Method for evaluating test substance in intestinal bacterial flora

Abstract

The present disclosure provides a method for evaluating, in vitro, the influence of a test substance such as a food or a drug candidate compound on intestinal bacterial flora in a mammal, particularly a human. More specifically, the present disclosure provides a method for increasing at least one of α-diversity (such as Shannon coefficient), OTU and the like with an increase in the content of intestinal bacterial flora added to a culture medium for culturing in a container in an original equilibrated state, the method being characterized by using no stirring device.

Description

Methods for evaluating test substances in the intestinal microbiota

[0001] The present disclosure relates to a method for evaluating a test substance in the intestinal microbiota.

[0002] A wide variety of bacteria constantly proliferate in the intestinal tract of mammals, and these bacteria are called the intestinal flora. In recent years, it has become clear that the intestinal flora has various effects on human health, and therefore, an in vitro method for evaluating the effects of test substances such as foods and drug candidate compounds on the intestinal environment has been reported (Patent Document 1).

[0003] Patent No. 7051175

[0004] The present inventors have previously developed an apparatus for maintaining human intestinal bacterial species in a culture system using human feces as an inoculum source. After extensive research, the present inventors discovered that restoring a culture solution containing intestinal microbiota to a state of original equilibrium is useful for evaluating test substances. Based on this finding, the present disclosure is as follows: [1] A method for increasing at least one selected from the group consisting of α-diversity (Shannon coefficient, Ciao1 coefficient, Simpson coefficient, etc.) and the number of OTUs with an increase in the content of intestinal microbiota added to a culture medium for culturing in a vessel at an original equilibrium state, the method being characterized by not using a stirring device. [2] A method for increasing at least one selected from the group consisting of α-diversity (Shannon coefficient, Ciao1 coefficient, Simpson coefficient, etc.) and the number of OTUs with an increase in the content of intestinal microbiota added to a culture medium for culturing in a vessel at an original equilibrium state, the method being characterized by not adjusting the pH. [3] A method for increasing at least one selected from the group consisting of α-diversity (Shannon coefficient, Ciao1 coefficient, Simpson coefficient, etc.) and the number of OTUs with an increase in the content of intestinal microbiota added to a culture medium for culturing in a vessel under a state of original equilibrium, wherein the vessel is a microwell type. [4] A method for increasing at least one selected from the group consisting of α-diversity (Shannon coefficient, Ciao1 coefficient, Simpson coefficient, etc.) and the number of OTUs with an increase in the content of intestinal microbiota added to a culture medium for culturing in a vessel under a state of original equilibrium, wherein the volume of the vessel is suitable for shaking culture. [5] A method for increasing at least one selected from the group consisting of α-diversity (Shannon coefficient, Ciao1 coefficient, Simpson coefficient, etc.) and the number of OTUs with an increase in the content of intestinal microbiota added to a culture medium for culturing in a vessel under a state of original equilibrium, wherein the vessel has a volume of 3 mL or less. [6] A method for increasing at least one selected from the group consisting of alpha diversity (Shannon coefficient, Ciao1 coefficient, Simpson coefficient, etc.) and the number of OTUs with an increase in the content of intestinal flora added to a medium for culturing in a container under an original equilibrium state, wherein the volume of the medium is 3 mL or less.[7] The method according to any one of the above items, wherein the original equilibrated medium is used to evaluate a test substance. [8] A container or culture well containing the medium for culturing the intestinal microbiota, so that the number of OTUs and / or the Shannon coefficient increases with an increase in the content of the intestinal microbiota relative to the medium in an original equilibrated state, wherein the volume of the container is suitable for shaking culture. [9] A container or culture well containing the medium for culturing the intestinal microbiota, so that the number of OTUs and / or the Shannon coefficient increases with an increase in the content of the intestinal microbiota relative to the medium in an original equilibrated state, wherein the volume of the container is 3 mL or less.

[10] A method for evaluating a test substance, comprising: A) adding the intestinal microbiota to a culture well containing a medium with a volume of 3 mL or less; B) culturing the intestinal microbiota until it reaches an original equilibrated state; C) adding a test substance to the container; and D) obtaining and evaluating evaluation items before and after adding the test substance.

[11] A system for evaluating a test substance, comprising: A) a container including a culture well capable of accommodating a medium having a volume of 3 mL or less; B) a means for adding an intestinal microbiota; C) a means for culturing until a state of original equilibrium is reached; D) a means for adding a test substance; and E) a means for acquiring and evaluating evaluation items before and after the addition of the test substance.

[12] The method, container, culture well, or system described in the above items, wherein the state of original equilibrium includes equilibrating the microbiota diversity to the state of a pre-inoculation sample of the intestinal microbiota.

[13] The method, container, culture well, or system described in the above items, wherein the evaluation item after the addition of the test substance is acquired while the state of original equilibrium is maintained.

[14] The method, container, culture well, or system described in the above items, wherein the evaluation item after the addition of the test substance is acquired 24 to 96 hours after the start of culture.

[15] The method, container, culture well, or system described in the above items, wherein the test substance is added 6 to 72 hours after the start of culture.

[16] The method, container, culture well, or system according to any one of the preceding items, wherein the evaluation item after addition of the test substance is obtained 12 hours to 96 hours after addition of the test substance.

[17] The method, container, culture well, or system described in the preceding items, wherein the original equilibrium state is determined based on structural analysis of the intestinal bacterial flora.

[18] The method, container, culture well, or system described in the preceding items, wherein the original equilibrium state is determined based on analysis of 16S or genome of the intestinal bacterial flora.

[19] The method, container, culture well, or system described in the preceding items, wherein the original equilibrium state is determined based on the Pearson product-moment correlation coefficient of the 16S or genome analysis of the intestinal bacterial flora.

[20] The method, container, culture well, or system described in the preceding items, wherein the timing of adding the test substance is determined by the following steps A and B: (Step A) culturing a stool specimen containing the intestinal bacteria in a medium and obtaining in advance time-course data on the Pearson product-moment correlation coefficient of the bacterial flora structure before and after culturing, and (Step B) determining in advance the timing of adding the test substance to the specimen containing the intestinal bacteria cultured in the medium based on the time-course data obtained in Step A.

[21] The method, container, culture well, or system described in the above items, wherein the timing of adding the test substance to the original equilibrium state is determined from a time range in which a state in which the Pearson product-moment correlation coefficient between the microbiota structure before and after culture of the intestinal bacteria is 0.70 or higher is maintained for 24 hours or more.

[22] The method, container, culture well, or system described in the above items, wherein the medium contains mucin.

[23] The method, container, culture well, or system described in the above items, wherein the mucin is contained in the medium at a concentration of 0.4% or more.

[24] The method, container, culture well, or system described in the above items, wherein the intestinal microbiota is obtained from stool.

[25] The method, container, culture well, or system described in the above items, wherein the intestinal microbiota is obtained from human stool.

[26] The method, container, culture well, or system described in the above items, wherein the amount of sample added is 0.1% or more and 1.25% or less of the medium.

[27] A kit for evaluating a test substance, comprising an incubator having a culture well for accommodating a medium with a volume of 3 mL or less, the medium, and a substance for promoting and maintaining original equilibrium.

[0005] The present disclosure improves the reproducibility of changes in the intestinal microbiota caused by the administration of a test substance to humans. The present disclosure can suppress events that can change the composition of the intestinal microbiota due to culture, making it possible to more accurately examine the influence and effect of a test substance on the intestinal microbiota.

[0006] As described above, the present disclosure provides a method for evaluating a test substance, which enables the in vivo effects of a test substance such as a food or a drug candidate compound on the intestinal environment to be reproduced in vitro.

[0007] The method of the present disclosure allows for more accurate evaluation of the effects of test substances, such as food and drug candidate compounds, on the intestinal microbiota of mammals, particularly humans. In particular, the use of microwells allows for more accurate and efficient evaluation of candidate substances, while saving dose and maintaining a richer, more "restored" state of equilibrium (alpha diversity, such as the Shannon coefficient, Ciao1 coefficient, Simpson coefficient, and OTU number).

[0008] The present disclosure will now be described with reference to the best mode. Throughout this specification, singular expressions should be understood to include the plural concept unless otherwise specified. Therefore, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept unless otherwise specified. Furthermore, it should be understood that terms used in this specification are used in the sense commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In the event of conflict, the present specification (including definitions) will prevail.

[0009] (Definition of Terms) The definitions of terms particularly used in this specification and / or basic technical content will be explained as appropriate below.

[0010] All numerical values ​​herein are assumed to be modified by the term "about," whether expressly stated or not. The term "about" generally refers to a range of numerical values ​​that one of ordinary skill in the art would consider equivalent to the recited value (i.e., having the same function or result). In many instances, the term "about" may include numbers that are rounded to the nearest significant figure.

[0011] As used herein, the term "intestinal flora" refers to a group of bacteria normally present in the intestines of animals with an intestinal tract (e.g., mammals such as humans). Examples of bacteria that constitute the intestinal flora of healthy humans include, at the phylum level, bacteria belonging to the following phyla: Verrucomicobiota, Pseudomonadota, Fusobacteriota, Bacillota, Bacteroidota, and Actinomycetota. Further, at the genus level, bacteria belonging to the following genera or families may be mentioned: Bifidobacterium, Collinsella, Bacteroides, Parabacteroides, Prevotella, Rikenellaceae, Lactobacillales, etc.

[0012] As used herein, "animals having an intestinal tract" refers to any animal having an intestinal tract, such as mammals (mammals), birds, reptiles, amphibians, and fish, with mammals being preferred. "Mammals" include humans; pet animals such as dogs and cats; research animals such as mice and rats; and livestock such as pigs. In the present disclosure, "mammals" are preferably humans.

[0013] As used herein, the term "test substance" is not particularly limited as long as it is a material that has the potential to affect the intestinal environment of an animal, and may be food, physiologically active substances derived from food, food additives, beverages, microorganisms (bacteria, fungi, etc., including killed cells and extracts derived from cells), physiologically active substances, pharmaceuticals, pharmaceutical-like compounds, and mixtures thereof.

[0014] The present disclosure relates to a technique for restoring the intestinal bacterial flora in a culture medium to a state of original equilibrium.

[0015] As used herein, the term "original state" refers to the state of the intestinal bacterial flora of a sample such as feces (sometimes referred to as the intestinal bacterial flora before the start of culture).

[0016] As used herein, "remaining in equilibrium" refers to a state in which the original state is in equilibrium, i.e., within a certain range of fluctuation, and such a state is referred to as a "state of original equilibrium." Whether or not original equilibrium has been achieved can be evaluated using the Pearson product-moment correlation coefficient, which is a coefficient used to evaluate the degree of similarity between the flora of two bacterial flora. For example, when the Pearson product-moment correlation coefficient between the intestinal flora before the start of culture and the intestinal flora after the start of culture is 0.50 or higher, or 0.60 or higher, usually 0.70 or higher, preferably 0.80 or higher, or 0.85 or higher, 0.90 or higher, or 0.95 or higher, this can be considered to be original equilibrium. Furthermore, when original equilibrium continues for a certain period of time, this is referred to as an original equilibrium state. The certain period of time is 6 hours or higher, 12 hours or higher, or 18 hours or higher, preferably 24 hours or higher, more preferably 36 hours or higher, even more preferably 48 hours or higher, and even more preferably 72 hours or higher. In this specification, the "stabilization determination index" is calculated as an index until the original state equilibrium is reached.

[0017] In this specification, substances that promote and / or maintain the state equilibrium are also referred to as "state equilibrium promoting substances" or "state equilibrium maintaining substances," respectively. When a substance has both functions, it may also be called a state equilibrium promoting / maintaining substance.

[0018] As used herein, the term "medium" refers to any medium in which a bacterial colony can grow.

[0019] As used herein, the term "medium components" refers to each component that constitutes a medium.

[0020] As used herein, the term "microwell" refers to a plate having a particularly small cavity, also known as a microtiter plate or microplate. Microwells that can be used include, but are not limited to, the following:

[0021] Representative well plates: Sarstedt Other commercially available deep well plates: * Thermo Scientific Nunc https: / / axel.as-1.co.jp / asone / g / NC2008033368 / * Eppendorf umables / Plates / Eppendorf-Deepwell-Plates-p-PF-55960 * Scientific Specialties 65084 * FCR&BIO * BM Equipment Co., Ltd. * Watson https: / / watson.co.jp / detail / product / plate / other-plate.html

[0022] As used herein, the term "agitation device" refers to a device that agitates a medium in a container, and is defined as being different from a shaker.

[0023] For the understanding of the various terms used in this specification, including those mentioned above, where appropriate, all references cited in this specification, including scientific literature, patents, patent applications, and the like, are hereby incorporated by reference in their entirety to the same extent as if each were specifically set forth.

[0024] (Preferred Embodiments) Preferred embodiments of the present disclosure will be described below. The embodiments provided below are provided for a better understanding of the present disclosure, and it is understood that the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that those skilled in the art can make appropriate modifications within the scope of the present disclosure in light of the description herein. It is also understood that the following embodiments of the present disclosure can be used alone or in combination.

[0025] (Technology for improving the efficiency of original state equilibration) In one aspect, the present disclosure provides a method for increasing at least one selected from the group consisting of alpha diversity (such as the Shannon coefficient) and the number of OTUs when increasing the content of intestinal microbiota added to a medium for culturing in a container in an original state equilibration, wherein the volume of the medium and / or container is suitable for shaking culture.

[0026] The present disclosure is based on the discovery that, when a state of equilibrium is achieved in a large volume suitable for agitated culture such as jar culture, and various evaluation methods are performed as needed, and the dose is reduced, and while not wishing to be bound by theory, an increase in the intestinal microbiota reduces the diversity index, particularly α diversity (such as the Shannon coefficient) and the number of OTUs, and that this objective can unexpectedly be achieved using a small volume such as a microwell and / or agitated culture.

[0027] In another aspect, the present disclosure provides a method for increasing at least one of α diversity (such as the Shannon coefficient) and the number of OTUs when increasing the content of gut microbiota added to a medium for culturing in a container under a state of original equilibrium, wherein the volume of the medium is 3 mL or less and / or the container is 3 mL or less, as well as a container, culture device, etc. used therein. It has been unexpectedly found that by reducing the volume of the medium and / or culture container and performing evaluation in an appropriate format, at least one of α diversity (such as the Shannon coefficient) and the number of OTUs can be increased.

[0028] In another aspect, the present disclosure provides a method for increasing at least one selected from the group consisting of α-diversity (such as the Shannon coefficient) and the number of OTUs in a vessel with an increased content of intestinal microbiota added to a medium for culturing in a vessel under a pristine equilibrium state, the method being characterized by not using a stirring device and / or not adjusting the pH, as well as a vessel, a culture device, etc. used therein. It was unexpectedly found that by performing evaluation in a format suitable for shaking culture and / or in which the pH is not adjusted, it is possible to increase at least one of α-diversity, the number of OTUs, and / or the Shannon coefficient.

[0029] In another aspect, the present disclosure provides a method for increasing at least one selected from the group consisting of α-diversity (such as the Shannon coefficient) and the number of OTUs as the content of gut microbiota added to a medium for culturing in a container under a pristine equilibrium state increases, the method being characterized in that the container is microwell-shaped, as well as a container, a culture device, etc. used therein. It has been discovered that a shape such as a microwell can unexpectedly increase at least one of α-diversity (such as the Shannon coefficient) and the number of OTUs.

[0030] In another aspect, the present disclosure provides a method for increasing at least one selected from the group consisting of α-diversity (such as the Shannon coefficient) and the number of OTUs as the content of gut microbiota added to a medium for culturing in a vessel under a pristine equilibrium state increases, wherein the volume of the vessel is suitable for shaking culture, as well as a vessel, a culture device, etc. used therefor. It was unexpectedly found that by performing the evaluation in a format suitable for shaking culture, it was possible to increase at least one of α-diversity (such as the Shannon coefficient) and the number of OTUs.

[0031] In one embodiment, the original equilibrated medium is used to evaluate a test substance. It has been found that, in the evaluation of a test substance, performing large-scale evaluation in a small volume in a format suitable for shaking culture, such as a microwell, can unexpectedly increase at least one of α diversity (such as the Shannon coefficient) and the number of OTUs.

[0032] In another aspect, the present disclosure provides a container containing a medium for culturing a gut microbiota to increase the number of OTUs and / or Shannon coefficient, or alpha diversity, when increasing the content of the gut microbiota in the medium in an original equilibrium state, wherein the volume of the medium is a volume suitable for shaking culture (or 3 mL or less). The appropriate volume may be 5 mL or less, 4.5 mL or less, 4 mL or less, 3.5 mL or less, 3 mL or less, 2.5 mL or less, etc. This is because appropriate shaking culture allows the bacterial flora to grow appropriately.

[0033] In one embodiment, the original equilibration condition comprises equilibrating the microbiota diversity to that of a pre-inoculation sample of the intestinal microbiota.

[0034] In another embodiment, the endpoints after addition of the test substance are obtained while the original equilibration conditions are maintained.

[0035] In another embodiment, the evaluation item after addition of the test substance is obtained no less than 24 hours and no more than 96 hours after the start of culture.

[0036] In another embodiment, the test substance is added 6 hours or more, preferably 12 hours or more and 72 hours or less, after the incubation.

[0037] In another embodiment, the post-test substance assessment is obtained 6 hours or more, preferably 12 hours or more and 96 hours or less, after addition of the test substance.

[0038] In another embodiment, the original equilibrium state is determined based on a structural analysis of the intestinal microbiota.

[0039] In another embodiment, the original equilibrium state is determined based on 16S or genomic analysis of the gut microbiota.

[0040] In another embodiment, the structural analysis advantageously uses the Pearson product-moment correlation coefficient.

[0041] In another embodiment, the timing of adding the test substance is determined by a method comprising the following steps A and B: (Step A) culturing a stool specimen containing the intestinal bacteria in a culture medium and obtaining in advance time-course data on the Pearson product-moment correlation coefficient of the bacterial flora structure before and after culturing; and (Step B) determining in advance the timing of adding the test substance to the specimen containing the intestinal bacteria cultured in a culture medium, based on the time-course data obtained in Step A.

[0042] In another embodiment, the original state equilibration state of the present disclosure is characterized in that the timing of adding the test substance is determined based on a time range in which a Pearson product-moment correlation coefficient between the intestinal bacterial flora before and after culture is maintained at 0.8 or greater for 24 hours or more. This can be modified as appropriate. For example, original state equilibration can be achieved when the Pearson product-moment correlation coefficient between the intestinal bacterial flora before and after culture is 0.5 or greater, or 0.6 or greater, typically 0.70 or greater, and preferably 0.8 or greater, or 0.85 or greater, 0.90 or greater, or 0.95 or greater. Furthermore, original state equilibration is defined as a state in which original state equilibration continues for a certain period of time. This certain period of time is 6 hours or greater, 12 hours or greater, or 18 hours or greater, preferably 24 hours or greater, more preferably 36 hours or greater, even more preferably 48 hours or greater, and even more preferably 72 hours or greater. As used herein, the "stabilization determination index" is calculated as an index indicating the period until original state equilibration is achieved.

[0043] In another embodiment, the medium used in the present disclosure comprises mucin. In a particular embodiment, the mucin is present in the medium at a concentration of 0.4% or more.

[0044] In another embodiment, the bacterial flora is obtained from stool, preferably the bacterial flora is obtained from human stool.

[0045] In another embodiment, the amount of the sample added to the medium is 0.05% or more, alternatively 0.1% or more, and 1.5% (w / v) or less, alternatively 1.25% or less (w / v), typically 0.1% or more and 1.25% or less (w / v), relative to the medium.

[0046] (Evaluation Technique) In another aspect, the present disclosure provides a method for evaluating a test substance, comprising the steps of: A) adding an intestinal bacterial flora to a culture well having a medium with a volume of 3 mL or less; B) culturing the culture medium until the culture medium reaches a state of original equilibrium; C) adding a test substance; and D) obtaining and evaluating evaluation items before and after the addition of the test substance.

[0047] In another aspect, the present disclosure provides a method for evaluating a test substance, comprising the steps of: A) adding an intestinal bacterial flora to a culture well containing a medium, without using an agitator, preferably in a container with a volume suitable for shaking culture, and / or without adjusting the pH; B) culturing the culture until the culture reaches a state of original equilibrium; C) adding a test substance; and D) obtaining and evaluating evaluation items before and after adding the test substance.

[0048] In another aspect, the present disclosure provides a method for evaluating a test substance, comprising the steps of: A) adding an intestinal bacterial flora to a medium having a volume of 3 mL or less and / or a culture well having a container volume of 3 mL; B) culturing the medium until the intestinal bacterial flora reaches a state of original equilibrium; C) adding a test substance; and D) obtaining and evaluating evaluation items before and after the addition of the test substance.

[0049] In another aspect, the present disclosure provides a method for evaluating a test substance, comprising the steps of: A) adding an intestinal bacterial flora to a culture well containing a medium in a microwell-type container; B) culturing the intestinal bacterial flora until the intestinal bacterial flora reaches a state of original equilibrium; C) adding a test substance; and D) obtaining and evaluating evaluation items before and after the addition of the test substance.

[0050] In another aspect, the present disclosure provides a system for evaluating a test substance, comprising: A) a container including a culture well capable of accommodating a medium having a volume of 3 mL or less; B) means for adding an intestinal bacterial flora; C) means for culturing the intestinal bacterial flora until the intestinal bacterial flora reaches a state of original equilibrium; D) means for adding a test substance; and E) means for obtaining and evaluating evaluation items before and after the addition of the test substance.

[0051] In another aspect, the present disclosure provides a system for evaluating a test substance, comprising: A) a container including a microwell-type culture well; B) means for adding intestinal bacterial flora; C) means for culturing until the intestinal bacterial flora reaches a state of original equilibrium; D) means for adding a test substance; and E) means for obtaining and evaluating evaluation items before and after the addition of the test substance.

[0052] In another aspect, the present disclosure provides a system for evaluating a test substance, comprising: A) a container including a culture well capable of accommodating a medium in a volume suitable for shaking culture or having a shape suitable for shaking culture; B) means for adding intestinal bacterial flora; C) means for culturing until a state of original equilibrium is reached; D) means for adding a test substance; and E) means for obtaining and evaluating evaluation items before and after the addition of the test substance.

[0053] In another aspect, the present disclosure provides a system for evaluating a test substance, comprising: A) a container including a culture well equipped with a means for culturing without using a stirrer; B) a means for adding intestinal bacterial flora; C) a means for culturing until a state of original equilibrium is reached; D) a means for adding a test substance; and E) a means for obtaining and evaluating evaluation items before and after the addition of the test substance.

[0054] In another aspect, the present disclosure provides a system for evaluating a test substance, comprising: A) a container including a culture well capable of accommodating a culture medium without adjusting the pH; B) means for adding an intestinal bacterial flora; C) means for culturing the culture medium until the culture medium reaches a state of original equilibrium; D) means for adding a test substance; and E) means for obtaining and evaluating evaluation items before and after the addition of the test substance.

[0055] (Test Substance Addition Timing) In one embodiment, the improved evaluation method of the present disclosure features an improved timing for adding a test substance to a sample containing intestinal microbiota. More specifically, the present disclosure is characterized in that the test substance is added during the original equilibrium state. The addition may be performed at any time during the original equilibrium period, but from the standpoint of enabling a longer-term evaluation of the effects of the test substance on the intestinal microbiota after addition and evaluation efficiency, it is preferable to add the test substance at the beginning of the original equilibrium state. That is, the test substance can be added 6 hours or more after the start of culturing the intestinal bacteria, for example, between 12 and 120 hours, more preferably between 15 and 96 hours, even more preferably between 24 and 96 hours, and most preferably between 24 and 72 hours after the start of culturing.

[0056] (Cultivation and Collection After Addition of Test Substance) In one embodiment, it may be advantageous to select the timing of sample acquisition for evaluation. After adding a test substance to a culture medium and further culturing, the culture medium is collected for evaluation. The sample may be collected at any time after adding the test substance to the culture medium, but it is preferably collected while the original equilibrium state is maintained. Specifically, the sample is collected 6-120 hours after the start of culturing the intestinal bacterial flora, and this time is at least 6 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 36 hours, at least 48 hours, and / or at most 120 hours, at most 114 hours, at most 108 hours, at most 102 hours, at most 96 hours, at most 90 hours, at most 84 hours, at most 78 hours, at most 72 hours, at most 66 hours, at most 60 hours, at most 54 hours, or at most 48 hours. The timing at which the culture medium is collected for evaluation is 6 hours or more, 12 hours or more, 18 hours or more, 24 hours or more, 36 hours or more, 48 hours or more, and / or 126 hours or less, 120 hours or less, 114 hours or less, 108 hours or less, 102 hours or less, 96 hours or less, 90 hours or less, 84 hours or less, 78 hours or less, 72 hours or less, 66 hours or less, 60 hours or less, 54 hours or less, or 48 hours or less after the test substance is added to the culture medium.

[0057] (Culture Medium) The culture medium used in the present disclosure is not particularly limited as long as it is a medium in which enterobacteria can grow, and examples thereof include GAM medium, YCFA medium, modified YCFA medium, BBL medium, SOC medium, LB medium, etc. Among these, GAM medium is preferred, and for example, GAM agar medium, modified GAM agar medium, GAM semi-solid high-layer medium, GAM bouillon, and modified GAM bouillon (all manufactured by Nippon Pharmaceutical Co., Ltd.) can be used. Two or more types of culture medium selected from these media may be mixed in any ratio and used.

[0058] (Additive Components) In a preferred embodiment, various additive components can be added to the medium. Examples of additive components added to the medium used in the present disclosure include high molecular weight glycoproteins. High molecular weight glycoproteins that can be used in the present disclosure include polypeptides comprising an amino acid sequence with a tandem repeat structure to which an O-linked glycan has been attached.

[0059] As used herein, a tandem repeat structure refers to a structure in which an amino acid sequence of one to several dozen amino acids in length is regularly repeated. Examples of O-linked sugar chains include, but are not limited to, O-mannose, O-N-acetylglucosamine, O-fucose, O-glucose, and O-galactose. In the high molecular weight glycoprotein of the present disclosure, GalNAc (N-acetylgalactomisan) in the sugar chain is bound to a hydroxyl group of serine or threonine in the polypeptide via an O-glycosidic bond. The molecular weight of the high molecular weight glycoprotein of the present disclosure is from about 500,000 to about 20 million, more preferably from about 1 million to about 10 million.

[0060] Examples of high molecular weight glycoproteins include secretory mucins and membrane-bound mucins. Examples of secretory mucins include MUC2, MUC5AC, MUC5B, MUC6, and MUC7, while examples of membrane-bound mucins include MUC1, MUC3, MUC4, MUC12, MUC13, MUC16, MUC17, MUC20, and MUC21. Other mucins that can be used include MUC8, 9, 10, 11, 14, 15, 18, and 19. Of these, secretory mucins such as MUC2, MUC5AC, MUC5B, MUC6, and MUC7 are preferably used.

[0061] The mucin may be of human or non-human origin, for example, porcine.

[0062] The amount of high molecular weight glycoprotein added to the medium is preferably 0.1% by mass or more, more preferably 0.2% by mass or more, and even more preferably 0.4% by mass or more, relative to the medium from the viewpoint of obtaining a pristine equilibrium state, and is preferably 4.0% by mass or less, more preferably 2.0% by mass or less, from the viewpoint of maintaining the bacterial colony structure.

[0063] In certain embodiments, additional components other than high molecular weight glycoproteins may be added to the medium, for example, carbon sources such as glucose, nitrogen sources such as ammonia, nutrient sources such as vitamins and inorganic salts, and scaffolds necessary for bacterial growth may be added as needed.

[0064] In the present disclosure, the medium may be sterilized, for example, by autoclaving, before culturing. Liquid culture is preferred, and the liquid culture is also referred to as a "culture medium." During culturing, the culture medium may be appropriately stirred.

[0065] (Intestinal Microbiota Used in the Present Disclosure and Sample Preparation) The intestinal microbiota used in the present disclosure may be obtained from a stool sample or from a sample other than stool. One or more specific intestinal bacteria may also be used. The sample may be from a human or a non-human animal, with human samples being preferred, and human stool being even more preferred. Furthermore, the stool sample may be obtained immediately after excretion from the intestine, may be frozen after collection, or may be collected from the intestine. After collection, the stool sample may be stored in a container such as an anaerobic culture swab until the start of culture. The collected stool may be mixed with phosphate buffer solution (PBS) to form a suspension. The phosphate buffer solution used to suspend the stool may also contain ascorbic acid or glycerin. The concentration of the stool in the stool suspension may be 0.01% (w / v) to 50% (w / v). From the viewpoint of bacterial flora diversity, the amount of stool sample to be inoculated into the culture medium is preferably 0.05% (w / v) or more in the culture solution, more preferably 0.1% (w / v) or more, and even more preferably 0.12% (w / v) or more. From the viewpoint of unintended bacterial growth, the amount is preferably 5.0% (w / v) or less, more preferably 2.5% (w / v) or less, and even more preferably 1.25% (w / v) or less. As used herein, "inoculation" refers to taking a fixed amount from a stool suspension and adding it to a culture medium.

[0066] (Culture Vessels and Apparatus Used in the Present Disclosure) The culture vessels used in the present disclosure may be flasks, commercially available culture vessels, multi-well plates, or the like. Multi-well plates are preferably used to increase the throughput of evaluation. Here, the shape of each well of the multi-well plate may be approximately hemispherical, approximately rectangular, or approximately cylindrical, and the bottom may be flat or round. When using a multi-well plate, the volume per well is preferably 5 mL or less, more preferably 3 mL or less, and even more preferably 2 mL or less. Furthermore, the volume per well is preferably 0.1 mL or more, more preferably 0.2 mL or more.

[0067] The culture in the culture vessel is stirred during cultivation, preferably using a shaking stirrer. The shaking speed when using a shaking stirrer is preferably 30 to 2000 rpm, more preferably 50 to 2000 rpm, and most preferably 100 to 1000 rpm, but is not limited to these ranges.

[0068] (Culture Conditions - Atmosphere) In the present disclosure, the culture is carried out in an anaerobic environment. The anaerobic culture environment can be created by aerating an anaerobic gas into the culture medium. The anaerobic gas can be, for example, nitrogen, nitrogen and carbon dioxide, or nitrogen, carbon dioxide, and hydrogen. The anaerobic gas is aerated continuously or intermittently at a predetermined flow rate (e.g., 0.1 to 1.0 dL / min). Furthermore, since intestinal gas may contain, for example, nitrogen or carbon dioxide, the anaerobic gas is preferably a mixed gas consisting of nitrogen and carbon dioxide. Note that, in order to maintain highly anaerobic conditions, it is preferable to aerate the anaerobic gas continuously.

[0069] (Culture Conditions - pH) In the present disclosure, the pH of the culture medium at the start of culture is preferably 6.2 to 6.7, more preferably 6.2 to 6.5. By adjusting the pH to within the above range at the start of culture (for example, when the culture medium containing a fecal sample is placed in an anaerobic environment), it is possible to match the pH in the large intestine of a mammal corresponding to the feces used. It is sufficient that the pH of the culture medium at the start of culture is within the above range, and thereafter, the pH may be left as is without any particular adjustment, or the pH may be adjusted to within the above range using a pH adjuster as necessary to prevent an extreme drop in pH.

[0070] (Culture conditions - temperature, agitation) The culture temperature is preferably set to a temperature close to the body temperature of the mammal corresponding to the feces used, in order to mimic the environment in the large intestine of the mammal. For example, when human feces is used, the culture temperature is 36°C to 38°C, preferably 36°C to 37°C, since this is a temperature close to that of a healthy human. The culture method is not particularly limited, but a single batch method is preferred. Furthermore, even when the culture vessel is a flask or the like, it is preferable to shake the culture vessel, and even when the culture vessel is a multi-well plate, it is preferable to agitate the culture medium using a shaker.

[0071] (Preparation and Amount of Test Substance Added) The test substance added in the present disclosure is preferably added in the range of 0.1 g to 50 g, more preferably 1 g to 20 g, per 1 L of culture solution. If the test substance is solid, it may be added after dissolving it in a solvent such as water.

[0072] (Acquisition and analysis of bacterial flora structure data) The collected culture solution is used to perform bacterial composition analysis and bacterial diversity analysis of the intestinal flora. Metagenomic analysis of the intestinal flora may be performed using 16sRNA gene sequences or whole genome sequences. For example, as in Reference Example 1 of JP 2021-153471 A, OTUs (Operational Taxonomic Units) that reach 97% similarity can be used to calculate the Shannon index and Pearson product-moment correlation coefficient. Details of the bacterial flora structure data analysis are described in the examples of this specification. Data such as the pH and short-chain fatty acid concentration of the collected culture solution can also be obtained as needed.

[0073] (When the timing of adding the test substance is determined in advance) The present disclosure can also include a step of determining the timing of adding the test substance in advance. The intestinal microbiota is cultured without adding the test substance, and the culture medium is periodically collected to obtain time-course data such as the Shannon index and Pearson coefficient of the intestinal microbiota. From the obtained time-course data, the above-mentioned original equilibrium state can be determined, and the timing of adding the test substance can be determined.

[0074] In another aspect, the present disclosure provides a kit for evaluating a test substance, the kit including an incubator having a culture well for accommodating a medium having a volume of 3 mL or less, the medium, and a substance for promoting and maintaining the original equilibrium, such as mucin.

[0075] A multi-well plate can be preferably used as the incubator having the culture wells in the kit of the present disclosure. The shape of each well of the multi-well plate may be approximately hemispherical, approximately rectangular, or approximately cylindrical, and the bottom may be flat or round. The material of the incubator is not particularly limited, and examples include glass, polyvinyl chloride, cellulose-based polymers, polystyrene, polymethyl methacrylate, polycarbonate, polysulfone, polyurethane, polyester, polyamide, polystyrene, polypropylene, and other plastics.

[0076] The kit of the present disclosure includes a culture medium. The culture medium is not particularly limited as long as it allows the growth of enterobacteria, and examples thereof include GAM medium, YCFA medium, modified YCFA medium, BBL medium, SOC medium, and LB medium. Among these, GAM medium is preferred, and for example, GAM agar medium, modified GAM agar medium, GAM semi-solid high-layer medium, GAM bouillon, and modified GAM bouillon (all manufactured by Nippon Pharmaceutical Co., Ltd.) can be used. Two or more types of culture medium selected from these media can also be mixed in any ratio.

[0077] The kit of the present disclosure further comprises a substance for promoting equilibration and facilitating the growth of a bacterial flora. The substance for promoting equilibration and facilitating the growth of a bacterial flora includes a high molecular weight glycoprotein. Examples of high molecular weight glycoproteins include the mucins described above. The substance for promoting equilibration and facilitating the growth of a bacterial flora in the kit is present in an amount of 0.1% by mass or more, more preferably 0.2% by mass or more, and even more preferably 0.4% by mass or more, based on the mass of the medium. Furthermore, from the viewpoint of maintaining the bacterial flora structure, the substance for promoting equilibration and facilitating the growth of a bacterial flora in the kit is present in an amount of 4.0% by mass or less, more preferably 2.0% by mass or less, based on the mass of the medium.

[0078] By using the kit of the present disclosure, it becomes possible to simply test the influence and effect of a test substance on the intestinal bacterial flora.

[0079] All references cited in this disclosure, including scientific literature, patents, patent applications, and the like, are incorporated by reference in their entirety into this disclosure to the same extent as if each were specifically set forth.

[0080] The present disclosure has been described above by showing preferred embodiments for ease of understanding. Hereinafter, the present disclosure will be described based on examples. However, the above description and the following examples are provided for illustrative purposes only and are not intended to limit the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments or examples specifically described herein, but is limited only by the scope of the claims.

[0081] Example 1 (Inulin Addition (Multiwell Plate Culture)) (Preparation of Fecal Suspension) Feces serving as an inoculum for intestinal bacterial flora were collected from healthy individuals on the day of culture. After collection, the fecal samples were stored in anaerobic culture swabs (212550 BD BBL Culture Swab; Becton, Tickinson & Company) and transported to the laboratory. To prepare the inoculum, 0.5 g of feces was suspended in 2 mL of 0.1 M phosphate buffer (PBS) buffer (pH 6.5, consisting of a 68.5:31.5 (molar ratio) mixture of 0.1 M NaH2PO4 and 0.1 M Na2HPO4) supplemented with 1.0% L-ascorbic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) to prepare a fecal suspension.

[0082] (Preparation of medium) A medium was prepared by mixing 59 g / L of Gifu University-prescribed anaerobic medium (GAM medium [Code 05422] (manufactured by Nissui Pharmaceutical Co., Ltd.), 8.0 g / L of mucin (manufactured by Sigma-Aldrich, derived from porcine stomach, Type III), and 50 μL / L of an antifoaming agent. The pH was adjusted to 6.5 with 0.1 M phosphate buffer, and the medium was sterilized in an autoclave at 115° C. for 15 minutes.

[0083] (Culture Conditions) 1.0 mL of medium was dispensed into the required number of wells in a clean bench. A 96-well multi-well plate (model number 82.1972.002, 2.2 mL capacity, manufactured by Sarstedt Co., Ltd.) was used (hereinafter sometimes referred to as 96-well). The multi-well plate was stirred at approximately 500 rpm using a 96-well shaking incubator (manufactured by Biosan, model number TS-DW) installed in an anaerobic chamber. The culture temperature was 37°C. Culture was initiated by inoculating 50 μL / well of fecal suspension (12.5 mg / mL feces per culture solution), and this was designated as culture time 0.

[0084] The culture medium was collected 6, 24, 30, 48, 72, and 96 hours after the start of culture, and subjected to bacterial flora analysis. The culture medium was collected without opening the anaerobic chamber.

[0085] (Bacterial Flora Analysis) Genomic DNA of the bacterial flora was extracted from the culture medium collected at various times before and after the start of cultivation. The V3-V4 region of the bacterial 16S rRNA gene was amplified from the extracted genomic DNA and sequenced using a next-generation sequencer to perform bacterial diversity analysis and bacterial composition analysis. The procedure is as follows:

[0086] The bacterial 16S rRNA gene was amplified using the primer pair S-D-Bact-0341-b-S-17 (SEQ ID NO: 1) and S-D-Bact-0785-a-A-21 (SEQ ID NO: 2) with extracted genomic DNA as a template. An Illumina adapter overhang nucleotide sequence (Illumina, Inc.) was added to the gene-specific sequence. PCR cycling reactions were performed according to the manufacturer's instructions. The confirmed amplicons were purified using AMPure XP DNA purification beads (Beckman Coulter, Inc.) and eluted in 25 μl of 10 mM Tris (pH 8.5). The amplicons were quantified on an Agilent Bioanalyzer 2100 DNA 1000 chip (Agilent Technologies, Inc.) and pooled at equimolar concentrations. The 16S rRNA gene product (together with an internal control (PhiX control V3; Illumina)) was subjected to paired-end sequencing using a MiSeq sequencer (Illumina) with a 600-cycle MiSeq reagent kit (Illumina).

[0087] Paired-end reads with a Q score of 20 or more obtained by extracting the PhiX sequence using Basespace Sequence Hub (https: / / basespace.illumina.com / ) were combined using QIIME 2 version 2022.2 and quality control and correction were performed in the DADA2 pipeline, after which the OTU was inferred. The obtained OTU was used to estimate alpha diversity and calculate the Shannon index. In addition, the obtained OTU was classified using a naive Bayes classifier trained on the Greengenes 13_8 99% OTU full-length sequence database, and bacterial species assignment was performed. Using Excel (Microsoft Japan Co., Ltd.), the relative occupancy rate was calculated from the genus-level classification data of bacterial species attribution, and the Pearson product-moment correlation coefficient was calculated based on the relative occupancy rate.

[0088] Real-time PCR was performed using a QuantStudio® 3 Real-Time PCR System (Thermo Fisher Scientific). Amplification was performed using a primer set targeting all enterobacteria, as described in Takagi, R. et al., PLoS One 11, e0160533 (2016). Total bacterial counts were calculated from a calibration curve prepared from known concentrations of E. coli.

[0089] The results of the Pearson product-moment correlation coefficient are shown in Table 1. The results in Table 1 show that the cells were in a state of equilibrium between 6 hours and 96 hours after the start of culture.

[0090]

[0091] Example 2 Cultivation was carried out in the same manner as in Example 1, except that inulin (BENEO, OraftiGR, derived from chicory; abbreviated as INU) was added as a test substance to the culture medium at 0.3% (w / v) 24 hours after the start of culture.

[0092] Comparative Example 1 Cultivation was carried out in the same manner as in Example 2, except that inulin (BENEO, OraftiGR, derived from chicory; abbreviated as INU) was added at the start of cultivation (0 hours into cultivation).

[0093] The percentage of bacteria belonging to the genus Bifidobacterium was determined for each culture medium 48 hours and 72 hours after the start of culture in Examples 1 and 2 and Comparative Example 1. The results are shown in Table 2.

[0094]

[0095] The culture solution to which the test substance inulin was not added is referred to as "CUL," the culture solution to which inulin was added at the start of culture is referred to as "INU (added 0 h)," and the culture solution to which inulin was added 24 hours after the start of culture is referred to as "INU (added 24 h)."

[0096] In Table 2, it can be seen that in Comparative Example 1, in which inulin was added at the start of culture, the presence rate of Bifidobacterium was reduced at all culture times compared to Example 1, in which inulin was not added. On the other hand, in Example 2, in which inulin was added 24 hours after the start of culture, the presence rate of Bifidobacterium was improved at all culture times compared to Example 1, in which inulin was not added. The result that the presence rate of Bifidobacterium increased by the addition of inulin is consistent with the test results in which humans ingested inulin (Daniel So, et al., Am. J. Clin. Nutr 2018(107), 965-983). In other words, it was shown that the evaluation results of the human intestinal flora can be reproduced in vitro by adding the test substance inulin after the original equilibrium state was reached.

[0097] Example 3 (Water-soluble, indigestible dextrin added 24 hours after the start of culture) Culture was carried out in the same manner as in Example 2, except that water-soluble, indigestible dextrin (Fibersol 2 (maltodextrin), manufactured by Matsutani Scientific; referred to as DEX) was added as the test substance to the culture medium at 0.3% (w / v) 24 hours after the start of culture.

[0098] Comparative Example 2 (Water-soluble, indigestible dextrin added at 0 hours after the start of culture) Culture was carried out in the same manner as in Example 3, except that water-soluble, indigestible dextrin (Fibersol 2 (maltodextrin), manufactured by Matsutani Scientific Co., Ltd.; referred to as DEX) was added as the test substance at 0.3% (w / v) to the culture medium at the start of culture.

[0099] The percentage of bacteria belonging to the genus Faecalibacterium in each culture medium was determined 48 hours, 72 hours, and 96 hours after the start of culture. The results are shown in Table 3.

[0100]

[0101] The culture solution to which dextrin was not added is referred to as "CUL," the culture solution to which dextrin was added at the start of culture is referred to as "DEX (added 0 h)," and the culture solution to which dextrin was added 24 hours after the start of culture is referred to as "DEX (added 24 h)."

[0102] In Table 3, it can be seen that in Comparative Example 2, in which dextrin was added at the start of culture, the presence rate of Faecali- bacterium was reduced at any culture time compared to Example 1, in which dextrin was not added. On the other hand, in Example 3, in which dextrin was added 24 hours after the start of culture, the presence rate of Faecali- bacterium was increased at any culture time compared to Example 1, in which dextrin was not added. This result is consistent with the test results in which rats were given dextrin (Takagaki R et al., Bioscience, Biotechnology, and Biochemistry, Vol. 84, Issue 4, 2020, pp. 824-831). In other words, it was shown that the test results obtained in vivo can be reproduced in vitro by adding the test substance dextrin during the original equilibration state.

[0103] Example 4 (Relationship between fecal inoculum amount and bacterial flora diversity (multiwell plate culture)) (Preparation of fecal suspension) Feces serving as an inoculum source for intestinal bacterial flora were collected from healthy individuals on the day of culture. After collection, the fecal samples were stored in anaerobic culture swabs (212550 BD BBL Culture Swab; Becton, Tickinson & Company) and transported to the laboratory. To prepare the inoculum, 0.5 g of feces was suspended in 2 mL of 0.1 M phosphate buffer (PBS) buffer (pH 6.5, consisting of a 68.5:31.5 (molar ratio) mixture of 0.1 M NaH2PO4 and 0.1 M Na2HPO4) supplemented with 1.0% L-ascorbic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) to prepare a fecal suspension.

[0104] (Preparation of medium) A medium was prepared by mixing 59 g / L of Gifu University-prescribed anaerobic medium (GAM medium [Code 05422] (manufactured by Nissui Pharmaceutical Co., Ltd.) and 50 μL / L of an antifoaming agent. The pH was adjusted to 6.5 with 0.1 M phosphate buffer, and the medium was sterilized in an autoclave at 115° C. for 15 minutes.

[0105] (Culture Conditions) 1.0 mL of medium was dispensed per well into the required number of wells in a clean bench. A 96-well multi-well plate, model number 82.1972.002, with a capacity of 2.2 mL, manufactured by Sarstedt Co., Ltd., was used. The multi-well plate was stirred at approximately 500 rpm using a 96-well shaking incubator (manufactured by Biosan, model number TS-DW) installed in an anaerobic chamber. The culture temperature was 37°C. Culture was initiated by inoculating 50 μL / well of fecal suspension (0.0125 g / 1.0 mL of feces per culture solution), and this was designated as culture time 0.

[0106] The culture medium was collected 6, 24, 30, 48, 72, and 96 hours after the start of culture, and subjected to bacterial flora analysis. The culture medium was collected without opening the anaerobic chamber.

[0107] Examples 5 to 7 Cultivation and bacterial flora analysis were carried out in the same manner as in Example 4, except that the amount of fecal suspension added was changed to the amount shown in Table 4.

[0108] Comparative Example 3 (Relationship between Fecal Inoculum Amount and Microbiota Diversity (Jar Culture)) (Preparation of Fecal Suspension) Feces serving as an inoculum for intestinal microbiota were collected from healthy individuals on the day of culture. After collection, the fecal samples were stored in anaerobic culture swabs (212550 BD BBL Culture Swab; manufactured by Becton, Tickinson and Company) and transported to the laboratory. To prepare the inoculum, 0.5 g of feces was added to a total volume of 2 mL of 0.1 M phosphate buffer (PBS) buffer (pH 6.5, consisting of a 68.5:31.5 (molar ratio) mixture of 0.1 M NaH2PO4 and 0.1 M Na2HPO4) supplemented with 1.0% L-ascorbic acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to prepare a fecal suspension.

[0109] (Preparation of Medium) A medium was prepared by mixing 59 g / L of Gifu University-prescribed anaerobic medium (GAM medium [Code 05422] (manufactured by Nissui Pharmaceutical Co., Ltd.) and 50 μL / L of an antifoaming agent.

[0110] The medium was adjusted to pH 6.5 with 0.1 M phosphate buffer, and 100 mL of the medium was added to a jar fermenter (Able Co., Ltd., BJR-25NAIS-8M) with a capacity of approximately 200 mL, and sterilized in an autoclave at 115°C for 15 minutes.

[0111] (Culture Conditions) After sterilization, anaerobic conditions were established in the culture vessel by aerating (15 mL / min) a mixed gas of nitrogen and carbon dioxide (N2:CO2 = 80:20 (volume ratio)) that had been sterilized by filtration through a 0.2 μm PTFE membrane (manufactured by Pall Corporation) for 1 hour at 37°C before culturing.

[0112] 200 μL of the fecal suspension was inoculated into a medium-containing vessel (0.05 g feces / 100 mL culture solution), and anaerobic culture was initiated (culture time 0). During the culture, the medium was constantly bubbled with a sterilized, filtered mixed gas (N2:CO2 = 80:20 (volume ratio)) to maintain anaerobic conditions in the culture tank. The culture temperature was set at 37°C, and the incubation was performed with continuous stirring at approximately 300 rpm.

[0113] The culture medium was collected 6 hours, 24 hours, 30 hours, 48 ​​hours, 72 hours, and 96 hours after the start of culture, and subjected to bacterial flora analysis. The culture medium was collected using a syringe without opening the culture vessel, without introducing air. The obtained culture medium was subjected to bacterial flora analysis in the same manner as in Example 1.

[0114] Comparative Examples 4 to 6 Cultivation and bacterial flora analysis were carried out in the same manner as in Comparative Example 3, except that the amount of fecal suspension added was changed to the amount shown in Table 4.

[0115]

[0116] (Evaluation Results) Table 5 shows the calculation results of OTU and Shannon index after 72 hours of culture in Examples 4 to 7 and Comparative Examples 3 to 6.

[0117]

[0118] In Comparative Examples 3 to 6, which involved jar culture, and Examples 4 to 7, which involved 96-well culture, the number of detected bacterial species (OTU) was compared between Comparative Example 4 and Example 4, Comparative Example 5 and Example 6, and Comparative Example 6 and Example 7, which involved the same fecal inoculation amount. In addition, when comparing samples with the same fecal inoculation amount, the Shannon index, which reflects the diversity of the bacterial flora, was higher in the 96-well culture than in the jar culture. Furthermore, when comparing Examples 4 to 7, which involved different fecal inoculation amounts in the 96-well culture, the higher the fecal inoculation amount, the higher both the OTU value and the Shannon index, which were closer to the FEC value.

[0119] Table 6 shows the abundance of Enterococcus bacteria during jar culture. Enterococcus bacteria are a bacterial species known to be pathogenic bacteria that easily grow in culture. In jar culture, when the fecal inoculum amount was 1.25% (w / v), the proportion of Enterococcus bacteria not detected before culture to the total bacteria was 0.238%. This represents a value 7.94 times higher than the abundance rate at a fecal inoculum amount of 0.05%. Meanwhile, the abundance rate of Enterococcus bacteria in 96-well culture is shown in Table 7. In 96-well culture, the abundance rate of Enterococcus bacteria was a maximum of 0.070%, which was a suppressed increase in the abundance rate compared to the maximum of 0.238% observed in jar culture.

[0120]

[0121]

[0122] The results of Examples 4 to 7 and Comparative Examples 3 to 6 above showed that by performing shaking culture using a multiwell plate with a capacity of 2 mL per well, the number of OTUs and the Shannon index could be increased compared to culture using a jar fermenter with a capacity of approximately 200 mL.

[0123] (Consideration of culture medium)

[0124] Example 8 (Multiwell Plate Culture) (Preparation of Fecal Suspension) Feces serving as an inoculum for intestinal bacterial flora were collected from healthy individuals on the day of culture. After collection, the fecal samples were stored in anaerobic culture swabs (212550 BD BBL Culture Swab; manufactured by Becton, Tickinson and Company) and transported to the laboratory. To prepare the inoculum, 0.5 g of feces was dissolved in 0.1 M phosphate buffer (PBS) buffer (pH 6.5, 0.1 M NaHCO3) containing 1.0% L-ascorbic acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). ２ P.O. ４ and 0.1 M Na ２ HPO ４ A fecal suspension was prepared by suspending the feces in 2 mL of a 68.5:31.5 (molar ratio) mixture of

[0125] (Preparation of medium) A medium was prepared by mixing 59 g / L of Gifu University-prescribed anaerobic medium (GAM medium [Code 05422] (manufactured by Nissui Pharmaceutical Co., Ltd.), 8.0 g / L of mucin (manufactured by Sigma-Aldrich, derived from porcine stomach, Type III), and 50 μL / L of an antifoaming agent. The pH was adjusted to 6.5 with 0.1 M phosphate buffer, and the medium was sterilized in an autoclave at 115° C. for 15 minutes.

[0126] (Culture Conditions) 1.0 mL of medium was dispensed into the required number of wells in a clean bench. A 96-well multi-well plate (model number 82.1972.002, 2.2 mL capacity, manufactured by Sarstedt Co., Ltd.) was used (hereinafter sometimes referred to as 96-well). The multi-well plate was stirred at approximately 500 rpm using a 96-well shaking incubator (manufactured by Biosan, model number TS-DW) installed in an anaerobic chamber. The culture temperature was 37°C. Culture was initiated by inoculating 50 μL / well of fecal suspension (12.5 mg / mL feces per culture solution), and this was designated as culture time 0.

[0127] The culture medium was collected 6, 24, 30, 48, 72, and 96 hours after the start of culture, and subjected to bacterial flora analysis. The culture medium was collected without opening the anaerobic chamber.

[0128] (Bacterial Flora Analysis) Genomic DNA of the bacterial flora was extracted from the culture medium collected at various times before and after the start of cultivation. The V3-V4 region of the bacterial 16S rRNA gene was amplified from the extracted genomic DNA and sequenced using a next-generation sequencer to perform bacterial diversity analysis and bacterial composition analysis. The procedure is as follows:

[0129] The bacterial 16S rRNA gene was amplified using the primer pair S-D-Bact-0341-b-S-17 (SEQ ID NO: 1) and S-D-Bact-0785-a-A-21 (SEQ ID NO: 2) with extracted genomic DNA as a template. An Illumina adapter overhang nucleotide sequence (Illumina, Inc.) was added to the gene-specific sequence. PCR cycling reactions were performed according to the manufacturer's instructions. The confirmed amplicons were purified using AMPure XP DNA purification beads (Beckman Coulter, Inc.) and eluted in 25 μl of 10 mM Tris (pH 8.5). The amplicons were quantified on an Agilent Bioanalyzer 2100 DNA 1000 chip (Agilent Technologies, Inc.) and pooled at equimolar concentrations. The 16S rRNA gene product (together with an internal control (PhiX control V3; Illumina)) was subjected to paired-end sequencing using a MiSeq sequencer (Illumina) with a 600-cycle MiSeq reagent kit (Illumina).

[0130] Paired-end reads with a Q score of 20 or more obtained by extracting the PhiX sequence using Basespace Sequence Hub (https: / / basespace.illumina.com / ) were combined using QIIME 2 version 2022.2 and quality control and correction were performed in the DADA2 pipeline, after which the OTU was inferred. The obtained OTU was used to estimate alpha diversity and calculate the Shannon index. In addition, the obtained OTU was classified using a naive Bayes classifier trained on the Greengenes 13_8 99% OTU full-length sequence database, and bacterial species assignment was performed. Using Excel (Microsoft Japan Co., Ltd.), the relative occupancy rate was calculated from the genus-level classification data of bacterial species attribution, and the Pearson product-moment correlation coefficient was calculated based on the relative occupancy rate.

[0131] Example 9 (Study on GAM medium containing mucin) Cultivation and bacterial flora analysis were carried out in the same manner as in Example 8, except that in preparing the GAM medium, 8.0 g / L or 4.0 g / L of mucin (Sigma-Aldrich, derived from porcine stomach, Type III) was added to the GAM medium.

[0132] Example 10 (Study on Modified GAM Medium) Cultivation and bacterial flora analysis were performed in the same manner as in Example 8, except that modified GAM medium was used instead of GAM medium in the preparation of GAM medium. Modified GAM medium was prepared by mixing 41.7 g / L of modified Gifu University prescribed anaerobic medium (modified GAM medium [Code 05433] (manufactured by Nissui Pharmaceutical Co., Ltd.) and 50 μL / L of an antifoaming agent.

[0133] Example 11 (Study on modified GAM medium containing mucin) In preparing the modified GAM medium, culture and bacterial flora analysis were carried out in the same manner as in Example 10, except that 8.0 g / L or 4.0 g / L of mucin (Sigma-Aldrich, derived from porcine stomach, Type III) was added to the modified GAM medium.

[0134] Example 12 (Study on YCFA medium) Culture and bacterial flora analysis were performed in the same manner as in Example 8, except that YCFA medium was used instead of GAM medium for medium preparation. YCFA medium contained 10.0 g / L casein hydrolysate, 2.5 g / L yeast extract, 4.0 g / L sodium bicarbonate, 2.0 g / L glucose, 2.0 g / L maltose, 2.0 g / L cellobiose, 1.0 g / L L-cysteine ​​HCl, 0.001 g / L resazurin, 0.45 g / L dipotassium hydrogen phosphate, 0.45 g / L potassium dihydrogen phosphate, 0.9 g / L ammonium sulfate, 0.9 g / L sodium chloride, 0.09 g / L magnesium sulfate, 0.09 g / L calcium chloride, and 0.01 g / L hemin, and contained volatile fatty acids per liter. The medium was prepared by mixing 3.1 ml (acetic acid 2.026 ml / L, propionic acid 0.715 ml / L, n-valeric acid 0.119 ml / L, isovaleric acid 0.119 ml / L, isovaleric acid 0.119 ml / L), vitamin mixture 1: 1 ml (biotin 1 mg / 100 ml, cyanocobalamin 1 mg / 100 ml, p-aminobenzoic acid 3 mg / 100 ml, folic acid 5 mg / 100 ml, pyridoxine 15 mg / 100 ml), vitamin mixture 2: 1 ml (thiamine 5 mg / 100 ml, riboflavin 5 mg / 100 ml) and 50 μL / L of antifoaming agent, and the pH was adjusted to 7.5 with a pH adjuster.

[0135] Example 13 (Study on YCFA medium containing mucin) Culture and bacterial flora analysis were carried out in the same manner as in Example 12, except that in preparing the YCFA medium, 8.0 g / L or 4.0 g / L of mucin (Sigma-Aldrich, derived from porcine stomach, Type III) was added to the YCFA medium.

[0136] Example 14 (modified YCFA medium) Cultivation and bacterial flora analysis were performed in the same manner as in Example 8, except that modified YCFA medium was used instead of GAM medium to prepare the medium. The modified YCFA medium contained 10.0 g / L of casein hydrolysate, 2.5 g / L of yeast extract, 5.0 g / L of glucose, 0.045 g / L of magnesium sulfate, 0.09 g / L of calcium chloride, 0.45 g / L of dipotassium hydrogen phosphate, 0.45 g / L of potassium dihydrogen phosphate, 0.9 g / L of sodium chloride, 0.001 g / L of resazurin, and L-cysteine ​​HCl. The medium contained 1.0 g / L of volatile fatty acids (acetic acid 2.026 ml / L, propionic acid 0.715 ml / L, n-valeric acid 0.119 ml / L, isovaleric acid 0.119 ml / L, and isovaleric acid 0.119 ml / L), 2.7 ml of volatile fatty acids (acetic acid 2.026 ml / L, propionic acid 0.715 ml / L, n-valeric acid 0.119 ml / L, isovaleric acid 0.119 ml / L), 10 ml of a vitamin mixture (biotin 2 mg / L, cyanocobalamin 0.1 mg / L, folic acid 2 mg / L, pyridoxine 10 mg / L, thiamine 5 mg / 100 ml, riboflavin 5 mg / L, nicotinic acid 5 mg / L, calcium pantothenate 5 mg / L, p-aminobenzoic acid 5 mg / L, and lipoic acid 5 mg / L), and 50 μL / L of an antifoaming agent. The pH was adjusted to 6.8 with a pH adjuster to prepare the medium.

[0137] Example 15 (Study on modified YCFA medium containing mucin) Cultivation and bacterial flora analysis were performed in the same manner as in Example 14, except that 8.0 g / L or 4.0 g / L of mucin (Sigma-Aldrich, derived from porcine stomach, Type III) was added to the modified YCFA medium in preparing the modified YCFA medium. Relative occupancy rates were calculated from genus-level classification data of bacterial species attribution, and Pearson product-moment correlation coefficients were calculated based on the relative occupancy rates.

[0138] Table 6 shows the Pearson product-moment correlation coefficients after 72 hours of culture, relative to the results of Examples 8, 9, 10, and 11 in various media set at 100, and the Pearson product-moment correlation coefficients of Examples 12, 13, 14, and 15 in mucin-supplemented media. The results in Table 8 clearly show that the addition of mucin is effective in maintaining the original equilibrium state in all media.

[0139]

[0140] Example 16 (Abundance of useful bacteria: Effect of mucin addition) Genomic DNA of bacteria in the bacterial flora was extracted from the culture medium collected before the start of culture and 72 hours after the start of culture in Examples 8 and 9. Specific primers for Faecalibacterium duncaniae (Fd bacteria) and Blautia wexlerae (Bw bacteria) targeting the 16S rRNA gene of each bacteria were used to quantify the target bacterial genes from the extracted genomic DNA using a quantitative PCR device (Table 9).

[0141] The results in Table 9 show that when cultured in each medium without mucin, the abundance of the useful bacteria Fd and Bw decreased, whereas the addition of mucin to all media maintained Fd and Bw bacteria during culture, and their abundance was equal to or greater than that of the original sample (Fec), demonstrating its effectiveness in maintaining these useful bacteria.

[0142]

[0143] (Study of Mucin) Example 17 (Study of Medium Containing Porcine Type II Mucin) Cultivation and bacterial flora analysis were carried out in the same manner as in Example 8, except that in preparing the medium, 8.0 g / L of mucin (Sigma-Aldrich, derived from porcine stomach, Type II) was added to the GAM medium.

[0144] Example 18 (Study on medium containing porcine mucin) Cultivation and bacterial flora analysis were carried out in the same manner as in Example 8, except that in preparing the medium, 8.0 g / L of mucin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., derived from porcine stomach) was added to the GAM medium.

[0145] Example 19 (Study on medium containing skate mucin) Cultivation and bacterial flora analysis were carried out in the same manner as in Example 8, except that 8.0 g / L of mucin (derived from skate, manufactured by Marukyo Suisan Co., Ltd.) was added to GAM medium in the preparation of the medium. Relative occupancy rates were calculated from the classification data at the genus level for bacterial species attribution, and the Pearson product-moment correlation coefficient was calculated based on the relative occupancy rates.

[0146] Comparative Example 7 (Study of Mucin-Free Medium) Culture and bacterial flora analysis were performed in the same manner as in Example 8 for medium preparation. Table 10 shows the Pearson product-moment correlation coefficients after 72 hours of culture, relative to the result of Comparative Example 7, which is set to 100, for the Pearson product-moment correlation coefficients of Examples 1, 2, 3, and 4. The results in Table 10 clearly demonstrate that the addition of all mucins of different origins was effective in maintaining the original equilibrium state.

[0147]

[0148] Example 20 (Administration of Culture Preparation to Mice) The human intestinal bacterial flora culture prepared in Example 9 was administered to SPF mice, and the mice were fed a high-fat diet to assess insulin sensitivity. The administration of the microorganism to mice and the efficacy evaluation test were performed according to the method described in Nature Communications (202) 13:4477.

[0149] Specifically, SPF mice (6 weeks old) were fed a high-fat diet (AIN-93G, Oriental Yeast Co., Ltd.) for 10 weeks, and 5 × 10 ９ CFU will be orally administered three times a week, and the weight of each individual will be measured. Eight weeks after administration of the human intestinal flora culture medium, serum will be collected, and HOMA-IR and insulin concentrations will be measured, as well as an IPGTT test will be performed.

[0150] (Note) As described above, the present disclosure has been illustrated using preferred embodiments of the present disclosure, but the present disclosure should not be construed as being limited to these embodiments. It is understood that the scope of the present disclosure should be interpreted solely by the scope of the claims. It is understood that a person skilled in the art can implement an equivalent scope based on the description of the present disclosure and common general technical knowledge from the description of specific preferred embodiments of the present disclosure. It is understood that the contents of patents, patent applications, and literature cited in this specification are incorporated by reference into this specification as if the contents themselves were specifically set forth in this specification. This application claims priority to Japanese Patent Application No. 2023-196983, filed with the Japan Patent Office on November 20, 2023, the contents of which are incorporated herein by reference in their entirety.

[0151] According to the method of the present disclosure, it is possible to evaluate in vitro the effects of test substances such as foods and candidate pharmaceutical compounds on the intestinal flora of mammals, particularly humans.

Claims

1. A method for increasing at least one selected from the group consisting of alpha diversity and OTU number with an increase in the content of intestinal flora added to a medium for culturing in a container under original equilibrium conditions, the method being characterized in that a stirring device is not used.

2. A method for increasing at least one selected from the group consisting of alpha diversity and OTU number with an increase in the content of intestinal flora added to a medium for culturing in a container under original equilibrium conditions, the method being characterized in that the pH is not adjusted.

3. A method for increasing at least one selected from the group consisting of alpha diversity and OTU number by increasing the content of intestinal flora added to a medium for culturing in a container under original equilibrium conditions, the container being characterized in that it is of a microwell type.

4. A method for increasing at least one selected from the group consisting of alpha diversity and OTU number by increasing the content of intestinal flora added to a medium for culturing in a container under original equilibrium conditions, wherein the volume of the container is suitable for shaking culture.

5. A method for increasing at least one selected from the group consisting of alpha diversity and OTU number by increasing the content of intestinal flora added to a medium for culturing in a container under original equilibrium conditions, wherein the container is 3 mL or less.

6. A method for increasing at least one selected from the group consisting of alpha diversity and OTU number by increasing the content of intestinal flora added to a medium for culturing in a container under original equilibrium conditions, wherein the volume of the medium is 3 mL or less.

7. The method according to any one of claims 1 to 6, wherein the original equilibrated medium is used to evaluate a test substance.

8. A container or culture well containing a medium for culturing intestinal flora so as to increase the number of OTUs and / or the Shannon coefficient with an increase in the content of intestinal flora in the medium under original equilibrium conditions, the volume of the container being suitable for shaking culture.

9. A container or culture well containing a culture medium for culturing the intestinal flora so as to increase the number of OTUs and / or the Shannon coefficient with an increase in the content of the intestinal flora in the culture medium under original equilibrium conditions, the volume of the container being 3 mL or less.

10. A method for evaluating a test substance, comprising the steps of: A) adding an intestinal flora to a culture well having a volume of medium of 3 mL or less; B) culturing the intestinal flora until it reaches a state of original equilibrium; C) adding a test substance to the container; and D) obtaining and evaluating evaluation items before and after the addition of the test substance.

11. A system for evaluating a test substance comprising: A) a container including a culture well capable of accommodating a medium having a volume of 3 mL or less; B) a means for adding intestinal bacterial flora; C) a means for culturing until a state of equilibrium is reached; D) a means for adding a test substance; and E) a means for acquiring and evaluating evaluation items before and after the addition of the test substance.

12. The method of claim 1 to 7 or 10, wherein the original equilibration condition comprises equilibrating the microbiota diversity to the state of a pre-inoculation sample of the intestinal microbiota.

13. The method of claims 1 to 7, 10 or 12, wherein the evaluation items after addition of the test substance are obtained while the original equilibration conditions are maintained.

14. The method according to claims 1 to 7, 10 or 12 to 13, wherein the evaluation item after addition of the test substance is obtained 24 to 96 hours after the start of culture.

15. The method according to claims 1 to 7, 10 or 12 to 14, wherein the test substance is added 6 to 72 hours after the incubation.

16. The method of claims 1 to 7, 10 or 12 to 15, wherein the evaluation item after addition of the test substance is obtained 12 to 96 hours after addition of the test substance.

17. The method according to claims 1 to 7, 10 or 12 to 16, wherein the original equilibrium state is determined based on a structural analysis of the intestinal flora.

18. The method of claims 1 to 7, 10 or 12 to 17, wherein the original equilibrium state is determined based on 16S or genomic analysis of the intestinal flora.

19. The method of claims 1 to 7, 10 or 12 to 18, wherein the original equilibrium state is determined based on the Pearson product moment correlation coefficient of 16S or genomic analysis of the intestinal microbiota.

20. The method according to claims 1 to 7, 10 or 12 to 19, wherein the timing of adding the test substance is decided by the following steps A and B: (Step A) culturing a stool specimen containing the enterobacteria in a culture medium and obtaining in advance time-course data on the Pearson product-moment correlation coefficient of the bacterial flora structure before and after the culture, and (Step B) determining in advance the timing of adding the test substance to the specimen containing the enterobacteria cultured in a culture medium from the time-course data obtained in Step A.

21. A method according to claims 1 to 7, 10 or 12 to 20, characterized in that the timing of adding the test substance is determined from a time range in which the original equilibrium state is maintained for 24 hours or more, with the Pearson product moment correlation coefficient between the bacterial flora structure before and after cultivation of the intestinal bacteria being 0.70 or higher.

22. The method of claims 1 to 7, 10 or 12 to 21, wherein the medium contains mucin.

23. The method according to claims 1 to 7, 10, or 12 to 22, wherein the mucin is contained in the medium at a concentration of 0.4% or more.

24. The method of claims 1 to 7, 10 or 12 to 23, wherein the intestinal flora is obtained from stool.

25. The method of claims 1 to 7, 10 or 12 to 24, wherein the intestinal flora is obtained from human stool.

26. The method according to any one of claims 1 to 7, 10, or 12 to 25, wherein the amount of the sample added is 0.1% or more and 1.25% or less relative to the medium.

27. A kit for evaluating a test substance, comprising an incubator having a culture well for accommodating a medium having a volume of 3 mL or less, the medium, and a substance for promoting and maintaining original equilibrium.

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