WO2025110155A1 - Polymeric glycoprotein-containing composition for culturing intestinal flora, and for promoting and/or maintaining original equilibration - Google Patents

Abstract

The present disclosure provides a composition useful for in vitro assessment of the effect of an analyte, such as a food or drug candidate compound, on mammal, particularly human, intestinal flora. Specifically, the present disclosure provides a polymeric glycoprotein-containing composition for promoting and/or maintaining original equilibration when culturing intestinal flora in a culture medium. The composition is used so that a mucin, for example, is included in a culture medium. A GAM culture medium or a modification thereof can be used as the culture medium.

Description

Composition for culturing intestinal flora to promote and/or maintain in situ equilibria containing high molecular weight glycoproteins

The present disclosure relates to a composition for obtaining an intestinal flora that is rich in flora diversity and maintains that flora diversity for a certain period of time, which is necessary for in vitro evaluation of the effects of test substances such as foods and candidate pharmaceutical compounds on the intestinal flora in the intestines of mammals.

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

As a result of extensive research, the inventors have discovered that certain components contribute to maintaining the diversity of bacterial flora and the diversity of intestinal bacterial flora. The present disclosure based on this finding is as follows.

[1] A composition for promoting and/or maintaining original equilibrium when culturing an intestinal bacterial flora in a medium, comprising a high molecular weight glycoprotein.
[2] The composition described in the above item, wherein the high molecular weight protein includes mucin.
[3] The composition according to any one of the preceding items, wherein the mucin is contained in a medium at a concentration of 0.4 w/v % or more.
[4] The composition according to any one of the above items, wherein the medium is GAM medium, YCFA medium, or a modified form thereof.
[4A] The composition according to any one of the preceding items, wherein the medium is GAM medium or a modified form thereof.
[5] The composition according to any one of the preceding items, wherein the equilibration is performed to evaluate a test substance in the intestinal bacterial flora.
[6] The composition according to any one of the preceding items, wherein the composition is for maintaining the original equilibrium.
[7] The composition according to any one of the preceding items, wherein the composition is intended to suppress the loss of original state equilibrium after the medium has achieved the original state equilibrium.
[8] An original equilibrated medium for intestinal bacterial flora, comprising a high molecular weight glycoprotein and a medium component.
[9] The original equilibrated medium according to any one of the preceding items, wherein the medium is for evaluating a test substance in intestinal bacterial flora.
[10] The medium according to any one of the preceding items, wherein the high molecular weight protein comprises mucin.
[11] The medium according to any one of the preceding items, wherein the mucin is contained in an amount of 0.4 w/v % or more relative to the medium components.
[12] The medium according to any one of the preceding items, wherein the medium component is GAM medium or a modified form thereof.
[13] A composition for maintaining beneficial bacteria in the intestinal flora, comprising a high molecular weight glycoprotein.
[14] The composition according to any one of the preceding items, wherein the useful bacteria include at least one selected from the group consisting of Faecalibacterium duncaniae (Fd bacteria) and Blautia wexlerae (Bw bacteria).
[15] The composition according to any one of the preceding items, wherein the high molecular weight glycoprotein is a mucin.
[16] The composition according to any one of the preceding items, which is used for producing a bacterial preparation.
[17] A method for producing a bacterial preparation enriched in useful bacteria, comprising the steps of:
A) culturing the intestinal flora in a medium containing a high molecular weight glycoprotein;
B) Collecting the grown gut microbiota;
C) A method of production comprising, optionally after washing the intestinal flora, adding the intestinal flora to a medium for administration to obtain a bacterial preparation.
[18] The method according to any one of the preceding items, wherein the high molecular weight glycoprotein is mucin.
[19] A bacterial preparation produced by the production method according to any one of the above items.

This disclosure makes it possible to more accurately reproduce changes in the intestinal flora caused by the administration of a test substance to humans. It is thought that one of the reasons why in vivo results of a test substance cannot be reproduced in vitro is that the composition of the intestinal flora can change due to cultivation, but this effect has been successfully suppressed.

The present disclosure makes it possible to reproduce in vitro the in vivo effects of test substances, such as foods and candidate pharmaceutical compounds, on the intestinal environment, and to provide an intestinal microbiota sample that is rich in microbiota diversity and maintains that diversity for a certain period of time.

This disclosure makes it possible to provide intestinal microbiota samples that can be used to evaluate in vitro the effects of test substances, such as foods and candidate pharmaceutical compounds, on the intestinal microbiota of mammals, particularly humans.

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

(Definition of terms)
All numerical values herein are intended to be modified by the term "about," whether or not expressly stated. 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 numerical values that are rounded to the nearest significant figure.

As used herein, the term "intestinal flora" refers to a group of bacteria that normally reside 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, etc. Further, at the genus level, examples include bacteria belonging to the following genera or families: Bifidobacterium, Collinsella, Bacteroides, Parabacteroides, Prevotella, Rikenellaceae, Lactobacillales, etc.

As used herein, the term "useful bacteria" refers to any bacteria in the intestinal flora that is useful to a host (e.g., humans). Examples include, but are not limited to, bacteria of the genera Bifidobacterium, Lactobacillus, Faecalis, Blautia, Ackermansia, Roseburia, Ruminococcus, Bacteroides, Enterococcus, and Clostridium. Specific examples of useful bacteria include, but are not limited to, Faecalibacterium duncaniae (Fd bacteria), Blautia wexlerae (Bw bacteria), Bifidibacterium genus (bifidobacterium: an example is Bifidibacterium longum), and Lactobacillus genus (lactic acid bacillus: an example is Lactobacillus casei).

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 "bacterial preparation" refers to a preparation composed of bacteria, and more specifically, a powder or liquid preparation containing a useful group of microorganisms, which is expected to exert a medicinal effect alone or in combination with other substances. Examples of the form of the bacterial preparation include, but are not limited to, oral capsules, tablets (for oral use), enema preparations, gargle liquids, skin smears (ointments), etc. In addition, the bacterial preparation may contain one or more types of microorganisms. Bacterial preparations are also a type of biological preparation, and representative examples include enterobacterial preparations (Live Biotherapeutic Products: LBPs) composed of enterobacteria and microbiome medicines. Other representative examples include live bacterial preparations (including single bacteria/cocktails), which can also be used for fecal microbiota transplantation (FMT), etc. For bacterial preparations, see, for example, FEMS Microbiology Reviews, 2023, 47, 1-18.

In this specification, "drug effect" is interpreted in the broadest sense and refers to some biological effect on a subject. A drug effect can be recognized by some biological change in a subject or a phenomenon caused by it. Typically, it includes, but is not limited to, improvement of a disease state and maintenance or improvement of health by biological components such as probiotics. The desired drug effect of a microorganism obtained by the method disclosed herein can be confirmed by various methods including the use of a drug gene resistance system, enzyme activity based on the drug effect, survival activity based on the drug effect, migration activity of the microorganism, fixation of the microorganism in the subject, cell adhesion activity of the microorganism, adhesion activity of the microorganism to a mucin layer, fixation activity of the microorganism in animal tissue, etc. The generation of a population containing a microorganism having a desired drug effect is not particularly limited as long as it is under conditions that allow the microorganism to grow and/or amplify. When producing a bacterial preparation, for example, by comparing the growth curve under aerobic and/or anaerobic conditions with that of a wild strain (parent strain), it is possible to avoid clones that are difficult to mass-culture, which is a problem in preparation.

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.

Compositions for Promoting and/or Maintaining Original Equilibria
In one aspect, the present disclosure provides a composition, active ingredient, compound, and other technology for promoting and/or maintaining original equilibrium when culturing an intestinal bacterial flora in a medium containing a high molecular weight glycoprotein.

In one embodiment, the polymeric protein of the present disclosure includes mucin.

In one embodiment, the mucin used in the present disclosure is used in a medium at a concentration that may be 0.1 w/v% or more, 0.2 w/v% or more, 0.3 w/v% or more, 0.4 w/v% or more, 0.5 w/v% or more, 0.6 w/v% or more, 0.7 w/v% or more, 0.8 w/v% or more, and/or 4.0 w/v% or less, 3.0 w/v% or less, 2.5 w/v% or less, 2.0 w/v% or less, 1.9 w/v% or less, 1.5 w/v% or less, 1.0 w/v% or less. Without wishing to be bound by theory, a concentration of more than 2.0 w/v% is not advantageous since the pH during culture may remain low.

In one embodiment, 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 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 media selected from these media may be mixed in any ratio and used. GAM medium or a modified medium thereof is preferred.

In one embodiment, the original state equilibration in the present disclosure is performed in order to evaluate a test substance in the intestinal bacterial flora. Here, the original state equilibration is used, without being limited thereto, for example, in a method for evaluating a test substance in an intestinal bacterial flora, which includes the following steps: A) culturing the intestinal bacterial flora in a medium for a time effective for the intestinal bacterial flora to reach an original state equilibration state after the start of culture by inoculating the intestinal bacterial flora into the medium, B) adding the test substance to the medium containing the intestinal bacterial flora after the time has elapsed, and C) acquiring and evaluating evaluation items before and after the addition of the test substance.

In one embodiment, the compositions disclosed herein are for maintaining the original equilibration. A promoter for the original equilibration may be important because when evaluating candidate substances, it is preferable to maintain the original equilibration state for a long period of time if an evaluation is required over a long period of time.

In one embodiment, the composition of the present disclosure is intended to suppress the loss of original equilibrium after the medium used has achieved original equilibrium (also expressed as a deviation or decline from original equilibrium). Such substances are advantageous in cases where a candidate substance requires evaluation over a long period of time and therefore requires the original equilibrium state to be maintained for a long period of time, since they can suppress the loss of original equilibrium after the medium has achieved original equilibrium.

(Medium for equilibration)
In one embodiment, the medium of the present disclosure may be an original equilibrated medium for intestinal flora, comprising high molecular weight glycoproteins and medium components.

In this embodiment, the medium used in this disclosure is for evaluating a test substance in the intestinal flora.

In a preferred embodiment, the high molecular weight protein includes mucin.

In a preferred embodiment, mucin is contained at a concentration (w/v%) of 0.1 w/v% or more, 0.2 w/v% or more, 0.3 w/v% or more, 0.4 w/v% or more, 0.5 w/v% or more, 0.6 w/v% or more, 0.7 w/v% or more, 0.8 w/v% or more, and/or 4.0 w/v% or less, 3.0 w/v% or less, 2.5 w/v% or less, 2.0 w/v% or less, 1.9 w/v% or less, 1.5 w/v% or less, 1.0 w/v% or less relative to the medium components.

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 include GAM medium, YCFA medium, modified YCFA medium, BBL medium, SOC medium, and LB medium. 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.

In one embodiment, the medium components used in the present disclosure are components constituting GAM medium. The components constituting GAM medium may be contained at 50-200%, for example, 75%-150%, 80-120%, etc.

In one embodiment, the medium of the present disclosure may contain peptone/hemin and vitamin K. Although not wishing to be bound by theory, many anaerobic bacteria are enhanced in their presence. In addition, since some bacteria require them, it is considered advantageous to include these components. Thus, the present disclosure provides a composition for promoting and maintaining equilibrium when culturing intestinal flora in a medium, comprising peptone/hemin/vitamin K and a medium containing it or a modified version thereof. A modified version of GAM medium can be easily made by a person skilled in the art. Examples of the modified version include, but are not limited to, those described in https://axel.as-1.co.jp/asone/d/65-9404-61/, such as Acudia ^TM modified GAM bouillon.

The present disclosure provides a composition for promoting and maintaining equilibrium when culturing intestinal bacterial flora in a medium, the composition including GAM medium or a modified form thereof.

In one embodiment, the GAM medium may advantageously be semi-liquid. A medium that is semi-liquid refers to a medium that contains an agar component but is not for liquid culture. Alternatively, the medium may be a modified version of such a medium. Thus, the present disclosure provides a composition for promoting and maintaining the original equilibrium when culturing the intestinal bacterial flora in a medium.

In a preferred embodiment, the medium of the present disclosure may contain fucoidan and/or sodium lactate, for example, fucoidan may be contained at 0.1-5.0% and sodium lactate may be contained at 1.0-50 mM.

In another embodiment, the present disclosure provides that the amount of the specimen added to the culture medium is 0.05% or more, or 0.1% or more, and 1.5% w/v or less, or 1.25% w/v or less.

In one embodiment, any mucin may be used, but gastrointestinal secretory mucins (MUC2, 5AC, 5B, 6, 7, etc.) may be advantageous. As for the fermentation substrate for gastrointestinal and oral bacteria, classifications known in the art may be adopted, but are not limited thereto, and any may be used as long as a composition for promoting and maintaining equilibrium in the original state is also used when culturing the intestinal flora by referring to this specification. Without wishing to be bound by theory, mucins include secretory mucins produced by epithelial cells, etc., and membrane-bound mucins that have a hydrophobic membrane-spanning site and exist in a state bound to the cell membrane. The core proteins of mucins are collectively called MUCs, and are numbered in the order of their discovery. The genes that code for these core proteins are known to be of at least 23 types of human mucins (MUC1, 2, 3A, 3B, 4, 5AC, 5B, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21), of which MUC2, 5AC, 5B, 6, and 7 are secreted mucins, and MUC1, 3A, 3B, 4, 11, 12, 13, 16, 17, 20, and 21 are membrane-bound mucins.

Substances that promote and/or maintain equilibrium
In this aspect, the present disclosure provides a composition for promoting and maintaining original equilibrium when culturing an intestinal bacterial flora in a medium, the composition comprising a high molecular weight glycoprotein.

In one aspect, the present disclosure provides a substance for promoting state equilibrium or a substance for maintaining state equilibrium. Typically, the present disclosure uses a high molecular weight glycoprotein as the substance for promoting state equilibrium.

When a high molecular weight glycoprotein is used as a substance for promoting equilibration in the present disclosure, the effect of promoting and/or maintaining equilibration in the original state can be achieved by adding the high molecular weight glycoprotein to the culture medium used. In this case, the high molecular weight glycoprotein can function as a substance for promoting equilibration in the original state and can also have the function of maintaining equilibration in the original state. Exemplary high molecular weight glycoproteins of the present disclosure include those in which an O-linked glycan is attached to a polypeptide that includes an amino acid sequence having a tandem repeat structure.

The 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 may be 0.1 w/v% or more, 0.2 w/v% or more, 0.3 w/v% or more, 0.4 w/v% or more, 0.5 w/v% or more, 0.6 w/v% or more, 0.7 w/v% or more, 0.8 w/v% or more, and/or 4.0 w/v% or less, 3.0 w/v% or less, 2.5 w/v% or less, 2.0 w/v% or less, 1.9 w/v% or less, 1.5 w/v% or less, or 1.0 w/v% or less, from the viewpoint of promoting and maintaining equilibration at the original state. Also, from the viewpoint of maintaining the bacterial flora structure, 2.0 w/v% or 1.9 w/v% or less is preferable, and 0.8 w/v% or less is more preferable.

In this embodiment, the medium used in the present disclosure is not particularly limited as long as it is a medium in which enterobacteria can grow and does not inhibit the return to normal equilibrium, and examples of such medium 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 medium selected from these media may be mixed in any ratio and used.

In another embodiment, the present disclosure uses GAM medium as the original equilibration material.

In one embodiment, the GAM medium may be, for example, GAM agar medium, modified GAM agar medium, GAM semi-fluid high layer medium, GAM bouillon, or modified GAM bouillon (all manufactured by Nippon Pharmaceutical Co., Ltd.), but is not limited thereto as long as it has the effect of promoting and/or maintaining the original equilibration.

In a preferred embodiment, the present disclosure can use a composition comprising at least a high molecular weight glycoprotein and a GAM medium, each of which can be combined with any of the specific aspects described elsewhere herein.

Exemplary high molecular weight glycoproteins are as described in (Substances promoting original state equilibration), but examples that can be used include mucin derived from pig stomach and mucin derived from skate. However, they are not limited to these.

Exemplary high molecular weight glycoproteins are contained in the medium at a concentration that may be 0.1 w/v% or more, 0.2 w/v% or more, 0.3 w/v% or more, 0.4 w/v% or more, 0.5 w/v% or more, 0.6 w/v% or more, 0.7 w/v% or more, or 0.8 w/v% or more, for example, 0.1 w/v% or more, more preferably 0.2 w/v% or more, and even more preferably 0.4 w/v% or more. On the other hand, from the viewpoint of maintaining the bacterial flora structure, they are contained in a concentration that may be 4.0 w/v% or less, 3.0 w/v% or less, 2.5 w/v% or less, 2.0 w/v% or less, 1.9 w/v% or less, 1.5 w/v% or less, or 1.0 w/v% or less, for example, 4.0 w/v% or less, more preferably 2.0% or less.

Exemplary compositions may further include a carbon source such as glucose, a nitrogen source such as ammonia, a source of nutrients such as vitamins and inorganic salts, and a scaffolding necessary for bacterial growth. In addition, pH adjusters, surfactants, thickeners, dispersants, preservatives, etc. may also be added.

By using this composition for culturing bacterial flora or by adding this composition to a culture medium, the return to the original state is promoted and the original state is maintained.

Additional components of the medium
If necessary, a carbon source such as glucose, a nitrogen source such as ammonia, nutrient sources such as vitamins and inorganic salts, and a scaffold necessary for cell growth may be added to the medium.

In one embodiment, the medium may be sterilized, for example by autoclaving, before culturing. Liquid culture is preferred, and the liquid culture is also called 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 sample, 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 in the stool suspension may be 0.01 w/v% to 50 w/v%, or 0.05 to 2.5 g/m, and may be 0.1 to 1.0 g/ml, for example, 0.25 g/ml.

The amount of stool sample to be inoculated into the culture medium is 0.05 w/v% or more, more preferably 0.10 w/v% or more, even more preferably 0.12 w/v% or more, based on the viewpoint of bacterial flora diversity, and is preferably 0.05 to 1.5% w/v, and from the viewpoint of inhibiting the growth of unwanted bacteria, is preferably 1.5 w/v% or less, and even more preferably 1.25 w/v% or less. In this specification, inoculation refers to taking a fixed amount from the stool suspension and adding it to the 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, the volume per well is preferably 5 mL or less, more preferably 3 mL or less, and even more preferably 2 mL or less. In addition, the volume per well is preferably 0.1 mL or more, more preferably 0.2 mL or more, and even more preferably 0.3 mL or more.

Cultivation equipment that can be used includes commercially available equipment such as jar fermenters and shaking mixers.

(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 the culture medium with an anaerobic gas. 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), 0.1 to 1.0 dL/min, for example, 0.15 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 usually 6.2 to 7.0, preferably 6.2 to 6.7, and 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. When the culture vessel is a flask or the like, it is preferred to use an agitator or stirrer, and when the culture vessel is a multi-well plate, a shaker is used to agitate the culture solution during culture.

(Preparation of test substance and amount added)
The intestinal flora sample according to the present disclosure can be used to test the effect of a test substance on the intestinal flora. The test substance is added in an amount of preferably 1 g to 50 g, more preferably 1 g to 20 g, per 1 L of culture solution. If the test substance is a solid, it may be dissolved in a solvent such as water and then added.

(Time of adding test substance)
The test substance may be added at any time while the intestinal flora is in the original equilibrium state, but from the viewpoint of longer-term evaluation of the effect of the test substance on the intestinal flora after addition of the test substance 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, 12 hours or more, 15 hours or more, 18 hours or more, 24 hours or more, 30 hours or more, 36 hours or more, 42 hours or more, 48 hours or more, or/and 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, 48 hours or less, for example, 6-90 hours, 12-48 hours, or 48-84 hours, in another embodiment, between 24 hours and 96 hours after the start of culture, and most preferably between 24 hours and 72 hours after the start of culture.

(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 between 6 and 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.

(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 microbiota. The metagenomic analysis of the intestinal microbiota may be performed using a 16sRNA gene sequence or a whole genome sequence. For example, as in Reference Example 1 of JP 2021-153471, 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 can also be obtained as necessary regarding the pH, short chain fatty acid concentration, etc. of the collected culture solution.

(When the timing of adding the test substance is determined in advance)
The method may also include a step of determining the timing of adding the test substance in advance. The intestinal flora is cultured without adding the test substance, the culture medium is periodically obtained, and time-course data such as the Shannon index and Pearson product-moment correlation coefficient of the intestinal flora is obtained, and the above-mentioned original equilibrium state is determined from the obtained time-course data, and the time of adding the test substance can be determined.

(Bacterial preparations)
In another aspect, the present disclosure provides a composition for maintaining beneficial bacteria in the intestinal flora, comprising a high molecular weight glycoprotein. Here, useful bacteria that can be used in the present disclosure include bacteria of the genera Bifidobacterium, Lactobacillus, Faecalis, Blautia, Ackermansia, Roseburia, Ruminococcus, Bacteroides, Enterococcus, and Clostridium, and in particular Faecalibacterium duncaniae (Fd bacteria) and Blautia. wexlerae (Bw bacteria), Bifidobacterium genus (bifidobacterium: for example, Bifidibacterium longum), Lactobacillus genus bacteria (lactic acid bacillus: for example, Lactobacillus casei), etc. These Faecalibacterium duncaniae (Fd bacteria) and Blautia wexlerae (Bw bacteria) are useful bacteria, as described in Fd bacteria: FEMS Microbiology Reviews, 2023, 47, 1-18, Bw bacteria: Nature Communications | (2022) 13: 4477, etc., but no technology has been provided that can maintain these as bacterial preparations.

It was therefore unexpected that these useful bacteria could be maintained using high molecular weight glycoproteins, as disclosed herein, and the effect of mucin in particular is particularly remarkable.

The composition of the present disclosure may be provided as a technique (method, medium, etc.) for producing a bacterial preparation. In this specification, a "bacterial preparation" is a composition containing bacteria, and is preferably a pharmaceutical composition. The bacterial preparation may be composed of a bacterial suspension. In this specification, a "bacterial suspension" includes medically and/or pharma- ceutically acceptable bacteria and medically and/or pharma-ceutical acceptable drugs. The type of bacteria is not particularly limited as long as it is a medically and/or pharma-ceutical acceptable bacterium, but it is advantageous if the type is a bacterium contained in the intestinal flora, particularly a useful bacterium.

In one aspect of the present disclosure, the present disclosure provides a method for producing a bacterial preparation enriched in useful bacteria, which comprises: A) culturing an intestinal flora in a medium containing a high molecular weight glycoprotein; B) collecting the grown intestinal flora; and C) washing the intestinal flora as necessary, and then adding the intestinal flora to a medium for administration (e.g., a liquid for administration, physiological saline, a suspension, etc.) to produce a bacterial preparation. The bacterial preparation produced by this production method has useful bacteria maintained or grown (this is called enriched). The high molecular weight glycoprotein and useful bacteria that are the subject of this specification can be any of those described herein. In this specification, "enriched in useful bacteria" means that the ratio or amount, preferably the ratio, of useful bacteria present in the raw intestinal flora is maintained or increased in the composition of the subject bacterial preparation, etc. Usually, when producing a bacterial preparation, the total amount of bacteria contained in the raw intestinal flora increases, so it can be said that the amount of useful bacteria is usually increased.

The present disclosure provides a bacterial preparation produced by the method for producing a bacterial preparation of the present disclosure. In the present disclosure, the ratio or amount, preferably the ratio, of naturally occurring useful bacteria is maintained or increased, and in that respect, it can be said that a new bacterial preparation is provided because the ratio or amount of useful bacteria is maintained or increased, or increased, compared to when the intestinal flora is simply used as a preparation.

The administration medium used in this disclosure may be any medium used in medicines or food and beverages (e.g., any liquid or solid excipient, etc.). Such a medium may be, but is not limited to, saline, etc.

In one embodiment, the high molecular weight glycoprotein is preferably a mucin.

(Bacterial preparations (probiotics) can be used not only in medicines, but also in foods, supplements, and food and beverage additives.

In one embodiment, the oral composition disclosed herein specifically includes, but is not limited to, foods (including foods in the narrow sense, beverages, etc.), additives for foods and beverages, supplements, feed, additives for feed, pharmaceuticals (including quasi-drugs), etc.

In this specification, the bacterial preparation of the present disclosure may be provided as a food. In this disclosure, "food" includes health foods, functional foods, health foods (foods for specified health uses, foods with nutrient functions, foods with functional claims, etc.), dietary supplements, nutritional supplements, etc. Furthermore, the shape of the food may be selected appropriately, such as solid, liquid, or paste. In a broad sense, food may include beverages, and in a narrow sense, the concept may be intended to exclude beverages.

In this specification, the bacterial preparation of the present disclosure may be provided as a beverage. In this disclosure, "beverage" includes soft drinks, dairy drinks, alcoholic drinks, etc. As a combined concept of food and beverage, it may also be referred to as food and beverage, or food and beverage.

In this specification, the bacterial formulation of the present disclosure may be provided as a supplement. In this disclosure, the "supplement" may be in any form, including but not limited to tablets, granules, powders, sugar-coated tablets, capsules, syrups, suspensions, liquids, emulsions, etc. In addition, the bacterial formulation may be an enteric-coated formulation that is coated with a coating that has a differential solubility at different pH levels to protect the lactic acid-producing bacteria and butyric acid-producing bacteria from gastric acid and bile acid and allow them to act in the intestine.

In this specification, the bacterial preparation disclosed herein may be provided as feed. In this disclosure, "feed" includes feed for livestock or pets, and the form of the feed may be selected appropriately, such as solid, liquid, or paste.

In this specification, the bacterial preparation of the present disclosure may be provided as an additive for food and beverages. In this disclosure, "additive for food and beverages" can be used as an additive for food and beverages. Furthermore, "additive for feed" can be used as an additive for livestock or pet feed. Furthermore, these may be in any form, such as tablets, granules, powders, sugar-coated tablets, capsules, syrups, suspensions, liquids, emulsions, etc.

In this specification, the bacterial preparation of the present disclosure may be provided as a pharmaceutical. In this disclosure, "pharmaceuticals (including quasi-drugs under Japanese law and equivalents in countries other than Japan)" may be in any form, including, but not limited to, tablets, granules, powders, sugar-coated tablets, capsules, syrups, suspensions, liquids, emulsions, etc. Liquid preparations such as liquids and suspensions may be provided in a freeze-dried and storable state, and may be used after dissolving in a buffer solution containing water or saline solution at the time of use to prepare an appropriate concentration. In addition, those having a solid dosage form such as tablets may be coated as necessary (for example, sugar-coated tablets, gelatin-encapsulated tablets, enteric-coated tablets, etc.), or may be prepared as a controlled-release preparation such as a sustained-release preparation, delayed-release preparation, or immediate-release preparation using known technology. In addition, in order to protect the lactic acid-producing bacteria and butyric acid-producing bacteria from gastric acid and bile acid and to allow them to act in the intestine, they may be prepared as enteric-coated preparations coated with a coating that has different solubility at different pH levels.

Diseases to which bacterial preparations can be applied include, but are not limited to, immune-related diseases, ischemic diseases, lower limb ischemia, cerebrovascular ischemia, renal ischemia, pulmonary ischemia, neurological diseases, graft-versus-host disease (GVHD), inflammatory bowel disease, Crohn's disease, ulcerative colitis, radiation enteritis, systemic lupus erythematosus, lupus erythematosus, collagen disease, stroke, cerebral infarction, intracerebral hematoma, cerebrovascular palsy, brain tumor, liver cirrhosis, atopic dermatitis, multiple sclerosis, psoriasis, epidermolysis bullosa, diabetes, mycosis fungoides (Alibert-Bazin syndrome), scleroderma, and alterations of connective tissues such as cartilage. The compound can be used as a therapeutic agent for diseases selected from diseases caused by inflammation and/or inflammation, articular cartilage defects, meniscus damage, osteochondrosis elastosis, avascular necrosis, osteoarthritis of the knee, inflammatory arthritis, rheumatoid arthritis, eye diseases, angiogenesis-related diseases, ischemic heart disease, coronary heart disease, hereditary muscle diseases, hereditary blood diseases, hereditary neurological diseases, myocardial infarction, angina pectoris, heart failure, cardiomyopathy, valvular disease, wounds, epithelial damage, fibrosis, lung diseases, muscular dystrophy, spinal muscular atrophy, chronic pancreatitis, chronic nephritis, mental diseases (dementia, etc.), and cancer.

The bacterial preparation of the present disclosure is not particularly limited as long as it can maintain the bacteria in a form that can be used as a medicine and can be administered to animals such as humans. The medically and/or pharmaceutical acceptable drug may be mixed with the bacterial pharmaceutical preparation at the stage of manufacturing the pharmaceutical preparation, or at the stage of administering the bacterial pharmaceutical preparation to a patient or subject. The medically and/or pharmaceutical acceptable drug is not particularly limited, and examples thereof include physiological saline, electrolyte solutions, Ringer's solution, high-calorie infusions, glucose solutions, water for injection, amino acid electrolytes, etc. In addition, the medically and/or pharmaceutical acceptable drug may include salts, vitamins, amino acids, polysaccharides, dimethyl sulfoxide, buffers, albumin, culture media, cell cryoprotectants, etc. as necessary. In addition, it may include immunosuppressants, antibiotics, albumin preparations, vitamin preparations, anti-inflammatory agents, etc., which are components that have pharmaceutical activity. In one embodiment, for example, after administering an antibiotic, the subject's original intestinal bacteria are killed or reduced, and then the bacterial preparation of the present disclosure is administered, which is particularly advantageous when the subject has an abnormal intestinal flora. For example, patients with inflammatory bowel disease (IBD), cancer, etc. are said to often have a disturbed intestinal flora, and it has been reported that the intestinal flora is also disturbed in other diseases, so such a treatment method may be advantageous for any disease described herein or other diseases. Since the period until the intestinal bacteria are killed or reduced may vary depending on the condition of the subject and the antibiotic, the bacterial preparation of the present disclosure may be administered after observing the condition of the intestinal flora, or the bacterial preparation of the present disclosure may be administered after an appropriate period depending on the condition. This period may be shortened or extended as appropriate, and may be about 3 days, 1 week, 2 weeks, 3 weeks, or 1 month, but is not limited thereto.

The salts include, but are not limited to, salts of alkali metals such as lithium, sodium, and potassium; salts of alkaline earth metals such as calcium, barium, and magnesium; salts of aluminum, zinc, copper, and iron; ammonium salts; quaternary ammonium salts such as tetraethylammonium, tetrabutylammonium, methyltributylammonium, cetyltrimethylammonium, benzylmethylhexyldecylammonium, and choline; salts with organic amines such as pyridine, triethylamine, diisopropylamine, ethanolamine, diolamine, tromethamine, meglumine, procaine, and chloroprocaine; and salts with amino acids such as glycine, alanine, and valine.

Examples of the vitamins include, but are not limited to, folic acid, niacinamide, pyridoxine hydrochloride, biotin, calcium D-pantothenate, riboflavin, vitamin B12, thiamine, vitamin A, vitamin E, etc.

The amino acids include, but are not limited to, all essential amino acids (L-tryptophan, L-leucine, L-lysine, L-phenylalanine, L-isoleucine, L-threonine, L-histidine, L-methionine, and L-valine), all non-essential amino acids (L-alanine, L-arginine, L-asparagine, L-aspartic acid, glycine, L-glutamine, L-glutamic acid, L-cysteine, L-serine, L-tyrosine, and L-proline), and other natural amino acids such as L-cystine.

The polysaccharides include, but are not limited to, water-insoluble polysaccharides such as cellulose, chitin, and chitosan, and water-soluble polysaccharides such as hyaluronic acid, gellan gum, deacylated gellan gum, rhamsan gum, diutan gum, xanthan gum, carrageenan, xanthan gum, hexuronic acid, fucoidan, pectin, pectic acid, pectinic acid, heparan sulfate, heparin, heparitin sulfate, keratosulfate, chondroitin sulfate, dermatan sulfate, rhamnan sulfate, alginic acid, and salts thereof.

The medium is not particularly limited as long as it is composed of a component composition in which bacteria can survive and can be administered to animals such as humans, and examples of the medium include GAM medium, YCFA medium, modified YCFA medium, BBL medium, SOC medium, and LB medium. Among them, GAM medium is preferable, and for example, media such as 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. When orally administered to humans, BSA-free medium or a medium without animal-derived components such as meat extract can be used. Alternatively, microorganisms (bacteria) can be cultured using the above-mentioned media, and the bacteria can be collected and washed, or suspended in a buffer such as PBS or TE buffer before administration. For FMT, see Nomura, K.; Ishikawa, D; et al. Bacteroidetes Species Are Correlated with Disease Activity in Ulcerative Colitis. J. Clin. Med. 2021, 10, 1749. https://doi. org/10.3390/jcm10081749, Okahara, K. et al. ,A. Matching between Donors and Ulcerative Colitis Patients Is Important for Long-Term Maintenance after Fecal Microbiota Transplantation. J. Clin. Med. 2020,9,1650. https://doi.org/10.3390/jcm9061650 and the like can be referred to for administration to humans.

The bacterial cryoprotectant is not particularly limited, but examples thereof include dimethyl sulfoxide (DMSO), glycerol, polyethylene glycol, propylene glycol, glycerin, polyvinylpyrrolidone, sorbitol, dextran, and trehalose.

Antibiotics that may be used in the present disclosure include, for example, sulfa preparations, penicillin, phenethicillin, methicillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, nafcillin, ampicillin, amoxicillin, cyclacillin, carbenicillin, ticarcillin, piperacillin, azlocillin, mexocillin, mecillinam, anginocillin, cephalosporin and its derivatives, oxolinic acid, amifloxacin, temafloxacin, nalidixic acid, piromidic acid, ciprofloxacin, cinoxacin, norfloxacin, perfloxacin, rozaxacin, ofloxacin, enoxacin, pipemid Examples of suitable antibacterial agents include, but are not limited to, phenylacetamidophosphonate ...

Anti-inflammatory agents that may be used in the present disclosure include, but are not limited to, 5-aminosalicylic acid preparations, steroid preparations, immunosuppressants, biological preparations, etc. Examples of the 5-aminosalicylic acid preparations include, but are not limited to, salazosulfapyridine, mesalazine, etc. Examples of the steroid preparations include, but are not limited to, cortisone, prednisolone, methylprednisolone, etc.

The bacterial formulation of the present disclosure may also contain various additives to increase storage stability, isotonicity, absorbency and/or viscosity, such as emulsifiers, dispersants, buffers, preservatives, humectants, antioxidants, chelating agents, thickeners, gelling agents, pH adjusters, etc.

Thickening agents that may be used in the present disclosure include, but are not limited to, hydroxyethyl starch, dextran, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, and the like. The concentration of the thickening agent will depend on the thickening agent selected, but can be set at any concentration within the range that is safe when administered to a patient or subject and achieves the desired viscosity.

The viscosity of the bacterial suspension is not particularly limited, but if the viscosity is too high, rectal administration cannot be performed properly. For example, when measured using a rotational viscometer TV-20 (manufactured by Toki Sangyo) at a rotation speed of 10 rpm, the viscosity under the temperature conditions at the time of administration is preferably 35 mPa·sec or less, more preferably 30 mPa·sec or less, and even more preferably 25 mPa·sec or less, for example, 24 mPa·sec or less, 21 mPa·sec or less, 20 mPa·sec or less, 19 mPa·sec or less. c or less, 16 mPa·sec or less, 15 mPa·sec or less, 14 mPa·sec or less, 11 mPa·sec or less, 10 mPa·sec or less, 9 mPa·sec or less, 8 mPa·sec or less, 7 mPa·sec or less, 6 mPa·sec or less, 5 mPa·sec or less, 4 mPa·sec or less, 3 mPa·sec or less, 2.5 mPa·sec or less, or 2 mPa·sec or less.

Regarding the bacterial concentration (cells/mL) of the bacterial preparation, if the bacterial concentration is too high, the bacteria may clot, and the osmotic pressure of the bacterial preparation may become too high, which may cause a physical burden on the patient or subject. On the other hand, if the bacterial concentration is too low, it takes a long time to administer an amount of bacteria that can provide a therapeutic effect, which may lead to a deterioration in the quality of the bacterial preparation or a burden such as a longer time spent confined to the patient or subject. Therefore, taking these factors into consideration, the preferred bacterial concentration is appropriately set and is not particularly limited, but may be, for example, 1×10 ⁴ cells/mL or more, 2×10 ⁴ cells/mL or more, 3×10 ⁴ cells/mL or more, 4×10 ⁴ cells/mL or more, 5×10 ⁴ cells/mL or more, 6×10 ⁴ cells/mL or more, 7×10 ⁴ cells/mL or more, 8×10 ⁴ cells/mL or more, 9×10 ⁴ cells/mL or more, 1×10 ⁵ cells/mL or more, 2×10 ⁵ cells/mL or more, 3×10 ⁵ cells/mL or more, 4×10 ⁵ cells/mL or more, 5×10 ⁵ cells/mL or more, 6×10 ⁵ cells/mL or more, 7×10 ⁵ cells/mL or more, 8×10 ⁵ cells/mL or more, 9×10 ⁵ cells/mL or more, 1×10 ⁶ cells/mL or more, 1.5×10 ⁶ cells/mL or more, 2×10 ⁶ cells/mL or more, and 1×10 ⁹ cells/mL or less, 9×10 ⁸ cells/mL or less, 8×10 ⁸ cells/mL or less, 7×10 ⁸ cells/mL or less, 6×10 ⁸ cells/mL or less, 5×10 ⁸ cells/mL or less, 4×10 ⁸ cells/mL or less, 3×10 ⁸ cells/mL or less, 2×10 ⁸ cells/mL or less, 1×10 ⁸ cells/mL or less, 9×10 ⁷ cells/mL or less, 8×10 ⁷ cells/mL or less, 7×10 ⁷ cells/mL or less, 6×10 ⁷ cells/mL or less, 5×10 ⁷ cells/mL or less, 4×10 ⁷ cells/mL or less, 3×10 ⁷ cells/mL or less, 2×10 ⁷ cells/mL or less, 1×10 ⁷ cells/mL or less, 9×10 ⁶ cells/mL or less, 8×10 ⁶ cells/mL or less, 7×10 ⁶ cells/mL or less, 6×10 ⁶ cells/mL or less, 5×10 ⁶ cells/mL or less, 4×10 ⁶ cells/mL or less, 3×10 ⁶ In addition to the above, the concentration can be appropriately determined depending on the administration form, the purpose of use, and ^the age, weight, symptoms ^, etc. of the patient or subject.

The administration method in which the bacterial preparation of the present disclosure is used (e.g., administration rate, characteristics of the tubing used in combination, etc.) may be described on the label, package insert, or instruction manual attached to the cell preparation.

The bacterial preparation of the present disclosure is administered to a patient or subject by the administration method described below. The route of administration to the subject (patient or subject) of internal administration is not particularly limited, but can be, for example, rectal administration.

The container used to provide the bacterial preparation of the present disclosure is not particularly limited, so long as it is a container in which the cells in the bacterial preparation can survive. For example, the bacterial preparation of the present disclosure may be provided in a container separate from the container used in the administration method described below, or may be provided in a container that can be used in the administration method described below as is. From the standpoint of hygiene and ease of operation, it is preferable to use the container used to provide the bacterial preparation as is for the administration method described below. In this case, the provided bacterial preparation may be administered as is, or if frozen, it may be administered after thawing, or it may be administered after diluting with physiological saline, Ringer's solution, etc., as necessary.

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 (Achievement of original equilibrated state (jar culture mucin 0.8 g))
(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 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 medium was adjusted to pH 6.5 with 0.1 M phosphate buffer, and then 100 mL of the medium was added to a jar fermenter (manufactured by Able Co., Ltd., BJR-25NAIS-8M, hereinafter sometimes referred to as jar) with a capacity of approximately 200 mL, and sterilized by autoclaving 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 (N ₂ :CO ₂ = 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.

500 μL of the fecal suspension was inoculated into the medium-containing vessel (0.125 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 (N ₂ :CO ₂ = 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 about 300 rpm.

The culture fluid was collected 6, 24, 30, 48, 72, 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, without introducing air.

(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 then joined using QIIME 2 version 2022.2. The OTUs were then quality-controlled and corrected using the DADA2 pipeline, after which OTUs were inferred. The resulting OTUs were used to estimate alpha diversity and calculate the Shannon index. The resulting OTUs were then 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.

Shannon index and Pearson product-moment correlation coefficient were calculated using the QIIME software package.

Comparative Example 1 (no mucin added)
Cultivation and analysis of the bacterial flora were carried out in the same manner as in Example 1, except that in the preparation of the medium in Example 1, mucin (Sigma-Aldrich, derived from porcine stomach, Type III): 8.0 g/L was not added.

Table 1 shows the change in the Pearson product moment correlation coefficient with culture time for Example 1 and Comparative Example 1.

In Example 1 of the present invention, it can be seen that the Pearson product moment correlation coefficient remains at 0.8 or more from 24 to 96 hours of culture time. On the other hand, in Comparative Example 1, the Pearson product moment correlation coefficient is 0.8 or more at 24 and 30 hours of culture time, but falls below 0.8 after 48 hours of culture time. Comparing Example 1 and Comparative Example 1, it is clear that the mucin added to the medium is effective in maintaining the original equilibrium state.

Example 2 (Original equilibration state up to 2 hours)
Cultivation and bacterial flora analysis were performed in the same manner as in Example 1, except that the time for collecting the culture medium after the start of cultivation was changed to 6 hours, 12 hours, 15 hours, 18 hours, 21 hours, and 24 hours.

Comparative Example 2 (no mucin added)
Cultivation and bacterial flora analysis were performed in the same manner as in Comparative Example 1, except that the time for collecting the culture medium after the start of cultivation was changed to 6 hours, 12 hours, 15 hours, 18 hours, 21 hours, and 24 hours.

The results of Example 2 and Comparative Example 2 are shown in Table 2.

In Example 2, the Pearson product moment correlation coefficient was 0.80 or more after 15 hours of culture, whereas in Comparative Example 2, the Pearson product moment correlation coefficient was 0.80 or more after 18 hours of culture. In other words, it was clear that the mucin added to the medium had the effect of promoting the return to the original equilibrium state.

Example 3 (feces added amount 0.05 g/100 mL, mucin 0.4 g)
Cultivation and bacterial flora analysis were performed in the same manner as in Example 1, except that the amount of fecal suspension inoculated into the medium was changed to 0.05 g of feces/100 mL of culture solution, and the amount of mucin (Sigma-Aldrich, derived from porcine stomach, Type III) added in the preparation of the medium was changed to 4.0 g/L.

Comparative Example 3
Cultivation and analysis of the bacterial flora were performed in the same manner as in Example 1, except that in the preparation of the medium in Example 3, mucin (Sigma-Aldrich, derived from porcine stomach, Type III): 4.0 g/L was not added.

The results of the bacterial flora analysis for Example 3 and Comparative Example 3 are shown in Table 3.

Table 3 shows the relative values of the Pearson product moment correlation coefficient for Example 3 after 72 hours of culture, with the value for Comparative Example 3 taken as 100. Even under different conditions of the inoculation amount of the fecal suspension and the amount of mucin added, a high Pearson product moment correlation coefficient was obtained with the present invention.

Reference Example 1 (Test substance inulin was added 24 hours after the start of culture)
Cultivation and evaluation were carried out in the same manner as in Example 1, except that inulin (BENEO, OraftiGR, derived from chicory; indicated as INU) was added as a test substance to the culture solution at 0.3 w/v % 24 hours after the start of culture. The culture solution was sampled 48 hours, 72 hours, and 96 hours after the start of culture, and the abundance rate of bacteria belonging to the genus Bifidobacterium in each culture solution was determined.

Reference Example 2 (Inulin added at the start of culture)
Cultivation and evaluation were carried out in the same manner as in Example 1, except that inulin (BENEO, OraftiGR, derived from chicory; indicated as INU) was added at 0.3 w/v% to the culture solution as a test substance at the start of cultivation (0 hours of cultivation). The culture solution was sampled 48 hours, 72 hours, and 96 hours after the start of cultivation, and the abundance rate of bacteria of the genus Bifidobacterium in each culture solution was determined.

(Experimental Results)
The presence rate of Bifidobacterium bacteria in the culture medium was compared for the culture medium of Example 1 (without inulin added), the culture medium of Reference Example 1 to which inulin was added 24 hours after the start of culture, and the culture medium of Reference Example 2 to which inulin was added at the start of culture. The results are shown in Table 4.

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 4, it can be seen that in Reference Example 2, 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 Reference Example 1, 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 Reference Example 2, in which inulin was not added. The result that the presence rate of Bifidobacterium increases with the addition of 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 is reached.

Reference Example 3 (Multi-well plate culture without addition of test substance)
(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 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 culture, and bacterial flora analysis was performed. The culture medium was collected without opening the anaerobic chamber. Based on the results of the Pearson product moment correlation coefficient, the original equilibrium state was determined to be between 24 and 96 hours after the start of culture.

Reference Example 4 (Test substance water-soluble indigestible dextrin was added 24 hours after the start of culture (multi-well plate))
Cultivation and evaluation were carried out in the same manner as in Reference 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 % 24 hours after the start of cultivation.

Reference Example 5 (Water-soluble indigestible dextrin added at the start of culture (multi-well plate))
Cultivation and evaluation were carried out in the same manner as in Reference Example 3, except that water-soluble, indigestible dextrin (Matsutani Scientific, Fibersol 2 (maltodextrin); referred to as DEX) was added as the test substance at 0.3 w/v% to the culture medium at the start of cultivation.

The results of Reference Examples 3, 4, and 5 are shown in Table 5.

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 5, it can be seen that in Reference Example 5, in which dextrin was added at the start of culture, the presence rate of Faecalibacterium was lower at all culture times compared to Reference Example 3, in which dextrin was not added. On the other hand, in Reference Example 4, 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 Reference Example 3, 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, p824-831). 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 (Multi-well plate culture)
In this example, we will show an example of multi-well plate culture, and in particular, we investigated the medium.

(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 5 (Study of GAM medium containing mucin)
Cultivation and bacterial flora analysis were performed in the same manner as in Example 4, 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 6 (Study of modified GAM medium)
Cultivation and bacterial flora analysis were performed in the same manner as in Example 4, 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 7 (Study of modified GAM medium containing mucin)
In preparing the modified GAM medium, the culture and bacterial flora analysis were carried out in the same manner as in Example 6, 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.

Example 8 (Study on YCFA medium)
Cultivation and bacterial flora analysis were performed in the same manner as in Example 4, except that YCFA medium was used instead of GAM medium in the preparation of the medium. 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 3.1 ml of volatile fatty acids (acetic acid, ... The medium was prepared by mixing 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 9 (Study of YCFA medium containing mucin)
Cultivation and bacterial flora analysis were performed in the same manner as in Example 8, 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 10 (modified YCFA medium)
Cultivation and bacterial flora analysis were performed in the same manner as in Example 4, 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, 1.0 g/L L-cysteine HCl, 4.0 g/L sodium bicarbonate, and 0.01 g/L hemin, and contained 2.7 ml of volatile fatty acids (2.026 ml/L acetic acid, 0.026 ml/L propionic acid) per liter. 715 ml/L, n-valeric acid 0.119 ml/L, isovaleric acid 0.119 ml/L, isovaleric acid 0.119 ml/L), vitamin mixture: 10 ml (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, lipoic acid 5 mg/L) and antifoaming agent 50 μL/L were mixed and the pH was adjusted to 6.8 with a pH adjuster to prepare a medium.

Example 11 (Study of modified YCFA medium containing mucin)
Cultivation and bacterial flora analysis were performed in the same manner as in Example 9, except that in the preparation of 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 4, 5, 6, and 7 in various media set at 100, and the relative values of the Pearson product moment correlation coefficients for Examples 8, 9, 10, and 11 in mucin-added media. The results in Table 6 clearly show that the addition of mucin is effective in maintaining the original equilibrium state in all media.

Example 12 (Amount of useful bacteria: Effect of adding mucin)
In Examples 4 and 5, genomic DNA of bacteria in the bacterial flora was extracted from the culture solution collected before the start of the culture and 72 hours after the start of the culture. The target bacterial genes were quantified using a quantitative PCR device using specific primers for Faecalibacterium duncaniae (Fd bacteria) and Blautia wexlerae (Bw bacteria) that targeted the 16S rRNA genes of each bacteria from the extracted genomic DNA (Table 7).
The results in Table 7 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 bacteria during culture, and their abundance was equal to or greater than that in the original sample (Fec), clearly demonstrating its effectiveness in maintaining these useful bacteria.

(Study of mucin)
Example 13 (Study of medium containing porcine Type II mucin)
Cultivation and bacterial flora analysis were carried out in the same manner as in Example 4, 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 14 (Study of medium containing porcine mucin)
Cultivation and bacterial flora analysis were performed in the same manner as in Example 4, except that in the preparation of 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.

Example 15 (Study on medium containing ganglia imucin)
Cultivation and bacterial flora analysis were carried out in the same manner as in Example 4, 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 4 (Study of mucin-free medium)
In preparing the medium, the culture and bacterial flora analysis were performed in the same manner as in Example 4.

Table 8 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 4 set at 100. The results in Table 8 make it clear that the addition of all mucins of different origins was effective in maintaining the original equilibrium state.

Example 16 (Administration of culture medium preparation to mice)
The human intestinal flora culture solution prepared in Example 5 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 drug efficacy evaluation test are performed by the method described in Nature Communications (202) 13:4477. Specifically, SPF mice (6 weeks old) are raised on a high-fat diet (AIN-93G, manufactured by Oriental Yeast Co., Ltd.) for 10 weeks, and the human intestinal flora culture solution prepared in Example 5 is orally administered at 5 x ¹⁰⁹ CFU three times a week. The weight of each mouse is measured, and serum is collected 8 weeks after administration of the human intestinal flora culture solution, and HOMA-IR and insulin concentration are measured, and an IPGTT test is 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. 2023-196982 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 present disclosure provides a composition that can be used 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 (19)

A composition for promoting and/or maintaining pristine equilibrium when culturing intestinal bacterial flora in a medium, comprising a high molecular weight glycoprotein. The composition of claim 1, wherein the high molecular weight protein comprises mucin. The composition according to claim 2, wherein the mucin is contained in the medium at 0.4 w/v% or more. The composition according to any one of claims 1 to 3, wherein the medium is GAM medium, YCFA medium or a modified form thereof. The composition according to any one of claims 1 to 4, wherein the equilibration is performed to evaluate a test substance in the intestinal flora. The composition according to any one of claims 1 to 5, wherein the composition is for maintaining the original equilibrium. The composition according to any one of claims 1 to 6, wherein the composition is intended to prevent the medium from losing its original equilibrium after the medium has achieved the original equilibrium. An equilibrated medium for intestinal bacterial flora containing high molecular weight glycoproteins and medium components. The original equilibrated medium according to claim 8, wherein the medium is for evaluating a test substance in intestinal flora. The medium according to claim 8 or 9, wherein the high molecular weight protein includes mucin. The medium according to any one of claims 8 to 10, wherein the mucin is contained in an amount of 0.4 w/v% or more of the medium components. The medium according to any one of claims 8 to 11, wherein the medium components are GAM medium or a modified form thereof. A composition for maintaining beneficial bacteria in the intestinal flora, comprising a high molecular weight glycoprotein. The composition according to claim 13, wherein the beneficial bacteria include at least one selected from the group consisting of Faecalibacterium duncaniae (Fd bacteria) and Blautia wexlerae (Bw bacteria). The composition according to claim 13 or 14, wherein the high molecular weight glycoprotein is a mucin. The composition according to any one of claims 13 to 15, which is used for producing a bacterial preparation. A method for producing a bacterial preparation enriched in useful bacteria, comprising the steps of:
A) culturing the intestinal flora in a medium containing a high molecular weight glycoprotein;
B) Collecting the grown gut microbiota;
C) A method of production comprising, optionally after washing the intestinal flora, adding the intestinal flora to a medium for administration to obtain a bacterial preparation. The production method according to claim 17, wherein the high molecular weight glycoprotein is mucin. A bacterial preparation produced by the production method described in claim 17.