WO2025110156A1 - Method for evaluating test substance in intestinal bacterial flora - Google Patents

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

This disclosure relates to a method for evaluating a test substance in the intestinal microflora.

A wide variety of bacteria constantly proliferates in the intestinal tract of mammals; these bacteria are known as the intestinal flora. In recent years, it has become clear that the intestinal flora has various effects on human health, and a method has been reported for in vitro evaluation of the effects of test substances, such as food and candidate pharmaceutical compounds, on the intestinal environment (Patent Document 1).

Patent No. 7051175

The present inventors have previously developed a device for maintaining human intestinal bacterial species in a culture system using human feces as an inoculum source.
As a result of intensive research, the present inventors have found that returning a culture solution containing an intestinal bacterial flora to an original equilibrium state is useful for evaluating a test substance. The present disclosure based on this finding is as follows.
[1] 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 culture medium for culturing in a container under original equilibrium conditions, the method being characterized in that it does not use a stirring device.
[2] A method for increasing at least one selected from the group consisting of alpha diversity (Shannon coefficient, Ciao1 coefficient, Simpson coefficient, etc.) and OTU number with an increase in the content of intestinal flora added to a culture 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 (Shannon coefficient, Ciao1 coefficient, Simpson coefficient, etc.) and the number of OTUs with an increase in the content of intestinal flora added to a culture medium for culturing in a container under original equilibrium conditions, wherein the container is a microwell type.
[4] 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 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 (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 container is 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 original equilibrium conditions, wherein the volume of the medium is 3 mL or less.
[7] The method according to any one of the preceding items, wherein the original equilibrated medium is used to evaluate a test substance.
[8] A container or culture well that contains a medium for culturing an 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 medium under original equilibrium conditions, the volume of the container being suitable for shaking culture.
[9] A container or culture well that contains a culture medium for culturing an 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 in an original equilibrium state, the container having a volume of 3 mL or less.
[10]
A) adding an intestinal bacterial flora to a culture well having a volume of medium of 3 mL or less; B) culturing the intestinal bacterial 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) a container including a culture well capable of accommodating a medium having a volume of 3 mL or less;
B) A means for adding gut flora;
C) a means for incubating the culture until the culture is in equilibrium;
D) a means for adding a test substance; and
E) A system for evaluating a test substance, comprising means for acquiring and evaluating evaluation items before and after the addition of the test substance.
[12] The method, container, culture well, or system according to the preceding items, wherein the original equilibration state 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 according to any one of the preceding items, wherein the evaluation item after addition of the test substance is acquired while the original equilibration state is maintained.
[14] 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 24 hours to 96 hours after the start of culture.
[15] The method, container, culture well, or system according to any of the preceding items, wherein the test substance is added 6 hours or more and 72 hours or less after the 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 according to the preceding items, wherein the original equilibrium state is determined based on a structural analysis of the intestinal bacterial flora.
[18] The method, container, culture well, or system according to any of the preceding items, wherein the original equilibrium state is determined based on analysis of 16S or a genome of the intestinal microbiota.
[19] The method, container, culture well, or system according to the above items, 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] A method comprising the following steps A and B, wherein the test substance is added at the time of addition:
The method, container, culture well, or system described in the above items is determined by: (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 the culture medium from 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 is determined from a time range in which a state in which the Pearson product moment correlation coefficient between the bacterial flora structure before and after culture of the enterobacteria is 0.70 or more is maintained for 24 hours or more in the original equilibrium state.
[22] The method, container, culture well, or system according to the above items, wherein the medium contains mucin.
[23] The method, container, culture well, or system according to the preceding items, wherein the mucin is contained in a medium at a concentration of 0.4% or more.
[24] The method, container, culture well, or system according to the preceding items, wherein the intestinal bacterial flora is obtained from stool.
[25] The method, container, culture well, or system according to the preceding items, wherein the intestinal bacterial flora is obtained from human stool.
[26] The method, container, culture well, or system according to any one of the preceding items, 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.

The present disclosure has improved the reproducibility of changes in the intestinal flora caused by the administration of a test substance to humans. The present disclosure can suppress events that can cause changes in the composition of the intestinal flora due to cultivation, making it possible to more accurately test the influence and effect of the test substance on the intestinal flora.

Based on the above, the present disclosure provides a method for evaluating test substances, such as food and drug candidate compounds, that makes it possible to reproduce in vitro the in vivo effects of the test substances on the intestinal environment.

The method disclosed herein allows for more accurate evaluation of the effects of test substances, such as food and drug candidate compounds, on the intestinal flora of mammals, particularly humans. In particular, the use of microwells allows for more accurate, original equilibrium states to be maintained in a richer way (alpha diversity, such as the Shannon coefficient, Ciao1 coefficient, Simpson coefficient, and OTU number) while conserving dosage, allowing for more efficient evaluation of candidate substances.

The present disclosure will be described below while showing the best mode. Throughout this specification, singular expressions should be understood to include the concept of the plural, unless otherwise specified. Thus, singular articles (e.g., in the case of English, "a," "an," "the," etc.) should be understood to include the concept of the plural, unless otherwise specified. In addition, terms used in this specification should be understood to be used in the sense commonly used in the field, unless otherwise specified. Thus, 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 case of conflict, the present specification (including definitions) will take precedence.

(Definition of terms)
The following provides definitions of terms particularly used in this specification and/or explains basic technical content as appropriate.

All numerical values herein are intended to be modified by the term "about," whether or not expressly indicated. The term "about" generally refers to a range of numerical values that one of 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.

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). Bacteria that constitute the intestinal flora of healthy humans include, for example, bacteria belonging to the following phyla at the phylum level:
Verrucomicrobiota, Pseudomonadota, Fusobacteriota, Bacillota, Bacteroidota, Actinomycetota, and the like. Further, at the genus level, bacteria belonging to the following genera or families can be included: Bifidobacterium, Collinsella, Bacteroides, Parabacteroides, Prevotella, Rikenellaceae, Lactobacillales, etc.

In this specification, "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 this disclosure, "mammals" are preferably humans.

In this specification, 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, food-derived physiologically active substances, food additives, beverages, microorganisms (bacteria, fungi, etc., including killed bacteria and extracts derived from bacteria), physiologically active substances, pharmaceuticals, pharmaceutical-like compounds, and mixtures thereof.

This disclosure relates to a technology that restores the intestinal flora in a culture medium to a state of original equilibrium.

In this specification, "original state" refers to the state of the intestinal flora of a sample such as feces (sometimes referred to as the intestinal flora before the start of cultivation).

In this specification, "original state equilibrium" refers to a state of equilibrium in the original state, i.e., a state within a certain range of fluctuation, and such a state is called "original state equilibrium state". In this specification, whether original state equilibrium has been achieved can be evaluated using the Pearson product moment correlation coefficient, which is a coefficient that evaluates the degree to which the two bacterial floras are similar. For example, when the Pearson product moment correlation coefficient of the intestinal bacterial flora before the start of culture and the intestinal bacterial flora after the start of culture is 0.50 or more, or 0.60 or more, usually 0.70 or more, preferably 0.80 or more, or 0.85 or more, 0.90 or more, or 0.95 or more, it can be said that the original state equilibrium is achieved. In addition, when original state equilibrium continues for a certain period of time, it is called original state equilibrium state. The certain period of time is 6 hours or more, 12 hours or more, or 18 hours or more, preferably 24 hours or more, more preferably 36 hours or more, even more preferably 48 hours or more, and even more preferably 72 hours or more. In this specification, the "stabilization judgment index" is calculated as an index until the original state equilibrium is reached.

In this specification, substances that promote and/or maintain the state equilibration are also referred to as "state equilibration promoting substances" and "state equilibration maintaining substances," respectively. If they have both functions, they may be referred to as state equilibration promoting/maintaining substances.

In this specification, "culture medium" refers to any medium in which a bacterial flora can grow.

In this specification, "medium components" refers to each component that makes up the medium.

In this specification, the term "microwell" refers to a plate with a particularly small cavity, also called a microtiter plate or microplate. Microwells that can be used include, but are not limited to, the following:

Representative well plate: Sarstedt Other commercially available deep well plates:
*Thermo Scientific Nunc https://axel.as-1.co.jp/asone/g/NC2008033368/
*Eppendorf
1731560163500_0
umables/Plates/Eppendorf-Deepwell-Plates-p-PF-55960
*Scientific Specialties
1731560163500_1
65084
*FCR & BIO
1731560163500_2.html

* BM Equipment Co., Ltd.
1731560163500_3

* Watson https://watson.co.jp/detail/product/plate/other-plate.html

In this specification, the term "agitation device" refers to a device that agitates the medium in a container, and is defined as being different from a shaker.

For the understanding of the various terms used in this specification, including those described above, and for the purposes of this specification, scientific literature, patents, patent applications, and other references cited herein are hereby incorporated by reference in their entirety to the same extent as if each were specifically set forth.

Preferred Embodiments
The preferred embodiments of the present disclosure are 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 a person skilled in the art can make appropriate modifications within the scope of the present disclosure in light of the description in this specification. It is also understood that the following embodiments of the present disclosure can be used alone or in combination.

(Technology for improving efficiency of balancing)
In one aspect, the present disclosure provides a method for increasing at least one selected from the group consisting of alpha diversity (such as Shannon's coefficient) and OTU number when increasing the content of gut microbiota added to a medium for culturing in a container under original equilibrium conditions, wherein the volume of the medium and/or container is suitable for shaking culture.

This 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 necessary, and the dosage is reduced, and while not wishing to be bound by theory, the present disclosure finds that increasing the intestinal flora reduces the diversity index, particularly alpha diversity (such as the Shannon coefficient) and the number of OTUs, and that this objective can be unexpectedly achieved by using small volumes such as in microwells and/or agitated culture.

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

In another 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 with an increase in the content of intestinal flora added to a culture medium for culturing in a container in an original equilibrium state, the method being characterized in that a stirring device is not used and/or the pH is not adjusted, as well as a container, a culture device, etc. used therein. It has been found that by performing an evaluation in a format suitable for shaking culture and/or in which the pH is not adjusted, it is possible to unexpectedly increase at least one of alpha diversity, the number of OTUs, and/or the Shannon coefficient.

In another 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 with an increase in the content of intestinal flora added to a culture medium for culturing in a container in an original equilibrium state, the method being characterized in that the container is of a microwell type, and a container, culture device, etc. used therein. It has been discovered that a shape such as a microwell can unexpectedly increase at least one of alpha diversity (such as the Shannon coefficient) and the number of OTUs.

In another 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 with an increase in the content of intestinal microbiota added to a culture medium for culturing in a container in an original equilibrium state, the container having a volume suitable for shaking culture, and a container, culture device, etc. used therefor. It has been unexpectedly found that by performing evaluation in a format suitable for shaking culture, at least one of alpha diversity (such as the Shannon coefficient) and the number of OTUs can be increased.

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

In another aspect, the present disclosure provides a container for containing a medium for culturing a gut microbiota to increase the OTU number and/or Shannon coefficient, or alpha diversity, when increasing the content of the gut microbiota in the medium in a pristine equilibrium state, the medium having 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 the flora grows appropriately by ensuring that shaking culture is performed appropriately.

In one embodiment, the original equilibration state includes equilibrating the microbiota diversity to the state of a pre-inoculation sample of the gut microbiota.

In another embodiment, the evaluation items after addition of the test substance are obtained while the original equilibration state is maintained.

In another embodiment, the evaluation items after addition of the test substance are obtained 24 to 96 hours after the start of culture.

In another embodiment, the test substance is added at least 6 hours after the incubation, preferably at least 12 hours and at most 72 hours.

In another embodiment, the evaluation items after addition of the test substance are obtained at least 6 hours, preferably at least 12 hours and at most 96 hours, after addition of the test substance.

In another embodiment, the original equilibrium state is determined based on a structural analysis of the intestinal flora.

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

In another embodiment, the structural analysis advantageously uses the Pearson product moment correlation coefficient.

In another embodiment, the timing of adding the test substance is determined by a method including the following steps A and B: (step A) culturing a stool specimen containing the enterobacteria 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 the culture, and (step B) determining in advance the timing of adding the test substance to the specimen containing the enterobacteria cultured in the medium from the time-course data obtained in step A.

In another embodiment, the original equilibrium state in the present disclosure is characterized in that the timing of adding the test substance is determined from the time range in which 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 maintained at 0.8 or more for 24 hours or more. This can be changed as appropriate, and for example, the original equilibrium state can be determined 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 0.5 or more, or 0.6 or more, usually 0.70 or more, and preferably 0.8 or more, or 0.85 or more, 0.90 or more, or 0.95 or more. In addition, the original equilibrium state is referred to as a state in which the original equilibrium state continues for a certain period of time. The certain period of time is 6 hours or more, 12 hours or more, or 18 hours or more, preferably 24 hours or more, more preferably 36 hours or more, even more preferably 48 hours or more, and even more preferably 72 hours or more. In this specification, the "stabilization judgment index" is calculated as an index until the original equilibrium state is reached.

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

In another embodiment, the flora is obtained from stool, preferably the flora is obtained from human stool.

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

(Evaluation Technology)
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 volume of 3 mL or less of medium; B) culturing the culture well until the culture reaches an equilibrium state; C) adding a test substance; and D) obtaining and evaluating evaluation items before and after the addition of the test substance.

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

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 intestinal bacterial flora until the intestinal bacterial flora reaches an equilibrium state; C) adding a test substance; and D) acquiring and evaluating evaluation items before and after the addition of the test substance.

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 in a microwell-type container; B) culturing the intestinal bacterial flora until the intestinal bacterial flora reaches an equilibrium state; C) adding a test substance; and D) acquiring and evaluating evaluation items before and after the addition of the test substance.

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) a means for adding an 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 acquiring and evaluating evaluation items before and after the addition of the test substance.

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) a means for adding intestinal 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 acquiring and evaluating evaluation items before and after the addition of the test substance.

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 of a volume suitable for shaking culture or having a shape suitable for shaking culture; 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 acquiring and evaluating evaluation items before and after the addition of the test substance.

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 the use of an agitator; B) a means for adding intestinal 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 acquiring and evaluating evaluation items before and after the addition of the test substance.

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) a means for adding an 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 acquiring and evaluating evaluation items before and after the addition of the test substance.

(Time of adding test substance)
In one embodiment, the improved evaluation method in the present disclosure is characterized in that the timing of adding the test substance to a sample containing intestinal flora is improved. More specifically, the present disclosure is characterized in that the test substance is added during the original equilibrium state as the addition timing. The addition timing may be any time as long as it is during the original equilibrium period, but it is preferable to add the test substance at the beginning of the original equilibrium state in terms of being able to evaluate the effect of the test substance on the intestinal flora for a longer period of time and evaluation efficiency. That is, the test substance can be added 6 hours or more after the start of culturing the intestinal bacteria, for example, between 12 hours and 120 hours, more preferably between 15 hours and 96 hours after the start of culturing, even more preferably between 24 hours and 96 hours after the start of culturing, and most preferably between 24 hours and 72 hours after the start of culturing.

(Cultivation and collection after addition of test substance)
In one embodiment, it may be advantageous to select the time of sample acquisition for evaluation. After the test substance is added to the culture medium and further cultured, the culture medium is collected for evaluation. The culture medium may be collected at any time after the test substance is added to the culture medium, but is preferably collected while the original equilibrated state is maintained. Specifically, the culture medium is collected 6-120 hours after the start of the culture of the intestinal flora, and the time 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 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. In addition, 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.

(Culture medium)
The 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 preferable, and for example, GAM agar medium, modified GAM agar medium, GAM semi-fluid high-layer medium, GAM bouillon, and modified GAM bouillon (all manufactured by Nippon Pharmaceutical Co., Ltd.) can be used. Two or more types of media selected from these media may be mixed in any ratio and used.

(Additional ingredients)
In a preferred embodiment, various additional components can be added to the medium. Examples of additional 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 those in which an O-linked glycan is added to a polypeptide that includes an amino acid sequence having a tandem repeat structure.

In this specification, a tandem repeat structure refers to a structure in which an amino acid sequence of one to a dozen amino acids in length is regularly repeated. Examples of O-linked glycans include, but are not limited to, O-mannose, O-N-acetylglucosamine, O-fucose, O-glucose, and O-galactose. In the polymeric glycoprotein of the present disclosure, GalNAc (N-acetylgalactomisan) of the glycan is bound to the hydroxyl group of serine or threonine of the polypeptide by an O-glycosidic bond. The molecular weight of the polymeric glycoprotein of the present disclosure is about 500,000 to about 20 million, and more preferably about 1 million to about 10 million.

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. In addition, mucins such as MUC8, 9, 10, 11, 14, 15, 18, and 19 can also be used. Among these, secretory mucins such as MUC2, MUC5AC, MUC5B, MUC6, and MUC7 are preferably used.

The mucin may be of human or non-human origin, such as porcine.

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, based on the medium in terms of obtaining an original equilibrium state. Also, in terms of maintaining the bacterial flora structure, the amount is preferably 4.0% by mass or less, and more preferably 2.0% by mass or less.

In certain embodiments, additional components other than high molecular weight glycoproteins may be added. For example, if necessary, a carbon source such as glucose, a nitrogen source such as ammonia, a source of nutrients such as vitamins and inorganic salts, or a scaffold necessary for bacterial growth may be added to the medium.

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 the "culture medium." During culturing, the culture medium may be appropriately stirred.

(Intestinal microbiota and sample preparation for use in this disclosure)
The intestinal flora 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 be used. The sample may be from a human or a non-human animal, but is preferably from a human, and more preferably, human stool is advantageously used. The stool sample may be one immediately after being discharged from the intestine, may be frozen after collection, or may be one 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 (PBS) to form a suspension. The phosphate buffer in which the stool is suspended may contain ascorbic acid or glycerin. The concentration of the stool may be 0.01% (w/v) to 50% (w/v) in the state of a stool suspension. 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 proliferation of bacteria, 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. In this specification, inoculation refers to taking a certain amount from a stool suspension and adding it to a culture medium.

(Culture vessel and device used in the present disclosure)
The culture vessel used in the present disclosure may be a flask, a commercially available culture vessel, a multi-well plate, or the like. A multi-well plate is preferably used from the viewpoint of increasing the throughput of the evaluation. Here, the shape of each well of the multi-well plate may be approximately hemispherical, approximately rectangular, or approximately cylindrical, and the bottom surface may be flat or round. When using a multi-well plate, it is preferable that the volume per well is 5 mL or less, more preferably 3 mL or less, and even more preferably 2 mL or less. In addition, a volume of 0.1 mL or more, more preferably 0.2 mL or more per well is preferable.

The culture in the culture vessel is stirred during cultivation, preferably using a shaking stirrer. When using a shaking stirrer, the shaking speed 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.

(Culture conditions - atmosphere)
In the present disclosure, the culture is performed in an anaerobic environment. The anaerobic environment for culture can be created by aerating an anaerobic gas into the culture medium. The anaerobic gas is, for example, nitrogen, nitrogen and carbon dioxide, or nitrogen, carbon dioxide and hydrogen. The aeration of the anaerobic gas is performed continuously or intermittently at a predetermined flow rate (for example, 0.1 to 1.0 dL/min). In addition, since intestinal gas may contain, for example, nitrogen and carbon dioxide, the anaerobic gas is preferably a mixed gas consisting of nitrogen and carbon dioxide. In addition, in order to maintain a highly anaerobic condition, it is preferable to aerate the anaerobic gas constantly.

(Culture conditions-pH)
In the present disclosure, the pH of the culture medium at the start of the 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 the culture (for example, when the culture medium containing the 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 the culture is within the above range, and thereafter, the pH may be left as it 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.

(Culture conditions - temperature, stirring)
The culture temperature is preferably close to the body temperature of the mammal to which the feces is used, since this mimics the environment in the large intestine of the mammal to which the feces is applied. 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 limited, but a single batch method is preferred. In addition, even when the culture vessel is a flask or the like, it is preferable to shake the culture vessel, and in the case of a multi-well plate, it is preferable to stir the culture liquid using a shaker.

(Preparation of test substance and amount 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. When the test substance is a solid, it may be added after being dissolved in a solvent such as water.

(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. The metagenomic analysis of the intestinal flora may be performed using a 16sRNA gene sequence or a whole genome sequence. For example, as in Reference Example 1 of JP 2021-153471 A, an OTU (Operational Taxonomic Unit) that has reached 97% similarity can be used to calculate the Shannon index and the 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 necessary.

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

(kit)
In another aspect, the present disclosure provides a kit for evaluating a test substance, the kit 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 the equilibrium in the original state, such as mucin.

As the incubator with the culture wells in the kit of the present disclosure, a multi-well plate can be preferably used. 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 rounded. 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.

The kit of the present disclosure includes a culture medium. There are no particular limitations on the culture medium as long as it is a medium in which enterobacteria can grow, and examples 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-fluid 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.

The kit of the present disclosure further comprises a substance for promoting and regulating the equilibration of the original state. The substance for promoting and regulating the original state includes a high molecular weight glycoprotein. An example of a high molecular weight glycoprotein is the mucin mentioned above. The substance for promoting and regulating the original state in the kit is provided at 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 and regulating the original state is provided at 4.0% by mass or less, more preferably 2.0% by mass or less, based on the mass of the medium.

By using the kit disclosed herein, it is possible to easily test the effects and impact of a test substance on the intestinal flora.

All references cited in this disclosure, including but not limited to scientific literature, patents, patent applications, etc., are incorporated by reference in their entirety into this disclosure to the same extent as if each was specifically set forth herein.

The present disclosure has been described above by showing preferred embodiments for ease of understanding. Below, the present disclosure will be described based on examples, but the above description and the following examples are provided for illustrative purposes only and are not provided for the purpose of limiting the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments or examples specifically described in this specification, but is limited only by the scope of the claims.

Example 1 (Inulin addition (multi-well plate culture))
(Preparation of fecal suspension)
Feces serving as an inoculum of the intestinal flora were collected from healthy subjects 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 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 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to prepare a fecal suspension.

(Preparation of medium)
The medium was prepared by mixing 59 g/L of Gifu University 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.

(Culture conditions)
In a clean bench, 1.0 mL of medium per well was dispensed into the required number of wells. A multi-well plate manufactured by Sarstedt Co., Ltd., model number 82.1972.002, capacity 2.2 mL, and 96-well multi-well plate was used (hereinafter sometimes referred to as 96-well). The multi-well plate was stirred at about 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. The culture was started by inoculating 50 μL/well of fecal suspension (12.5 mg/mL of feces per culture solution), and this was designated as culture 0 h.

The culture medium was collected 6, 24, 30, 48, 72, and 96 hours after the start of cultivation, and bacterial flora analysis was performed. The culture medium was collected without opening the anaerobic chamber.

(Bacteria flora analysis)
Genomic DNA of the bacteria in the bacterial flora was extracted from the culture solution collected at each time before and after the start of the culture. 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.

The extracted genomic DNA was used as a template to amplify the bacterial 16S rRNA gene 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). 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). Amplicons were quantified on an Agilent Bioanalyzer 2100 DNA 1000 chip (Agilent Technologies, Inc.) and pooled in equimolar concentrations. The 16S rRNA gene product (along with an internal control (PhiX control V3; Illumina, Inc.)) was subjected to paired-end sequencing using a MiSeq sequencer (Illumina, Inc.) together with a 600-cycle MiSeq reagent kit (Illumina, Inc.).

Paired-end reads with a Q score of 20 or more were extracted from PhiX sequences using Basespace Sequence Hub (https://basespace.illumina.com/), and were then joined using QIIME 2 version 2022.2, quality controlled and corrected using the DADA2 pipeline, and OTUs were inferred. The resulting OTUs were used to estimate alpha diversity and calculate the Shannon index. The resulting OTUs were also classified using a naive Bayes classifier trained on the Greengenes 13_8 99% OTU full-length sequence database for species assignment. Using Excel (Microsoft Japan Co., Ltd.), the relative occupancy was calculated from the genus-level classification data of the bacterial species attribution, and the Pearson product-moment correlation coefficient was calculated based on the relative occupancy.

Real-time PCR was performed using the 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). The total number of bacteria was calculated from a standard curve created from known concentrations of E. coli.

The results of the Pearson product moment correlation coefficient are shown in Table 1. The results in Table 1 show that the original equilibrium state was reached between 6 and 96 hours after the start of culture.

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

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

The percentage of Bifidobacterium bacteria present in the culture fluids 48 and 72 hours after the start of culture in Examples 1 and 2 and Comparative Example 1 was determined. The results are shown in Table 2.

The culture medium to which the test substance inulin was not added is designated "CUL," the culture medium to which inulin was added at the start of culture is designated "INU (added at 0 h)," and the culture medium to which inulin was added 24 hours after the start of culture is designated "INU (added at 24 h)."

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 lower 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 adding inulin is consistent with the test results in which humans were given 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.

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

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

The percentage of Faecalibacterium bacteria present in each culture was determined 48 hours, 72 hours, and 96 hours after the start of culture. The results are shown in Table 3.

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 at 0 h)", and the culture solution to which dextrin was added 24 hours after the start of culture is referred to as "DEX (added at 24 h)".

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 Faecalibacterium was lower at all culture times 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 Faecalibacterium was higher at all culture times 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, p. 824-831). In other words, it was shown that by adding the test substance dextrin during the original equilibration state, it was possible to reproduce the in vivo test results in vitro.

Example 4 (Relationship between fecal inoculum amount and bacterial flora diversity (multi-well plate culture))
(Preparation of fecal suspension)
Feces serving as an inoculum of the intestinal flora were collected from healthy subjects 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 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 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to prepare a fecal suspension.

(Preparation of medium)
The medium was prepared by mixing 59 g/L of Gifu University 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.

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

The culture medium was collected 6, 24, 30, 48, 72, and 96 hours after the start of cultivation, and bacterial flora analysis was performed. The culture medium was collected without opening the anaerobic chamber.

Examples 5 to 7
Cultivation and bacterial flora analysis were performed 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.

Comparative Example 3 (Relationship between fecal inoculation amount and bacterial flora diversity (jar culture))
(Preparation of fecal suspension)
Feces serving as an inoculum of the intestinal flora were collected from healthy subjects 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 with 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) containing 1.0% L-ascorbic acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to a total of 2 mL to prepare a fecal suspension.

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

After adjusting the medium to pH 6.5 with 0.1 M phosphate buffer, 100 mL of 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.

(Culture conditions)
After sterilization, anaerobic conditions were created in the culture vessel by aerating (15 mL/min) with 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 cultivation.

200 μL of the fecal suspension was inoculated into the medium-containing vessel (0.05 g feces per 100 mL of culture solution), and anaerobic culture was started (culture time 0 hours). 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 incubation was performed with continuous stirring at approximately 300 rpm.

The culture fluid was collected 6 hours, 24 hours, 30 hours, 48 hours, 72 hours, and 96 hours after the start of cultivation, and bacterial flora analysis was performed. The culture fluid was collected using a syringe without opening the cultivation tank, and without mixing in air. The bacterial flora analysis was performed on the obtained culture fluid in the same manner as in Example 1.

Comparative Examples 4 to 6
Cultivation and bacterial flora analysis were performed 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.

(Evaluation Results)
The calculation results of OTU and Shannon index after 72 hours of culture in Examples 4 to 7 and Comparative Examples 3 to 6 are shown in Table 5.

Comparing 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 higher in Examples 4, 6, and 7, which involved 96-well culture, when the fecal inoculation amounts were the same, with Comparative Example 4 and Example 4, Comparative Example 5 and Example 6, and Comparative Example 6 and Example 7. In addition, when comparing the Shannon index, which reflects the diversity of the bacterial flora, between samples with the same fecal inoculation amount, it was higher in the 96-well culture than in the jar culture. In addition, 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 the OTU value and Shannon index, and the closer it was to the FEC value.

Table 6 shows the presence rate of Enterococcus bacteria during jar culture. Enterococcus bacteria are a species of bacteria 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%. If the presence rate when the fecal inoculum amount was 0.05% is taken as 1, this shows a value of 7.94 times. On the other hand, the presence rate of Enterococcus bacteria in 96-well culture is shown in Table 7. In 96-well culture, the presence rate of Enterococcus bacteria was at most 0.070%, which was a suppressed increase in the presence rate of Enterococcus bacteria compared to the maximum of 0.238% observed in jar culture.

The results of Examples 4 to 7 and Comparative Examples 3 to 6 show that shaking culture using a multi-well plate with a capacity of 2 mL per well can increase the number of OTUs and the Shannon index compared to culture using a jar fermenter with a capacity of approximately 200 mL.

(Consideration of culture medium)

Example 8 (Multi-well plate culture)
(Preparation of fecal suspension)
Feces serving as an inoculum of the intestinal flora were collected from healthy subjects 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 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 NaH ₂ PO ₄ and 0.1 M Na ₂ HPO ₄ ) supplemented with 1.0% L-ascorbic acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to prepare a fecal suspension.

(Preparation of medium)
The medium was prepared by mixing 59 g/L of Gifu University 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.

(Culture conditions)
In a clean bench, 1.0 mL of medium per well was dispensed into the required number of wells. A multi-well plate manufactured by Sarstedt Co., Ltd., model number 82.1972.002, capacity 2.2 mL, and 96-well multi-well plate was used (hereinafter sometimes referred to as 96-well). The multi-well plate was stirred at about 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. The fecal suspension was inoculated at 50 μL/well (12.5 mg/mL feces per culture solution) to start the culture, and this was designated as culture 0 h.

The culture medium was collected 6, 24, 30, 48, 72, and 96 hours after the start of cultivation, and bacterial flora analysis was performed. The culture medium was collected without opening the anaerobic chamber.

(Bacteria flora analysis)
Genomic DNA of the bacteria in the bacterial flora was extracted from the culture solution collected at each time before and after the start of the culture. 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.

The extracted genomic DNA was used as a template to amplify the bacterial 16S rRNA gene 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). 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). Amplicons were quantified on an Agilent Bioanalyzer 2100 DNA 1000 chip (Agilent Technologies, Inc.) and pooled in equimolar concentrations. The 16S rRNA gene product (along with an internal control (PhiX control V3; Illumina, Inc.)) was subjected to paired-end sequencing using a MiSeq sequencer (Illumina, Inc.) together with a 600-cycle MiSeq reagent kit (Illumina, Inc.).

Paired-end reads with a Q score of 20 or more were extracted from PhiX sequences using Basespace Sequence Hub (https://basespace.illumina.com/), and were then joined using QIIME 2 version 2022.2, quality controlled and corrected using the DADA2 pipeline, and OTUs were inferred. The resulting OTUs were used to estimate alpha diversity and calculate the Shannon index. The resulting OTUs were also classified using a naive Bayes classifier trained on the Greengenes 13_8 99% OTU full-length sequence database for species assignment. Using Excel (Microsoft Japan Co., Ltd.), the relative occupancy was calculated from the genus-level classification data of the bacterial species attribution, and the Pearson product-moment correlation coefficient was calculated based on the relative occupancy.

Example 9 (Study of GAM medium containing mucin)
Cultivation and bacterial flora analysis were performed 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.

Example 10 (Study of 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 modified Gifu University prescribed anaerobic medium (modified GAM medium [Code 05433] (manufactured by Nissui Pharmaceutical Co., Ltd.): 41.7 g/L and antifoaming agent: 50 μL/L.

Example 11 (Study of modified GAM medium containing mucin)
Cultivation and bacterial flora analysis were performed in the same manner as in Example 10, except that in preparing the modified 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 modified GAM medium.

Example 12 (Study on YCFA medium)
Cultivation and bacterial flora analysis were performed in the same manner as in Example 8, except that YCFA medium was used instead of GAM medium in the preparation of the medium. The 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 antifoaming agent 50 μL/L, and adjusting the pH to 7.5 with a pH adjuster.

Example 13 (Study on YCFA medium containing mucin)
Cultivation and bacterial flora analysis were performed 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.

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 in the preparation of the medium. Modified YCFA medium contained 10.0 g/L casein hydrolysate, 2.5 g/L yeast extract, 5.0 g/L glucose, 0.045 g/L magnesium sulfate, 0.09 g/L calcium chloride, 0.45 g/L dipotassium hydrogen phosphate, 0.45 g/L potassium dihydrogen phosphate, 0.9 g/L sodium chloride, 0.001 g/L resazurin, and L-cysteine HCl. The medium contained 1.0 g/L, 4.0 g/L sodium bicarbonate, and 0.01 g/L hemin, and was mixed with 2.7 ml of volatile fatty acids (2.026 ml/L acetic acid, 0.715 ml/L propionic acid, 0.119 ml/L n-valeric acid, 0.119 ml/L isovaleric acid, and 0.119 ml/L isovaleric acid), 10 ml of vitamin mixture (2 mg/L biotin, 0.1 mg/L cyanocobalamin, 2 mg/L folic acid, 10 mg/L pyridoxine, 5 mg/L thiamine, 5 mg/100 ml riboflavin, 5 mg/L nicotinic acid, 5 mg/L calcium pantothenate, 5 mg/L p-aminobenzoic acid, and 5 mg/L lipoic acid) and 50 μL/L of antifoaming agent per 1 L, and the pH was adjusted to 6.8 with a pH adjuster to prepare a medium.

Example 15 (Study of modified YCFA medium containing mucin)
Cultivation and bacterial flora analysis were performed in the same manner as in Example 14, except that in preparing the modified 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 modified YCFA medium. Relative occupancy rates were 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 rates.

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

Example 16 (Amount of useful bacteria: Effect of adding mucin)
Genomic DNA of bacteria in the bacterial flora was extracted from the culture fluids collected before and 72 hours after the start of the culture in Examples 8 and 9. The target bacterial genes were quantified from the extracted genomic DNA using specific primers for Faecalibacterium duncaniae (Fd bacteria) and Blautia wexlerae (Bw bacteria) targeting the 16S rRNA genes of each bacteria using a quantitative PCR device (Table 9).

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

(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 the preparation of the medium, 8.0 g/L of mucin (Sigma-Aldrich, derived from porcine stomach, Type II) was added to the GAM medium.

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

Example 19 (Study on medium containing ganglia imucin)
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 the GAM medium in the preparation of the medium. Relative occupancy rates were 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 rates.

Comparative Example 7 (Study of mucin-free medium)
In preparing the medium, the culture and bacterial flora analysis were performed in the same manner as in Example 8.
Table 10 shows the relative values of the Pearson product-moment correlation coefficients for Examples 1, 2, 3, and 4 for the Pearson product-moment correlation coefficients after 72 hours of culture, with the result for Comparative Example 7 set at 100. It is clear from the results in Table 10 that the addition of all mucins of different origins was effective in maintaining the original equilibrium state.

Example 20 (Administration of culture medium formulation to mice)
The human intestinal flora culture solution prepared in Example 9 is administered to SPF mice, and insulin sensitivity is measured by subjecting them to a high-fat diet. The administration of the microorganism to the mice and the efficacy evaluation test are performed according to the method described in Nature Communications (202) 13:4477.

Specifically, SPF mice (6 weeks old) were fed a high-fat diet (AIN-93G, Oriental Yeast) for 10 weeks and orally administered 5 x ¹⁰⁹ CFU of the human intestinal flora culture medium prepared in Example 9 three times a week. The mice were weighed and serum was collected 8 weeks after administration of the human intestinal flora culture medium. HOMA-IR and insulin concentrations were measured and an IPGTT test was performed.

(Note)
As described above, the present disclosure has been illustrated using a preferred embodiment of the present disclosure, but the present disclosure should not be interpreted as being limited to this embodiment. It is understood that the scope of the present disclosure should be interpreted only 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 technical common sense from the description of the specific preferred embodiment of the present disclosure. It is understood that the patents, patent applications and literature cited in this specification should be incorporated by reference to the present specification in the same manner as the contents themselves are specifically described 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 by reference in their entirety in this specification.

The method disclosed herein makes it possible to evaluate in vitro the effects of test substances, such as food or candidate pharmaceutical compounds, on the intestinal flora of mammals, particularly humans.

Claims (27)

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 vessel under original equilibrium conditions, the method being characterized in that no stirring device is used. 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 an original equilibrium state, the method being characterized in that the pH is not adjusted. 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 a state of original equilibrium, the method being characterized in that the container is of a microwell type. 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 a pristine equilibrium state, the container having a volume suitable for shaking culture. 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 an original equilibrium state, the container being 3 mL or less. 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 an original equilibrium state, the method comprising: a medium having a volume of 3 mL or less. The method according to any one of claims 1 to 6, wherein the original equilibrated medium is used to evaluate a test substance. A container or culture well that contains a medium for culturing intestinal flora, in order to increase the number of OTUs and/or the Shannon coefficient with an increase in the content of the intestinal flora in the medium in a pristine equilibrium state, the volume of the container being suitable for shaking culture. A container or culture well that contains a culture medium for culturing intestinal flora, in order 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 in a state of original equilibrium, the container having a volume of 3 mL or less. A) adding the gut microbiota to a culture well having a volume of medium of 3 mL or less;
B) culturing the intestinal flora until it reaches an equilibrium state;
C) adding a test substance to the container;
D) obtaining and evaluating evaluation items before and after the addition of the test substance. 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 gut flora;
C) a means for incubating the culture until the culture is in equilibrium;
D) a means for adding a test substance; and
E) A system for evaluating a test substance, comprising means for acquiring and evaluating evaluation items before and after the addition of the test substance. The method according to claims 1 to 7 or 10, wherein the original equilibration state includes equilibrating the microbiota diversity to the state of a pre-inoculation sample of the intestinal microbiota. The method according to claims 1 to 7, 10 or 12, wherein the evaluation items after the addition of the test substance are obtained while the original equilibrium state is maintained. The method according to claims 1 to 7, 10, or 12 to 13, wherein the evaluation items after the addition of the test substance are obtained 24 to 96 hours after the start of culture. 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. The method according to claims 1 to 7, 10, or 12 to 15, wherein the evaluation items after addition of the test substance are obtained 12 to 96 hours after addition of the test substance. 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. The method according to claims 1 to 7, 10, or 12 to 17, wherein the original equilibrium state is determined based on analysis of the 16S or genome of the intestinal flora. The method according to 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 the 16S or genome analysis of the intestinal microbiota. The timing of adding the test substance is a method including the following steps A and B:
The method according to claims 1 to 7, 10, or 12 to 19, comprising: (Step A) culturing a stool specimen containing the enterobacteria 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 the culture; and (Step B) determining in advance the timing of adding a test substance to the specimen containing the enterobacteria cultured in the medium, based on the time-course data obtained in Step A. The 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 maintains a state in which the Pearson product moment correlation coefficient between the enterobacteria flora structure before and after cultivation of 0.70 or more for 24 hours or more. The method according to claims 1 to 7, 10, or 12 to 21, wherein the medium contains mucin. 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. The method according to claims 1 to 7, 10, or 12 to 23, wherein the intestinal flora is obtained from feces. The method according to claims 1 to 7, 10, or 12 to 24, wherein the intestinal flora is obtained from human feces. The method according to 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. A kit for evaluating a test substance, comprising an incubator with a culture well that can hold a medium with a volume of 3 mL or less, the medium, and a substance that promotes and maintains equilibrium in the original state.