Composition for the suppression of irritable bowel syndrome

Abstract

To provide agents and food that improve, or suppress, intestinal permeability, thereby treating various diseases and conditions including IBS, alleviating symptoms, and preventing their onset.SOLUTION: A composition for suppressing intestinal permeability contains bacterial cells belonging to Dialister or a culture product thereof as an active ingredient. The inventive composition can be used to suppress diseases or conditions caused by elevated intestinal permeability and suppress abdominal discomfort symptoms.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a composition for suppressing intestinal permeability and improving diseases or conditions caused by increased intestinal permeability, such as irritable bowel syndrome. The present invention also relates to an effective method for diagnosing irritable bowel syndrome. [Background technology]

[0002] It is known that there are approximately 100 trillion diverse bacteria in the intestines of humans and animals. These bacterial groups are also called intestinal bacteria, intestinal flora, or gut microbiota. Intestinal bacteria are bacteria that have been ingested orally and have proliferated in the intestinal environment. Therefore, they originate from food and beverages that the host actively consumes, as well as bacteria that have entered the digestive tract through infection and proliferated. Some intestinal bacteria are excreted from the body, but they are also constantly present in healthy hosts, and their composition can be influenced by the host's diet and environment.

[0003] In recent years, it has become increasingly known that the types and proportions of gut bacteria are related to the health and diseases of the host humans and animals. For example, it has been reported that individuals who develop irritable bowel syndrome (IBS) after infectious enteritis have a different gut bacterial composition than healthy individuals, that stress can alter gut bacterial composition, and that gut bacterial composition may also be associated with mental illnesses such as depression (Non-Patent Documents 1 and 2). Furthermore, Non-Patent Document 3 reports that germ-free mice transplanted with feces from IBS patients exhibited IBS symptoms.

[0004] Therefore, maintaining a healthy balance of gut bacteria is considered important for preserving the physical and mental health of the host. In addition to consuming probiotics (bacteria) such as lactic acid bacteria and bifidobacteria as food and beverages, taking them as supplements has also been suggested. On the other hand, Patent Document 1 discloses a method for testing IBS using intestinal microbiota profiles of 15 types of bacteria, including the genus Dialister. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] WO2020 / 213732 [Non-patent literature]

[0006] [Non-Patent Document 1] Journal of Intestinal Microbiota, 2018, Vol. 32, No. 1, pp. 1-6. [Non-Patent Document 2] Liu et al. BMC Microbiology (2020) 20:168 [Non-Patent Document 3] De Palma et al., Sci. Transl. Med. 9, eaaf6397 (2017) [Overview of the project] [Problems that the invention aims to solve]

[0007] It has been reported that intestinal permeability is increased in patients with gastrointestinal disorders such as irritable bowel syndrome (IBS) (Therap Adv Gastroenterol. 2021; 14: 1756284821993586), and it has also been reported that stress increases intestinal permeability (Exp Neurol, November 2021, 345, 113841). Furthermore, it has been reported that intestinal bacteria and their metabolites that invade tissues from the intestinal tract may cause diseases such as cancer, arteriosclerosis, hypertension, liver damage, and aging (Journal of Intestinal Microbiology, 2018, Vol. 32, No. 1, pp. 1-6). A report (Gastroenterology & Hepatology, Vol. 5, Issue 11, p996-1007, November 2020) that lubiprostone, a drug used to treat constipation-predominant IBS, improves intestinal permeability and thus improves non-alcoholic fatty liver disease suggests a link between intestinal permeability and non-alcoholic fatty liver disease.

[0008] Therefore, it is believed that improving intestinal permeability, or in other words, inhibiting intestinal permeability, can treat, alleviate symptoms of, and prevent the onset of various diseases and conditions, including IBS, and there is a demand for drugs and foods that can be used for this purpose. [Means for solving the problem]

[0009] In view of the above problems, the inventors conducted various studies and found that bacteria of the genus Dialister, a type of intestinal bacteria, reduce intestinal permeability, and that the number of Dialister bacteria is significantly lower in IBS patients compared to healthy individuals. Based on these findings, the inventors completed the present invention. Furthermore, the inventors also found that the likelihood of developing IBS in a subject can be detected with high accuracy by using a specific combination of bacteria, including Dialister bacteria.

[0010] In other words, the present invention provides the following: 1. A composition for inhibiting intestinal permeability, comprising bacterial cells or culture products of bacteria of the genus Dialister as an active ingredient. 2. A composition for suppressing diseases or conditions caused by increased intestinal permeability, comprising bacterial cells or culture products of the genus Dialister as an active ingredient. 3. A composition for suppressing unpleasant abdominal symptoms, comprising bacterial cells or culture products of the genus Dialister as an active ingredient. 4. A composition comprising bacterial cells or culture products of the genus Dialister as an active ingredient for suppressing irritable bowel syndrome (IBS), inflammatory bowel disease, diabetes, chronic renal failure, obesity, cancer, cardiovascular disease, or non-alcoholic fatty liver disease. 5. The composition according to any one of items 1 to 4 above, wherein the bacterium belonging to the genus Dialister is Dialister invisus, Dialister succinatiphilus, Dialister hominis, and / or Dialister propionicifaciens. 6. The composition according to any one of items 1 to 5 above, wherein the culture product is a culture product of Dialister invisus and / or Dialister succinatiphilus. 7. The composition according to any one of items 1 to 6 above, wherein the culture product is a molecular weight fraction of less than 1000 kDa. 8. A method for obtaining data on the likelihood of developing irritable bowel syndrome in subjects, including calculating the proportion of one or more, five or more, ten or more, fifteen or more, or all 20 of the following bacteria (1) to (20) in stool samples obtained from subjects as a percentage of the total gut flora, and comparing these proportions with pre-established reference data: (1) Bacteria of the genus Parabacteroides, (2) Bacteroides bacteria, (3) Bacteria of the genus Escherichia Shigella (4) Bacteria of the genus Odoribacter, (5) Bacteria of the genus Roseburia, (6) Lachnospiraceae bacterium UCG.004, (7) Bacteria of the genus Anaerotruncus, (8) Bacteria of the genus Dialister, (9) Ruminococcusae bacterium UCG.009, (10) Bacteria of the genus Parasutterella, (11) Bacteria of the genus Lactobacillus, (12) Eubacterium eligens, (13) Bacteria of the genus Peptoclostridium, (14) Bacteria of the genus Holdemania, (15) Clostridiales.Family.XIII UCG.001, (16) Bacteria of the genus Holdemanella, (17) Bacteria of the genus Bacillus, (18) Bacteria of the genus Peptostreptococcus, (19) Bacteria of the genus Klebsiella, and (20) Bacteria of the genus Mitsuokella. 9. The method according to 8 above, wherein the data on the bacteria randomly selected from the bacteria (1) to (20) above are repeatedly compared with the reference data.

Advantages of the Invention

[0011] According to the present invention, intestinal permeability can be suppressed, and thereby diseases or conditions caused by enhanced intestinal permeability can be suppressed. Further, according to the present invention, the likelihood of developing IBS can be detected with higher accuracy, leading to prevention of onset and early treatment.

Brief Description of the Drawings

[0012] [Figure 1] Shows the change in TEER at 5 hours after stimulation with fecal supernatant (10% or 25%) when the value before stimulation is set to 100%. IBS: IBS patient group, Ctrl: control group. *: p < 0.05. [Figure 2]This shows the number of reads (number of reads detected out of 10,000 reads) of *Bacteria genus *Dialister* detected in stool samples from the IBS patient group and the control group. IBS: IBS patient group, Ctrl: control group. [Figure 3] The graph shows the change in TEER 3 hours after bacterial stimulation, with the value before stimulation set to 100%. Bacteria (-): No stimulation, B. dorei: Bacteroides dorei, D. invisus: Dialister invisus, D. succinatiphilus: Dialister succinatiphilus, D. hominis: Dialister hominis, D. propionicifaciens: Dialister propionicifaciens. [Figure 4] The graph shows the change in TEER 24 hours after stimulation of the culture supernatant, with the pre-stimulation value set to 100%. GAM + succinic acid: culture medium stimulation, B. dorei: Bacteroides dorei, D. invisus: Dialister invisus, D. succinatiphilus: Dialister succinatiphilus, D. hominis: Dialister hominis, D. propionicifaciens: Dialister propionicifaciens. [Figure 5] This shows the change in TEER 24 hours after stimulation of the Dialister-Invisas culture supernatant fraction sample, with the pre-stimulation value set to 100%. Stock: Unfractionated culture supernatant, >50k: ≥50kDa, <50k: <50kDa, <30k: <30 kDa, <10k: <10 kDa, <3k: <3 kDa. [Figure 6] This shows the change in TEER at 24 hours after stimulation of the Dialister succinatiphilus culture supernatant fraction sample, with the pre-stimulation value set to 100%. Stock: Unfractionated culture supernatant, >50k: ≥50kDa, <50k: <50kDa, <30k: <30 kDa, <10k: <10 kDa, <3k: <3 kDa. [Figure 7] The ROC curve for the IBS diagnosis model created using Random Forest is shown. [Modes for carrying out the invention]

[0013] The present invention provides a composition for inhibiting intestinal permeability, comprising bacterial cells or culture products of the genus Dialister as an active ingredient.

[0014] Bacteria of the genus Dialister are anaerobic, Gram-negative bacteria belonging to the phylum Firmicutes, class Negativicutes, order Selenomonadales, and family Veillonellaceae.

[0015] Any species of the genus Dialister can be suitably used in the present invention, but for example, Dialister invisus, Dialister succinatiphilus, Dialister hominis, Dialister propionicifaciens, Dialister micraerophilus, Dialister pneumosintes, and Dialister massiliensis can be suitably used, and these Dialister bacteria can be used individually or in combination. More preferably, Dialister invisus, Dialister succinatiphilus, Dialister hominis, or Dialister propionicifaciens can be used individually or in combination.

[0016] These bacteria can all be obtained from mammalian feces or from distribution and depositary institutions such as the BioResource Research Center for Microbial Materials Development (RIKEN BRC-JCM) and the American Type Culture Collection (ATCC). They can be propagated using known methods, and the method of acquisition is not limited. For example, *Dialister invisus* can be obtained as JCM17566 or ATCC-51894, *Dialister succinatiphilus* as JCM15077, *Dialister hominis* as JCM33369, and *Dialister propionisifaciens* as JCM17568.

[0017] One embodiment of the composition of the present invention contains, as is or cultured and grown, the bacteria of the genus Dialister obtained as described above as an active ingredient. When preparing the composition, operations such as the removal of impurities may be included as appropriate, if necessary.

[0018] A preferred embodiment of the present invention is a composition for inhibiting intestinal permeability, comprising the cells of Dialister inviscus, Dialister succinatiphilus, Dialister hominis, and / or Dialister propionisifaciens as an active ingredient.

[0019] Another aspect of the composition of the present invention contains a culture product of bacteria of the genus Dialister as an active ingredient. The method for obtaining the culture product is not particularly limited, but it can be obtained, for example, by the method described in the examples below. Any culture product may be used, but among the culture products, for example, a fraction that has been confirmed to have an effect of improving intestinal permeability, i.e., a molecular weight fraction of less than 1000 kDa, preferably less than 500 kDa, and more preferably less than 50 kDa, can be suitably used.

[0020] A preferred embodiment of the present invention is a composition for inhibiting intestinal permeability, wherein the culture product contains a culture product of Dialister inviscius and / or Dialister succinatiphilus as an active ingredient.

[0021] The composition of the present invention has the effect of suppressing intestinal permeability and can therefore be used to suppress diseases or conditions caused by increased intestinal permeability. In this specification, "diseases or conditions caused by increased intestinal permeability" are not limited to, but include, for example, irritable bowel syndrome (IBS); inflammatory bowel diseases of the intestines such as leaky gut syndrome, inflammatory bowel disease, and celiac disease; and systemic diseases such as diabetes mellitus, chronic renal failure, obesity, cancer, cardiovascular disease, and non-alcoholic fatty liver disease. Irritable bowel syndrome (IBS) is known to have constipation-predominant, diarrhea-predominant, mixed, and unclassifiable types, but the composition of the present invention can provide therapeutic or symptom-relieving effects for any type of IBS.

[0022] The compositions of the present invention can also be used to suppress unpleasant abdominal symptoms. Unpleasant abdominal symptoms include, but are not limited to, abdominal pain, constipation, diarrhea, bloating, and the like.

[0023] The composition of the present invention may be a pharmaceutical composition. When used as a pharmaceutical composition, the method of administration may be intra-gastrointestinal administration such as oral administration or enteral administration. Furthermore, the composition of the present invention may be a food, health food, functional food, supplement, etc. In this case, administration (ingestion) may be orally.

[0024] The composition of the present invention, which contains bacterial cells or culture products as an active ingredient, can be prepared as a liquid composition. Alternatively, the composition of the present invention can be prepared as a solid composition. In this case, the bacterial cells or culture products can be dried by means commonly used in the art, such as freeze-drying.

[0025] The composition can be prepared in liquid or solid form. Depending on the dosage form, excipients, binders, gelling agents, disintegrants, lubricants, sweeteners, flavoring agents, fragrances, etc., may be added to the composition as appropriate.

[0026] The compositions of the present invention may be administered to mammals, such as humans, monkeys, dogs, cats, rabbits, guinea pigs, mice, rats, cattle, horses, sheep, goats, hippos, elephants, tigers, lions, etc. The subjects of administration may be humans, pets, livestock, or mammals kept in zoos. The subjects of administration may also be non-human experimental animals used to predict and evaluate the effects in humans.

[0027] The compositions of the present invention can be administered (ingested) to subjects suffering from diseases caused by increased intestinal permeability, or subjects in a state caused by increased intestinal permeability. Specifically, the compositions of the present invention can be administered to patients suffering from, for example, irritable bowel syndrome (IBS); inflammatory bowel disease such as leaky gut syndrome, inflammatory bowel disease, and celiac disease; or systemic diseases such as diabetes mellitus, chronic renal failure, obesity, cancer, cardiovascular disease, and non-alcoholic fatty liver disease. The compositions of the present invention can also be administered (ingested) to suppress uncomfortable abdominal symptoms in subjects.

[0028] The above-mentioned diseases or conditions in the subjects may be chronic or transient. The compositions of the present invention can also be administered (ingested) prophylactically to subjects who are likely to develop the above-mentioned diseases or conditions. For example, the compositions of the present invention can be administered prophylactically to undiagnosed subjects with irritable bowel syndrome, or to improve transient conditions such as increased intestinal permeability in healthy individuals.

[0029] The dosage (intake) of the composition of the present invention may vary depending on the condition of the human or animal being studied, and is therefore not limited, but for example, the dosage of bacterial cells may be 10 per day. 5 ~10 15 pieces, 10 7 ~10 13 pieces, 10 9 ~10 11The dosage can be within a range of one individual. The dosage of the culture product can be appropriately determined by calculating from the above bacterial cell volume. The frequency of administration (intake frequency) can also vary depending on the condition of the subject being administered to, and is not limited, but for example, it can be three times a day, twice a day, once a day, once every two days, once every three days, once a week, etc. Administration may be continued for a long period of time, as long as the symptoms or condition of the disease caused by increased intestinal permeability persist, until the uncomfortable abdominal symptoms are reduced or disappear. The effects of the present invention can be confirmed by the reduction or disappearance of symptoms in the recipient.

[0030] Alternatively, the effects of the present invention can also be confirmed in vitro. For example, human colon cancer-derived cells Caco-2 can differentiate into a monolayer similar to intestinal epithelium, and are therefore widely used to evaluate intestinal barrier function such as intestinal permeability. Intestinal permeability can be evaluated by measuring the transepithelial electrical resistance (TEER) of Caco-2. Since intestinal permeability is known to be increased in IBS patients (Therap Adv Gastroenterol. 2021; 14: 1756284821993586), the change in TEER value when the composition of the present invention is added using Caco-2 can be compared with the case where it is not added to evaluate whether or not there is an improvement in intestinal permeability.

[0031] The inventors have also shown that human gut microbiota data, specifically 29 genera of gut bacteria narrowed down through intergroup comparisons, and more specifically, 20 genera of gut bacteria extracted as IBS characteristics, are useful as biomarkers for IBS, and suggest that these gut bacteria are associated with the onset and symptoms of IBS.

[0032] Accordingly, the present invention also provides a method for obtaining data for diagnosing the likelihood of a subject suffering from irritable bowel syndrome, using the amount of any of the following bacteria (1) to (20) in a sample obtained from the subject as an indicator. (1) Bacteria of the genus Parabacteroides, (2) Bacteroides bacteria, (3) Bacteria of the genus Escherichia Shigella (4) Bacteria of the genus Odoribacter, (5) Bacteria of the genus Roseburia, (6) Lachnospiraceae bacterium UCG.004, (7) Bacteria of the genus Anaerotruncus, (8) Bacteria of the genus Dialister, (9) Ruminococcusae bacterium UCG.009, (10) Bacteria of the genus Parasutterella, (11) Lactobacillus bacteria, (12) Eubacterium eligens, a bacterium of the genus Eubacterium. (13) Peptoclostridium bacteria, (14) Bacteria of the genus Holdemania, (15) Clostridiales Family XIII (UCG.001) (16) Bacteria of the genus Holdemanella, (17) Bacillus bacteria, (18) Bacteria of the genus Peptostreptococcus, (19) Bacteria of the genus Klebsiella, (20) Bacteria of the genus Mitsuokella.

[0033] The method of the present invention can provide information on whether a sample obtained from an individual subject or a group of subjects is associated with irritable bowel syndrome (IBS). In the above method, the subject is a human subject who needs information about the likelihood of developing IBS and may be an IBS patient. The subject may also be a patient suffering from a disease other than IBS, or a healthy individual.

[0034] As described in the examples below, the inventors first obtained stool samples from IBS patients and healthy individuals, respectively, and obtained genus-level intestinal microbiota data from the DNA present in the samples. By statistically analyzing this data, they selected the following 29 types of bacteria that differed in abundance between IBS patients and healthy individuals. 1. Bacteria of the genus Parabacteroides, 2. Bacteria of the genus Peptostreptococcus, 3. Bacteria of the genus Parasutterella, 4. Bacteroides bacteria, 5. Ruminococcusae bacterium UCG.009 6. Bacteria of the genus Roseburia, 7. Bacteria of the genus Holdemanella, 8. Bacteria of the genus Pseudomonas, 9. Bacteria of the genus Escherichia Shigella 10. Bacteria of the genus Klebsiella, 11. Bacillus bacteria, 12. Campylobacter bacteria, 13. Bacteria of the genus Granulicatella, 14 Bacteria of the genus Papillibacter, 15 Coprococcus bacteria 2, 16. Bacteria of the genus Mitsuokella, 17 Bacteria of the genus Atopobium, 18. Lachnospiraceae bacteria, FE2018 group. 19. Bacteria of the family Flavobacteriaceae, 20. Eubacterium genus bacteria, eligens. 21. Bacteria of the genus Dialister, 22. Bacteria of the genus Odoribacter, 23. Bacteria of the genus Lactobacillus, 24. Lachnospiraceae bacterium UCG.004, 25 Clostridiales Family XIII (UCG.001) 26 Peptoclostridium bacteria, 27. Bacteria of the genus Holdemania, 28. Cloacibacillus bacteria, 29. Bacteria of the genus Anaerotruncus.

[0035] The bacteria listed above (1-29) include those present in greater quantities in the IBS patient group compared to the healthy control group, as well as those present in smaller quantities. The bacteria listed above (1-29) are listed in descending order of p-value when comparing the IBS patient group and the healthy control group.

[0036] Therefore, by focusing on one or more bacteria selected from the above-mentioned bacteria and evaluating the microbiome data in the subject's sample, predictive data regarding the likelihood of the subject suffering from IBS can be provided. Here, "microbiome data" is not limited to, but could be, for example, the ratio of the intended bacterial number to the total number of bacteria (DNA copy number) in the sample.

[0037] The evaluation can yield predictive data by comparing the results with pre-prepared standard datasets for IBS patients and healthy individuals. The data can be for one or more species of bacteria selected from the above-mentioned bacteria.

[0038] As described in the examples, the inventors further repeated the step of creating and evaluating an IBS diagnosis prediction model using machine learning (random forest) from the gut microbiota data selected above, and found that the following 20 types of bacteria are particularly important in determining the likelihood of developing IBS. (1) Bacteria of the genus Parabacteroides, (2) Bacteroides bacteria, (3) Bacteria of the genus Escherichia Shigella (4) Bacteria of the genus Odoribacter, (5) Bacteria of the genus Roseburia, (6) Lachnospiraceae bacterium UCG.004, (7) Bacteria of the genus Anaerotruncus, (8) Bacteria of the genus Dialister, (9) Ruminococcusae bacterium UCG.009, (10) Bacteria of the genus Parasutterella, (11) Lactobacillus bacteria, (12) Eubacterium eligens, a bacterium of the genus Eubacterium. (13) Peptoclostridium bacteria, (14) Bacteria of the genus Holdemania, (15) Clostridiales Family XIII (UCG.001) (16) Bacteria of the genus Holdemanella, (17) Bacillus bacteria, (18) Bacteria of the genus Peptostreptococcus, (19) Bacteria of the genus Klebsiella, (20) Bacteria of the genus Mitsuokella. The bacteria listed above (1) to (20) are listed in order of their contribution to prediction accuracy, based on the results of evaluating multiple IBS diagnosis prediction models.

[0039] Therefore, by using stool samples collected from subjects, measuring the proportion of intestinal bacteria, applying the proportions to the IBS diagnosis prediction model described above, and comparing the proportions of each bacterial genus, it is possible to determine whether or not the stool originates from an IBS patient.

[0040] The bacteria used for evaluation may be one or more, two or more, three or more, four or more, five or more, ten or more, fifteen or more, or all 20 of the bacteria listed in (1) to (20) above. The abundance of each bacterium can be calculated as a percentage of the total gut flora, and the likelihood of developing IBS can be assessed by comparing this with pre-prepared reference data for that bacterium. Alternatively, data for one or more bacteria randomly selected from the above bacteria can be repeatedly compared with the reference data. There is no particular limit to the number of repetitions, but for example, evaluation can be performed 2, 3, 5, or 10 times. The reference data may be a standard dataset for IBS patients as described above, or a standard dataset for healthy individuals.

[0041] The method of the present invention can automatically perform steps such as DNA extraction, sequencing, creation of gut microbiota data, and comparison with an IBS diagnosis prediction model after obtaining a stool sample from a subject. Therefore, the process can be carried out quickly even when evaluating a large number of bacteria.

[0042] For example, the method of the present invention specifically, Steps include entering the subject's gut bacteria data, and For the 29 genera or all 20 genera mentioned above, the step is to compare their relative abundance with the IBS diagnosis prediction model. It is also possible to program a computer to output results indicating that the higher the degree of agreement with the model example, the more likely the subject is to have IBS. [Examples]

[0043] The present invention will be further described below with reference to examples, but the present invention is not limited to these examples.

[0044] [Example 1: Verification of the effect of gut microbiota on intestinal permeability] <Method> We investigated the effect of human colon cancer-derived cells, Caco-2, on intestinal permeability of stool from IBS patients. Stool samples were collected from five IBS patients (constipation-predominant or mixed type) and five healthy individuals (Ctrl), and frozen on the same day of collection. The frozen stool samples were suspended in PBS to a concentration of 100 mg / mL, and insoluble components were removed by centrifugation. The collected supernatant was then filtered through a 0.22 μm filter to prepare stool supernatant samples.

[0045] Human colon cancer-derived cells Caco-2 (obtained from ATCC) were divided into 1.0 × 10⁻⁶ cells. 4 Cells were suspended in EMEM medium (Wako, #055-08975, containing L-glutamine, phenol red, sodium pyruvate, non-essential amino acids, and 1,500 mg / L sodium bicarbonate) containing 20% ​​FBS and penicillin / streptomycin to a concentration of cells / mL. 200 μL of the resulting cell suspension was seeded into the apical side of an insert well (Falcon® Culture Insert 24-well type, pore size 1.0 μm), and 700 μL of EMEM medium was added to the basal side. The cells were cultured in a CO2 incubator at 37°C and 5% CO2, with the medium changed every 2 or 3 days.

[0046] Insert wells containing Caco-2 cells that were 17 days or older after seeding were stimulated by adding 1 μg / mL LPS (INVIVOGEN, #tlrl-eblps) to the apical side and 20 ng / mL IFN-γ (Pepro Tech, #AF-300-02), 20 ng / mL TNF-α (R&D SYSTEMS, #210-TA-005), and 10 ng / mL IL-1β (Pepro Tech, #AF-200-01B) to the basal side. After 24 hours, the cells were stimulated again by adding the stool supernatant sample prepared above to the apical side at a concentration of 10% or 25%. The TEER of the cells was measured before stimulation with the stool supernatant and 5 hours after stimulation. A t-test was used as the statistical method.

[0047] <Result> Figure 1 shows the change in TEER at 5 hours after stool supernatant stimulation, with the value before stool supernatant stimulation set to 100%. In both the IBS patient group and the Ctrl group, TEER decreased with stool supernatant stimulation, but with 10% stool supernatant stimulation, the decrease was greater in IBS patients compared to Ctrl patients. Furthermore, with 25% stool supernatant stimulation, TEER decreased significantly more in IBS patients compared to Ctrl patients, indicating that differences in gut bacteria between IBS patients and healthy individuals are related to differences in Caco-2 cell TEER, i.e., affect intestinal permeability.

[0048] [Example 2: Identification of IBS-related bacterial candidates] <Method> Stool samples were collected from five IBS patients (constipation-predominant or mixed type) and five healthy individuals (Ctrl), immediately inactivated, and stored. Microbial analysis utilized 16S rRNA gene sequence information (SILVA rRNA database), widely used for classifying microbial species in populations containing many types of microorganisms, to identify and measure the relative abundance of microorganisms present in each stool sample. Specifically, DNA derived from intestinal bacteria was extracted from the stored stool samples, and the V3-V4 region of the 16S rRNA gene was amplified using the extracted DNA as a template. The amplified sequences were then sequenced using MiSeq, and 10,000 reads from each sample were used for microbial analysis. QIIME was used as the analysis tool to identify bacteria and calculate their relative abundance from the obtained sequences. The Mann-Whitney U test was used as the statistical method.

[0049] <Result> Microbial analysis identified bacterial genera that differed between the IBS and Ctrl groups. Among the extracted genera, *Dialister* bacteria were significantly less abundant in the IBS group compared to the Ctrl group. Figure 2 shows the number of *Dialister* bacteria reads (number of reads detected per 10,000 reads) in the IBS and Ctrl groups. *Dialister* bacteria were relatively more abundant in the Ctrl group (mean: approximately 25 reads, median: 10 reads) and less abundant in the IBS group (maximum less than 10 reads).

[0050] [Example 3: Verification of the effect of *Dialister* bacteria on intestinal permeability] <Method> As strains belonging to the genus Dialister, Dialister invisus (JCM17566), Dialister succinatiphilus (JCM15077), Dialister hominis (JCM33369), or Dialister propionisifaciens (JCM17568) were cultured under anaerobic conditions in a modified GAM medium (Nissui Pharmaceutical) containing 1% succinic acid. For comparison, Bacteroides dorei (JCM13471), one of the most dominant bacteria in the intestinal flora, was cultured similarly under anaerobic conditions. All strains were obtained from the RIKEN Resource Center, and bacterial growth was confirmed by measuring OD600. Bacterial samples were prepared by centrifuging the bacterial culture solution (1,500 × g, 10 minutes), removing the culture medium components from the supernatant, and then resuspending the cells in PBS.

[0051] Human colon cancer-derived Caco-2 cells 1.0 × 10 4 Cells were suspended in EMEM medium (Wako, #055-08975, containing L-glutamine, phenol red, sodium pyruvate, non-essential amino acids, and 1,500 mg / L sodium bicarbonate) containing 20% ​​FBS and penicillin / streptomycin to a concentration of cells / mL. 200 μL of the resulting cell suspension was seeded into the apical side of an insert well (Falcon® Culture Insert 24-well type, pore size 1.0 μm), and 700 μL of EMEM medium was added to the basal side. The cells were cultured in a CO2 incubator at 37°C and 5% CO2, with the medium changed every 2 or 3 days.

[0052] Insert wells containing Caco-2 cells that were 17 days or older after seeding were stimulated with 1 μg / mL LPS on the apical side and 20 ng / mL IFN-γ, 20 ng / mL TNF-α, and 10 ng / mL IL-1β on the basal side.

[0053] Forty-eight hours after stimulation, the cells were moved to anaerobic conditions, and a bacterial sample prepared to achieve an OD600 of 1 was added to the apical side for stimulation. The TEER (transepithelial electrical resistance) was measured before bacterial stimulation and 3 hours after stimulation. Dunnett's test was used as the statistical method.

[0054] <Result> Figure 3 shows the change in TEER at 3 hours after bacterial stimulation, with the pre-stimulation value set to 100%. Compared to no bacterial stimulation (-), stimulation with Dialister inviscus, Dialister succinatiphilus, Dialister hominis, and Dialister propionisifaciens all significantly suppressed the decrease in TEER from the pre-stimulation level. On the other hand, stimulation with Bacteroides dorei did not show a significant change compared to no bacterial stimulation. These results indicate that the presence of Dialister bacteria suppresses the decrease in TEER, i.e., suppresses the increase in intestinal permeability.

[0055] [Example 4: Verification of the effect of culture supernatant of *Dialister* bacteria on intestinal permeability] <Method> In the same manner as in Example 3, Dialister invisus (JCM17566), Dialister succinatiphyllus (JCM15077), Dialister hominis (JCM33369), or Dialister propionysifaciens (JCM17568) were cultured under anaerobic conditions in GAM medium containing 1% succinic acid. As a control, Bacteroides dorei (JCM13471) was also cultured under anaerobic conditions in GAM medium containing 1% succinic acid.

[0056] The culture medium was collected and its OD600 was measured. All culture media were then diluted in GAM medium containing 1% succinic acid to an OD600 of 0.17. The diluted culture media were centrifuged (1,500 × g, 10 min), the supernatant was collected, and bacterial components were removed by passing the supernatant through a 0.22 μm filter to prepare culture supernatant samples.

[0057] Human colon cancer-derived Caco-2 cells 1.0 × 104 Suspended in EMEM medium (manufactured by Wako, #055-08975, containing 20% FBS, penicillin-streptomycin, L-glutamine, phenol red, sodium pyruvate, non-essential amino acids, and 1,500 mg / L sodium bicarbonate) to a concentration of cells / mL. 200 μL of the obtained cell suspension was seeded on the apical side of an insert well (for Falcon® Culture Insert 24-well, pore size 1.0 μm), 700 μL of EMEM medium was added to the basal side, and the cells were cultured in a CO2 incubator at 37 °C and 5% CO2, with the medium changed once every 2 days or 3 days.

[0058] On the apical side of an insert well containing Caco-2 cells 17 days or more after seeding, 1 μg / mL of LPS was added, and on the basal side, 20 ng / mL of IFN-γ, 20 ng / mL of TNF-α, and 10 ng / mL of IL-1β were added for stimulation. 24 hours after stimulation, the culture supernatant sample prepared above was added to the apical side at a concentration of 10% for stimulation. The TEER (trans-epithelial electrical resistance) was measured before and 24 hours after the culture supernatant stimulation. The Dunnett test was used as a statistical method.

[0059] <Results> Figure 4 shows the change in TEER at 24 hours after the culture supernatant stimulation when the value before stimulation was set to 100%. Compared with the medium stimulation (GAM medium containing 1% succinic acid), the culture supernatants of Dialister invisus, Dialister succinatiphilus, Dialister hominis, and Dialister propionisiphaciens showed high TEER values, and particularly the culture supernatants of Dialister invisus and Dialister succinatiphilus showed significantly higher TEER values. On the other hand, no significant change was observed in the stimulation with the culture supernatant of Bacteroides dorei compared with the medium stimulation. From the above, it was suggested that the culture supernatants of Dialister bacteria increase TEER.

[0060] [Example 5 Verification of the effect of Dialister culture supernatant fraction samples on intestinal permeability] <Method> In the same manner as in Example 3, Dialister invisus (JCM17566) and Dialister succinatiphyllus (JCM15077) were cultured under anaerobic conditions in GAM medium containing 1% succinic acid.

[0061] The culture medium was collected and its OD600 was measured. All culture media were diluted in GAM medium containing 1% succinic acid to an OD600 of 0.17. The diluted culture media were centrifuged (1,500 × g, 10 min) and the supernatant was collected. The collected supernatant was filtered through a 0.22 μm filter to remove cellular components and prepare culture supernatant samples.

[0062] The obtained culture supernatant sample was fractionated into fractions of less than 50 kDa, less than 30 kDa, less than 10 kDa, and less than 3 kDa using a centrifugal filter (Amicon Ultra, Merck), and culture supernatant fraction samples were prepared.

[0063] Human colon cancer-derived Caco-2 cells 1.0 × 10 4 Cells were suspended in EMEM medium (Wako, #055-08975, containing L-glutamine, phenol red, sodium pyruvate, non-essential amino acids, and 1,500 mg / L sodium bicarbonate) containing 20% ​​FBS and penicillin / streptomycin to a concentration of cells / mL. 200 μL of the resulting cell suspension was seeded into the apical side of an insert well (Falcon® Culture Insert 24-well type, pore size 1.0 μm), and 700 μL of EMEM medium was added to the basal side. The cells were cultured in a CO2 incubator at 37°C and 5% CO2, with the medium changed every 2 or 3 days.

[0064] Insert wells containing Caco-2 cells that were 17 days or older after seeding were stimulated by adding 1 μg / mL of LPS to the apical side and 20 ng / mL of IFN-γ, 20 ng / mL of TNF-α, and 10 ng / mL of IL-1β to the basal side. 24 hours after stimulation, the culture supernatant fraction sample prepared above was added to the apical side to a concentration of 10% and the cells were stimulated again. TEER (transepithelial electrical resistance) was measured before stimulation and 24 hours after stimulation. Dunnett's test was used as the statistical method.

[0065] <Result> Figures 5 and 6 show the change in TEER at 24 hours after stimulation of the culture supernatant fraction sample, with the pre-stimulation value set to 100%. Dialister invissus stimulation showed significantly higher TEER in all fractions (<50 kDa, <30 kDa, <10 kDa, <3 kDa) and <3 kDa) compared to the medium stimulation (GAM medium containing 1% succinic acid) (Figure 5). Similarly, Dialister succinatiphilus stimulation also showed significantly higher TEER in all fractions (<50 kDa, <30 kDa, <10 kDa, <3 kDa) and <3 kDa compared to the medium stimulation (GAM medium containing 1% succinic acid) (Figure 6).

[0066] [Example 6: Relationship between gut microbiota data and irritable bowel syndrome (IBS)] <Method> The gut microbiota data was obtained according to previously reported procedures (Hosomi et al Sci Rep 7(1):4339, 2017; Mohsen et al BMC Bioinformatics 20(1):581, 2019). Specifically, DNA was extracted from the stool of human subjects using an automated nucleic acid extractor (PI-80X, Kurabo), sequenced using Illumina's Miseq, referenced the SILVA database, and analyzed using the Qiime1 pipeline to obtain gut microbiota data.

[0067] Next, we narrowed down the gut microbiota data to be used in constructing the IBS diagnostic model. Using a Student's t-test (two-tailed, unequal-variance two-sample test), we compared the gut microbiota data of 24 IBS patients diagnosed according to the ROME IV diagnostic criteria and 43 non-IBS control subjects, and selected gut bacteria (genera) with a p-value of less than 0.15.

[0068] Next, an IBS diagnosis model was constructed using the gut bacteria selected above. A random forest prediction algorithm was used, and 20 IBS patients and 35 non-IBS control subjects were randomly selected from 24 IBS patients and 43 non-IBS control subjects as training data to create the diagnosis model. The remaining 4 IBS patients and 8 control subjects were used as test data to evaluate the accuracy of the diagnosis model.

[0069] The evaluation method involved creating a Receiver Operating Characteristic (ROC) curve using the sensitivity and specificity of the prediction model, with AUC (Area Under the Curve) as the criterion. The process from randomly generating training and test data to evaluating the prediction model was repeated 10 times for examination. In addition, IBS features were extracted from this prediction model.

[0070] <Result> Based on the above intergroup comparisons, the following 29 genera of intestinal bacteria were selected. The numbers below indicate the selected bacteria, ordered from smallest to largest p-value. 1. Bacteria of the genus Parabacteroides, 2. Bacteria of the genus Peptostreptococcus, 3. Bacteria of the genus Parasutterella, 4. Bacteroides bacteria, 5. Ruminococcusae bacterium UCG.009 6. Bacteria of the genus Roseburia, 7. Bacteria of the genus Holdemanella, 8. Bacteria of the genus Pseudomonas, 9. Bacteria of the genus Escherichia Shigella 10. Bacteria of the genus Klebsiella, 11. Bacillus bacteria, 12. Campylobacter bacteria, 13. Bacteria of the genus Granulicatella, 14 Bacteria of the genus Papillibacter, 15 Coprococcus bacteria 2, 16. Bacteria of the genus Mitsuokella, 17 Bacteria of the genus Atopobium, 18. Lachnospiraceae bacteria, FE2018 group. 19. Bacteria of the family Flavobacteriaceae, 20. Eubacterium genus bacteria, eligens. 21. Bacteria of the genus Dialister, 22. Bacteria of the genus Odoribacter, 23. Bacteria of the genus Lactobacillus, 24. Lachnospiraceae bacterium UCG.004, 25 Clostridiales Family XIII (UCG.001) 26 Peptoclostridium bacteria, 27. Bacteria of the genus Holdemania, 28. Cloacibacillus bacteria, 29. Bacteria of the genus Anaerotruncus. Tables 1 and 2 show the detailed classifications of the above bacteria and the results of statistical analysis comparing them by group.

[0071] [Table 1]

[0072] [Table 2]

[0073] The AUCs for 10 test data in the random forest-based classification model were 0.875, 0.750, 0.728, 0.812, 0.688, 0.812, 0.750, 0.750, 0.750, and 0.812, with a mean of 0.773, a standard deviation of 0.054, and a standard error of 0.017. Among these, the model with the highest AUC was the IBS classification model with a test data AUC of 0.875, sensitivity of 100%, and specificity of 75% (Figure 7).

[0074] Furthermore, the following 20 genera of intestinal bacteria were extracted as IBS features, as they contributed significantly to the prediction accuracy of the IBS diagnosis model. The numbers below indicate the extracted bacteria, ranked in order of importance. (1) Bacteria of the genus Parabacteroides, (2) Bacteroides bacteria, (3) Bacteria of the genus Escherichia Shigella (4) Bacteria of the genus Odoribacter, (5) Bacteria of the genus Roseburia, (6) Lachnospiraceae bacterium UCG.004, (7) Bacteria of the genus Anaerotruncus, (8) Bacteria of the genus Dialister, (9) Ruminococcusae bacterium UCG.009, (10) Bacteria of the genus Parasutterella, (11) Lactobacillus bacteria, (12) Eubacterium eligens, (13) Peptoclostridium bacteria, (14) Bacteria of the genus Holdemania, (15) Clostridiales Family XIII (UCG.001) (16) Bacteria of the genus Holdemanella, (17) Bacillus bacteria, (18) Bacteria of the genus Peptostreptococcus, (19) Bacteria of the genus Klebsiella, (20) Bacteria of the genus Mitsuokella. Table 3 shows the detailed classification of the above bacteria.

[0075] [Table 3]

[0076] This example demonstrates that human gut microbiota data, specifically 29 genera of gut bacteria narrowed down through intergroup comparison, and more preferably 20 genera of gut bacteria extracted as IBS characteristics, are useful as biomarkers for IBS. Furthermore, it suggests that these gut bacteria are associated with the onset and symptoms of IBS. [Industrial applicability]

[0077] The present invention can suppress intestinal permeability, thereby suppressing diseases or conditions caused by increased intestinal permeability. Furthermore, the present invention can predict the likelihood of developing IBS with greater accuracy, leading to prevention of onset and early treatment.

Claims

1. A composition for inhibiting intestinal permeability, comprising bacterial cells or culture supernatant of bacteria of the genus Dialister as an active ingredient.

2. The composition according to claim 1, wherein the bacterium belonging to the genus Dialister is Dialister invisus, Dialister succinatiphilus, Dialister hominis, and / or Dialister propionicifaciens.

3. The composition according to claim 1 or 2, wherein the culture supernatant is a culture product of Dialister invisus and / or Dialister succinatiphilus.

4. The composition according to any one of claims 1 to 3, wherein the culture supernatant is a molecular weight fraction of less than 1000 kDa.

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