Method for concentrating microorganisms and concentration container

Abstract

Provided are a concentration method and vessel which make it possible to quickly obtain a highly concentrated product of bacteria or the like by using a simple means. The present invention pertains to a method for concentrating a microorganism in a sample liquid which contains a microorganism in a solvent by using porous cellulose fine particles which have a plurality of voids therein, said method comprising a step for adhering the microorganism to the surface of the porous cellulose fine particles by contacting the sample liquid to the porous cellulose fine particles, and a liquid removal step for concentrating the microorganism in the voids of the porous cellulose fine particles by removing the solvent of the sample liquid, wherein: the allowed water content C, which is the mass in tare weight of PBS(-) in swollen porous cellulose fine particles obtained by swelling 100 mg of dry particles in PBS(-) until the PBS(-) has stopped falling therefrom, is 1.9 g or higher; and the free water ratio, which is obtained by first obtaining the drying amount (C-E) obtained by subtracting the mass E of the PBS(-) after centrifuging the swollen porous cellulose fine particles for 1 minute at 1000G from said allowed water content C, and then dividing said drying amount by E, is 2.9 or higher. The present invention also pertains to a vessel.

Description

Method for concentrating microorganisms and concentration container

[0001] The present invention relates to a method for concentrating microorganisms, such as bacteria or viruses, in a sample liquid containing the microorganisms in a solvent using porous cellulose microparticles having a large number of pores therein, and to a concentration container used in the concentration method.

[0002] In infectious disease diagnosis, environmental testing, and food microbiology testing, there is a need for rapid and simple methods to detect, identify, and / or enumerate pathogens or harmful microorganisms that impair quality in samples. For example, in infectious disease diagnosis, point-of-care (POCT) tests are used to identify pathogenic bacteria or viruses so that doctors and nurses can make appropriate clinical decisions at the patient's side. In environmental testing and food microbiology testing, tests are conducted to target environmental indicator bacteria such as common bacteria and coliform bacteria, as well as harmful microorganisms such as food poisoning bacteria and viruses, for employee or environmental hygiene management and quality control of raw materials, intermediate products, and final products. In both cases, the smaller the business or the more on-site the business, the more specialized equipment and simple operating methods that do not require knowledge of microbiology are required.

[0003] Testing methods for these microorganisms can be broadly divided into culture methods, which identify the microorganisms based on their biochemical properties after isolation and culture; genetic methods, which amplify and detect genes specific to the microorganisms using PCR, LAMP, or other methods; and immunological methods, which detect the microorganisms by utilizing a specific reaction between an antigen marker specific to the microorganism and an antibody. Each of these testing methods requires a small sample volume of approximately 0.01 to 1 mL, making it impossible to utilize a significant portion of the test substance (the bacteria or virus itself, or its surface or internal components) contained in approximately 0.01 to 1 L of a patient specimen (e.g., urine or body fluid), food sample (e.g., liquid sample or bacterial or viral extract obtained by stomacher), or environmental sample (e.g., water quality). This limits the detection accuracy or sensitivity of genetic methods and immunological methods used as simple and rapid tests. Furthermore, when microorganisms are present in trace amounts, long periods of enrichment culture (e.g., 24 hours) are required to avoid false positives during sampling, which compromises the ease and speed of the testing. To solve this problem and ensure sufficient detection material without using large-scale equipment, concentration is performed by adsorbing, separating, and recovering microorganisms using an adsorption carrier.

[0004] For example, in Patent Document 1 below, E. coli is adsorbed by shaking inorganic microparticles (amorphous metal silicate) and a sample for 1 to 20 minutes. However, the particles described in Patent Document 1 are not crosslinked cellulose particles, and shaking for 10 minutes or more is required to achieve an adsorption rate of 50% or more. Furthermore, 10 mg of inorganic microparticles are used for 10 mL of E. coli sample at 1e3 cfu / mL (hereinafter, 1eM represents 10 to the Mth power). Furthermore, Patent Document 1 does not disclose the allowable water content, porosity, etc. per certain mass of dry particles.

[0005] The following Patent Document 2 describes a method for detecting bacterial mastitis by contacting aminosilica with a specimen containing a mixture of pathogenic bacteria and cells, and recovering only the pathogenic bacteria. However, the particles described in Patent Document 2 are not cross-linked cellulose particles, and it takes one hour to mix the aminosilica with the specimen. Furthermore, the purpose is not to concentrate the pathogenic bacteria, but to separate them from the cells, and there is no description of the allowable moisture content per certain mass of dried particles, porosity, etc.

[0006] On the other hand, Patent Document 3 listed below describes an adsorption / separation agent for viruses and cells that is made of calcium phosphate porous granules having two types of continuous pore structure: continuous fine pores with an average pore size of 20 to 500 nm and continuous small pores with an average pore size of 1 to 50 μm. However, the particles described in Patent Document 3 are not crosslinked cellulose particles, and there is no description regarding the allowable water content per certain mass of dry particles, porosity, etc.

[0007] Patent Document 4 below describes a virus concentration method that includes the steps of adding particles having cationic groups on their surface and a particle size of 0.5 to 300 μm and antibodies against the surface antigen of the target virus to a sample that may contain the virus, thereby causing the virus to adhere to the particles, and separating and collecting the particles with the virus attached from the sample. It is stated that this virus concentration method allows for convenient processing of multiple samples simultaneously using a simple means, is easily adaptable to automation, and provides a virus concentration method that does not adversely affect nucleic acid amplification testing. However, the particles described are non-porous synthetic polymers obtained by polymerizing various monomers, and there is no description of their application to bacterial concentration methods, let alone the allowable water content per given mass of dry particles, porosity, etc.

[0008] Patent Document 5 listed below describes a method for removing viruses from a virus-containing sample by contacting the sample with an adsorbent having a structure in which polyethyleneimine is bound to a base carrier containing porous particles. Crosslinked cellulose particles having a specific ligand are cited as the base carrier, but there is no description about their application to a method for concentrating bacteria, let alone the allowable water content per certain mass of dry particles, porosity, etc.

[0009] Patent Document 6 listed below describes that porous cellulose microparticles modified with polycations can be used industrially as microcarriers for culturing bacteria, yeast, and animal and plant cells. However, this document is based on the premise of adhesion and proliferation, and does not describe a liquid extraction process for removing the solvent or concentrating microorganisms in the pores, much less the allowable water content per certain mass of dry particles, porosity, etc.

[0010] Patent No. 5972174 Patent No. 4889263 Patent No. 2543766 Patent No. 4161167 JP 2019-194547 JP 4-91142

[0011] Although Patent Documents 1, 2, 3, and 4 describe adsorbents for cells or viruses, the materials used are non-porous synthetic polymers, porous amorphous metal silicates, porous calcium phosphate, etc., and are not cellulose microparticles with high porosity and permeability, which have the characteristics of allowable water content per a certain mass of dry particles, porosity, etc., as described below, making it difficult to obtain high-density bodies of microorganisms. Patent Document 5 also describes crosslinked cellulose particles to which polyethyleneimine is bound as an adsorbent, but this adsorbent has the above-mentioned specific polyethyleneimine bound as a ligand and is specialized for adsorbing and removing viruses. Furthermore, Patent Documents 1, 2, 4, and 5 describe conditions for contacting the adsorbent with a microbial sample, but require at least 10 minutes of stirring and shaking to fully adsorb 10 mL of specimen sample onto the adsorbent, which is not a simple operating method.

[0012] In view of the current state of the prior art described above, the problem to be solved by the present invention is to provide a method for concentrating microorganisms, which allows highly concentrated microorganisms to be obtained quickly and conveniently, and a concentration container for use in said concentration method.

[0013] As a result of extensive research and experimentation to solve this problem, the inventors unexpectedly discovered that the problem can be solved by using porous cellulose microparticles as an adsorbent in such a way that 100 mg of dry particles are swollen in phosphate-buffered saline(-) (hereinafter also referred to as PBS(-)), and the porous cellulose microparticles in a swollen state at which the PBS(-) no longer falls off under their own weight have an allowable water content C of 1.9 or more, which is the mass of PBS(-) of the PBS(-) of the porous cellulose microparticles in the swollen state, when the particles are swollen and the mass E of the PBS(-) of the porous cellulose microparticles is obtained by centrifuging the porous cellulose microparticles in the swollen state at 1000 G for 1 minute, to obtain a dehydrated amount (C-E), which is then divided by E, so that the free water ratio ((C-E) / E) is 2.9 or more, which led to the completion of the present invention.

[0014] That is, the present invention is as follows. [1] A method for concentrating microorganisms in a sample liquid containing microorganisms in a solvent using porous cellulose microparticles having a large number of pores therein, the method comprising the following steps: a step of bringing the sample liquid into contact with the porous cellulose microparticles to adsorb the microorganisms contained in the sample liquid onto the surface of the porous cellulose microparticles; and a step of removing the solvent from the sample liquid to concentrate the microorganisms on the surface and / or in the pores of the porous cellulose microparticles; wherein the porous cellulose microparticles have an allowable water content C of 1.9 g or more, which is the mass of PBS(-) of the porous cellulose microparticles in a swollen state where 100 mg of dry particles are swollen with PBS(-) and the PBS(-) no longer falls off under their own weight, and the free water ratio ((C-E) / E), obtained by subtracting the mass E of PBS(-) obtained by centrifuging the porous cellulose microparticles in the swollen state at 1000 G for 1 minute from the allowable water content C, is 2.9 or more. [2] The method according to [1] above, further comprising the following step: after the liquid-removing step, using an eluent to recover microorganisms concentrated on the surface and / or in the pores of the porous cellulose microparticles into the eluent. [3] The method according to [1] or [2] above, further comprising a step of washing the porous cellulose microparticles with a liquid or gas between each of the steps. [4] The method according to any of [1] to [3] above, wherein the average particle size of the porous cellulose microparticles is 100 μm or more and 1000 μm or less. [5] The method according to any of [1] to [4] above, wherein the average open pore size of the pores of the porous cellulose microparticles is 1 μm or more and 100 μm or less. [6] The method according to any of [1] to [5] above, wherein the porous cellulose microparticles form a continuous pore structure in which adjacent pores are interconnected by openings in the membrane separating them. [7] The surface area (specific surface area) per 1 g of dry particles of the porous cellulose microparticles is 0.3 m or more. 2 / g or more 4.4m 2 / g or less. [8] The method according to any one of [1] to [7], wherein the cellulose constituting the porous cellulose microparticles has a cationic functional group and a charge capacity of 0.5 mmol / g or more and 5.0 mmol / g or less. [9] The method according to [8], wherein the cationic functional group is at least one selected from the group consisting of a primary amino group, a secondary amino group, and a tertiary amino group.

[10] The method according to [9], wherein the cationic functional group is N,N-diethylaminoethyl (DEAE).

[11] The method according to any one of [1] to

[10] , wherein the cellulose constituting the porous cellulose microparticles is intermolecularly crosslinked.

[12] The method according to any one of [1] to

[11] , wherein the cellulose constituting the porous cellulose microparticles is cuprammonium regenerated cellulose.

[13] The method according to any one of [1] to

[12] , wherein the microorganism is a bacterium or a virus.

[14] A concentration container for use in a method for concentrating microorganisms in a sample liquid containing microorganisms in a solvent using porous cellulose microparticles having a large number of pores therein, the concentration container comprising: a cylindrical body having an inlet opening and an outlet opening; a filter provided on the outlet opening side; and porous cellulose microparticles filled in the space above the filter; wherein the sample liquid supplied from the inlet opening side comes into contact with the porous cellulose microparticles, and the microorganisms contained in the sample liquid are adsorbed onto the surfaces of the porous cellulose microparticles, while the solvent of the sample liquid is removed from the outlet opening side, and the microorganisms are concentrated in the pores of the porous cellulose microparticles, and The porous cellulose microparticles have an allowable water content C, which is the mass of PBS(-) of the porous cellulose microparticles in a swollen state at which 100 mg of dry particles are swollen with PBS(-) and the PBS(-) no longer falls off under their own weight, of 1.9 g or more, and a free water ratio ((C-E) / E), obtained by subtracting the mass E of PBS(-) obtained by centrifuging the porous cellulose microparticles in the swollen state at 1000 G for 1 minute from the allowable water content C, from the allowable water content C, is 2.9 or more.

[15] The concentrating container according to

[14] , wherein the average particle size of the porous cellulose microparticles is 100 μm or more and 1000 μm or less.

[16] The concentrating container according to

[14] or

[15] , wherein the average pore size of the pores of the porous cellulose microparticles is 1 μm or more and 100 μm or less.

[17] The concentrating container according to any of

[14] to

[16] , wherein the porous cellulose microparticles form a continuous pore structure in which adjacent pores are connected to each other by openings in the membrane that separate them.

[18] The surface area (specific surface area) of the porous cellulose microparticles per 1 g of dry particles is 0.3 m. 2 / g or more 4.4m 2 / g or less.

[19] The concentrating container according to any one of

[14] to

[18] , wherein the cellulose constituting the porous cellulose microparticles has a cationic functional group and a charge capacity of 0.5 mmol / g or more and 5.0 mmol / g or less.

[20] The concentrating container according to

[19] , wherein the cationic functional group is at least one selected from the group consisting of a primary amino group, a secondary amino group, and a tertiary amino group.

[21] The method according to

[20] , wherein the cationic functional group is N,N-diethylaminoethyl (DEAE).

[22] The concentrating container according to any one of

[14] to

[21] , wherein the cellulose constituting the porous cellulose microparticles is intermolecularly crosslinked.

[23] The concentrating container according to any one of

[14] to

[22] , wherein the cellulose constituting the porous cellulose microparticles is cuprammonium regenerated cellulose.

[24] The concentration container according to any one of

[14] to

[23] , wherein the microorganisms are bacteria or viruses.

[25] A method for concentrating microorganisms in a sample liquid containing microorganisms in a solvent using porous cellulose microparticles having a large number of pores therein, the method comprising the following steps: a step of bringing the sample liquid into contact with the porous cellulose microparticles in a concentration container equipped with a cylindrical body having an inlet opening and an outlet opening, a filter provided on the outlet opening side, and porous cellulose microparticles filled in the space above the filter, thereby adsorbing the microorganisms contained in the sample liquid onto the surfaces of the porous cellulose microparticles; and a step of aerating the sample liquid to remove the solvent and concentrate the microorganisms on the surfaces and / or in the pores of the porous cellulose microparticles. The porous cellulose microparticles have an allowable water content C, which is the mass of PBS(-) of the porous cellulose microparticles in a swollen state where 100 mg of dry particles are swollen with PBS(-) and the PBS(-) no longer falls off under their own weight, of 1.9 g or more, and the free water ratio ((C-E) / E), obtained by subtracting the mass E of PBS(-) obtained by centrifuging the porous cellulose microparticles in the swollen state at 1000 G for 1 minute from the allowable water content C, from the allowable water content C, is 2.9 or more.

[26] The method for concentrating microorganisms according to

[25] , wherein the aeration is performed by aerating air through an inlet opening of the concentration container using a syringe.

[27] The method for concentrating microorganisms according to

[26] , wherein the volume of the air is 0.5 mL or more and 1 mL or less per 1 mg of the porous cellulose microparticles.

[0015] The concentration method and concentration container of the present invention can concentrate microorganisms using specific porous cellulose microparticles with high porosity and dehydration rate, which are indicated by the allowable water content and free water ratio, and therefore can obtain a high amount of microorganisms adsorbed per dry weight of particles, a high-density mass of microorganisms, and can also recover high concentrations of microorganisms from the obtained high-density mass of microorganisms. Therefore, the concentration method and concentration container of the present invention can be suitably used in microbial testing, etc.

[0016] Photographs of porous cellulose microparticles (labeled cross-linked cellulose) and other concentration carriers. A conceptual diagram of "allowable moisture content" and "free moisture ratio," and an explanatory diagram of how to calculate them. Particle suspensions of porous cellulose microparticles (labeled particles of the present invention) and comparative particles, circle 1 to circle 4. An electron microscope image of porous cellulose microparticles after adsorption of Escherichia coli. A schematic diagram of a concentration container for holding porous cellulose microparticles.

[0017]

[0013] The first embodiment of the present invention is a method for concentrating microorganisms in a sample liquid containing microorganisms in a solvent by using porous cellulose microparticles having a large number of pores therein, the method comprising the steps of: bringing the sample liquid into contact with the porous cellulose microparticles to adsorb the microorganisms contained in the sample liquid onto the surface of the porous cellulose microparticles; and removing the solvent from the sample liquid to concentrate the microorganisms on the surface and / or in the pores of the porous cellulose microparticles. The porous cellulose microparticles have an allowable water content C, which is the mass of PBS(-) of the porous cellulose microparticles in a swollen state where 100 mg of dry particles are swollen with PBS(-) and the PBS(-) no longer falls off under their own weight, of 1.9 g or more, and the free water ratio ((C-E) / E), obtained by subtracting the mass E of PBS(-) obtained by centrifuging the porous cellulose microparticles in the swollen state at 1000 G for 1 minute from the allowable water content C, is obtained by dividing the obtained amount of dehydration (C-E) by E, is 2.9 or more.

[0018] [Allowable Water Content (g) and Free Water Ratio] Figure 2 shows a conceptual diagram of "allowable water content" and "free water ratio," as well as an explanatory diagram of how to calculate them. The allowable water content is the mass of PBS(-) of a particle when 100 mg of dry particles are swollen with PBS(-) and the particle no longer loses PBS(-) under its own weight. This is an absolute indicator of how much liquid a particle can absorb. The larger this value, the more voids the particle forms internally upon swelling. Therefore, a high allowable water content indicates a larger pore size upon swelling and a higher porosity. It is generally known that the higher the porosity, the easier water permeates through a porous material. A high allowable water content indicates a high water permeability. A large pore size allows microorganisms to adsorb to the surface inside the pores, while high water permeability facilitates the penetration of microorganisms into the pores. Furthermore, it also facilitates the elution (detachment) of microorganisms during microorganism recovery. The higher the allowable water content, the better, and it is preferably 1.9 g or more, more preferably 2.1 g or more, even more preferably 2.4 g or more, and most preferably 2.6 g or more. As shown in Figure 1, the porous cellulose microparticles (referred to as crosslinked cellulose) used in this embodiment have many pores inside the microparticles when swollen, and have a high porosity compared to other concentration carriers.

[0019] The free moisture ratio is the ratio ((C-E) / E) of the mass of PBS(-) remaining in the particle (E: bound moisture) to the mass of PBS(-) removed from the particle (C-E: free moisture) when a centrifugal force of 1000 G is applied for 1 minute from the allowable moisture content C in the swollen state. This is a relative index indicating how much liquid the swollen particle dehydrates. The larger this value, the greater the particle's free moisture content relative to its bound moisture content, i.e., the greater the relative amount of dehydration. A large amount of dehydration allows for the removal of excess moisture, resulting in a high density of microorganisms (a high number of adsorbed microorganisms per particle weight (mg)). The higher the free moisture ratio, the more preferable it is, preferably 2.9 or more, more preferably 5.7 or more, even more preferably 6.4 or more, and most preferably 7.1 or more. The removal of contained moisture in the liquid-draining step can be achieved by known methods. Examples include vacuum drying, centrifugation, replacement by aeration with air, removal using a water-absorbing material, etc., but are not limited thereto. An appropriate method may be selected depending on the application. For example, centrifugation is suitable for controlling the force for removing contained moisture, while a method of aerating and replacing air using a syringe, etc., is suitable for removing contained moisture by a simple operation without using special equipment. In one embodiment, centrifugation at 1000 G for 1 minute corresponds to the aeration of 1 to 2 mL of air.

[0020] The type of cellulose constituting the porous cellulose microparticles of this embodiment is not particularly limited, but examples include known celluloses such as cuprammonium regenerated cellulose, viscose regenerated cellulose, cotton, pulp, and polynosic, and cuprammonium regenerated cellulose and viscose regenerated cellulose are preferred, and cuprammonium regenerated cellulose is more preferred. Cuprammonium regenerated cellulose has a low degree of crystallinity, making it highly reactive when modified with various functional groups, and even porous particles can be homogeneously modified to the inside of the particles, and also has excellent particle shape stability.

[0021] [Average Particle Diameter (μm)] The average particle diameter of the porous cellulose microparticles of this embodiment is preferably 100 μm or more and 1000 μm or less, more preferably 100 μm or more and 500 μm or less, even more preferably 200 μm or more and 400 μm or less, and even more preferably 200 μm or more and 280 μm or less. If the average particle diameter is smaller than 100 μm, the filter openings on the inlet and outlet sides of the concentration container for holding the porous cellulose microparticles must be made smaller, which increases the pressure loss when supplying a sample liquid containing microorganisms or when removing the solvent from the sample liquid, resulting in reduced operability and restrictions on the amount of liquid to be treated. On the other hand, if the average particle diameter is larger than 1000 μm, the resistance on the particles increases, and flow between the particles is prioritized over the interior of the particles, resulting in non-uniform (or reduced) water permeability. In this specification, the average particle diameter refers to the average particle diameter of porous particles measured by swelling them with water or physiological saline, removing excess water, and measuring 20 or more particles using an optical microscope at an appropriate magnification.

[0022] [Average pore size (μm) of pores] The pore size of the porous cellulose microparticles of this embodiment is preferably 1 μm or more and 100 μm or less, more preferably 10 μm or more and 80 μm or less, and even more preferably 10 μm or more and 50 μm or less. If the average pore size is smaller than 1 μm, free movement of the sample liquid containing microorganisms within the porous cellulose microparticles may not be achieved. Furthermore, the particle size of viruses is generally approximately 0.02 μm, and the particle size of bacteria is approximately 1 μm to 5 μm. In the case of viruses, the particle size is extremely small, so Brownian motion dominates the movement (displacement) within the pores. On the other hand, for bacteria, the particle size dominates. Therefore, for both microorganisms, if the average pore size of the porous cellulose microparticles is smaller than 1 μm, adsorption of the microorganisms into the interior of the particles may be significantly limited. On the other hand, if the average pore size is larger than 100 μm, sufficient surface area for adsorption of microorganisms cannot be secured, and efficient concentration of the microorganisms may not be achieved. The average pore size in this specification refers to the average pore size obtained by swelling porous particles with water or physiological saline, removing excess water, and measuring the interlaminar distance for 20 or more particles using an electron microscope at an appropriate magnification. Furthermore, when the pore size of each pore is not a perfect sphere or circle, the pore size is defined as the shortest diameter.

[0023] The porous cellulose microparticles of this embodiment preferably have a continuous pore structure in which the pores are interconnected by openings in the membrane that separate the pores. Such an interconnected structure allows microorganisms to be adsorbed efficiently even to the interior of the porous cellulose microparticles, and also increases the surface area for microorganism adsorption.

[0024] [Specific surface area (m 2 The surface area (specific surface area) of the porous cellulose microparticles of this embodiment when dried is 0.3 m 2 / g or more 4.4m 2 / g or less, and more preferably 0.6m 2 / g or more 3.3m 2 / g or less, more preferably 0.8m 2 / g or more 1.7m 2 / g or less. Surface area is 0.3 m 2If the surface area is less than 4.4 m / g, the surface area required for adsorption of microorganisms is small, and the microorganisms may not be concentrated efficiently. 2 If the surface area is more than 1 / g, the charge per unit area will be low, which may result in a decrease in the rate of adhesion of microorganisms. In this specification, the surface area is the surface area measured by the BET method.

[0025] [Charge Capacity (mmol / g)] Unlike particles such as polystyrene and gelatin, the charge capacity of the porous cellulose microparticles of this embodiment must also take into account the influence of hydroxyl groups contained in the cellulose substrate. Furthermore, compared to polysaccharide particles such as dextran particles, the intramolecular hydrogen bonds and crystallinity are significantly different, so these must be taken into account. The charge capacity of the porous particles of this embodiment is preferably 0.5 mmol / g to 5.0 mmol / g, more preferably 0.5 mmol / g to 3.0 mmol / g, even more preferably 0.6 mmol / g to 3.0 mmol / g, even more preferably 0.6 mmol / g to 2.5 mmol / g, even more preferably 0.7 mmol / g to 2.5 mmol / g, and particularly preferably 0.9 mmol / g to 2.0 mmol / g. When the charge capacity is within the above range, combined with the specific range of the surface area of ​​the pores, the charge per unit surface area is increased, thereby improving the adhesion rate of microorganisms. Furthermore, when recovering microorganisms with an eluent, they are not inhibited by the charge, and can be recovered.The charge capacity in this specification refers to the average charge capacity measured for three or more samples of a solution obtained by adsorbing chlorine to the amine in the porous particles with hydrochloric acid, washing away the adsorbed chlorine with a sodium sulfate solution, and titrating the solution with silver nitrate using potassium chromate as an indicator.

[0026] The cationic substituent constituting the porous cellulose microparticles of this embodiment is not particularly limited, but is preferably at least one selected from the group consisting of a primary amino group, a secondary amino group, and a tertiary amino group, more preferably a tertiary amino group, and even more preferably a diethylaminoethyl group.

[0027] The porous cellulose microparticles of this embodiment can be produced by a method in which a solution containing dissolved cellulose is formed into a desired shape while being cooled to below the solidification temperature of the solution to freeze it, and then the solvent is extracted and removed or the dissolving ability is lost. In general, the freezing temperature is preferably not set to be more than 40°C lower than the freezing temperature of the solvent, and is usually selected in the range of 0 to 20°C lower than the freezing temperature.

[0028] The frozen cellulose solution or cellulose derivative solution is then subjected to extraction and removal of the solvent dissolving the cellulose or cellulose derivative, or to reducing its dissolving power (hereinafter, these processes are collectively referred to as "solvent removal, etc."), resulting in a solidified porous cellulose body. The conditions for solvent removal, etc., are not particularly limited. Typically, the frozen body can be quickly placed in a coagulation bath or regeneration bath, but it is preferable and recommended that the coagulation bath or regeneration bath be kept below the freezing temperature of the solution. However, in the case of cellulose derivatives, a cellulose regeneration process is required, and this regeneration is carried out simultaneously with or sequentially (i.e., after solvent removal, etc.). The regeneration itself can be carried out by conventional methods. Using the above production method, there is no need to add foreign substances such as porosifying agents to the polymer solution during production, making it easy to produce droplets of uniform diameter, and particle size can be freely controlled. The size and shape of the pores are essentially determined by the size and shape of the solvent crystals formed when the solvent in the solution freezes and solidifies. Therefore, the shape and size of the pores can be adjusted by changing the type of polymer solution, temperature, and other freeze-solidification conditions. When the above-mentioned production method is used, the porous cellulose microparticles obtained have a continuous pore structure in which the pores communicate with each other via openings in the membrane that separate the pores.

[0029] As the porous cellulose microparticles used in the concentration method of this embodiment, granulated cellulose particles can be used as they are, but it is preferable to crosslink them before use. There are no limitations on the crosslinking method, but any crosslinking agent having two or more functional groups capable of reactively bonding with the hydroxyl groups of cellulose can be used for intermolecular crosslinking of porous cellulose materials. Examples include bifunctional organic substances. Examples include X-R-Z type compounds (where R represents an aliphatic residue containing a carbon atom, and X and Z are various halogens, epoxy, etc., bonded to the carbon atoms of the aliphatic residue) that react relatively easily in the presence of an alkaline reactant. Examples of bifunctional compounds suitable for the above reaction include epichlorohydrin, dichlorohydrin, 1,2- or 3,4-diepoxybutane, bisepoxypropyl ether, ethylene glycol-bis-epoxypropyl ether, 1,4-butanediol-bis-epoxypropyl ether, and compounds closely related to these, but are not limited to these. Crosslinking allows the crystalline structure of cellulose to be maintained even when modified with many cationic substituents, thereby keeping the particle structure stable.

[0030] There are no particular limitations on the method for modifying the cellulose constituting the porous cellulose microparticles used in the concentration method of this embodiment with a cationic substituent, but multiple methods are possible, such as direct modification with hydroxyl groups or modification via a functional group having an epoxy group. A preferred method is to directly modify the hydroxyl groups of the cellulose using 2-(diethylamino)ethyl chloride hydrochloride or the like. Because cellulose has many hydroxyl groups, direct modification with hydroxyl groups makes it possible to modify a large amount of cationic substituents uniformly, even to the interior of the particles. On the other hand, when introducing the cationic substituent via a third substituent such as an epoxy group, the amount and variation of the introduced cationic substituent depends on the amount of the third substituent introduced, making it difficult to control the amount introduced into the interior of the particles. Furthermore, there are no particular limitations on the reaction conditions for introducing a cationic substituent using 2-(diethylamino)ethyl chloride hydrochloride. However, the charge density inside the pores can be controlled by setting an optimal pH taking into account changes in the pH of the solution caused by the 2-(diethylamino)ethyl chloride hydrochloride itself, and by adding an additive such as Glauber's salt to minimize the effect of the charge on the hydroxyl groups of the cellulose. In this embodiment, the preferred cationic functional group is an N,N-diethylaminoethyl (DEAE) group.

[0031] [Dry particle weight (mg) per swollen volume (mL)] The porous cellulose microparticles of this embodiment preferably have a dry particle weight (mg) per mL of particle volume (swollen volume) swollen with PBS(-) of 70 mg or less, more preferably 55 mg or less, and even more preferably 40 mg or less. A low dry particle weight per swollen volume means that the porous particles have a large pore size in liquid and a high porosity. When the dry particle weight per swollen volume is within the above range, water permeability is increased and free movement of microorganisms within the pores is achieved, allowing efficient adsorption to the surface inside the pores. Furthermore, during microorganism recovery, the mobility of the eluent and eluted microorganisms out of the pores is increased, making it easier to elute the microorganisms.

[0032] [Adsorption rate (%)] The porous cellulose microparticles used in the adsorption method of this embodiment utilize as much of the detection substance in the sample (bacteria or viruses themselves, or their surface or internal components) as possible, so the higher the adsorption rate of microorganisms on the surface of the porous cellulose microparticles, the better, and preferably 50% or more, more preferably 60% or more, even more preferably 70% or more, and most preferably 80% or more. Here, the surface of the porous cellulose microparticles refers to the combination of both the outermost surface and the pore surface of the microparticles.

[0033] [Amount of microorganism-containing sample (mL)] In the adsorption method of this embodiment, the amount of the microorganism-containing sample to be contacted can be increased or decreased depending on the amount of porous cellulose microparticles used, but in the vortex method (the method for contacting particles and microorganisms by uniform dispersion described in Example 3), the larger the liquid volume, the lower the probability of contact between the particles and microorganisms during stirring, so it is preferably 10 mL or less for a dry weight of 2 mg, more preferably 5 mL or less, even more preferably 1 mL or less, and most preferably 0.1 mL or less. On the other hand, in the column method (the method for contacting particles and microorganisms by solid phase separation described in Example 4), there is no limit to the amount of liquid passed, but if the sample is too large, it takes time to pass the liquid and is not a simple operation method, so it is preferably 0.1 mL or more and 10,000 mL or less, more preferably 1 mL or more and 10,000 mL or less, even more preferably 1 mL or more and 1,000 mL or less.

[0034] A second embodiment of the present invention is a concentration container for use in a method for concentrating microorganisms in a sample liquid containing microorganisms in a solvent using porous cellulose microparticles having a large number of pores therein, the concentration container comprising: a cylindrical body having an inlet opening and an outlet opening; two filters provided at a predetermined distance on the inlet opening side and the outlet opening side, respectively; and porous cellulose microparticles filled in the space defined by the two filters; wherein the sample liquid supplied from the inlet opening side comes into contact with the porous cellulose microparticles, and the microorganisms contained in the sample liquid are adsorbed onto the surfaces of the porous cellulose microparticles while the solvent of the sample liquid is removed from the outlet opening side, so that the microorganisms are concentrated on the surfaces and / or in the pores of the porous cellulose microparticles; and The porous cellulose microparticles are characterized in that, when 100 mg of dry particles are swollen in PBS(-), the porous cellulose microparticles in a swollen state at which the PBS(-) no longer falls off under their own weight have an allowable water content C of 1.9 g or more, which is the mass of PBS(-) of the porous cellulose microparticles, and the free water ratio ((C-E) / E), obtained by subtracting the mass E of PBS(-) obtained by centrifuging the porous cellulose microparticles in the swollen state at 1000 G for 1 minute from the allowable water content C, is 2.9 or more. The porous cellulose microparticles used in this second embodiment can be the same as those used in the first embodiment described above. A schematic diagram of this concentration container is shown in Figure 5.

[0035] As used herein, the term "microorganism" is not particularly limited and broadly encompasses various bacteria, such as Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella, various viruses, fungi, protozoa, and rickettsia. In this embodiment, these microorganisms are the subject of concentration. Furthermore, sample liquids containing bacteria, viruses, and the like are not particularly limited and broadly encompass patient specimens (e.g., urine and body fluids), food samples (e.g., liquid samples and bacterial or viral extracts obtained by stomachers), and environmental samples (e.g., water quality).

[0036] In this specification, the term "concentration carrier" refers to a carrier used to concentrate a target substance from a solution containing the target substance by attaching the target substance to the carrier and reducing the supernatant. The target substance to be removed may include bacteria, various viruses, proteins, etc. other than the target substance to be concentrated, but is not particularly limited.

[0037] [Adsorption amount per particle weight (pieces / mg)] In this specification, the adsorption amount per particle weight refers to the number of microorganisms adsorbed per dry weight of the carrier after the microorganisms are attached (pieces / mg), and the larger this value, the higher the density of the microorganisms obtained. There is no limitation on whether this high-density microorganism is used as the carrier (complex) after the microorganisms are attached, or whether the adsorption carrier and the microorganism are separated and used. In addition, there is no limitation on whether the moisture content is removed from the high-density microorganism or not.

[0038] [Concentration of Microorganisms] In one embodiment, to obtain a concentrated bacterial solution, an eluent can be passed through the high-density body to recover only the microorganisms. The lower the amount of water content carried over from the high-density body, the higher the concentration of the concentrate obtained. Known methods can be used to remove the water content. Examples include vacuum drying, centrifugation, replacement by aeration with air, and removal using a water-absorbing body. These methods are not limited to these, and are appropriately selected depending on the application. For example, centrifugation is preferred when controlling the force required to remove the water content. However, when removing the water content through simple operations without using specialized equipment, a method of aerating and replacing air using a syringe or the like is preferred. In this embodiment, centrifugation at 1000 G for 1 minute corresponds to the aeration of 1 to 2 mL of air. An appropriate eluent can be selected to obtain a live bacterial solution.

[0039] The eluent can be selected based on its salt concentration, preferably 0.1 M or higher, more preferably 0.3 M or higher. However, because too high a salt concentration can adversely affect the recovered bacteria, it is preferably 1 M or lower. Second, the ionic species of the eluent can be selected from cations or anions. Generally, the higher the valence, the higher the elution ability. However, for example, sodium borate loses elution ability, so the selection of the ionic species is important. On the other hand, it is necessary to consider whether the solubility is such that an ion concentration within the above range can be obtained in the environment in which the present invention is implemented (e.g., 0°C to 40°C). In one embodiment taking the above into consideration, a salt containing magnesium ions is preferred, more preferably magnesium chloride. Third, the pH of the eluent is important for maintaining the survival of microorganisms and for inducing electrostatic interactions between the microorganisms and the surface charge of the adsorption carrier in a direction favorable for dissociation. A buffer solution can also be added to the eluent to maintain a constant pH. In one embodiment, for Escherichia coli and Staphylococcus aureus, a pH of 2 to 10 is preferred, more preferably a pH of 4 to 8. However, the pH should be appropriately selected depending on the microbial species, and is not limited to this range. The eluent may contain surfactants, enzymes, chaotropic salts, sugars, amino acids, polymers, etc. depending on the type of use of the recovered bacteria.

[0040] [Concentration of Microbial Components] In another embodiment, to obtain concentrated surface or internal components of bacteria or viruses, the high-density body can be contacted with a lytic component and the target eluted component can be recovered. The lower the moisture content carried over from the high-density body, the higher the concentration of the target component can be obtained, and the moisture content can be removed using the methods described above. The lytic component is appropriately selected based on the bacterial or viral species and is not limited to specific species. Taking advantage of the excellent chemical strength (e.g., hydrolysis resistance, chemical resistance) of porous cellulose microparticles, it can contain, for example, alkaline solutions, organic solvents, surfactants, enzymes, chaotropic salts, sugars, amino acids, polymers, etc. Furthermore, taking advantage of the excellent physical strength (e.g., heat resistance, abrasion resistance) of porous cellulose microparticles, the target component can be eluted by subjecting the high-density body to ultrasonic or heat treatment.

[0041] [Separation of impurity components by washing] High-density bodies of microorganisms can be washed with an appropriate solution in any step. One example of washing can be performed for the purpose of removing unwanted impurity components from patient samples or food components. Alternatively, washing can be performed for the purpose of selectively removing only specific microbial species or cell types by selecting an appropriate eluent. Furthermore, washing can be performed for the purpose of B / F separation of the detection reagent to be used when the high-density bodies are directly subjected to testing (e.g., bacterial detection by immunoassay). The washing solution is not limited to a specific bacterial or viral species and is selected appropriately. However, taking into consideration the selection range of the eluent and / or lytic component described above, a composition that does not elute or lyse the microorganisms to be tested is preferred.

[0042] The present invention will be specifically described below with reference to examples and comparative examples, but the present invention is not limited to these examples. First, methods for measuring the physical properties of the structures used in the examples and comparative examples, and methods for preparing the bacterial suspension will be described.

[0043] (1) Allowable Water Content (g) and Free Water Ratio The tare weight A (g) of an empty column (PolyPrep (registered trademark), manufactured by Bio-Rad Laboratories, Inc.) is measured. The moisture content of the porous particle sample is measured using a heat-drying moisture meter (MX-50, manufactured by A&D), and the porous particle sample is packed into the column so that the dry weight is 100 mg. 20 mL of PBS(-) (166-23555, manufactured by Fujifilm Wako Pure Chemical Industries) is added, and the PBS(-) drips from the bottom of the column due to gravity. The column weight B (g) is measured at the point when the PBS(-) stops dripping. The allowable water content C (g) per 100 mg of particles is calculated using the following formula (1): Allowable water content C (g) per 100 mg of particles = B - (0.1 + A)...Equation (1) Next, the above column is centrifuged at 1000 G for 1 minute, and the column weight D (g) after dehydration by centrifugal force is measured. The mass (g) of PBS(-) after centrifugation (E: bound water content) per 100 mg of particles is calculated using the following equation (2): Bound water content E (g) = D - (0.1 + A)...Equation (2) The free water ratio F is calculated using the following equation (3): F = (C - E) / E...Equation (3) An overview of the method for measuring the allowable water content (g) and free water ratio is shown in Figure 2.

[0044] (3) Average Particle Diameter (μm) The average particle diameter is the average particle diameter of at least 20 porous particles measured under a microscope at an appropriate magnification after swelling the porous particles with water or physiological saline and removing excess water.

[0045] (4) Average pore size (μm) of pores The average pore size of pores is the average pore size obtained by swelling porous particles with water or physiological saline, removing excess water, and measuring the interlaminar distance of 20 or more particles using a microscope at an appropriate magnification. When the pore size of each pore is not a perfect sphere or circle, the pore size is defined as the shortest diameter.

[0046] (5) Specific surface area (m 2 / g) The surface area is the surface area measured by the BET method, and the specific surface area is the surface area per 1 g of dry particles.

[0047] (6) Charge capacity: 5.0 g of sample was weighed out and washed twice with 30 mL of distilled water using a funnel and a suction filter, and then substituted with water. -4 The sample is washed four times with 50 mL of 10% sodium sulfate solution using a funnel and a suction filter. The filtrate is then washed four times with 50 mL of 10% sodium sulfate solution, and the filtrate is recovered. Two mL of 0.1 N potassium chromate solution is added to the recovered filtrate and stirred. The sample is then titrated with a 1 N silver nitrate solution to determine the titer A (mL). Separately, the sample that was washed with the 10% sodium sulfate solution in the above process is washed four times with 50 mL of pure water, dried at 105°C for 15 hours or more, and weighed on a balance to determine the dry weight B (g). The charge capacity is calculated using the measured titer G (mL) and the dry weight H (g) of the sample using the following formula (4): Charge capacity (mmol / g) = 1 (N) (mol / L) × G / H... formula (4)

[0048] (7) Dry particle weight (mg) per swollen volume (mL) The moisture content of a porous particle sample is measured using a heat-drying moisture meter (MX-50, manufactured by A&D), and the porous particle sample is packed into an empty column (PolyPrep (registered trademark), manufactured by Bio-Rad Laboratories, Inc.) so that the dry weight is 100 mg. 20 mL of PBS(-) is added, and the PBS(-) drips from the bottom of the column due to gravity. The particle packing volume I (mL) at the point when it stops dripping is measured using the column scale. The dry particle weight J (mg / mL) per swollen volume is calculated using the following formula (5): Dry particle weight J (mg / mL) per swollen volume = 100 / I... formula (5)

[0049] (8) Method for preparing various bacterial solutions (Preparation of E. coli solution) 15 μL of E. coli strain (ATCC No. 25922) was inoculated into 1 mL of autoclave-sterilized 3% Trypticase Soy Broth liquid medium (211768, manufactured by BD) and cultured with shaking at 37°C under aerobic conditions for 16 hours. Centrifuged at 2000 × g for 5 minutes, the supernatant was removed, and 1 mL of distilled water was added to obtain a bacterial solution with an OD (600 nm) of 1 (approximately 1e9 cfu / mL). The resulting E. coli solution was diluted appropriately with physiological saline for use. (Preparation of Staphylococcus aureus Solution) 15 μL of Staphylococcus aureus strain (ATCC No. 25923) was inoculated into 1 mL of autoclave-sterilized 3% Trypticase Soy Broth liquid medium (211768, manufactured by BD) and cultured with shaking at 37°C under aerobic conditions for 16 hours. Centrifuged at 2000 × g for 5 minutes, the supernatant was removed, and 1 mL of distilled water was added to obtain a bacterial solution with an OD (600 nm) of 1 (approximately 1e9 cfu / mL). The resulting Staphylococcus aureus solution was diluted appropriately with physiological saline for use. (Preparation of E. coli T1 phage) 15 μL of E. coli strain (JM109 No. 25922) was inoculated into 10 mL of autoclave-sterilized 3% Trypticase Soy Broth liquid medium (211768, manufactured by BD), and after 16 hours of shaking culture at 37 ° C. under aerobic conditions, 100 μL of E. coli T1 phage (T1, NBRC20001) diluted with 1% Proteose Peptone aqueous solution (SIGMA, P0431-250G) was inoculated and further cultured for 7 hours. After removing bacterial cells from the culture medium using a 0.22 μm filter, molecular weights of 100 kDa or less were excluded by ultrafiltration, and the number of phage plaques was measured using the double agar plate method, which was 3.7e9 pfu / mL. The resulting E. coli T1 phage solution is diluted appropriately with physiological saline before use.

[0050] (9) Preparation of porous cellulose microparticles and comparative particles The names, sources of purchase, physical properties, etc. of comparative particles (circle 1) to (circle 4) prepared as comparative examples are shown in Table 1 below.

[0051]

[0052] [Preparation of Porous Cellulose Microparticles (also referred to herein as "Particles of the Invention") (A)] A cellulose-copper-ammonium solution with a viscosity of 2 poise at 5°C, a cellulose concentration of 2%, a copper concentration of 1.2%, and an ammonia concentration of 4%, prepared using purified linters as a raw material, was sprayed in an amount of 500 mL with nitrogen gas using a two-fluid nozzle and stirred into 10 L of silicone oil (SH200, 1.5 cs, Toray Industries, Inc.) cooled to -40°C. The silicone oil was heated to -20°C and stirred for 20 minutes, after which 10 L of 40% sulfuric acid cooled to -35°C was added. After adding the sulfuric acid, stirring was continued for 8 hours, the temperature was raised to 20°C, the silicone oil was removed, and the remainder was extracted and washed with water to obtain porous cellulose microparticles. A particle sample with an average particle diameter of 200 μm to 280 μm was obtained using a classification sieve. Next, 6.66 g of particle sample was weighed and added to a reaction solution (pure water: 267.77 g, sodium dodecyl sulfate: 0.016 g, 48% sodium hydroxide aqueous solution: 13.05 g, epichlorohydrin: 3.845 g) and stirred for 1 h in a thermostatic bath at 60 ° C. The sample was washed three times with 8000 mL of pure water by suction filtration to obtain crosslinked cellulose microparticles. The entire amount of the obtained crosslinked cellulose microparticles was added to a reaction solution (pure water: 217.30 g, anhydrous sodium sulfate: 88.83 g, sodium dodecyl sulfate: 0.016 g, 2-(diethylamino)ethyl chloride hydrochloride aqueous solution (50%): 11.08 g) and stirred. Then, a reaction initiation solution (48% sodium hydroxide aqueous solution: 5.33 g, pure water: 6.83 g) was added and gently stirred, and the reaction was allowed to proceed for 1 h while stirring in a thermostatic bath at 70 ° C. After 1 hour, a neutralization solution (3.14 g of 36% hydrochloric acid, 3.14 g of pure water) was added and stirred, and the sample was washed three times with 8000 mL of pure water by suction filtration to obtain particles of the present invention. The obtained particles had an average particle size of 240 μm, an average pore size of 30 μm, through-holes with an average pore size of 30 μm or less, and a surface area (specific surface area) per 1 g of dried particles of 1.1 m 2 / g, and the charge capacity was 1.80 mmol / g. The porous cellulose microparticles prepared in this manner are designated (A). [Preparation of porous cellulose microparticles (B) to (D) with different charge capacities] After obtaining crosslinked cellulose microparticles using the same procedure as above, 6.66 g of the particles were added to a reaction solution (pure water: 217.30 g, anhydrous sodium sulfate: 88.83 g, sodium dodecyl sulfate: 0.016 g, 2-(diethylamino)ethyl chloride hydrochloride aqueous solution (50%): 2.325 g) and stirred. Then, a reaction initiation solution (48% sodium hydroxide aqueous solution: 1.930 g, pure water: 6.83 g) was added and gently stirred, and the mixture was allowed to react for 1 hour while stirring in a thermostatic chamber at 70 °C. After 1 hour, a neutralization solution (3.14 g of 36% hydrochloric acid, 3.14 g of pure water) was added and stirred. The sample was washed three times with 8000 mL of pure water by suction filtration to obtain particles with a charge capacity of 0.55 mmol / g. The porous cellulose microparticles thus prepared are designated (B). Next, after obtaining particles with a charge capacity of 0.55 mmol / g again, 6.66 g of the particles were added to a reaction solution (217.30 g of pure water, 88.83 g of anhydrous sodium sulfate, 0.016 g of sodium dodecyl sulfate, 2.325 g of 50% aqueous 2-(diethylamino)ethyl chloride hydrochloride solution) and stirred. Then, a reaction initiation solution (1.930 g of 48% aqueous sodium hydroxide solution, 6.83 g of pure water) was added and gently stirred, and the reaction was allowed to proceed for 1 hour while stirring in a constant temperature bath at 70 °C. After 1 hour, a neutralization solution (3.14 g of 36% hydrochloric acid, 3.14 g of pure water) was added and stirred. The sample was washed three times with 8000 mL of pure water by suction filtration to obtain particles with a charge capacity of 1.04 mmol / g. The porous cellulose microparticles thus prepared are designated (C). After further obtaining particles with a charge capacity of 1.80 mmol / g, 6.66 g of particles were added to a reaction solution (217.30 g of pure water, 88.83 g of anhydrous sodium sulfate, 0.016 g of sodium dodecyl sulfate, 11.08 g of 50% aqueous 2-(diethylamino)ethyl chloride hydrochloride solution) and stirred. A reaction initiation solution (5.33 g of 48% aqueous sodium hydroxide solution, 6.83 g of pure water) was then added and gently stirred, followed by a reaction with stirring for 1 hour in a constant temperature bath at 70 °C.After 1 hour, a neutralization solution (3.14 g of 36% hydrochloric acid, 3.14 g of pure water) was added and stirred, and the sample was washed three times with 8000 mL of pure water by suction filtration to obtain particles with a charge capacity of 2.60 mmol / g. The porous cellulose microparticles prepared in this manner are designated as (2). From the above, four porous cellulose microparticles with different charge capacities were obtained.

[0053] [Example 1: Measurement of dry particle weight per swollen volume] The dry particle weight (mg) per swollen volume (mL) was measured for the particles of the present invention (A) and the comparative particles (circle 1) to (circle 4). The results are shown in Table 2 below.

[0054]

[0055] The particles of the present invention had the smallest dry particle weight per swollen volume compared to the comparative particles, and even at a small weight, they absorbed water and swelled significantly. This significant difference was observed even when compared to the comparative particles (Maruichi), which had a similar average pore size upon swelling, suggesting that the characteristics of the crosslinked cellulose material and structure contributed to this.

[0056] [Example 2: Measurement of allowable moisture content C and free moisture ratio F] To measure the ease with which liquid or gas permeates porous particles, the allowable moisture content C and free moisture ratio F were measured for the particles (a) to (b) of the present invention and the comparative particles (circle 1) to (circle 4). The results are shown in Table 3 below.

[0057]

[0058] A comparison of the allowable moisture content shows that the particles of the present invention retain significantly more moisture when packed into an empty column than the comparative particles (circle 1) to (circle 4). On the other hand, a gentle centrifugal force of 1000 G for 1 minute easily liberates moisture from the particles and replaces it with gas, revealing that the particles have the physical property of having a significantly higher free moisture ratio. In other words, the particles of the present invention are highly permeable to liquids or gases due to their high porosity.

[0059] Example 3: Measurement of Escherichia coli adsorption performance by vortex method Based on the dry particle weight per swollen volume determined in Example 1, the particles of the present invention (A) and comparative particles (Circle 1) to (Circle 4) were weighed so that the swollen particle volume was uniform as shown in Table 4 below (for particles in a wet or suspended state, the weight was measured divided by the water content). 10 mL of PBS(-) was added to each particle, and the mixture was sterilized in an autoclave at 121°C for 20 minutes. The mixture was centrifuged at 1000 G for 1 minute, the supernatant was aspirated off, and 10 mL of sterilized PBS(-) was added. After washing and charge balancing of the particles twice, the mixture was diluted to a final volume of 4 mL. The suspensions of the particles of the present invention (A) and comparative particles (Circle 1) to (Circle 4) prepared in this manner are shown in Figure 3.

[0060]

[0061] 0.2 mL of the thus-prepared uniformly dispersed particle suspension (corresponding to a swollen particle volume of 0.056 mL) was measured into a 2 mL sterile sampling tube, and 0.8 mL of a sample prepared by diluting the E. coli solution prepared in (8) above at 1e4 or 1e6 times was added. The mixture was then vortexed at maximum strength for 30 seconds or 2 minutes using a TUBE MIXER (eccentric vibration type, MT-360, manufactured by TOMY). After centrifugation at 400 G for 1 minute, the supernatant was diluted with saline to a dilution rate K (times) suitable for plate culture, and 100 μL was inoculated onto a standard agar medium (8-MR22, manufactured by Poremedia). Culture was performed at 35°C for 48 hours, and the colony count L (cfu / mL) was measured. The same procedure as above was repeated except that 0.2 mL of saline was used instead of the particle suspension, and the colony count M (cfu / mL) was measured. The amount of adsorbed E. coli per dry particle weight and the adsorption rate were calculated using the following formulas (5) and (6), respectively. Note that the values ​​shown in Table 4 are used for the dry particle weight in the formulas below. Adsorption amount per dry particle weight N (number / mg) = K × (M - L) / (dry particle weight / 20) ... formula (5) Adsorption rate (%) = (M - L) / M × 100 ... formula (6) The results are shown in Table 5 below.

[0062]

[0063] Compared with the comparative particles (circle 1) to (circle 4), the particles of the present invention (A) maintained a high adsorption rate over a wide range of E. coli concentrations (1e3 to 1e5 cfu / mL). To achieve high adsorption capacity, E. coli must be adsorbed deep into the particles, which requires the particles to have a sufficiently large pore size. Even compared to the comparative particles (circle 1), which have a similar average pore size upon swelling, the particles of the present invention (A) exhibited a significantly higher adsorption rate. This indicates that the particles of the present invention (A) achieve a highly efficient adsorption of E. coli not possible with conventional particles, due not only to their pore size but also to the high permeability achieved by their high porosity. Furthermore, the comparative particles (circle 3) and (circle 4), which exhibited adsorption rates of 70% or higher after 2 minutes of vortexing, exhibited a significant drop in adsorption rate after 30 seconds, whereas the particles of the present invention (A) maintained the same performance even after 30 seconds, demonstrating that the structural features described above also contribute to rapid adsorption. Figure 4 shows an example of an SEM image of porous cellulose microparticles after E. coli adsorption. Furthermore, although the particles of the present invention (i) have a high porosity, they exhibit highly efficient adsorption performance when compared with the comparative particles (circle 1) to (circle 4) assuming a uniform swollen particle volume, and therefore it is possible to obtain a high-density body of microorganisms with a significantly large adsorption amount N (units / mg) per dry particle weight.

[0064] Example 4: Measurement of E. coli adsorption performance by column method An empty column (Presh-SPE®, ASTY) was filled with 0.2 mL of the uniformly dispersed particle suspension prepared in Example 3, and excess PBS(-) was removed by suction from the outlet to prepare a particle-filled column. 10 mL of a sample prepared by diluting the E. coli solution prepared in (8) above 1e3 times (hereinafter referred to as the 1e3-fold diluted E. coli solution or E. coli sample) was passed through the column at 100 μL / sec using a disposable syringe, and the liquid that passed through the column (hereinafter referred to as the flow-through) was collected. Next, 1 mL of saline was passed through the column to wash the E. coli adsorption column, and then 10 mL of air was passed through to remove free moisture. The above-mentioned E. coli sample and flow-through were diluted with physiological saline to a dilution rate suitable for plate smear culture, and 100 μL was inoculated onto a standard agar medium (8-MR22, Poremedia). Culture was performed at 35°C for 48 hours, and the number of colonies was counted. The amount of E. coli adsorbed per dry particle weight, T, and the adsorption rate, S, were calculated using the following formulas (7) and (8), respectively. Note that the values ​​shown in Table 4 were used for the dry particle weight in the formula below. E. coli sample: bacterial concentration O (cfu / mL) = number of inoculated colonies (cfu / mL) × dilution rate Q Flow-through: bacterial concentration P (cfu / mL) = number of inoculated colonies (cfu / mL) × dilution rate R Adsorption amount per dry particle weight S (pieces / mg) = (O - P) × 10 (mL) / (dry particle weight / 20) ... formula (7) Adsorption rate T (%) = (O - P) / O × 100 ... formula (8) The results are shown in Table 6 below.

[0065]

[0066] In the column method, the particles of the present invention (A) also achieved a higher adsorption rate T than the comparative particles (Circle 1) to (Circle 4), demonstrating that they can exhibit high adsorption performance regardless of whether they are used in the vortex method (i.e., a method of contacting particles with microorganisms by uniform dispersion) or the column method (i.e., a method of contacting particles with microorganisms by solid-phase separation). Furthermore, it was confirmed that in the column method, the particles can be washed after adsorption of E. coli, and that free moisture remaining on the particles can be easily removed by aeration.

[0067] Example 5: Measurement of Adsorption Performance of Microorganisms Other Than E. coli Using the Column Method Using the same procedures as in Examples 3 and 4, the adsorption amount S and adsorption rate T of the microorganism per dry particle weight were calculated for the E. coli, Staphylococcus aureus, and E. coli T1 phage (virus) prepared in (8) above. However, for the measurement of E. coli T1 phage, 10 μL of the diluted solution was mixed with 30 μL of 1e9 cfu / mL JM109 E. coli and 5 mL of soft agar, solidified on a standard agar medium, and the number of plaques after 24 hours of incubation at 35°C was measured. The adsorption rate T and adsorption amount S per dry particle weight of E. coli T1 phage were calculated by substituting cfu / mL for pfu / mL. The results are shown in Table 7 below.

[0068]

[0069] The results shown in Table 7 demonstrate that the particles of the present invention (A) can adsorb Escherichia coli, a Gram-negative bacterium, and Staphylococcus aureus, a Gram-positive bacterium, with high efficiency regardless of the bacterial species. Furthermore, it was shown that the particles of the present invention (A) can adsorb not only bacteria but also viruses as well.

[0070] Example 6: Method for recovering bacteria from E. coli adsorption particles (high-density bodies of microorganisms) obtained by the column method and obtaining a concentrate of E. coli. 1 mL of 1 M magnesium chloride was passed through the E. coli adsorption column from which the free water had been removed, finally obtained in Example 4, as an eluent, and the flow-through of the eluent was collected. The flow-through was diluted with physiological saline to a dilution rate V, a concentration suitable for plate smear culture, and 100 μL was inoculated onto a standard agar medium (8-MR22, Poremedia). The medium was cultured at 35°C for 48 hours, and the number of colonies was counted. The concentration factor W of E. coli was calculated using the following formula (9): Eluent flow-through: bacterial concentration U (cfu / mL) = number of inoculated colonies (cfu / mL) × dilution rate V Concentration factor W (fold) = bacterial concentration U of eluent flow-through / bacterial concentration O of E. coli sample... formula (9) The results are shown in Table 8 below.

[0071]

[0072] The particles of the present invention (a) achieved a concentration factor of 1 or more, demonstrating that bacteria can be concentrated by passing an eluent through them.

[0073] Example 7: Measurement of adsorption rate T (%) and concentration factor W (times) of particles (a) to (d) of the present invention having different charge capacities

[0074] Using the same procedures as in Examples 4 and 5, the adsorption rate T (%) and concentration factor W (times) were measured for the four particles (a) to (d) of the present invention having different charge capacities. The results are shown in Table 9 below.

[0075]

[0076] The results shown in Table 9 indicate that an adsorption rate of 50% or more can be obtained when the charge capacity of the porous cellulose microparticles is 0.55 to 2.6 mmol / g. It was also found that the concentration factor W is more preferably when the eluent is 1 M magnesium chloride and the charge capacity is 1.04 to 2.6 mmol.

[0077] Example 8: Measurement of adsorption rate T (%) and concentration factor W (times) of particles (a) of the present invention depending on the bacterial concentration, flow rate, and eluent volume of different E. coli samples Using a column packed with 0.2 mL of particle suspension of particles (a) of the present invention (2 mg dry particle weight), the adsorption rate T (%) and concentration factor W (times) depending on the bacterial concentration O, flow rate, and eluent volume of different E. coli samples were measured using the same procedures as in Examples 4 and 5. The results are shown in Table 10. Note that the volume of eluent in Table 10 refers to the volume (mL) of 1 M magnesium chloride passed through as the eluent.

[0078]

[0079] The results shown in Table 10 indicate that a column packed with 0.2 mL of particle suspension of the particles (a) of the present invention (corresponding to 2 mg of dry particle weight and 0.056 mL of swollen particle volume) can pass an E. coli sample through at a flow rate of 10 to 1000 mL and can recover the E. coli sample with an eluent volume of 0.1 to 1 mL, and that within this range, a high adsorption rate T and concentration factor W similar to those of Examples 4 and 5 can be maintained. Furthermore, this column can adsorb an E. coli sample with a bacterial concentration O of up to 1e8 cfu / mL, and a particularly high adsorption rate T was obtained at a concentration of 1e1 to 1e7 cfu / mL.

[0080] The concentration method and concentration container of the present invention can concentrate microorganisms using specific porous cellulose microparticles with high porosity and dehydration rate, which are indicated by the allowable water content and free water ratio, and therefore can obtain a high amount of microorganisms adsorbed per dry weight of particles, a high-density mass of microorganisms, and can also recover high concentrations of microorganisms from the obtained high-density mass of microorganisms. Therefore, the concentration method and concentration container of the present invention can be suitably used in microbial testing, etc.

Claims

1. A method for concentrating microorganisms in a sample liquid containing microorganisms in a solvent by using porous cellulose microparticles having a large number of pores therein, the method comprising the following steps: a step of bringing the sample liquid into contact with the porous cellulose microparticles to adsorb the microorganisms contained in the sample liquid onto the surface of the porous cellulose microparticles; and a liquid extraction step of removing the solvent from the sample liquid to concentrate the microorganisms on the surface and / or within the pores of the porous cellulose microparticles; wherein the porous cellulose microparticles have an allowable water content C, which is the mass of PBS(-) of the porous cellulose microparticles in a swollen state where 100 mg of dry particles are swollen with PBS(-) and the PBS(-) no longer falls off under their own weight, of 1.9 g or more, and a free water ratio ((C-E) / E), obtained by subtracting the mass E of PBS(-) after centrifuging the porous cellulose microparticles in the swollen state at 1000 G for 1 minute from the allowable water content C, divided by E, of 2.9 or more.

2. The method according to claim 1, further comprising the following step: after the liquid removal step, recovering the microorganisms concentrated on the surface and / or within the pores of the porous cellulose microparticles in the elution liquid using the elution liquid.

3. The method according to claim 1 or 2, further comprising the step of washing the porous cellulose microparticles with a liquid or gas between each of said steps.

4. The method according to claim 1 or 2, wherein the average particle diameter of the porous cellulose microparticles is 100 μm or more and 1000 μm or less.

5. The method according to claim 1 or 2, wherein the average pore size of the pores in the porous cellulose microparticles is 1 μm or more and 100 μm or less.

6. The method according to claim 1 or 2, wherein the porous cellulose microparticles form a continuous pore structure in which adjacent pores are interconnected by openings in the membrane that separate them.

7. The surface area (specific surface area) of the porous cellulose microparticles per 1 g of dry particles is 0.3 m 2 / g or more 4.4m 2 The method according to claim 1 or 2, wherein the molecular weight is 1 / g or less.

8. The method according to claim 1 or 2, wherein the cellulose constituting the porous cellulose microparticles has a cationic functional group and a charge capacity of 0.5 mmol / g or more and 5.0 mmol / g or less.

9. The method according to claim 8, wherein the cationic functional group is at least one selected from the group consisting of a primary amino group, a secondary amino group, and a tertiary amino group.

10. The method of claim 9, wherein the cationic functional group is an N,N-diethylaminoethyl (DEAE) group.

11. The method according to claim 1 or 2, wherein the cellulose constituting the porous cellulose microparticles is intermolecularly crosslinked.

12. The method according to claim 1 or 2, wherein the cellulose constituting the porous cellulose microparticles is made of cuprammonium regenerated cellulose.

13. The method of claim 1 or 2, wherein the microorganism is a bacterium or a virus.

14. A concentration container for use in a method for concentrating microorganisms in a sample liquid containing microorganisms in a solvent by using porous cellulose microparticles having a large number of pores therein, the concentration container comprising: a cylindrical body having an inlet opening and an outlet opening; a filter provided on the outlet opening side; and porous cellulose microparticles filled in the space above the filter; wherein the sample liquid supplied from the inlet opening side comes into contact with the porous cellulose microparticles, and the microorganisms contained in the sample liquid are adsorbed onto the surface of the porous cellulose microparticles while the solvent of the sample liquid is removed from the outlet opening side, so that the microorganisms are concentrated on the surface and / or within the pores of the porous cellulose microparticles, and The porous cellulose microparticles are characterized in that, when 100 mg of dry particles are swollen with PBS(-), the porous cellulose microparticles in a swollen state at which the PBS(-) no longer falls off under their own weight have an allowable water content C of 1.9 g or more, and the free water ratio ((C-E) / E) obtained by subtracting the mass E of PBS(-) obtained by centrifuging the porous cellulose microparticles in the swollen state at 1000 G for 1 minute from the allowable water content C is 2.9 or more.

15. A concentration container according to claim 14, wherein the average particle diameter of the porous cellulose microparticles is 100 μm or more and 1000 μm or less.

16. A concentration container according to claim 14 or 15, wherein the average pore size of the pores in the porous cellulose microparticles is 1 μm or more and 100 μm or less.

17. A concentration container as described in claim 14 or 15, characterized in that the porous cellulose microparticles form a continuous pore structure in which adjacent pores are interconnected by openings in the membrane that separate them.

18. The surface area (specific surface area) of the porous cellulose microparticles per 1 g of dry particles is 0.3 m 2 / g or more 4.4m 2 The concentration container according to claim 14 or 15, wherein the concentration is 0.1 to 1.5% by mass %.

19. A concentration container as described in claim 14 or 15, wherein the cellulose constituting the porous cellulose microparticles has a cationic functional group and a charge capacity of 0.5 mmol / g or more and 5.0 mmol / g or less.

20. The concentration container according to claim 19, wherein the cationic functional group is at least one selected from the group consisting of a primary amino group, a secondary amino group, and a tertiary amino group.

21. The concentrator vessel of claim 20, wherein the cationic functional group is an N,N-diethylaminoethyl (DEAE) group.

22. A concentration container as described in claim 14 or 15, wherein the cellulose constituting the porous cellulose microparticles is intermolecularly crosslinked.

23. A concentrating container as described in claim 14 or 15, wherein the cellulose constituting the porous cellulose microparticles is made of cuprammonium regenerated cellulose.

24. The concentration container according to claim 14 or 15, wherein the microorganism is a bacterium or a virus.

25. A method for concentrating microorganisms in a sample liquid containing microorganisms in a solvent by using porous cellulose microparticles having a large number of pores therein, comprising the following steps: a step of bringing the sample liquid into contact with the porous cellulose microparticles in a concentration vessel equipped with a cylindrical body having an inlet opening and an outlet opening; a filter provided on the outlet opening side; and porous cellulose microparticles filled in the space above the filter, thereby adsorbing the microorganisms contained in the sample liquid onto the surface of the porous cellulose microparticles; and a step of aerating the sample liquid to remove the solvent and concentrate the microorganisms on the surface and / or in the pores of the porous cellulose microparticles; The porous cellulose microparticles have an allowable water content C, which is the mass of PBS(-) of the porous cellulose microparticles in a swollen state at which 100 mg of dry particles are swollen with PBS(-) and the PBS(-) no longer falls off under its own weight, of 1.9 g or more, and a free water ratio ((C-E) / E), which is obtained by subtracting the mass E of PBS(-) obtained by centrifuging the porous cellulose microparticles in the swollen state at 1000 G for 1 minute from the allowable water content C, by E, is 2.9 or more.

26. The method for concentrating microorganisms according to claim 25, wherein the aeration is performed by aerating air through an inlet opening of the concentration container using a syringe.

27. A method for concentrating microorganisms as described in claim 26, wherein the volume of the air is 0.5 mL or more and 1 mL or less per 1 mg of the porous cellulose microparticles.

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