasmic components. Therefore, 400 μL of zirconia beads (φ0.5 mm), 400 μL of ultrapure water, and cultured cells (the amount recovered with a 5 μL loop) were placed in a 1.5 mL screw-cap tube and disrupted using a bead-type cell disrupter (product name: MS-100, manufactured by Tomy Seiko Co., Ltd.) at 4000 rpm for 3 minutes. 1 μL of the solution after bead crushing treatment was dropped into a well of a MALDI-MS sample plate, and 1 μL of CHCA solution prepared at 10 mg / mL in a 50% aqueous solution of ACN containing 1% TFA was then dropped and dried to prepare a sample / matrix mixed crystal.

[0055] 2. Mass spectrometry Mass spectrometry was carried out in the same manner as in Comparative Example 1.

[0056] 3. Results Figure 5(a) shows the MALDI mass spectrum in the m / z range of 2000-12000 obtained by mass spectrometry in Comparative Example 2. In Figure 5(a), peaks derived from intracellular cytoplasmic components such as proteins were observed in the m / z range of 4000-10000. Furthermore, peaks derived from biopolymers were observed in the m / z range of 2000-6000. In Figure 5(a), the peak derived from cytoplasmic components near m / z 4400 was observed with high intensity. As a result, the peak intensity derived from biopolymers was relatively low, making the peak distribution difficult to see.

[0057] Example 2 1. Preparation of Analytical Samples Gordonia sputi (NBRC 100414T) was cultured using the same method as in Comparative Example 2, and a suspension containing the bacteria was subjected to bead disruption. The bead-disrupted solution was centrifuged at 15,000 g for 5 min. The resulting supernatant was then ultrafiltered at 14,000 g for 10 min using an Amicon Ultra (NMWL = 100 kDa). 1 μL of the filtrate (filtered fraction) obtained by ultrafiltration was added to a well of a MALDI-MS sample plate. 1 μL of CHCA solution (prepared at 10 mg / mL in 50% ACN aqueous solution containing 1% TFA) was then added and dried to prepare a sample / matrix mixed crystal.

[0058] 2. Mass spectrometry Mass spectrometry was carried out in the same manner as in Comparative Example 1.

[0059] 3. Results Figure 5(b) shows the MALDI mass spectrum in the m / z range of 2000-12000 obtained by the mass spectrometry in Example 1. In Figure 5(b), the peaks derived from microbial components, which were observed in the m / z range of 4000-10000 in Figure 5(a), were not observed, and peaks derived mainly from biopolymers were clearly observed in the m / z range of 2000-6000.

[0060] [Experimental Example 3] In Experimental Example 3, an analytical sample (Comparative Example 3) was prepared by subjecting a solution containing a sample to a bead-crushing treatment, without centrifuging or ultrafiltration, and an analytical sample (Example 3) was prepared by subjecting a solution containing a sample to a bead-crushing treatment, then centrifuging and ultrafiltration, and using the residue obtained by ultrafiltration. Mass spectrometry was then performed on each of these samples to evaluate the separation effect of biopolymers and microbial components.

[0061] (Comparative Example 3) 1. Preparation of analytical sample Pseudarthrobacter sulfonivor (JCM 13520T) was cultured for 3 days at 25°C on a 3% trypticase soy agar medium containing 1.5% agar. The cultured cells were subjected to a bead-crushing treatment in the same manner as in Comparative Example 2. Using the solution after the bead-crushing treatment, a sample / matrix mixed crystal for MALDI-MS was prepared in the same manner as in Comparative Example 2.

[0062] 2. Mass spectrometry Mass spectrometry was carried out in the same manner as in Comparative Example 1.

[0063] 3. Results Figure 6(a) shows the MALDI mass spectrum in the m / z range of 2000-12000 obtained by mass spectrometry in Comparative Example 3. Note that in Figure 6(a), the range of m / z 3000 or higher is shown with the relative intensity magnified five-fold (the same applies to Figure 6(b) described below). In Figure 6(a), a peak derived from the biopolymer was observed in the m / z range of 3500-9500. In addition, peaks derived from protein-derived microbial components were observed in the m / z range of 4000-8000, overlapping with the biopolymer peak.

[0064] Example 3 1. Preparation of Analytical Samples Pseudarthrobacter sulfonivor (JCM 13520T) was cultured using the same method as in Comparative Example 3, and a suspension containing the bacteria was subjected to bead disruption. The solution after bead disruption was centrifuged at 15,000 g for 5 minutes. The resulting supernatant was then ultrafiltered at 14,000 g for 10 minutes using an Amicon Ultra (NMWL = 100 kDa). The same matrix solution as in Comparative Example 1 was added to the residue (captured fraction) obtained by ultrafiltration. 1 μL of this solution was then dropped into a well of a MALDI-MS sample plate and dried. The sample / matrix mixed crystals were then prepared as analytical samples.

[0065] 2. Mass spectrometry Mass spectrometry was carried out in the same manner as in Comparative Example 1.

[0066] 3. Results Figure 6(b) shows the MALDI mass spectrum from m / z 2000 to 12000 obtained by the mass spectrometry in Example 3. In Figure 6(b), the peaks derived from biopolymers observed in Figure 6(a) were not observed, and a group of peaks derived from ribosomal proteins (peaks labeled L36, L33, L32, S18, and S10 in Figure 6(b)), which are known to be effective biomarker proteins for identifying microorganisms, were clearly observed.

[0067] Aspects It will be apparent to those skilled in the art that the exemplary embodiments described above are examples of the following aspects.

[0068] (Item 1) A method for preparing an analytical sample of a component derived from a microorganism that produces a biopolymer according to one embodiment of the present invention comprises the steps of: preparing a sample solution containing the biopolymer and components of the microorganism that produces the biopolymer; centrifuging the sample solution; ultrafiltering the supernatant obtained by the centrifugation; and preparing an analytical sample using the filtrate obtained by the ultrafiltration.

[0069] (Item 5) Another aspect of the present invention provides a method for preparing a sample for analysis of components derived from a microorganism that produces a biopolymer, the method comprising the steps of: preparing a sample solution containing a biopolymer and components of the microorganism that produces the biopolymer; centrifuging the sample solution; ultrafiltering the supernatant obtained by the centrifugation; and preparing a sample for analysis using the residue obtained by the ultrafiltration.

[0070] According to the methods for preparing samples for analyzing components derived from microorganisms that produce biopolymers according to paragraphs 1 and 5, the biopolymers and the components of the microorganisms that produce the biopolymers can be separated without much effort or time.

[0071] (2) In the method for preparing a sample for analysis of a component derived from a microorganism that produces a biopolymer according to the first aspect, the microorganism may be a gram-negative bacterium, and the sample solution may be prepared by disrupting the cell wall of the microorganism by contacting the microorganism with an organic solvent.

[0072] According to the method for preparing a sample for analyzing components derived from microorganisms that produce biopolymers, which relates to paragraph 2, a sample solution containing the biopolymer and components of the microorganisms that produce the biopolymers can be prepared by simple processing.

[0073] (Item 3) In the method for preparing a sample for analysis of a component derived from a microorganism that produces a biopolymer according to item 1, the microorganism may be a gram-negative bacterium or a gram-positive bacterium, and the cell wall of the microorganism may be destroyed by subjecting the microorganism to a bead crushing treatment or ultrasonic treatment, thereby preparing the sample solution.

[0074] According to the method for preparing a sample for analyzing components derived from microorganisms that produce biopolymers as described in paragraph 3, a sample solution containing a biopolymer and components of the microorganisms that produce the biopolymers can be prepared by simple processing, regardless of whether the microorganisms are gram-negative or gram-positive bacteria.

[0075] (4) In the method for preparing a sample for analysis of a component derived from a microorganism that produces a biopolymer according to the third aspect, the microorganism may be a gram-positive bacterium, and the biopolymer produced on the cell surface of the microorganism may be extracted with the organic solvent by contacting the microorganism with the organic solvent, and after removing the organic solvent, the microorganism may be subjected to the bead crushing treatment or the ultrasonic treatment.

[0076] According to the method for preparing a sample for analysis of components derived from microorganisms that produce biopolymers according to the fourth aspect, it is possible to separate biopolymers produced outside the cells of Gram-positive bacteria from biopolymers produced inside the cells.

[0077] (Item 6) In the method for preparing a sample for analysis of a component derived from a microorganism that produces a biopolymer according to any one of Items 1 to 5, the ultrafiltration may be performed using a membrane with a molecular weight cutoff of 3 kDa or more and 100 kDa or less.

[0078] According to the method for preparing a sample for analyzing components derived from a microorganism that produces a biopolymer according to item 6, the biopolymer can be reliably separated from the components of the microorganism that produces the biopolymer.

[0079] (Item 7) An analytical method according to one aspect of the present invention involves analyzing an analytical sample prepared by the method for preparing an analytical sample of a component derived from a microorganism that produces a biopolymer described in any one of Items 1 to 6.

[0080] According to the analytical method of the seventh aspect, the constituent components of a biopolymer and a microorganism that produces the biopolymer can be analyzed with high accuracy.

[0081] (Item 8) In the analysis method according to item 7, the analysis may be mass spectrometry.

[0082] (Item 9) In the analytical method according to item 8, the mass spectrometry may be such that the analytical sample is ionized by matrix-assisted laser desorption ionization.

[0083] According to the analytical method of the eighth or ninth aspect, the constituent components of a biopolymer and a microorganism that produces the biopolymer can be analyzed with higher accuracy.

[0084] (Item 10) A method for identifying a microorganism that produces a biopolymer according to one embodiment of the present invention identifies the type of the microorganism based on the presence or absence of a peak corresponding to the biopolymer or the magnitude of the peak in a mass spectrum obtained by the analytical method according to item 8 or 9.

[0085] According to the method for identifying a microorganism that produces a biopolymer according to the tenth aspect, the microorganism that produces a biopolymer can be identified with high accuracy.

Claims

1. A step of preparing a sample solution containing a biopolymer and components of a microorganism that produces the biopolymer; a step of centrifuging the sample solution; a step of ultrafiltering the supernatant obtained by the centrifugation; and a step of preparing an analytical sample using the filtrate obtained by the ultrafiltration. A method for preparing an analytical sample of components derived from a microorganism that produces a biopolymer, which has these steps.

2. The method for preparing an analytical sample of components derived from a microorganism that produces a biopolymer according to claim 1, wherein the microorganism is a gram-negative bacterium, and the cell wall of the microorganism is disrupted by bringing the microorganism into contact with an organic solvent to prepare the sample solution.

3. The method for preparing an analytical sample of components derived from a microorganism that produces a biopolymer according to claim 1, wherein the microorganism is a gram-negative bacterium or a gram-positive bacterium, and the cell wall of the microorganism is disrupted by subjecting the microorganism to bead milling treatment or ultrasonic treatment to prepare the sample solution.

4. The method for preparing an analytical sample of components derived from a microorganism that produces a biopolymer according to claim 3, wherein the microorganism is a gram-positive bacterium, and the biopolymer produced on the cell surface of the microorganism is extracted with the organic solvent by bringing the microorganism into contact with the organic solvent. After removing the organic solvent, the microorganism is subjected to the bead milling treatment or the ultrasonic treatment.

5. A step of preparing a sample solution containing a biopolymer and components of a microorganism that produces the biopolymer; a step of centrifuging the sample solution; a step of ultrafiltering the supernatant obtained by the centrifugation; and a step of preparing an analytical sample using the residue obtained by the ultrafiltration. A method for preparing an analytical sample of components derived from a microorganism that produces a biopolymer, which has these steps.

6. The method for preparing an analytical sample of components derived from a microorganism that produces a biopolymer according to any one of claims 1 to 5, wherein the ultrafiltration is performed using a membrane having a molecular weight cut-off of 3 kDa or more and 100 kDa or less.

7. An analytical method for analyzing an analytical sample prepared by the method for preparing an analytical sample of components derived from a microorganism that produces a biopolymer according to any one of claims 1 to 5.

8. The analytical method according to claim 7, wherein the analysis is mass spectrometry.

9. The analytical method according to claim 8, wherein in the mass spectrometry, the analytical sample is ionized by matrix-assisted laser desorption ionization.

10. A method for identifying a microorganism that produces a biopolymer, which comprises identifying the type of the microorganism based on the presence or absence of a peak corresponding to the biopolymer or the magnitude of the peak in the mass spectrum obtained by the analysis method according to claim 9.

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