Target bacteria and uses thereof

Abstract

Provided is a method for evaluating the intestinal barrier function of a subject, the method comprising the following steps: (1a) determining the occupancy of target bacteria in feces derived from the subject, obtaining a measured value of the occupancy of the target bacteria; (1b) comparing the measured value obtained in step 1a with a reference value, obtaining a comparison result; and (1c) evaluating the intestinal barrier function of the subject based on the comparison result obtained in step 1b, wherein the occupancy of the target bacteria comprises the occupancy of one or more bacteria selected from a predetermined group.

Description

Technical Field

[0001] This invention relates to target bacteria and their uses.

[0002] Specifically, the present invention relates to a method for evaluating the intestinal barrier function of a subject, target bacteria for the aforementioned evaluation, use of the target bacteria for the aforementioned evaluation, a kit for the aforementioned evaluation, and a computer program for causing a computer to perform the aforementioned method.

[0003] In addition, the present invention relates to a method for diagnosing diseases or symptoms caused by reduced intestinal barrier function, target bacteria for the aforementioned diagnosis, use of the target bacteria for the aforementioned diagnosis, a reagent kit for the aforementioned diagnosis, and a computer program for causing a computer to perform the aforementioned method.

[0004] In addition, the present invention relates to a method for treating diseases or symptoms caused by decreased intestinal barrier function in a subject.

[0005] In addition, the present invention relates to a method for evaluating the risk of a subject developing a disease or symptom caused by a decrease in intestinal barrier function or the likelihood of developing the aforementioned disease or symptom, a target bacterium for the aforementioned evaluation, the use of the target bacterium for the aforementioned evaluation, a kit for the aforementioned evaluation, and a computer program for causing a computer to perform the aforementioned method.

[0006] In addition, the present invention relates to a method for acquiring data on diseases or symptoms caused by reduced intestinal barrier function for diagnostic subjects, target bacteria for the acquisition, use of the target bacteria for the acquisition, a reagent kit for the acquisition, and a computer program for causing a computer to perform the aforementioned method.

[0007] In addition, the present invention relates to a method for screening substances or compositions that have an effect on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by reduced intestinal barrier function, target bacteria for the aforementioned screening, use of the target bacteria for the aforementioned screening, a kit for the aforementioned screening, and a computer program for causing a computer to perform the aforementioned method.

[0008] In addition, the present invention relates to a method for evaluating the effect of candidate substances or candidate compositions on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by reduced intestinal barrier function, target bacteria for the aforementioned evaluation, use of target bacteria for the aforementioned evaluation, kits for the aforementioned evaluation, and computer programs for enabling a computer to perform the aforementioned methods.

[0009] In addition, the present invention relates to a method for manufacturing food or pharmaceutical compositions. Background Technology

[0010] The intestinal lumen is a space surrounded by a layer of mucosal epithelial cells, which divide the intestinal lumen (outside the organism) from the body. The intestine is exposed to foreign substances (such as harmful microorganisms, toxins, and allergens) entering from the outside, making it an important organ not only for nutrient absorption but also for biological defense. The intestinal barrier function prevents foreign substances (such as harmful microorganisms, toxins, and allergens) from entering the intestinal lumen and invading the body.

[0011] The intestinal barrier function involves physical and chemical barriers. Physical barriers, as the name suggests, act as physical barriers to prevent the invasion of foreign substances. These include the mucus layer covering intestinal epithelial cells, glycocalyx (aggregates of sugar chains on the surface of intestinal epithelial cells), and tight junctions (adhesion mechanisms between intestinal epithelial cells). Chemical barriers are groups of molecules that inhibit the invasion of foreign substances by inducing chemical changes, exerting antibacterial activity, or neutralizing activity. These molecules include, for example, antimicrobial peptides produced by intestinal epithelial cells such as Panette cells (e.g., defensin family molecules, Reg3 family molecules, lactoferrin, lysozyme, etc.). M cells, which take up antigens and present them to dendritic cells, and immunoglobulin A (IgA), which exerts neutralizing activity by secreting into the intestine to prevent the invasion of foreign substances, are also types of chemical barriers.

[0012] Decreased intestinal barrier function can be a cause of various diseases or symptoms (e.g., enteritis, allergic diseases, mental illness, non-alcoholic fatty liver disease, type 2 diabetes, metabolic syndrome, obesity, etc.).

[0013] As a method for assessing intestinal barrier function, the lactulose-mannitol test (glucose tolerance test) is known. In the lactulose-mannitol test, intestinal permeability is measured; increased intestinal permeability is an indicator of deterioration in intestinal barrier function (especially physical barrier functions such as tight junction structures). The lactulose-mannitol test has drawbacks such as requiring prolonged restraint and fasting time for the subject, and placing a burden on the subject's digestive tract due to the ingested sugar alcohol.

[0014] The concentration of ligin in the blood, which is known to be related to intestinal barrier function (Non-Patent Literature 1), is sometimes used in methods for evaluating intestinal barrier function. However, there are issues such as diurnal variation in ligin concentration and low reproducibility (Non-Patent Literature 2).

[0015] Previously, there were no known biomarkers that could easily and accurately assess intestinal barrier function.

[0016] Existing technical documents

[0017] Non-patent literature

[0018] Non-patent literature 1: Fasana A et al., Zonulin, “a newly discovered modulator of intestinal permeability, and its expression in coeliac disease.”, THELANCET., 2000; 355: pp.1518-1519

[0019] Non-patent literature 2: Aristo Vojdani et al., “Fluctuation of zonulin levels in blood vs stability of antibodies.”, World Journal of Gastroenterology., 2017; 23(31): pp. 5669-5679 Summary of the Invention

[0020] The problem the invention aims to solve

[0021] The object of the present invention is to provide a method for evaluating the intestinal barrier function of a subject, target bacteria for the aforementioned evaluation, uses of the target bacteria for the aforementioned evaluation, a kit for the aforementioned evaluation, and a computer program for causing a computer to perform the aforementioned method.

[0022] In addition, the present invention aims to provide a method for diagnosing diseases or symptoms caused by reduced intestinal barrier function, target bacteria for the aforementioned diagnosis, use of the target bacteria for the aforementioned diagnosis, a reagent kit for the aforementioned diagnosis, and a computer program for causing a computer to perform the aforementioned method.

[0023] In addition, the present invention aims to provide a method for treating diseases or symptoms caused by decreased intestinal barrier function in a subject.

[0024] In addition, the present invention aims to provide a method for evaluating the risk of a subject developing a disease or symptom caused by a decrease in intestinal barrier function or the possibility of developing the aforementioned disease or symptom, a target bacterium for the aforementioned evaluation, the use of the target bacterium for the aforementioned evaluation, a kit for the aforementioned evaluation, and a computer program for causing a computer to perform the aforementioned method.

[0025] In addition, the present invention aims to provide a method for acquiring data on diseases or symptoms caused by reduced intestinal barrier function in a diagnostic subject, target bacteria for the acquisition, use of the target bacteria for the acquisition, a reagent kit for the acquisition, and a computer program for causing a computer to perform the aforementioned method.

[0026] In addition, the present invention aims to provide a method for screening substances or compositions that have an effect on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by reduced intestinal barrier function, target bacteria for the aforementioned screening, use of the target bacteria for the aforementioned screening, a kit for the aforementioned screening, and a computer program for causing a computer to perform the aforementioned method.

[0027] In addition, the present invention aims to provide a method for evaluating the effect of a candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by reduced intestinal barrier function, a target bacterium for the aforementioned evaluation, the use of the target bacterium for the aforementioned evaluation, a kit for the aforementioned evaluation, and a computer program for causing a computer to perform the aforementioned method.

[0028] In addition, the present invention aims to provide a method for manufacturing food or pharmaceutical compositions.

[0029] Solution for solving the problem

[0030] In this invention, group A consists of the following bacteria. It should be noted that the bacterial names in group A are family names.

[0031] [Group A]

[0032] Rumenococcal bacteria

[0033] Peptostreptococcal bacteria

[0034] Institute of Physics and Chemistry, Bacteriology

[0035] Eubacteriaceae

[0036] Bacillus family bacteria

[0037] Group 1 bacteria of the Clostridium family

[0038] In this invention, group B consists of the following bacteria. It should be noted that the bacterial names in group B are genus names.

[0039] Group B

[0040] Bacteria of the genus *Lombocytosporum* in the family Peptostreptococci

[0041] Allergens of Bacteriology, Institute of Physics and Chemistry

[0042] UCG-010 bacteria of the family Trichophyceae

[0043] Ruminococcal bacteria of the genus *Ruminococcus* (family Ruminococci)

[0044] Group Gaucher bacteria of the family Trichophyceae [Ruminococcus genus]

[0045] Group 1 bacteria of the genus *Ruminococcus* in the family Ruminococciaceae

[0046] Ruminococci (Ruminococci) NK4A214 group of bacteria

[0047] Ruminococci (family Ruminococci) UCG-002 bacteria

[0048] Active group of bacteria in the family Trichophyceae [Ruminococcus genus]

[0049] Ruminococci (family Ruminococci) UCG-003 bacteria

[0050] Ruminococci (family Ruminococci) UCG-009 bacteria

[0051] Bacteria of the genus *Eubacterium* (family Eubacteriales)

[0052] Ruminococcal bacteria of the genus *Flavobacterium*

[0053] Bacteria of the genus Bacillus in the family Bacillusaceae

[0054] Ruminococci (family Eubacterium, genus *Eubacterium*) coprosterol-producing bacteria

[0055] Enterobacteriaceae (Peptostreptococci)

[0056] Anaerobic bacteria of the genus *Corynebacterium* in the family Ruminococci.

[0057] Marine fungi of the genus *Ostomella* (family Laminariaceae).

[0058] Clostridium bacteria, Group 1 of the Clostridium family, specifically the genus Clostridium.

[0059] The present invention includes the following technical solutions.

[0060] [1] A method for evaluating intestinal barrier function in subjects, wherein,

[0061] The method includes the following steps:

[0062] (1a) The step of determining the occupancy of target bacteria in feces derived from the subject and obtaining a measured value of the occupancy of the target bacteria;

[0063] (1b) The step of comparing the measured value obtained in step 1a with the reference value to obtain the comparison result; and

[0064] (1c) The step of evaluating the intestinal barrier function of the subject based on the comparison results obtained in step 1b.

[0065] The occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B.

[0066] [2] A method for diagnosing diseases or symptoms caused by decreased intestinal barrier function in a subject, wherein,

[0067] The method includes the following steps:

[0068] (2a) The step of determining the occupancy of target bacteria in feces derived from the subject and obtaining a measured value of the occupancy of the target bacteria;

[0069] (2b) The step of comparing the measured value obtained in step 2a with the reference value to obtain the comparison result; and

[0070] (2c) A step of diagnosing the disease or symptom based on the comparison results obtained in step 2b.

[0071] The occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B.

[0072] [3] A method for treating a disease or symptoms caused by decreased intestinal barrier function in a subject, wherein...

[0073] The method includes the following steps:

[0074] (3a) The step of determining the occupancy of target bacteria in feces derived from the subject and obtaining a measured value of the occupancy of the target bacteria;

[0075] (3b) The step of comparing the measured value obtained in step 3a with the reference value to obtain the comparison result;

[0076] (3c) The step of diagnosing the disease or symptom based on the comparison results obtained in step 3b; and

[0077] (3d) In the event that the subject is diagnosed with the disease or symptoms, the step of administering to the subject a substance or composition for treating the disease or symptoms.

[0078] The occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B.

[0079] [4] A method for evaluating the risk or likelihood of a subject developing a disease or symptom caused by decreased intestinal barrier function, wherein...

[0080] The method includes the following steps:

[0081] (4a) The step of determining the occupancy of target bacteria in feces derived from the subject and obtaining a measured value of the occupancy of the target bacteria;

[0082] (4b) The step of comparing the measured value obtained in step 4a with the reference value to obtain the comparison result; and

[0083] (4c) The step of evaluating the risk or the likelihood based on the comparison results obtained in step 4b.

[0084] The occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B.

[0085] [5] A method for acquiring data on diseases or symptoms caused by decreased intestinal barrier function in a diagnostic subject, wherein...

[0086] The method includes the following steps:

[0087] (5a) The step of determining the occupancy of target bacteria in feces derived from the subject, and obtaining a measured value of the occupancy of the target bacteria; and

[0088] (5b) The step of comparing the measured value obtained in step 5a with the reference value and obtaining the comparison result as the data.

[0089] The occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B.

[0090] [6] A method for screening substances or compositions that have an effect on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by decreased intestinal barrier function, wherein,

[0091] The method includes the following steps:

[0092] (6a) The step of administering a candidate substance or candidate composition to a subject whose intestinal barrier function has been reduced;

[0093] (6b) The step of determining the occupancy of target bacteria in feces obtained from the subject after administration of the candidate substance or candidate composition, and obtaining a measured value of the occupancy of the target bacteria;

[0094] (6c) The step of comparing the measured value obtained in step 6b with the reference value to obtain the comparison result; and

[0095] (6d) Based on the comparison results obtained in step 6c, the step of evaluating the effect of the candidate substance or the candidate composition on improving the intestinal barrier function or on preventing or improving the disease or symptoms.

[0096] The occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B.

[0097] [7] A method for evaluating the effect of a candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by decreased intestinal barrier function, wherein,

[0098] The method includes the following steps:

[0099] (7a) The step of administering the candidate substance or the candidate composition to a subject whose intestinal barrier function has been reduced;

[0100] (7b) The step of determining the occupancy of target bacteria in feces obtained from the subject after administration of the candidate substance or the candidate composition, and obtaining a measured value of the occupancy of the target bacteria;

[0101] (7c) The step of comparing the measured value obtained in step 7b with the reference value to obtain the comparison result; and

[0102] (7d) The step of evaluating the effect of the candidate substance or the candidate composition on improving the intestinal barrier function or on preventing or improving the disease or symptoms, based on the comparison results obtained in step 7c.

[0103] The occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B.

[0104] [8] The method according to any one of [1] to [7], wherein the intestinal barrier function is the barrier function of the small intestine.

[0105] [9] The method according to any one of [1] to [8], wherein the occupancy rate of the target bacteria includes the occupancy rate of two, three or more bacteria selected from group A and group B.

[0106]

[10] The method according to any one of [1] to [9], wherein the occupancy rate of the target bacteria includes the occupancy rate of one or more selected from the occupancy rate of Ruminococcus, the occupancy rate of Peptostreptococcus, the occupancy rate of Peptococcus spp., the occupancy rate of Peptostreptococcus spp., and the occupancy rate of Peptostreptococcus spp. Allergenia spp.

[0107]

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

[10] , wherein the occupancy of the target bacteria is determined by microbial community analysis using 16S rRNA amplicon sequencing.

[0108]

[12] A target bacterium for evaluating the intestinal barrier function of a subject, the target bacterium being a target bacterium derived from the subject’s feces, the target bacterium comprising one or more bacteria selected from groups A and B.

[0109]

[13] A target bacterium for diagnosing a disease or symptom caused by a decrease in intestinal barrier function in a subject, the target bacterium being a target bacterium derived from the subject’s feces, the target bacterium comprising one or more bacteria selected from groups A and B.

[0110]

[14] A target bacterium for evaluating the risk or likelihood of a subject developing a disease or symptom caused by a decrease in intestinal barrier function, the target bacterium being a target bacterium derived from the subject’s feces, the target bacterium comprising one or more bacteria selected from groups A and B.

[0111]

[15] A target bacterium for obtaining data on diseases or symptoms caused by reduced intestinal barrier function in a subject for diagnosis, the target bacterium being a target bacterium derived from the subject’s feces, the target bacterium comprising one or more bacteria selected from groups A and B.

[0112]

[16] A target bacterium for screening substances or compositions that have an effect on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by reduced intestinal barrier function, said target bacterium being a target bacterium in feces obtained from a subject with reduced intestinal barrier function after administering the candidate substance or candidate composition to said subject, said target bacterium comprising one or more bacteria selected from groups A and B.

[0113]

[17] A target bacterium for evaluating the effect of a candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving disease or symptoms caused by reduced intestinal barrier function, said target bacterium being a target bacterium in feces obtained from a subject with reduced intestinal barrier function after the subject is given said candidate substance or candidate composition, said target bacterium comprising one or more bacteria selected from groups A and B.

[0114]

[18] The target bacteria according to any one of

[12] to

[17] , wherein the intestinal barrier function is the barrier function of the small intestine.

[0115]

[19] The target bacteria according to any one of

[12] to

[18] , wherein the target bacteria comprises two, three or more bacteria selected from group A and group B.

[0116]

[20] The target bacteria according to any one of

[12] to

[19] , wherein the target bacteria comprises one or more bacteria selected from Ruminococcus, Peptostreptococcus, Pharmacopoeia, Lombocytia spp. of Peptostreptococcus and Allergan spp. of Pharmacopoeia.

[0117]

[21] Use of target bacteria in evaluating the intestinal barrier function of subjects, wherein the target bacteria are target bacteria derived from the feces of the subjects, and the target bacteria include one or more bacteria selected from groups A and B.

[0118]

[22] Use of target bacteria in diagnosing diseases or symptoms caused by reduced intestinal barrier function in a subject, wherein the target bacteria are target bacteria derived from the subject’s feces and the target bacteria comprise one or more bacteria selected from groups A and B.

[0119]

[23] Use of target bacteria in evaluating the risk or likelihood of a subject developing a disease or symptom caused by a decrease in intestinal barrier function, wherein the target bacteria are target bacteria derived from the subject’s feces and the target bacteria comprise one or more bacteria selected from groups A and B.

[0120]

[24] Use of target bacteria in obtaining data for diagnosing diseases or symptoms caused by reduced intestinal barrier function in subjects, wherein the target bacteria are target bacteria derived from the feces of the subjects and the target bacteria comprise one or more bacteria selected from groups A and B.

[0121]

[25] Use of target bacteria in screening for substances or compositions that have an effect on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by reduced intestinal barrier function, wherein the target bacteria are target bacteria in feces obtained from a subject with reduced intestinal barrier function after administering the candidate substance or candidate composition to the subject, and the target bacteria comprise one or more bacteria selected from groups A and B.

[0122]

[26] Use of target bacteria in evaluating the effect of a candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving disease or symptoms caused by reduced intestinal barrier function, wherein the target bacteria are target bacteria in feces obtained from a subject with reduced intestinal barrier function after administration of the candidate substance or candidate composition to the subject, and the target bacteria comprise one or more bacteria selected from groups A and B.

[0123]

[27] The use according to any one of

[21] to

[26] , wherein the intestinal barrier function is the barrier function of the small intestine.

[0124]

[28] The use according to any one of

[21] to

[27] , wherein the target bacteria comprises two, three or more bacteria selected from groups A and B.

[0125]

[29] The use according to any one of

[21] to

[28] , wherein the target bacteria comprises one or more bacteria selected from Ruminococcus, Peptostreptococcus, Pharmacopoeia, Lombocytella, and Allergan.

[0126]

[30] A kit for evaluating the intestinal barrier function of a subject, wherein the kit comprises one or more reagents for determining the occupancy of target bacteria in feces derived from the subject, the occupancy of target bacteria comprising the occupancy of one or more bacteria selected from groups A and B.

[0127]

[31] A kit for diagnosing a disease or symptom caused by a decrease in intestinal barrier function in a subject, wherein the kit comprises one or more reagents for determining the occupancy of target bacteria in feces derived from the subject, the occupancy of target bacteria comprising the occupancy of one or more bacteria selected from groups A and B.

[0128]

[32] A kit for evaluating the risk or likelihood of a subject developing a disease or symptom caused by a decrease in intestinal barrier function, wherein the kit comprises one or more reagents for determining the occupancy of target bacteria in feces derived from the subject, the occupancy of target bacteria comprising the occupancy of one or more bacteria selected from groups A and B.

[0129]

[33] A kit for obtaining data on a disease or symptom caused by a decrease in intestinal barrier function in a subject for diagnosis, wherein the kit comprises one or more reagents for determining the occupancy of target bacteria in feces derived from the subject, the occupancy of target bacteria comprising the occupancy of one or more bacteria selected from groups A and B.

[0130]

[34] A kit for screening substances or compositions that have an effect on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by reduced intestinal barrier function, wherein the kit comprises one or more reagents for determining the occupancy of target bacteria in feces obtained from subjects with reduced intestinal barrier function after administration of the candidate substance or candidate composition to the subjects, the occupancy of target bacteria comprising the occupancy of one or more bacteria selected from groups A and B.

[0131]

[35] A kit for evaluating the effect of a candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by reduced intestinal barrier function, wherein the kit comprises one or more reagents for determining the occupancy of target bacteria in feces obtained from a subject with reduced intestinal barrier function after administration of the candidate substance or candidate composition to the subject, the occupancy of target bacteria comprising the occupancy of one or more bacteria selected from groups A and B.

[0132]

[36] The kit according to any one of

[30] to

[35] , wherein the intestinal barrier function is the barrier function of the small intestine.

[0133]

[37] The kit according to any one of

[30] to

[36] , wherein the occupancy rate of the target bacteria includes the occupancy rate of two, three or more bacteria selected from group A and group B.

[0134]

[38] The kit according to any one of

[30] to

[37] , wherein the occupancy rate of the target bacteria includes the occupancy rate of one or more selected from Ruminococcal bacteria, Peptostreptococcal bacteria, Pharmacopoeia bacteria, Peptostreptococcal bacteria, and Peptostreptococcal bacteria.

[0135]

[39] The kit according to any one of

[30] to

[38] , wherein the occupancy of the target bacteria is determined by microbial community analysis using 16S rRNA amplicon sequencing.

[0136]

[40] A method for manufacturing a food or pharmaceutical composition, comprising the step of mixing a substance or composition selected by the method described in [6] with one or more ingredients constituting the food or pharmaceutical composition.

[0137]

[41] A computer program for causing a computer to perform a method for evaluating the intestinal barrier function of a subject.

[0138] The method includes the following steps:

[0139] (S101) A step of obtaining a measurement of the occupancy of target bacteria in feces derived from the object;

[0140] (S102) The step of comparing the measured value obtained in step S101 with the reference value to obtain a comparison result; and

[0141] (S103) The step of evaluating the intestinal barrier function of the subject based on the comparison results obtained in step S102.

[0142] The occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B.

[0143]

[42] A computer program for causing a computer to perform a method for diagnosing a disease or symptoms caused by reduced intestinal barrier function in a subject.

[0144] The method includes the following steps:

[0145] (S201) A step of obtaining a measurement value of the occupancy of target bacteria in feces derived from the object;

[0146] (S202) The step of comparing the measured value obtained in step S201 with the reference value to obtain the comparison result;

[0147] (S203) A step of diagnosing the disease or symptom based on the comparison results obtained in step S202.

[0148] The occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B.

[0149]

[43] A computer program for causing a computer to perform a method for evaluating the risk of a subject developing a disease or symptoms caused by reduced intestinal barrier function, or the likelihood of developing said disease or symptoms.

[0150] The method includes the following steps:

[0151] (S301) A step of obtaining a measurement value of the occupancy of target bacteria in feces derived from the object;

[0152] (S302) The step of comparing the measured value obtained in step S301 with the reference value to obtain a comparison result; and

[0153] (S303) The step of evaluating the risk or the probability based on the comparison results obtained in step S302.

[0154] The occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B.

[0155]

[44] A computer program for causing a computer to perform a method for acquiring data on a disease or symptoms caused by reduced intestinal barrier function in a diagnostic subject.

[0156] The method includes the following steps:

[0157] (S401) The step of obtaining a measurement value of the occupancy of target bacteria in feces derived from said object; and

[0158] (S402) The step of comparing the measured value obtained in step S401 with the reference value and obtaining the comparison result as the data.

[0159] The occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B.

[0160]

[45] A computer program for causing a computer to perform a method for screening substances or compositions that have an effect on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by reduced intestinal barrier function.

[0161] The method includes the following steps:

[0162] (S501) A step of obtaining a measurement value of the occupancy of target bacteria in feces obtained from a subject with reduced intestinal barrier function after administering a candidate substance or candidate composition to the subject.

[0163] (S502) The step of comparing the measured value obtained in step S501 with the reference value to obtain a comparison result; and

[0164] (S503) Based on the comparison results obtained in step S502, the step of evaluating the effect of the candidate substance or the candidate composition on improving the intestinal barrier function or on preventing or improving the disease or symptoms.

[0165] The occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B.

[0166]

[46] A computer program for causing a computer to perform a method for evaluating the effect of a candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by reduced intestinal barrier function.

[0167] The method includes the following steps:

[0168] (S601) A step of obtaining a measurement of the occupancy of target bacteria in feces obtained from a subject with reduced intestinal barrier function after administering a candidate substance or candidate composition to the subject.

[0169] (S602) The step of comparing the measured value obtained in step S601 with the reference value to obtain a comparison result; and

[0170] (S603) Based on the comparison results obtained in step S602, the step of evaluating the effect of the candidate substance or the candidate composition on improving the intestinal barrier function or on preventing or improving the disease or symptoms.

[0171] The occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B.

[0172]

[47] The computer program according to any one of

[41] to

[46] , wherein the intestinal barrier function is the barrier function of the small intestine.

[0173]

[48] ​​The computer program according to any one of

[41] to

[47] , wherein the occupancy rate of the target bacteria includes the occupancy rate of two, three or more bacteria selected from group A and group B.

[0174]

[49] The computer program according to any one of

[41] to

[48] , wherein the occupancy rate of the target bacteria includes the occupancy rate of one or more selected from the occupancy rates of Ruminococcus, Peptostreptococcus, Pharmacopoeia, Rhodotorula spp., and Allergan spp.

[0175]

[50] The computer program according to any one of

[41] to

[49] , wherein the occupancy of the target bacteria is determined by microbial community analysis using 16S rRNA amplicon sequencing.

[0176]

[51] A computer-readable recording medium having a computer program recorded in any one of

[41] to

[50] .

[0177] The effects of the invention

[0178] According to the present invention, a method for evaluating the intestinal barrier function of a subject, target bacteria for the aforementioned evaluation, use of the target bacteria for the aforementioned evaluation, a kit for the aforementioned evaluation, and a computer program for causing a computer to perform the aforementioned method are provided.

[0179] In addition, according to the present invention, a method for diagnosing diseases or symptoms caused by decreased intestinal barrier function is provided, target bacteria for the aforementioned diagnosis, use of the target bacteria for the aforementioned diagnosis, a reagent kit for the aforementioned diagnosis, and a computer program for causing a computer to perform the aforementioned method are provided.

[0180] In addition, according to the present invention, a method is provided for treating diseases or symptoms caused by decreased intestinal barrier function in a subject.

[0181] In addition, according to the present invention, a method for evaluating the risk of a subject developing a disease or symptom caused by a decrease in intestinal barrier function or the possibility of suffering from the aforementioned disease or symptom is provided, a target bacterium for the aforementioned evaluation, the use of the target bacterium for the aforementioned evaluation, a kit for the aforementioned evaluation, and a computer program for causing a computer to perform the aforementioned method are provided.

[0182] In addition, according to the present invention, a method for obtaining data on diseases or symptoms caused by reduced intestinal barrier function for a diagnostic subject is provided, target bacteria for the aforementioned acquisition, use of the aforementioned target bacteria, a reagent kit for the aforementioned acquisition, and a computer program for causing a computer to perform the aforementioned method are provided.

[0183] In addition, according to the present invention, a method for screening substances or compositions having an effect on improving intestinal barrier function or preventing or improving diseases or symptoms caused by reduced intestinal barrier function is provided, target bacteria for the aforementioned screening, use of the target bacteria for the aforementioned screening, a kit for the aforementioned screening, and a computer program for causing a computer to perform the aforementioned method.

[0184] In addition, according to the present invention, a method for evaluating the effect of a candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by reduced intestinal barrier function is provided, a target bacterium for the aforementioned evaluation, the use of the target bacterium for the aforementioned evaluation, a kit for the aforementioned evaluation, and a computer program for causing a computer to perform the aforementioned method are provided.

[0185] In addition, according to the present invention, a method for manufacturing a food or pharmaceutical composition is provided.

[0186] The technology provided by this invention can personalize the intestinal barrier status of an individual, which is helpful for precision nutrition (especially precision prebiotics and precision nutrition), precision healthcare, etc. Attached Figure Description

[0187] Figure 1 This is a schematic diagram of an apparatus according to one embodiment of the present invention.

[0188] Figure 2 This is a block diagram illustrating the hardware configuration of an apparatus according to one embodiment of the present invention.

[0189] Figure 3 This is a flowchart illustrating the processing steps of an apparatus according to one embodiment of the present invention.

[0190] Figure 4 This is a flowchart illustrating the processing steps of an apparatus according to one embodiment of the present invention.

[0191] Figure 5This is a flowchart illustrating the processing steps of an apparatus according to one embodiment of the present invention. Detailed Implementation

[0192] The present invention will now be described. It should be noted that two or more embodiments described in this specification can be combined, and these combinations are also included in the present invention.

[0193] Method 1

[0194] Method 1 of the present invention relates to a method for evaluating the intestinal barrier function of a subject.

[0195] In Method 1, biomarkers derived from the subject's sample are used as indicators to evaluate the subject's intestinal barrier function.

[0196] In Method 1, the specimen used is feces derived from the subject. The feces can be dry or wet. By using feces as the specimen, the intestinal barrier function of the subject can be easily evaluated.

[0197] The subject can be any animal that excretes feces; there are no particular limitations. Animals can include vertebrates such as mammals, reptiles, birds, amphibians, and fish, but mammals and birds are preferred, with mammals being more preferred. Mammals can include primates (e.g., humans, gorillas, chimpanzees, orangutans), rodents (e.g., mice, rats, hamsters, guinea pigs, rabbits), livestock (e.g., cattle, pigs, sheep, goats, horses), and pets (e.g., dogs, cats), but humans are preferred. Birds can include poultry (e.g., chickens, ducks, domestic ducks).

[0198] Feces are excretions that pass through the anus. Feces from the subject can be collected from the subject's feces using standard methods and can be used as specimens.

[0199] Based on the mass of the dried feces, the moisture content in the dried feces is preferably 20% by mass or less, more preferably 10% by mass or less, and even more preferably 5% by mass or less. The lower limit for the moisture content in the dried feces is zero. Dried feces can be obtained by drying feces that have just been expelled from the anus. The drying process can be carried out, for example, using a commercially available freeze dryer. In the freeze drying process, the processing temperature is, for example, -80°C or higher and 0°C or lower, and the processing time is, for example, 24 hours or higher and 72 hours or lower.

[0200] Based on the mass of wet feces, the water content in the wet feces is preferably 40% by mass or more, more preferably 50% by mass or more, and even more preferably 60% by mass or more. There is no particular upper limit, such as 95% by mass or less, 90% by mass or less, 80% by mass or less, or 70% by mass or less. These upper limits can be combined with any of the lower limits mentioned above. The wet feces can be feces freshly expelled from the anus, or feces obtained by freezing freshly expelled feces and then thawing it. The freezing temperature is preferably -100°C or higher and -10°C or lower, more preferably -80°C or higher and -15°C or lower, and even more preferably -80°C or higher and -60°C or lower. Freezing can be performed, for example, by collecting freshly expelled feces in a special container for fecal collection (e.g., a Faeces tube with a blade and screw cap (manufactured by Sarstedt)). The cryopreservation period is preferably within 180 days from the date of collection, more preferably within 60 days from the date of collection, and even more preferably within 30 days from the date of collection. That is, it is preferable to use wet feces within 180 days from the date of collection for biomarker determination, more preferably within 60 days from the date of collection, and even more preferably within 30 days from the date of collection.

[0201] The biomarkers used in Method 1 are target bacteria derived from the subjects' feces. Specifically, the occupancy of target bacteria derived from the subjects' feces was used as an indicator to evaluate the subjects' intestinal barrier function.

[0202] The occupancy rate of target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B. By using the occupancy rate of one or more bacteria selected from groups A and B as an indicator, the intestinal barrier function of subjects can be evaluated with high accuracy.

[0203] The occupancy rate of target bacteria can include the occupancy rate of one type of bacteria selected from groups A and B, or it can include the occupancy rate of two or more types of bacteria selected from groups A and B. From the viewpoint of improving the accuracy of the evaluation of the subject's intestinal barrier function, it is preferable to include the occupancy rate of two or more types of bacteria selected from groups A and B (specifically, two, three, or four or more types). When the occupancy rate of target bacteria includes the occupancy rate of two or more types of bacteria selected from groups A and B, the occupancy rates of these two or more types of bacteria can be used separately as indicators for evaluating the subject's intestinal barrier function.

[0204] In one implementation, the occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from group A, but does not include the occupancy rate of any bacteria from group B.

[0205] In another embodiment, the occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from group B, but does not include the occupancy rate of any bacteria from group A.

[0206] In another embodiment, the occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from group A and the occupancy rate of one or more bacteria selected from group B.

[0207] "The prevalence of one or more bacteria selected from groups A and B" refers to the prevalence of one or more bacteria selected from groups C and D below. It should be noted that group C corresponds to group A, and group D corresponds to group B.

[0208] [Group C]

[0209] Prevalence of Ruminococci

[0210] Prevalence of Peptostreptococci

[0211] Prevalence of Mycology in the Institute of Physics and Chemistry

[0212] Prevalence of Eubacteriaceae

[0213] Prevalence of Bacillus species

[0214] Prevalence of Group 1 bacteria in the Clostridium family

[0215] [Group D]

[0216] Prevalence of bacteria in the genus *Lombocytozoon* of the family Peptostreptococcus

[0217] Prevalence of Allergens genus in the Mycology family of the Institute of Physics and Chemistry

[0218] Prevalence of UCG-010 bacteria in the family Trichophyceae

[0219] Prevalence of Ruminococcal Negative Bacillus genus

[0220] Prevalence of Group Gaucher bacteria in the family Trichophyceae [Ruminococcus genus]

[0221] Prevalence of Group 1 bacteria in the genus *Ruminococcus* of the family Ruminococciaceae

[0222] Prevalence of Ruminococci (NK4A214 group)

[0223] Prevalence of UCG-002 bacteria in the Ruminococcal family (Ruminococcal family)

[0224] Prevalence of active bacteria in the family Trichophyceae [Ruminococcus genus]

[0225] Prevalence of UCG-003 bacteria in the Ruminococcalae family

[0226] Prevalence of UCG-009 bacteria in the Ruminococcalae family

[0227] Prevalence of bacteria in the genus *Eubacterium* of the family Eubacteriae

[0228] Prevalence of *Bacillus faecalis* in the Ruminococcal family

[0229] Prevalence of Bacillus genus in the Bacillusaceae family

[0230] Prevalence of coprosterol-producing bacteria in the Rumenococcal family [Eubacterium genus]

[0231] Prevalence of Enterobacteriaceae (Peptostreptococci)

[0232] Prevalence of anaerobic bacteria in the genus *Corynebacterium* of the family Ruminococci.

[0233] Prevalence of bacteria in the genus *Ostomyces* of the family Nematomycetes

[0234] Prevalence of Clostridium species in the narrow sense of Group 1 of the Clostridiumceae family

[0235] Ruminococcal bacteria is a collective term for bacteria belonging to the Ruminococcal family. Ruminococcal bacteria include the following bacteria.

[0236] Ruminococcal family, genus *Bacillus*

[0237] Group 1 of the genus *Ruminococcus* in the family Ruminococciaceae

[0238] Ruminococci (Ruminococciidae) NK4A214 group

[0239] Ruminococciidae UCG-002

[0240] Ruminococciidae UCG-003

[0241] Ruminococciidae UCG-009

[0242] Ruminococci (family Ruminococci) and genus *Aureobacter*

[0243] Ruminococci (family Eubacterium) coprosterol-producing group

[0244] Anaerobic Species of the Ruminococcal Family

[0245] Ruminococciidae UCG-005

[0246] Ruminococcal family DTU089

[0247] Group 6 of Clostridium rumeniformis in the family Ruminococci

[0248] Ruminococci (Ruminococci) UCG-013

[0249] Ruminococciidae UCG-010

[0250] Group 2 of the genus Ruminococcus in the family Ruminococci

[0251] Ruminococci (family Ruminococci) and genus Flavonoids

[0252] Group 5 of Clostridium rumeniformis in the family Ruminococci

[0253] Ruminococcal family, *Vibrio spp.*

[0254] Clostridium rumeniaceae

[0255] Ruminococciidae UCG-004

[0256] Butyric acid cocci of the Ruminococcal family

[0257] Ruminococcal family GCA-900066225

[0258] Ruminococciidae UCG-014

[0259] Candidatus Soleaferrea, a candidate genus of Ruminococcalae.

[0260] Ruminococcal family, Hydrogen anaerobic bacillus

[0261] Ruminococcal family, genus *Vibrio*

[0262] Ruminococciidae UBA1819

[0263] Group 9 of Clostridium rumeniformis in the family Ruminococci

[0264] Anaerobic Clusia of the Ruminococcal Family

[0265] Lactobacillus spp., Ruminococcalae

[0266] Ruminococci (Ruminococci family, genus Fourniger)

[0267] Ruminococcal CAG-352

[0268] Ruminococci V9D2013 group

[0269] Rare Micrococcus genus in Ruminococci

[0270] Ruminococci (Ruminococci) UCG-007

[0271] Group 1 of Clostridium rumeniformis in the family Ruminococci

[0272] Ruminococci (family Ruminococci) and genus Desmophora

[0273] Ruminococci (family Ruminococci) and genus *Anaerobicaceae*

[0274] Ruminococci (family Ruminococci) and genus *Tempylobacter*

[0275] Anaerobic rod-shaped bacteria of the Ruminococcal family

[0276] Anaerobic Clusia of the Ruminococcal Family

[0277] Anaerobic Species of the Ruminococcal Family

[0278] Ruminococcal family, genus Bacillus

[0279] Ruminococciaceae, European Research Association of Bacteria

[0280] Ethanol-producing bacteria (Ruminococci family)

[0281] Ruminococcal family, *Vibrio spp.*

[0282] Lactobacillus spp., Ruminococcalae

[0283] Ruminococci (family Ruminococci) and *Pseudomonas* genus

[0284] Ruminococci (family Ruminococci)

[0285] Ruminococci (family Ruminococci) genus *Sporidiformes*

[0286] Rare Micrococcus genus in Ruminococci

[0287] Ruminococci (family Ruminococci) and genus *Liriobacterium*

[0288] The prevalence of Ruminococci is the prevalence of all bacteria belonging to the Ruminococci family. The prevalence of Ruminococci is calculated based on the formula: (Number of all Ruminococci in feces) / (Total number of bacteria from all families in feces).

[0289] Peptostreptococci are a collective term for bacteria belonging to the family Peptostreptococci. Peptostreptococci include the following bacteria.

[0290] Peptostreptococcus family, genus Lombozychomycosis

[0291] Enterobacteriaceae (Peptostreptococci)

[0292] Peptostreptococcus genus

[0293] Peptostreptococcus family, Terremycosis

[0294] Peptostreptococcus (family Peptostreptococci) genus

[0295] Peptostreptococcus family, genus *Peptostreptococcus*

[0296] Peptostreptococcus family Clostridium genus

[0297] Peptostreptococcus Infectious Disease Center, genus *Bacillus*

[0298] Peptostreptococcus family, genus *Streptococcus*

[0299] Fusobacterium genus, family Peptostreptococcus

[0300] Peptostreptococcus family Maldive bacillus

[0301] Peptostreptococcus family, Paramaldactylus genus

[0302] Peptostreptococcus spp.

[0303] Peptostreptococcus spp.

[0304] Peptostreptococcus family, *Streptococcus proteans* genus

[0305] Peptococciaceae family, spore-forming acetylcholine bacilli

[0306] Peptostreptococcus family, genus *Warmobacter*

[0307] Peptostreptococcus family, genus *Peptostreptococcus*

[0308] The prevalence of Peptostreptococci is the prevalence of all bacteria belonging to the Peptostreptococci family. The prevalence of Peptostreptococci is calculated based on the formula: (Number of all bacteria belonging to the Peptostreptococci family in feces) / (Total number of bacteria from all families in feces).

[0309] The term "Bacteria of the Institute of Physics and Chemistry" is a collective term for bacteria belonging to the Bacteria family of the Institute of Physics and Chemistry. The Bacteria family of the Institute of Physics and Chemistry includes the following bacteria.

[0310] Institute of Physics and Chemistry, Bacteriaceae, Allergenia

[0311] Institute of Physics and Chemistry, Department of Mycology, RC9 Intestinal Group

[0312] Mycology, Institute of Physics and Chemistry

[0313] Anaerobic bacteria of the Bacteriology family, Institute of Physics and Chemistry

[0314] Institute of Physics and Chemistry, Mycology, Allergenia cambogia

[0315] Mycology of the Institute of Physics and Chemistry, genus *Mycoplasma*

[0316] The percentage of bacteria belonging to the Mycaceae family in feces is the percentage of all bacteria belonging to the Mycaceae family in feces. This percentage is calculated using the formula: (Number of all bacteria belonging to the Mycaceae family in feces) / (Total number of bacteria from all families in feces).

[0317] Eubacteriaceae is a collective term for bacteria belonging to the family Eubacteriaceae. Eubacteriaceae includes the following bacteria.

[0318] Eubacteriaceae, Eubacteria genus

[0319] Eubacteriaceae, Corynebacterium genus

[0320] Eubacteriaceae, genus Acetobacter

[0321] Alkalibae, family Eubacteriaceae

[0322] Alkalibacteria (family Eubacteriaceae)

[0323] Eubacteriaceae, Aminoacidae

[0324] Eubacteriaceae, Garcia genus

[0325] Enterobacteriaceae, genus *Intestinibacillus*

[0326] Eubacteriaceae, genus Irregular Bacillus

[0327] Eubacterialaceae, genus Difficultivar

[0328] Eubacteriaceae, Pseudomonas genus

[0329] Eubacteriaceae, genus *Bacillus*

[0330] The prevalence of Eubacteria is the prevalence of all bacteria belonging to the Eubacteria family. The prevalence of Eubacteria is calculated based on the formula: (Number of all Eubacteria in feces) / (Total number of bacteria from all families in feces).

[0331] Bacillus family is a collective term for bacteria belonging to the Bacillus family. Bacillus family includes the following bacteria.

[0332] Bacillus genus of Bacillus family

[0333] Bacillus genus, Bacillus family

[0334] Bacillus genus of Bacillus family Bacillus

[0335] Bacillus family, genus Bacillus

[0336] Bacillus genus, Bacillus family

[0337] Bacillus family, facultative Bacillus genus

[0338] Bacillus genus, Bacillus family

[0339] Bacillus spp., Family Bacillusaceae

[0340] Bacillus spp., Bacillus family

[0341] Bacillus family, Microbacteria genus

[0342] Bacillus genus, Bacillus family

[0343] Bacillus genus, Bacillus family

[0344] Bacillus family, genus *Bacillus*

[0345] Bacillus family, genus Halophilus

[0346] Bacillus family, Lactobacillus genus

[0347] Bacillus family, Bacillus genus

[0348] Bacillus genus, Bacillus family

[0349] Bacillus family, Lysine Bacillus genus

[0350] Bacillus family, Bacillus genus

[0351] Bacillus genus of Bacillus in the family Bacillusaceae

[0352] Bacillus family, Ornithine Bacillus genus

[0353] Bacillus family, Bacillus genus

[0354] Bacillus spp., Bacillus family

[0355] Bacillus genus of Bacillus family Bacillus

[0356] Bacillus family, Bacillus spp.

[0357] Bacillus genus of Bacillus in the family Bacillusaceae

[0358] Bacillus family, sacchariformis

[0359] Bacillus spp. (family Bacillusaceae)

[0360] Bacillus family, genus *Salmonella*

[0361] Bacillus salivarius, family Bacillusaceae

[0362] Bacillus family, genus *Haloxylon*

[0363] Bacillus family, Bacillus sacchariformis genus

[0364] Bacillus genus, Bacillus family

[0365] Bacillus spp., Bacillus family

[0366] Bacillus spp., Bacillus family

[0367] Bacillus family, Bacillus urea genus

[0368] Bacillus family, Mycobacterium genus

[0369] Bacillus spp., Bacillus family

[0370] The prevalence of Bacillus species is the prevalence of all bacteria belonging to the Bacillus family. The prevalence of Bacillus species is calculated based on the formula: (Number of all Bacillus species in feces) / (Total number of bacteria from all families in feces).

[0371] Clostridium group 1 bacteria is a collective term for bacteria belonging to group 1 of the family Clostridium. Group 1 of Clostridium includes the following bacteria.

[0372] Group 1 of the family Clostridium, and Group 1 of the genus Clostridium.

[0373] The prevalence of Clostridium Group 1 bacteria is the prevalence of all bacteria belonging to Clostridium Group 1. The prevalence of Clostridium Group 1 bacteria is calculated based on the formula: (Number of all bacteria belonging to Clostridium Group 1 in feces) / (Total number of bacteria from all families in feces).

[0374] Bacteria of the genus *Rombocytospora* in the family Peptostreptococci are a collective term for bacteria belonging to the genus *Rombocytospora* within the family Peptostreptococci. The prevalence of *Rombocytospora* bacteria in the family Peptostreptococci is the prevalence of all bacteria belonging to the genus *Rombocytospora* in the family Peptostreptococci. This prevalence is calculated using the formula: (Number of all bacteria belonging to the genus *Rombocytospora* in feces) / (Total number of bacteria of all genera in feces).

[0375] The genus *Allergenia* in the family *Bacteriaceae* of the Institute of Physics and Chemistry is a collective term for bacteria belonging to the genus *Allergenia* in the family *Bacteriaceae* of the Institute of Physics and Chemistry. The prevalence of *Allergenia* bacteria in the family *Bacteriaceae* of the Institute of Physics and Chemistry* is the prevalence of all bacteria belonging to this genus. The prevalence of *Allergenia* bacteria in the family *Bacteriaceae* of ...

[0376] The bacteria belonging to the family UCG-010 in the family Trichophyceae is a collective term for bacteria within this family. The prevalence of UCG-010 bacteria is the prevalence of all bacteria belonging to this family. This prevalence is calculated using the formula: (Number of all bacteria belonging to UCG-010 in feces) / (Total number of bacteria from all genera in feces).

[0377] Ruminococcal bacteria (genus *Ruminococcus*) is a collective term for bacteria belonging to the genus *Ruminococcus* within the family Ruminococcalaceae. The prevalence of *Ruminococcal* bacteria represents the prevalence of all bacteria belonging to the genus *Ruminococcus* in the family Ruminococcalaceae. The prevalence of *Ruminococcal* bacteria is calculated using the formula: (Number of all *Ruminococcal* bacteria in feces) / (Total number of bacteria from all genera in feces).

[0378] The Lachnospiraceae [Ruminococcus] gauvreauii group is a collective term for bacteria belonging to this group within the Lachnospiraceae family. The prevalence of Lachnospiraceae [Ruminococcus] gauvreauii group represents the prevalence of all bacteria belonging to this group. This prevalence is calculated using the formula: (Number of all bacteria belonging to the Lachnospiraceae [Ruminococcus] gauvreauii group in feces) / (Total number of bacteria from all genera in feces).

[0379] Ruminococcus genus Group 1 bacteria in the family Ruminococciaceae is a collective term for bacteria belonging to Group 1 of the genus Ruminococcus. The prevalence of Ruminococcus genus Group 1 bacteria in the family Ruminococciaceae is the prevalence of all bacteria belonging to Group 1 of the genus Ruminococcus in the family Ruminococciaceae. The prevalence of Ruminococcus genus Group 1 bacteria in the family Ruminococciaceae is calculated based on the formula: (Number of all bacteria belonging to Group 1 of the genus Ruminococcus in feces) / (Total number of bacteria of all genera in feces).

[0380] The NK4A214 group of Ruminococci is a collective term for bacteria belonging to the NK4A214 group of Ruminococci. The prevalence of NK4A214 group bacteria is the prevalence of all bacteria belonging to this group in Ruminococci. This prevalence is calculated using the formula: (Number of all bacteria belonging to the NK4A214 group in feces) / (Total number of bacteria from all genera in feces).

[0381] Ruminococcal family, specifically Ruminococcal family UCG-002 bacteria, is a collective term for bacteria belonging to the Ruminococcal family, specifically UCG-002. The prevalence of Ruminococcal family UCG-002 bacteria is the prevalence of all bacteria belonging to the Ruminococcal family, specifically UCG-002. This prevalence is calculated based on the formula: (Number of all Ruminococcal family UCG-002 bacteria in feces) / (Total number of bacteria from all genera in feces).

[0382] The active group of Lachnospiraceae [Ruminococcus] gnavusgroup is a collective term for bacteria belonging to this group. The prevalence of Lachnospiraceae [Ruminococcus] gnavusgroup represents the prevalence of all bacteria belonging to this group. The prevalence of Lachnospiraceae [Ruminococcus] gnavusgroup is calculated using the formula: (Number of all bacteria belonging to the active group of Lachnospiraceae [Ruminococcus] in feces) / (Total number of bacteria from all genera in feces).

[0383] Ruminococcal family, specifically Ruminococcal family UCG-003 bacteria, is a collective term for bacteria belonging to the Ruminococcal family, specifically UCG-003. The prevalence of Ruminococcal family UCG-003 bacteria is the prevalence of all bacteria belonging to the Ruminococcal family, specifically UCG-003. This prevalence is calculated based on the formula: (Number of all Ruminococcal family UCG-003 bacteria in feces) / (Total number of bacteria from all genera in feces).

[0384] Ruminococcal family, specifically the UCG-009 family, is a collective term for bacteria belonging to the UCG-009 genus within the Ruminococcal family. The prevalence of Ruminococcal family UCG-009 bacteria represents the prevalence of all bacteria belonging to the UCG-009 genus in the Ruminococcal family. This prevalence is calculated using the formula: (Number of all Ruminococcal family UCG-009 bacteria in feces) / (Total number of bacteria from all genera in feces).

[0385] Bacteria of the genus *Eubacterium* in the family Eubacteriaceae is a collective term for bacteria belonging to the genus *Eubacterium*. The prevalence of bacteria in the genus *Eubacterium* is the prevalence of all bacteria belonging to the genus *Eubacterium* in the family Eubacteriaceae. The prevalence of bacteria in the genus *Eubacterium* is calculated based on the formula: (Number of all bacteria belonging to the genus *Eubacterium* in feces) / (Total number of bacteria from all genera in feces).

[0386] Bacteria of the genus *Femtobacter* in the family Ruminococci are a collective term for bacteria belonging to the genus *Femtobacter* in the family Ruminococci. The prevalence of *Femtobacter* bacteria in the family Ruminococci is the prevalence of all bacteria belonging to the genus *Femtobacter* in the family Ruminococci. The prevalence of *Femtobacter* bacteria in the family Ruminococci is calculated based on the formula: (Number of all bacteria belonging to the genus *Femtobacter* in feces) / (Total number of bacteria from all genera in feces).

[0387] Bacteria of the genus *Bacillus* in the family Bacillusaceae is a collective term for bacteria belonging to the genus *Bacillus* in the family Bacillusaceae. The prevalence of bacteria in the genus *Bacillus* is the prevalence of all bacteria belonging to the genus *Bacillus* in the family Bacillusaceae. The prevalence of bacteria in the genus *Bacillus* is calculated based on the formula: (Number of all bacteria belonging to the genus *Bacillus* in feces) / (Total number of bacteria of all genera in feces).

[0388] The coprostanoligenes group of Ruminococaceae [Eubacterium] is a collective term for bacteria belonging to this group. The prevalence of coprostanoligenes bacteria in Ruminococaceae [Eubacterium] is the prevalence of all bacteria belonging to this group. The prevalence of coprostanoligenes bacteria in Ruminococaceae [Eubacterium] is calculated using the formula: (Number of all bacteria belonging to the coprostanoligenes group in feces) / (Total number of bacteria of all genera in feces).

[0389] Enterobacteriaceae is a collective term for bacteria belonging to the genus Enterobacteriaceae within the family Peptostreptococci. The prevalence of Enterobacteriaceae bacteria represents the prevalence of all Enterobacteriaceae bacteria within this genus. This prevalence is calculated using the formula: (Number of all Enterobacteriaceae bacteria in feces) / (Total number of bacteria from all genera in feces).

[0390] The genus *Anaerobacter* in the family Ruminococci is a collective term for bacteria belonging to the genus *Anaerobacter* within the family Ruminococci. The prevalence of *Anaerobacter* bacteria in Ruminococci is the prevalence of all bacteria belonging to this genus. The prevalence of *Anaerobacter* bacteria in Ruminococci is calculated based on the formula: (Number of all bacteria belonging to the genus *Anaerobacter* in feces) / (Total number of bacteria from all genera in feces).

[0391] Marine Nematomycetes (family Natriiaceae) and *Orocinobacter* genus are collectively referred to as bacteria belonging to the genus *Orocinobacter* within the family Natriiaceae. The prevalence of *Orocinobacter* bacteria in the family Natriiaceae is the prevalence of all bacteria belonging to the genus *Orocinobacter*. This prevalence is calculated using the formula: (Number of all bacteria belonging to the genus *Orocinobacter* in feces) / (Total number of bacteria from all genera in feces).

[0392] Clostridium genus (narrow sense) of Group 1 of the family Clostridium is a collective term for bacteria belonging to the genus Clostridium of Group 1 of the family Clostridium. The prevalence of Clostridium genus (narrow sense) is the prevalence of all bacteria belonging to this genus in Group 1 of the family Clostridium. This prevalence is calculated using the formula: (Number of all bacteria belonging to the genus Clostridium of Group 1 of the family Clostridium) / (Total number of bacteria from all genera in the feces).

[0393] In formulas used to calculate the occupancy of each bacterium, "number of bacteria" and "total number of bacteria" can be absolute or relative, but are usually relative. For example, the "number of bacteria" and "total number of bacteria" obtained in the following microbial community analysis using 16S rRNA amplicon sequencing are relative.

[0394] The accuracy of evaluating intestinal barrier function using the prevalence of Ruminococcus, Peptostreptococcus, Pharmacopoeia, *Peptostreptococcus* spp., and *Allergenia* spp. from the Pharmacopoeia is particularly high. Therefore, from the viewpoint of improving the accuracy of evaluating intestinal barrier function, the prevalence of target bacteria preferably includes the prevalence of one or more species selected from the group consisting of Ruminococcus, Peptostreptococcus, Pharmacopoeia, *Peptostreptococcus* spp., and *Allergenia* spp. from the Pharmacopoeia. More preferably, it includes the prevalence of two or more species selected from that group (specifically, two, three, four, or five species). In addition to the prevalence of one or more species selected from that group, the prevalence of target bacteria may also include the prevalence of one or more other species selected from groups A and B.

[0395] In one embodiment, the occupancy rate of the target bacteria includes one occupancy rate selected from the group consisting of occupancy rates of Ruminococci, Peptostreptococci, Pharmacopoeia, *Leptococcus spp.* (from the Institute of Physicochemical Sciences), *Leptococcus rubescens* (from the Institute of Physicochemical Sciences), and *Allergenia* (from the Institute of Physicochemical Sciences). In addition to one occupancy rate selected from this group, the target bacteria occupancy rate may also include one or more other occupancy rates selected from groups A and B. In one example, the occupancy rate selected from this group is the occupancy rate of Ruminococci. In another example, the occupancy rate selected from this group is the occupancy rate of *Peptostreptococci*. In another example, the occupancy rate selected from this group is the occupancy rate of *Pharmacopoeia*. In another example, the occupancy rate selected from this group is the occupancy rate of *Leptococcus spp.* (from the Institute of Physicochemical Sciences). In yet another example, the occupancy rate selected from this group is the occupancy rate of *Allergenia* (from the Institute of Physicochemical Sciences).

[0396] In another embodiment, the occupancy rate of the target bacteria includes 2, 3, 4, or 5 occupancy rates selected from the group consisting of the occupancy rates of Ruminococcus, Peptostreptococcus, Pharmacopoeia, Rhodotorula spp. (Peptostreptococcus), and Allergan spp. (Pharmacopoeia). In addition to the 2, 3, 4, or 5 occupancy rates selected from this group, the occupancy rate of the target bacteria may also include 1 or more occupancy rates selected from groups A and B.

[0397] Method 1 includes the following steps:

[0398] (1a) The step of determining the occupancy of target bacteria in feces derived from the subject, and obtaining a measured value of the target bacteria occupancy; and

[0399] (1b) The step of comparing the measured value obtained in step 1a with the reference value to obtain the comparison result; and

[0400] (1c) The step of evaluating the intestinal barrier function of the subject based on the comparison results obtained in step 1b.

[0401] <Step 1a>

[0402] Step 1a is to determine the occupancy of target bacteria in feces derived from the subject and to obtain the measured value of the target bacteria occupancy.

[0403] In the case where the occupancy of the target bacteria includes the occupancy of one type of bacteria selected from groups A and B, the occupancy of that one type of bacteria is determined in step 1a.

[0404] If the occupancy rate of the target bacteria includes the occupancy rate of two or more bacteria selected from groups A and B, then in step 1a, the occupancy rate of these two or more bacteria is determined. "Determining the occupancy rate of two or more bacteria" includes separately determining the occupancy rate of two or more bacteria and calculating a combined variable as a statistical analysis value based on the occupancy rate of the two or more bacteria.

[0405] The occupancy of target bacteria can be determined using conventional methods. Preferably, the occupancy of target bacteria is determined by microbial community analysis using 16S rRNA amplicon sequencing. That is, the values ​​obtained in step 1a are preferably obtained by microbial community analysis (metagenomic analysis) using 16S rRNA amplicon sequencing. Microbial community analysis using 16S rRNA amplicon sequencing can be performed, for example, as follows.

[0406] First, a sample is prepared from feces derived from the subject. The sample is, for example, a diluted fecal solution obtained by suspending the feces in a solvent (e.g., ultrapure water) and then crushing it as needed.

[0407] Genomic DNA from bacteria is extracted from the sample. After amplifying a predetermined region of the 16S rRNA gene by PCR, the PCR amplification product is purified as needed. DNA extraction can be performed using commercially available kits. Primer setting, amplification conditions, and purification of the PCR amplification product can be performed according to standard methods. The predetermined region of the 16S rRNA gene to be amplified is not particularly limited as long as it allows for the identification and compositional analysis of bacteria in the bacterial community; for example, regions containing the variable region of the 16S rRNA gene can be listed. Regions containing the variable region of the 16S rRNA gene include, for example, regions containing the V3-V4 region, regions containing the V1-V2 region, regions containing the V1-V4 region, and regions containing the V4 region. Regions containing the V3-V4 region are preferred.

[0408] For the obtained PCR amplification products (amplifiers), index PCR was performed using primers with inherent base sequences (indexes) appended to each sample. Sample identification indexes were then appended to the PCR amplification products of each sample. Primer setup and amplification conditions can be performed according to standard methods.

[0409] Sequence analysis was performed on the indexed PCR amplification products (libraries). Sequence analysis was preferably performed using a next-generation sequencer (e.g., Illumina MiSeq). After quality filtering, the sequences were clustered together for amplicon sequence variant (ASV) analysis. An ASV is an operational taxonomic unit that groups identical sequences into the same bacterial species after correcting erroneous sequences to true sequences. The sequences of each ASV were compared with bacterial sequence databases (reference databases) to identify and analyze the composition of the bacterial community contained in the sample.

[0410] The number of ASVs indicates the number of bacterial species that make up the bacterial community in a sample, and the number of reads belonging to the same ASV indicates the relative abundance of a specific bacterial species. Therefore, the occupancy rate of a specific bacterial species in the bacterial community contained in a sample can be calculated based on the number of reads belonging to the same ASV.

[0411] The occupancy of target bacteria based on ASV analysis can be calculated using commercially available 16S rRNA system analysis software. For example, the occupancy of target bacteria can be calculated by analyzing files (in fastq format) obtained through sequence analysis using a next-generation microbiome bioinformatics platform such as QIIME2 (https: / / qiime2.org) using a next-generation sequencer (e.g., Illumina's MiSeq).

[0412] When using 16S rRNA amplicon sequencing for bacterial community analysis, you can refer to information such as Sanschagrin S, Yergeau E. Next-generation sequencing of 16S ribosomal RNA gene amplicons. J VisExp. 2014; (90): 51709. Published 2014 Aug 29. doi: 10.3791 / 51709.

[0413] When determining the occupancy of target bacteria through microbial community analysis using 16S rRNA amplicon sequencing, step 1a only needs to include sequence analysis of the indexed PCR amplification product (library) and subsequent processing. Step 1a may include processing prior to sequence analysis of the indexed PCR amplification product, such as collection of feces from the subject (i.e., collection of feces used as the test sample from feces excreted by the subject), preparation of the sample from the subject's feces, extraction of bacterial genomic DNA from the sample, amplification of a predetermined region of the 16S rRNA gene (e.g., the region containing the V3-V4 region) by PCR, purification of the PCR amplification product, and indexing of the PCR amplification product by Index PCR, or it may not include these processes.

[0414] When determining the occupancy rate (first occupancy rate, second occupancy rate, ..., nth occupancy rate (n is an integer of 2 or more)) of two or more bacteria selected from group A and group B, and calculating the combined variable as the statistical analysis value based on the occupancy rate of the two or more bacteria, the combined variable is preferably calculated according to the following formula (1).

[0415] Combined variable = (coefficient 1 × (first occupancy rate)) + (coefficient 2 × (second occupancy rate)) + ... + (coefficient n × (nth occupancy rate)) + constant term ... (1)

[0416] There are no special restrictions on n as long as it is an integer greater than or equal to 2, such as an integer greater than or equal to 2 and less than or equal to 6.

[0417] The coefficients and constants vary depending on the quantity and type of the combined occupancy rates. The coefficients and constants can be obtained through multinomial logistic regression analysis. The coefficients are partial regression coefficients obtained through multinomial logistic regression analysis. The method for obtaining the coefficients and constants through multinomial logistic regression analysis is well known to those skilled in the art. For references regarding this method, see Metab Brain Dis, DOI 10.1007 / s11011-017-0029-x, Published online: 11 May 2017, PNAS, October 14, 2003, vol.100, no.21, pp.12343-12348 (www.pnas.org / cgi / doi / 10.1073 / pnas.2033602100, Ann Transl Med, 2019, 7(16):388, p.1-10 (http: / / dx.doi.org / 10.21037 / atm.2019.07.102). The coefficients used to derive the combined variables can be derived from the measured data, ensuring that the combined variables are close to 1 in the group with reduced intestinal barrier function and close to 0 in the group with normal intestinal barrier function.

[0418] In the case of the combined prevalence of Ruminococcus, Peptostreptococcus, Peptostreptococcus spp., and the Institute of Physics and Chemistry Bacteria, for example, the combined variables can be obtained from the prevalence of these four bacteria based on the above equation (1) using the coefficients and constants shown in Table 1. In Table 1, “R1” represents the prevalence of Ruminococcus, “R2” represents the prevalence of Peptostreptococcus, “R3” represents the prevalence of Peptostreptococcus spp., and “R4” represents the prevalence of Institute of Physics and Chemistry Bacteria.

[0419] [Table 1]

[0420]

[0421] <Step 1b>

[0422] Step 1b is the step of comparing the measured value obtained in step 1a with the benchmark value and obtaining the comparison result.

[0423] In step 1a, if the occupancy rate of one bacterium selected from groups A and B is determined, in step 1b, the determined occupancy rate of that bacterium is compared with a baseline occupancy rate for that bacterium to obtain a comparison result. If the determined occupancy rate of that bacterium is obtained through microbial community analysis using 16S rRNA amplicon sequencing, then the baseline occupancy rate of that bacterium is also obtained through microbial community analysis using 16S rRNA amplicon sequencing.

[0424] If, in step 1a, the occupancy rate of two or more bacteria selected from groups A and B is determined, then in step 1b, the measured occupancy rate of each of the two or more bacteria is compared with the baseline occupancy rate of each of the two or more bacteria to obtain a comparison result. If the measured occupancy rate of each of the two or more bacteria is obtained through microbial community analysis using 16S rRNA amplicon sequencing, then the baseline occupancy rate of each of the two or more bacteria is also obtained through microbial community analysis using 16S rRNA amplicon sequencing.

[0425] In step 1a, if a combined variable is obtained based on the occupancy rate of two or more bacteria selected from group A and group B, preferably based on the combined variable obtained by equation (1) above, then in step 1b, the combined variable is compared with a baseline value of the combined variable to obtain a comparison result. If the combined variable is based on a value obtained by microbial community analysis using 16S rRNA amplicon sequencing, then the baseline value of the combined variable is also based on a value obtained by microbial community analysis using 16S rRNA amplicon sequencing.

[0426] <Step 1c>

[0427] Step 1c is a step to evaluate the intestinal barrier function of the subject based on the comparison results obtained in step 1b.

[0428] In Method 1, "positive" means that the subject's intestinal barrier function is reduced, and "negative" means that the subject's intestinal barrier function is not reduced.

[0429] The intestinal barrier function to be evaluated can be the intestinal barrier function of the small intestine or the intestinal barrier function of the large intestine, with the small intestine being the preferred candidate.

[0430] The intestinal barrier function, particularly the physical barrier function such as tight junction structures, is the subject of evaluation.

[0431] When the comparison result obtained in step 1b is a comparison between the measured value of the occupancy of one type of bacteria selected from groups A and B and the baseline value of the occupancy of that one type of bacteria, the comparison result is used to evaluate the intestinal barrier function of the subject.

[0432] When the comparison result obtained in step 1b is a comparison between the measured values ​​of the occupancy of two or more bacteria selected from group A and group B and the baseline values ​​of the occupancy of the two or more bacteria, it is preferable to use the comparison result related to the occupancy of all bacteria included in the comparison result to evaluate the intestinal barrier function of the subject. However, it is also possible to use the comparison result related to the occupancy of a subset of bacteria (one or more bacteria) arbitrarily selected from the comparison result to evaluate the intestinal barrier function of the subject.

[0433] When the comparison result obtained in step 1b is a comparison of the combined variable, which is calculated based on the occupancy of two or more bacteria selected from group A and group B, with the baseline value of the combined variable, the comparison result is used to evaluate the intestinal barrier function of the subject.

[0434] Regarding the occupancy rate of each bacterium, the intestinal barrier function of the subject can be evaluated based on whether the comparison results obtained in step 1b meet the criteria shown in Table 2. Specifically, for the occupancy rate of each bacterium with a "high" criterion shown in Table 2, if the measured value obtained in step 1a is higher than the criterion value (i.e., the comparison results obtained in step 1b meet the criteria shown in Table 2), it can be evaluated as positive; if the measured value obtained in step 1a is lower than the criterion value (i.e., the comparison results obtained in step 1b do not meet the criteria shown in Table 2), it can be evaluated as negative. Similarly, for the occupancy rate of each bacterium with a "low" criterion shown in Table 2, if the measured value obtained in step 1a is lower than the criterion value (i.e., the comparison results obtained in step 1b meet the criteria shown in Table 2), it can be evaluated as positive; if the measured value obtained in step 1a is higher than the criterion value (i.e., the comparison results obtained in step 1b do not meet the criteria shown in Table 2), it can be evaluated as negative. It should be noted that the criteria shown in Table 2 can also be applied to methods other than Method 1.

[0435] In step 1a, if the occupancy rate of one type of bacteria selected from group A and group B is determined, the result of the comparison of the occupancy rate of the one type of bacteria can be evaluated as positive if it meets the criteria shown in Table 2, and the result of the comparison of the occupancy rate of the one type of bacteria can be evaluated as negative if it does not meet the criteria shown in Table 2.

[0436] In step 1a, if the occupancy rate of two or more bacteria selected from groups A and B is measured, a positive result can be evaluated if the occupancy rate of at least one of these two or more bacteria meets the criteria shown in Table 2; a negative result can be evaluated if none of the comparison results for the occupancy rate of these two or more bacteria meet the criteria shown in Table 2. The more bacteria whose occupancy rates meet the criteria shown in Table 2, the higher the probability of a positive result. Therefore, in step 1a, by measuring the occupancy rate of two or more bacteria selected from groups A and B (e.g., two, three, or four or more), the accuracy of the assessment of the subject's intestinal barrier function can be improved.

[0437] [Table 2]

[0438]

[0439] The baseline value can be set using conventional methods.

[0440] In one embodiment, the occupancy rate of each bacterium selected from group A or group B can be used as a cutoff value obtained from the receiver operating characteristic (ROC) curve of the occupancy rate of each bacterium as a baseline value. This embodiment (hereinafter referred to as "Embodiment 1-1") will be described below.

[0441] ROC curves for the occupancy of each bacterium can be constructed using conventional methods based on the occupancy of each bacterium in feces obtained from individuals of the same species as the subject (e.g., humans if the subject is human) into a negative group (individuals with unreduced intestinal barrier function) and a positive group (individuals with reduced intestinal barrier function). In the ROC curves, the vertical axis represents sensitivity, and the horizontal axis represents (1-specificity). When constructing the negative and positive groups, the distinction between negative and positive can be made using known methods for detecting intestinal barrier function (e.g., the lactulose-mannitol test).

[0442] A cutoff value is a value that satisfies both high sensitivity and high specificity when evaluated against a certain benchmark. The cutoff value can be appropriately set considering the balance between sensitivity and specificity. For example, a cutoff value can be set to a value that shows a high positive rate in the positive group and a high negative rate in the negative group. Methods for setting cutoff values ​​are well known to those skilled in the art. For example, methods are known to use the point on the ROC curve that is closest to the upper left corner of the ROC curve as the cutoff value, or the point on the ROC curve where the value of (sensitivity + specificity - 1) is the maximum value as the cutoff value. It should be noted that the point on the ROC curve where the value of (sensitivity + specificity - 1) is the maximum value is called the Younden index.

[0443] Table 3 provides specific examples of the cutoff values ​​for the occupancy of each bacterium selected from groups A and B.

[0444] [Table 3]

[0445]

[0446] Regarding the occupancy rate of Ruminococci, the preferred cutoff value is in the range of 15.0 to 25.0, the more preferred cutoff value is in the range of 17.0 to 23.0, and the even more preferred cutoff value is in the range of 20.0 to 22.0.

[0447] Regarding the percentage of Streptococcus bacteria, the preferred cutoff value is in the range of 0.500 to 1.300, the more preferred cutoff value is in the range of 0.600 to 1.280, and the even more preferred cutoff value is in the range of 0.700 to 0.800.

[0448] Regarding the occupancy rate of Mycaceae bacteria from the Institute of Physics and Chemistry, the preferred cutoff value is in the range of 0.400 to 1.300, the more preferred cutoff value is in the range of 0.500 to 1.200, and the even more preferred cutoff value is in the range of 0.600 to 0.800.

[0449] Regarding the occupancy rate of Eubacteriaceae, the preferred cutoff value is in the range of 0.010 to 0.040, the more preferred cutoff value is in the range of 0.015 to 0.030, and the even more preferred cutoff value is in the range of 0.020 to 0.025.

[0450] Regarding the occupancy rate of Bacillus species, the preferred cutoff value is in the range of 0.005 to 0.030, the more preferred cutoff value is in the range of 0.007 to 0.020, and the even more preferred cutoff value is in the range of 0.010 to 0.020.

[0451] Regarding the occupancy of Group 1 bacteria of the Clostridium family, the preferred cutoff value is in the range of 0.010 to 0.050, the more preferred cutoff value is in the range of 0.020 to 0.040, and the even more preferred cutoff value is in the range of 0.030 to 0.035.

[0452] Regarding the percentage of bacteria in the genus *Lombocytozoon* of the family Peptostreptococcus, the preferred cutoff value is in the range of 0.400 to 0.800, the more preferred cutoff value is in the range of 0.450 to 0.700, and the even more preferred cutoff value is in the range of 0.500 to 0.550.

[0453] Regarding the percentage of Allergenia spp. in the mycology family of the Institute of Physics and Chemistry, the preferred cutoff value is in the range of 0.500 to 1.000, the more preferred cutoff value is in the range of 0.550 to 0.900, and the even more preferred cutoff value is in the range of 0.600 to 0.800.

[0454] Regarding the occupancy rate of UCG-010 bacteria from the family Trichophyceae, the preferred cutoff value is in the range of 0.005 to 0.020, the more preferred cutoff value is in the range of 0.008 to 0.015, and the even more preferred cutoff value is in the range of 0.009 to 0.013.

[0455] Regarding the occupancy rate of Ruminococcus-negative spore-forming bacteria, the preferred cutoff value is in the range of 0.020 to 0.060, the more preferred cutoff value is in the range of 0.030 to 0.050, and the even more preferred cutoff value is in the range of 0.039 to 0.045.

[0456] Regarding the occupancy of the Gossypium group of bacteria in the family Ruminococcus, the preferred cutoff value is in the range of 0.010 to 0.090, the more preferred cutoff value is in the range of 0.015 to 0.060, and the even more preferred cutoff value is in the range of 0.019 to 0.025.

[0457] Regarding the occupancy rate of Group 1 Ruminococcus bacteria in the Ruminococcus genus of the Ruminococcus family, the preferred cutoff value is in the range of 0.060 to 0.230, the more preferred cutoff value is in the range of 0.070 to 0.100, and the even more preferred cutoff value is in the range of 0.080 to 0.090.

[0458] Regarding the occupancy rate of Ruminococcus NK4A214 group bacteria, the preferred cutoff value is in the range of 0.040 to 0.140, the more preferred cutoff value is in the range of 0.050 to 0.100, and the even more preferred cutoff value is in the range of 0.050 to 0.090.

[0459] Regarding the occupancy rate of UCG-002 bacteria from the Ruminococcus family, the preferred cutoff value is in the range of 0.009 to 0.450, the more preferred cutoff value is in the range of 0.100 to 0.300, and the even more preferred cutoff value is in the range of 0.120 to 0.150.

[0460] Regarding the occupancy rate of the active group of bacteria in the family Trichophytonceae [Ruminococcus genus], the preferred cutoff value is in the range of 0.100 to 0.400, the more preferred cutoff value is in the range of 0.150 to 0.300, and the even more preferred cutoff value is in the range of 0.170 to 0.280.

[0461] Regarding the occupancy rate of Ruminococcus family UCG-003 bacteria, the preferred cutoff value is in the range of 0.008 to 0.020, the more preferred cutoff value is in the range of 0.009 to 0.015, and the even more preferred cutoff value is in the range of 0.010 to 0.013.

[0462] Regarding the occupancy rate of UCG-009 bacteria from the Ruminococcus family, the preferred cutoff value is in the range of 0.008 to 0.020, the more preferred cutoff value is in the range of 0.009 to 0.015, and the even more preferred cutoff value is in the range of 0.010 to 0.013.

[0463] Regarding the percentage of bacteria in the genus *Eubacterium* of the family Eubacteriaceae, the preferred cutoff value is in the range of 0.008 to 0.020, the more preferred cutoff value is in the range of 0.009 to 0.015, and the even more preferred cutoff value is in the range of 0.010 to 0.013.

[0464] Regarding the percentage of bacteria in the genus *Bacillus* of the family Ruminococci, the preferred cutoff value is in the range of 5.00 to 7.80, a more preferred cutoff value is in the range of 6.00 to 6.70, and an even more preferred cutoff value is in the range of 6.20 to 6.50.

[0465] Regarding the occupancy rate of Bacillus genus in the Bacillusaceae family, the preferred cutoff value is in the range of 0.005 to 0.030, the more preferred cutoff value is in the range of 0.007 to 0.020, and the even more preferred cutoff value is in the range of 0.010 to 0.020.

[0466] Regarding the occupancy of coprosterol-producing bacteria in the Ruminococcus family [Eubacterium genus], the preferred cutoff value is in the range of 0.90 to 1.40, the more preferred cutoff value is in the range of 1.00 to 1.37, and the even more preferred cutoff value is in the range of 1.22 to 1.26.

[0467] Regarding the occupancy rate of Enterobacteriaceae (Peptostreptococci), the preferred cutoff value is in the range of 0.070 to 0.160, the more preferred cutoff value is in the range of 0.080 to 0.140, and the even more preferred cutoff value is in the range of 0.090 to 0.120.

[0468] Regarding the occupancy rate of anaerobic bacteria of the genus *Corynebacterium* in the family Ruminococci, the preferred cutoff value is in the range of 0.010 to 0.035, the more preferred cutoff value is in the range of 0.015 to 0.030, and the even more preferred cutoff value is in the range of 0.020 to 0.025.

[0469] Regarding the percentage of bacteria in the genus *Ostomyces* of the family Laminariaceae, the preferred cutoff value is in the range of 0.030 to 0.085, the more preferred cutoff value is in the range of 0.040 to 0.080, and the even more preferred cutoff value is in the range of 0.045 to 0.060.

[0470] Regarding the occupancy of Clostridium genus in the narrow sense of Group 1 of Clostridiumceae, the preferred cutoff value is a value in the range of 0.015 to 0.045, a more preferred cutoff value is a value in the range of 0.020 to 0.040, and an even more preferred cutoff value is a value in the range of 0.025 to 0.035.

[0471] In another embodiment, regarding the occupancy rate of two or more bacteria selected from group A or group B, the occupancy rate of these two or more bacteria in feces obtained from a negative group (a group of individuals whose intestinal barrier function is not reduced) and a positive group (a group of individuals whose intestinal barrier function is reduced) consisting of individuals of the same species as the subject (e.g., if the subject is a human, then a human). Based on the above formula (1), a combination variable is obtained from the occupancy rate of these two or more bacteria. An ROC curve is generated based on the obtained combination variable, and the cutoff value obtained from the generated ROC curve is used as a reference value for the occupancy rate of these two or more bacteria. Hereinafter, this embodiment (hereinafter referred to as "Embodiment 1-2") will be described.

[0472] Implementation methods 1-2 are applicable to cases where the measured values ​​obtained in step 1a are based on the combined variables obtained from the occupancy rates of two or more bacteria selected from group A or group B using the above formula (1).

[0473] When forming negative and positive groups, the distinction between negative and positive can be performed using a known method for detecting intestinal barrier function (e.g., lactulose-mannitol test). ROC curves can be constructed using conventional methods based on the combined variables obtained from the negative and positive groups. In the ROC curve, the vertical axis represents sensitivity, and the horizontal axis represents (1-specificity). When forming negative and positive groups, the distinction between negative and positive can be performed using a known method for detecting intestinal barrier function (e.g., lactulose-mannitol test). The explanation of the cutoff value is the same as in Embodiment 1-1.

[0474] Table 1 shows the specific examples of the coefficients and constants for the combined prevalence of Ruminococcus, Peptostreptococcus, Peptostreptococcus spp., and the prevalence of Physicochemical Bacteria. Table 4 shows the specific examples of the cutoff values. In Table 4, "R1", "R2", "R3" and "R4" are synonyms with those in Table 1.

[0475] [Table 4]

[0476]

[0477] Regarding No. 1 in Table 4, the preferred cutoff value is a value in the range of -1.300 to -0.090, the more preferred cutoff value is a value in the range of -1.240 to -0.100, and the even more preferred cutoff value is a value in the range of -0.142 to -0.140.

[0478] Regarding No. 2 in Table 4, the preferred cutoff value is a value in the range of -1.300 to -0.050, the more preferred cutoff value is a value in the range of -1.240 to -0.090, and the even more preferred cutoff value is a value in the range of -0.151 to -0.100.

[0479] Regarding No. 3 in Table 4, the preferred cutoff value is a value in the range of -1.000 to -0.100, the more preferred cutoff value is a value in the range of -0.410 to -0.150, and the even more preferred cutoff value is a value in the range of -0.300 to -0.254.

[0480] Regarding No. 4 in Table 4, the preferred cutoff value is a value in the range of -0.470 to -0.030, the more preferred cutoff value is a value in the range of -0.250 to -0.040, and the even more preferred cutoff value is a value in the range of -0.072 to -0.060.

[0481] Regarding No. 5 in Table 4, the preferred cutoff value is a value in the range of -0.300 to 0.600, the more preferred cutoff value is a value in the range of 0.070 to 0.500, and the even more preferred cutoff value is a value in the range of 0.100 to 0.441.

[0482] Regarding No. 6 in Table 4, the preferred cutoff value is a value in the range of -1.000 to -0.100, the more preferred cutoff value is a value in the range of -0.420 to -0.150, and the even more preferred cutoff value is a value in the range of -0.200 to -0.254.

[0483] Regarding No. 7 in Table 4, the preferred cutoff value is a value in the range of -1.000 to -0.050, the more preferred cutoff value is a value in the range of -0.500 to -0.070, and the even more preferred cutoff value is a value in the range of -0.420 to -0.090.

[0484] Regarding No. 8 in Table 4, the preferred cutoff value is a value in the range of -0.090 to 0.500, the more preferred cutoff value is a value in the range of 0.220 to 0.450, and the even more preferred cutoff value is a value in the range of 0.343 to 0.400.

[0485] Regarding No. 9 in Table 4, the preferred cutoff value is a value in the range of -0.600 to -0.300, the more preferred cutoff value is a value in the range of -0.570 to -0.350, and the even more preferred cutoff value is a value in the range of -0.480 to -0.441.

[0486] Regarding No. 10 in Table 4, the preferred cutoff value is a value in the range of -0.390 to 0.600, the more preferred cutoff value is a value in the range of 0.100 to 0.450, and the even more preferred cutoff value is a value in the range of 0.130 to 0.411.

[0487] Regarding No. 11 in Table 4, the preferred cutoff value is a value in the range of 0.030 to 0.350, the more preferred cutoff value is a value in the range of 0.080 to 0.250, and the even more preferred cutoff value is a value in the range of 0.100 to 0.225.

[0488] In another embodiment, regarding the occupancy rate of each bacterium selected from group A or group B, the average occupancy rate of each bacterium in feces obtained from a negative group (a group of individuals whose intestinal barrier function is not reduced) consisting of individuals of the same species as the subject (e.g., humans if the subject is a human) can be used as a baseline value for the occupancy rate of each bacterium. This embodiment (hereinafter referred to as "Embodiments 1-3") will be described below.

[0489] When forming a negative group, the distinction between negative and positive can be made using well-known methods for detecting intestinal barrier function (e.g., lactulose-mannitol test).

[0490] Regarding the occupancy rate of each bacteria with a baseline of "high" shown in Table 2, a positive result can be evaluated if the measured value obtained in step 1a is higher than the average value. Preferably, a positive result is evaluated if the measured value obtained in step 1a is 1.05 times or more of the average value, more preferably if the measured value obtained in step 1a is 1.1 times or more of the average value, and even more preferably if the measured value obtained in step 1a is 1.5 times or more of the average value.

[0491] Regarding the occupancy rate of each bacteria with a baseline of "low" shown in Table 2, a positive result can be evaluated if the measured value obtained in step 1a is lower than the average value. Preferably, a positive result is evaluated if the measured value obtained in step 1a is less than 0.9 times the average value; more preferably, a positive result is evaluated if the measured value obtained in step 1a is less than 0.7 times the average value; and even more preferably, a positive result is evaluated if the measured value obtained in step 1a is less than 0.5 times the average value.

[0492] In another embodiment, regarding the occupancy rate of each bacterium selected from group A or group B, the median value of the occupancy rate of each bacterium in feces obtained from a negative group (a group of individuals whose intestinal barrier function is not reduced) consisting of individuals of the same species as the subject (e.g., humans if the subject is a human) can be used as a baseline value for the occupancy rate of each bacterium. This embodiment (hereinafter referred to as "Embodiments 1-4") will be described below.

[0493] The descriptions of embodiments 1-3 also apply to embodiments 1-4, unless otherwise specified. In application, "average value" is replaced with "median value".

[0494] Method 2

[0495] Embodiment 2 of the present invention relates to a method for diagnosing diseases or symptoms caused by decreased intestinal barrier function in a subject. Hereinafter, "diseases or symptoms caused by decreased intestinal barrier function" will sometimes be referred to as "diseases or symptoms".

[0496] The method of Embodiment 2 of the present invention includes the following steps:

[0497] (2a) The steps of determining the occupancy of target bacteria in feces derived from the subject and obtaining the measured value of the occupancy of target bacteria.

[0498] (2b) The step of comparing the measured value obtained in step 2a with the reference value to obtain the comparison result; and

[0499] (2c) Based on the comparison results obtained in step 2b, the steps for diagnosing the disease or symptoms caused by decreased intestinal barrier function in the subject.

[0500] The aforementioned occupancy rate of target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B.

[0501] The explanation of Method 1 also applies to Method 2, unless otherwise specified. When applying this method, replace "Step 1a" with "Step 2a", "Step 1b" with "Step 2b", "Step 1c" with "Step 2c", "evaluate the intestinal barrier function of the subject" with "diagnose the disease or symptoms of the subject", "accuracy of the evaluation of the intestinal barrier function of the subject" with "accuracy of the diagnosis of the disease or symptoms of the subject", "positive evaluation" with "positive diagnosis", and "negative evaluation" with "negative diagnosis".

[0502] In Method 2, "positive" means that the subject has a disease or symptoms, and "negative" means that the subject does not have a disease or symptoms.

[0503] The disease or symptom being diagnosed can be caused by a decrease in the intestinal barrier function of the small intestine or the large intestine, preferably caused by a decrease in the intestinal barrier function of the small intestine.

[0504] Diseases or symptoms caused by a decrease in intestinal barrier function include, for example, enteritis, allergic diseases, mental illness, non-alcoholic fatty liver disease, type 2 diabetes, metabolic syndrome, and obesity.

[0505] Examples of enteritis include irritable bowel syndrome, ulcerative colitis, and Crohn's disease. Examples of allergic diseases include food allergies, atopic dermatitis, and asthma. Examples of mental illnesses include anxiety disorders and depression. Examples of non-alcoholic fatty liver disease include non-alcoholic fatty liver disease and non-alcoholic steatohepatitis.

[0506] Diagnosis is typically performed by a physician. Based on the comparison results obtained in step 2b, the physician diagnoses whether the subject has a disease or symptoms caused by decreased intestinal barrier function. If the subject does have a disease or symptoms caused by decreased intestinal barrier function, the physician can determine whether prevention or treatment of the disease or symptoms is necessary, select appropriate methods for prevention or treatment, and implement preventative or therapeutic measures. "Prevention" includes preventing, suppressing, and delaying the onset of a disease or symptom. "Treatment" includes suppressing the progression or worsening of a disease or symptom, delaying the progression or worsening of a disease or symptom, and alleviating, reducing, improving, and curing a disease or symptom.

[0507] The method of Embodiment 2 of the present invention can also be used to assist in the diagnosis of diseases or symptoms caused by decreased intestinal barrier function. The act of assisting in the diagnosis of diseases or symptoms is the act of providing the comparison results obtained in step 2b as data useful for the diagnosis of the disease or symptoms; it can be a medical act or a non-medical act, but is usually a non-medical act. In addition to the data provided through the acts assisting in the diagnosis of diseases or symptoms, a doctor's diagnosis can also be based on one or more other types of information. Furthermore, the doctor's experience, know-how, etc., can also participate in the doctor's diagnosis.

[0508] Method 3

[0509] Embodiment 3 of the present invention relates to a method for treating a disease or symptom caused by a decrease in intestinal barrier function in a subject. Hereinafter, "disease or symptom caused by a decrease in intestinal barrier function" will sometimes be referred to as "disease or symptom".

[0510] The method of embodiment 3 of the present invention includes the following steps:

[0511] (3a) The step of determining the occupancy of target bacteria in feces derived from the subject and obtaining the measured value of the occupancy of target bacteria.

[0512] (3b) The step of comparing the measured value obtained in step 3a with the reference value to obtain the comparison result;

[0513] (3c) Based on the comparison results obtained in step 3b, the step of diagnosing the disease or symptoms caused by decreased intestinal barrier function in the subject; and

[0514] (3d) In cases where the subject is diagnosed with a disease or symptoms caused by decreased intestinal barrier function, the procedure of administering to the subject a substance or composition for treating the disease or symptoms caused by decreased intestinal barrier function.

[0515] The aforementioned occupancy rate of target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B.

[0516] The explanation of Method 2 also applies to Method 3, unless otherwise specified. When applying, "Step 2a" should be replaced with "Step 3a", "Step 2b" with "Step 3b", and "Step 2c" with "Step 3c".

[0517] A substance or composition for treating a disease or symptom, administered to a subject diagnosed with a disease or symptom caused by a weakened intestinal barrier, may be appropriately selected based on the type of disease or symptom the subject has. "Treatment" includes the suppression of the progression or worsening of a disease or symptom, the delay of the progression or worsening of a disease or symptom, and the relief, reduction, improvement, and cure of a disease or symptom.

[0518] Examples of substances or compositions for treating irritable bowel syndrome include, for example, high molecular weight polymers and serotonin receptor antagonists. Examples of substances or compositions for treating ulcerative colitis or Crohn's disease include, for example, 5-aminosalicylic acid preparations and corticosteroids. Examples of substances or compositions for treating allergic diseases include, for example, antihistamines. Examples of substances or compositions for treating anxiety disorders or depression include, for example, selective serotonin reuptake inhibitors and norepinephrine antidepressants. Examples of substances or compositions for treating non-alcoholic fatty liver disease include, for example, antioxidants, diabetes medications, and medications for treating lipid metabolism disorders.

[0519] Substances or compositions used to treat diseases or symptoms may be administered in the form of food compositions containing such substances or compositions. Examples of food compositions include yogurt, probiotics, prebiotics, and supplements. Food compositions may also be, for example, foods for specific health purposes, functionally labeled foods, or foods for patients.

[0520] Routes of administration for substances or compositions used to treat diseases or symptoms may include, for example, oral, non-oral (e.g., intranasal, ophthalmic, ocular, transdermal, respiratory, rectal, urinary, subcutaneous, intramuscular, intravenous, etc.). Substances or compositions used to treat diseases or symptoms can be formulated into administration forms suitable for the route of administration. Administration forms may include, for example, tablets and injections. During formulation, additives suitable for oral administration (e.g., excipients, disintegrants, lubricants, binders, etc.) or additives suitable for non-oral administration (e.g., diluents, solvents, etc.) may be used. The dosage and frequency of administration of substances or compositions used to treat diseases or symptoms can be appropriately adjusted considering the method of administration, the age of the recipient, weight, etc. The number of times of administration per day may be once or more than twice. The administration period may be one day or more than two days (e.g., more than one week, more than two weeks, more than three weeks, or more than four weeks).

[0521] Method 4

[0522] Embodiment 4 of the present invention relates to a method for evaluating the risk of a subject developing a disease or symptom caused by reduced intestinal barrier function, or the likelihood of a subject suffering from such a disease or symptom. Hereinafter, the "risk of a subject developing a disease or symptom caused by reduced intestinal barrier function" is sometimes referred to as "risk of disease", and the "likelihood of a subject suffering from a disease or symptom caused by reduced intestinal barrier function" is sometimes referred to as "likelihood of suffering".

[0523] Method 4 includes the following steps:

[0524] (4a) The step of determining the occupancy of target bacteria in feces derived from the subject, and obtaining the measured value of the target bacteria occupancy; and

[0525] (4b) The step of comparing the measured value obtained in step 4a with the reference value to obtain the comparison result; and

[0526] (4c) The step of evaluating the risk of disease or the likelihood of developing the disease based on the comparison results obtained in step 4b.

[0527] The aforementioned occupancy rate of target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B.

[0528] The explanation of Method 1 also applies to Method 4, unless otherwise specified. When applying, "Step 1a" is replaced with "Step 4a", "Step 1b" is replaced with "Step 4b", "Step 1c" is replaced with "Step 4c", "evaluate the intestinal barrier function of the subject" is replaced with "evaluate the risk of disease or the likelihood of developing the disease", and "accuracy of the evaluation of the intestinal barrier function of the subject" is replaced with "accuracy of the evaluation of the risk of disease or the likelihood of developing the disease".

[0529] In Method 4, "positive" means there is a high risk or possibility of developing the disease, while "negative" means there is a low risk or possibility of developing the disease.

[0530] The risk of disease in the subject being evaluated can be the risk of the subject developing a disease or symptoms caused by a decrease in the intestinal barrier function of the small intestine, or the risk of the subject developing a disease or symptoms caused by a decrease in the intestinal barrier function of the large intestine, preferably the risk of the subject developing a disease or symptoms caused by a decrease in the intestinal barrier function of the small intestine.

[0531] The likelihood of a subject being evaluated can be the likelihood of the subject having a disease or symptom caused by a decrease in the intestinal barrier function of the small intestine, or the likelihood of the subject having a disease or symptom caused by a decrease in the intestinal barrier function of the large intestine, preferably the likelihood of the subject having a disease or symptom caused by a decrease in the intestinal barrier function of the small intestine.

[0532] The explanation of diseases or symptoms caused by decreased intestinal barrier function is the same as in Method 2.

[0533] Method 5

[0534] Embodiment 5 of the present invention relates to a method for obtaining data on diseases or symptoms caused by decreased intestinal barrier function in a diagnostic subject. Hereinafter, "diseases or symptoms caused by decreased intestinal barrier function" will sometimes be referred to as "diseases or symptoms".

[0535] Method 5 includes the following steps:

[0536] (5a) The step of determining the occupancy of target bacteria in feces derived from the subject, and obtaining a measured value of the target bacteria occupancy; and

[0537] (5b) The step of comparing the measured values ​​obtained in step 5a with the baseline values ​​and obtaining the comparison results as data for diagnosing diseases or symptoms caused by decreased intestinal barrier function in the target population.

[0538] The aforementioned occupancy rate of target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B.

[0539] The explanation of Method 1 also applies to Method 5, unless otherwise specified. When applying, "Step 1a" is replaced with "Step 5a", "Step 1b" is replaced with "Step 5b", "evaluate the intestinal barrier function of the subject" is replaced with "obtain data for diagnosing the disease or symptoms of the subject", and "accuracy of the evaluation of the intestinal barrier function of the subject" is replaced with "accuracy of the diagnosis of the disease or symptoms of the subject".

[0540] In Method 5, "positive" means that the subject has a disease or symptoms, and "negative" means that the subject does not have a disease or symptoms.

[0541] The disease or symptom being diagnosed can be caused by a decrease in the intestinal barrier function of the small intestine or the large intestine, preferably caused by a decrease in the intestinal barrier function of the small intestine.

[0542] The explanation of diseases or symptoms caused by decreased intestinal barrier function is the same as in Method 2.

[0543] Regarding the occupancy rates of each bacteria selected from groups A and B, comparison results that meet the criteria shown in Table 2 can be used as data for a positive diagnosis, while comparison results that do not meet the criteria shown in Table 2 can be used as data for a negative diagnosis. Specifically, regarding the occupancy rates of each bacteria for which the criteria shown in Table 2 are "high," comparison results where the measured value obtained in step 5a is higher than the reference value can be used as data for a positive diagnosis, while comparison results where the measured value obtained in step 5a is lower than the reference value can be used as data for a negative diagnosis. Similarly, regarding the occupancy rates of each bacteria for which the criteria shown in Table 2 are "low," comparison results where the measured value obtained in step 5a is lower than the reference value can be used as data for a positive diagnosis, while comparison results where the measured value obtained in step 5a is higher than the reference value can be used as data for a negative diagnosis.

[0544] In step 5a, when determining the occupancy rate of one type of bacteria selected from group A and group B, if the comparison result of the occupancy rate of the one type of bacteria meets the criteria shown in Table 2, the comparison result can be obtained as data for diagnosis of positive; if the comparison result of the occupancy rate of the one type of bacteria does not meet the criteria shown in Table 2, the comparison result can be obtained as data for diagnosis of negative.

[0545] In step 5a, when determining the occupancy rate of two or more bacteria selected from groups A and B, if the comparison result of the occupancy rate of at least one of the two or more bacteria meets the criteria shown in Table 2, the comparison result can be obtained as data for a positive diagnosis; if the comparison result of the occupancy rate of any of the two or more bacteria does not meet the criteria shown in Table 2, the comparison result can be obtained as data for a negative diagnosis. The more bacteria in the comparison result that meet the criteria shown in Table 2, the higher the probability of a positive result. Therefore, in step 5a, by determining the occupancy rate of two or more bacteria selected from groups A and B (e.g., two, three, or four or more), the accuracy of the diagnosis of the subject's disease or symptoms can be improved.

[0546] The data obtained in step 5b can be used to diagnose diseases or symptoms caused by decreased intestinal barrier function in the subject, or to assist in the diagnosis of diseases or symptoms caused by decreased intestinal barrier function in the subject.

[0547] Diagnosis is typically performed by a physician. Based on the data obtained in step 5b, the physician diagnoses whether the subject has a disease or symptoms caused by decreased intestinal barrier function. If the subject does have such a disease or symptom, the physician can determine whether prevention or treatment is necessary, select appropriate methods for prevention or treatment, and implement preventative or therapeutic measures. "Prevention" includes preventing, suppressing, and delaying the onset of a disease or symptom. "Treatment" includes suppressing the progression or worsening of a disease or symptom, delaying its progression or worsening, and alleviating, reducing, improving, and curing the disease or symptom.

[0548] Actions that assist in the diagnosis of a disease or symptom are actions that provide the data acquired in step 5b as data for the diagnosis of a disease or symptom. These actions can be medical or non-medical, but are usually non-medical. A doctor's diagnosis may be based on one or more other types of data in addition to the data provided through actions that assist in the diagnosis of a disease or symptom.

[0549] Method 6

[0550] Embodiment 6 of the present invention relates to a method for screening substances or compositions that have an effect on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by reduced intestinal barrier function. Hereinafter, "the effect on preventing or improving diseases or symptoms caused by reduced intestinal barrier function" is sometimes referred to as "the effect on preventing or improving diseases or symptoms".

[0551] Method 6 includes the following steps:

[0552] (6a) The step of administering a candidate substance or candidate composition to a subject whose intestinal barrier function has been reduced;

[0553] (6b) The step of determining the occupancy of target bacteria in feces obtained from the subject after administration of the candidate substance or candidate composition, and obtaining a measured value of the occupancy of target bacteria; and

[0554] (6c) The step of comparing the measured value obtained in step 6b with the reference value to obtain the comparison result; and

[0555] (6d) Based on the comparison results obtained in step 6c, the step of evaluating the effect of the candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving disease or symptoms.

[0556] The aforementioned occupancy rate of target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B.

[0557] The description of Method 1 also applies to Method 6, unless otherwise specified. When applying this method, "Step 1a" is replaced with "Step 6b", "Step 1b" with "Step 6c", "Step 1c" with "Step 6d", "evaluate the intestinal barrier function of the subject" is replaced with "evaluate the improvement effect of the candidate substance or candidate composition on the intestinal barrier function, or the prevention or improvement effect on the disease or symptoms", and "accuracy of the evaluation of the intestinal barrier function of the subject" is replaced with "accuracy of the evaluation of the improvement effect of the candidate substance or candidate composition on the intestinal barrier function, or the prevention or improvement effect on the disease or symptoms".

[0558] In Method 6, "positive" means that the candidate substance or composition has an effect on improving intestinal barrier function or on preventing or improving disease or symptoms, while "negative" means that the candidate substance or composition does not have an effect on improving intestinal barrier function or on preventing or improving disease or symptoms. "Prevention" includes preventing, inhibiting, and delaying the onset of disease or symptoms. "Improvement" includes inhibiting the progression or worsening of disease or symptoms, delaying the progression or worsening of disease or symptoms, and alleviating, reducing, improving, and curing disease or symptoms.

[0559] The improvement effect on intestinal barrier function used for evaluation can be the improvement effect on the intestinal barrier function of the small intestine or the intestinal barrier function of the large intestine, with the improvement effect on the intestinal barrier function of the small intestine being preferred.

[0560] The preventive or ameliorative effect of the disease or symptom being evaluated can be the preventive or ameliorative effect of the disease or symptom caused by the decreased intestinal barrier function of the small intestine, or the preventive or ameliorative effect of the disease or symptom caused by the decreased intestinal barrier function of the large intestine, preferably the preventive or ameliorative effect of the disease or symptom caused by the decreased intestinal barrier function of the small intestine.

[0561] The explanation of diseases or symptoms caused by decreased intestinal barrier function is the same as in Method 2.

[0562] In the method of Method 6, candidate substances or candidate compositions that are evaluated as having an effect on improving intestinal barrier function or preventing or improving disease or symptoms can be screened as substances or compositions that have an effect on improving intestinal barrier function or preventing or improving disease or symptoms.

[0563] Step 6a is the step of administering the candidate substance or candidate composition to a subject whose intestinal barrier function has been reduced.

[0564] A decrease in the intestinal barrier function of an individual can be confirmed by well-known methods for detecting intestinal barrier function (e.g., lactulose-mannitol test) or by method 1.

[0565] Method 6 may also include, prior to step 6a, a step of evaluating the subject's intestinal barrier function using the method of Method 1 and confirming that the subject's intestinal barrier function has been reduced. When Method 6 includes this step, the types of bacteria present in step 1a of Method 1 may be the same as or different from the types of bacteria present in step 6b, but from the viewpoint of improving the accuracy of evaluating the effect of the candidate substance or candidate composition on improving intestinal barrier function or on the prevention or improvement of disease or symptoms, they are preferably the same.

[0566] Method 6 may also include a step of screening for subjects with reduced intestinal barrier function using the method of Method 1 before step 6a. When Method 6 includes this step, the types of bacteria whose occupancy is measured in step 1a of Method 1 may be the same as or different from the types of bacteria whose occupancy is measured in step 6b, but from the viewpoint of improving the accuracy of evaluating the effect of the candidate substance or candidate composition on improving intestinal barrier function or on the prevention or improvement of disease or symptoms, they are preferably the same.

[0567] The recipient of the candidate substance or candidate composition is not particularly limited as long as the intestinal barrier function is reduced. It can be a person with reduced intestinal barrier function or a model animal (excluding humans) with reduced intestinal barrier function, preferably a model animal (excluding humans) with reduced intestinal barrier function. Examples of model animals include vertebrates such as mammals, reptiles, birds, amphibians, and fish, but mammals and birds are preferred, and mammals are more preferred. Examples of mammals include primates (e.g., gorillas, chimpanzees, orangutans), rodents (e.g., mice, rats, hamsters, guinea pigs, rabbits), livestock (e.g., cattle, pigs, sheep, goats, horses), and pet animals (e.g., dogs, cats). Examples of birds include poultry (e.g., chickens, ducks, domestic ducks).

[0568] There are no particular limitations on the candidate substances or candidate combinations to be given to subjects with impaired intestinal barrier function. For example, they can be selected from high molecular weight compounds, low molecular weight compounds, cell cultures, cell extracts, antibodies, proteins, peptides, nucleic acids, sugars, inorganic salts, metal complexes, microorganisms (bacteria (probiotics), yeast, etc.), dietary fiber, indigestible sugars, indigestible proteins, fermented products (including fermented foods), prebiotics, and combinations of two or more of them.

[0569] Routes of administration for candidate substances or compositions may include, for example, oral and non-oral administration (e.g., intranasal, ophthalmic, ocular, transdermal, intrainhalation, rectal, urinary tract, subcutaneous, intramuscular, intravenous, etc.). Candidate substances or compositions may be formulated into administration forms suitable for the route of administration. Examples of administration methods include tablets and injections. During formulation, additives suitable for oral administration (e.g., excipients, disintegrants, lubricants, binders, etc.) or additives suitable for non-oral administration (e.g., diluents, solvents, etc.) may be used. The dosage and frequency of administration of the candidate substance or composition may be appropriately adjusted considering the route of administration, the age and weight of the subject. The number of administrations per day may be once or more than twice. The administration period may be one day or more than two days (e.g., more than one week, more than two weeks, more than three weeks, or more than four weeks).

[0570] Step 6b is a step of determining the occupancy of target bacteria in feces obtained from the subject after administration of the candidate substance or candidate composition, and obtaining a measured value of the occupancy of target bacteria.

[0571] Step 6b can be performed in the same manner as step 1a of method 1, except that it uses feces obtained from the subject after administering the candidate substance or candidate composition to the subject whose intestinal barrier function has been reduced.

[0572] The feces used in step 6b are obtained from the subject after administering the candidate substance or candidate composition to a subject with reduced intestinal barrier function. Feces can be obtained from the subject at multiple time points after administration of the candidate substance or candidate composition. The time point at which feces are obtained can be appropriately adjusted considering the administration method, the subject's age, weight, etc. In one embodiment, the time point at which feces are obtained is, for example, any time point between 0.5 hours and 24 hours after administration, or any time point between 0.5 hours and 12 hours after administration, or any time point between 1 hour and 10 hours after administration. Here, "after administration" can refer to a single administration or to two or more consecutive administrations. Two or more consecutive administrations could be, for example, the final administration after four weeks or more of consecutive administration, or the final administration after one day to four weeks of consecutive administration, or the final administration after three days to three weeks of consecutive administration, or the final administration after one week to two weeks of consecutive administration.

[0573] Step 6c is to compare the measured value obtained in step 6b with the benchmark value and obtain the comparison result.

[0574] Step 6c can be performed in the same way as step 1b of method 1, except that it uses the measured values ​​obtained in step 6b.

[0575] Step 6d is a step of evaluating the effect of candidate substances or candidate compositions on improving intestinal barrier function or on preventing or improving disease or symptoms, based on the comparison results obtained in step 6c.

[0576] Step 6d can be performed in the same manner as step 1c of method 1, except that it evaluates the effect of the candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving disease or symptoms.

[0577] Regarding the occupancy rates of each bacterium selected from groups A and B, the effectiveness of the candidate substance or candidate composition in improving intestinal barrier function or preventing or improving diseases or symptoms can be evaluated based on whether the comparison results obtained in step 6c meet the criteria shown in Table 2. Specifically, for the occupancy rates of each bacterium with a "high" criterion shown in Table 2, if the measured value obtained in step 6b is higher than the criterion value (i.e., the comparison results obtained in step 6c meet the criteria shown in Table 2), it can be evaluated as negative; if the measured value obtained in step 6b is lower than the criterion value (i.e., the comparison results obtained in step 6c do not meet the criteria shown in Table 2), it can be evaluated as positive. Similarly, for the occupancy rates with a "low" criterion shown in Table 2, if the measured value obtained in step 6b is lower than the criterion value (i.e., the comparison results obtained in step 6c meet the criteria shown in Table 2), it can be evaluated as negative; if the measured value obtained in step 6b is higher than the criterion value (i.e., the comparison results obtained in step 6c do not meet the criteria shown in Table 2), it can be evaluated as positive.

[0578] In step 6b, when determining the occupancy rate of one type of bacteria selected from groups A and B, if the comparison result of the occupancy rate of the one type of bacteria meets the criteria shown in Table 2, it can be evaluated as negative; if the comparison result of the occupancy rate of the one type of bacteria does not meet the criteria shown in Table 2, it can be evaluated as positive.

[0579] In step 6b, when determining the occupancy rate of two or more bacteria selected from groups A and B, a negative result can be evaluated if any comparison result of the occupancy rate of these two or more bacteria meets the criteria shown in Table 2, and a positive result can be evaluated if the comparison result of the occupancy rate of at least one of the two or more bacteria does not meet the criteria shown in Table 2. The more bacteria whose comparison results do not meet the criteria shown in Table 2, the higher the probability of a positive result. Therefore, in step 6b, by determining the occupancy rate of two or more bacteria selected from groups A and B (e.g., two, three, or four or more), the accuracy of evaluating the effect of the candidate substance or candidate composition on improving intestinal barrier function or on the prevention or improvement of disease or symptoms can be improved.

[0580] Method 7

[0581] Embodiment 7 of the present invention relates to a method for evaluating the effect of a candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by reduced intestinal barrier function. Hereinafter, the "preventive or improving effect on diseases or symptoms caused by reduced intestinal barrier function" will sometimes be referred to as the "preventive or improving effect on diseases or symptoms".

[0582] Method 7 includes the following steps:

[0583] (7a) The step of administering a candidate substance or candidate composition to a subject whose intestinal barrier function has been reduced;

[0584] (7b) The step of determining the occupancy of target bacteria in feces obtained from the subject after administration of the candidate substance or candidate composition, and obtaining a measured value of the occupancy of target bacteria; and

[0585] (7c) The step of comparing the measured value obtained in step 7b with the reference value to obtain the comparison result; and

[0586] (7d) Based on the comparison results obtained in step 7c, the step of evaluating the effect of the candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving disease or symptoms.

[0587] The aforementioned occupancy rate of target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B.

[0588] The explanation of method 6 also applies to method 7, unless otherwise specified. When applying, "step 6a" should be replaced with "step 7a", "step 6b" with "step 7b", "step 6c" with "step 7c", and "step 6d" with "step 7d".

[0589] Method 8

[0590] Method 8 of the present invention relates to a target bacterium for evaluating the intestinal barrier function of a subject, the target bacterium being a target bacterium derived from the subject's feces, the target bacterium comprising one or more bacteria selected from groups A and B.

[0591] The explanation of Method 1 also applies to Method 8, unless otherwise specified.

[0592] Method 9

[0593] Method 9 of the present invention relates to a target bacterium for diagnosing a disease or symptom caused by a decrease in intestinal barrier function in a subject, the target bacterium being a target bacterium derived from the subject's feces, the target bacterium comprising one or more bacteria selected from groups A and B.

[0594] The explanation of Method 2 also applies to Method 9, unless otherwise specified.

[0595] Method 10

[0596] Method 10 of the present invention relates to a target bacterium for evaluating the risk or likelihood of a subject developing a disease or symptom caused by a decrease in intestinal barrier function, wherein the target bacterium is a target bacterium derived from the subject's feces and comprises one or more bacteria selected from groups A and B.

[0597] The explanation of Method 4 also applies to Method 10, unless otherwise specified.

[0598] Method 11

[0599] Method 11 of the present invention relates to a target bacterium for obtaining data on diseases or symptoms caused by reduced intestinal barrier function in a subject for diagnosis, wherein the target bacterium is a target bacterium derived from the subject's feces and comprises one or more bacteria selected from groups A and B.

[0600] The explanation of method 5 also applies to method 11, unless otherwise specified.

[0601] Method 12

[0602] Method 12 of the present invention relates to a target bacterium for screening substances or compositions that have an effect on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by reduced intestinal barrier function. The target bacterium is a target bacterium in feces obtained from a subject with reduced intestinal barrier function after administering a candidate substance or candidate composition to the subject. The target bacterium comprises one or more bacteria selected from groups A and B.

[0603] The explanation of method 6 also applies to method 12, unless otherwise specified.

[0604] Method 13

[0605] Method 13 of the present invention relates to a target bacterium for evaluating the effect of a candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by reduced intestinal barrier function. The target bacterium is a target bacterium in feces obtained from a subject with reduced intestinal barrier function after administering the candidate substance or candidate composition to the subject. The target bacterium comprises one or more bacteria selected from groups A and B.

[0606] The explanation of method 7 also applies to method 13, unless otherwise specified.

[0607] Method 14

[0608] Method 14 of the present invention relates to the use of target bacteria in evaluating the intestinal barrier function of a subject, wherein the target bacteria are target bacteria derived from the feces of the subject, and the target bacteria comprise one or more bacteria selected from groups A and B.

[0609] The explanation of Method 1 also applies to Method 14, unless otherwise specified.

[0610] Method 15

[0611] Method 15 of the present invention relates to the use of target bacteria in a diagnostic subject for a disease or symptom caused by a decrease in intestinal barrier function, wherein the target bacteria are target bacteria derived from the subject’s feces, and the target bacteria comprise one or more bacteria selected from groups A and B.

[0612] The explanation of Method 2 also applies to Method 15, unless otherwise specified.

[0613] Method 16

[0614] The present invention relates to the use of target bacteria in evaluating the risk or likelihood of a subject developing a disease or symptom caused by a decrease in intestinal barrier function, wherein the target bacteria are target bacteria derived from the subject's feces, and the target bacteria comprise one or more bacteria selected from groups A and B.

[0615] The explanation of method 4 also applies to method 16, unless otherwise specified.

[0616] Method 17

[0617] Method 17 of the present invention relates to the use of target bacteria in obtaining data for diagnosing diseases or symptoms caused by reduced intestinal barrier function in the subject, wherein the target bacteria are target bacteria derived from the subject's feces, and the target bacteria comprise one or more bacteria selected from groups A and B.

[0618] The explanation of method 5 also applies to method 17, unless otherwise specified.

[0619] Method 18

[0620] Method 18 of the present invention relates to the use of target bacteria in screening for substances or compositions that have an effect on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by reduced intestinal barrier function, wherein the target bacteria are target bacteria in feces obtained from a subject with reduced intestinal barrier function after administering the candidate substance or candidate composition to the subject, and the target bacteria comprise one or more bacteria selected from groups A and B.

[0621] The explanation of Method 6 also applies to Method 18, unless otherwise specified.

[0622] Method 19

[0623] Method 19 of the present invention relates to the use of target bacteria in evaluating the effect of a candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by reduced intestinal barrier function, wherein the target bacteria are target bacteria in feces obtained from a subject with reduced intestinal barrier function after administration of the candidate substance or candidate composition to the subject, and the target bacteria comprise one or more bacteria selected from groups A and B.

[0624] The explanation of method 7 also applies to method 19, unless otherwise specified.

[0625] Method 20

[0626] The present invention, in aspect 20, relates to a kit for evaluating the intestinal barrier function of a subject, wherein the kit comprises one or more reagents for determining the occupancy of target bacteria in feces derived from the subject, the occupancy of target bacteria comprising the occupancy of one or more bacteria selected from groups A and B.

[0627] The explanation of Method 1 also applies to Method 20, unless otherwise specified.

[0628] The kit may contain one reagent or two or more reagents.

[0629] The type of reagent can be appropriately selected according to the method for determining the occupancy of target bacteria. When the occupancy of target bacteria is determined by microbial community analysis using 16S rRNA amplicon sequencing, the kit contains one or more reagents for microbial community analysis using 16S rRNA amplicon sequencing. As reagents for microbial community analysis using 16S rRNA amplicon sequencing, examples include reagents for preparing samples from feces derived from the subject (e.g., solvents for suspending feces, etc.), reagents for extracting bacterial-derived genomic DNA from samples (e.g., reagents for dissolving bacteria, reagents for denaturing or decomposing impurities such as proteins, reagents for separating DNA from impurities, reagents for precipitating DNA, etc.), reagents for amplifying a predetermined region of the 16S rRNA gene (e.g., a region containing the V3-V4 region) by PCR (e.g., primers, polymerases, dNTPs, buffers, etc.), reagents for purifying PCR amplification products, reagents for indexing PCR amplification products by Index PCR (e.g., indexed primers, polymerases, dNTPs, buffers, etc.), and devices for performing sequence analysis of indexed PCR amplification products (e.g., next-generation sequencers, etc.).

[0630] Method 21

[0631] The present invention relates to a kit for diagnosing diseases or symptoms caused by reduced intestinal barrier function in a subject, wherein the kit comprises one or more reagents for measuring the occupancy of target bacteria in feces derived from the subject, the occupancy of target bacteria comprising the occupancy of one or more bacteria selected from groups A and B.

[0632] The explanation of Method 2 also applies to Method 21, unless otherwise specified.

[0633] The instructions for the reagents are the same as in Method 20.

[0634] Method 22

[0635] The present invention, embodiment 22, relates to a kit for evaluating the risk of a subject developing a disease or symptom caused by a decrease in intestinal barrier function, or the likelihood of suffering from such a disease or symptom, wherein the kit comprises one or more reagents for determining the occupancy of target bacteria in feces derived from the subject, the occupancy of target bacteria comprising the occupancy of one or more bacteria selected from groups A and B.

[0636] The explanation of method 4 also applies to method 22, unless otherwise specified.

[0637] The instructions for the reagents are the same as in Method 20.

[0638] Method 23

[0639] The present invention, in aspect 23, relates to a kit for obtaining data on a disease or symptom caused by a reduced intestinal barrier function in a subject for diagnosis, wherein the kit comprises one or more reagents for determining the occupancy of target bacteria in feces derived from the subject, the occupancy of target bacteria comprising the occupancy of one or more bacteria selected from groups A and B.

[0640] The explanation of method 5 also applies to method 23, unless otherwise specified.

[0641] The instructions for the reagents are the same as in Method 20.

[0642] Method 24

[0643] The present invention, embodiment 24, relates to a kit for screening substances or compositions that have an effect on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by reduced intestinal barrier function, wherein the kit comprises one or more reagents for determining the occupancy of target bacteria in feces obtained from a subject with reduced intestinal barrier function after administration of the candidate substance or candidate composition to the subject, the occupancy of target bacteria comprising the occupancy of one or more bacteria selected from groups A and B.

[0644] The explanation of method 6 also applies to method 24, unless otherwise specified.

[0645] The instructions for the reagents are the same as in Method 20.

[0646] Method 25

[0647] The present invention relates to a kit for evaluating the effect of a candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by reduced intestinal barrier function, wherein the kit comprises one or more reagents for determining the occupancy of target bacteria in feces obtained from a subject with reduced intestinal barrier function after administration of the candidate substance or candidate composition to the subject, wherein the occupancy of target bacteria includes the occupancy of one or more bacteria selected from groups A and B.

[0648] The explanation of method 7 also applies to method 25, unless otherwise specified.

[0649] The instructions for the reagents are the same as in Method 20.

[0650] Method 26

[0651] Method 26 of the present invention relates to a method for manufacturing a food or pharmaceutical composition, comprising the steps of: mixing a substance or composition selected by the method of method 6 with one or more ingredients constituting the food or pharmaceutical composition.

[0652] There are no particular limitations on one or more components that can be mixed with the substances or compositions screened by method 6, as long as they are permitted to be components of the food or pharmaceutical composition. They can be appropriately selected according to the type of food or pharmaceutical composition that is to be manufactured.

[0653] Method 26 may include the step of screening substances or compositions by method 6.

[0654] Method 27

[0655] Method 27 of the present invention relates to a computer program for causing a computer to perform a method for evaluating the intestinal barrier function of a subject.

[0656] Method 27, which enables a computer to execute a computer program, includes the following steps:

[0657] (S101) Step of obtaining a measurement of the occupancy of target bacteria in feces derived from the object;

[0658] (S102) The step of comparing the measured value obtained in step S101 with the reference value to obtain the comparison result; and

[0659] (S103) The step of evaluating the intestinal barrier function of the subject based on the comparison results obtained in step S102.

[0660] The aforementioned occupancy rate of target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B.

[0661] The explanation of Method 1 also applies to Method 27, unless otherwise specified.

[0662] When executed by a computer, the computer program of method 27 causes the computer to perform a method for evaluating the intestinal barrier function of a subject. The computer program of method 27 contains instructions for causing the computer to perform a method for evaluating the intestinal barrier function of a subject.

[0663] Hereinafter, one embodiment of mode 27 will be described with reference to the accompanying drawings. Figure 1 This is a schematic diagram of the apparatus used in this embodiment. Figure 2 This is a block diagram illustrating the hardware configuration of the device according to this embodiment. Figures 3-5 This is a flowchart illustrating the processing steps of the apparatus in this embodiment.

[0664] like Figure 1 As shown, the device 10 includes a computer system 30. (As indicated...) Figure 1 As shown, the device 10 may also include a measuring device 20 connected to the computer system 30. The computer system 30 may be a device independent of the measuring device 20, or it may be a device with the measuring device 20 built into it. The device 10 may also be a device in which the measuring device 20 and the computer system 30 are integrated.

[0665] The measuring device 20 performs a measurement of the occupancy rate of target bacteria in feces derived from the subject, obtains the measured value of the target bacteria occupancy rate or the measured value required to calculate the target bacteria occupancy rate, and sends it to the computer system 30. The descriptions of the feces derived from the subject, the measurement of the target bacteria occupancy rate, and the measured value of the target bacteria occupancy rate are the same as in Method 1.

[0666] The assay device 20 is, for example, an automated assay device that performs the determination of the occupancy of target bacteria in feces derived from the subject (preferably a determination performed by microbial community analysis using 16S rRNA amplicon sequencing), obtaining a value for the occupancy of target bacteria or a value required to calculate the occupancy of target bacteria. When the assay device 20 performs a determination using microbial community analysis with 16S rRNA amplicon sequencing, the assay device 20 only needs to be able to perform sequence analysis of indexed PCR amplification products (libraries) and subsequent processing. The processing prior to sequence analysis of the indexed PCR amplification products, such as the collection of feces from the subject (i.e., collecting feces from the subject's excrement to be used as the test sample), sample preparation from the subject's feces, extraction of bacterial genomic DNA from the sample, amplification of a predetermined region of the 16S rRNA gene (e.g., the region containing the V3-V4 region) by PCR, purification of the PCR amplification products, indexing the PCR amplification products by IndexPCR, and setting the indexed PCR amplification products (library) on a sequencer (e.g., a next-generation sequencer), can be performed by a human or by the assay device 20. In one embodiment, the processing prior to sequence analysis of the indexed PCR amplification products is performed by a human.

[0667] like Figure 2 As shown, the computer system 30 includes a computer main body 300, an input unit 301, and a display unit 302 for displaying information about the object to be examined, evaluation results, etc.

[0668] The processor of computer system 30 executes a computer program installed on hard disk 313 for evaluating the intestinal barrier function of the subject, based on the measured value of the occupancy of target bacteria.

[0669] like Figure 2As shown, the computer main body 300 includes a central processing unit (CPU) 310, a read-only memory (ROM) 311, a random access memory (RAM) 312, a hard disk 313, an input / output interface 314, a reading device 315, a communication interface 316, and an image output interface 317. The CPU 310, ROM 311, RAM 312, hard disk 313, input / output interface 314, reading device 315, communication interface 316, and image output interface 317 are connected via a bus 318 for data communication. The measuring device 20 is connected to the computer system 30 via the communication interface 316 for data communication.

[0670] CPU 310 can execute programs stored in ROM 311 or hard disk 313, as well as programs loaded into RAM 312. By executing programs, CPU 330 enables device 10 to function as a device for evaluating the intestinal barrier function of a subject.

[0671] ROM311 is composed of mask ROM, PROM, EPROM, EEPROM, etc. ROM311 records the computer program executed by CPU310 and the data used to execute the computer program.

[0672] RAM312 is composed of SRAM, DRAM, etc. RAM312 is used to read programs recorded in ROM311 and hard disk 313. In addition, RAM312 is used as the working area of ​​CPU310 when executing programs.

[0673] The hard disk 313 contains computer programs such as the operating system and application programs that are executed by the CPU 310, as well as data used to execute those computer programs.

[0674] The reading device 315 comprises a floppy disk drive, a CD-ROM drive, a DVD-ROM drive, a USB port, an SD card reader, a CF card reader, a Memory Stick reader, a solid-state drive, etc. The reading device 315 can read programs or data recorded on the portable recording medium 400.

[0675] The input / output interface 314 comprises serial interfaces such as USB, IEEE 1394, and RS-232C; parallel interfaces such as SCSI, IDE, and IEEE 1284; and analog interfaces such as D / A converters and A / D converters. Input units 301, such as a keyboard and mouse, are connected to the input / output interface 314. The operator can input various commands to the computer main unit 300 through the input units 301.

[0676] The communication interface 316 is, for example, an Ethernet (registered trademark) interface. The computer body 300 can send data to printers, computers of the target or service providers, smartphone terminals of the target, etc., through the communication interface 316.

[0677] The image output interface 317 is connected to a display unit 302, which may be composed of an LCD, CRT, or the like. Thus, the display unit 302 can output image signals based on image data provided by the CPU 310. The display unit 302 displays an image (screen) based on the input image signal. The display unit 302 can display the raw data itself, or it can convert the raw data into illustrations, diagrams, etc., for easier understanding by the viewer.

[0678] The following is for reference Figures 3-5 The processing steps using the device 10 will be explained.

[0679] like Figure 3 As shown, in step S101, CPU 310 acquires a measurement value of the occupancy rate of target bacteria in feces derived from the object. The descriptions of the feces derived from the object, the measurement of the occupancy rate of target bacteria, and the measurement value of the occupancy rate of target bacteria are the same as in Method 1.

[0680] In one embodiment, the measuring device 20 performs a determination of the occupancy rate of target bacteria in feces derived from the subject (preferably a determination performed using microbial community analysis via 16S rRNA amplicon sequencing), obtains the target bacteria occupancy rate value, and sends it to the computer system 30. The description of the feces derived from the subject, the determination of the target bacteria occupancy rate, and the target bacteria occupancy rate value is the same as in Method 1. The CPU 310 stores the target bacteria occupancy rate value sent from the measuring device 20 in the hard disk 313. In step S301, the CPU 310 obtains the target bacteria occupancy rate value stored in the hard disk 313.

[0681] In another embodiment, the measuring device 20 performs a measurement of the occupancy rate of target bacteria in the feces of the subject (preferably a measurement performed by microbial community analysis using 16S rRNA amplicon sequencing), obtains the measurement value required to calculate the occupancy rate of target bacteria, and sends it to the computer system 30. The description of the feces from the subject, the measurement of the occupancy rate of target bacteria, and the measurement value of the occupancy rate of target bacteria are the same as in Method 1. Based on the measurement value sent from the measuring device 20, the CPU 310 calculates the measurement value of the occupancy rate of target bacteria and stores it in the hard disk 313. In step S301, the CPU 310 obtains the measurement value of the occupancy rate of target bacteria stored in the hard disk 313.

[0682] In another embodiment, the operator inputs from the input unit 301 a target bacteria occupancy value obtained in advance through the determination of the occupancy rate of target bacteria in the feces of the subject (preferably a determination performed by microbial community analysis using 16S rRNA amplicon sequencing). The descriptions of the feces from the subject, the determination of the target bacteria occupancy rate, and the target bacteria occupancy rate value are the same as in Method 1. The CPU 310 stores the input target bacteria occupancy rate value in the hard disk 313. In step S301, the CPU 310 acquires the target bacteria occupancy rate value stored in the hard disk 313.

[0683] In another embodiment, the operator inputs the measurement value required to calculate the occupancy rate of target bacteria from the input unit 301. This measurement value is pre-obtained by measuring the occupancy rate of target bacteria in the feces of the subject (preferably a measurement performed using microbial community analysis via 16S rRNA amplicon sequencing). The descriptions of the feces from the subject, the measurement of the target bacteria occupancy rate, and the measurement value of the target bacteria occupancy rate are the same as in Method 1. The CPU 310 calculates the measurement value of the target bacteria occupancy rate based on the input measurement value and stores it in the hard disk 313. In step S301, the CPU 310 acquires the measurement value of the target bacteria occupancy rate stored in the hard disk 313.

[0684] like Figure 3 As shown, in step S102, the CPU 310 compares the measured value of the target bacteria occupancy rate with the reference value stored in the hard disk 313 to obtain a comparison result. The descriptions of the reference value, comparison, and comparison result are the same as in Method 1. The CPU 310 stores the obtained comparison result in the hard disk 313. The CPU 310 may also output the obtained comparison result, displaying it on the display unit 302, printing it, or transmitting it to another computer or application (such as a smartphone). Alternatively, when outputting the comparison result, the CPU 310 may also use the measured value of the target bacteria occupancy rate, the reference value, etc., as reference information, displaying them on the display unit 302, printing them, or transmitting them to another computer or application (such as a smartphone). Furthermore, the CPU 310 may also display lifestyle suggestions for improving intestinal barrier function, recommended foods (including functional foods), etc., along with the comparison result on the display unit 302, printing them, or transmitting them to another computer or application (such as a smartphone). This allows for the provision of indicators to doctors, nurses, nursing staff, testing personnel, testing institutions, service providers, and the individuals themselves for evaluating the intestinal barrier function.

[0685] like Figure 3As shown, in step S103, CPU 310 evaluates the intestinal barrier function of the subject based on the comparison results obtained in step S102. The evaluation process is the same as in Method 1. CPU 310 stores the evaluation results in hard disk 313. CPU 310 may also output the evaluation results and display them on display unit 302, print them, or transmit them to other computers or applications (such as smartphones). Furthermore, when outputting the evaluation results, CPU 310 may also display the measured value of the target bacteria occupancy rate, benchmark value, etc., as reference information on display unit 302, print them, or transmit them to a computer or application (such as a smartphone). Additionally, CPU 310 may display lifestyle suggestions for improving intestinal barrier function, recommended foods (including functional foods, etc.), etc., along with the evaluation results on display unit 302, print them, or transmit them to other computers or applications (such as smartphones). Thus, indicators for evaluating the intestinal barrier function of the subject can be provided to doctors, nurses, nursing staff, testing personnel, testing institutions, service providers, and the subject themselves.

[0686] Regarding the occupancy rates of each bacterium selected from groups A and B, CPU310 evaluates the intestinal barrier function of the subjects based on whether the comparison results obtained in step S102 meet the benchmarks shown in Table 2. Specifically, regarding the occupancy rates of each bacterium whose benchmarks are "high" as shown in Table 2, such as... Figure 4 As shown, the CPU 310 determines whether the measured value obtained in step 101 is higher than the benchmark value (i.e., whether the comparison result meets the benchmark shown in Table 2) (step S103a). If the measured value obtained in step 101 is higher than the benchmark value (i.e., the comparison result meets the benchmark shown in Table 2), it is evaluated as positive (step S103b). If the measured value obtained in step 101 is lower than the benchmark value (i.e., the comparison result does not meet the benchmark shown in Table 2), it is evaluated as negative (step S103c). Furthermore, regarding the occupancy rate of each bacteria with a benchmark of "low" shown in Table 2, as follows... Figure 5 As shown, the CPU310 determines whether the measured value obtained in step 101 is lower than the benchmark value (i.e., whether the comparison result meets the benchmark shown in Table 2) (step S103d). If the measured value obtained in step 101 is lower than the benchmark value (i.e., the comparison result meets the benchmark shown in Table 2), it is evaluated as positive (step S103e). If the measured value obtained in step 101 is higher than the benchmark value (i.e., the comparison result does not meet the benchmark shown in Table 2), it is evaluated as negative (step S103f).

[0687] Method 28

[0688] Embodiment 28 of the present invention relates to a computer program for causing a computer to perform a method for diagnosing a disease or symptom caused by reduced intestinal barrier function in a subject. Hereinafter, "disease or symptom caused by reduced intestinal barrier function" will sometimes be referred to as "disease or symptom".

[0689] Method 28, which enables a computer to execute a computer program, includes the following steps:

[0690] (S201) Step of obtaining a measurement of the occupancy of target bacteria in feces derived from the object;

[0691] (S202) The step of comparing the measured value obtained in step S201 with the reference value to obtain the comparison result;

[0692] (S203) Based on the comparison results obtained in step S202, the steps for diagnosing the disease or symptoms caused by decreased intestinal barrier function in the subject.

[0693] The aforementioned occupancy rate of target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B.

[0694] The explanation of Method 2 also applies to Method 28, unless otherwise specified. Similarly, the explanation of Method 27 also applies to Method 28, unless otherwise specified. In application, "Step S101" is replaced with "Step S201", "Step S102" with "Step S202", "Step S103" with "Step S203", "evaluate the intestinal barrier function of the subject" with "diagnose the disease or symptoms of the subject", "accuracy of the evaluation of the intestinal barrier function of the subject" with "accuracy of the diagnosis of the disease or symptoms of the subject", "positive evaluation" with "positive diagnosis", and "negative evaluation" with "negative diagnosis".

[0695] Method 29

[0696] Embodiment 29 of the present invention relates to a computer program for causing a computer to execute a method for evaluating the risk of a subject developing a disease or symptom caused by reduced intestinal barrier function, or the likelihood of a subject suffering from such a disease or symptom. Hereinafter, the "risk of a subject developing a disease or symptom caused by reduced intestinal barrier function" is sometimes referred to as "risk of disease", and the "likelihood of a subject suffering from a disease or symptom caused by reduced intestinal barrier function" is sometimes referred to as "likelihood of disease".

[0697] Method 29, which enables a computer to execute a computer program, includes the following steps:

[0698] (S301) A step of obtaining a measurement of the occupancy of target bacteria in feces derived from the object;

[0699] (S302) The step of comparing the measured value obtained in step S301 with the reference value to obtain the comparison result; and

[0700] (S303) The step of evaluating the risk or the likelihood based on the comparison results obtained in step S302.

[0701] The aforementioned occupancy rate of target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B.

[0702] The explanation of Method 4 also applies to Method 29, unless otherwise specified. Similarly, the explanation of Method 27 also applies to Method 29, unless otherwise specified. When applying this method, "Step S101" is replaced with "Step S301", "Step S102" with "Step S302", "Step S103" with "Step S303", "evaluating the intestinal barrier function of the subject" is replaced with "evaluating the risk or likelihood of developing the disease", and "accuracy of the evaluation of the subject's intestinal barrier function" is replaced with "accuracy of the evaluation of the risk or likelihood of developing the disease".

[0703] Method 30

[0704] Embodiment 30 of the present invention relates to a computer program for causing a computer to execute a method for acquiring data on a disease or symptom caused by reduced intestinal barrier function in a diagnostic subject. Hereinafter, "disease or symptom caused by reduced intestinal barrier function" will sometimes be referred to as "disease or symptom".

[0705] Method 30, which enables a computer to execute a computer program, includes the following steps:

[0706] (S401) The step of obtaining a measurement value of the occupancy of target bacteria in feces derived from the object; and

[0707] (S402) The step of comparing the measured value obtained in step S401 with the baseline value and obtaining the comparison result as data for diagnosing the disease or symptoms caused by the decreased intestinal barrier function of the subject.

[0708] The aforementioned occupancy rate of target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B.

[0709] The explanation of Method 5 also applies to Method 30, unless otherwise specified. Similarly, the explanation of Method 27 also applies to Method 30, unless otherwise specified. In application, "Step S101" is replaced with "Step S401", "Step S102" is replaced with "Step S402", "evaluate the intestinal barrier function of the subject" is replaced with "obtain data for diagnosing the subject's disease or symptoms", and "accuracy of the evaluation of the subject's intestinal barrier function" is replaced with "accuracy of the diagnosis of the subject's disease or symptoms".

[0710] Method 31

[0711] Embodiment 31 of the present invention relates to a computer program for causing a computer to execute a method for screening substances or compositions having an effect on improving intestinal barrier function or having a preventive or improving effect on diseases or symptoms caused by reduced intestinal barrier function. Hereinafter, "the preventive or improving effect on diseases or symptoms caused by reduced intestinal barrier function" is sometimes referred to as "the preventive or improving effect on diseases or symptoms".

[0712] Method 31, which enables a computer to execute a computer program, includes the following steps:

[0713] (S501) A step of obtaining a measurement of the occupancy of target bacteria in feces obtained from subjects with reduced intestinal barrier function after administration of a candidate substance or candidate composition to the subjects.

[0714] (S502) The step of comparing the measured value obtained in step S501 with the reference value to obtain the comparison result; and

[0715] (S503) Based on the comparison results obtained in step S502, the step of evaluating the effect of the candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving disease or symptoms.

[0716] The aforementioned occupancy rate of target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B.

[0717] The explanation of Method 6 also applies to Method 31, unless otherwise specified. Similarly, the explanation of Method 27 also applies to Method 31, unless otherwise specified. In application, "Step S101" is replaced with "Step S501", "Step S102" with "Step S502", "Step S103" with "Step S503", "evaluating the intestinal barrier function of the subject" is replaced with "evaluating the improvement effect of the candidate substance or candidate composition on the intestinal barrier function, or the prevention or improvement effect on the disease or symptoms", and "accuracy of the evaluation of the intestinal barrier function of the subject" is replaced with "accuracy of the evaluation of the improvement effect of the candidate substance or candidate composition on the intestinal barrier function, or the prevention or improvement effect on the disease or symptoms".

[0718] Method 32

[0719] Embodiment 32 of the present invention relates to a computer program for causing a computer to execute a method for evaluating the effect of a candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by reduced intestinal barrier function. Hereinafter, the "prevention or improvement effect on diseases or symptoms caused by reduced intestinal barrier function" is sometimes referred to as the "prevention or improvement effect on diseases or symptoms".

[0720] Method 32, which enables a computer to execute a computer program, includes the following steps:

[0721] (S601) A step of obtaining a measurement of the occupancy of target bacteria in feces obtained from subjects with reduced intestinal barrier function after administration of a candidate substance or candidate composition to the subjects.

[0722] (S602) The step of comparing the measured value obtained in step S601 with the reference value to obtain the comparison result; and

[0723] (S603) Based on the comparison results obtained in step S602, the step of evaluating the effect of the candidate substance or the aforementioned candidate composition on improving intestinal barrier function or on preventing or improving disease or symptoms.

[0724] The aforementioned occupancy rate of target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B.

[0725] The explanation of Method 7 also applies to Method 32, unless otherwise specified. Similarly, the explanation of Method 27 also applies to Method 32, unless otherwise specified. In application, "Step S101" is replaced with "Step S601", "Step S102" with "Step S602", "Step S103" with "Step S603", "evaluating the intestinal barrier function of the subject" is replaced with "evaluating the improvement effect of the candidate substance or candidate composition on the intestinal barrier function, or the prevention or improvement effect on the disease or symptoms", and "accuracy of the evaluation of the intestinal barrier function of the subject" is replaced with "accuracy of the evaluation of the improvement effect of the candidate substance or candidate composition on the intestinal barrier function, or the prevention or improvement effect on the disease or symptoms".

[0726] Method 33

[0727] The present invention relates to a computer-readable recording medium having a computer program as described in any one of embodiments 27 to 32.

[0728] Non-transitory recording media that contain programs and are readable by a computer include, for example, ROM, floppy disk (registered trademark), hard disk, optical disk, optical disc, CD-ROM, magnetic tape, non-volatile memory card, and other non-transitory recording media.

[0729] Example

[0730] The present invention will now be described based on embodiments.

[0731] The lactulose-mannitol test, a well-known method for assessing intestinal barrier function, was performed on 108 subjects. The lactulose-mannitol test was conducted as follows: Subjects were allowed to eat before 9 PM the day before the test, and were permitted to drink water until bedtime. On the day of the test, a glucose tolerance test was performed without allowing subjects to eat or drink. Subjects ingested a glucose solution (10g lactulose, 5g mannitol, and 20g sucrose dissolved in 100mL of water). At 2–3 hours, 3–4 hours, 4–5 hours, and 5–6 hours after glucose ingestion, subjects ingested at least 150mL of water. After water ingestion, urine samples were collected from 0–6 hours after ingestion, and the amounts of lactulose and mannitol in the urine were determined using liquid chromatography-tandem mass spectrometry (LC-MS / MS). The LC-MS / MS was performed as follows.

[0732] The assay sample used in LC-MS / MS was prepared from urine as follows: 400 μL of cooled acetonitrile was added to 50 μL of urine and stirred thoroughly. The mixture was then incubated on ice for 1 hour. The resulting mixture was further stirred, and 800 μL of 75% (w / w) acetonitrile was added and stirred thoroughly. The mixture was then filtered through a Millex FH 0.45 μm filter and used as the assay sample. No internal standard was used.

[0733] LC-MS / MS determinations and subsequent data analysis were performed using a QTRAP 4500 LC-MS / MS system (Thermo Fisher Scientific). Agilent AdvanceBio MS SpentMedium 120Å 2.1x100mm column (Agilent Technologies) was used as the column. The LC-MS / MS determination conditions are described below.

[0734] Mobile phase A: 10 mM ammonium formate (pH 10.5)

[0735] Mobile phase B: Acetonitrile (90%) / 100mM ammonium formate (pH 10.5) (10%)

[0736] Mobile phase conditions: 0-15 minutes B 97%-89%, 15-18 minutes B 89%

[0737] Flow rate: 0.4 mL / min

[0738] Injection volume: 5 μL

[0739] Temperature: 35℃

[0740] Ion spray voltage: -4500V

[0741] After determining the amount of lactulose and mannitol in urine using LC-MS / MS (hereinafter referred to as "excretion"), their respective excretion rates (%) are calculated based on the following formula.

[0742] Excretion rate = Excretion volume (μmol / mL) × Molecular weight (μg / μmol) × Urine volume (mL) / Sugar intake (μg) × 100

[0743] After calculating the excretion rate (%), the lactulose / mannitol ratio is calculated based on the following formula.

[0744] Lactulose / Mannitol ratio = Lactulose excretion rate / Mannitol excretion rate

[0745] Twenty-seven subjects with a lactulose / mannitol ratio of 0.025 or higher were designated as the positive group, and 27 subjects with a lactulose / mannitol ratio of 0.015 or lower were designated as the negative group.

[0746] Feces and serum were obtained from the negative and positive groups and used in the following comparative examples and embodiments.

[0747] Serum was obtained as follows: On the day of the lactulose-mannitol test, 20 mL of blood was collected from the subject, a portion of serum was separated, and stored in an ultra-low temperature freezer (-80°C) until use.

[0748] Fecal samples were obtained as follows: Two days prior to the lactulose-mannitol test, at any time on the day of the test, subjects were instructed to collect approximately 10g of freshly excreted feces into a collection container (Faeces tube, with blade, screwcap (Sarstedt)) and store it in a freezer until submission. On the day of the test, to maintain the frozen state, the collection container containing the feces was placed in an insulated container and carried and submitted. After submission, it was stored in an ultra-low temperature freezer (-80°C) until use.

[0749] Comparative Example 1

[0750] For each serum sample obtained from the negative and positive groups, the concentration of conjugated protein in the serum was determined by ELISA. The ELISA method was performed using the Zonuline ELISA kit (Immundiagnostik AG, Germany) according to the manufacturer's protocol.

[0751] Using the serum ligin concentrations in the negative and positive groups, receiver operating characteristic (ROC) curves were constructed. The area under the curve (AUC) and cutoff value were then calculated from the ROC curves. The ROC curves, AUC, and cutoff value were obtained using the statistical analysis software "EXCEL Statistics" (manufactured by Social Information Services Co., Ltd.). The ROC curve is a curve plotted with sensitivity on the vertical axis and (1-specificity) on the horizontal axis when plotting the serum ligin concentrations in the negative and positive groups. The AUC is the area under the ROC curve. The point on the ROC curve that is closest to the upper left corner of the ROC curve was used as the cutoff value. The results are shown in Table 5.

[0752] [Table 5]

[0753]

[0754] Example 1

[0755] (1) Analysis of fecal microbiota

[0756] Microbial communities in feces obtained from both the negative and positive groups were analyzed using 16S rRNA amplicon sequencing. The microbial community analysis using 16S rRNA amplicon sequencing was performed as follows.

[0757] 20 mg of fecal matter was suspended in ultrapure water and disrupted using Fastprep (MP-Biomedicals) (physical disruption using 1 mm diameter zirconia beads). Bacterial genomic DNA was extracted using the QIAamp Fast DNA Stool Mini kit (QIAGEN). Approximately 460 bp of the variable regions V3 and V4 of the 16S rRNA gene, containing the gene, was amplified by PCR. The PCR products were purified using AMPure XP beads (Beckman Coulter). After purification, index PCR was performed on the PCR products using primers containing the inherent base sequence (index) of each sample to add a sample identification index. The indexed PCR products (library) were purified using AMPure XP beads (Beckman Coulter) and dissolved in 10 mM Tris-HCl (pH 8.0). Quantification of the library was performed using the Quant-iT ds DNA Assay kit (Invitrogen). The library diluted to 4 nM and 4 nM PhiX control DNA (manufactured by Illumina Corporation) were denatured using 0.2N NaOH. Sequence analysis of the denatured library was performed using the next-generation sequencer MiSeq (manufactured by Illumina Corporation) to obtain 16S rRNA gene sequence data. The sequence data output from MiSeq were processed using QIIME2 (version 2020.8) and Silva (version 138) as reference databases to obtain the constituent bacteria and their occupancy at each taxonomic level.

[0758] The occupancy rate of each bacterium was calculated as follows: Amplicon Sequence Variant (ASV) analysis was performed, and the occupancy rate was calculated based on the number of reads belonging to each ASV. Then, the occupancy rate of bacteria belonging to a particular family was calculated using the formula: (Number of all bacteria belonging to that family in feces) / (Total number of bacteria from all families in feces). Furthermore, the occupancy rate of bacteria belonging to a particular genus was calculated using the formula: (Number of all bacteria belonging to that genus in feces) / (Total number of bacteria from all genera in feces).

[0759] Receiver Operating Characteristic (ROC) curves were constructed using the prevalence of bacteria from each family or genera in feces. The area under the curve (AUC) and cutoff value were then calculated from the ROC curves. The ROC curves, AUC, and cutoff values ​​were obtained using the statistical analysis software "EXCEL Statistics" (manufactured by Social Information Services Co., Ltd.). The ROC curve plots the prevalence of bacteria from each family or genera in feces, with sensitivity on the ordinate and (1-specificity) on the lateral. The AUC is the area under the ROC curve. The point on the ROC curve closest to the top-left corner was used as the cutoff value. The results are shown in Tables 6 and 7. A value closer to 1 indicates a higher accuracy in assessing intestinal barrier function. An AUC value less than 0.60 indicates low accuracy; an AUC value greater than 0.60 and less than 0.64 indicates slightly low accuracy; an AUC value greater than 0.64 and less than 0.68 indicates slightly high accuracy; and an AUC value greater than 0.68 indicates high accuracy.

[0760] Table 6 shows the percentage of bacteria from families with an AUC of 0.64 or higher in the comprehensive bacterial survey, and Table 7 shows the percentage of bacteria from genera with an AUC of 0.64 or higher in the comprehensive bacterial survey.

[0761] Regarding the occupancy rates recorded in Tables 6 and 7, the trends in the positive groups were clarified based on ROC curves. The results are shown in Table 8. In Table 8, "increase" indicates an increasing trend in the positive groups, and "decrease" indicates a decreasing trend in the positive groups. It should be noted that in the ROC curves related to the occupancy rates with an increasing trend in the positive groups, when the cutoff value = 0, the sensitivity (i.e., true positive rate) = 1 and (1-specificity) (i.e., false positive rate) = 1; in the ROC curves related to the occupancy rates with a decreasing trend in the positive groups, when the cutoff value = -1, the sensitivity (i.e., true positive rate) = 0 and (1-specificity) (i.e., false positive rate) = 0.

[0762] The results shown in Tables 6 and 7 indicate that, when the sample is feces, the intestinal barrier function can be evaluated with high accuracy by using the prevalence of one or more bacteria selected from groups A and B as an indicator. Since the closer the AUC is to 1, the higher the accuracy of the intestinal barrier function evaluation, the prevalence of Ruminococcus, Peptostreptococcus, Brugia, Peptostreptococcus spp., and Brugia allergenia spp. in assessing intestinal barrier function is particularly accurate.

[0763] [Table 6]

[0764]

[0765] [Table 7]

[0766]

[0767] [Table 8]

[0768]

[0769] (2) Combination variables (statistical analysis values)

[0770] Regarding the prevalence of Ruminococci, Peptostreptococci, Institute of Physicochemical Biology, and *Rombocytozoon* spp. (Peptostreptococci), combined variables were calculated based on the following formula. ROC curves were then constructed based on these combined variables, and the AUC and cutoff values ​​were determined from the ROC curves. The results are shown in Table 9. The coefficients and constants were obtained through multinomial logistic regression analysis. The coefficients and constants are shown in Table 1. In Table 9, "R1" represents the prevalence of Ruminococci, "R2" represents the prevalence of Peptostreptococci, "R3" represents the prevalence of *Rombocytozoon* spp. (Peptostreptococci), and "R4" represents the prevalence of Institute of Physicochemical Biology.

[0771] Statistical analysis value = (coefficient 1 × (first-ranked market share)) + (coefficient 2 × (second-ranked market share)) + ... + (coefficient n × (nth-ranked market share)) + constant term

[0772] As shown in Table 9, it was determined that combining the proportions of two or more species selected from Ruminococcal bacteria, Peptostreptococcal bacteria, physicochemical bacteria, and Peptostreptococcal bacteria can increase the AUC of the ROC curve, thus improving the accuracy of the evaluation of intestinal barrier function.

[0773] [Table 9]

[0774]

Claims

1. A method for evaluating intestinal barrier function in subjects, wherein, The method includes the following steps: (1a) The step of determining the occupancy of target bacteria in feces derived from the subject and obtaining a measured value of the occupancy of the target bacteria; (1b) The step of comparing the measured value obtained in step 1a with the reference value to obtain the comparison result; and (1c) The step of evaluating the intestinal barrier function of the subject based on the comparison results obtained in step 1b. The occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B below: [Group A] Rumenococcal bacteria Peptostreptococcal bacteria Institute of Physics and Chemistry, Bacteriology Eubacteriaceae Bacillus family bacteria Group 1 bacteria of the Clostridium family Group B Bacteria of the genus *Lombocytosporum* in the family Peptostreptococci Allergens of Bacteriology, Institute of Physics and Chemistry UCG-010 bacteria of the family Trichophyceae Ruminococcal bacteria of the genus *Ruminococcus* (family Ruminococci) Group Gaucher bacteria of the family Trichophyceae [Ruminococcus genus] Group 1 bacteria of the genus *Ruminococcus* in the family Ruminococciaceae Ruminococci (Ruminococci) NK4A214 group of bacteria Ruminococci (family Ruminococci) UCG-002 bacteria Active group of bacteria in the family Trichophyceae [Ruminococcus genus] Ruminococci (family Ruminococci) UCG-003 bacteria Ruminococci (family Ruminococci) UCG-009 bacteria Bacteria of the genus *Eubacterium* (family Eubacteriales) Ruminococcal bacteria of the genus *Flavobacterium* Bacteria of the genus Bacillus in the family Bacillusaceae Ruminococci (family Eubacterium, genus *Eubacterium*) coprosterol-producing bacteria Enterobacteriaceae (Peptostreptococci) Anaerobic bacteria of the genus *Corynebacterium* in the family Ruminococci. Marine fungi of the genus *Ostomella* (family Laminariaceae). Clostridium bacteria, Group 1 of the Clostridiumceae family, in the narrow sense.

2. A method for diagnosing diseases or symptoms caused by decreased intestinal barrier function in a subject, wherein, The method includes the following steps: (2a) The step of determining the occupancy of target bacteria in feces derived from the subject and obtaining a measured value of the occupancy of the target bacteria; (2b) The step of comparing the measured value obtained in step 2a with the reference value to obtain the comparison result; and (2c) A step of diagnosing the disease or symptom based on the comparison results obtained in step 2b. The occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B as described in claim 1.

3. A method for treating a disease or symptoms caused by decreased intestinal barrier function in a subject, wherein, The method includes the following steps: (3a) The step of determining the occupancy of target bacteria in feces derived from the subject and obtaining a measured value of the occupancy of the target bacteria; (3b) The step of comparing the measured value obtained in step 3a with the reference value to obtain the comparison result; (3c) The step of diagnosing the disease or symptom based on the comparison results obtained in step 3b; and (3d) In the event that the subject is diagnosed with the disease or symptoms, the step of administering to the subject a substance or composition for treating the disease or symptoms. The occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B as described in claim 1.

4. A method for evaluating the risk or likelihood of a subject developing a disease or symptoms caused by decreased intestinal barrier function, wherein, The method includes the following steps: (4a) The step of determining the occupancy of target bacteria in feces derived from the subject and obtaining a measured value of the occupancy of the target bacteria; (4b) The step of comparing the measured value obtained in step 4a with the reference value to obtain the comparison result; and (4c) The step of evaluating the risk or the likelihood based on the comparison results obtained in step 4b. The occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B as described in claim 1.

5. A method for acquiring data on diseases or symptoms caused by decreased intestinal barrier function in a diagnostic subject, wherein, The method includes the following steps: (5a) The step of determining the occupancy of target bacteria in feces derived from the subject, and obtaining a measured value of the occupancy of the target bacteria; and (5b) The step of comparing the measured value obtained in step 5a with the reference value and obtaining the comparison result as the data. The occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B as described in claim 1.

6. A method for screening substances or compositions that have an effect on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by decreased intestinal barrier function, wherein, The method includes the following steps: (6a) The step of administering a candidate substance or candidate composition to a subject whose intestinal barrier function has been reduced; (6b) The step of determining the occupancy of target bacteria in feces obtained from the subject after administration of the candidate substance or candidate composition, and obtaining a measured value of the occupancy of the target bacteria; (6c) The step of comparing the measured value obtained in step 6b with the reference value to obtain the comparison result; and (6d) Based on the comparison results obtained in step 6c, the step of evaluating the effect of the candidate substance or the candidate composition on improving the intestinal barrier function or on preventing or improving the disease or symptoms. The occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B as described in claim 1.

7. A method for evaluating the effect of a candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by decreased intestinal barrier function, wherein, The method includes the following steps: (7a) The step of administering the candidate substance or the candidate composition to a subject whose intestinal barrier function has been reduced; (7b) The step of determining the occupancy of target bacteria in feces obtained from the subject after administration of the candidate substance or the candidate composition, and obtaining a measured value of the occupancy of the target bacteria; (7c) The step of comparing the measured value obtained in step 7b with the reference value to obtain the comparison result; and (7d) The step of evaluating the effect of the candidate substance or the candidate composition on improving the intestinal barrier function or on preventing or improving the disease or symptoms, based on the comparison results obtained in step 7c. The occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B as described in claim 1.

8. The method according to any one of claims 1 to 7, wherein, The intestinal barrier function mentioned refers to the barrier function of the small intestine.

9. The method according to any one of claims 1 to 7, wherein, The occupancy rate of the target bacteria includes the occupancy rate of two, three, or four or more bacteria selected from Group A and Group B as described in claim 1.

10. The method according to any one of claims 1 to 7, wherein, The occupancy rate of the target bacteria includes the occupancy rate of one or more of the following: Ruminococcal bacteria, Peptostreptocpccaceae bacteria, Pharmacopoeia bacteria, Peptostreptocpccaceae bacteria, and Allergan bacteria.

11. The method according to any one of claims 1 to 7, wherein, The occupancy of the target bacteria was determined by microbial community analysis using 16S rRNA amplicon sequencing.

12. A target bacterium for evaluating the intestinal barrier function of a subject, said target bacterium being derived from the subject's feces. The target bacteria comprise one or more bacteria selected from groups A and B as described in claim 1.

13. A target bacterium for diagnosing diseases or symptoms caused by decreased intestinal barrier function in a subject, said target bacterium being derived from the subject's feces. The target bacteria comprise one or more bacteria selected from groups A and B as described in claim 1.

14. A target bacterium for evaluating the risk or likelihood of a subject developing a disease or symptom caused by reduced intestinal barrier function, wherein the target bacterium is derived from the subject's feces. The target bacteria comprise one or more bacteria selected from groups A and B as described in claim 1.

15. A target bacterium for acquiring data on diseases or symptoms caused by reduced intestinal barrier function in a subject for diagnosis, said target bacterium being derived from the subject's feces. The target bacteria comprise one or more bacteria selected from groups A and B as described in claim 1.

16. A target bacterium for screening substances or compositions that have an effect on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by reduced intestinal barrier function, said target bacterium being a target bacterium in feces obtained from a subject with reduced intestinal barrier function after administration of the candidate substance or candidate composition to said subject. The target bacteria comprise one or more bacteria selected from groups A and B as described in claim 1.

17. A target bacterium for evaluating the effect of a candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by reduced intestinal barrier function, said target bacterium being a target bacterium in feces obtained from a subject with reduced intestinal barrier function after administration of said candidate substance or candidate composition to said subject. The target bacteria comprise one or more bacteria selected from groups A and B as described in claim 1.

18. The target bacteria according to any one of claims 12 to 17, wherein, The intestinal barrier function mentioned refers to the barrier function of the small intestine.

19. The target bacteria according to any one of claims 12 to 17, wherein, The target bacteria comprise two, three, or four or more bacteria selected from groups A and B as described in claim 1.

20. The target bacteria according to any one of claims 12 to 17, wherein, The target bacteria include one or more bacteria selected from the following families: Ruminococci, Peptostreptococci, Pharmacopoeia, Rampobacillus, and Allergan.

21. The use of target bacteria in evaluating intestinal barrier function, among which, The target bacteria are derived from the feces of the subject. The target bacteria comprise one or more bacteria selected from groups A and B as described in claim 1.

22. The use of target bacteria in the diagnosis of diseases or symptoms caused by decreased intestinal barrier function in the subject, among which, The target bacteria are derived from the feces of the subject. The target bacteria comprise one or more bacteria selected from groups A and B as described in claim 1.

23. The use of target bacteria in evaluating the risk or likelihood of a subject developing a disease or symptom caused by a weakened intestinal barrier, wherein, The target bacteria are derived from the feces of the subject. The target bacteria comprise one or more bacteria selected from groups A and B as described in claim 1.

24. The use of target bacteria in obtaining data for diagnostic purposes regarding diseases or symptoms caused by impaired intestinal barrier function, wherein, The target bacteria are derived from the feces of the subject. The target bacteria comprise one or more bacteria selected from groups A and B as described in claim 1.

25. The use of target bacteria in screening substances or compositions that have an effect on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by decreased intestinal barrier function, wherein, The target bacteria are target bacteria in the feces obtained from subjects with impaired intestinal barrier function after administration of the candidate substance or candidate composition. The target bacteria comprise one or more bacteria selected from groups A and B as described in claim 1.

26. The use of target bacteria in evaluating the effects of candidate substances or candidate compositions on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by decreased intestinal barrier function, wherein, The target bacteria are target bacteria in feces obtained from subjects with impaired intestinal barrier function after administration of the candidate substance or the candidate composition to the subjects. The target bacteria comprise one or more bacteria selected from groups A and B as described in claim 1.

27. The use according to any one of claims 21 to 26, wherein, The intestinal barrier function mentioned refers to the barrier function of the small intestine.

28. The use according to any one of claims 21 to 26, wherein, The target bacteria comprise two, three, or four or more bacteria selected from groups A and B as described in claim 1.

29. The use according to any one of claims 21 to 26, wherein, The target bacteria include one or more bacteria selected from the following families: Ruminococci, Peptostreptococci, Pharmacopoeia, Rampobacillus, and Allergan.

30. A kit for evaluating intestinal barrier function in subjects, wherein, The kit contains one or more reagents for determining the occupancy of target bacteria in feces derived from the subject. The occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B as described in claim 1.

31. A kit for diagnosing diseases or symptoms caused by decreased intestinal barrier function in a subject, wherein, The kit contains one or more reagents for determining the occupancy of target bacteria in feces derived from the subject. The occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B as described in claim 1.

32. A kit for evaluating the risk or likelihood of a subject developing a disease or symptom caused by decreased intestinal barrier function, wherein, The kit contains one or more reagents for determining the occupancy of target bacteria in feces derived from the subject. The occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B as described in claim 1.

33. A kit for acquiring data on diseases or symptoms caused by decreased intestinal barrier function in a diagnostic subject, wherein, The kit contains one or more reagents for determining the occupancy of target bacteria in feces derived from the subject. The occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B as described in claim 1.

34. A kit for screening substances or compositions that have an effect on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by decreased intestinal barrier function, wherein, The kit contains one or more reagents for determining the occupancy of target bacteria in feces obtained from subjects with impaired intestinal barrier function after administration of a candidate substance or candidate composition to the subjects. The occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B as described in claim 1.

35. A kit for evaluating the effect of a candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by decreased intestinal barrier function, wherein, The kit contains one or more reagents for determining the occupancy of target bacteria in feces obtained from subjects with impaired intestinal barrier function after administration of the candidate substance or the candidate composition to the subjects. The occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B as described in claim 1.

36. The kit according to any one of claims 30 to 35, wherein, The intestinal barrier function mentioned refers to the barrier function of the small intestine.

37. The kit according to any one of claims 30 to 35, wherein, The occupancy rate of the target bacteria includes the occupancy rate of two, three, or four or more bacteria selected from Group A and Group B as described in claim 1.

38. The kit according to any one of claims 30 to 35, wherein, The occupancy rate of the target bacteria includes the occupancy rate of one or more of the following: Ruminococcus family, Peptostreptococcus family, Institute of Physicochemical Sciences family, Peptostreptococcus spp., and Allergan spp.

39. The kit according to any one of claims 30 to 35, wherein, The occupancy of the target bacteria was determined by microbial community analysis using 16S rRNA amplicon sequencing.

40. A method for manufacturing a food or pharmaceutical composition, comprising the step of: mixing a substance or composition selected by the method of claim 6 with one or more ingredients constituting the food or pharmaceutical composition.

41. A computer program for causing a computer to perform a method for evaluating the intestinal barrier function of a subject. The method includes the following steps: (S101) A step of obtaining a measurement of the occupancy of target bacteria in feces derived from the object; (S102) The step of comparing the measured value obtained in step S101 with the reference value to obtain a comparison result; and (S103) The step of evaluating the intestinal barrier function of the subject based on the comparison results obtained in step S102. The occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B as described in claim 1.

42. A computer program for causing a computer to perform a method for diagnosing a disease or symptoms caused by decreased intestinal barrier function in a subject. The method includes the following steps: (S201) A step of obtaining a measurement value of the occupancy of target bacteria in feces derived from the object; (S202) The step of comparing the measured value obtained in step S201 with the reference value to obtain the comparison result; (S203) A step of diagnosing the disease or symptom based on the comparison results obtained in step S202. The occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B as described in claim 1.

43. A computer program for causing a computer to perform a method for evaluating the risk or likelihood of a subject developing a disease or symptoms caused by reduced intestinal barrier function. The method includes the following steps: (S301) A step of obtaining a measurement value of the occupancy of target bacteria in feces derived from the object; (S302) The step of comparing the measured value obtained in step S301 with the reference value to obtain a comparison result; and (S303) The step of evaluating the risk or the probability based on the comparison results obtained in step S302. The occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B as described in claim 1.

44. A computer program for causing a computer to perform a method for acquiring data on a disease or symptoms caused by impaired intestinal barrier function in a diagnostic subject. The method includes the following steps: (S401) A step of obtaining a measurement of the occupancy of target bacteria in feces derived from the object; as well as (S402) The step of comparing the measured value obtained in step S401 with the reference value and obtaining the comparison result as the data. The occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B as described in claim 1.

45. A computer program for causing a computer to perform a method for screening substances or compositions that have effects of improving intestinal barrier function or preventing or improving diseases or symptoms caused by reduced intestinal barrier function. The method includes the following steps: (S501) A step of obtaining a measurement value of the occupancy of target bacteria in feces obtained from a subject with reduced intestinal barrier function after administering a candidate substance or candidate composition to the subject. (S502) The step of comparing the measured value obtained in step S501 with the reference value to obtain a comparison result; and (S503) Based on the comparison results obtained in step S502, the step of evaluating the effect of the candidate substance or the candidate composition on improving the intestinal barrier function or on preventing or improving the disease or symptoms. The occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B as described in claim 1.

46. ​​A computer program for causing a computer to perform a method for evaluating the effect of a candidate substance or candidate composition on improving intestinal barrier function or on preventing or improving diseases or symptoms caused by reduced intestinal barrier function. The method includes the following steps: (S601) A step of obtaining a measurement of the occupancy of target bacteria in feces obtained from a subject with reduced intestinal barrier function after administering a candidate substance or candidate composition to the subject. (S602) The step of comparing the measured value obtained in step S601 with the reference value to obtain a comparison result; and (S603) Based on the comparison results obtained in step S602, the step of evaluating the effect of the candidate substance or the candidate composition on improving the intestinal barrier function or on preventing or improving the disease or symptoms. The occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from groups A and B as described in claim 1.

47. The computer program according to any one of claims 41 to 46, wherein, The intestinal barrier function mentioned refers to the barrier function of the small intestine.

48. The computer program according to any one of claims 41 to 46, wherein, The occupancy rate of the target bacteria includes the occupancy rate of two, three, or four or more bacteria selected from Group A and Group B as described in claim 1.

49. The computer program according to any one of claims 41 to 46, wherein, The occupancy rate of the target bacteria includes the occupancy rate of one or more of the following: Ruminococcus family, Peptostreptococcus family, Institute of Physicochemical Sciences family, Peptostreptococcus spp., and Allergan spp.

50. The computer program according to any one of claims 41 to 46, wherein, The occupancy of the target bacteria was determined by microbial community analysis using 16S rRNA amplicon sequencing.

51. A computer-readable recording medium having recorded the computer program of any one of claims 41 to 46.

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