Target bacteria and use therefor

Abstract

The purpose of the present invention is to provide a method for evaluating the intestinal barrier function of a subject. The present invention provides a method for evaluating the intestinal barrier function of a subject comprising the following steps: (1a) a step in which the occupancy rate of target bacteria in feces originating from the subject is measured and a measurement value for the occupancy rate of the target bacteria is acquired; (1b) a step in which the measurement value acquired in step 1a is compared with a reference value and a comparison result is acquired; and (1c) a step in which the intestinal barrier function of the subject is evaluated on the basis of the comparison result acquired in step 1b. The occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from a predetermined group.

Description

Target bacteria and their uses

[0001] The present invention relates to target bacteria and uses thereof.

[0002] Specifically, the present invention relates to a method for assessing the intestinal barrier function of a subject, a target bacterium for said assessment, use of the target bacterium for said assessment, a kit for said assessment, and a computer program for causing a computer to execute said method.

[0003] The present invention also relates to a method for diagnosing a disease or symptom caused by a decrease in intestinal barrier function in a subject, a target bacterium for said diagnosis, use of the target bacterium for said diagnosis, a kit for said diagnosis, and a computer program for causing a computer to execute said method.

[0004] The present invention also relates to a method for treating a disease or condition caused by a compromised intestinal barrier function in a subject.

[0005] The present invention also relates to a method for assessing the risk of a subject developing a disease or symptom caused by a decrease in intestinal barrier function, or the possibility of the subject suffering from said disease or symptom, a target bacterium for said assessment, use of the target bacterium for said assessment, a kit for said assessment, and a computer program for causing a computer to execute said method.

[0006] The present invention also relates to a method for obtaining data for diagnosing a disease or symptom caused by a decrease in intestinal barrier function in a subject, a target bacterium for said obtaining, use of the target bacterium for said obtaining, a kit for said obtaining, and a computer program for causing a computer to execute said method.

[0007] The present invention also relates to a method for screening for a substance or composition that has the effect of improving intestinal barrier function or the effect of preventing or improving diseases or symptoms caused by a decrease in intestinal barrier function, a target bacterium for said screening, use of the target bacterium for said screening, a kit for said screening, and a computer program for causing a computer to execute said method.

[0008] The present invention also relates to a method for evaluating the effect of a candidate substance or candidate composition in improving intestinal barrier function or the effect of preventing or improving diseases or symptoms caused by a decrease in intestinal barrier function, a target bacterium for said evaluation, use of the target bacterium for said evaluation, a kit for said evaluation, and a computer program for causing a computer to execute said method.

[0009] The present invention also relates to a method for producing a food or pharmaceutical composition.

[0010] The intestinal lumen is a space surrounded by a single layer of mucosal epithelial cells, which separate the intestinal lumen (outside the body) from the inside of the body. Because the intestinal tract is exposed to foreign substances (e.g., harmful microorganisms, toxins, allergens, etc.) introduced into the intestinal lumen from the outside world, it is an important organ not only in terms of nutrient absorption but also in terms of biological defense. The intestinal barrier function prevents foreign substances (e.g., harmful microorganisms, toxins, allergens, etc.) introduced into the intestinal lumen from the outside world from entering the body.

[0011] Intestinal barrier function involves physical and chemical barriers. Physical barriers literally function as a physical barrier to prevent the intrusion of foreign substances. Physical barriers include the mucus layer that covers intestinal epithelial cells, the glycocalyx, which is an aggregate of glycans present on the surface of intestinal epithelial cells, and the tight junction structure, which is an intercellular adhesion mechanism of intestinal epithelial cells. Chemical barriers are a group of molecules that chemically change foreign substances and exert antibacterial and neutralizing activities, thereby inhibiting their invasion. Examples of such molecules include antibacterial peptides (e.g., defensin family molecules, Reg3 family molecules, lactoferrin, lysozyme, etc.) produced by intestinal epithelial cells such as Paneth cells. M cells, which take up antigens and present them to dendritic cells, and immunoglobulin A (IgA), which is secreted into the intestinal tract and exerts neutralizing activity to prevent the intrusion of foreign substances, are also types of chemical barriers.

[0012] A decrease in intestinal barrier function causes various diseases or symptoms (for example, enteritis, allergic diseases, psychiatric disorders, non-alcoholic fatty liver disease, type 2 diabetes, metabolic syndrome, obesity, etc.).

[0013] The lactulose-mannitol test (glucose tolerance test) is a known method for testing intestinal barrier function. In the lactulose-mannitol test, intestinal permeability is examined, and an increase in intestinal permeability is an indicator of a deterioration in intestinal barrier function (particularly physical barrier functions such as tight junction structures). The lactulose-mannitol test has drawbacks, such as requiring subjects to undergo long periods of restraint and fasting, and placing a strain on the subjects' digestive tract due to the ingested sugar alcohols.

[0014] Blood zonulin concentration, which is known to be related to intestinal barrier function (Non-Patent Document 1), is sometimes used in testing methods to evaluate intestinal barrier function, but there are issues such as daily variations in blood zonulin concentration and low reproducibility (Non-Patent Document 2).

[0015] To date, no biomarkers have been known that can easily and accurately evaluate intestinal barrier function.

[0016] Fasana A et al., Zonulin, “a newly discovered modulator of intestinal permeability, and its expression in coeliac disease.”, THE LANCET., 2000; 355: pp.1518-1519Aristo Vojdani et al., “Fluctuation of zonulin levels in blood vs stability of antibodies.”, World Journal of Gastroenterology., 2017; 23(31): pp.5669-5679

[0017] The present invention aims to provide a method for evaluating the intestinal barrier function of a subject, a target bacterium for said evaluation, use of the target bacterium for said evaluation, a kit for said evaluation, and a computer program for causing a computer to execute said method.

[0018] The present invention also aims to provide a method for diagnosing a disease or symptom caused by a decrease in intestinal barrier function in a subject, a target bacterium for said diagnosis, use of said target bacterium for said diagnosis, a kit for said diagnosis, and a computer program for causing a computer to execute said method.

[0019] Another object of the present invention is to provide a method for treating a disease or symptom caused by a decrease in intestinal barrier function in a subject.

[0020] The present invention also aims to provide a method for assessing the risk of a subject developing a disease or symptom caused by a decrease in intestinal barrier function, or the possibility of the subject suffering from said disease or symptom, a target bacterium for said assessment, use of the target bacterium for said assessment, a kit for said assessment, and a computer program for causing a computer to execute said method.

[0021] The present invention also aims to provide a method for obtaining data for diagnosing a disease or symptom caused by a decrease in intestinal barrier function in a subject, a target bacterium for said obtaining, use of the target bacterium for said obtaining, a kit for said obtaining, and a computer program for causing a computer to execute said method.

[0022] The present invention also aims to provide a method for screening for substances or compositions that have the effect of improving intestinal barrier function or the effect of preventing or improving diseases or symptoms caused by a decrease in intestinal barrier function, a target bacterium for said screening, use of the target bacterium for said screening, a kit for said screening, and a computer program for causing a computer to execute said method.

[0023] Another object of the present invention is to provide a method for evaluating the effect of a candidate substance or candidate composition on improving intestinal barrier function or the effect of preventing or improving diseases or symptoms caused by a decrease in intestinal barrier function, a target bacterium for said evaluation, use of the target bacterium for said evaluation, a kit for said evaluation, and a computer program for causing a computer to execute said method.

[0024] Another object of the present invention is to provide a method for producing a food or pharmaceutical composition.

[0025] In the present invention, Group A is composed of the following bacteria. The names of bacteria in Group A are family names. [Group A] Ruminococcaceae bacteria Peptostreptococcaceae bacteria Rikenellaceae bacteria Eubacteriaceae bacteria Bacillaceae bacteria Clostridiaceae bacteria

[0026] In the present invention, group B consists of the following bacteria: The names of bacteria in group B are genus names. [Group B] Peptostreptococcaceae Romboutsia bacteria Rikenellaceae Alistipes bacteria Lachnospiraceae Lachnospiraceae UCG-010 bacteria Ruminococcaceae Negativibacillus bacteria Lachnospiraceae [Ruminococcus] gauvreauii group bacteria Ruminococcaceae Ruminococcus 1 bacteria Ruminococcaceae Ruminococcaceae NK4A214 group bacteria Ruminococcaceae Ruminococcaceae UCG-002 bacteria Lachnospiraceae [Ruminococcus] gnavus group bacteria Ruminococcaceae Ruminococcaceae UCG-003 bacteria Ruminococcaceae Ruminococcaceae UCG-009 bacteria Eubacteriaceae Eubacterium bacteria Ruminococcaceae Faecalibacterium bacteria Bacillusae Bacillus bacteria Ruminococcaceae [Eubacterium] coprostanoligenes group bacteria Peptostreptococcaceae Intestinibacter bacteria Ruminococcaceae Anaerotruncus bacteria Marinifilaceae Odoribacter bacteria Clostridiaceae 1 Clostridium sensu stricto bacteria

[0027] The present invention encompasses the following inventions: [1] A method for evaluating the intestinal barrier function of a subject, the method comprising the following steps: (1a) measuring the occupancy rate of target bacteria in feces derived from the subject to obtain a measurement value of the occupancy rate of the target bacteria; (1b) comparing the measurement value obtained in step 1a with a reference value to obtain 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 rate of the target bacteria includes the occupancy rate of one or more bacteria selected from group A and group B. [2] A method for diagnosing a disease or symptom caused by a decrease in intestinal barrier function in a subject, the method comprising the following steps: (2a) measuring the occupancy rate of target bacteria in feces derived from the subject and obtaining a measurement value of the occupancy rate of the target bacteria; (2b) comparing the measurement value obtained in step 2a with a reference value and obtaining a comparison result; and (2c) diagnosing the disease or symptom based on the comparison result obtained in step 2b, wherein the occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from group A and group B. [3] A method for treating a disease or symptom caused by a decrease in intestinal barrier function in a subject, the method comprising the following steps: (3a) measuring the occupancy rate of target bacteria in feces derived from the subject and obtaining a measurement value of the occupancy rate of the target bacteria; (3b) comparing the measurement value obtained in step 3a with a reference value and obtaining a comparison result; (3c) diagnosing the disease or symptom based on the comparison result obtained in step 3b; and (3d) administering to the subject a substance or composition for treating the disease or symptom if the subject is diagnosed as suffering from the disease or symptom, wherein the occupancy rate of the target bacteria includes an occupancy rate of one or more bacteria selected from group A and group B.[4] A method for assessing the risk of a subject developing a disease or symptom caused by a decrease in intestinal barrier function, or the possibility of the subject suffering from said disease or symptom, the method comprising the following steps: (4a) measuring the occupancy rate of target bacteria in feces derived from the subject and obtaining a measurement value of the occupancy rate of the target bacteria; (4b) comparing the measurement value obtained in step 4a with a reference value and obtaining a comparison result; and (4c) assessing the risk or the possibility based on the comparison result obtained in step 4b, wherein the occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from group A and group B. [5] A method for obtaining data for diagnosing a disease or symptom caused by a decrease in intestinal barrier function in a subject, the method comprising the following steps: (5a) measuring the occupancy of target bacteria in feces derived from the subject and obtaining a measurement value of the occupancy of the target bacteria; and (5b) comparing the measurement value obtained in step 5a with a reference value and obtaining the comparison result as the data, wherein the occupancy of the target bacteria includes the occupancy of one or more bacteria selected from group A and group B. [6] A method for screening for a substance or composition that has an effect of improving intestinal barrier function or an effect of preventing or improving a disease or symptom caused by a decrease in intestinal barrier function, the method comprising the following steps: (6a) a step of administering a candidate substance or candidate composition to a subject with a decreased intestinal barrier function; (6b) a step of measuring the occupancy rate of target bacteria in stool obtained from the subject after administration of the candidate substance or candidate composition, and obtaining a measurement value of the occupancy rate of the target bacteria; (6c) a step of comparing the measurement value obtained in step 6b with a reference value and obtaining a comparison result; and (6d) a step of evaluating the effect of the candidate substance or candidate composition in improving the intestinal barrier function or the effect of preventing or improving the disease or symptom based on the comparison result obtained in step 6c, wherein the occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from group A and group B.[7] A method for evaluating the effect of a candidate substance or candidate composition on improving intestinal barrier function, or the effect of preventing or improving a disease or symptom caused by a decrease in intestinal barrier function, the method comprising the following steps: (7a) administering the candidate substance or the candidate composition to a subject with a decreased intestinal barrier function; (7b) measuring the occupancy rate of target bacteria in stool obtained from the subject after administration of the candidate substance or the candidate composition, and obtaining a measurement value of the occupancy rate of the target bacteria; (7c) comparing the measurement value obtained in step 7b with a reference value and obtaining a comparison result; and (7d) evaluating the effect of the candidate substance or the candidate composition on improving the intestinal barrier function, or the effect of preventing or improving the disease or symptom, based on the comparison result obtained in step 7c, wherein the occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from group A and group B. [8] The method according to any one of [1] to [7], wherein the intestinal barrier function is a barrier function of the small intestine. [9] The method according to any one of [1] to [8], wherein the occupancy rate of the target bacteria comprises an occupancy rate of two, three, or four or more bacteria selected from group A and group B.

[10] The method according to any one of [1] to [9], wherein the occupancy rate of the target bacteria comprises one or more occupancies selected from an occupancy rate of Ruminococcaceae bacteria, an occupancy rate of Peptostreptococcaceae bacteria, an occupancy rate of Rikenellaceae bacteria, an occupancy rate of Peptostreptococcaceae Romboutsia bacteria, and an occupancy rate of Rikenellaceae Alistipes bacteria.

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

[10] , wherein the occupancy rate of the target bacteria is measured by microbiota analysis using 16S rRNA amplicon sequencing.

[12] Target bacteria in feces derived from a subject for evaluating the intestinal barrier function of the subject, the target bacteria comprising one or more bacteria selected from group A and group B.

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

[14] Target bacteria in stool derived from a subject, for assessing the risk of the subject developing a disease or symptom caused by a decline in intestinal barrier function, or the possibility of the subject suffering from said disease or symptom, the target bacteria comprising one or more bacteria selected from Group A and Group B.

[15] Target bacteria in stool derived from a subject, for obtaining data for diagnosing a disease or symptom caused by a decline in intestinal barrier function in the subject, the target bacteria comprising one or more bacteria selected from Group A and Group B.

[16] Target bacteria in stool obtained from a subject with a decline in intestinal barrier function after administering a candidate substance or candidate composition to the subject, for screening for a substance or composition having an effect of improving intestinal barrier function or an effect of preventing or improving a disease or symptom caused by a decline in intestinal barrier function, the target bacteria comprising one or more bacteria selected from Group A and Group B.

[17] Target bacteria in feces obtained from a subject with impaired intestinal barrier function after administration of a candidate substance or candidate composition to the subject, for evaluating the effect of the candidate substance or candidate composition in improving intestinal barrier function or the effect of preventing or improving a disease or symptom caused by impaired intestinal barrier function, the target bacteria comprising one or more bacteria selected from Group A and Group B.

[18] The target bacterium according to any one of

[12] to

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

[19] The target bacterium according to any one of

[12] to

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

[20] The target bacterium according to any one of

[12] to

[19] , wherein the target bacteria comprises one or more bacteria selected from Ruminococcaceae bacteria, Peptostreptococcaceae bacteria, Rikenellaceae bacteria, Peptostreptococcaceae Romboutsia bacteria, and Rikenellaceae Alistipes bacteria.

[21] Use of target bacteria in feces derived from a subject to evaluate the intestinal barrier function of the subject, wherein the target bacteria include one or more bacteria selected from group A and group B.

[22] Use of target bacteria in stool derived from a subject for diagnosing a disease or symptom caused by a decline in intestinal barrier function in the subject, wherein the target bacteria comprise one or more bacteria selected from Group A and Group B.

[23] Use of target bacteria in stool derived from a subject for assessing the risk of the subject developing a disease or symptom caused by a decline in intestinal barrier function in the subject, or the possibility of the subject suffering from said disease or symptom, wherein the target bacteria comprise one or more bacteria selected from Group A and Group B.

[24] Use of target bacteria in stool derived from a subject for obtaining data for diagnosing a disease or symptom caused by a decline in intestinal barrier function in the subject, wherein the target bacteria comprise one or more bacteria selected from Group A and Group B.

[25] Use of target bacteria in stool obtained from a subject with a decline in intestinal barrier function after administration of a candidate substance or composition to the subject, for screening for a substance or composition having an effect of improving intestinal barrier function or an effect of preventing or ameliorating a disease or symptom caused by a decline in intestinal barrier function, wherein the target bacteria comprise one or more bacteria selected from Group A and Group B.

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

[27] The use according to any one of

[21] to

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

[28] The use according to any one of

[21] to

[27] , wherein the target bacteria comprise two, three, or four or more bacteria selected from Group A and Group B.

[29] The use according to any one of

[21] to

[28] , wherein the target bacteria comprise one or more bacteria selected from Ruminococcaceae bacteria, Peptostreptococcaceae bacteria, Rikenellaceae bacteria, Peptostreptococcaceae Romboutsia bacteria, and Rikenellaceae Alistipes bacteria.

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

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

[32] A kit for assessing the risk of a subject developing a disease or symptom caused by a decrease in intestinal barrier function, or the possibility that a subject is suffering from the disease or symptom, the kit comprising one or more reagents for measuring the occupancy rate of target bacteria in feces derived from the subject, wherein the occupancy rate of the target bacteria comprises the occupancy rate of one or more bacteria selected from Group A and Group B.

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

[34] A kit for screening a substance or composition that has an effect of improving intestinal barrier function or an effect of preventing or improving a disease or symptom caused by a decrease in intestinal barrier function, the kit comprising one or more reagents for measuring the occupancy rate of target bacteria in feces obtained from a subject with a decrease in intestinal barrier function after administering a candidate substance or candidate composition to the subject, wherein the occupancy rate of the target bacteria comprises the occupancy rate of one or more bacteria selected from group A and group B.

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

[36] The kit according to any one of

[30] to

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

[37] The kit according to any one of

[30] to

[36] , wherein the occupancy rate of the target bacteria comprises an occupancy rate of two, three, or four or more bacteria selected from Group A and Group B.

[38] The kit according to any one of

[30] to

[37] , wherein the occupancy rate of the target bacteria comprises one or more occupancies selected from the occupancy rate of Ruminococcaceae bacteria, the occupancy rate of Peptostreptococcaceae bacteria, the occupancy rate of Rikenellaceae bacteria, the occupancy rate of Peptostreptococcaceae Romboutsia bacteria, and the occupancy rate of Rikenellaceae Alistipes bacteria.

[39] The kit according to any one of

[30] to

[38] , wherein the occupancy rate of the target bacteria is measured by bacterial flora analysis using 16S rRNA amplicon sequencing.

[40] A method for producing a food or pharmaceutical composition, comprising a step of blending a substance or composition screened by the method described in [6] with one or more ingredients that constitute the food or pharmaceutical composition.

[41] A computer program for causing a computer to execute a method for evaluating the intestinal barrier function of a subject, the method comprising the following steps: (S101) acquiring a measurement value of the occupancy rate of target bacteria in stool derived from the subject; (S102) comparing the measurement value acquired in step S101 with a reference value and acquiring a comparison result; and (S103) evaluating the intestinal barrier function of the subject based on the comparison result acquired in step S102, wherein the occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from group A and group B.

[42] A computer program for causing a computer to execute a method for diagnosing a disease or symptom caused by a decrease in intestinal barrier function in a subject, the method comprising the following steps: (S201) a step of obtaining a measurement value of the occupancy rate of target bacteria in feces derived from the subject; (S202) a step of comparing the measurement value obtained in step S201 with a reference value and obtaining a comparison result; (S203) a step of diagnosing the disease or symptom based on the comparison result obtained in step S202, wherein the occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from group A and group B.

[43] A computer program for causing a computer to execute a method for assessing the risk of a subject developing a disease or symptom caused by a decrease in intestinal barrier function, or the possibility of the subject suffering from said disease or symptom, the method comprising the following steps: (S301) a step of acquiring a measurement value of the occupancy rate of target bacteria in stool derived from the subject; (S302) a step of comparing the measurement value acquired in step S301 with a reference value and acquiring a comparison result; and (S303) a step of assessing the risk or the possibility based on the comparison result acquired in step S302, wherein the occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from group A and group B.

[44] A computer program for causing a computer to execute a method for acquiring data for diagnosing a disease or symptom caused by a decrease in intestinal barrier function in a subject, the method comprising the following steps: (S401) acquiring a measurement value of the occupancy rate of target bacteria in feces derived from the subject; and (S402) comparing the measurement value acquired in step S401 with a reference value and acquiring the comparison result as the data, wherein the occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from group A and group B.

[45] A computer program for causing a computer to execute a method for screening for a substance or composition that has an effect of improving intestinal barrier function or an effect of preventing or improving a disease or symptom caused by a decrease in intestinal barrier function, the method comprising the following steps: (S501) a step of obtaining a measurement value of the occupancy rate of target bacteria in feces obtained from a subject with decreased intestinal barrier function after administering a candidate substance or candidate composition to the subject; (S502) a step of comparing the measurement value obtained in step S501 with a reference value and obtaining a comparison result; and (S503) a step of evaluating the effect of the candidate substance or candidate composition in improving the intestinal barrier function or the effect of preventing or improving the disease or symptom based on the comparison result obtained in step S502, wherein the occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from group A and group B.

[46] A computer program causing a computer to execute a method for evaluating the effect of a candidate substance or candidate composition on improving intestinal barrier function, or the effect of preventing or improving a disease or symptom caused by a decrease in intestinal barrier function, the method comprising the following steps: (S601) acquiring a measurement value of the occupancy rate of target bacteria in feces obtained from a subject with decreased intestinal barrier function after administration of the candidate substance or candidate composition to the subject; (S602) comparing the measurement value acquired in step S601 with a reference value and acquiring a comparison result; and (S603) evaluating the effect of the candidate substance or candidate composition on improving the intestinal barrier function, or the effect of preventing or improving the disease or symptom, based on the comparison result acquired in step S602, wherein the occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from group A and group B.

[47] The computer program according to any one of

[41] to

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

[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 four or more bacteria selected from group A and group B.

[49] The computer program according to any one of

[41] to

[48] , wherein the occupancy rate of the target bacteria comprises one or more occupancies selected from the group consisting of the occupancy rate of Ruminococcaceae bacteria, the occupancy rate of Peptostreptococcaceae bacteria, the occupancy rate of Rikenellaceae bacteria, the occupancy rate of Peptostreptococcaceae Romboutsia bacteria, and the occupancy rate of Rikenellaceae Alistipes bacteria.

[50] The computer program according to any one of

[41] to

[49] , wherein the occupancy rate of the target bacteria is measured by bacterial flora analysis using 16S rRNA amplicon sequencing.

[51] A computer-readable recording medium having the computer program according to any one of

[41] to

[50] recorded thereon.

[0028] According to the present invention, there are provided a method for assessing the intestinal barrier function of a subject, a target bacterium for said assessment, use of the target bacterium for said assessment, a kit for said assessment, and a computer program for causing a computer to execute said method.

[0029] The present invention also provides a method for diagnosing a disease or symptom caused by a decrease in intestinal barrier function in a subject, a target bacterium for said diagnosis, use of said target bacterium for said diagnosis, a kit for said diagnosis, and a computer program for causing a computer to execute said method.

[0030] The present invention also provides a method for treating a disease or symptom caused by a decrease in intestinal barrier function in a subject.

[0031] The present invention also provides a method for assessing the risk of a subject developing a disease or symptom caused by a decrease in intestinal barrier function, or the possibility of the subject suffering from said disease or symptom, a target bacterium for said assessment, use of the target bacterium for said assessment, a kit for said assessment, and a computer program for causing a computer to execute said method.

[0032] The present invention also provides a method for obtaining data for diagnosing a disease or symptom caused by a decrease in intestinal barrier function in a subject, a target bacterium for said obtaining, use of the target bacterium for said obtaining, a kit for said obtaining, and a computer program for causing a computer to execute said method.

[0033] The present invention also provides a method for screening for a substance or composition that has the effect of improving intestinal barrier function or the effect of preventing or improving diseases or symptoms caused by a decrease in intestinal barrier function, a target bacterium for the screening, use of the target bacterium for the screening, a kit for the screening, and a computer program for causing a computer to execute the method.

[0034] The present invention also provides a method for evaluating the effect of a candidate substance or candidate composition in improving intestinal barrier function or the effect of preventing or improving diseases or symptoms caused by a decrease in intestinal barrier function, a target bacterium for said evaluation, use of the target bacterium for said evaluation, a kit for said evaluation, and a computer program for causing a computer to execute said method.

[0035] The present invention also provides a method for producing a food or pharmaceutical composition.

[0036] The technology provided by the present invention enables a personal understanding of the state of the intestinal barrier of a subject, and can contribute to precision nutrition (particularly precision prebiotics and precision nutrient), precision healthcare, and the like.

[0037] Fig. 1 is a schematic diagram of an apparatus according to an embodiment of the present invention. Fig. 2 is a block diagram showing the hardware configuration of an apparatus according to an embodiment of the present invention. Fig. 3 is a flowchart showing a processing procedure performed by an apparatus according to an embodiment of the present invention. Fig. 4 is a flowchart showing a processing procedure performed by an apparatus according to an embodiment of the present invention. Fig. 5 is a flowchart showing a processing procedure performed by an apparatus according to an embodiment of the present invention.

[0038] The present invention will be described below. Two or more of the embodiments described in this specification can be combined, and such combinations are also encompassed by the present invention.

[0039] Aspect 1 Aspect 1 of the present invention relates to a method for evaluating the intestinal barrier function of a subject.

[0040] In the method according to Aspect 1, a biomarker in a sample derived from a subject is used as an indicator for evaluating the intestinal barrier function of the subject.

[0041] The sample used in Aspect 1 is feces derived from a subject. The feces may be dry or wet. By using feces as a sample, the intestinal barrier function of a subject can be easily evaluated.

[0042] The subject is not particularly limited as long as it is an animal that excretes feces. Examples of animals include vertebrates such as mammals, reptiles, birds, amphibians, and fish, with mammals and birds being preferred, and mammals being more preferred. Examples of mammals include primates (e.g., humans, gorillas, chimpanzees, orangutans, etc.), rodents (e.g., mice, rats, hamsters, guinea pigs, rabbits, etc.), livestock (e.g., cows, pigs, sheep, goats, horses, etc.), and pets (e.g., dogs, cats, etc.), with humans being preferred. Examples of birds include poultry (e.g., chickens, ducks, geese, etc.).

[0043] Feces is excreted from the anus. Feces from a subject can be collected from feces excreted by the subject according to a conventional method, and the collected feces can be used as a specimen.

[0044] The moisture content in dried feces is preferably 20% by mass or less, more preferably 10% by mass or less, and more preferably 5% by mass or less, based on the mass of the dried feces. The lower limit of the moisture content in dried feces is zero. Dried feces can be obtained by drying feces immediately after it has been excreted 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, from -80°C to 0°C, and the processing time is, for example, from 24 hours to 72 hours.

[0045] The moisture content of wet feces is preferably 40% by mass or more, more preferably 50% by mass or more, and more preferably 60% by mass or more, based on the mass of the wet feces. The upper limit is not particularly limited, but is, for example, 95% by mass or less, 90% by mass or less, 80% by mass or less, or 70% by mass or less. Each of these upper limits may be combined with any of the above-mentioned lower limits. The wet feces may be feces immediately after being excreted from the anus, or feces immediately after being excreted from the anus and then frozen and stored and thawed. The temperature for frozen storage 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. Frozen storage can be performed, for example, by placing feces immediately after being excreted from the anus in a container specifically for collecting feces (e.g., a Faeces tube with a blade and a screw cap (manufactured by Sarstedt)). The period of frozen storage is preferably within 180 days from collection, more preferably within 60 days from collection, and even more preferably within 30 days from collection. That is, wet feces collected within 180 days from collection is preferably used for biomarker measurement, more preferably within 60 days from collection, and even more preferably within 30 days from collection.

[0046] The biomarker used in Aspect 1 is a target bacterium in feces derived from a subject. Specifically, the occupancy rate of the target bacterium in feces derived from a subject is used as an index for evaluating the intestinal barrier function of the subject.

[0047] The occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from group A and group B. By using the occupancy rate of one or more bacteria selected from group A and group B as an index, the intestinal barrier function of a subject can be evaluated with high accuracy.

[0048] The occupancy rate of the target bacteria may include the occupancy rate of one bacterium selected from Group A and Group B, or may include the occupancy rate of two or more bacteria selected from Group A and Group B. However, from the viewpoint of improving the accuracy of the evaluation of the intestinal barrier function of the subject, it preferably includes the occupancy rate of two or more (specifically, two, three, or four or more) bacteria selected from Group A and Group B. When the occupancy rate of the target bacteria includes the occupancy rate of two or more bacteria selected from Group A and Group B, each of the occupancy rates of the two or more bacteria can be used as an index for evaluating the intestinal barrier function of the subject.

[0049] In one embodiment, the occupancy of the target bacteria includes the occupancy of one or more bacteria selected from Group A and does not include the occupancy of any bacteria in Group B.

[0050] In another embodiment, the occupancy of the target bacteria includes an occupancy of one or more bacteria selected from Group B and does not include an occupancy of any bacteria in Group A.

[0051] In yet another embodiment, the occupancy of the target bacteria comprises an occupancy of one or more bacteria selected from Group A and an occupancy of one or more bacteria selected from Group B.

[0052] The "occupancy rate of one or more bacteria selected from group A and group B" means the occupancy rate of one or more bacteria selected from the following group C and group D. Group C corresponds to group A, and group D corresponds to group B.[Group C] Ruminococcaceae bacteria occupancy Peptostreptococcaceae bacteria occupancy Rikenellaceae bacteria occupancy Eubacteriaceae bacteria occupancy Bacillaceae bacteria occupancy Clostridiaceae 1 bacteria occupancy [Group D] Peptostreptococcaceae Romboutsia bacteria occupancy Rikenellaceae Alistipes bacteria occupancy Lachnospiraceae Lachnospiraceae UCG-010 bacteria occupancy Ruminococcaceae Negativibacillus bacteria occupancy Lachnospiraceae [Ruminococcus] gauvreauii group bacteria occupancy Ruminococcaceae Ruminococcus 1 bacteria occupancy Ruminococcaceae Ruminococcaceae NK4A214 group bacteria occupancy Ruminococcaceae Ruminococcaceae UCG-002 bacteria occupancy Lachnospiraceae [Ruminococcus] Occupancy of gnavus group bacteria Ruminococcaceae Occupancy of Ruminococcaceae UCG-003 bacteria Ruminococcaceae Occupancy of Ruminococcaceae UCG-009 bacteria Eubacteriaceae Occupancy of Eubacterium bacteria Ruminococcaceae Occupancy of Faecalibacterium bacteria Bacillaceae Occupancy of Bacillus bacteria Ruminococcaceae [Eubacterium] Occupancy of coprostanoligenes group bacteria Peptostreptococcaceae Occupancy of Intestinibacter bacteria Ruminococcaceae Occupancy of Anaerotruncus bacteria Marinifilaceae Occupancy of Odoribacter bacteria Clostridiaceae 1 Occupancy of Clostridium sensu stricto bacteria.

[0053] Ruminococcaceae bacteria are the general term for bacteria belonging to the family Ruminococcaceae. The following bacteria are included in Ruminococcaceae bacteria. Ruminococcaceae Negativibacillus Ruminococcaceae Ruminococcus 1 Ruminococcaceae Ruminococcaceae NK4A214 group Ruminococcaceae Ruminococcaceae UCG-002 Ruminococcaceae Ruminococcaceae UCG-003 Ruminococcaceae Ruminococcaceae UCG-009 Ruminococcaceae Faecalibacterium Ruminococcaceae [Eubacterium] coprostanoligenes group Ruminococcaceae Anaerotruncus Ruminococcaceae Ruminococcaceae UCG-005 Ruminococcaceae DTU089 Ruminococcaceae Ruminiclostridium 6 Ruminococcaceae Ruminococcaceae UCG-013 Ruminococcaceae Ruminococcaceae UCG-010 Ruminococcaceae Ruminococcus 2 Ruminococcaceae Flavonifractor Ruminococcaceae Ruminiclostridium 5 Ruminococcaceae Oscillospira Ruminococcaceae Ruminiclostridium Ruminococcaceae Ruminococcaceae UCG-004 Ruminococcaceae Butyricicoccus Ruminococcaceae GCA-900066225 Ruminococcaceae Ruminococcaceae UCG-014 Ruminococcaceae Candidatus Soleaferrea RuminococcaceaeHydrogenoanaerobacterium Ruminococcaceae Oscillibacter Ruminococcaceae UBA1819 Ruminococcaceae Ruminiclostridium 9 Ruminococcaceae Anaerofilum Ruminococcaceae Papillibacter Ruminococcaceae Fournierella Ruminococcaceae CAG-352 Ruminococcaceae V9D2013 group Ruminococcaceae Subdoligranulum Ruminococcaceae Ruminococcaceae UCG-007 Ruminococcaceae Ruminiclostridium 1 Ruminococcaceae Fastidiosipila Ruminococcaceae Acetanaerobacterium Ruminococcaceae Acutalibacter Ruminococcaceae Anaerobacterium Ruminococcaceae Anaerofilum Ruminococcaceae Anaerotruncus Ruminococcaceae Dysosmobacter Ruminococcaceae Ercella Ruminococcaceae Ethanoligenens Ruminococcaceae Oscillospira Ruminococcaceae Papillibacter Ruminococcaceae Pseudobacteroides Ruminococcaceae Ruminococcus Ruminococcaceae Sporobacter Ruminococcaceae Subdoligranulum Ruminococcaceae Youngiibacter

[0054] The occupancy rate of Ruminococcaceae bacteria is the occupancy rate of all bacteria belonging to the family Ruminococcaceae. The occupancy rate of Ruminococcaceae bacteria is calculated based on the formula: (total number of bacteria belonging to the family Ruminococcaceae in feces) / (total number of bacteria from all families in feces).

[0055] Peptostreptococcaceae bacteria is a general term for bacteria belonging to the family Peptostreptococcaceae. Peptostreptococcaceae bacteria include the following bacteria: Peptostreptococcaceae Romboutsia Peptostreptococcaceae Intestinibacter Peptostreptococcaceae Peptoclostridium Peptostreptococcaceae Terrisporobacter Peptostreptococcaceae Peptostreptococcus Peptostreptococcaceae Acetoanaerobium Peptostreptococcaceae Clostridioides Peptostreptococcaceae Criibacterium Peptostreptococcaceae Filifactor Peptostreptococcaceae Fusibacter Peptostreptococcaceae Maledivibacter Peptostreptococcaceae Paramaledivibacter Peptostreptococcaceae Peptacetobacter Peptostreptococcaceae Peptoanaerobacter Peptostreptococcaceae Proteocatella Peptostreptococcaceae Sporacetigenium Peptostreptococcaceae Tepidibacter Peptostreptococcaceae Wukongibacter

[0056] The occupancy rate of Peptostreptococcaceae bacteria is the occupancy rate of all bacteria belonging to the Peptostreptococcaceae family. The occupancy rate of Peptostreptococcaceae bacteria is calculated based on the formula: (total number of bacteria belonging to the Peptostreptococcaceae family in feces) / (total number of bacteria from all families in feces).

[0057] Rikenellaceae bacteria is a general term for bacteria belonging to the family Rikenellaceae. Rikenellaceae bacteria include the following bacteria: Rikenellaceae Alistipes Rikenellaceae Rikenellaceae RC9 gut group Rikenellaceae Rikenella Rikenellaceae Anaerocella Rikenellaceae Gallalistipes Rikenellaceae Mucinivorans

[0058] The occupancy rate of Rikenellaceae bacteria is the occupancy rate of all bacteria belonging to the Rikenellaceae family. The occupancy rate of Rikenellaceae bacteria is calculated based on the formula: (total number of bacteria belonging to the Rikenellaceae family in feces) / (total number of bacteria from all families in feces).

[0059] Eubacteriaceae bacteria is a general term for bacteria belonging to the Eubacteriaceae family. Eubacteriaceae bacteria include the following bacteria: Eubacteriaceae Eubacterium Eubacteriaceae Anaerofustis Eubacteriaceae Acetobacterium Eubacteriaceae Alkalibacter Eubacteriaceae Alkalibaculum Eubacteriaceae Aminicella Eubacteriaceae Garciella Eubacteriaceae Intestinibacillus Eubacteriaceae Irregularibacter Eubacteriaceae Mogibacterium Eubacteriaceae Pseudoramibacter Eubacteriaceae Rhabdanaerobium

[0060] The occupancy rate of Eubacteriaceae bacteria is the occupancy rate of all bacteria belonging to the Eubacteriaceae family. The occupancy rate of Eubacteriaceae bacteria is calculated based on the formula: (total number of bacteria belonging to the Eubacteriaceae family in feces) / (total number of bacteria from all families in feces).

[0061] Bacillaceae bacteria are a general term for bacteria belonging to the Bacillaceae family. Bacillaceae bacteria include the following bacteria: Bacillaceae Bacillus Bacillaceae Caldibacillus Bacillaceae Geobacillus Bacillaceae Aeribacillus Bacillaceae Alkalibacillus Bacillaceae Amphibacillus Bacillaceae Anoxybacillus Bacillaceae Caldalkalibacillus Bacillaceae Cerasibacillus Bacillaceae Exiguobacterium Bacillaceae Filobacillus Bacillaceae Gracilibacillus Bacillaceae Halalkalibacillus Bacillaceae Halobacillus Bacillaceae Halolactibacillus Bacillaceae Jeotgalibacillus Bacillaceae Lentibacillus Bacillaceae Lysinibacillus Bacillaceae Marinibacillus Bacillaceae Oceanobacillus Bacillaceae Ornithinibacillus Bacillaceae Paraliobacillus Bacillaceae Paucisalibacillus Bacillaceae Pelagibacillus Bacillaceae Piscibacillus Bacillaceae Pontibacillus Bacillaceae Saccharococcus Bacillus Salibacillus Bacillaceae Salimicrobium Bacillaceae Salinibacillus Bacillaceae Salirhabdus Bacillus Bacillaceae Salsuginibacillus Bacillaceae TenuibacillusBacillaceae Terribacillus Bacillaceae Thalassobacillus Bacillaceae Ureibacillus Bacillaceae Virgibacillus Bacillaceae Vulcanibacillus

[0062] The Bacillaceae occupancy rate is the occupancy rate of all bacteria belonging to the Bacillaceae family. The Bacillaceae occupancy rate is calculated based on the formula: (total number of bacteria belonging to the Bacillaceae family in feces) / (total number of bacteria from all families in feces).

[0063] Clostridiaceae 1 bacteria is a general term for bacteria belonging to the family Clostridiaceae 1. Clostridiaceae 1 bacteria include the following bacteria: Clostridiaceae 1 Clostridium sensu stricto 1

[0064] The Clostridiaceae 1 bacterial occupancy is the percentage of all bacteria belonging to the family Clostridiaceae 1. The Clostridiaceae 1 bacterial occupancy is calculated based on the formula: (total number of bacteria belonging to the family Clostridiaceae 1 in feces) / (total number of bacteria from all families in feces).

[0065] Peptostreptococcaceae Romboutsia bacteria is a general term for bacteria belonging to the family Peptostreptococcaceae Romboutsia. The occupancy rate of Peptostreptococcaceae Romboutsia bacteria is the occupancy rate of all bacteria belonging to the family Peptostreptococcaceae Romboutsia. The occupancy rate of Peptostreptococcaceae Romboutsia bacteria is calculated based on the formula: (total number of bacteria belonging to Peptostreptococcaceae Romboutsia in feces) / (total number of bacteria of all genera in feces).

[0066] Rikenellaceae Alistipes bacteria is a general term for bacteria belonging to the Rikenellaceae Alistipes family. The occupancy rate of Rikenellaceae Alistipes bacteria is the occupancy rate of all bacteria belonging to Rikenellaceae Alistipes. The occupancy rate of Rikenellaceae Alistipes bacteria is calculated based on the formula: (total number of bacteria belonging to Rikenellaceae Alistipes in feces) / (total number of bacteria of all genera in feces).

[0067] Lachnospiraceae UCG-010 bacteria is a collective term for bacteria belonging to the family Lachnospiraceae UCG-010. The occupancy rate of Lachnospiraceae UCG-010 bacteria is the occupancy rate of all bacteria belonging to Lachnospiraceae UCG-010. The occupancy rate of Lachnospiraceae UCG-010 bacteria is calculated based on the formula: (total number of bacteria belonging to Lachnospiraceae UCG-010 in feces) / (total number of bacteria of all genera in feces).

[0068] Ruminococcaceae Negativibacillus bacteria is a general term for bacteria belonging to the Ruminococcaceae Negativibacillus family. The occupancy rate of Ruminococcaceae Negativibacillus bacteria is the occupancy rate of all bacteria belonging to Ruminococcaceae Negativibacillus. The occupancy rate of Ruminococcaceae Negativibacillus bacteria is calculated based on the formula: (total number of bacteria belonging to Ruminococcaceae Negativibacillus in feces) / (total number of bacteria of all genera in feces).

[0069] Lachnospiraceae [Ruminococcus] gauvreauii group bacteria is a collective term for bacteria belonging to the Lachnospiraceae [Ruminococcus] gauvreauii group. The occupancy rate of Lachnospiraceae [Ruminococcus] gauvreauii group bacteria is the occupancy rate of all bacteria belonging to the Lachnospiraceae [Ruminococcus] gauvreauii group. The occupancy rate of Lachnospiraceae [Ruminococcus] gauvreauii group bacteria is calculated using the formula: (total number of bacteria belonging to the Lachnospiraceae [Ruminococcus] gauvreauii group in feces) / (total number of bacteria of all genera in feces).

[0070] Ruminococcaceae Ruminococcus 1 bacteria is a general term for bacteria belonging to the Ruminococcaceae Ruminococcus 1. The occupancy rate of Ruminococcaceae Ruminococcus 1 bacteria is the occupancy rate of all bacteria belonging to the Ruminococcaceae Ruminococcus 1. The occupancy rate of Ruminococcaceae Ruminococcus 1 bacteria is calculated based on the formula: (total number of bacteria belonging to the Ruminococcaceae Ruminococcus 1 in feces) / (total number of bacteria of all genera in feces).

[0071] Ruminococcaceae NK4A214 group bacteria is a collective term for bacteria belonging to the Ruminococcaceae NK4A214 group. The occupancy rate of Ruminococcaceae NK4A214 group bacteria is the occupancy rate of all bacteria belonging to the Ruminococcaceae NK4A214 group. The occupancy rate of Ruminococcaceae NK4A214 group bacteria is calculated using the formula: (total number of bacteria belonging to the Ruminococcaceae NK4A214 group in feces) / (total number of bacteria of all genera in feces).

[0072] Ruminococcaceae UCG-002 bacteria is a collective term for bacteria belonging to the family Ruminococcaceae UCG-002. The occupancy rate of Ruminococcaceae UCG-002 bacteria is the occupancy rate of all bacteria belonging to Ruminococcaceae UCG-002. The occupancy rate of Ruminococcaceae UCG-002 bacteria is calculated based on the formula: (total number of bacteria belonging to Ruminococcaceae UCG-002 in feces) / (total number of bacteria of all genera in feces).

[0073] Lachnospiraceae [Ruminococcus] gnavus group bacteria is a collective term for bacteria belonging to the Lachnospiraceae [Ruminococcus] gnavus group. The percentage of Lachnospiraceae [Ruminococcus] gnavus group bacteria is the percentage of all bacteria belonging to the Lachnospiraceae [Ruminococcus] gnavus group. The percentage of Lachnospiraceae [Ruminococcus] gnavus group bacteria is calculated using the formula: (total number of bacteria belonging to the Lachnospiraceae [Ruminococcus] gnavus group in feces) / (total number of bacteria of all genera in feces).

[0074] Ruminococcaceae UCG-003 bacteria is a collective term for bacteria belonging to the family Ruminococcaceae UCG-003. The occupancy rate of Ruminococcaceae UCG-003 bacteria is the occupancy rate of all bacteria belonging to Ruminococcaceae UCG-003. The occupancy rate of Ruminococcaceae UCG-003 bacteria is calculated based on the formula: (total number of bacteria belonging to Ruminococcaceae UCG-003 in feces) / (total number of bacteria of all genera in feces).

[0075] Ruminococcaceae UCG-009 bacteria is a collective term for bacteria belonging to the family Ruminococcaceae UCG-009. The occupancy rate of Ruminococcaceae UCG-009 bacteria is the occupancy rate of all bacteria belonging to Ruminococcaceae UCG-009. The occupancy rate of Ruminococcaceae UCG-009 bacteria is calculated based on the formula: (total number of bacteria belonging to Ruminococcaceae UCG-009 in feces) / (total number of bacteria of all genera in feces).

[0076] Eubacteriaceae Eubacterium bacteria is a general term for bacteria belonging to the Eubacteriaceae Eubacterium family. The occupancy rate of Eubacteriaceae Eubacterium bacteria is the occupancy rate of all bacteria belonging to the Eubacteriaceae Eubacterium family. The occupancy rate of Eubacteriaceae Eubacterium bacteria is calculated based on the formula: (total number of bacteria belonging to the Eubacteriaceae Eubacterium family in feces) / (total number of bacteria of all genera in feces).

[0077] Ruminococcaceae Faecalibacterium bacteria is a general term for bacteria belonging to the Ruminococcaceae Faecalibacterium family. The occupancy rate of Ruminococcaceae Faecalibacterium bacteria is the occupancy rate of all bacteria belonging to the Ruminococcaceae Faecalibacterium family. The occupancy rate of Ruminococcaceae Faecalibacterium bacteria is calculated based on the formula: (total number of bacteria belonging to the Ruminococcaceae Faecalibacterium family in feces) / (total number of bacteria of all genera in feces).

[0078] Bacillaceae Bacillus is a general term for bacteria belonging to the Bacillaceae family. The occupancy rate of Bacillaceae Bacillus is the occupancy rate of all bacteria belonging to the Bacillaceae family. The occupancy rate of Bacillaceae Bacillus is calculated based on the formula: (total number of bacteria belonging to the Bacillaceae Bacillus in feces) / (total number of bacteria of all genera in feces).

[0079] Ruminococcaceae [Eubacterium] coprostanoligenes group bacteria is a collective term for bacteria belonging to the Ruminococcaceae [Eubacterium] coprostanoligenes group. The occupancy rate of Ruminococcaceae [Eubacterium] coprostanoligenes group bacteria is the occupancy rate of all bacteria belonging to the Ruminococcaceae [Eubacterium] coprostanoligenes group. The occupancy rate of Ruminococcaceae [Eubacterium] coprostanoligenes group bacteria is calculated using the formula: (total number of bacteria belonging to the Ruminococcaceae [Eubacterium] coprostanoligenes group in feces) / (total number of bacteria of all genera in feces).

[0080] Peptostreptococcaceae Intestinibacter bacteria is a general term for bacteria belonging to the Peptostreptococcaceae Intestinibacter family. The occupancy rate of Peptostreptococcaceae Intestinibacter bacteria is the occupancy rate of all bacteria belonging to the Peptostreptococcaceae Intestinibacter family. The occupancy rate of Peptostreptococcaceae Intestinibacter bacteria is calculated based on the formula: (total number of bacteria belonging to Peptostreptococcaceae Intestinibacter in feces) / (total number of bacteria of all genera in feces).

[0081] Ruminococcaceae Anaerotruncus bacteria is a general term for bacteria belonging to the family Ruminococcaceae Anaerotruncus. The occupancy rate of Ruminococcaceae Anaerotruncus bacteria is the occupancy rate of all bacteria belonging to Ruminococcaceae Anaerotruncus. The occupancy rate of Ruminococcaceae Anaerotruncus bacteria is calculated based on the formula: (total number of bacteria belonging to Ruminococcaceae Anaerotruncus in feces) / (total number of bacteria of all genera in feces).

[0082] Marinifilaceae Odoribacter bacteria is a general term for bacteria belonging to the family Marinifilaceae Odoribacter. The occupancy rate of Marinifilaceae Odoribacter bacteria is the occupancy rate of all bacteria belonging to Marinifilaceae Odoribacter. The occupancy rate of Marinifilaceae Odoribacter bacteria is calculated based on the formula: (total number of bacteria belonging to Marinifilaceae Odoribacter in feces) / (total number of bacteria of all genera in feces).

[0083] Clostridiaceae 1 Clostridium sensu stricto bacteria is a collective term for bacteria belonging to Clostridiaceae 1 Clostridium sensu stricto. The occupancy rate of Clostridiaceae 1 Clostridium sensu stricto bacteria is the occupancy rate of all bacteria belonging to Clostridiaceae 1 Clostridium sensu stricto. The occupancy rate of Clostridiaceae 1 Clostridium sensu stricto bacteria is calculated based on the formula: (total number of bacteria belonging to Clostridiaceae 1 Clostridium sensu stricto in feces) / (total number of bacteria of all genera in feces).

[0084] In the formula used to calculate the occupancy rate of each bacterium, the "number of bacteria" and the "total number of bacteria" may be absolute or relative amounts, but are usually relative amounts. For example, the "number of bacteria" and the "total number of bacteria" obtained by bacterial flora analysis using 16S rRNA amplicon sequencing, which will be described later, are relative amounts.

[0085] The occupancy rates of Ruminococcaceae bacteria, Peptostreptococcaceae bacteria, Rikenellaceae bacteria, Peptostreptococcaceae Romboutsia bacteria, and Rikenellaceae Alistipes bacteria are particularly highly accurate in evaluating intestinal barrier function. Therefore, from the viewpoint of improving the accuracy of evaluating the intestinal barrier function of a subject, the occupancy rate of the target bacteria preferably includes one or more occupancies selected from the group consisting of Ruminococcaceae bacteria, Peptostreptococcaceae bacteria, Rikenellaceae bacteria, Peptostreptococcaceae Romboutsia bacteria, and Rikenellaceae Alistipes bacteria, and more preferably includes two or more (specifically, 2, 3, 4, or 5) occupancies selected from the group. The occupancy rate of the target bacteria may include one or more occupancies selected from the group in question, as well as one or more occupancies of other bacteria selected from groups A and B.

[0086] In one embodiment, the occupancy of the target bacteria includes an occupancy of one selected from the group consisting of an occupancy of Ruminococcaceae bacteria, an occupancy of Peptostreptococcaceae bacteria, an occupancy of Rikenellaceae bacteria, an occupancy of Peptostreptococcaceae Romboutsia bacteria, and an occupancy of Rikenellaceae Alistipes bacteria. In addition to the occupancy of one selected from this group, the occupancy of the target bacteria may also include an occupancy of one or more other bacteria selected from Group A and Group B. In one example, the occupancy of one selected from this group is an occupancy of Ruminococcaceae bacteria. In another example, the occupancy of one selected from this group is an occupancy of Peptostreptococcaceae bacteria. In yet another example, the occupancy of one selected from this group is an occupancy of Rikenellaceae bacteria. In yet another example, the occupancy of one selected from this group is an occupancy of Peptostreptococcaceae Romboutsia bacteria. In yet another example, the occupancy of a selected one from the group is the occupancy of Rikenellaceae Alistipes bacteria.

[0087] In another embodiment, the occupancy of the target bacteria includes 2, 3, 4, or 5 occupancies selected from the group consisting of occupancy of Ruminococcaceae bacteria, occupancy of Peptostreptococcaceae bacteria, occupancy of Rikenellaceae bacteria, occupancy of Peptostreptococcaceae Romboutsia bacteria, and occupancy of Rikenellaceae Alistipes bacteria. In addition to 2, 3, 4, or 5 occupancies selected from the group, the occupancy of the target bacteria may also include occupancy of one or more other bacteria selected from Groups A and B.

[0088] The method according to aspect 1 includes the following steps: (1a) measuring the occupancy rate of target bacteria in feces derived from a subject to obtain a measurement value of the occupancy rate of the target bacteria; and (1b) comparing the measurement value obtained in step 1a with a reference value to obtain a comparison result; and (1c) evaluating the intestinal barrier function of the subject based on the comparison result obtained in step 1b.

[0089] <Step 1a> Step 1a is a step of measuring the occupancy rate of target bacteria in feces derived from a subject and obtaining a measurement value of the occupancy rate of target bacteria.

[0090] When the occupancy rate of the target bacteria includes the occupancy rate of one bacterium selected from group A and group B, in step 1a, the occupancy rate of the one bacterium is measured.

[0091] When the occupancy rate of the target bacteria includes the occupancy rates of two or more bacteria selected from Group A and Group B, Step 1a involves measuring the occupancy rates of the two or more bacteria. "Measuring the occupancy rates of two or more bacteria" encompasses measuring the occupancy rates of each of the two or more bacteria and determining a combined variable, which is a statistical analysis value, from the occupancy rates of the two or more bacteria.

[0092] The occupancy rate of the target bacteria can be measured according to a conventional method. The occupancy rate of the target bacteria is preferably measured by bacterial flora analysis using 16S rRNA amplicon sequencing. That is, the measured value obtained in step 1a is preferably a value obtained by bacterial flora analysis (metagenomic analysis) using 16S rRNA amplicon sequencing. Bacterial flora analysis using 16S rRNA amplicon sequencing can be performed, for example, as follows.

[0093] First, a sample is prepared from feces derived from a subject. The sample is, for example, a diluted feces solution obtained by suspending feces in a solvent (e.g., ultrapure water) and, if necessary, subjecting the feces to disruption treatment.

[0094] Bacterial genomic DNA is extracted from a sample, a predetermined region of the 16S rRNA gene is amplified by PCR, and the PCR amplification product is purified as needed. DNA extraction can be performed using a commercially available kit. Primer design, amplification condition setting, PCR amplification product purification, and the like 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 enables identification and composition analysis of bacteria in the bacterial flora, and examples include regions containing the variable region of the 16S rRNA gene. Examples of regions containing the variable region of the 16S rRNA gene include regions containing the V3-V4 region, regions containing the V1-V2 region, regions containing the V1-V4 region, and regions containing the V4 region. Of these, regions containing the V3-V4 region are preferred.

[0095] The resulting PCR amplification products (amplicons) are subjected to index PCR using primers with a unique base sequence (index) for each sample, and a sample identification index is added to the PCR amplification products of each sample. Primer selection, amplification conditions, etc. can be performed according to standard methods.

[0096] The indexed PCR amplification products (library) are sequenced. Sequence analysis is preferably performed using a next-generation sequencer (e.g., Illumina MiSeq). After quality filtering, the sequences are clustered and ASV (Amplicon Sequence Variant) analysis is performed. An ASV is an operational classification unit that treats identical sequences as a single bacterial species after correcting error sequences to the true sequence. The sequence of each ASV is compared with a bacterial sequence database (reference database) to identify and analyze the composition of the bacteria in the bacterial flora contained in the sample.

[0097] The number of ASVs represents the number of types of bacteria that make up the bacterial flora contained in the sample, and the number of reads belonging to the same ASV represents the relative abundance of a specific type of bacteria. Therefore, the occupancy rate of a specific type of bacteria in the bacterial flora contained in the sample can be calculated based on the number of reads belonging to the same ASV.

[0098] The occupancy rate of the target bacterium based on ASV analysis can be calculated using commercially available 16S rRNA phylogenetic analysis software. For example, the occupancy rate of the target bacterium can be calculated by analyzing a file (fastq format) obtained by sequence analysis using a next-generation sequencer (e.g., Illumina MiSeq) using a next-generation microbiome bioinformatics platform such as QIIME2 (https: / / qiime2.org).

[0099] When performing bacterial flora analysis using 16S rRNA amplicon sequencing, Sanschagrin S, Yergeau E. Next-generation sequencing of 16S ribosomal RNA gene amplicons. J Vis Exp. 2014; (90): 51709. Published 2014 Aug 29. doi: 10.3791 / 51709 and other information can be referenced.

[0100] When the occupancy rate of the target bacteria is measured by microbiota analysis using 16S rRNA amplicon sequencing, step 1a may include sequence analysis of the indexed PCR amplification product (library) and subsequent processing. Step 1a may or may not 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 to be used as a specimen from feces excreted by the subject), preparation of a sample from the feces from the subject, extraction of bacterial genomic DNA from the sample, amplification of a predetermined region of the 16S rRNA gene by PCR (e.g., a region including the V3-V4 region), purification of the PCR amplification product, addition of an index to the PCR amplification product by index PCR, etc.

[0101] When measuring the occupancy rates of two or more bacteria selected from group A and group B (first occupancy rate, second occupancy rate, ..., nth occupancy rate (n is an integer of 2 or more)), and determining a combined variable, which is a statistical analysis value, from the occupancy rates of the two or more bacteria, it is preferable to determine the combined variable from the following formula (1): Combined variable = (coefficient 1 x (first occupancy rate)) + (coefficient 2 x (second occupancy rate)) + ... + (coefficient n x (nth occupancy rate)) + constant term ... (1)

[0102] There are no particular limitations on n as long as it is an integer of 2 or more, but it is, for example, an integer of 2 or more and 6 or less.

[0103] Each coefficient and constant term varies depending on the number and type of occupancies to be combined. Each coefficient and constant term can be determined by multinomial logistic regression analysis. Each coefficient is a partial regression coefficient determined by multinomial logistic regression analysis. A method for determining each coefficient and constant term by multinomial logistic regression analysis is well known to those skilled in the art. Regarding this method, for example, 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, pp. 1-10 (http: / / dx.doi.org / 10.21037 / atm.2019.07.102) and other literature can be referenced. The coefficients used to determine the combination variables can be derived from the measurement data so that the combination variables are close to 1 for groups with impaired intestinal barrier function and close to 0 for groups without impaired intestinal barrier function (groups with normal intestinal barrier function).

[0104] When the occupancy rates of Ruminococcaceae bacteria, Peptostreptococcaceae bacteria, Peptostreptococcaceae Romboutsia bacteria, and Rikenellaceae bacteria are combined, a combination variable can be determined from the four occupancies based on the above formula (1) using, for example, the coefficients and constant terms shown in Table 1. In Table 1, "R1" represents the occupancy rate of Ruminococcaceae bacteria, "R2" represents the occupancy rate of Peptostreptococcaceae bacteria, "R3" represents the occupancy rate of Peptostreptococcaceae Romboutsia bacteria, and "R4" represents the occupancy rate of Rikenellaceae bacteria.

[0105]

[0106] <Step 1b> Step 1b is a step of comparing the measurement value obtained in step 1a with a reference value and obtaining the comparison result.

[0107] When the occupancy rate of one bacterium selected from group A and group B is measured in step 1a, the measured value of the occupancy rate of the one bacterium is compared with a reference value of the occupancy rate of the one bacterium to obtain a comparison result in step 1b. When the measured value of the occupancy rate of the one bacterium is a value obtained by bacterial flora analysis using 16S rRNA amplicon sequencing, the reference value of the occupancy rate of the one bacterium is also a value obtained by bacterial flora analysis using 16S rRNA amplicon sequencing.

[0108] When the occupancy rates of two or more bacteria selected from group A and group B are measured in step 1a, the measured values ​​of the occupancy rates of the two or more bacteria are compared with the reference values ​​of the occupancy rates of the two or more bacteria to obtain a comparison result in step 1b. When the measured values ​​of the occupancy rates of the two or more bacteria are values ​​obtained by bacterial biota analysis using 16S rRNA amplicon sequencing, the reference values ​​of the occupancy rates of the two or more bacteria are also values ​​obtained by bacterial biota analysis using 16S rRNA amplicon sequencing.

[0109] In step 1a, when a combination variable is determined from the occupancy rates of two or more bacteria selected from group A and group B, preferably a combination variable determined based on the above formula (1), in step 1b, the combination variable is compared with a reference value for the combination variable to obtain a comparison result. If the combination variable is based on a value obtained by bacterial biota analysis using 16S rRNA amplicon sequencing, the reference value for the combination variable is also based on a value obtained by bacterial biota analysis using 16S rRNA amplicon sequencing.

[0110] <Step 1c> Step 1c is a step of evaluating the intestinal barrier function of the subject based on the comparison results obtained in step 1b.

[0111] In Aspect 1, "positive" means that the intestinal barrier function of the subject is impaired, and "negative" means that the intestinal barrier function of the subject is not impaired.

[0112] The intestinal barrier function to be evaluated may be the intestinal barrier function of the small intestine or the intestinal barrier function of the large intestine, but is preferably the intestinal barrier function of the small intestine.

[0113] The intestinal barrier function to be evaluated is, in particular, the physical barrier function such as the tight junction structure.

[0114] If the comparison result obtained in step 1b is a comparison result between the measured occupancy rate of one bacterium selected from group A and group B and the reference value for the occupancy rate of that one bacterium, the comparison result is used to evaluate the intestinal barrier function of the subject.

[0115] When the comparison result obtained in step 1b is a comparison result between the respective measured values ​​of the occupancy rates of two or more bacteria selected from group A and group B and the respective reference values ​​of the occupancy rates of the two or more bacteria, it is preferable to evaluate the intestinal barrier function of the subject using the comparison result regarding the occupancy rates of all bacteria included in the comparison result, but the intestinal barrier function of the subject may also be evaluated using the comparison result regarding the occupancy rate of a portion of bacteria (one or two or more bacteria) arbitrarily selected from the comparison result.

[0116] If the comparison result obtained in step 1b is a comparison result between a combination variable calculated from the occupancy rate of two or more bacteria selected from group A and group B and a reference value of the combination variable, the comparison result is used to evaluate the intestinal barrier function of the subject.

[0117] The intestinal barrier function of a subject can be evaluated based on whether the comparison results obtained in step 1b for the occupancy rate of each bacterium satisfy the criteria shown in Table 2. Specifically, for the occupancy rate of each bacterium for which the criteria shown in Table 2 are "high," a positive evaluation can be obtained if the measured value obtained in step 1a is higher than the reference value (i.e., the comparison result obtained in step 1b satisfies the criteria shown in Table 2). A negative evaluation can be obtained if the measured value obtained in step 1a is equal to or lower than the reference value (i.e., the comparison result obtained in step 1b does not satisfy the criteria shown in Table 2). Furthermore, for the occupancy rate of each bacterium for which the criteria shown in Table 2 are "low," a positive evaluation can be obtained if the measured value obtained in step 1a is lower than the reference value (i.e., the comparison result obtained in step 1b satisfies the criteria shown in Table 2). A negative evaluation can be obtained if the measured value obtained in step 1a is equal to or higher than the reference value (i.e., the comparison result obtained in step 1b does not satisfy the criteria shown in Table 2). The criteria shown in Table 2 can also be applied to aspects other than Aspect 1.

[0118] In step 1a, when the occupancy rate of one bacterium selected from group A and group B is measured, if the comparison result of the occupancy rate of that one bacterium satisfies the criteria shown in Table 2, it can be evaluated as positive, and if the comparison result of the occupancy rate of that one bacterium does not satisfy the criteria shown in Table 2, it can be evaluated as negative.

[0119] In step 1a, when the occupancy rates of two or more bacteria selected from group A and group B are measured, if the comparison result of the occupancy rate of at least one of the two or more bacteria satisfies the criteria shown in Table 2, the result can be evaluated as positive, and if the comparison result of none of the two or more bacteria satisfies the criteria shown in Table 2, the result can be evaluated as negative. The more occupancies of bacteria whose comparison results meet the criteria shown in Table 2, the more likely the result is positive. Therefore, by measuring the occupancy rates of two or more (e.g., two, three, or four or more) bacteria selected from group A and group B in step 1a, the accuracy of the evaluation of the subject's intestinal barrier function can be improved.

[0120]

[0121] The reference value can be set according to a conventional method.

[0122] In one embodiment, regarding the occupancy rate of each bacterium selected from group A or group B, a cutoff value obtained from an ROC (Receiver Operating Characteristic) curve of the occupancy rate of each bacterium can be used as a reference value for the occupancy rate of each bacterium. This embodiment (hereinafter referred to as "Embodiment 1-1") will be described below.

[0123] The ROC curve for the occupancy rate of each bacterium can be created according to standard methods based on the occupancy rate of each bacterium in feces obtained from a negative group (a group of individuals without impaired intestinal barrier function) and a positive group (a group of individuals with impaired intestinal barrier function) consisting of individuals of the same species as the subject (for example, humans if the subject is human). In the ROC curve, the vertical axis represents sensitivity and the horizontal axis represents (1 - specificity). When forming the negative and positive groups, discrimination between negative and positive can be performed using a known method for testing intestinal barrier function (for example, the lactulose-mannitol test).

[0124] The cutoff value is a value that can satisfy both high sensitivity and high specificity when evaluation is performed using that value as a standard. The cutoff value can be set appropriately taking into consideration the balance between sensitivity and specificity. For example, a value that shows a high positive rate in the positive group and a high negative rate in the negative group can be set as the cutoff value. Methods for setting cutoff values ​​are well known to those skilled in the art. For example, a method is known in which the point on the ROC curve that is the shortest distance from the upper left corner of the ROC curve is set as the cutoff value, or a method is known in which the point on the ROC curve where the value of (sensitivity + specificity - 1) is the maximum is set as the cutoff value. The point on the ROC curve where the value of (sensitivity + specificity - 1) is the maximum is called the Younden index.

[0125] Specific examples of cutoff values ​​for the occupancy rates of each of the bacteria selected from group A and group B are shown in Table 3.

[0126]

[0127] For the occupancy rate of Ruminococcaceae bacteria, the preferred cutoff value is a value within the range of 15.0 to 25.0, the more preferred cutoff value is a value within the range of 17.0 to 23.0, and the even more preferred cutoff value is a value within the range of 20.0 to 22.0.

[0128] For the occupancy of Peptostreptococcaceae bacteria, the preferred cutoff value is a value within the range of 0.500 to 1.300, the more preferred cutoff value is a value within the range of 0.600 to 1.280, and the even more preferred cutoff value is a value within the range of 0.700 to 0.800.

[0129] For the occupancy of Rikenellaceae bacteria, the preferred cutoff value is a value within the range of 0.400 to 1.300, the more preferred cutoff value is a value within the range of 0.500 to 1.200, and the even more preferred cutoff value is a value within the range of 0.600 to 0.800.

[0130] For the occupancy of Eubacteriaceae bacteria, the preferred cutoff value is a value within the range of 0.010 to 0.040, the more preferred cutoff value is a value within the range of 0.015 to 0.030, and the even more preferred cutoff value is a value within the range of 0.020 to 0.025.

[0131] For the occupancy of Bacillaceae bacteria, the preferred cutoff value is a value within the range of 0.005 to 0.030, the more preferred cutoff value is a value within the range of 0.007 to 0.020, and the even more preferred cutoff value is a value within the range of 0.010 to 0.020.

[0132] For Clostridiaceae 1 bacterial occupancy, the preferred cutoff value is a value within the range of 0.010 to 0.050, the more preferred cutoff value is a value within the range of 0.020 to 0.040, and the even more preferred cutoff value is a value within the range of 0.030 to 0.035.

[0133] For the occupancy of Peptostreptococcaceae Romboutsia bacteria, the preferred cutoff value is a value within the range of 0.400 to 0.800, the more preferred cutoff value is a value within the range of 0.450 to 0.700, and the even more preferred cutoff value is a value within the range of 0.500 to 0.550.

[0134] For the occupancy of Rikenellaceae Alistipes bacteria, the preferred cutoff value is a value within the range of 0.500 to 1.000, the more preferred cutoff value is a value within the range of 0.550 to 0.900, and the even more preferred cutoff value is a value within the range of 0.600 to 0.800.

[0135] For the occupancy of Lachnospiraceae Lachnospiraceae UCG-010 bacteria, the preferred cutoff value is a value within the range of 0.005 to 0.020, the more preferred cutoff value is a value within the range of 0.008 to 0.015, and the even more preferred cutoff value is a value within the range of 0.009 to 0.013.

[0136] For the occupancy rate of Ruminococcaceae Negativibacillus bacteria, the preferred cutoff value is a value within the range of 0.020 to 0.060, the more preferred cutoff value is a value within the range of 0.030 to 0.050, and the even more preferred cutoff value is a value within the range of 0.039 to 0.045.

[0137] With respect to the occupancy rate of Lachnospiraceae [Ruminococcus] gauvreauii group bacteria, a preferred cutoff value is a value within the range of 0.010 to 0.090, a more preferred cutoff value is a value within the range of 0.015 to 0.060, and an even more preferred cutoff value is a value within the range of 0.019 to 0.025.

[0138] For the occupancy rate of Ruminococcaceae Ruminococcus 1 bacteria, the preferred cutoff value is a value within the range of 0.060 to 0.230, the more preferred cutoff value is a value within the range of 0.070 to 0.100, and the even more preferred cutoff value is a value within the range of 0.080 to 0.090.

[0139] With respect to the occupancy rate of Ruminococcaceae Ruminococcaceae NK4A214 group bacteria, the preferred cutoff value is within the range of 0.040 to 0.140, the more preferred cutoff value is within the range of 0.050 to 0.100, and the even more preferred cutoff value is within the range of 0.050 to 0.090.

[0140] For the occupancy of Ruminococcaceae Ruminococcaceae UCG-002 bacteria, the preferred cutoff value is a value within the range of 0.009 to 0.450, the more preferred cutoff value is a value within the range of 0.100 to 0.300, and the even more preferred cutoff value is a value within the range of 0.120 to 0.150.

[0141] For the occupancy rate of Lachnospiraceae [Ruminococcus] gnavus group bacteria, a preferred cutoff value is a value within the range of 0.100 to 0.400, a more preferred cutoff value is a value within the range of 0.150 to 0.300, and an even more preferred cutoff value is a value within the range of 0.170 to 0.280.

[0142] For the occupancy of Ruminococcaceae Ruminococcaceae UCG-003 bacteria, the preferred cutoff value is a value within the range of 0.008 to 0.020, the more preferred cutoff value is a value within the range of 0.009 to 0.015, and the even more preferred cutoff value is a value within the range of 0.010 to 0.013.

[0143] For the occupancy of Ruminococcaceae Ruminococcaceae UCG-009 bacteria, the preferred cutoff value is a value within the range of 0.008 to 0.020, the more preferred cutoff value is a value within the range of 0.009 to 0.015, and the even more preferred cutoff value is a value within the range of 0.010 to 0.013.

[0144] For the occupancy of Eubacteriaceae Eubacterium bacteria, the preferred cutoff value is a value within the range of 0.008 to 0.020, the more preferred cutoff value is a value within the range of 0.009 to 0.015, and the even more preferred cutoff value is a value within the range of 0.010 to 0.013.

[0145] For the occupancy of Ruminococcaceae Faecalibacterium bacteria, the preferred cutoff value is a value within the range of 5.00 to 7.80, the more preferred cutoff value is a value within the range of 6.00 to 6.70, and the even more preferred cutoff value is a value within the range of 6.20 to 6.50.

[0146] For the occupancy rate of Bacillaceae Bacillus bacteria, the preferred cutoff value is a value within the range of 0.005 to 0.030, the more preferred cutoff value is a value within the range of 0.007 to 0.020, and the even more preferred cutoff value is a value within the range of 0.010 to 0.020.

[0147] For the occupancy rate of Ruminococcaceae [Eubacterium] coprostanoligenes group bacteria, the preferred cutoff value is a value within the range of 0.90 to 1.40, the more preferred cutoff value is a value within the range of 1.00 to 1.37, and the even more preferred cutoff value is a value within the range of 1.22 to 1.26.

[0148] For the occupancy of Peptostreptococcaceae Intestinibacter bacteria, the preferred cutoff value is a value within the range of 0.070 to 0.160, the more preferred cutoff value is a value within the range of 0.080 to 0.140, and the even more preferred cutoff value is a value within the range of 0.090 to 0.120.

[0149] For the occupancy of Ruminococcaceae Anaerotruncus bacteria, the preferred cutoff value is a value within the range of 0.010 to 0.035, the more preferred cutoff value is a value within the range of 0.015 to 0.030, and the even more preferred cutoff value is a value within the range of 0.020 to 0.025.

[0150] For the occupancy of Marinifilaceae Odoribacter bacteria, the preferred cutoff value is a value within the range of 0.030 to 0.085, the more preferred cutoff value is a value within the range of 0.040 to 0.080, and the even more preferred cutoff value is a value within the range of 0.045 to 0.060.

[0151] For the prevalence of Clostridiaceae 1 Clostridium sensu stricto bacteria, the preferred cutoff value is a value within the range of 0.015 to 0.045, the more preferred cutoff value is a value within the range of 0.020 to 0.040, and the even more preferred cutoff value is a value within the range of 0.025 to 0.035.

[0152] In another embodiment, the occupancy rates of two or more bacteria selected from Group A or Group B are measured in feces obtained from a negative group (a group of individuals whose intestinal barrier function is not impaired) and a positive group (a group of individuals whose intestinal barrier function is impaired) consisting of individuals of the same species as the subject (for example, if the subject is human, then humans), and a combination variable is determined from the occupancy rates of the two or more bacteria based on the above formula (1). An ROC curve is created based on the determined combination variable, and the cutoff value determined from the created ROC curve can be used as a reference value for the occupancy rates of the two or more bacteria. This embodiment (hereinafter referred to as "Embodiment 1-2") will be described below.

[0153] Embodiment 1-2 is applied when the measurement value obtained in step 1a is a combination variable obtained from the occupancy rate of two or more bacteria selected from group A or group B based on the above formula (1).

[0154] When forming the negative group and the positive group, the discrimination between negative and positive can be performed using a known test method for intestinal barrier function (e.g., lactulose-mannitol test). The ROC curve can be created in a standard manner based on the combined variables obtained from the negative group and the positive group. In the ROC curve, the vertical axis represents sensitivity and the horizontal axis represents (1-specificity). When forming the negative group and the positive group, the discrimination between negative and positive can be performed using a known test method for intestinal barrier function (e.g., lactulose-mannitol test). The explanation regarding the cutoff value is the same as in embodiment 1-1.

[0155] When the occupancy rates of Ruminococcaceae bacteria, Peptostreptococcaceae bacteria, Peptostreptococcaceae Romboutsia bacteria, and Rikenellaceae bacteria are combined, specific examples of coefficients and constant terms are as shown in Table 1, and specific examples of cutoff values ​​are as shown in Table 4. In Table 4, "R1," "R2," "R3," and "R4" have the same meanings as in Table 1.

[0156]

[0157] For No. 1 in Table 4, the preferred cutoff value is within the range of −1.300 to −0.090, the more preferred cutoff value is within the range of −1.240 to −0.100, and the even more preferred cutoff value is within the range of −0.142 to −0.140.

[0158] For No. 2 in Table 4, the preferred cutoff value is within the range of −1.300 to −0.050, the more preferred cutoff value is within the range of −1.240 to −0.090, and the even more preferred cutoff value is within the range of −0.151 to −0.100.

[0159] For No. 3 in Table 4, a preferred cutoff value is within the range of −1.000 to −0.100, a more preferred cutoff value is within the range of −0.410 to −0.150, and an even more preferred cutoff value is within the range of −0.300 to −0.254.

[0160] For No. 4 in Table 4, the preferred cutoff value is a value within the range of −0.470 to −0.030, the more preferred cutoff value is a value within the range of −0.250 to −0.040, and the even more preferred cutoff value is a value within the range of −0.072 to −0.060.

[0161] For No. 5 in Table 4, the preferred cutoff value is a value within the range of −0.300 to 0.600, the more preferred cutoff value is a value within the range of 0.070 to 0.500, and the even more preferred cutoff value is a value within the range of 0.100 to 0.441.

[0162] For No. 6 in Table 4, the preferred cutoff value is within the range of −1.000 to −0.100, the more preferred cutoff value is within the range of −0.420 to −0.150, and the even more preferred cutoff value is within the range of −0.200 to −0.254.

[0163] For No. 7 in Table 4, the preferred cutoff value is within the range of −1.000 to −0.050, the more preferred cutoff value is within the range of −0.500 to −0.070, and the even more preferred cutoff value is within the range of −0.420 to −0.090.

[0164] For No. 8 in Table 4, the preferred cutoff value is a value within the range of −0.090 to 0.500, the more preferred cutoff value is a value within the range of 0.220 to 0.450, and the even more preferred cutoff value is a value within the range of 0.343 to 0.400.

[0165] For No. 9 in Table 4, a preferred cutoff value is within the range of −0.600 to −0.300, a more preferred cutoff value is within the range of −0.570 to −0.350, and an even more preferred cutoff value is within the range of −0.480 to −0.441.

[0166] For No. 10 in Table 4, the preferred cutoff value is a value within the range of −0.390 to 0.600, the more preferred cutoff value is a value within the range of 0.100 to 0.450, and the even more preferred cutoff value is a value within the range of 0.130 to 0.411.

[0167] For No. 11 in Table 4, the preferred cutoff value is within the range of 0.030 to 0.350, the more preferred cutoff value is within the range of 0.080 to 0.250, and the even more preferred cutoff value is within the range of 0.100 to 0.225.

[0168] In yet another embodiment, with regard to the occupancy rate of each bacterium selected from Group A or Group B, the average 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 impaired) consisting of individuals of the same species as the subject (for example, if the subject is human, then humans) can be used as a reference value for the occupancy rate of each bacterium. This embodiment (hereinafter referred to as "Embodiments 1-3") will be described below.

[0169] When forming a negative group, discrimination between negative and positive results can be carried out by a known method for testing intestinal barrier function (e.g., lactulose-mannitol test).

[0170] For the occupancy rate of each bacteria with a "high" standard shown in Table 2, if the measured value obtained in step 1a is higher than the average value, it can be evaluated as positive. It is preferable to evaluate the measured value obtained in step 1a as positive if it is 1.05 times or more the average value, more preferably to evaluate the measured value obtained in step 1a as positive if it is 1.1 times or more the average value, and even more preferably to evaluate the measured value obtained in step 1a as positive if it is 1.5 times or more the average value.

[0171] For the occupancy rate of each bacteria with a "low" standard shown in Table 2, if the measured value obtained in step 1a is lower than the average value, it can be evaluated as positive. It is preferable to evaluate the measured value obtained in step 1a as positive if it is 0.9 times or less the average value, more preferably to evaluate the measured value obtained in step 1a as positive if it is 0.7 times or less the average value, and even more preferably to evaluate the measured value obtained in step 1a as positive if it is 0.5 times or less the average value.

[0172] In yet another embodiment, with regard to the occupancy rate of each bacterium selected from Group A or Group B, the median of the occupancy rate of each bacterium in feces obtained from a negative group (a group of individuals whose intestinal barrier function is not impaired) consisting of individuals of the same species as the subject (for example, if the subject is human, then humans) can be used as a reference value for the occupancy rate of each bacterium. This embodiment (hereinafter referred to as "Embodiments 1-4") will be described below.

[0173] Unless otherwise specified, the explanations regarding Embodiments 1 to 3 also apply to Embodiments 1 to 4. When applying, "average value" is read as "median value."

[0174] Aspect 2 of the present invention relates to a method for diagnosing a disease or symptom caused by a decrease in intestinal barrier function in a subject. Hereinafter, a "disease or symptom caused by a decrease in intestinal barrier function" may be referred to as a "disease or symptom".

[0175] The method according to aspect 2 of the present invention comprises the following steps: (2a) measuring the occupancy rate of target bacteria in feces derived from a subject to obtain a measurement value of the occupancy rate of the target bacteria; (2b) comparing the measurement value obtained in step 2a with a reference value to obtain a comparison result; and (2c) diagnosing a disease or symptom caused by a decrease in intestinal barrier function in the subject based on the comparison result obtained in step 2b, wherein the occupancy rate of the target bacteria comprises the occupancy rate of one or more bacteria selected from group A and group B.

[0176] Unless otherwise specified, the explanations regarding Aspect 1 also apply to Aspect 2. When applying, "step 1a" should be read as "step 2a," "step 1b" as "step 2b," "step 1c" as "step 2c," "assessing the intestinal barrier function of a subject" as "diagnosing a disease or symptom in a subject," "accuracy of assessing the intestinal barrier function of a subject" as "accuracy of diagnosing a disease or symptom in a subject," "assessing as positive" as "diagnosing as positive," and "assessing as negative" as "diagnosing as negative."

[0177] In Aspect 2, "positive" means that the subject is suffering from the disease or condition, and "negative" means that the subject is not suffering from the disease or condition.

[0178] The disease or symptom to be diagnosed may be a disease or symptom caused by a decrease in the intestinal barrier function of the small intestine, or a disease or symptom caused by a decrease in the intestinal barrier function of the large intestine, but is preferably a disease or symptom caused by a decrease in the intestinal barrier function of the small intestine.

[0179] Examples of diseases or symptoms caused by a decrease in intestinal barrier function include enteritis, allergic diseases, psychiatric disorders, non-alcoholic fatty liver disease, type 2 diabetes, metabolic syndrome, and obesity.

[0180] Examples of enteritis include irritable bowel syndrome, ulcerative colitis, Crohn's disease, etc. Examples of allergic diseases include food allergies, atopic dermatitis, asthma, etc. Examples of mental diseases include anxiety disorders, depression, etc. Examples of non-alcoholic fatty liver diseases include non-alcoholic fatty liver, non-alcoholic steatohepatitis, etc.

[0181] Diagnosis is typically performed by a physician. Based on the comparison results obtained in step 2b, the physician diagnoses whether the subject is suffering from a disease or symptom caused by impaired intestinal barrier function. If the subject is suffering from a disease or symptom caused by impaired intestinal barrier function, the physician may determine whether or not the subject needs to be prevented or treated for the disease or symptom, select a method for preventing or treating the disease or symptom, or implement the prevention or treatment of the disease or symptom for the subject. "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, mitigating, improving, and curing a disease or symptom.

[0182] The method according to aspect 2 of the present invention may be a method for assisting in the diagnosis of a disease or symptom caused by a decrease in intestinal barrier function in a subject. The act of assisting in the diagnosis of a disease or symptom is an act of providing the comparison results obtained in step 2b as data useful for diagnosing the disease or symptom, and may be a medical or non-medical act, but is typically a non-medical act. A doctor's diagnosis may be based on one or more other types of information in addition to the data provided by the act of assisting in the diagnosis of a disease or symptom. Furthermore, a doctor's diagnosis may also involve the doctor's experience, tips, etc.

[0183] Aspect 3 Aspect 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, a "disease or symptom caused by a decrease in intestinal barrier function" may be referred to as a "disease or symptom".

[0184] The method according to aspect 3 of the present invention comprises the following steps: (3a) measuring the occupancy rate of target bacteria in feces derived from a subject to obtain a measurement value of the occupancy rate of the target bacteria; (3b) comparing the measurement value obtained in step 3a with a reference value to obtain a comparison result; (3c) diagnosing a disease or symptom caused by a decrease in intestinal barrier function in the subject based on the comparison result obtained in step 3b; and (3d) if the subject is diagnosed as suffering from a disease or symptom caused by a decrease in intestinal barrier function, administering to the subject a substance or composition for treating the disease or symptom caused by a decrease in intestinal barrier function, wherein the occupancy rate of the target bacteria comprises the occupancy rate of one or more bacteria selected from group A and group B.

[0185] Unless otherwise specified, the explanations for Aspect 2 also apply to Aspect 3. When applying, "Step 2a" shall be read as "Step 3a", "Step 2b" as "Step 3b", and "Step 2c" as "Step 3c".

[0186] A substance or composition for treating a disease or symptom to be administered to a subject diagnosed with a disease or symptom caused by impaired intestinal barrier function can be appropriately selected depending on the type of disease or symptom the subject is suffering from. "Treatment" includes suppressing the progression or worsening of a disease or symptom, delaying the progression or worsening of a disease or symptom, as well as alleviating, mitigating, ameliorating, and curing a disease or symptom.

[0187] Examples of substances or compositions for treating irritable bowel syndrome include high molecular weight polymers and serotonin receptor antagonists. Examples of substances or compositions for treating ulcerative colitis or Crohn's disease include 5-aminosalicylic acid preparations and corticosteroids. Examples of substances or compositions for treating allergic diseases include antihistamines. Examples of substances or compositions for treating anxiety disorders or depression include selective serotonin reuptake inhibitors and noradrenergic antidepressants. Examples of substances or compositions for treating non-alcoholic fatty liver disease include antioxidants, antidiabetic drugs, and drugs for treating dyslipidemia.

[0188] A substance or composition for treating a disease or symptom may be administered in the form of a food composition containing the substance or composition. Examples of food compositions include yogurt, probiotics, prebiotics, supplements, etc. The food composition may be, for example, a food for specified health uses, a food with functional claims, a food for patients, etc.

[0189] The administration route of a substance or composition for treating a disease or symptom can be, for example, oral or parenteral (e.g., intranasal, ophthalmic, otic, transdermal, intratracheal, rectal, urinary, subcutaneous, intramuscular, intravenous, etc.). The substance or composition for treating a disease or symptom can be formulated into a dosage form suitable for the administration route. Examples of dosage forms include tablets, injections, etc. When formulating the substance or composition, additives suitable for oral administration (e.g., excipients, disintegrants, lubricants, binders, etc.) or additives suitable for parenteral administration (e.g., diluents, solvents, etc.) can be used. The dosage and frequency of administration of the substance or composition for treating a disease or symptom can be appropriately adjusted taking into account the dosage form, the age, weight, etc. of the subject. The number of administrations per day can be one or two or more times. The administration period can be one day or two or more days (e.g., one week or more, two weeks or more, three weeks or more, or four weeks or more).

[0190] Aspect 4 Aspect 4 of the present invention relates to a method for assessing the risk of a subject developing a disease or symptom caused by a decrease in intestinal barrier function, or the possibility of the subject suffering from said disease or symptom. Hereinafter, the "risk of a subject developing a disease or symptom caused by a decrease in intestinal barrier function" may be referred to as the "risk of development," and the "possibility of a subject suffering from a disease or symptom caused by a decrease in intestinal barrier function" may be referred to as the "possibility of suffering from."

[0191] The method according to aspect 4 comprises the following steps: (4a) measuring the occupancy of target bacteria in feces derived from the subject to obtain a measurement value of the occupancy of the target bacteria; and (4b) comparing the measurement value obtained in step 4a with a reference value to obtain a comparison result; and (4c) assessing the risk of developing or the possibility of being susceptible to the disease based on the comparison result obtained in step 4b, wherein the occupancy of the target bacteria comprises the occupancy of one or more bacteria selected from group A and group B.

[0192] Unless otherwise specified, the explanations regarding Aspect 1 also apply to Aspect 4. When applying, "Step 1a" shall be read as "Step 4a", "Step 1b" as "Step 4b", "Step 1c" as "Step 4c", "assessing the subject's intestinal barrier function" as "assessing the risk of developing or the likelihood of being affected", and "accuracy of the assessment of the subject's intestinal barrier function" as "accuracy of the assessment of the risk of developing or the likelihood of being affected".

[0193] In Aspect 4, "positive" means that there is or is a high risk of developing or possibility of being affected, and "negative" means that there is or is a low risk of developing or possibility of being affected.

[0194] The risk of onset to be evaluated may be the risk of the subject developing a disease or symptom caused by a decrease in the intestinal barrier function of the small intestine, or the risk of the subject developing a disease or symptom caused by a decrease in the intestinal barrier function of the large intestine, but is preferably the risk of the subject developing a disease or symptom caused by a decrease in the intestinal barrier function of the small intestine.

[0195] The susceptibility to be evaluated may be the possibility that the subject is suffering from a disease or symptom caused by a decrease in the intestinal barrier function of the small intestine, or the possibility that the subject is suffering from a disease or symptom caused by a decrease in the intestinal barrier function of the large intestine, but is preferably the possibility that the subject is suffering from a disease or symptom caused by a decrease in the intestinal barrier function of the small intestine.

[0196] The explanation regarding the disease or symptom caused by the impaired intestinal barrier function is the same as in Aspect 2.

[0197] Aspect 5 Aspect 5 of the present invention relates to a method for obtaining data for diagnosing a disease or symptom caused by a decrease in intestinal barrier function in a subject. Hereinafter, a "disease or symptom caused by a decrease in intestinal barrier function" may be referred to as a "disease or symptom".

[0198] The method according to aspect 5 comprises the following steps: (5a) measuring the occupancy rate of target bacteria in feces derived from a subject to obtain a measurement value of the occupancy rate of the target bacteria; and (5b) comparing the measurement value obtained in step 5a with a reference value to obtain a comparison result as data for diagnosing a disease or symptom caused by a decrease in intestinal barrier function in the subject, wherein the occupancy rate of the target bacteria comprises the occupancy rate of one or more bacteria selected from group A and group B.

[0199] Unless otherwise specified, the explanations regarding Aspect 1 also apply to Aspect 5. When applied, "Step 1a" should be read as "Step 5a," "Step 1b" as "Step 5b," "assessing the intestinal barrier function of a subject" as "obtaining data for diagnosing a disease or symptom of a subject," and "accuracy of assessing the intestinal barrier function of a subject" as "accuracy of diagnosing a disease or symptom of a subject."

[0200] In Aspect 5, "positive" means that the subject is suffering from the disease or condition, and "negative" means that the subject is not suffering from the disease or condition.

[0201] The disease or symptom to be diagnosed may be a disease or symptom caused by a decrease in the intestinal barrier function of the small intestine, or a disease or symptom caused by a decrease in the intestinal barrier function of the large intestine, but is preferably a disease or symptom caused by a decrease in the intestinal barrier function of the small intestine.

[0202] The explanation regarding the disease or symptom caused by the impaired intestinal barrier function is the same as in Aspect 2.

[0203] With respect to the occupancy rates of each bacterium selected from group A and group B, comparison results that satisfy the criteria shown in Table 2 can be obtained as data for diagnosing a positive result, and comparison results that do not satisfy the criteria shown in Table 2 can be obtained as data for diagnosing a negative result. Specifically, with respect to the occupancy rates of each bacterium for which the criteria shown in Table 2 are "high," a comparison result in which the measurement value obtained in step 5a is higher than the reference value can be obtained as data for diagnosing a positive result, and a comparison result in which the measurement value obtained in step 5a is equal to or lower than the reference value can be obtained as data for diagnosing a negative result. Furthermore, with respect to the occupancy rates of each bacterium for which the criteria shown in Table 2 are "low," a comparison result in which the measurement value obtained in step 5a is lower than the reference value can be obtained as data for diagnosing a positive result, and a comparison result in which the measurement value obtained in step 5a is equal to or higher than the reference value can be obtained as data for diagnosing a negative result.

[0204] In step 5a, when the occupancy rate of one bacterium selected from group A and group B is measured, if the comparison result of the occupancy rate of the one bacterium satisfies the criteria shown in Table 2, the comparison result can be obtained as data for diagnosing a positive result, and if the comparison result of the occupancy rate of the one bacterium does not satisfy the criteria shown in Table 2, the comparison result can be obtained as data for diagnosing a negative result.

[0205] In step 5a, when the occupancy rates of two or more bacteria selected from group A and group B are measured, if the comparison result of the occupancy rate of at least one of the two or more bacteria satisfies the criteria shown in Table 2, the comparison result can be obtained as data for diagnosing a positive result, and if the comparison result of none of the two or more bacteria satisfies the criteria shown in Table 2, the comparison result can be obtained as data for diagnosing a negative result. The more occupancy rates of bacteria whose comparison results meet the criteria shown in Table 2, the more likely the result is positive. Therefore, by measuring the occupancy rates of two or more (e.g., two, three, or four or more) bacteria selected from group A and group B in step 5a, the accuracy of diagnosing the target disease or symptom can be improved.

[0206] The data obtained in step 5b can be used to diagnose a disease or condition caused by a decrease in intestinal barrier function in a subject, or to assist in the diagnosis of a disease or condition caused by a decrease in intestinal barrier function in a subject.

[0207] Diagnosis is typically performed by a physician. Based on the data obtained in step 5b, the physician diagnoses whether the subject is suffering from a disease or symptom caused by impaired intestinal barrier function. If the subject is suffering from a disease or symptom caused by impaired intestinal barrier function, the physician may determine whether or not the subject needs to be prevented or treated for the disease or symptom, select a method for preventing or treating the disease or symptom, or implement the prevention or treatment of the disease or symptom for the subject. "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, mitigating, improving, and curing a disease or symptom.

[0208] The act of assisting in the diagnosis of a disease or symptom is an act of providing the data acquired in step 5b as data for diagnosing a disease or symptom, and may be a medical act or a non-medical act, but is usually a non-medical act. A doctor's diagnosis may be based on one or more other types of data in addition to the data provided by the act of assisting in the diagnosis of a disease or symptom.

[0209] Aspect 6 Aspect 6 of the present invention relates to a method for screening for a substance or composition having an effect of improving intestinal barrier function or an effect of preventing or improving a disease or symptom caused by a decrease in intestinal barrier function. Hereinafter, the "effect of preventing or improving a disease or symptom caused by a decrease in intestinal barrier function" may also be referred to as the "effect of preventing or improving a disease or symptom."

[0210] The method of Aspect 6 comprises the following steps: (6a) administering a candidate substance or candidate composition to a subject with reduced intestinal barrier function; (6b) measuring the occupancy rate of target bacteria in stool obtained from the subject after administration of the candidate substance or candidate composition to obtain a measurement value of the occupancy rate of the target bacteria; and (6c) comparing the measurement value obtained in step 6b with a reference value to obtain a comparison result; and (6d) evaluating the effect of the candidate substance or candidate composition on improving intestinal barrier function or the effect of preventing or improving a disease or symptom based on the comparison result obtained in step 6c, wherein the occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from group A and group B.

[0211] Unless otherwise specified, the explanations regarding Aspect 1 also apply to Aspect 6. When applied, "Step 1a" should be read as "Step 6b," "Step 1b" as "Step 6c," "Step 1c" as "Step 6d," "assessing the intestinal barrier function of a subject" as "assessing the effect of a candidate substance or candidate composition on improving the intestinal barrier function or on preventing or improving a disease or symptom," and "accuracy of assessing the intestinal barrier function of a subject" as "accuracy of assessing the effect of a candidate substance or candidate composition on improving the intestinal barrier function or on preventing or improving a disease or symptom."

[0212] In Aspect 6, "positive" means that the candidate substance or candidate composition has an effect of improving intestinal barrier function or an effect of preventing or improving a disease or symptom, and "negative" means that the candidate substance or candidate composition does not have an effect of improving intestinal barrier function or an effect of preventing or improving a disease or symptom. "Prevention" includes prevention, suppression, and delay of the onset of a disease or symptom. "Improvement" includes suppression of the progression or worsening of a disease or symptom, delay of the progression or worsening of a disease or symptom, and alleviation, relief, improvement, and cure of a disease or symptom.

[0213] The effect of improving the intestinal barrier function to be evaluated may be the effect of improving the intestinal barrier function of the small intestine or the effect of improving the intestinal barrier function of the large intestine, but is preferably the effect of improving the intestinal barrier function of the small intestine.

[0214] The preventive or ameliorative effect on the disease or symptom to be evaluated may be the preventive or ameliorative effect on a disease or symptom caused by a decrease in the intestinal barrier function of the small intestine, or the preventive or ameliorative effect on a disease or symptom caused by a decrease in the intestinal barrier function of the large intestine, but is preferably the preventive or ameliorative effect on a disease or symptom caused by a decrease in the intestinal barrier function of the small intestine.

[0215] The explanation regarding the disease or symptom caused by the impaired intestinal barrier function is the same as in Aspect 2.

[0216] In the method according to Aspect 6, a candidate substance or composition evaluated to have an effect of improving intestinal barrier function or an effect of preventing or improving a disease or symptom can be screened as a substance or composition having an effect of improving intestinal barrier function or an effect of preventing or improving a disease or symptom.

[0217] Step 6a is a step of administering a candidate substance or composition to a subject with impaired intestinal barrier function.

[0218] The impairment of the intestinal barrier function of a subject can be confirmed by a known method for testing intestinal barrier function (e.g., lactulose-mannitol test) or by the method according to Aspect 1.

[0219] The method of Aspect 6 may include, before step 6a, a step of evaluating the intestinal barrier function of the subject by the method of Aspect 1 and confirming that the intestinal barrier function of the subject is impaired. When the method of Aspect 6 includes this step, the type of bacterial occupancy measured in step 1a of Aspect 1 may be the same as or different from the type of bacterial occupancy measured in step 6b, but is preferably the same from the viewpoint of improving the accuracy of the evaluation of the effect of a candidate substance or composition on improving intestinal barrier function or the effect of preventing or ameliorating a disease or symptom.

[0220] The method of Aspect 6 may include, before step 6a, a step of selecting a subject with reduced intestinal barrier function by the method of Aspect 1. When the method of Aspect 6 includes this step, the type of bacterial occupancy measured in step 1a of Aspect 1 may be the same as or different from the type of bacterial occupancy measured in step 6b, but from the viewpoint of improving the accuracy of evaluation of the effect of a candidate substance or composition on improving intestinal barrier function or the effect of preventing or ameliorating a disease or symptom, it is preferable that the types of bacterial occupancy measured in step 1a of Aspect 1 are the same.

[0221] The subject to which the candidate substance or candidate composition is administered is not particularly limited as long as the subject has impaired intestinal barrier function. The subject may be a human with impaired intestinal barrier function or a model animal (excluding humans) with impaired intestinal barrier function. Preferably, the subject is a model animal (excluding humans) with impaired intestinal barrier function. Model animals include, for example, vertebrates such as mammals, reptiles, birds, amphibians, and fish. Mammals and birds are preferred, with mammals being more preferred. Examples of mammals include primates (e.g., gorillas, chimpanzees, orangutans), rodents (e.g., mice, rats, hamsters, guinea pigs, rabbits), livestock (e.g., cows, pigs, sheep, goats, horses), and pets (e.g., dogs, cats). Examples of birds include poultry (e.g., chickens, ducks, and geese).

[0222] The candidate substance or composition to be administered to a subject with reduced intestinal barrier function is not particularly limited, and can be selected from, for example, high molecular weight compounds, low molecular weight compounds, cell cultures, cell extracts, antibodies, proteins, peptides, nucleic acids, carbohydrates, inorganic salts, metal complexes, microorganisms (bacteria (probiotics), yeast, etc.), dietary fiber, indigestible carbohydrates, indigestible proteins, fermented products (including fermented foods), prebiotics, and combinations of two or more of these.

[0223] Examples of routes of administration of a candidate substance or candidate composition to a subject include oral and parenteral administration (e.g., intranasal, ophthalmic, otic, transdermal, intratracheal, rectal, urinary, subcutaneous, intramuscular, intravenous, etc.). The candidate substance or candidate composition may be formulated into a dosage form suitable for the administration route. Examples of dosage forms include tablets and injections. When formulating the candidate substance or candidate composition, additives suitable for oral administration (e.g., excipients, disintegrants, lubricants, binders, etc.) or additives suitable for parenteral administration (e.g., diluents, solvents, etc.) can be used. The dosage and frequency of administration of the candidate substance or candidate composition can be appropriately adjusted taking into account the dosage form, the age, weight, etc. of the subject. The number of administrations per day may be one or two or more times. The administration period may be one day or two or more days (e.g., one week or more, two weeks or more, three weeks or more, or four weeks or more).

[0224] Step 6b is a step of measuring the occupancy of the target bacteria in stool obtained from the subject after administration of the candidate substance or composition to obtain a measurement of the occupancy of the target bacteria.

[0225] Step 6b can be performed in the same manner as step 1a of aspect 1, except that feces obtained from a subject having reduced intestinal barrier function after administering a candidate substance or composition to the subject is used.

[0226] The feces used in step 6b are obtained from a subject with impaired intestinal barrier function after administration of a candidate substance or candidate composition. The feces may be obtained from the subject at multiple time points after administration of the candidate substance or candidate composition. The time point at which the feces are obtained can be adjusted appropriately, taking into account the dosage form, the subject's age, body weight, etc. In one embodiment, the time point at which the feces are obtained is, for example, any time point between 0.5 hours and 24 hours after administration, 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" may be after a single administration or after two or more consecutive administrations. Examples of the time point after two or more consecutive administrations include, for example, after the final administration of four weeks or more of consecutive administrations, after the final administration of one day to four weeks of consecutive administrations, after the final administration of three days to three weeks of consecutive administrations, or after the final administration of one week to two weeks of consecutive administrations.

[0227] Step 6c is a step of comparing the measurement value obtained in step 6b with a reference value and obtaining a comparison result.

[0228] Step 6c can be performed in the same manner as step 1b of aspect 1, except that the measurement values ​​obtained in step 6b are used.

[0229] Step 6d is a step of evaluating the effect of the candidate substance or candidate composition in improving the intestinal barrier function or the effect of preventing or improving a disease or symptom based on the comparison result obtained in step 6c.

[0230] Step 6d can be performed in the same manner as step 1c of aspect 1, except that the effect of the candidate substance or candidate composition on improving the intestinal barrier function or the effect of preventing or improving a disease or symptom is evaluated.

[0231] The effect of the candidate substance or composition on improving intestinal barrier function or the effect on preventing or improving a disease or symptom can be evaluated based on whether the comparison result obtained in step 6c for the occupancy rate of each bacterium selected from group A and group B satisfies the criteria shown in Table 2. Specifically, for the occupancy rate of each bacterium for which the criteria shown in Table 2 are "high," the result can be evaluated as negative if the measured value obtained in step 6b is higher than the reference value (i.e., the comparison result obtained in step 6c satisfies the criteria shown in Table 2), and the result can be evaluated as positive if the measured value obtained in step 6b is equal to or lower than the reference value (i.e., the comparison result obtained in step 6c does not satisfy the criteria shown in Table 2). Furthermore, for each occupancy rate for which the standard shown in Table 2 is "low," if the measurement value obtained in step 6b is lower than the standard value (i.e., the comparison result obtained in step 6c meets the standard shown in Table 2), it can be evaluated as negative, and if the measurement value obtained in step 6b is equal to or greater than the standard value (i.e., the comparison result obtained in step 6c does not meet the standard shown in Table 2), it can be evaluated as positive.

[0232] In step 6b, when the occupancy rate of one bacterium selected from group A and group B is measured, if the comparison result of the occupancy rate of that one bacterium satisfies the criteria shown in Table 2, it can be evaluated as negative, and if the comparison result of the occupancy rate of that one bacterium does not satisfy the criteria shown in Table 2, it can be evaluated as positive.

[0233] In step 6b, when the occupancy rates of two or more bacteria selected from group A and group B are measured, if the comparison results of all of the occupancy rates of the two or more bacteria satisfy the criteria shown in Table 2, the result can be evaluated as negative, and if the comparison result of at least one of the occupancy rates of the two or more bacteria does not satisfy the criteria shown in Table 2, the result can be evaluated as positive. The more occupancies of bacteria whose comparison results do not satisfy the criteria shown in Table 2, the more likely the result is positive. Therefore, by measuring the occupancy rates of two or more (e.g., two, three, or four or more) bacteria selected from group A and group B in step 6b, the accuracy of evaluating the effect of a candidate substance or composition on improving intestinal barrier function or the effect of preventing or improving a disease or symptom can be improved.

[0234] Aspect 7 Aspect 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 the effect of preventing or improving a disease or symptom caused by a decrease in intestinal barrier function, wherein the "effect of preventing or improving a disease or symptom caused by a decrease in intestinal barrier function" may be referred to as the "effect of preventing or improving a disease or symptom."

[0235] The method of Aspect 7 comprises the following steps: (7a) administering a candidate substance or candidate composition to a subject with reduced intestinal barrier function; (7b) measuring the occupancy rate of target bacteria in feces obtained from the subject after administration of the candidate substance or candidate composition to obtain a measurement value of the occupancy rate of the target bacteria; and (7c) comparing the measurement value obtained in step 7b with a reference value to obtain a comparison result; and (7d) evaluating the effect of the candidate substance or candidate composition on improving intestinal barrier function or the effect of preventing or improving a disease or symptom based on the comparison result obtained in step 7c, wherein the occupancy rate of the target bacteria comprises the occupancy rate of one or more bacteria selected from group A and group B.

[0236] Unless otherwise specified, the explanations for Aspect 6 also apply to Aspect 7. When applying, "step 6a" should be read as "step 7a", "step 6b" as "step 7b", "step 6c" as "step 7c", and "step 6d" as "step 7d".

[0237] Aspect 8 Aspect 8 of the present invention relates to target bacteria in feces derived from a subject for evaluating the intestinal barrier function of the subject, the target bacteria comprising one or more bacteria selected from Group A and Group B.

[0238] The statements regarding aspect 1 also apply to aspect 8, except where otherwise specified.

[0239] Aspect 9 Aspect 9 of the present invention relates to a target bacterium in feces derived from a subject for diagnosing a disease or symptom caused by a decrease in intestinal barrier function of the subject, the target bacterium comprising one or more bacteria selected from Group A and Group B.

[0240] The explanations regarding aspect 2 also apply to aspect 9, unless otherwise specified.

[0241] Aspect 10 Aspect 10 of the present invention relates to target bacteria in feces derived from a subject, for assessing the risk of the subject developing a disease or symptom caused by a decrease in intestinal barrier function, or the possibility that the subject has the disease or symptom, wherein the target bacteria include one or more bacteria selected from group A and group B.

[0242] The statements regarding aspect 4 also apply to aspect 10, except where otherwise specified.

[0243] Aspect 11 Aspect 11 of the present invention relates to target bacteria in feces derived from a subject, for obtaining data for diagnosing a disease or symptom caused by a decrease in intestinal barrier function of the subject, the target bacteria comprising one or more bacteria selected from Group A and Group B.

[0244] The statements regarding aspect 5 also apply to aspect 11, except where otherwise specified.

[0245] Aspect 12 Aspect 12 of the present invention relates to target bacteria in feces obtained from a subject with impaired intestinal barrier function after administration of a candidate substance or candidate composition to the subject, for screening for a substance or composition having an effect of improving intestinal barrier function or an effect of preventing or improving a disease or symptom caused by impaired intestinal barrier function, the target bacteria comprising one or more bacteria selected from Group A and Group B.

[0246] The statements regarding aspect 6 also apply to aspect 12, except where otherwise specified.

[0247] Aspect 13 Aspect 13 of the present invention relates to target bacteria in feces obtained from a subject with reduced intestinal barrier function after administration of a candidate substance or candidate composition to the subject, for the purpose of evaluating the effect of the candidate substance or candidate composition on improving intestinal barrier function or the effect of the candidate substance or candidate composition on preventing or improving a disease or symptom caused by reduced intestinal barrier function, wherein the target bacteria include one or more bacteria selected from Group A and Group B.

[0248] The statements regarding aspect 7 also apply to aspect 13, except where otherwise specified.

[0249] Aspect 14 Aspect 14 of the present invention relates to use of target bacteria in feces derived from a subject for assessing intestinal barrier function of the subject, wherein the target bacteria comprise one or more bacteria selected from Group A and Group B.

[0250] The statements regarding aspect 1 also apply to aspect 14, except where otherwise specified.

[0251] Aspect 15 Aspect 15 of the present invention relates to use of target bacteria in feces derived from a subject for diagnosing a disease or symptom caused by a decrease in intestinal barrier function in the subject, wherein the target bacteria comprise one or more bacteria selected from Group A and Group B.

[0252] The statements regarding aspect 2 also apply to aspect 15, unless otherwise specified.

[0253] Aspect 16 Aspect 16 of the present invention relates to use of target bacteria in stool from a subject to assess the risk of the subject developing a disease or symptom caused by a decrease in intestinal barrier function, or the possibility that the subject has the disease or symptom, wherein the target bacteria comprise one or more bacteria selected from group A and group B.

[0254] The statements regarding aspect 4 also apply to aspect 16, except where otherwise specified.

[0255] Aspect 17 Aspect 17 of the present invention relates to use of target bacteria in feces derived from a subject for obtaining data for diagnosing a disease or symptom caused by a decrease in intestinal barrier function in the subject, wherein the target bacteria comprise one or more bacteria selected from Group A and Group B.

[0256] The statements regarding aspect 5 also apply to aspect 17, except where otherwise specified.

[0257] Aspect 18 Aspect 18 of the present invention relates to use of target bacteria in feces obtained from a subject with impaired intestinal barrier function after administration of a candidate substance or candidate composition to the subject, for screening for a substance or composition having an effect of improving intestinal barrier function or an effect of preventing or improving a disease or symptom caused by impaired intestinal barrier function, wherein the target bacteria comprise one or more bacteria selected from Group A and Group B.

[0258] The statements regarding aspect 6 also apply to aspect 18, except where otherwise specified.

[0259] Aspect 19 Aspect 19 of the present invention relates to use of target bacteria in feces obtained from a subject with reduced intestinal barrier function after administration of a candidate substance or candidate composition to the subject, for evaluating the effect of the candidate substance or candidate composition on improving intestinal barrier function or the effect of the candidate substance or candidate composition on preventing or improving a disease or symptom caused by reduced intestinal barrier function, wherein the target bacteria comprise one or more bacteria selected from Group A and Group B.

[0260] The statements regarding aspect 7 also apply to aspect 19, except where otherwise specified.

[0261] Aspect 20 Aspect 20 of the present invention relates to a kit for assessing intestinal barrier function of a subject, the kit comprising one or more reagents for measuring an occupancy rate of target bacteria in feces derived from the subject, wherein the occupancy rate of the target bacteria comprises an occupancy rate of one or more bacteria selected from group A and group B.

[0262] The discussion regarding aspect 1 also applies to aspect 20, except where otherwise specified.

[0263] The kit may contain one reagent or two or more reagents.

[0264] The type of reagent can be appropriately selected depending on the method for measuring the occupancy rate of the target bacteria. When the occupancy rate of the target bacteria is measured by bacterial flora analysis using 16S rRNA amplicon sequencing, the kit includes one or more reagents for bacterial flora analysis using 16S rRNA amplicon sequencing. Examples of reagents for bacterial flora analysis using 16S rRNA amplicon sequencing include reagents for preparing samples from feces derived from a subject (e.g., solvents for suspending feces), reagents for extracting bacterial genomic DNA from a sample (e.g., reagents for lysing bacteria, reagents for denaturing or decomposing contaminants such as proteins, reagents for separating DNA from contaminants, reagents for precipitating DNA), reagents for amplifying a specific region of the 16S rRNA gene by PCR (e.g., a region containing the V3-V4 region) (e.g., primers, polymerase, dNTPs, buffer solutions, etc.), reagents for purifying PCR amplification products, reagents for adding indexes to PCR amplification products by index PCR (e.g., indexed primers, polymerase, dNTPs, buffer solutions, etc.), and devices for performing sequence analysis of indexed PCR amplification products (e.g., next-generation sequencers, etc.).

[0265] Aspect 21 Aspect 21 of the present invention relates to a kit for diagnosing a disease or symptom caused by a decrease in intestinal barrier function in a subject, the kit comprising one or more reagents for measuring an occupancy rate of target bacteria in feces derived from the subject, wherein the occupancy rate of the target bacteria comprises an occupancy rate of one or more bacteria selected from group A and group B.

[0266] The statements regarding aspect 2 also apply to aspect 21, unless otherwise specified.

[0267] The explanation regarding the reagents is the same as in Aspect 20.

[0268] Aspect 22 Aspect 22 of the present invention relates to a kit for assessing the risk of a subject developing a disease or symptom caused by a decrease in intestinal barrier function, or the possibility of the subject suffering from said disease or symptom, the kit comprising one or more reagents for measuring the occupancy rate of target bacteria in feces derived from the subject, wherein the occupancy rate of the target bacteria comprises the occupancy rate of one or more bacteria selected from group A and group B.

[0269] The statements regarding aspect 4 also apply to aspect 22, except where otherwise specified.

[0270] The explanation regarding the reagents is the same as in Aspect 20.

[0271] Aspect 23 Aspect 23 of the present invention relates to a kit for obtaining data for diagnosing a disease or symptom caused by a decrease in intestinal barrier function in a subject, the kit comprising one or more reagents for measuring an occupancy rate of target bacteria in feces derived from the subject, wherein the occupancy rate of the target bacteria comprises an occupancy rate of one or more bacteria selected from group A and group B.

[0272] The statements regarding aspect 5 also apply to aspect 23, except where otherwise specified.

[0273] The explanation regarding the reagents is the same as in Aspect 20.

[0274] Aspect 24 Aspect 24 of the present invention relates to a kit for screening for a substance or composition having an effect of improving intestinal barrier function or an effect of preventing or improving a disease or symptom caused by a decrease in intestinal barrier function, the kit comprising one or more reagents for measuring the occupancy rate of target bacteria in feces obtained from a subject with decreased intestinal barrier function after administration of a candidate substance or candidate composition to the subject, wherein the occupancy rate of the target bacteria comprises the occupancy rate of one or more bacteria selected from group A and group B.

[0275] The statements regarding aspect 6 also apply to aspect 24, except where otherwise specified.

[0276] The explanation regarding the reagents is the same as in Aspect 20.

[0277] Aspect 25 Aspect 25 of the present invention relates to a kit for evaluating the effect of a candidate substance or candidate composition on improving intestinal barrier function or the effect of preventing or improving a disease or symptom caused by a decrease in intestinal barrier function, the kit comprising one or more reagents for measuring the occupancy rate of target bacteria in feces obtained from a subject with a decreased intestinal barrier function after administration of the candidate substance or the candidate composition to the subject, wherein the occupancy rate of the target bacteria comprises the occupancy rate of one or more bacteria selected from group A and group B.

[0278] The statements regarding aspect 7 also apply to aspect 25, except where otherwise specified.

[0279] The explanation regarding the reagents is the same as in Aspect 20.

[0280] Aspect 26 Aspect 26 of the present invention relates to a method for producing a food or pharmaceutical composition, the method comprising the step of blending a substance or composition screened by the method according to Aspect 6 with one or more ingredients constituting the food or pharmaceutical composition.

[0281] The one or more ingredients to be blended with the substance or composition screened by the method of Aspect 6 are not particularly limited as long as they are acceptable ingredients for food or pharmaceutical compositions, and can be selected appropriately depending on the type of food or pharmaceutical composition to be produced.

[0282] The method according to Aspect 26 may comprise the step of screening a substance or composition by the method according to Aspect 6.

[0283] Aspect 27 Aspect 27 of the present invention relates to a computer program for causing a computer to execute a method for evaluating the intestinal barrier function of a subject.

[0284] A method executed by a computer according to Aspect 27 includes the following steps: (S101) obtaining a measurement value of the occupancy rate of target bacteria in feces derived from a subject; (S102) comparing the measurement value obtained in step S101 with a reference value and obtaining a comparison result; and (S103) evaluating the intestinal barrier function of the subject based on the comparison result obtained in step S102, wherein the occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from group A and group B.

[0285] The statements regarding aspect 1 also apply to aspect 27, except where otherwise specified.

[0286] A computer program according to Aspect 27, when executed by a computer, causes the computer to execute a method for assessing intestinal barrier function in a subject. The computer program according to Aspect 27 includes instructions for causing a computer to execute the method for assessing intestinal barrier function in a subject.

[0287] An embodiment of Aspect 27 will be described below with reference to the drawings. Fig. 1 is a schematic diagram of an apparatus used in this embodiment, Fig. 2 is a block diagram showing the hardware configuration of the apparatus of this embodiment, and Figs. 3 to 5 are flowcharts showing the processing procedures performed by the apparatus of this embodiment.

[0288] As shown in Fig. 1, the device 10 includes a computer system 30. As shown in Fig. 1, the device 10 may also include a measurement device 20 connected to the computer system 30. The computer system 30 may be a device separate from the measurement device 20, or may include the measurement device 20. The device 10 may also be a device in which the measurement device 20 and the computer system 30 are integrated.

[0289] The measurement device 20 measures the occupancy rate of the target bacteria in the feces derived from the subject, obtains the measured value of the occupancy rate of the target bacteria or the measured value necessary for calculating the measured value of the occupancy rate of the target bacteria, and transmits it to the computer system 30. The description of the feces derived from the subject, the measurement of the occupancy rate of the target bacteria, and the measured value of the occupancy rate of the target bacteria is the same as in Aspect 1.

[0290] The measurement device 20 is, for example, an automatic measurement device that measures the occupancy rate of target bacteria in feces derived from a subject (preferably, measurement by microbiota analysis using 16S rRNA amplicon sequencing) and acquires a measurement value of the occupancy rate of the target bacteria or a measurement value necessary for calculating the measurement value of the occupancy rate of the target bacteria. When the measurement device 20 performs measurement by microbiota analysis using 16S rRNA amplicon sequencing, it is sufficient for the measurement device 20 to be able to perform sequence analysis of the indexed PCR amplification product (library) and subsequent processing. Processing prior to sequence analysis of the indexed PCR amplification products, such as collection of feces from the subject (i.e., collection of feces to be used as a specimen from feces excreted by the subject), preparation of a sample from the feces from the subject, extraction of bacterial genomic DNA from the sample, amplification of a predetermined region of the 16S rRNA gene by PCR (e.g., a region including the V3-V4 region), purification of the PCR amplification products, addition of indexes to the PCR amplification products by index PCR, loading of the indexed PCR amplification products (library) into a sequencer (e.g., a next-generation sequencer), etc., may be performed by a human or by measurement device 20. In one embodiment, processing prior to sequence analysis of the indexed PCR amplification products is performed by a human.

[0291] As shown in FIG. 2, the computer system 30 includes a computer main body 300, an input unit 301, and a display unit 302 that displays sample information, evaluation results, and the like.

[0292] The processor of the computer system 30 executes a computer program installed on the hard disk 313 for evaluating the intestinal barrier function of a subject based on the measured occupancy rate of the target bacteria.

[0293] 2 , the computer main body 300 includes a CPU (Central Processing Unit) 310, a ROM (Read Only Memory) 311, a RAM (Random Access Memory) 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 to each other via a bus 318 so as to enable data communication. The measuring device 20 is connected to the computer system 30 via the communication interface 316 so as to be able to communicate with each other.

[0294] The CPU 310 can execute programs stored in the ROM 311 or the hard disk 313 and programs loaded into the RAM 312. When the CPU 310 executes the programs, the device 10 functions as a device for evaluating the intestinal barrier function of a subject.

[0295] The ROM 311 is configured by a mask ROM, a PROM, an EPROM, an EEPROM, etc. The ROM 311 stores computer programs executed by the CPU 310 and data used for executing the computer programs.

[0296] The RAM 312 is composed of SRAM, DRAM, etc. The RAM 312 is used to read out programs recorded in the ROM 311 and the hard disk 313. The RAM 312 is also used as a working area for the CPU 310 when executing a program.

[0297] The hard disk 313 has installed thereon an operating system to be executed by the CPU 310, computer programs such as application programs, and data used to execute the computer programs.

[0298] The reading device 315 is configured by a flexible 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.

[0299] The input / output interface 314 is composed of, for example, a serial interface such as USB, IEEE1394, or RS-232C, a parallel interface such as SCSI, IDE, or IEEE1284, and an analog interface consisting of a D / A converter, an A / D converter, etc. An input unit 301 such as a keyboard or a mouse is connected to the input / output interface 314. An operator can input various commands to the computer main body 300 through the input unit 301.

[0300] The communication interface 316 is, for example, an Ethernet (registered trademark) interface, etc. The communication interface 316 enables the computer main unit 300 to transmit data to a printer, a target or service provider's computer, a target smartphone terminal, etc.

[0301] The image output interface 317 is connected to a display unit 302 that is configured with an LCD, CRT, or the like. This allows the display unit 302 to output a video signal corresponding to the image data provided by the CPU 310. The display unit 302 displays an image (screen) according to the input video signal. The display unit 302 may display the raw data itself, or may convert the raw data into illustrations, drawings, or the like to make it easier for the subject to understand.

[0302] The processing procedure performed by the device 10 will be described below with reference to FIGS.

[0303] 3 , in step S101, the CPU 310 acquires a measurement value of the occupancy rate of the target bacteria in the feces derived from the subject. The description of the feces derived from the subject, the measurement of the occupancy rate of the target bacteria, and the measurement value of the occupancy rate of the target bacteria are the same as in aspect 1.

[0304] In one embodiment, measurement device 20 measures the occupancy rate of target bacteria in feces derived from the subject (preferably by microbiota analysis using 16S rRNA amplicon sequencing), obtains the measured value of the occupancy rate of target bacteria, and transmits it to computer system 30. The description of the feces derived from the subject, the measurement of the occupancy rate of target bacteria, and the measured value of the occupancy rate of target bacteria is the same as in aspect 1. CPU 310 stores the measured value of the occupancy rate of target bacteria transmitted from measurement device 20 in hard disk 313. In step S301, CPU 310 obtains the measured value of the occupancy rate of target bacteria stored in hard disk 313.

[0305] In another embodiment, measurement device 20 measures the occupancy rate of target bacteria in feces derived from a subject (preferably by microbiota analysis using 16S rRNA amplicon sequencing), acquires measurement values ​​necessary for calculating the target bacterial occupancy rate measurement value, and transmits these to computer system 30. The description of the subject's feces, the measurement of the target bacterial occupancy rate, and the target bacterial occupancy rate measurement value are the same as in aspect 1. CPU 310 calculates the target bacterial occupancy rate measurement value based on the measurement values ​​transmitted from measurement device 20, and stores the target bacterial occupancy rate measurement value on hard disk 313. In step S301, CPU 310 acquires the target bacterial occupancy rate measurement value stored on hard disk 313.

[0306] In yet another embodiment, the operator inputs, via input unit 301, a measurement value of the occupancy rate of the target bacteria previously obtained by measuring the occupancy rate of the target bacteria in feces derived from a subject (preferably, measurement by bacterial flora analysis using 16S rRNA amplicon sequencing). The description of the feces derived from a subject, the measurement of the occupancy rate of the target bacteria, and the measurement value of the occupancy rate of the target bacteria are the same as in aspect 1. CPU 310 stores the input measurement value of the occupancy rate of the target bacteria in hard disk 313. In step S301, CPU 310 acquires the measurement value of the occupancy rate of the target bacteria stored in hard disk 313.

[0307] In yet another embodiment, the operator inputs, via input unit 301, measurement values ​​necessary for calculating a measurement value of the occupancy rate of the target bacteria previously obtained by measuring the occupancy rate of the target bacteria in feces derived from a subject (preferably, measurement by bacterial flora analysis using 16S rRNA amplicon sequencing). The description of the feces derived from the subject, the measurement of the occupancy rate of the target bacteria, and the measurement value of the occupancy rate of the target bacteria are the same as in aspect 1. CPU 310 calculates the measurement value of the occupancy rate of the target bacteria based on the input measurement values ​​and stores them on hard disk 313. In step S301, CPU 310 acquires the measurement value of the occupancy rate of the target bacteria stored on hard disk 313.

[0308] As shown in FIG. 3 , in step S102, the CPU 310 compares the measured value of the occupancy rate of the target bacteria with a reference value stored on the hard disk 313 and obtains the comparison result. The description of the reference value, comparison, and comparison result is the same as in aspect 1. The CPU 310 stores the obtained comparison result on the hard disk 313. The CPU 310 may output the obtained comparison result and display it on the display unit 302, print it on a printer, or transfer it to another computer or app (e.g., a smartphone). When outputting the comparison result, the CPU 310 may display the measured value of the occupancy rate of the target bacteria, the reference value, etc. on the display unit 302, print it on a printer, or transfer it to another computer or app (e.g., a smartphone) as reference information. The CPU 310 may also display lifestyle advice for improving intestinal barrier function, recommended foods (including functional foods), etc., together with the comparison result on the display unit 302, print it on a printer, or transfer it to another computer or app (e.g., a smartphone). This makes it possible to provide doctors, nurses, caregivers, examiners, testing companies, service providers, subjects themselves, and others with indicators for evaluating the intestinal barrier function of the subject.

[0309] As shown in FIG. 3 , in step S103, the CPU 310 evaluates the intestinal barrier function of the subject based on the comparison results obtained in step S102. The description of the evaluation is the same as in aspect 1. The CPU 310 stores the evaluation results in the hard disk 313. The CPU 310 may output the evaluation results and display them on the display unit 302, print them on a printer, or transfer them to another computer or app (e.g., a smartphone). When outputting the evaluation results, the CPU 310 may display the measured values, reference values, etc. of the occupancy rate of the target bacteria on the display unit 302, print them on a printer, or transfer them to a computer or app (e.g., a smartphone) as reference information. The CPU 310 may also display lifestyle advice for improving intestinal barrier function, recommended foods (including functional foods), etc., together with the evaluation results on the display unit 302, print them on a printer, or transfer them to another computer or app (e.g., a smartphone). This makes it possible to provide doctors, nurses, caregivers, examiners, testing companies, service providers, subjects themselves, and others with indicators for evaluating the intestinal barrier function of the subject.

[0310] With respect to the occupancy rate of each bacterium selected from group A and group B, the CPU 310 evaluates the intestinal barrier function of the subject based on whether the comparison result obtained in step S102 satisfies the criteria shown in Table 2. Specifically, with respect to the occupancy rate of each bacterium for which the criteria shown in Table 2 are "high," as shown in Fig. 4, the CPU 310 determines whether the measurement value obtained in step 101 is higher than the reference value (i.e., whether the comparison result satisfies the criteria shown in Table 2) (step S103a). If the measurement value obtained in step 101 is higher than the reference value (i.e., the comparison result satisfies the criteria shown in Table 2), the result is evaluated as positive (step S103b). If the measurement value obtained in step 101 is equal to or lower than the reference value (i.e., the comparison result does not satisfy the criteria shown in Table 2), the result is evaluated as negative (step S103c). Furthermore, with regard to the occupancy rate of each bacterium for which the standard shown in Table 2 is "low," as shown in FIG. 5, the CPU 310 determines whether the measurement value obtained in step 101 is lower than the standard value (i.e., whether the comparison result satisfies the standard shown in Table 2) (step S103d), and if the measurement value obtained in step 101 is lower than the standard value (i.e., the comparison result satisfies the standard shown in Table 2), it evaluates it as positive (step S103e), and if the measurement value obtained in step 101 is equal to or greater than the standard value (i.e., the comparison result does not satisfy the standard shown in Table 2), it evaluates it as negative (step S103f).

[0311] Aspect 28 of the present invention relates to a computer program for causing a computer to execute a method for diagnosing a disease or symptom caused by a decrease in intestinal barrier function in a subject. Hereinafter, a "disease or symptom caused by a decrease in intestinal barrier function" may be referred to as a "disease or symptom."

[0312] A method executed by a computer according to Aspect 28 includes the following steps: (S201) obtaining a measurement value of the occupancy rate of target bacteria in feces derived from a subject; (S202) comparing the measurement value obtained in step S201 with a reference value and obtaining a comparison result; (S203) diagnosing a disease or symptom caused by a decrease in intestinal barrier function in the subject based on the comparison result obtained in step S202, wherein the occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from group A and group B.

[0313] The explanation regarding Aspect 2 also applies to Aspect 28, unless otherwise specified. Furthermore, the explanation regarding Aspect 27 also applies to Aspect 28, unless otherwise specified. When applied, "step S101" is replaced with "step S201," "step S102" is replaced with "step S202," "step S103" is replaced with "step S203," "assessing the intestinal barrier function of the subject" is replaced with "diagnosing a disease or symptom of the subject," "accuracy of assessing the intestinal barrier function of the subject" is replaced with "accuracy of diagnosing a disease or symptom of the subject," "assess as positive" is replaced with "diagnose as positive," and "assess as negative" is replaced with "diagnose as negative."

[0314] Aspect 29 Aspect 29 of the present invention relates to a computer program for causing a computer to execute a method for assessing the risk of a subject developing a disease or symptom caused by a decrease in intestinal barrier function, or the possibility of the subject suffering from said disease or symptom. Hereinafter, the "risk of a subject developing a disease or symptom caused by a decrease in intestinal barrier function" may be referred to as the "risk of development," and the "possibility of a subject suffering from a disease or symptom caused by a decrease in intestinal barrier function" may be referred to as the "possibility of suffering from."

[0315] A method executed by a computer according to Aspect 29 includes the following steps: (S301) obtaining a measurement value of the occupancy rate of target bacteria in feces derived from a subject; (S302) comparing the measurement value obtained in step S301 with a reference value and obtaining a comparison result; and (S303) evaluating the risk or the possibility based on the comparison result obtained in step S302, wherein the occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from group A and group B.

[0316] The explanation regarding Aspect 4 also applies to Aspect 29, unless otherwise specified. Furthermore, the explanation regarding Aspect 27 also applies to Aspect 29, unless otherwise specified. When applying, "Step S101" is replaced with "Step S301", "Step S102" is replaced with "Step S302", "Step S103" is replaced with "Step S303", "assessing the intestinal barrier function of a subject" is replaced with "assessing the risk of onset or likelihood of susceptibility", and "accuracy of assessing the intestinal barrier function of a subject" is replaced with "accuracy of assessing the risk of onset or likelihood of susceptibility".

[0317] Aspect 30 of the present invention relates to a computer program for causing a computer to execute a method for acquiring data for diagnosing a disease or symptom caused by a decrease in intestinal barrier function in a subject. Hereinafter, a "disease or symptom caused by a decrease in intestinal barrier function" may be referred to as a "disease or symptom."

[0318] A method executed by a computer according to Aspect 30 includes the following steps: (S401) acquiring a measurement value of the occupancy rate of target bacteria in feces derived from a subject; and (S402) comparing the measurement value acquired in step S401 with a reference value, and acquiring the comparison result as data for diagnosing a disease or symptom caused by a decrease in intestinal barrier function in the subject, wherein the occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from group A and group B.

[0319] Unless otherwise specified, the explanation regarding Aspect 5 also applies to Aspect 30. Furthermore, unless otherwise specified, the explanation regarding Aspect 27 also applies to Aspect 30. When applying, "step S101" should be read as "step S401," "step S102" as "step S402," "evaluating the intestinal barrier function of a subject" as "acquiring data for diagnosing a disease or symptom of a subject," and "accuracy of evaluating the intestinal barrier function of a subject" as "accuracy of diagnosing a disease or symptom of a subject."

[0320] Aspect 31 Aspect 31 of the present invention relates to a computer program for causing a computer to execute a method for screening for a substance or composition that has an effect of improving intestinal barrier function or an effect of preventing or improving a disease or symptom caused by a decrease in intestinal barrier function. Hereinafter, the "effect of preventing or improving a disease or symptom caused by a decrease in intestinal barrier function" may be referred to as the "effect of preventing or improving a disease or symptom."

[0321] The method executed by a computer according to Aspect 31 includes the following steps: (S501) obtaining a measurement value of the occupancy rate of target bacteria in feces obtained from a subject with reduced intestinal barrier function after administration of a candidate substance or candidate composition to the subject; (S502) comparing the measurement value obtained in step S501 with a reference value and obtaining a comparison result; and (S503) evaluating the effect of the candidate substance or candidate composition on improving intestinal barrier function or the effect of preventing or improving a disease or symptom based on the comparison result obtained in step S502, wherein the occupancy rate of target bacteria includes the occupancy rate of one or more bacteria selected from group A and group B.

[0322] The explanation regarding Aspect 6 also applies to Aspect 31, unless otherwise specified. Furthermore, the explanation regarding Aspect 27 also applies to Aspect 31, unless otherwise specified. When applied, "step S101" is replaced with "step S501," "step S102" with "step S502," "step S103" with "step S503," "assessing the intestinal barrier function of a subject" with "assessing the effect of a candidate substance or candidate composition on improving intestinal barrier function or the effect of preventing or improving a disease or symptom," and "accuracy of assessing the intestinal barrier function of a subject" with "accuracy of assessing the effect of a candidate substance or candidate composition on improving intestinal barrier function or the effect of preventing or improving a disease or symptom."

[0323] Aspect 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 the effect of preventing or improving a disease or symptom caused by a decrease in intestinal barrier function. Hereinafter, the "effect of preventing or improving a disease or symptom caused by a decrease in intestinal barrier function" may be referred to as the "effect of preventing or improving a disease or symptom."

[0324] The method executed by a computer according to Aspect 32 includes the following steps: (S601) obtaining a measurement value of the occupancy rate of target bacteria in feces obtained from a subject with reduced intestinal barrier function after administration of a candidate substance or candidate composition to the subject; (S602) comparing the measurement value obtained in step S601 with a reference value and obtaining a comparison result; and (S603) evaluating the effect of the candidate substance or the candidate composition on improving intestinal barrier function or the effect of preventing or improving a disease or symptom based on the comparison result obtained in step S602, wherein the occupancy rate of target bacteria includes the occupancy rate of one or more bacteria selected from group A and group B.

[0325] The explanation regarding Aspect 7 also applies to Aspect 32, unless otherwise specified. Furthermore, the explanation regarding Aspect 27 also applies to Aspect 32, unless otherwise specified. When applied, "Step S101" is replaced with "Step S601," "Step S102" is replaced with "Step S602," "Step S103" is replaced with "Step S603," "evaluating the intestinal barrier function of a subject" is replaced with "evaluating the effect of a candidate substance or candidate composition on improving intestinal barrier function or the effect of preventing or improving a disease or symptom," and "accuracy of evaluating the intestinal barrier function of a subject" is replaced with "accuracy of evaluating the effect of a candidate substance or candidate composition on improving intestinal barrier function or the effect of preventing or improving a disease or symptom."

[0326] Aspect 33 of the present invention relates to a computer-readable recording medium having the computer program according to any one of Aspects 27 to 32 recorded thereon.

[0327] Examples of computer-readable recording media on which a program is recorded include non-transitory recording media such as ROM, floppy disks (registered trademark), hard disks, optical disks, magneto-optical disks, CD-ROMs, magnetic tapes, and non-volatile memory cards.

[0328] The present invention will be described below based on examples.

[0329] A lactulose-mannitol test, a known method for testing intestinal barrier function, was performed on 108 subjects. The lactulose-mannitol test was conducted as follows. Subjects were instructed to finish their meals by 9:00 PM the day before the test and were allowed to drink water until bedtime. On the day of the test, a glucose tolerance test was conducted without allowing the subjects to eat or drink water. Subjects ingested a sugar solution (10 g of lactulose, 5 g of mannitol, and 20 g of sucrose dissolved in 100 mL of water). After ingestion of the sugar solution, subjects ingested at least 150 mL of water between 2 and 3 hours, 3 and 4 hours, 4 and 5 hours, and 5 and 6 hours. After water ingestion, all urine samples were collected from 0 to 6 hours, and the amounts of lactulose and mannitol in the urine were measured using liquid chromatography-tandem mass spectrometry (LC-MS / MS). LC-MS / MS was performed as follows.

[0330] The measurement sample used for LC-MS / MS was prepared from urine as follows: 400 μL of chilled acetonitrile was added to 50 μL of urine, the mixture was thoroughly stirred, and the mixture was allowed to stand on ice for 1 hour. The resulting mixture was further stirred, and 800 μL of 75% by mass acetonitrile was added, the mixture was thoroughly stirred, and the mixture was filtered through a Millex FH 0.45 μm filter to be used as the measurement sample. No internal standard was used.

[0331] LC-MS / MS measurements and subsequent data analysis were performed using a QTRAP 4500 LC-MS / MS system (Thermo Fisher Scientific). The column used was an Agilent AdvanceBio MS Spent Media 120 Å 2.1 x 100 mm (Agilent). The LC-MS / MS measurement conditions were as follows: Mobile phase A: 10 mM ammonium formate (pH 10.5) Mobile phase B: acetonitrile (90%) / 100 mM ammonium formate (pH 10.5) (10%) Mobile condition: 0-15 min B 97%-89%, 15-18 min B 89% Flow: 0.4 mL / min Injection volume: 5 μL Temperature: 35°C IonSpray Voltage: -4500 V

[0332] The amounts of lactulose and mannitol in urine (hereinafter referred to as "excretion amounts") were measured by LC-MS / MS, and then the excretion rates (%) of each were calculated based on the following formula: Excretion rate = excretion amount (μmol / mL) × molecular weight (μg / μmol) × urine volume (mL) / sugar intake (μg) × 100.

[0333] After calculating the excretion rate (%), the lactulose / mannitol ratio was calculated based on the following formula: Lactulose / mannitol ratio = lactulose excretion rate / mannitol excretion rate

[0334] The 27 subjects with a lactulose / mannitol ratio of 0.025 or greater were classified as the positive group, and the 27 subjects with a lactulose / mannitol ratio of 0.015 or less were classified as the negative group.

[0335] Feces and serum were obtained from the negative and positive groups and used in the following comparative examples and examples.

[0336] Serum was obtained as follows: 20 mL of blood was collected from each subject on the day of the lactulose-mannitol test, and a portion was separated into serum, which was then stored in a deep freezer (-80°C) until use.

[0337] Feces were obtained as follows. At any time between two days before and the day of the lactulose-mannitol test, subjects were asked to collect approximately 10 g of feces immediately after voiding into a stool collection container (Faeces tube, with blade, screw cap (Sarstedt)) and store it in a freezer until submission. On the day of the test, subjects were asked to bring the stool collection container containing the feces in an insulated container to keep the feces frozen. After submission, the feces were stored in a deep freezer (-80°C) until use.

[0338] Comparative Example 1 The serum zonulin concentration in each of the sera obtained from the negative and positive groups was measured by ELISA. The ELISA was performed using a Zonulin ELISA kit (Immundiagnostik AG, Germany) according to the manufacturer's protocol.

[0339] Using the serum zonulin concentrations of the negative and positive groups, a receiver operating characteristic (ROC) curve was created, and the area under the curve (AUC) and cutoff value were calculated from the created ROC curve. The ROC curve, AUC, and cutoff value were calculated using the statistical analysis software "Excel Statistics" (manufactured by Social Information Services Co., Ltd.). The ROC curve is a curve drawn by plotting the serum zonulin concentrations of the negative and positive groups, with sensitivity on the vertical axis and (1-specificity) on the horizontal axis. AUC is the area under the ROC curve. The point on the ROC curve that is the shortest distance from the upper left corner of the ROC curve was adopted as the cutoff value. The results are shown in Table 5.

[0340]

[0341] Example 1 (1) Analysis of Fecal Bacteria The bacterial flora in each fecal sample obtained from the negative and positive groups was analyzed using 16S rRNA amplicon sequencing. The bacterial flora analysis using 16S rRNA amplicon sequencing was performed as follows.

[0342] 20 mg of feces was suspended in ultrapure water and subjected to disruption treatment (physical disruption using 1 mm diameter zirconia beads) using Fastprep (manufactured by MP-Biomedicals). Bacterial genomic DNA was extracted using a QIAamp Fast DNA Stool Mini Kit (manufactured by QIAGEN), and an approximately 460 bp region containing the variable regions V3 and V4 of the 16S rRNA gene was amplified by PCR. The resulting PCR amplification product was purified using AMPure XP beads (manufactured by Beckman Coulter). After purification, index PCR was performed on the PCR product using primers containing a base sequence (index) unique to each sample, and a sample identification index was added to the PCR amplification product of each sample. The indexed PCR amplification 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 (Illumina) were denatured using 0.2 N NaOH. The denatured library was sequenced using a next-generation sequencer, Miseq (Illumina), to obtain 16S rRNA gene sequence data. The sequence data output from Miseq was processed using QIIME2 (version 2020.8) and silva (version 138) as the reference database to obtain the constituent bacteria of each taxonomic hierarchy and their occupancy.

[0343] The occupancy rate of each bacterium was calculated based on the number of reads belonging to each ASV (Amplicon Sequence Variant) analysis. In this case, the occupancy rate of a certain family of bacteria was calculated based on the formula: (total number of bacteria belonging to that family in feces) / (total number of bacteria of all families in feces). In addition, the occupancy rate of a certain genus of bacteria was calculated based on the formula: (total number of bacteria belonging to that genus in feces) / (total number of bacteria of all genera in feces).

[0344] A receiver operating characteristic (ROC) curve was created using the occupancy rate of bacteria of each family or genus in each stool sample, and the area under the curve (AUC) and cutoff value were calculated from the created ROC curve. The ROC curve, AUC, and cutoff value were calculated using the statistical analysis software "Excel Statistics" (manufactured by Social Information Services Co., Ltd.). The ROC curve is a curve drawn by plotting the occupancy rate of bacteria of each family or genus in each stool sample, with sensitivity on the vertical axis and (1-specificity) on the horizontal axis. AUC is the area under the ROC curve. The point on the ROC curve with the smallest distance from the upper left corner of the ROC curve was adopted as the cutoff value. The results are shown in Tables 6 and 7. The closer the AUC value is to 1, the higher the accuracy of the assessment of intestinal barrier function. When the AUC value is less than 0.60, the accuracy of the evaluation is low; when the AUC value is 0.60 or more but less than 0.64, the accuracy of the evaluation is somewhat low; when the AUC value is 0.64 or more but less than 0.68, the accuracy of the evaluation is somewhat high; and when the AUC value is 0.68 or more, the accuracy of the evaluation is high.

[0345] Table 6 shows the results of the occupancy rate of bacteria in families with an AUC of 0.64 or more among the occupancy rates of bacteria in each comprehensively measured family, and Table 7 shows the results of the occupancy rate of bacteria in genera with an AUC of 0.64 or more among the occupancy rates of bacteria in each comprehensively measured genera.

[0346] For each occupancy listed in Tables 6 and 7, trends in the positive group were clarified based on the ROC curve. The results are shown in Table 8. In Table 8, "increase" indicates a tendency to increase in the positive group, and "decrease" indicates a tendency to decrease in the positive group. In the ROC curve for occupancies that tend to increase in the positive group, when the cutoff value is 0, the sensitivity (i.e., true positive rate) = 1, and (1 - specificity) (i.e., false positive rate) = 1. In the ROC curve for occupancies that tend to decrease in the positive group, when the cutoff value is -1, the sensitivity (i.e., true positive rate) = 0, and (1 - specificity) (i.e., false positive rate) = 0.

[0347] The results shown in Tables 6 and 7 demonstrate that when the sample is feces, the intestinal barrier function of a subject can be evaluated with high accuracy by using the occupancy rate of one or more bacteria selected from Group A and Group B as an index. The closer the AUC is to 1, the higher the accuracy of the evaluation of intestinal barrier function. Therefore, the occupancy rates of Ruminococcaceae bacteria, Peptostreptococcaceae bacteria, Rikenellaceae bacteria, Peptostreptococcaceae Romboutsia bacteria, and Rikenellaceae Alistipes bacteria are particularly accurate in evaluating intestinal barrier function.

[0348]

[0349]

[0350]

[0351] (2) Combination Variables (Statistical Analysis Values) The combination variables for the occupancy rate of Ruminococcaceae bacteria, the occupancy rate of Peptostreptococcaceae bacteria, the occupancy rate of Rikenellaceae bacteria, and the occupancy rate of Peptostreptococcaceae Romboutsia bacteria were calculated based on the following formula. An ROC curve was created based on the calculated combination variables, and the AUC and cutoff value were calculated from the created ROC curve. The results are shown in Table 9. Each coefficient and constant term was calculated using multinomial logistic regression analysis. The coefficients and constant terms are as shown in Table 1. In Table 9 and Table 1, "R1" represents the occupancy rate of Ruminococcaceae bacteria, "R2" represents the occupancy rate of Peptostreptococcaceae bacteria, "R3" represents the occupancy rate of Peptostreptococcaceae Romboutsia bacteria, and "R4" represents the occupancy rate of Rikenellaceae bacteria. Statistical analysis value = (coefficient 1 × (first occupancy rate)) + (coefficient 2 × (second occupancy rate)) + ... + (coefficient n × (nth occupancy rate)) + constant term

[0352] As shown in Table 9, it was found that by combining two or more occupancies selected from the occupancy rate of Ruminococcaceae bacteria, the occupancy rate of Peptostreptococcaceae bacteria, the occupancy rate of Rikenellaceae bacteria, and the occupancy rate of Peptostreptococcaceae Romboutsia bacteria, the AUC of the ROC curve can be increased, i.e., the accuracy of the assessment of intestinal barrier function can be improved.

[0353]

Claims

1. A method for assessing intestinal barrier function of a subject, the method comprising the steps of: (1a) measuring the occupancy rate of target bacteria in feces derived from the subject and obtaining a measurement value of the occupancy rate of the target bacteria; (1b) comparing the measurement value obtained in step 1a with a reference value and 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 rate of the target bacteria is determined to be the following groups A and B: [Group A] Ruminococcaceae bacteria Peptostreptococcaceae bacteria Rikenellaceae bacteria Eubacteriaceae bacteria Bacillaceae bacteria Clostridiaceae 1 bacteria [Group B] Peptostreptococcaceae Romboutsia bacteria Rikenellaceae Alistipes bacteria Lachnospiraceae Lachnospiraceae UCG-010 bacteria Ruminococcaceae Negativibacillus bacteria Lachnospiraceae [Ruminococcus] gauvreauii group bacteria Ruminococcaceae Ruminococcus 1 bacteria Ruminococcaceae Ruminococcaceae NK4A214 group bacteria Ruminococcaceae Ruminococcaceae UCG-002 bacteria Lachnospiraceae the method comprising an occupancy of one or more bacteria selected from: [Ruminococcus] gnavus group bacteria, Ruminococcaceae Ruminococcaceae UCG-003 bacteria, Ruminococcaceae Ruminococcaceae UCG-009 bacteria, Eubacteriaceae Eubacterium bacteria, Ruminococcaceae Faecalibacterium bacteria, Bacillaceae Bacillus bacteria, Ruminococcaceae [Eubacterium] coprostanoligenes group bacteria, Peptostreptococcaceae Intestinibacter bacteria, Ruminococcaceae Anaerotruncus bacteria, Marinifilaceae Odoribacter bacteria, Clostridiaceae Clostridium sensu stricto bacteria; 2. A method for diagnosing a disease or symptom caused by a decrease in intestinal barrier function in a subject, the method comprising the following steps: (2a) measuring the occupancy rate of target bacteria in feces derived from the subject and obtaining a measurement value of the occupancy rate of the target bacteria; (2b) comparing the measurement value obtained in step 2a with a reference value and obtaining a comparison result; and (2c) diagnosing the disease or symptom based on the comparison result obtained in step 2b, wherein the occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from group A and group B described in claim 1.

3. A method for treating a disease or symptom caused by a decrease in intestinal barrier function in a subject, the method comprising the following steps: (3a) measuring the occupancy rate of target bacteria in feces derived from the subject and obtaining a measurement value of the occupancy rate of the target bacteria; (3b) comparing the measurement value obtained in step 3a with a reference value and obtaining a comparison result; (3c) diagnosing the disease or symptom based on the comparison result obtained in step 3b; and (3d) if the subject is diagnosed as suffering from the disease or symptom, administering to the subject a substance or composition for treating the disease or symptom, wherein the occupancy rate of the target bacteria comprises an occupancy rate of one or more bacteria selected from group A and group B described in claim 1.

4. A method for assessing the risk of a subject developing a disease or symptom caused by a decrease in intestinal barrier function, or the possibility of the subject suffering from said disease or symptom, said method comprising the following steps: (4a) measuring the occupancy rate of target bacteria in stool derived from said subject and obtaining a measurement value of the occupancy rate of said target bacteria; (4b) comparing the measurement value obtained in step 4a with a reference value and obtaining a comparison result; and (4c) assessing said risk or possibility based on the comparison result obtained in step 4b, wherein said occupancy rate of target bacteria comprises the occupancy rate of one or more bacteria selected from group A and group B described in claim 1.

5. A method for obtaining data for diagnosing a disease or symptom caused by a decrease in intestinal barrier function in a subject, the method comprising the following steps: (5a) measuring the occupancy rate of target bacteria in feces derived from the subject and obtaining a measurement value of the occupancy rate of the target bacteria; and (5b) comparing the measurement value obtained in step 5a with a reference value and obtaining the comparison result as the data, wherein the occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from group A and group B described in claim 1.

6. A method for screening for a substance or composition that has the effect of improving intestinal barrier function or the effect of preventing or improving diseases or symptoms caused by a decrease in intestinal barrier function, the method comprising the following steps: (6a) a step of administering a candidate substance or candidate composition to a subject with a decreased intestinal barrier function; (6b) a step of measuring the occupancy rate of target bacteria in stool obtained from the subject after administration of the candidate substance or candidate composition, and obtaining a measurement value of the occupancy rate of the target bacteria; (6c) a step of comparing the measurement value obtained in step 6b with a reference value and obtaining a comparison result; and (6d) a step of evaluating the effect of the candidate substance or candidate composition in improving intestinal barrier function or the effect of preventing or improving the disease or symptom based on the comparison result obtained in step 6c, wherein the occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from group A and group B described in claim 1.

7. A method for evaluating the effect of a candidate substance or candidate composition on improving intestinal barrier function or the effect of preventing or improving a disease or symptom caused by a decrease in intestinal barrier function, the method comprising the following steps: (7a) administering the candidate substance or candidate composition to a subject with decreased intestinal barrier function; (7b) measuring the occupancy rate of target bacteria in stool obtained from the subject after administration of the candidate substance or candidate composition, and obtaining a measurement value of the occupancy rate of the target bacteria; (7c) comparing the measurement value obtained in step 7b with a reference value and obtaining a comparison result; and (7d) evaluating the effect of the candidate substance or candidate composition on improving intestinal barrier function or the effect of preventing or improving the disease or symptom based on the comparison result obtained in step 7c, wherein the occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from group A and group B described in claim 1.

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

9. The method of any one of claims 1 to 7, wherein the occupancy of the target bacteria comprises an occupancy of two, three, or four or more bacteria selected from groups A and B of claim 1.

10. The method of any one of claims 1 to 7, wherein the occupancy of the target bacteria comprises one or more occupancies selected from the occupancy of Ruminococcaceae bacteria, the occupancy of Peptostreptococcaceae bacteria, the occupancy of Rikenellaceae bacteria, the occupancy of Peptostreptococcaceae Romboutsia bacteria, and the occupancy of Rikenellaceae Alistipes bacteria.

11. The method of any one of claims 1 to 7, wherein the occupancy of the target bacteria is measured by bacterial flora analysis using 16S rRNA amplicon sequencing.

12. Target bacteria in feces from a subject for evaluating the intestinal barrier function of the subject, the target bacteria comprising one or more bacteria selected from Group A and Group B of claim 1.

13. A target bacterium in feces derived from a subject for diagnosing a disease or symptom caused by a decrease in the intestinal barrier function of the subject, the target bacterium comprising one or more bacteria selected from group A and group B described in claim 1.

14. Target bacteria in feces from a subject for assessing the risk of the subject developing a disease or symptom caused by a decrease in intestinal barrier function or the possibility of the subject suffering from said disease or symptom, wherein said target bacteria comprises one or more bacteria selected from group A and group B described in claim 1.

15. Target bacteria in feces derived from a subject for obtaining data for diagnosing a disease or symptom caused by a decrease in the intestinal barrier function of the subject, the target bacteria comprising one or more bacteria selected from group A and group B described in claim 1.

16. Target bacteria in feces obtained from a subject with impaired intestinal barrier function after administration of a candidate substance or candidate composition to the subject, for screening for a substance or composition that has the effect of improving intestinal barrier function or the effect of preventing or improving diseases or symptoms caused by impaired intestinal barrier function, wherein the target bacteria include one or more bacteria selected from group A and group B described in claim 1.

17. Target bacteria in feces obtained from a subject with impaired intestinal barrier function after administration of a candidate substance or candidate composition to the subject, for the purpose of evaluating the effect of the candidate substance or candidate composition in improving intestinal barrier function or the effect of preventing or improving diseases or symptoms caused by impaired intestinal barrier function, wherein the target bacteria include one or more bacteria selected from group A and group B described in claim 1.

18. The target bacterium according to any one of claims 12 to 17, wherein the intestinal barrier function is a barrier function of the small intestine.

19. The target bacterium according to any one of claims 12 to 17, wherein the target bacterium comprises two, three, or four or more bacteria selected from Groups A and B of claim 1.

20. The target bacterium of any one of claims 12 to 17, wherein the target bacterium comprises one or more bacteria selected from Ruminococcaceae bacteria, Peptostreptococcaceae bacteria, Rikenellaceae bacteria, Peptostreptococcaceae Romboutsia bacteria, and Rikenellaceae Alistipes bacteria.

21. Use of target bacteria in feces from a subject to assess the intestinal barrier function of the subject, wherein the target bacteria comprises one or more bacteria selected from groups A and B of claim 1.

22. Use of target bacteria in feces from a subject for diagnosing a disease or condition caused by a decrease in intestinal barrier function in the subject, wherein the target bacteria comprises one or more bacteria selected from groups A and B described in claim 1.

23. Use of target bacteria in stool from a subject to assess the risk of the subject developing or the likelihood of the subject suffering from a disease or condition caused by impaired intestinal barrier function, wherein the target bacteria comprises one or more bacteria selected from groups A and B of claim 1.

24. Use of target bacteria in feces from a subject to obtain data for diagnosing a disease or condition caused by a decrease in intestinal barrier function in the subject, wherein the target bacteria comprises one or more bacteria selected from groups A and B described in claim 1.

25. Use of target bacteria in feces obtained from a subject with impaired intestinal barrier function after administration of a candidate substance or candidate composition to the subject, for screening for a substance or composition that has the effect of improving intestinal barrier function or the effect of preventing or improving diseases or symptoms caused by impaired intestinal barrier function, wherein the target bacteria include one or more bacteria selected from group A and group B described in claim 1.

26. Use of target bacteria in feces obtained from a subject with impaired intestinal barrier function after administration of a candidate substance or candidate composition to the subject, for evaluating the effect of the candidate substance or candidate composition in improving intestinal barrier function or the effect of the candidate substance or candidate composition in preventing or improving diseases or symptoms caused by impaired intestinal barrier function, wherein the target bacteria comprise one or more bacteria selected from group A and group B described in claim 1.

27. The use according to any one of claims 21 to 26, wherein the intestinal barrier function is a 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 according to claim 1.

29. The use of any one of claims 21 to 26, wherein the target bacteria comprises one or more bacteria selected from Ruminococcaceae bacteria, Peptostreptococcaceae bacteria, Rikenellaceae bacteria, Peptostreptococcaceae Romboutsia bacteria, and Rikenellaceae Alistipes bacteria.

30. A kit for assessing intestinal barrier function of a subject, the kit comprising one or more reagents for measuring the occupancy of target bacteria in feces derived from the subject, wherein the occupancy of the target bacteria comprises the occupancy of one or more bacteria selected from groups A and B described in claim 1.

31. A kit for diagnosing a disease or symptom caused by a decrease in intestinal barrier function in a subject, the kit comprising one or more reagents for measuring the occupancy of target bacteria in feces derived from the subject, wherein the occupancy of the target bacteria comprises the occupancy of one or more bacteria selected from group A and group B described in claim 1.

32. A kit for assessing the risk of a subject developing a disease or symptom caused by a decrease in intestinal barrier function, or the possibility of the subject suffering from said disease or symptom, the kit comprising one or more reagents for measuring the occupancy of target bacteria in feces derived from the subject, wherein the occupancy of the target bacteria comprises the occupancy of one or more bacteria selected from group A and group B described in claim 1.

33. A kit for obtaining data for diagnosing a disease or symptom caused by a decrease in intestinal barrier function in a subject, the kit comprising one or more reagents for measuring the occupancy of target bacteria in feces derived from the subject, the occupancy of the target bacteria comprising the occupancy of one or more bacteria selected from group A and group B described in claim 1.

34. A kit for screening for a substance or composition that has the effect of improving intestinal barrier function or the effect of preventing or improving diseases or symptoms caused by a decrease in intestinal barrier function, the kit comprising one or more reagents for measuring the occupancy of target bacteria in feces obtained from a subject with a decreased intestinal barrier function after administering a candidate substance or candidate composition to the subject, the occupancy of the target bacteria comprising the occupancy of one or more bacteria selected from group A and group B described in claim 1.

35. A kit for evaluating the effect of a candidate substance or candidate composition on improving intestinal barrier function or the effect of preventing or improving diseases or symptoms caused by a decrease in intestinal barrier function, the kit comprising one or more reagents for measuring the occupancy rate of target bacteria in feces obtained from a subject with a decreased intestinal barrier function after administration of the candidate substance or candidate composition to the subject, the occupancy rate of the target bacteria comprising the occupancy rate of one or more bacteria selected from group A and group B described in claim 1.

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

37. A kit according to any one of claims 30 to 35, wherein the occupancy of the target bacteria comprises an occupancy of two, three, or four or more bacteria selected from groups A and B of claim 1.

38. The kit of any one of claims 30 to 35, wherein the occupancy of the target bacteria comprises one or more occupancies selected from the occupancy of Ruminococcaceae bacteria, the occupancy of Peptostreptococcaceae bacteria, the occupancy of Rikenellaceae bacteria, the occupancy of Peptostreptococcaceae Romboutsia bacteria, and the occupancy of Rikenellaceae Alistipes bacteria.

39. The kit of any one of claims 30 to 35, wherein the occupancy of the target bacteria is measured by bacterial flora analysis using 16S rRNA amplicon sequencing.

40. A method for producing a food or pharmaceutical composition, comprising the step of blending a substance or composition screened by the method of claim 6 with one or more ingredients that constitute the food or pharmaceutical composition.

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

42. A computer program for causing a computer to execute a method for diagnosing a disease or symptom caused by a decrease in intestinal barrier function in a subject, the method comprising the following steps: (S201) a step of obtaining a measurement value of the occupancy rate of target bacteria in feces derived from the subject; (S202) a step of comparing the measurement value obtained in step S201 with a reference value and obtaining a comparison result; (S203) a step of diagnosing the disease or symptom based on the comparison result obtained in step S202, wherein the occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from group A and group B described in claim 1.

43. A computer program for causing a computer to execute a method for assessing a subject's risk of developing a disease or symptom caused by a decrease in intestinal barrier function, or the possibility of suffering from said disease or symptom, the method comprising the following steps: (S301) a step of obtaining a measurement value of the occupancy rate of target bacteria in stool derived from the subject; (S302) a step of comparing the measurement value obtained in step S301 with a reference value and obtaining a comparison result; and (S303) a step of evaluating the risk or the possibility based on the comparison result obtained in step S302, wherein the occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from group A and group B described in claim 1.

44. A computer program for causing a computer to execute a method for obtaining data for diagnosing a disease or symptom caused by a decrease in intestinal barrier function in a subject, the method comprising the following steps: (S401) obtaining a measurement value of the occupancy rate of target bacteria in feces derived from the subject; and (S402) comparing the measurement value obtained in step S401 with a reference value and obtaining the comparison result as the data, wherein the occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from group A and group B described in claim 1.

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

46. ​​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 the effect of preventing or improving diseases or symptoms caused by a decrease in intestinal barrier function, the method comprising the following steps: (S601) obtaining a measurement value of the occupancy rate of target bacteria in feces obtained from a subject with decreased intestinal barrier function after administering a candidate substance or candidate composition to the subject; (S602) comparing the measurement value obtained in step S601 with a reference value and obtaining a comparison result; and (S603) evaluating the effect of the candidate substance or candidate composition on improving intestinal barrier function or the effect of preventing or improving diseases or symptoms based on the comparison result obtained in step S602, wherein the occupancy rate of the target bacteria includes the occupancy rate of one or more bacteria selected from group A and group B described in claim 1.

47. A computer program according to any one of claims 41 to 46, wherein the intestinal barrier function is a barrier function of the small intestine.

48. A computer program according to any one of claims 41 to 46, wherein the occupancy of the target bacteria comprises the occupancy of two, three, or four or more bacteria selected from groups A and B of claim 1.

49. The computer program of any one of claims 41 to 46, wherein the occupancy of the target bacteria comprises one or more occupancies selected from the occupancy of Ruminococcaceae bacteria, the occupancy of Peptostreptococcaceae bacteria, the occupancy of Rikenellaceae bacteria, the occupancy of Peptostreptococcaceae Romboutsia bacteria, and the occupancy of Rikenellaceae Alistipes bacteria.

50. The computer program of any one of claims 41 to 46, wherein the occupancy of the target bacteria is measured by bacterial flora analysis using 16S rRNA amplicon sequencing.

51. A computer-readable recording medium on which the computer program according to any one of claims 41 to 46 is recorded.

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