Screening method and solution

Abstract

Provided is a screening method with which a microorganism having a desired activity can be selected with high throughput. A screening method for searching for a microorganism (12) having a desired activity includes: a particle formation step for forming at least one detection layer (13) comprising a gel or an aqueous solution and containing a desired activity detector body (14) on the outside of a core particle (11) comprising a solid culture medium containing the microorganism (12), thereby producing a multi-layer particle (10); and a selection step for selecting a multi-layer particle (10) on the basis of a signal indicating a desired activity detection result by the detector body (14). In the method, when the detection layer (13) is formed as a layer comprising the aqueous solution, the detection layer (13) is formed as an outermost layer of the multi-layer particle (10) in a hydrophobic solvent (20).

Description

Screening methods and solutions

[0001] The present invention relates to a method for screening microorganisms using multi-layered particles and a solution containing multi-layered particles.

[0002] The development of new compounds useful for drug discovery such as new antibacterial substances, enzymes that enable new reactions, and unused bioresources such as the microorganisms that produce them are recognized as important challenges. As an example, it is expected that unused microorganisms present in the environment that have not been discovered because they have not been cultured, and microorganisms that have been modified from the properties of existing microorganisms that have already been cultivated, can be used as resources.

[0003] However, the proportion of microorganisms having a specific target activity among the microorganisms present in the environment is usually extremely small, and large-scale screening of the order of one million samples is required to isolate microorganisms having the target activity from the environment and to screen the cultured microorganisms for the purpose of modifying or improving the properties of the cultured microorganisms.

[0004] For example, Non-Patent Document 1 discloses a method for culturing microorganisms using gel microparticles. According to this culturing method, different microorganisms can be cultured in each of a large number of gel microparticles dispersed in a hydrogel.

[0005] Patent Literature 1 also describes a method of agglomerating hydrogel microparticles in oil. This method allows dissolved substances and ions to pass between the hydrogel microparticles. This strengthens intercellular interactions while maintaining the separate culture of microorganisms within each hydrogel microparticle, improving culture efficiency.

[0006] Japanese Patent Application Publication No. 2017-063764

[0007] Karsten Zengler et. al., PNAS, Vol.99, No.24, p15681-15686, 2002

[0008] However, in the conventional methods using these gel particles, it is difficult to add a detection body such as cells and a substrate for detecting a target activity in a high-throughput manner after culturing the microorganisms.

[0009] An object of one aspect of the present invention is to realize a screening method that allows high-throughput selection of microorganisms having a target activity.

[0010] In order to solve the above-mentioned problems, a screening method according to one embodiment of the present invention is a screening method for searching for a target microorganism having a target activity in a population of microorganisms, and includes a particle formation step in which one or more detection layers of gel or aqueous solution containing a detector of the target activity are formed on the outside of a core particle of a solid medium containing the microorganism to obtain multi-layer particles, and a selection step in which the multi-layer particles containing the target microorganism are selected based on a signal indicating the detection result of the target activity by the detector, and in the particle formation step, when the detection layer is formed as a layer of aqueous solution, the layer of aqueous solution is formed as the outermost layer of the multi-layer particle in a hydrophobic solvent.

[0011] In order to solve the above-mentioned problems, one embodiment of the present invention provides a solution comprising multi-layer particles and a hydrophobic solvent for searching for target microorganisms having a target activity in a population of microorganisms, wherein the multi-layer particles have a core particle of a solid medium containing the microorganism, and a detection layer of a gel or aqueous solution containing a detector of the target activity, formed in one or more layers on the outside of the core particle, and when the detection layer is an aqueous solution, the detection layer is the outermost layer of the multi-layer particles.

[0012] According to one aspect of the present invention, a screening method that allows for high-throughput selection of microorganisms having a target activity can be realized.

[0013] 14 is a schematic diagram showing a solution containing multi-layer particles according to one embodiment of the present invention. FIG. 15 is a flowchart showing a screening method according to one embodiment of the present invention. FIG. 16 is a diagram showing a microscopic image of core particles containing lactic acid bacteria. FIG. 17 is a diagram showing a microscopic image of the core particles shown in FIG. 3 after cultivation. FIG. 18 is a diagram showing a microscopic image of multi-layer particles containing lactic acid bacteria and E. coli as a detection body. FIG. 19 is a diagram showing a microscopic image of multi-layer particles not containing E. coli as a detection body. FIG. 19 is a diagram showing a microscopic image of the multi-layer particles shown in FIG. 5 after cultivation. FIG. 19 is a diagram showing a dot plot and set areas in a cell sorter for a sample containing the multi-layer particles shown in FIG. 6. FIG. 20 is a diagram showing microscopic images of multi-layer particles and core particles sorted from area B shown in FIG. 8. FIG. 21 is a diagram showing a dot plot and set areas in a cell sorter for a sample containing the multi-layer particles shown in FIG. 7. FIG. 21 is a diagram showing a microscopic image of multi-layer particles sorted from area B shown in FIG. 9. FIG. 22 is a diagram showing a microscopic image of multi-layer particles sorted from area C shown in FIG. 23 is a diagram showing a microscopic image of core particles containing cellulase-producing bacteria. FIG. 23 is a diagram showing a microscopic image of multi-layer particles in which a detection layer is formed from an aqueous solution. FIG. 24 is a diagram showing microscopic images of the multi-layer particles shown in FIG. 14 before and after cultivation. FIG. 1 shows microscopic images of multi-layer particles containing soil bacteria in the core particle before and after cultivation.

[0014] Hereinafter, one embodiment of the present invention will be described. In this specification, the numerical range "A to B" means A or more and B or less.

[0015] [Outline of Screening Method] A screening method according to one embodiment of the present invention is a method for searching for a target microorganism having a target activity in a population of microorganisms.

[0016] It is believed that there exist microorganisms in the environment that have useful activities that have not been discovered until now because they are difficult to cultivate artificially. However, there have been major bottlenecks in the search for and acquisition of useful microorganisms that produce useful enzymes or compounds. One of the bottlenecks is that the proportion of microorganisms in the environment that have a specific target activity is extremely low.

[0017] In other words, isolating a single type of microorganism from the environment or screening mutant strains for the purpose of modifying and improving the properties of a cultured microbial strain requires a scale of one million samples. Furthermore, analyzing the detailed substance production capabilities of microorganisms (such as the type and amount of substances produced, as well as the level and tendency of gene expression) is extremely difficult to do efficiently using existing technologies because there are so many types of microorganisms to target.

[0018] Therefore, there is a need for a technology for screening microorganisms having a target activity at an ultra-high throughput that far exceeds existing technologies. Similarly, there is a need for ultra-high throughput analysis and screening methods for breeding selected useful microorganisms, i.e., for creating mutant strains and further screening for mutant strains with improved functions and / or activities.

[0019] However, simply increasing the number of samples that can be measured is not sufficient because detecting the specific activity of a candidate microorganism requires the addition of a separate additive containing the analyte required for detecting the specific activity, which requires high-throughput operations.

[0020] In particular, it is often necessary to culture a microorganism for a certain period of time to allow the expression of a specific activity. Furthermore, a single cell is not sufficient to detect the expression of a specific activity, and a step of growing the same microorganism and then detecting it is often necessary. Furthermore, there are many cases where an additive containing a detection substance required for detection cannot be added simultaneously with the culture of the target microorganism, for example, the additive has a negative effect on the growth and culture of the microorganism.

[0021] In such cases, it is preferable to add the additive containing the detectable substance after culturing the microorganism for a predetermined period of time. When using a container such as a multi-well microplate generally used for screening (for 100 to 1000 samples), the additive can be added relatively easily at the desired timing using existing equipment and tools.

[0022] However, in ultra-high-throughput screening of one million or more samples, the liquid volume is extremely small, making it extremely difficult to add additives to the container later. This is particularly true when using microdroplets with a diameter of 1 mm or less. For this reason, the scope of application of high-throughput screening using microdroplet technology is limited, and it has rarely been put to practical use.

[0023] According to a screening method of one embodiment of the present invention, it is possible to rapidly and relatively easily detect and select microorganisms having a target activity (target microorganisms) from a variety of microorganisms or a large number of mutant strains in the environment.

[0024] In particular, one embodiment of the present invention provides a screening method that allows for the addition of an additive containing a detectable substance necessary for detecting a specific activity at a desired timing in a screening system using microdroplet technology. This screening method is an extremely groundbreaking method in that it enables high-throughput screening while still taking advantage of the advantage of microdroplet technology, which is that it can cultivate many microorganisms that are difficult to culture on plate media.

[0025] This configuration effectively promotes the utilization of the beneficial activities of microorganisms, i.e., the effective utilization of natural resources, which contributes to the achievement of, for example, Goal 12.2 "sustainable management and efficient use of natural resources" of the Sustainable Development Goals (SDGs) advocated by the United Nations.

[0026] [Multi-layer particles] A screening method according to one embodiment of the present invention is carried out using multi-layer particles 10 shown in Fig. 1. Fig. 1 is a schematic diagram showing an example of a solution 1 containing multi-layer particles 10.

[0027] The multi-layer particle 10 has a core particle 11 and a detection layer 13. In this embodiment, an example in which the multi-layer particle 10 has these two layers will be described, but the present invention is not limited to this, and the multi-layer particle 10 may have three or more layers. For example, the multi-layer particle 10 may have a plurality of core particles 11, a plurality of detection layers 13, or may further have layers other than the core particle 11 and the detection layer 13.

[0028] The size of the multilayer particle 10 is not particularly limited, but may be, for example, 1 μm to 1 mm in diameter. When the multilayer particle 10 has a shape other than a sphere, it is preferable that the major axis, which indicates the longest distance between two imaginary points on the multilayer particle 10, is within the above-mentioned range of diameter values.

[0029] (Core Particle) The core particle 11 is formed from a solid medium containing the microorganism 12. The core particle 11 constitutes the innermost layer of the multi-layer particle 10. The size of the core particle 11 is not particularly limited, but the diameter or major axis may be, for example, 1 μm to 1 mm.

[0030] The microorganism 12 contained in the core particle 11 is not particularly limited and may be a prokaryote or a eukaryote such as a bacterium or archaea. The microorganism 12 is preferably a unicellular organism, but may also be a multicellular organism. The microorganism 12 may be a microorganism present in the environment, an artificially cultured microorganism, or a microorganism that has been artificially mutated. When microorganisms present in the environment are the target of screening, the microorganism 12 may be obtained from, for example, river water, soil, biofilm, etc.

[0031] Preferably, the core particle 11 initially contains a single microorganism 12. The microorganism 12 contained in the core particle 11 can be grown by culturing the multi-layer particle 10. The core particle 11 can contain, for example, 100 to 1000 microorganisms 12, depending on the size.

[0032] The solid medium for forming the core particle 11 may be configured as a hydrogel containing, for example, agarose, agar, gelatin, etc. The solid medium may contain a nutrient source used for culturing the microorganism 12, and may further contain other components such as a buffer.

[0033] The nutrient source contained in the solid medium is not particularly limited, but if the nutrient source required by the microorganism 12 having the target activity can be estimated, it is preferable that the solid medium contains the nutrient source. Furthermore, if the nutrient source required by the microorganism 12 having the target activity is unknown, the nutrient source contained in the solid medium may be selected according to a commonly known medium composition for microorganisms. Furthermore, if it can be estimated that the microorganism 12 having the target activity can be cultured with water even without the nutrient source, the solid medium may not contain the nutrient source.

[0034] (Detection Layer) The detection layer 13 is formed as one or more layers of a gel or aqueous solution containing a detector 14 having a target activity on the outside of the core particle 11 .

[0035] The detection layer 13 may be configured as a hydrogel containing agarose, agar, gelatin, or the like, similar to the core particle 11. Furthermore, the detection layer 13 may further contain components such as a nutrient source, similar to the core particle 11.

[0036] The detector 14 functions as a sensor for detecting the microorganism 12 having the target activity. The target activity may be the activity of a substance secreted by the microorganism 12, or may be an activity exhibited by the microorganism 12 itself. The activity of the substance secreted by the microorganism 12 may be, for example, antibacterial activity due to an antibacterial substance, cellulase activity that cleaves glycosidic bonds in cellulose or the like, or physiological activity of a substance with other physiological activity. The activity of the substance secreted by the microorganism 12 may be, for example, catalytic activity of a specific reaction due to an enzyme or the like secreted by the microorganism 12. Furthermore, an example of the activity exhibited by the microorganism 12 itself is the production activity of an industrially useful substance by the microorganism 12.

[0037] The detector 14 may be selected appropriately depending on the target activity. For example, if the target activity is antibacterial activity, the detector 14 may be a cell different from the microorganism 12 having the target activity. The cell different from the microorganism 12 having the target activity is a cell that is expected to be sensitive to the antibacterial activity of the microorganism 12, and examples of such cells include microbial and animal cells. If the detector 14 is such a cell, the microorganism 12 having the target activity can be selected by detecting whether the cell is alive or dead.

[0038] Furthermore, when the target activity is antibacterial activity that is effective against existing drug-resistant bacteria, the detection body 14 may be a known drug-resistant bacterium. The drug-resistant bacterium may be a bacterium that is resistant to a single antibacterial substance, and from the perspective of discovering new antibacterial substances, multidrug-resistant bacteria are preferable. This configuration makes it possible to screen for microorganisms 12 that exhibit antibacterial activity that is effective against existing drug-resistant bacteria.

[0039] When the detector 14 is a cell, the cell is preferably a cell into which a fluorescent substance or a luminescent substance has been introduced. In this case, a decrease in the fluorescence of the fluorescent substance or the luminescence of the luminescent substance due to a decrease in the number of cells that are the detector 14 can be used as a signal indicating that the detector 14 has detected the target activity. Furthermore, the cells used as the detector 14 may be such that the production of the introduced fluorescent substance or luminescent substance is promoted or suppressed by the target activity of the microorganism 12.

[0040] According to this configuration, when cell viability or proliferation is used as an indicator of the target activity, the target activity can be easily detected using the fluorescence emitted by the fluorescent substance or the luminescence of the luminescent substance as a signal. The cell into which the fluorescent substance or luminescent substance has been introduced may be, for example, a cell into which a gene expressing a fluorescent protein or a luminescent protein has been introduced. The luminescent protein may be a luminescent enzyme or an enzyme that catalyzes the conversion of a luminescent substance precursor into a luminescent substance.

[0041] Furthermore, whether cells are alive or dead or whether they are proliferating may be detected using a reagent or the like that can detect live or dead cells. As such, the detector 14 may be any substance that is affected in some way by the target activity possessed by the microorganism 12, and it is not essential that the detector 14 itself emits a signal that visualizes the effect. The signal that visualizes whether the detector 14 has been affected may be generated indirectly using a reagent or the like.

[0042] Such a reagent or the like for visualizing the detection result of the detection body 14 may be added to the solution in which the multi-layer particle 10 is dispersed, or another layer containing the reagent or the like may be further formed on the multi-layer particle 10. Furthermore, the detection layer 13 may contain, apart from the detection body 14, a substance for visualizing the effect on the detection body 14.

[0043] When the detection bodies 14 are cells, the detection layer 13 preferably contains a nutrient source or the like used for the proliferation of the cells. If the cells that become the detection bodies 14 can proliferate in the detection layer 13, the presence or absence of proliferation of the cells can be used as an indicator of the target activity.

[0044] When the target activity is a specific enzyme activity, the detector 14 may be a substrate for the enzyme. When the detector 14 is an enzyme substrate, the substrate is preferably labeled with a fluorescent substance, a luminescent substance, or the like, but is not limited to this. A substrate labeled with a fluorescent substance, a luminescent substance, or the like may not fluoresce or emit light as it is, but may be converted into a substance that emits fluorescence or luminescence due to the target activity of the microorganism 12. Such a target activity of the microorganism 12 may be, for example, an enzyme activity that decomposes the substrate. For example, when the detector 14 is an unlabeled substrate, the effect of the enzyme on the substrate may be detected using a reagent or the like.

[0045] Furthermore, when the target activity is a specific enzyme activity, the enzyme activity is not particularly limited, and may include, for example, industrially used enzymes such as amylase, cellulase, protease, lipase, glycosidase, and transferase.

[0046] When the target activity is cellulase activity, the detector 14 may be a fluorescent substance that emits fluorescence under the influence of cellulase activity or a luminescent substance that emits light under the influence of cellulase activity. The fluorescent substance may be a fluorescent substrate that has a glycosidic bond that serves as a substrate for cellulase and emits fluorescence upon cleavage of the glycosidic bond.

[0047] Examples of such fluorescent substrates include compounds formed by binding a fluorescent substance to cellulose, such as fluorescein di-beta-D-cellobioside. Furthermore, the luminescent substance may be a luminescent substrate that has a glycosidic bond that serves as a substrate for cellulase and emits light upon cleavage of the glycosidic bond. Examples of such luminescent substrates include compounds formed by binding a cellulose to a luminescent substance, such as resorufin β-D-cellobioside.

[0048] When using such fluorescent or luminescent substances, an increase in the fluorescence of the fluorescent substance or the luminescence of the luminescent substance can be used as a signal indicating that the detector 14 has detected the target activity when selecting microorganisms 12 having cellulase activity.

[0049] The thickness of the detection layer 13 is not particularly limited, but may be, for example, 1 μm to 1 mm. The detection layer 13 is preferably formed to have a substantially uniform thickness, but if the thickness varies depending on the position on the multi-layer particle 10, it is preferable that the average thickness be within the above-mentioned numerical range.

[0050] Two or more detection layers 13 may be formed on the outside of core particle 11. In this case, each detection layer 13 may be a layer containing a different type of detection body 14. For example, when multiple target activities are set from the perspective of efficiency, etc., it is conceivable that the period for culturing microorganisms 12 varies depending on the target activity. In this case, one detection layer 13 may be formed first to detect the corresponding target activity, and the unselected multi-layer particles 10 may be further cultured, and another detection layer 13 may be formed to redetect the corresponding target activity.

[0051] The multi-layer particle 10 may have a single core particle 11 inside the detection layer 13, or may have multiple core particles 11. If the multi-layer particle 10 has a single core particle 11, the accuracy of selecting the microorganisms 12 having the target activity can be improved. Furthermore, if the multi-layer particle 10 has multiple core particles 11, the throughput of selecting the microorganisms 12 having the target activity can be improved.

[0052] Furthermore, the detection layer 13 may be a layer formed of an aqueous solution instead of a gel. When the multi-layer particle 10 has the aqueous solution detection layer 13, the aqueous solution detection layer 13 is formed as the outermost layer of the multi-layer particle 10. Such a multi-layer particle 10 can be formed by dispersing it in a hydrophobic solvent 20. If the aqueous solution detection layer 13 is used as the outermost layer of the multi-layer particle 10, for example, the particle diameter of the multi-layer particle 10 can be easily adjusted.

[0053] Furthermore, when the outermost detection layer 13 of the multi-layer particle 10 is formed from an aqueous solution, the detection body 14 contained in the detection layer 13 may be a water-soluble compound. Such a multi-layer particle 10 is maintained in a dispersed state in the hydrophobic solvent 20. Therefore, the detection body 14, which is a water-soluble compound, hardly leaks from the detection layer 13 into the hydrophobic solvent 20.

[0054] On the other hand, when the outermost detection layer 13 of the multi-layer particle 10 is formed of a gel, the multi-layer particle 10 can be dispersed in water or an aqueous solution. In this case, if the detector 14 contained in the outermost detection layer 13 is a water-soluble compound, leakage of the detector 14 from the detection layer 13 into the water or aqueous solution may occur depending on the size of the detector 14. This phenomenon is likely to occur when the size of the detector 14 is smaller than the molecular network of the gel in the detection layer 13, for example.

[0055] Thus, when the detector 14 is a water-soluble compound, it is preferable to keep the multi-layer particle 10 dispersed in a hydrophobic solvent from the viewpoint of effectively detecting the signal of the detector 14. In such a case, the outermost detector layer of the multi-layer particle 10 can be an aqueous solution instead of a gel. An example of the detector 14 that is a water-soluble compound is an enzyme substrate. As described above, examples of such enzyme substrates include fluorescent substances that emit fluorescence under the influence of cellulase activity and luminescent substances that emit light under the influence of cellulase activity.

[0056] (Solution) The solution 1 is used to search for target microorganisms having a target activity in a population of microorganisms 12, and contains the multi-layer particles 10 and a hydrophobic solvent 20.

[0057] The hydrophobic solvent 20 is intended to be a low-polarity solvent and a liquid that is immiscible or almost immiscible with water. The hydrophobic solvent 20 is preferably a liquid with low cytotoxicity. The hydrophobic solvent 20 is preferably an oil, such as a mineral oil, a silicone oil, or a fluorine-based oil.

[0058] Furthermore, if the multi-layer particle 10 is dispersed in the hydrophobic solvent 20, the outermost detection layer 13 of the multi-layer particle 10 can be formed as a layer of aqueous solution. By forming at least one of the detection layers 13 as a layer of aqueous solution, for example, the dispersion efficiency of both the substance secreted by the microorganism 12 and the detection body 14 and the efficiency of preventing elution from the multi-layer particle 10 in the aqueous solution detection layer 13 are greatly improved compared to when it is a gel. This makes it possible to improve the detection sensitivity of the detection body 14 and shorten the incubation time after forming the detection layer 13. Furthermore, by forming the outermost detection layer 13 of the multi-layer particle 10 as a layer of aqueous solution, which has a lower viscosity than a gel, it is easy to control the particle size of the multi-layer particle 10 to be uniform.

[0059] The amount of multi-layer particles 10 contained in solution 1 is 10 ２ ~10 １０ 10 ６ ~10 ８ A concentration in the range of cells / mL is preferred from the viewpoint of promoting cell-cell interactions and screening efficiency.

[0060] The screening method according to one embodiment of the present invention can be carried out even when the multi-layer particle 10 is dispersed in an aqueous solution instead of the hydrophobic solvent 20. In this case, the detection layers 13 formed on the multi-layer particle 10 are all gel layers.

[0061] [Steps of Screening Method] Next, a screening method according to one embodiment of the present invention, which is realized by the above-described multi-layer particle 10, will be described. As shown in Figure 2, the screening method includes particle formation steps (S1 to S3) and selection steps (S4 to S5). Figure 2 is a flowchart showing an example of the screening method according to one embodiment of the present invention.

[0062] (Particle formation process) The particle formation process is a process of forming one or more detection layers 13 of gel or aqueous solution containing a detector 14 with a target activity on the outside of a core particle 11 of a solid medium containing a microorganism 12, thereby obtaining a multi-layer particle 10.

[0063] Specifically, first, a core particle 11 containing a microorganism 12 is formed (S1). To form the core particle 11, a general method for forming a water / oil (W / O) emulsion, such as that described in Patent Document 1, can be used.

[0064] For example, an aqueous solution containing a solid medium in a sol state before solidification, microorganisms 12, and a surfactant for the aqueous phase is mixed with hydrophobic solvent 20 and vigorously stirred to form a state in which aqueous phase particles are dispersed in hydrophobic solvent 20. The aqueous phase particles are gelled by refrigeration or the like to form core particles 11.

[0065] It is preferable to adjust the number (density) of the microorganisms 12 in the aqueous solution so that each core particle 11 contains a single-cell microorganism 12. In this way, a colony in which a single microorganism grows is formed in each multi-layer particle 10.

[0066] Next, it is preferable to culture the microorganisms 12 in the core particles 11 for a predetermined period of time (S2). This culture allows the microorganisms 12 to grow, thereby improving the detection sensitivity of the target activity. Furthermore, even if the target activity is an activity that is not expressed unless the microorganisms 12 grow and form colonies, it can be detected without any problems.

[0067] The core particles 11 may be cultured in a state where they are dispersed in the hydrophobic solvent 20 used when forming the core particles 11, or the solvent may be replaced with another type of hydrophobic solvent 20 or an aqueous solution. In each of the subsequent steps, the solvent may also be replaced as appropriate.

[0068] When the type of microorganism 12 having the target activity can be estimated, the predetermined culture period may be a period appropriate for that type. Furthermore, the predetermined period may be appropriately determined while observing the microorganism 12 in the core particle 11, or may be empirically determined. Similarly, the culture temperature may be appropriately determined. For example, the culture period may be 1 to 1000 hours, and the culture temperature may be 4 to 70°C.

[0069] Next, a detection layer 13 containing a detection body 14 is formed on the outside of the core particle 11 to obtain a multi-layer particle 10 (S3). The detection layer 13 can be formed by a method of forming a W / O emulsion using the core particle 11 as a core.

[0070] For example, core particles 11 that form the inner layer are mixed with a gel solution or aqueous solution in a sol state before solidification that contains detection body 14. Then, by forming a W / O emulsion using the method described above, multi-layer particles 10 are obtained in which detection layer 13 is formed on the outside of core particles 11. At this time, the number of core particles 11 contained in one multi-layer particle 10 in each formed W / O emulsion can be adjusted by adjusting the number (density) of core particles 11 relative to the volume of the gel solution or aqueous solution. For example, when forming multi-layer particles 10 having a single core particle 11, the number (density) of core particles 11 can be adjusted to form multi-layer particles 10 so that zero or one core particle 11 is encapsulated in detection layer 13.

[0071] Furthermore, the multi-layer particle 10 may further have another layer formed thereon in addition to the core particle 11 and the detection layer 13. In this case, a gel solution or an aqueous solution containing a substance to be contained in the other layer is mixed with the core particle 11 or the multi-layer particle 10 to form a W / O emulsion, thereby obtaining the multi-layer particle 10 on which the other layer is formed.

[0072] When the detection layer 13 is formed as a layer of an aqueous solution, the layer of the aqueous solution is formed as the outermost layer of the multi-layer particle 10 in the hydrophobic solvent 20. The detection layer 13 of the aqueous solution can be stably maintained as the outermost layer of the multi-layer particle 10 in the hydrophobic solvent 20. The outermost layer of the multi-layer particle 10 may be another layer formed from an aqueous solution.

[0073] (Selection Step) The selection step is a step of selecting multi-layer particles 10 containing microorganisms 12 having the target activity based on a signal indicating the result of detection of the target activity by the detector 14 .

[0074] First, it is preferable to culture the multi-layer particle 10 formed in the particle formation step for a predetermined period of time (S4). This culture allows the secretory substances of the microorganism 12 contained in the core particle 11 to react with the detector 14 contained in the detection layer 13. The culture conditions, such as the predetermined period and culture temperature, may be determined appropriately depending on the target activity and the type of detector 14.

[0075] In culturing the multilayer particles 10, the multilayer particles 10 formed in the hydrophobic solvent 20 may be cultured in a state in which they are dispersed in the hydrophobic solvent 20 as they are, or the multilayer particles 10 may be cultured after being dispersed in another type of hydrophobic solvent 20 or an aqueous solution.

[0076] Next, the multi-layer particles 10 are selected based on the signal from the detector 14 (S5). Here, an example will be described in which the target activity is antibacterial activity and the detector 14 is a cell into which a fluorescent protein has been introduced, but this is not limiting. The screening method according to one embodiment of the present invention can accommodate various functions and activities of the microorganisms 12, such as antibacterial activity, specific enzyme activity such as cellulase activity, or activity for producing useful substances, depending on the type of detector 14.

[0077] Here, the multi-layer particles 10 after the culture in step S4 are analyzed using a cell sorter, droplet sorter, or the like. Depending on the specifications of the cell sorter or droplet sorter, the multi-layer particles 10 may be analyzed while dispersed in the hydrophobic solvent 20, or may be analyzed after being dispersed in an aqueous solution. Then, multi-layer particles 10 in which the fluorescence emitted by the fluorescent protein expressed in the detector 14 has disappeared are selected as multi-layer particles 10 containing microorganisms 12 having the target activity.

[0078] The selected multi-layer particles 10 may be used for mass cultivation of microorganisms 12 having the target activity. For example, the microorganisms 12 may be inoculated from the selected multi-layer particles 10 into a medium having a larger cultivation scale than the multi-layer particles 10 to obtain a culture strain of the microorganisms 12 having the target activity. According to such a method, the microorganisms 12 having the target activity can be artificially grown and used in a manner suitable for their activity.

[0079] Furthermore, the selected multi-layer particles 10 can be used to search for genes that express a target activity, for example, by performing genome sequencing on the encapsulated microorganisms 12. If the gene in question can be estimated or identified, for example, even if it is difficult to artificially mass-culture the microorganisms 12 that have the target activity, the target activity can be easily reproduced by introducing the gene into cells that are relatively easy to culture.

[0080] According to the above-described configuration, the target activity of the microorganism 12 can be detected using a large number of multi-layer particles 10, and therefore, it becomes possible to screen for microorganisms having the target activity with efficiency far exceeding that of existing technologies using a cell sorter or the like.

[0081] In particular, in a screening system using gel microdroplet (GMD) technology, cells, substrates, reagents, and the like required for detecting a target activity can be added at a desired timing, that is, even after the step of culturing the microorganism 12. Therefore, it becomes possible to evaluate various activities that could not be detected in a screening system using GMD technology in the past.

[0082] For example, when cells themselves are used as the detector 14 for antibacterial activity, or when the substrate that serves as the detector 14 for cellulase activity or the like is a polymer, it is often difficult to add the detector 14 from outside the GMD. According to a screening method according to one embodiment of the present invention, after culturing and growing microorganisms, a layer of gel or aqueous solution containing the detector 14 is formed to create multi-layer particles 10, and the target activity can be screened with high throughput using a large number of multi-layer particles 10.

[0083] [Summary] The screening method according to aspect 1 of the present invention is a screening method for searching for a target microorganism having a target activity in a population of microorganisms, and includes a particle formation step of forming one or more detection layers of gel or aqueous solution containing a detector of the target activity on the outside of a core particle of a solid medium containing the microorganism to obtain multi-layer particles, and a selection step of selecting the multi-layer particles containing the target microorganism based on a signal indicating the detection result of the target activity by the detector, wherein in the particle formation step, when the detection layer is formed as a layer of aqueous solution, the layer of aqueous solution is formed as the outermost layer of the multi-layer particle in a hydrophobic solvent.

[0084] In the screening method according to aspect 2 of the present invention, in the above aspect 1, the detection substance may be a cell different from the target microorganism.

[0085] A screening method according to a third aspect of the present invention may be the screening method according to the second aspect, wherein the detection body is the cell introduced with a fluorescent substance or a luminescent substance.

[0086] A screening method according to aspect 4 of the present invention may be the same as that according to aspect 3, wherein the target activity is antibacterial activity, and in the selection step, a decrease in the fluorescence of the fluorescent substance or the luminescence of the luminescent substance due to a decrease in the cells that are the detection body may be used as the signal indicating that the detection body has detected the target activity.

[0087] A screening method according to aspect 5 of the present invention is the same as that according to aspect 1, wherein the outermost detection layer is formed as a layer of aqueous solution, and the detection element contained in the detection layer may be a water-soluble compound.

[0088] A screening method according to aspect 6 of the present invention is such that, in aspect 1 or 5, the target activity is cellulase activity, the detector is a fluorescent substance that emits fluorescence under the influence of the cellulase activity or a luminescent substance that emits light under the influence of the cellulase activity, and in the selection step, an increase in the fluorescence of the fluorescent substance or the luminescence of the luminescent substance may be used as the signal indicating that the detector has detected the target activity.

[0089] The solution according to aspect 7 of the present invention is a solution containing multi-layer particles and a hydrophobic solvent for searching for a target microorganism having a target activity in a population of microorganisms, wherein the multi-layer particles have a core particle of a solid medium containing the microorganism, and one or more detection layers of a gel or aqueous solution containing a detector of the target activity formed on the outside of the core particle, and when the detection layer is an aqueous solution, the detection layer is the outermost layer of the multi-layer particles.

[0090] An embodiment of the present invention will now be described.

[0091] Using model microorganisms, it was confirmed whether or not the use of multi-layer particles 10, which are GMDs having multiple layers, enables rapid detection and selection of microorganisms 12 having the target activity from among a large number of microorganisms 12.

[0092] In this example, the target activity was antibacterial activity. Multilayer particles 10 were formed, with lactic acid bacteria (microorganisms 12) producing antibacterial substances in the inner core particle 11, and fluorescent protein-expressing E. coli as detectors 14 for detecting the antibacterial substances in the second detection layer 13. Multilayer particles 10 in which the E. coli fluorescence was reduced due to the antibacterial substances produced by the lactic acid bacteria were selected using a cell sorter, and the selected multilayer particles 10 were confirmed by culturing. Details of this example are described below.

[0093] 1. Screening of microorganisms with antibacterial activity 1-1. Formation of core particles First, lactic acid bacteria (Lactobacillus paracasei subsp. paracasei NBRC 15889 strain) were cultured to be encapsulated in core particles 11. This lactic acid bacteria strain formed an inhibition zone in a susceptibility test using Escherichia coli (Escherichia coli BL21 strain).

[0094] Lactic acid bacteria were inoculated onto MRS (de Man-Rogosa-Sharpe) agar medium containing 1% calcium carbonate, and pre-cultured for 24 hours at 28°C under anaerobic conditions. Calcium carbonate in the medium dissolved due to the production of lactic acid, forming a transparent halo around the grown lactic acid bacteria colonies. On the day before forming core particles 11, a colony with a halo formed was scraped with a microloop and inoculated into 5 mL of MRS liquid medium, followed by static culture at 28°C for 16 hours.

[0095] The cells were collected by centrifugation and suspended in PBS. A portion of the cell suspension was subjected to nucleic acid staining in the presence of 1x SYBR Green I, and the cells were counted using a fluorescence microscope to calculate the cell concentration of the cell suspension.

[0096] SeaPlaque (registered trademark) Agarose was used as the hydrogel for forming the core particle 11. 500 μL of 1.5 w / v% agarose was heated to 70°C to melt, and after melting, it was kept at 40°C. 25 μL of Poloxamer 188 solution, 50 μL of 10x concentrated MRS medium, and 5.0 × 10 ８ 100 μL of a PBS cell suspension containing lactic acid bacteria cells was added and mixed well by pipetting.

[0097] Mineral oil containing a 1% concentration of the surfactant Span 80 was kept at 40°C, and the agarose mixture was added to it and mixed by inversion, followed by stirring at 1,300 rpm for 2 minutes using a CellSys 100 Microdrop Maker to form micelles that would become core particles 11. The rotation speed was then reduced to 150 rpm, and the mixture was stirred for 7 minutes while cooling with ice water to solidify the gel.

[0098] Some of the formed core particles 11 were washed with PBS to remove the oil and observed under a microscope. As shown in Figure 3, core particles 11 with diameters of 5 to 30 μm were formed, and each core particle 11 contained zero to multiple lactic acid bacteria cells.

[0099] The resulting mixture of core particles 11 and oil was sealed and cultured for 2 days at room temperature while stirring at 10 rpm using a rotator. After culture, the oil and core particles 11 were separated by centrifugation, and the core particles 11 were recovered and washed with MRS liquid medium to remove the oil.

[0100] When the collected core particles 11 were observed under a microscope, proliferation of lactic acid bacteria within the core particles 11 was confirmed, as shown in FIG.

[0101] <1-2. Formation of multi-layer particles> Escherichia coli BL21 strain (hereinafter referred to as pRSET-EmGFP E. coli) expressing green fluorescent protein by introducing the pRSET-EmGFP Bacterial Expression Vector (Invitrogen) was prepared as the detector 14 to be added to the detection layer 13. The excitation wavelength / fluorescence wavelength of the green fluorescence of this E. coli is 487 nm / 509 nm.

[0102] The pRSET-EmGFP E. coli was inoculated onto a Luria-Bertani (LB) agar medium containing 100 μg / mL of ampicillin as a preculture and cultured for 16 hours to form colonies. On the day before forming the multi-layer particle 10, a single colony was inoculated into 5 mL of LB liquid medium containing the same concentration of ampicillin and cultured at 37° C. for 16 hours with shaking at 112 rpm.

[0103] The pRSET-EmGFP E. coli cells were collected by centrifugation and suspended in PBS. A portion of the cell suspension was subjected to nucleic acid staining in the presence of 1x SYBR® Green I, and the cells were counted using a fluorescence microscope to calculate the cell concentration of the cell suspension.

[0104] The hydrogel of the detection layer 13 was SeaPlaque Agarose, similar to that of the core particle 11. After melting, 1 mL of 1.5 w / v % agarose was kept at 40°C, and 50 μL of Poloxamer 188 solution, 100 μL of 10-fold concentrated MRS medium, 4.0 × 10 ９ 100 μL of a PBS cell suspension containing pRSET-EmGFP E. coli cells and the above-mentioned lactic acid bacteria-containing core particles 11 were added in a volume of 100 μL and mixed well by pipetting.

[0105] Mineral oil containing 1% Span 80 was kept at 40°C, to which the agarose mixture was added and mixed by inversion, followed by stirring for 2 minutes at 1,300 rpm using a CellSys 100 Microdrop Maker to produce micelles encapsulating the lactic acid bacteria-containing core particles 11. The rotation speed was then reduced to 150 rpm, and the mixture was stirred for 7 minutes while cooling with ice water to solidify the gel.

[0106] To detect the loss of fluorescence from pRSET-EmGFP E. coli due to lactic acid bacteria using a cell sorter, multi-layer particles that did not contain pRSET-EmGFP E. coli in the outer layer of core particle 11 were prepared as a positive control. 100 μL of PBS was added instead of the pRSET-EmGFP E. coli suspension, and multi-layer particles were prepared in the same manner as multi-layer particles 10.

[0107] A portion of the prepared multi-layer particles 10 was washed with PBS to remove the oil, and the resulting image observed under a microscope is shown in Figure 5. Approximately 10% of the multi-layer particles 10 and single-layer particles of pRSET-EmGFP E. coli were observed. In Figure 5, the multi-layer particles 10 are indicated by arrows. The diameter of the multi-layer particles 10 was 20 to 40 μm, and almost all of the multi-layer particles 10 contained pRSET-EmGFP E. coli, which served as the detection body 14. Figure 6 shows a microscopic image of a positive control multi-layer particle that does not contain pRSET-EmGFP E. coli in the outer layer, with the arrow indicating the particle containing lactic acid bacteria in the core particle 11.

[0108] The mixture of multi-layer particles 10 and oil was sealed and cultured at room temperature for 6 days while stirring at 10 rpm using a rotator. After the culture, the oil and multi-layer particles 10 were separated by centrifugation, and the multi-layer particles 10 were collected and washed with PBS to remove the oil.

[0109] A microscopic image of the multi-layer particle 10 containing pRSET-EmGFP E. coli in the outer layer after cultivation is shown in Figure 7. As indicated by the arrow in Figure 7, colonies of lactic acid bacteria were confirmed to have developed inside the multi-layer particle 10.

[0110] <1-3. Selection of Multilayer Particles> The multilayer particles 10 washed with PBS were passed through a nylon membrane filter with a pore size of 20 μm to prevent clogging of the flow path of the cell sorter.

[0111] The multi-layer particle 10 was stained with LDS 751 (excitation / fluorescence wavelengths = 543 nm / 712 nm), a cell membrane permeable nucleic acid staining reagent for staining the whole bacteria, at a final concentration of 10 μM.

[0112] The cell sorter used was a Sony Cell Sorter SH800S, with a sorting tip having an orifice diameter of 130 μm. A 488 nm laser was used as the fluorescence excitation light source, and forward scattered light (FSC) and backscattered light (BSC) were used to detect the multilayer particles 10. Sensors with optical filters FL1 (500-550 nm) and FL5 (690-750 nm) were used to detect the green fluorescence of EmGFP and the red fluorescence from LDS 751, respectively.

[0113] First, positive control multilayer particles that did not contain pRSET-EmGFP E. coli in the outer layer were measured using a cell sorter. First, the vertical and horizontal axes of the dot plot were set to BSC and FSC, respectively, to display the measured values ​​of each multilayer particle, and Area A was designated as the region where the multilayer particle or core particle 11 population was distributed. Next, for the multilayer particles or core particles 11 distributed in Area A, their fluorescence intensities were displayed in a dot plot with FL5 (LDS 751 fluorescence intensity) and FL1 (EmGFP fluorescence intensity) set as the vertical and horizontal axes. Multilayer particles with high LDS 751 red fluorescence intensity were targeted, and Area B was designated as the region where this population was distributed.

[0114] Figure 8 shows the dot plots of the measured multi-layer particles or core particles 11 and the set areas, and Figure 9 shows an image of particles sorted from area B. In Figure 9, the fluorescent areas (white areas inside the particles) are colonies of lactic acid bacteria.

[0115] Next, multi-layer particles 10 having a detection layer 13 containing pRSET-EmGFP E. coli were measured under the same conditions as above. For groups of multi-layer particles 10 that did not contain lactic acid bacteria or showed little growth of lactic acid bacteria, a new area C was set on the dot plots of FL1 and FL5. The dot plots of the cell sorter under these conditions are shown in Figure 10.

[0116] The multilayer particles 10 distributed in the target areas B and C were each sorted and observed under a fluorescence microscope. The multilayer particles 10 sorted from area B are shown in Figure 11, and the multilayer particles 10 sorted from area C are shown in Figure 12.

[0117] In Figure 11, the white areas indicated by arrows represent colonies of lactic acid bacteria inside the multi-layer particle 10, and fluorescence from pRSET-EmGFP E. coli was not detected. Furthermore, in Figure 12, the white areas within the multi-layer particle 10 represent pRSET-EmGFP E. coli, and fluorescence from lactic acid bacteria colonies was not detected. Thus, in the multi-layer particle 10 sorted from area B, the presence of lactic acid bacteria colonies in the core particle 11 and the disappearance of fluorescence from the pRSET-EmGFP E. coli in the detection layer 13 were confirmed. In the multi-layer particle 10 sorted from area C, pRSET-EmGFP E. coli emitting green fluorescence was confirmed.

[0118] The multi-layer particles 10 distributed in areas B and C were sorted, inoculated onto a microplate-type petri dish containing MRS agar medium containing 1% calcium carbonate, and cultured for 6 days at 28°C under anaerobic conditions. Lactic acid bacteria colonies were confirmed by the presence or absence of halos around the colonies.

[0119] While 87% of lactic acid bacteria colonies emerged from the multi-layer particles 10 in area B, only 4% emerged from the multi-layer particles 10 in area C. This result indicates that the screening method according to one embodiment of the present invention was able to accurately select multi-layer particles 10 containing lactic acid bacteria. It is also believed that this screening method was able to select multi-layer particles 10 containing lactic acid bacteria with a high ability to produce antibacterial substances from a population of lactic acid bacteria.

[0120] This example confirmed that by using the multi-layer particles 10, it is possible to quickly identify and obtain microorganisms having the target activity from among a large number of microorganisms.

[0121] 2. Screening of Microorganisms Having Cellulase Activity 2-1. Formation of Core Particles First, a cellulase-producing bacterium (Cellulomonas sp. 16064 strain) to be encapsulated in the core particles 11 was cultured.

[0122] Agar medium (2g / L Hipolypepton, 0.4g / L Yeast extract, 0.2g / L MgSO ４ ・7H ２The cellulase-producing fungus was inoculated into the core particle 11 (0) and pre-cultured for 24 hours. On the day before the formation of the core particle 11, a colony of the cellulase-producing fungus was scraped with a microloop and inoculated into 5 mL of a liquid medium (0.5 g / L KH ２ P.O. ４ ,0.1g / L CaCl ２ , 6.0g / L NaCl, 0.5g / L K ２ HPO ４ , 0.1g / L NaNO ３ , 1.0g / L (NH ４ ) ２ SO ４ ,0.1g / L MgSO ４ , 1.0 g / L yeast extract) was inoculated and cultured at 28°C for 24 hours with shaking at 200 rpm.

[0123] The cells were collected by centrifugation and suspended in PBS. A portion of the cell suspension was subjected to nucleic acid staining in the presence of 1x SYBR Green I, and the cells were counted using a fluorescence microscope to calculate the cell concentration of the cell suspension.

[0124] SeaPlaque (registered trademark) Agarose was used as the hydrogel for forming the core particle 11. 500 μL of 1.5 w / v% agarose was heated to 70° C. to melt it, and after melting, it was kept at 40° C. 50 μL of the liquid medium containing no CMC and 1.1 × 10 ８ 100 μL of a PBS cell suspension containing cellulase-producing bacteria was added and mixed well by pipetting.

[0125] Mineral oil containing a 1% concentration of the surfactant Span 80 was kept at 40°C, and the agarose mixture was added to it and mixed by inversion, followed by stirring at 1,300 rpm for 2 minutes using a CellSys 100 Microdrop Maker to form micelles that would become core particles 11. The rotation speed was then reduced to 150 rpm, and the mixture was stirred for 7 minutes while cooling with ice water to solidify the gel.

[0126] A portion of the formed core particles 11 was washed with the liquid medium containing 1% CMC to remove the oil. The resulting mixture of core particles 11 and oil was sealed and cultured at room temperature for 6 days while stirring at 175 rpm using a rotator. After culture, the oil and core particles 11 were separated by centrifugation, and the core particles 11 were recovered and washed with PBS to remove the oil.

[0127] When the collected core particles 11 were observed under a microscope, cellulase-producing bacteria were confirmed to have grown within the core particles 11 having a diameter of 5 to 30 μm, as shown in FIG.

[0128] <2-2. Formation of multi-layer particles> A GMD in WODL (water in oil droplet) was prepared, which is one embodiment of the multi-layer particle 10, in which the detection layer 13 is formed from an aqueous solution. Fluorescein di-beta-D-cellobioside (AAT Bioquest), a fluorescent substrate for cellulase, was used as the detection body 14 added to the detection layer 13.

[0129] The cellulase-producing bacteria-containing core particles 11 were mixed at 6.1 × 10 ３ The mixture and fluorescein di-beta-D-cellobioside were added to 1 mL of the liquid medium (with the exception that the final concentration of CMC was 0.1%) to serve as detection layer 13, and mixed thoroughly by pipetting. Fluorescein di-beta-D-cellobioside was added to a final concentration of 40 μM.

[0130] The resulting mixture and fluorinated oil containing 008-FluoroSurfactant-5wtH (On-chip Biotechnologies) at a concentration of 2 w / w% were fed to an On-chip (registered trademark) Droplet Generator (On-chip Biotechnologies) to obtain GMD in WODL in which a detection layer 13 was formed in the fluorinated oil from the liquid medium, which was an aqueous solution. Microscopic observation revealed GMD in WODL as multi-layer particles 10 comprising a cellulase-producing bacteria-containing core particle 11 and a detection layer 13 formed from an aqueous solution, as shown in Figure 14.

[0131] 2-3. Selection of multilayer particles using a model system The prepared GMD in WODL was left in the dark and cultured at room temperature for 24 or 48 hours. The GMD in WODL after 24 or 48 hours of culture was observed under a microscope in bright field and fluorescent field, and the results are shown in Figure 15. For observation under a fluorescent field, blue excitation light of 460-495 nm was used as the fluorescence excitation light source, and an optical filter that transmits light of 510 nm or longer was used to detect green fluorescence.

[0132] As indicated by the arrow in the bright-field microscope image in Figure 15, GMD in WODL containing cellulase-producing bacteria was observed. Furthermore, in the fluorescent-field microscope image, green fluorescence was detected both after 24 and 48 hours of culture. This indicates that in GMD in WODL having cellulase-producing bacteria-containing core particles 11, fluorescein di-beta-D-cellobioside in detection layer 13 was cleaved by cellulase produced by the cellulase-producing bacteria to fluorescein, which emitted green fluorescence.

[0133] This result indicates that GMD in WODL, which is a multi-layer particle 10 containing cellulase-producing bacteria, can be accurately selected by the screening method according to one embodiment of the present invention. Furthermore, this result indicates that multi-layer particle 10 having an aqueous solution as detection layer 13 can also be suitably used in the screening method according to one embodiment of the present invention.

[0134] This example confirmed that by using the multi-layer particles 10, it is possible to quickly identify and obtain microorganisms having the target activity from among a large number of microorganisms.

[0135] <2-4. Selection of multi-layer particles containing environmental microorganisms> Screening of cellulase-producing bacteria was attempted from soil bacteria. Here, GMD in WODL was prepared in accordance with the methods described above in <2-1. Formation of core particles> and <2-2. Formation of multi-layer particles>, except that the microorganisms 12 to be encapsulated in core particles 11 were soil bacteria collected from forest soil on the Hiroshima University campus.

[0136] The prepared GMD in WODL was left in the dark and cultured at room temperature for 24 or 48 hours. The results of bright-field and fluorescent microscopic observation of the GMD in WODL after 24 or 48 hours of culture are shown in Figure 16. The fluorescent microscopic observation conditions were the same as those described above in <2-3. Selection of multilayer particles using a model system>.

[0137] As indicated by the filled and open arrows in the bright-field microscope image in Figure 16, GMD in WODL containing microorganism 12 was observed. Furthermore, in the fluorescent microscope image, GMD in WODL emitting green fluorescence was detected after both 24 and 48 hours of culture. The GMD in WODL emitting green fluorescence is indicated by the open arrow in the bright-field microscope image.

[0138] This result indicates that GMD in WODL containing cellulase-producing bacteria with cellulase activity could be clearly detected from among GMD in WODL containing a variety of soil bacteria.

[0139] This example suggests that by using the multi-layer particles 10, it is possible to rapidly identify and obtain microorganisms having a target activity from among a large number of microorganisms present in the environment, for example.

[0140] [Additional Notes] The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments or examples are also included in the technical scope of the present invention.

[0141] One embodiment of the present invention can be used to screen for microorganisms having a target activity that is useful in drug discovery and the like.

[0142] REFERENCE SIGNS LIST 1 Solution 10 Multilayer particle 11 Core particle 12 Microorganism 13 Detection layer 14 Detection body 20 Hydrophobic solvent

Claims

1. A screening method for searching for a target microorganism having a target activity in a population of microorganisms, comprising: a particle formation step of forming one or more detection layers of gel or aqueous solution containing a detector for the target activity on the outside of a core particle of a solid medium containing the microorganism to obtain a multi-layer particle; and a selection step of selecting the multi-layer particle containing the target microorganism based on a signal indicating the detection result of the target activity by the detector, wherein in the particle formation step, when the detection layer is formed as a layer of aqueous solution, the layer of aqueous solution is formed as the outermost layer of the multi-layer particle in a hydrophobic solvent.

2. The screening method according to claim 1, wherein the detection subject is a cell different from the target microorganism.

3. The screening method according to claim 2, wherein the detection body is the cell into which a fluorescent substance or a luminescent substance has been introduced.

4. The screening method according to claim 3, wherein the target activity is antibacterial activity, and in the selection process, a decrease in the fluorescence of the fluorescent substance or the luminescence of the luminescent substance due to a decrease in the cells that are the detection body is used as the signal indicating that the detection body has detected the target activity.

5. A screening method according to claim 1, wherein the outermost detection layer is formed as a layer of an aqueous solution, and the detection substance contained in the detection layer is a water-soluble compound.

6. A screening method according to claim 1 or 5, wherein the target activity is cellulase activity, the detector is a fluorescent substance that emits fluorescence under the influence of the cellulase activity or a luminescent substance that emits light under the influence of the cellulase activity, and in the selection process, an increase in the fluorescence of the fluorescent substance or the luminescence of the luminescent substance is used as the signal indicating that the detector has detected the target activity.

7. A solution comprising multi-layer particles and a hydrophobic solvent for searching for a target microorganism having a target activity in a population of microorganisms, wherein the multi-layer particles have a core particle of a solid medium containing the microorganism, and a detection layer of a gel or aqueous solution containing a detector of the target activity formed in one or more layers on the outside of the core particle, and when the detection layer is an aqueous solution, the detection layer is the outermost layer of the multi-layer particles.

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