Method and apparatus for inspecting ulcerative colitis, and screening method of therapeutic drug

Abstract

To provide a method for inspecting ulcerative colitis which is useful for determining a disease state of ulcerative colitis.SOLUTION: A method for producing a strain sample derived from a subject in which the strain diversity and / or the number of strains in the sample of the subject are maintained includes: 1) a process of adding the sample of the subject to a culture medium; 2) a process of performing an operation to bring pH of the culture medium to 6.2 to 6.7 in starting the cultivation if necessary; and 3) a process of culturing the sample after starting the cultivation without performing an operation intended to maintain pH of the culture medium. Preferably, the method includes A) a process of performing sterilization of the culture medium and aeration to anaerobic gas and / or B) a process of suspending the sample to ascorbic acid-added phosphate buffer, before 1).SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method and device for testing ulcerative colitis, and a method for screening a therapeutic agent for ulcerative colitis. [Background technology]

[0002] Ulcerative colitis is widespread worldwide, and the number of patients is also increasing dramatically in Japan. The current main treatment for ulcerative colitis is the administration of mesalazine (5-aminosalicylic acid (5-ASA)), a non-steroidal drug. However, this treatment only temporarily alleviates the disease, but does not eliminate the possibility of recurrence or bring the disease into remission. Therefore, in order to cure ulcerative colitis, there is a demand for tests to determine the disease state of ulcerative colitis and for treatment of the disease.

[0003] When examining the intestinal environment of patients with ulcerative colitis, the proliferation of Escherichia coli is increased compared to healthy individuals, indicating an abnormality. It is believed that correcting the intestinal environment is necessary to cure ulcerative colitis.

[0004] Meanwhile, in the human intestinal tract, a wide variety of bacteria continues to grow, and these are called intestinal flora. In research into intestinal flora, culture is performed using an intestinal tract mimicking culture device that reproduces the human intestinal environment. A culture method and device that can culture intestinal flora while maintaining the composition balance of the intestinal flora have been proposed (Patent Document 1 and Non-Patent Document 1). In this culture method, individual intestinal flora are cultured in an anaerobic environment using a medium supplemented with lower fatty acids, while adjusting the pH of the medium to a range of 6.0 to 7.0.

[0005] Testing for ulcerative colitis and screening for therapeutic drugs requires a culture method and device that mimics the intestinal environment of an ulcerative colitis patient. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2015 / 136916 [Non-patent literature]

[0007] [Non-Patent Document 1] Takagi, R. et al., PLoS One 11, e0160533 (2016) Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention is intended to solve the above problems, and has an object to provide a method and device for testing ulcerative colitis, and a method for screening therapeutic drugs, which are useful for determining the disease state of ulcerative colitis. [Means for solving the problem]

[0009] The present invention provides a method for testing ulcerative colitis, the method comprising: performing anaerobic culture using a culture medium containing a fecal sample collected from the subject; obtaining data on indicator factors for ulcerative colitis from the culture solution; and comparing said data with standards for said indicator factors in healthy individuals and / or individuals affected by ulcerative colitis; Including, The anaerobic culture is carried out such that the pH of the culture medium is 6.2 to 6.7 at the start of the culture, and then the culture is allowed to stand.

[0010] In one embodiment, the subject is a subject suffering from or suspected of suffering from ulcerative colitis.

[0011] In one embodiment, the indicator factor for ulcerative colitis is at least one selected from the group consisting of butyric acid amount, Lachnospiraceae bacterial amount, and culture medium pH profile.

[0012] In one embodiment, the pH of the culture medium is 6.2 to 6.5 at the initiation of the culture.

[0013] The present invention provides a method for screening a therapeutic agent for ulcerative colitis, the method comprising: performing anaerobic culture using a culture medium containing a fecal sample collected from an individual suffering from ulcerative colitis and to whom a candidate therapeutic agent has been administered; obtaining data on indicator factors for ulcerative colitis from the culture solution; and comparing said data to standards of said indicator factors in healthy individuals and / or individuals affected by ulcerative colitis to assess the efficacy of said potential therapeutic agent; Including, The anaerobic culture is carried out such that the pH of the culture medium is 6.2 to 6.7 at the start of the culture, and then the culture is allowed to stand.

[0014] The present invention provides a testing device for ulcerative colitis, the testing device comprising: (a) an anaerobic culture means for performing an anaerobic culture using a culture medium containing a fecal sample collected from a subject; (b) a measuring means for measuring data of an index factor for ulcerative colitis from the culture solution of the anaerobic culture means; and (c) a comparison means for storing in advance standards for indicator factors of healthy individuals and / or individuals affected by ulcerative colitis, and for comparing data on the indicator factors obtained from the measurement means with the standards for the indicator factors; Equipped with. Effect of the Invention

[0015] According to the present invention, a culture method simulating the intestinal environment of an ulcerative colitis patient is provided, which provides a method and an apparatus for testing ulcerative colitis. The culture method is also useful for screening therapeutic agents for ulcerative colitis. [Brief description of the drawings]

[0016] [Figure 1] FIG. 1 is a diagram illustrating an example of an ulcerative colitis testing device according to the present invention. [Diagram 2] Graph showing pH profiles of fecal samples and culture fluid from 13 healthy individuals (A) and 11 patients with ulcerative colitis (B). [Diagram 3] Principal coordinate analysis (PCoA) of 16S rRNA gene sequencing data of samples collected at the start of culture (fecal samples) and after 30 hours of culture (culture fluid samples) for healthy individuals (HS) and ulcerative colitis (UC) patients, respectively. [Figure 4] This is a graph showing the abundance ratio of various Enterobacteriaceae relative to the total enterobacteria in samples collected at the start of culture (fecal samples) and samples collected 30 hours after culture (culture fluid samples) for healthy individuals (HS) and ulcerative colitis (UC) patients. [Diagram 5] This is a box plot showing the abundance ratio of Lachnospiraceae bacteria to the total intestinal bacteria in samples collected at the start of culture (fecal samples) and samples collected 30 hours after culture (culture medium samples) for healthy individuals and ulcerative colitis (UC) patients. [Figure 6] Box plots showing the amounts of acetate, propionate, butyrate, and SCFA (the sum of succinate, lactate, acetate, propionate, and butyrate) in samples (culture medium samples) collected after 30 hours of culture for healthy individuals and ulcerative colitis (UC) patients. [Figure 7] FIG. 1 shows a graph showing the results of plotting the abundance ratio of Lachnospiraceae bacteria in samples (culture medium samples) collected 30 hours after culture from patients with ulcerative colitis against the amount of butyric acid produced by culture (A), and the results of plotting the abundance ratio of Lachnospiraceae bacteria in fecal samples (B) and culture medium samples (C) from patients with ulcerative colitis against the partial Mayo score. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] (Testing methods for ulcerative colitis) The present invention provides a method for testing ulcerative colitis. The method for testing ulcerative colitis of the present invention can be used to determine the morbid state of ulcerative colitis. The "morbid state" includes the presence or absence of morbidity and the severity of the disease. The testing method of the present invention can be used to determine whether a subject is morbid with ulcerative colitis, whether the symptoms of ulcerative colitis in a subject are progressing, or whether the symptoms of ulcerative colitis in a subject undergoing treatment have been alleviated.

[0018] The subject is any animal with an intestinal tract, for example, a mammal, for example, includes humans, dogs, cats, cows, pigs, etc., and is preferably humans. The subject may be a livestock animal or a pet animal. In one embodiment, the subject is a subject that suffers from or is suspected of suffering from ulcerative colitis.

[0019] The culture medium may be prepared by adding a fecal sample collected from a subject to a culture medium. The culture medium may be any medium that can be used for anaerobic culture. The fecal sample may be prepared in advance as a suspension (e.g., suspended in 0.1 M phosphate buffer containing 1% L-ascorbic acid) and added, for example, at 100 μL per 100 mL of culture medium. The anaerobic culture medium may be, for example, Gifu University Formulated Anaerobic Medium (GAM medium), such as GAM agar medium, modified GAM agar medium, GAM semi-liquid high-layer medium, GAM bouillon, and modified GAM bouillon (for example, all manufactured by Nissui Pharmaceutical Co., Ltd.). Before culture, the medium may be sterilized (for example, autoclave sterilization). Liquid culture is preferred, and the culture medium may be appropriately stirred during culture. After collection, the fecal sample may be stored in a container such as an anaerobic culture swab until the start of the test.

[0020] The anaerobic environment for culture can be created by aerating the medium with an anaerobic gas. The anaerobic gas is, for example, nitrogen, nitrogen and carbon dioxide, or nitrogen, carbon dioxide and hydrogen. The anaerobic gas is aerated constantly or intermittently at a flow rate of, for example, 0.1 to 1.0 dL / min. The anaerobic gas is preferably a mixed gas of nitrogen and carbon dioxide. It is preferable to aerate the anaerobic gas constantly.

[0021] In the present invention, anaerobic culture is performed with the culture medium having a pH of 6.2 to 6.7 at the start of culture, and then left to stand. Preferably, the pH at the start is 6.2 to 6.5. At the start of culture (for example, when the culture medium containing a fecal sample is placed in an anaerobic environment), the pH of the culture medium may be within the above range, and the pH is adjusted to within the above range using a pH regulator as necessary. After the start of culture, the pH is left to stand. During culture, no operation intended to maintain the pH within the above range (for example, pH adjustment by addition of a pH regulator or alkali) is performed.

[0022] The culture period is preferably a period during which data on the index factor for ulcerative colitis can be obtained. The culture period depends on the index factor for ulcerative colitis for which data is to be obtained, and is, for example, 24 to 48 hours, preferably 24 to 30 hours. The culture temperature is near the body temperature of the subject, and when the subject is a human, for example, the temperature is 36°C to 38°C, preferably 37°C. The culture method is not limited, but is preferably a single batch type.

[0023] Data on indicator factors for ulcerative colitis is obtained from the culture medium. An indicator factor for ulcerative colitis refers to a factor that can be used to determine the morbid state of ulcerative colitis (e.g., the presence or absence of morbidity or the severity of the disease). Examples of indicator factors for ulcerative colitis include the amount of butyric acid, the amount of Lachnospiraceae bacteria, and a culture medium pH profile, and may be a combination of at least two of these indicator factors. Data on indicator factors for ulcerative colitis is obtained from the culture medium at the start of culture, during culture, and after culture is completed, depending on the indicator factor.

[0024] The amount of butyric acid can be measured from the culture medium by a method commonly used by those skilled in the art (e.g., high performance liquid chromatography (HPLC)). The culture medium can be collected at any time point as long as the amount of butyric acid produced by the enterobacteria that may be present in the collected feces can be measured, but is preferably collected from the latter half of the culture to around the end of the culture.

[0025] The pH profile is represented by the variation of pH over the culture period, during which the pH may be monitored continuously or may be measured intermittently multiple times.

[0026] The amount of Lachnospiraceae bacteria is the amount of bacteria classified as belonging to the Lachnospiraceae family, regardless of the genus. The amount of Lachnospiraceae bacteria may be any value that can represent the abundance of Lachnospiraceae bacteria in a sample, such as the number of bacteria in a sample, or the ratio of the amount to the total enterobacteria in a sample. Preferably, the ratio of the amount to the total enterobacteria. The amount of Lachnospiraceae bacteria can be determined using techniques (e.g., 16S rRNA gene sequencing, real-time PCR, etc.) that are commonly used by those skilled in the art for detecting, identifying, and quantifying enterobacteria. The primer set used in this case may be prepared based on known sequence information, or a commercially available primer set may be used. A culture solution collected at any time point during culture may be used as a sample for measuring the amount of Lachnospiraceae bacteria. For example, a culture solution sample (fecal sample) at the start of culture, a culture solution sample during or after culture, or a combination of these may be used.

[0027] The data of the indicator factor is compared with the standard of the same indicator factor of healthy individuals and / or individuals suffering from ulcerative colitis. For example, if the indicator factor of ulcerative colitis is butyric acid amount, the data obtained for butyric acid amount is compared with the standard of butyric acid amount of healthy individuals and / or individuals suffering from ulcerative colitis. The data obtained from the above culture can be used in creating the standard. The "standard" can be created according to the presence or absence of ulcerative colitis and the severity.

[0028] The individual may also be an animal similar to the subject described above. A "healthy individual" refers to an individual that is at least not affected by ulcerative colitis. A "healthy individual" may also be an individual that previously suffered from ulcerative colitis disease but has been cured. An "individual affected by ulcerative colitis" refers to an individual currently suffering from ulcerative colitis. An "individual affected by ulcerative colitis" may also be an individual currently undergoing treatment as long as the individual suffers from ulcerative colitis. A "healthy individual" or an "individual affected by ulcerative colitis" may be the same individual as the subject.

[0029] The "standard" of the indicator factor for healthy individuals and / or individuals suffering from ulcerative colitis includes a case where a standard prepared based on data previously obtained from the relevant individuals (e.g., individual population) is used, and a case where data previously obtained from the subject itself is used as the standard. In the latter case, for example, the test method of the present invention can be used as a means for verifying the effect of treatment on the subject.

[0030] The amount of butyric acid is lower in ulcerative colitis patients than in healthy individuals. The amount of Lachnospiraceae bacteria is lower in ulcerative colitis patients than in healthy individuals. Regarding the pH profile, the pH fluctuation during the culture period is different when comparing healthy individuals and ulcerative colitis patients. For example, as shown in FIG. 2, in the pH profile of a healthy individual, the pH fluctuation of the culture medium can reproduce the normal pH in the human large intestine, whereas the pH profile of an ulcerative colitis patient can show abnormalities.

[0031] (Method of screening for therapeutic agents for ulcerative colitis) The present invention also provides a method for screening a therapeutic agent for ulcerative colitis. In this method, a fecal sample is collected from an individual suffering from ulcerative colitis and administered a therapeutic agent candidate. The culturing step and the data acquisition step are as described above. To evaluate the effectiveness of the therapeutic agent candidate, the acquired data are compared with the standard of indicator factors of healthy individuals and / or individuals suffering from ulcerative colitis. This comparison step is also as described above. It can be determined whether the acquired data meets the standard of healthy individuals or the standard of less severely affected individuals rather than the standard of more severely affected individuals.

[0032] The screening method of the present invention can determine whether a "candidate therapeutic agent" is effective in treating ulcerative colitis by using as a "standard" a standard created based on data previously obtained from a relevant individual (e.g., a population of individuals). The screening method of the present invention can also be used to select a therapeutic agent suitable for an individual by using the "standard" individual as an affected individual before administration of the candidate therapeutic agent (an "individual with ulcerative colitis") and comparing data of an individual after administration of the candidate therapeutic agent (an "individual with ulcerative colitis and administered the candidate therapeutic agent") against this standard.

[0033] (Ulcerative colitis testing device) The present invention also provides a method for testing for ulcerative colitis.The testing device of the present invention will be described with reference to the drawings.

[0034] FIG. 1 is a schematic diagram showing an example of an ulcerative colitis testing device of the present invention.

[0035] The testing device 100 of the present invention comprises anaerobic culturing means 110 , measuring means 140 , and comparing means 170 .

[0036] In the testing device 100 of the present invention, the anaerobic culturing means 110 includes a culture tank 112 and a lid 114 capable of sealing the culture tank 112. The culture tank 112 contains a culture solution 102 containing a fecal specimen collected from a subject, and anaerobic culturing is performed. In the embodiment shown in FIG. 1, a shaft 118 extends from a motor 116 disposed above the lid 114 to a position where the shaft 118 is immersed in the culture solution 102 in the culture tank 112, and at least one stirring blade 120 is provided at the end of the shaft 118. The motor 116 rotates the shaft 118, and the culture solution 102 is stirred by the stirring blade 120.

[0037] In addition, although the motor 116 is provided above the lid 114 in Fig. 1, the present invention is not limited to such an arrangement. The motor may be provided below the culture tank 112. Alternatively, the shaft 118 may be separated from the motor by forming the agitator blades 120 from a magnetic material. In this case, a member made of another magnetic material is provided around the shaft core of the motor, and the agitator blades 120 may rotate in conjunction with the rotation of this member.

[0038] Furthermore, in the anaerobic culture means 110 shown in FIG. 1, an air supply pipe 122 is disposed in the culture tank 112, for example, passing through the upper part of the lid 114. The air supply pipe 122 can introduce an inert gas (for example, nitrogen gas, carbon dioxide gas, and a combination thereof) into the culture tank 112 by opening and closing a valve 124, thereby making the culture tank 112 anaerobic. An exhaust pipe 126, preferably equipped with a check valve (not shown), is also provided in the culture tank 112, and unnecessary gas in the culture tank 112 is exhausted to the outside of the culture tank 112 through the exhaust pipe 126. In FIG. 1, both the air supply pipe 122 and the exhaust pipe 126 are described as penetrating the lid 114, and their ends are disposed in the culture tank 112, but the present invention is not necessarily limited to such a configuration. For example, both or either one of the exhaust pipe 122 and the exhaust pipe 126 may be disposed so as to penetrate the culture tank 112.

[0039] The anaerobic culturing means 110 is also provided with a temperature adjusting means 128 such as a thermostat in the culturing tank 112. The temperature adjusting means 128 makes it possible to maintain the culture solution 102 in the culturing tank 112 at a desired temperature.

[0040] In the present invention, the anaerobic culture means 100 may be, for example, a commercially available jar fermenter.

[0041] In the testing device 100 of the present invention, the measuring means 140 plays a role in acquiring data corresponding to the temperature of the culture solution 102 in the anaerobic culturing means 110 and index factors of ulcerative colitis from the culture solution 102 .

[0042] In the embodiment shown in FIG. 1, a temperature sensor 142 and a chemical sensor 144 are provided as the measuring means 140 at positions where they are immersed in the culture solution 102 of the culture tank 112. Examples of the chemical sensor 144 include, but are not limited to, ion sensors such as a hydrogen ion electrode (pH electrode) and an ion selective electrode. Such chemical sensors can selectively detect indicator factors of ulcerative colitis contained in the culture solution 102 (for example, the content of organic acids such as butyric acid, and the pH profile of the culture solution). Note that, although FIG. 1 shows an example in which two sensors, a temperature sensor 142 and a chemical sensor 144, are provided as the measuring means 140, the present invention is not limited thereto. In the detection device of the present invention, at least one measuring means is provided.

[0043] In the testing device 100 of the present invention, the comparison means 170 plays a role in comparing the data of the indicator factors obtained from the measurement means 140 with pre-stored standards of the indicator factors of healthy individuals and / or individuals affected by ulcerative colitis.

[0044] In the embodiment shown in FIG. 1, the comparison means 170 is composed of a control unit 172, a storage unit 174, and a display unit 176.

[0045] 1, the control unit 172 is electrically connected to the valve 124 of the air supply pipe 122 in the anaerobic culture means 110, the motor 116, and the temperature adjustment means 128 via cables 181, 182, and 183. This allows the control unit 172 to control the opening and closing of the valve 124 of the air supply pipe 122, the rotation speed of the motor 116, and the set temperature of the temperature adjustment means 128. The control unit 172 is also electrically connected to the measurement means 140 (i.e., the temperature sensor 142 and the chemical sensor 144) via cables 184 and 185. This allows the temperature data and index factor data detected by the temperature sensor 142 and the chemical sensor 144 to be transmitted to the control unit 172 as electrical signals via the cables 184 and 184.

[0046] 1 shows an example of wired connections between the control unit 172, the valve 124, the motor 116, the temperature adjustment means 128, the temperature sensor 142, and the chemical sensor 144 via cables 181, 182, 183, 184, and 185, but the present invention is not limited to this. Instead of the wired connections, wireless connections may be made between these through communication means (not shown).

[0047] The memory unit 174 pre-stores criteria for index factors of healthy individuals and / or individuals suffering from ulcerative colitis as information, reads out the criteria in response to a command from the control unit 172, and transmits the criteria to the control unit 172 as appropriate. The memory unit 174 can also save data on index factors transmitted from the control unit 172.

[0048] Examples of the storage unit 174 include an external or internal hard disk drive (HDD), a magnetic disk such as a floppy disk; an optical disk such as a CD-ROM, a CD-R, a CD-RW, a DVD-ROM, a DVD±R, a DVD-RW, a DVD-RAM, a Blu-ray disk (e.g., BD-ROM, BD-R, BD-R BDXL, and BD-RE), and a laser disk (registered trademark); a magneto-optical disk; and a USB memory, a solid-state drive (SSD), an SD memory card, a mini SD card, a micro SD card, and an SD HC memory card, mini SDHC card, micro SDHC card, SDXC memory card, micro SDXC card and other flash memory;

[0049] In FIG. 1, the display unit 176 is electrically connected to the control unit 172 via a cable 188. The display unit 176 displays various information such as the open / close state of the valve 124 in the anaerobic culture means 110 controlled by the control unit 172, the number of revolutions of the motor 116, the set temperature of the temperature control means 128, and the temperature of the culture solution 102 obtained from the measurement means 140 and the acquired data of the acquired index factors so that the operator can relatively recognize them. Examples of such a display unit 176 include a display and a printer. Note that, in FIG. 1, an example of a wired connection between the display unit 176 and the control unit 172 via a cable 188 is shown, but the present invention is not limited to this. Instead of the wired connection, a wireless connection may be made between them via a communication means not shown.

[0050] In the testing device 100 of the present invention, the control unit 172 compares data on the index factors for ulcerative colitis relating to the fecal sample in the culture solution 102 obtained from the chemical sensor 144 with the standards for the index factors of healthy individuals and / or individuals suffering from ulcerative colitis stored in advance in the memory unit 174. The comparison results transmitted from the control unit 172 are then displayed on the display unit 176. Furthermore, the data on the index factors for ulcerative colitis obtained from the chemical sensor 144 and / or the comparison results obtained from the control unit 172 may be separately stored in the memory unit 174 as necessary. In the testing device 100 of the present invention, the control unit 172 may be an electronic calculator such as a personal computer.

[0051] In this manner, the testing device 100 of the present invention can test for ulcerative colitis from a stool sample collected from a subject. EXAMPLES

[0052] The present invention will be described in detail below with reference to examples, although the present invention is not limited to these.

[0053] Example 1: Collection and culture of fecal samples Fecal samples were collected from 13 healthy subjects and 11 patients with ulcerative colitis. All participants had not received any antibiotics for at least 2 months. Written informed consent was obtained from the participants. Disease activity was determined by the Mayo clinical score and partial Mayo score. Endoscopic Mayo clinical scores were determined for patients who had undergone endoscopy within 1 month prior to fecal collection (n = 9). Partial Mayo clinical scores were determined at the time of fecal collection (n = 11). This study was performed in accordance with the guidelines of the Kobe University Hospital and approved by the Kobe University Institutional Review Board. All methods used in this study were in accordance with the Kobe University Medical Ethics Approval Guidelines. After collection, fecal samples were stored in anaerobic culture swabs (212550 BD BBL Culture Swab; Becton, Tickinson and Company) and sent to the laboratory.

[0054] A single-batch culture apparatus mimicking the human large intestine was constructed using a multichannel incubator (Bio Jr.8; Able Co., Ltd.). This in vitro model apparatus consisted of eight parallel independent vessels, each of which contained 100 mL of Gifu University anaerobic medium (GAM medium [Code 05422]; Nissui Pharmaceutical Co., Ltd.) that had been autoclaved (115°C for 15 min) and the pH was adjusted to 6.5 at the start of the culture. Anaerobic conditions were created in the culture vessels by aerating (15 mL / min) a nitrogen and carbon dioxide mixture (N2:CO2 = 80:20) that had been sterilized by filtration through a 0.2 μm PTFE membrane (Pall Corporation) for 1 h at 37°C. To prepare the inoculum, each fecal sample was suspended in 2 mL of 0.1 M phosphate buffer (pH 6.5, consisting of a 2:1 mixture of NaH2PO4 and 0.1 M Na2HPO4) supplemented with 1% L-ascorbic acid (Wako Pure Chemical Industries, Ltd.). 100 μL of the fecal suspension was inoculated into each medium-containing vessel to initiate anaerobic culture. Anaerobic conditions were maintained during culture by aerating the medium with a filter-sterilized mixed gas.

[0055] At the start of cultivation, and after 6, 9, 12, 24, and 30 hours, a portion of the culture medium was collected from the protruding part on the side of the container. pH data was collected every 5 minutes during the cultivation period. The sample collected at the start of cultivation (fecal sample) and the sample collected after 30 hours of cultivation (culture medium) were also used for bacterial flora evaluation. The sample collected after 30 hours of cultivation (culture medium) was also used for short-chain fatty acid (SCFA) quantification. The fecal samples and culture medium were stored at -20°C until used for measurement.

[0056] Example 2: Evaluation of pH profile of fecal culture fluid from healthy subjects and patients with ulcerative colitis According to Example 1, pH profiles were created based on pH data collected from samples derived from healthy subjects (n=13) and ulcerative colitis patients (n=11).

[0057] Figure 2 is a graph showing the pH profiles of fecal samples and culture fluid from 13 healthy subjects (A) and 11 patients with ulcerative colitis (B). The vertical axis shows the measured pH value, and the horizontal axis shows the incubation time (hours). As a result, in most of the healthy subjects, the pH dropped to about 5.5-6.0 after 6 hours of incubation, then gradually rose and approached pH 6.5 (pH of the large intestine) in the later stages of incubation (Figure 1(A)). In contrast, in the patients with ulcerative colitis, the pH dropped to about 6.0 after 6 hours of incubation, then the pH rose sharply to around 6.5 and then further decreased, and then the pH was maintained after the decrease or gradually increased, but the pH showed a clearly abnormal movement compared to that of the healthy subjects (Figure 1(B)). In this way, the condition of the patient can be roughly predicted just by looking at the pH profile.

[0058] Example 3: Evaluation of the bacterial flora in fecal cultures from healthy subjects and patients with ulcerative colitis (3-1: Extraction of microbial DNA) DNA extraction of bacteria from the large intestinal flora was performed for the samples collected at the start of the culture (fecal samples) and the samples collected 30 hours after the culture (culture fluid) as follows. The extraction solution used for DNA extraction was 500 μL of Tris-EDTA (TE) saturated phenol, 250 μL of lysis buffer (1 M Tris pH 8.0, 0.5 M EDTA), 50 μL of 10% SDS, and 0.3 g of glass beads (φ0.1 mm). 200 μL of the extraction solution was added to the sample, and the bacteria were disrupted by vigorously shaking vertically for 30 seconds at power level 5.0 using FastPrep24 (MP). The supernatant was then collected by centrifugation, and the proteins were denatured using phenol, chloroform, and isoamyl alcohol, followed by isopropanol precipitation to precipitate the DNA. The precipitated DNA was dissolved in 100 μL of TE and used as template DNA. The purified DNA was stored at -20°C until use.

[0059] (3-2: 16S rRNA gene sequencing) Genomic DNA was subjected to amplification of the V3-V4 region of the bacterial 16S rRNA gene using the primer pair SD-Bact-0341-bS-17 (SEQ ID NO: 1) and SD-Bact-0785-aA-21 (SEQ ID NO: 2). Illumina adapter overhang nucleotide sequences (Illumina, Inc.) were added to the gene-specific sequences. PCR cycling reactions were performed according to the manufacturer's instructions. Verified amplicons were purified using AMPure XP DNA purification beads (Beckman Coulter, Inc.) and eluted in 25 μl of 10 mM Tris (pH 8.5). Amplicons were quantified on an Agilent Bioanalyzer 2100 DNA 1000 chip (Agilent Technologies, Inc.) and pooled in equimolar concentrations. 16S rRNA gene products (along with an internal control (PhiX control V3; Illumina)) were subjected to paired-end sequencing using a MiSeq sequencer (Illumina) with a 600-cycle MiSeq reagent kit (Illumina). PhiX sequences were excised and paired-end reads with a Q score of 20 or more were joined using the software package QIIME version 1.9.1. Chimeric sequences were identified and excluded from the libraries. Paired-end reads were phylogenetically classified by the GreenGenes phylogenetic database using the Ribosomal Database Project (RDP) Classifier. Operational Taxonomic Units (OTUs) that reached 97% similarity were used for Shannon-Wiener and Simpson index diversity comparisons and Chao species number estimation. Principal coordinate analysis (PCoA) was performed using OTU information from each sample and calculated based on unweighted UniFrac distances. All raw sequence data generated in this experiment were deposited in MG-RAST and were assigned accession numbers.

[0060] (3-3: Real-time PCR) Real-time PCR was performed using a TP700 Thermal Cycler Dice Real Time System Lite (Takara Bio Inc.) As described in Non-Patent Document 1, amplification was performed using a primer set targeting all enterobacteria.

[0061] (3-4: Bioinformatics and statistical analysis) Shannon-Wiener diversity index, Simpson index and Chao1 were calculated using the QIIME software package. Nonparametric Kruskal-Wallis tests were used to determine significant differences for OTU numbers, Shannon-Wiener diversity index, Simpson index, Chao1, and bacterial 16S rRNA gene and SCFA concentrations. Differences in microbial community composition as described in unweighted UniFrac were tested using MANOVA tests. A P value of less than 0.05 was considered significant.

[0062] (3-5:Result) The 16S rRNA gene sequencing data for samples collected at the start of culture (fecal samples) and after 30 hours of culture (culture fluid samples) for healthy individuals and ulcerative colitis (UC) patients are shown in Table 1 below.

[0063] [Table 1]

[0064] The number of reads is an index of the number of bacteria, and the number of OTUs is an index of the species number. The Shannon-Wiener diversity index ("Shannon") and the Simpson index ("Simpson") are indices of species diversity, and Chao1 is an index of species richness.

[0065] The number of OTUs in fecal samples (collected at the start of culture) from healthy subjects was maintained in the culture medium samples (collected 30 hours after culture). Similarly, the number of OTUs in fecal samples from ulcerative colitis patients was maintained in the culture medium samples. For healthy subjects, there were no significant differences in the Shannon-Wiener diversity index, Simpson's index, and Chao1 between fecal samples and culture medium samples. For ulcerative colitis patients, there were no significant differences in the Shannon-Wiener diversity index, Simpson's index, and Chao1 between fecal samples and culture medium samples. Therefore, it can be seen that the bacterial species diversity and number of bacterial species in the bacterial flora in the fecal samples were maintained in the culture medium samples.

[0066] There was no significant difference in the number of OTUs in fecal samples between healthy subjects and patients with ulcerative colitis, and this tendency was also reproduced in the culture fluid samples. Furthermore, there was no significant difference in the Shannon-Wiener diversity index, Simpson's index, and Chao1 diversity index between healthy subjects and patients with ulcerative colitis in fecal samples, and the tendency of no significant difference in all these indices was also reproduced in the culture fluid samples. Thus, the relationship between the bacterial species diversity and the number of bacterial species between healthy subjects and patients with ulcerative colitis was similar in both fecal and culture fluid samples.

[0067] Figure 3 is a graph showing principal coordinate analysis (PCoA) of 16S rRNA gene sequencing data of samples collected at the start of culture (fecal samples) and samples collected 30 hours after culture (culture fluid samples) for healthy subjects (HS) and ulcerative colitis (UC) patients, respectively. In Figure 3, "HS stool" represents the healthy subject stool sample, "UC stool" represents the ulcerative colitis patient stool sample, "HS culture fluid" represents the healthy subject culture fluid sample, and "UC culture fluid" represents the ulcerative colitis patient culture fluid sample.

[0068] As shown in Figure 3, the UC stool plots formed different clusters than the HS stool plots, indicating that the UC patient's microbiota was distinct from that of healthy subjects in the stool samples. Figure 3 shows that such differences in the microbiota between UC patients and healthy subjects were maintained in the comparison of the cluster formation of the culture sample plots.

[0069] Figure 4 is a graph showing the abundance ratio of various Enterobacteriaceae relative to the total enterobacteria in samples collected at the start of culture (fecal samples) and samples collected 30 hours after culture (culture fluid samples) for healthy subjects (HS) and ulcerative colitis (UC) patients. In Figure 4, "HS feces" represents the fecal samples from healthy subjects, "UC feces" represents the fecal samples from ulcerative colitis patients, "HS culture fluid" represents the culture fluid samples from healthy subjects, and "UC culture fluid" represents the culture fluid samples from ulcerative colitis patients.

[0070] As shown in FIG. 4, it is interesting to note that the abundance ratio of Lachnospiraceae bacteria was significantly lower in both fecal samples and culture samples from ulcerative colitis patients than in healthy subjects. This point is more clearly shown in FIG. 5. FIG. 5 is a box-and-whisker plot showing the abundance ratio of Lachnospiraceae bacteria to the total intestinal bacteria in the samples at the start of culture (fecal samples) and the samples collected 30 hours after culture (culture samples) for each of healthy subjects and ulcerative colitis (UC) patients. The vertical axis of FIG. 5 shows the abundance ratio (%) of Lachnospiraceae bacteria to the total intestinal bacteria, and the horizontal axis shows the measured samples. By this culture, the decrease in the abundance ratio of Lachnospiraceae bacteria in ulcerative colitis patients compared to healthy subjects, which was observed in the fecal samples, was also observed in the culture samples.

[0071] The abundance ratio of species (genus level) belonging to the Lachnospiraceae family to the total intestinal bacteria was also determined in samples collected at the start of culture (fecal samples) and 30 hours after culture (culture fluid samples) from healthy subjects and ulcerative colitis (UC) patients. The results are shown in Table 2 below.

[0072] [Table 2]

[0073] Table 2 shows the results for the top five species of Lachnospiraceae with the highest abundance ratios: Blautia spp., Coprococcus spp., Dorea spp., Lachnospira spp., and Roseburia spp. In Table 2, asterisks (*) indicate significant differences between HS and UC patients in fecal samples and corresponding culture samples (*P<0.05, **P<0.01). For Coprococcus spp., a significantly lower abundance ratio was observed in E. ulcerative colitis patients than in healthy subjects in both fecal and culture samples.

[0074] Example 4: Quantification of SCFAs in fecal cultures from healthy subjects and patients with ulcerative colitis The amounts of various acids (succinic acid, lactic acid, acetic acid, propionic acid, and butyric acid) contained in short-chain fatty acids (SCFAs) in the samples (culture medium) collected after 30 hours of culture were measured using high performance liquid chromatography (HPLC) (Shimadzu Corporation) equipped with an Aminex HPX-87H column (Bio-Rad Laboratories) and a RID-10A refractive index detector (Shimadzu Corporation). The HPLC was operated at 65°C with a flow rate of 0.6 mL / min and 5 mM H2SO4 as the mobile phase.

[0075] FIG. 6 is a box plot showing the amounts of acetic acid, propionic acid, butyric acid, and SCFA (total of succinic acid, lactic acid, acetic acid, propionic acid, and butyric acid) in samples (culture fluid samples) collected after 30 hours of culture for each of healthy subjects and ulcerative colitis (UC) patients. The vertical axis of each graph in FIG. 6 shows the concentration (mM) of each acid in the sample, and the horizontal axis shows the sample measured. In both healthy subjects and ulcerative colitis patients, acetic acid, propionic acid, and butyric acid were mainly produced. Little production of lactic acid and succinic acid was observed. A significant decrease in the amount of butyric acid produced was observed in the culture fluid samples of ulcerative colitis patients compared to the culture fluid samples of healthy subjects.

[0076] Figure 7A shows the results of plotting the abundance ratio (%) of Lachnospiraceae bacteria in culture samples from ulcerative colitis patients (horizontal axis) against the amount of butyric acid (mM) produced by culture (vertical axis). The abundance ratio of Lachnospiraceae bacteria in the culture samples was highly correlated with the amount of butyric acid produced by culture (Figure 7A). In other words, the lower the abundance ratio of Lachnospiraceae bacteria in the culture samples, the lower the amount of butyric acid produced.

[0077] Figure 7 also shows the results of plotting the percentage (%) (horizontal axis) of Lachnospiraceae bacteria in fecal samples (B) and culture samples (C) from ulcerative colitis patients against the partial Mayo score (vertical axis). There was no correlation between the partial Mayo score and the percentage of Lachnospiraceae bacteria in either the fecal or culture samples (Figures 7B and C).

[0078] Ulcerative colitis is characterized by repeated periods of exacerbation and remission, so a low abundance of Lachnospiraceae bacteria and the resulting low amount of butyrate produced may lead to relapse of colitis. Therefore, considering that butyrate induces differentiation of regulatory T cells in peripheral tissues and has anti-inflammatory effects (Furusawa, Y. et al., Nature 10.1038 / nature12721. 2013), increasing butyrate-producing bacteria in the intestine of ulcerative colitis patients is important for maintaining remission. Furthermore, the above results indicate that this culture model can be used to evaluate the role of prebiotics and probiotics on commensal bacteria and metabolite production in ulcerative colitis patients in a high-throughput manner. [Explanation of symbols]

[0079] 100 Inspection Equipment 102 Culture solution 110 Anaerobic Cultivation Methods 112 Culture tank 114 Lid 116 Motor 118 Shaft 120 Mixing blade 122 Air supply pipe 124 Valve 126 Exhaust pipe 128 Temperature control means 140 Measurement means 142 Temperature Sensor 144 Chemical Sensors 170 Means of comparison 172 Control section 174 Memory section 176 Display section

Claims

1. A method for producing a bacterial species sample derived from a subject, in which the number of bacterial species in the sample from the subject is maintained, comprising: 1) adding a subject sample to a medium; 2) at the start of culture, performing an operation so that the pH of the medium is 6.2 to 6.7; 3) culturing the sample after the start of the culture without any manipulation intended to maintain the pH of the medium; wherein the medium is aerated with a nitrogen and carbon dioxide mixture.

2. Before 1) A) sterilizing the medium and aerating it with a nitrogen and carbon dioxide mixed gas; and / or B) suspending the sample in a phosphate buffer containing ascorbic acid The method of claim 1 , comprising:

3. The method according to claim 2, wherein the sterilization is by autoclave.

4. A method according to any one of claims 1 to 3, further comprising a step of measuring the pH of the culture medium.

5. A method according to any one of claims 1 to 4, wherein the bacterial species sample derived from the subject maintains bacterial species diversity and number of bacterial species.

6. A method for culturing a subject sample so as to maintain the number of bacterial species, comprising: 1) adding a subject sample to a medium; 2) at the start of culture, performing an operation so that the pH of the medium is 6.2 to 6.7; 3) culturing the sample after the start of the culture without any manipulation intended to maintain the pH of the medium; wherein the medium is aerated with a nitrogen and carbon dioxide mixture.

7. Before 1) A) sterilizing the medium and aerating it with a nitrogen and carbon dioxide mixed gas; and / or B) suspending the sample in a phosphate buffer containing ascorbic acid The method of claim 6, comprising:

8. The method according to claim 7, wherein the sterilization is by autoclave.

9. The method described in any one of claims 6 to 8, further comprising a step of measuring the pH of the culture medium.

10. A method according to any one of claims 6 to 9, wherein the culturing method is one that maintains bacterial species diversity and number of bacterial species.

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