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

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 microbiota to promote and / or maintain pristine equilibrium containing high molecular weight glycoproteins

[0001] The present disclosure relates to a composition for obtaining an intestinal microbiota that is rich in microbiota diversity and maintains that microbiota 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 microbiota in the intestines of mammals.

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

[0003] Patent No. 7051175

[0004] As a result of extensive research, the present inventors have found that certain components contribute to maintaining the diversity of the microbial flora and the intestinal bacterial flora. Based on this finding, the present disclosure is as follows.

[0005] [1] A composition for promoting and / or maintaining original state equilibration when culturing intestinal bacterial flora in a medium, comprising a high molecular weight glycoprotein. [2] The composition described in any one of the above items, wherein the high molecular weight protein comprises mucin. [3] The composition described in any one of the above items, wherein the mucin is contained in the medium at a concentration of 0.4 w / v% or more. [4] The composition described in any one of the above items, wherein the medium is GAM medium, YCFA medium, or a modified version thereof. [4A] The composition described in any one of the above items, wherein the medium is GAM medium or a modified version thereof. [5] The composition described in any one of the above items, wherein the original state equilibration is performed to evaluate a test substance in the intestinal bacterial flora. [6] The composition described in any one of the above items, wherein the composition is for maintaining the original state equilibration. [7] The composition described in any one of the above items, wherein the composition is for preventing the original state equilibration from being lost after the medium has achieved original state equilibration. [8] A medium for original state equilibration of intestinal bacterial flora, comprising a high molecular weight glycoprotein and medium components. [9] The original equilibrated medium according to any one of the above items, wherein the medium is for evaluating a test substance in intestinal bacterial flora.

[10] The medium according to any one of the above items, wherein the high molecular weight protein comprises mucin.

[11] The medium according to any one of the above 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 above items, wherein the medium components are GAM medium or a modified form thereof.

[13] A composition for maintaining useful bacteria in intestinal bacterial flora, comprising a high molecular weight glycoprotein.

[14] The composition according to any one of the above 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 above items, wherein the high molecular weight glycoprotein is mucin.

[16] The composition according to any one of the above items, wherein the composition is used for producing a bacterial preparation.

[17] A method for producing a bacterial preparation enriched with beneficial bacteria, comprising: A) culturing intestinal bacterial flora in a medium containing a high molecular weight glycoprotein; B) collecting the grown intestinal bacterial flora; and C) optionally washing the intestinal bacterial flora, and then adding the intestinal bacterial flora to a vehicle for administration to obtain a bacterial preparation.

[18] The production method according to any one of the above 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.

[0006] According to the present disclosure, it is possible to more accurately reproduce changes in the intestinal microbiota caused by the administration of a test substance to a human. The composition of the intestinal microbiota can change during culture, which may be one of the reasons why in vivo results of a test substance cannot be reproduced in vitro. However, this effect has been successfully suppressed.

[0007] The present disclosure makes it possible to reproduce in vitro the in vivo effects of test substances such as foods and drug candidate compounds on the intestinal environment, and to provide an intestinal microbiota sample that is rich in microbiota diversity and maintains that microbiota diversity for a certain period of time.

[0008] The present disclosure makes it possible to provide an intestinal microbiota sample 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.

[0009] The present disclosure will now be described with reference to the best mode. Throughout this specification, singular expressions should be understood to include the plural concept unless otherwise specified. Therefore, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept unless otherwise specified. Furthermore, it should be understood that terms used in this specification are used in the sense commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In the event of conflict, the present specification (including definitions) will prevail.

[0010] (Definitions) The definitions of terms particularly used in this specification and / or basic technical content will be explained as appropriate below.

[0011] (Definition of Terms) All numerical values ​​herein, whether explicitly stated or not, are assumed to be modified by the term "about." The term "about" generally refers to a range of numerical values ​​that one of ordinary skill in the art would consider equivalent to the recited value (i.e., having the same function or result). In many instances, the term "about" may include numbers that are rounded to the nearest significant figure.

[0012] As used herein, the term "intestinal microbiota" refers to a group of bacteria normally present in the intestines of animals with an intestinal tract (e.g., mammals such as humans). Examples of bacteria that constitute the intestinal microbiota of healthy humans include, at the phylum level, bacteria belonging to the following phyla: Verrucomicobiota, Pseudomonadota, Fusobacteriota, Bacillota, Bacteroidota, and Actinomycetota. Further, at the genus level, bacteria belonging to the following genera or families may be mentioned: Bifidobacterium, Collinsella, Bacteroides, Parabacteroides, Prevotella, Rikenellaceae, Lactobacillales, etc.

[0013] As used herein, the term "useful bacteria" refers to any bacteria that are useful to a host (e.g., humans) in the intestinal flora, etc. Examples include, but are not limited to, bacteria of the genera Bifidobacterium, Lactobacillus, Faecalis, Blautia, Akkermansia, 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 of which is Bifidibacterium longum), and Lactobacillus genus (lactic acid bacillus: an example of which is Lactobacillus casei).

[0014] As used herein, "animals having an intestinal tract" refers to any animal having an intestinal tract, such as mammals (mammals), birds, reptiles, amphibians, and fish, with mammals being preferred. "Mammals" include humans; pet animals such as dogs and cats; research animals such as mice and rats; and livestock such as pigs. In the present disclosure, "mammals" are preferably humans.

[0015] As used herein, the term "test substance" is not particularly limited as long as it is a material that has the potential to affect the intestinal environment of an animal, and may be food, physiologically active substances derived from food, food additives, beverages, microorganisms (bacteria, fungi, etc., including killed cells and extracts derived from cells), physiologically active substances, pharmaceuticals, pharmaceutical-like compounds, and mixtures thereof.

[0016] The present disclosure relates to a technique for restoring the intestinal bacterial flora in a culture medium to a state of original equilibrium.

[0017] As used herein, the term "original state" refers to the state of the intestinal bacterial flora of a sample such as feces (sometimes referred to as the intestinal bacterial flora before the start of culture).

[0018] As used herein, "remaining in equilibrium" refers to a state in which the original state is in equilibrium, i.e., within a certain range of fluctuation, and such a state is referred to as a "state of original equilibrium." Whether or not original equilibrium has been achieved can be evaluated using the Pearson product-moment correlation coefficient, which is a coefficient used to evaluate the degree of similarity between the flora of two bacterial flora. For example, when the Pearson product-moment correlation coefficient between the intestinal flora before the start of culture and the intestinal flora after the start of culture is 0.50 or higher, or 0.60 or higher, usually 0.70 or higher, preferably 0.80 or higher, or 0.85 or higher, 0.90 or higher, or 0.95 or higher, this can be considered to be original equilibrium. Furthermore, when original equilibrium continues for a certain period of time, this is referred to as an original equilibrium state. The certain period of time is 6 hours or higher, 12 hours or higher, or 18 hours or higher, preferably 24 hours or higher, more preferably 36 hours or higher, even more preferably 48 hours or higher, and even more preferably 72 hours or higher. In this specification, the "stabilization determination index" is calculated as an index until the original state equilibrium is reached.

[0019] In this specification, substances that promote and / or maintain the state equilibrium are also referred to as "state equilibrium promoting substances" or "state equilibrium maintaining substances," respectively. When a substance has both functions, it may also be called a state equilibrium promoting / maintaining substance.

[0020] As used herein, the term "medium" refers to any medium in which a bacterial colony can grow.

[0021] As used herein, the term "medium components" refers to each component that constitutes a medium.

[0022] As used herein, the term "bacterial preparation" refers to a preparation composed of bacteria, and more specifically, to a powder or liquid preparation containing a useful group of microorganisms that is expected to exert its medicinal effects alone or in combination with other substances. Examples of bacterial preparations include, but are not limited to, oral capsules, tablets (for oral use), enema preparations, gargle solutions, and skin smears (ointments). Furthermore, bacterial preparations may contain one or more types of microorganisms. Bacterial preparations are also a type of biological preparation, and representative examples include live biotherapeutic products (LBPs) composed of intestinal bacteria and microbiome medicines. Other representative examples include live bacterial preparations (including single-bacterial and cocktail-based preparations), which may also be used in fecal microbiota transplantation (FMT). For bacterial preparations, see, for example, FEMS Microbiology Reviews, 2023, 47, 1-18).

[0023] As used herein, "drug efficacy" is interpreted in the broadest sense to refer to any biological effect on a subject. A drug efficacy can be recognized by a change in some biological phenomenon or a phenomenon resulting from such a change in the subject. Typical examples include, but are not limited to, improvement of a disease state and maintenance or improvement of health through the use of biological components such as probiotics. The desired drug efficacy of a microorganism obtained by the method of the present disclosure can be confirmed by various techniques, including the use of a drug gene resistance system, enzyme activity based on the drug efficacy, survival activity based on the drug efficacy, chemotactic activity of the microorganism, colonization of the microorganism in the subject, cell adhesion activity of the microorganism, adhesion activity of the microorganism to mucin layers, and colonization activity of the microorganism in animal tissue. The generation of a population containing a microorganism with the desired drug efficacy is not particularly limited, as long as the conditions allow the microorganism to grow and / or amplify. When producing a bacterial formulation, for example, by comparing the growth curve under aerobic and / or anaerobic conditions with that of a wild-type strain (parent strain), clones that are difficult to mass-cultivate, which pose a challenge in formulation, can be avoided.

[0024] For the understanding of the various terms used in this specification, including those mentioned above, where appropriate, all references cited in this specification, including scientific literature, patents, patent applications, and the like, are hereby incorporated by reference in their entirety to the same extent as if each were specifically set forth.

[0025] (Preferred Embodiments) Preferred embodiments of the present disclosure will be described below. The embodiments provided below are provided for a better understanding of the present disclosure, and it is understood that the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that those skilled in the art can make appropriate modifications within the scope of the present disclosure in light of the description herein. It is also understood that the following embodiments of the present disclosure can be used alone or in combination.

[0026] (Composition for Promoting and / or Maintaining Original State Equilibrium) In one aspect, the present disclosure provides a composition, active ingredient, compound, and other technology therefor for promoting and / or maintaining original state equilibrium when culturing intestinal bacterial flora in a medium containing a high molecular weight glycoprotein.

[0027] In one embodiment, the macromolecular protein of the present disclosure comprises a mucin.

[0028] In one embodiment, the mucin used in the present disclosure is contained in a culture 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, or 1.0 w / v% or less. Without wishing to be bound by theory, a concentration greater than 2.0 w / v% is not advantageous because the pH during culture may remain low.

[0029] 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-solid high-layer medium, GAM bouillon, and modified GAM bouillon (all manufactured by Nippon Pharmaceutical Co., Ltd.) can be used. Two or more media selected from these media may be mixed in any ratio and used. GAM medium or a modified medium thereof is preferred.

[0030] In one embodiment, the original state equilibration in the present disclosure is performed to evaluate a test substance in the intestinal microbiota. Here, the original state equilibration is used for, but is not limited to, a method for evaluating a test substance in the intestinal microbiota, the method comprising the following steps: A) culturing the intestinal microbiota in a medium for a time period effective for the intestinal microbiota to reach an original state equilibration after the start of culture by inoculating the intestinal microbiota into the medium, B) adding the test substance to the medium containing the intestinal microbiota after the elapse of the time period, and C) obtaining and evaluating evaluation items before and after adding the test substance.

[0031] In one embodiment, the compositions of the present disclosure are for maintaining the original equilibration. A promoter for the original equilibration may be important because, when evaluating a candidate substance, it is preferable to maintain the original equilibration state for a long period of time if long-term evaluation is required.

[0032] In one embodiment, the composition of the present disclosure is intended to prevent the medium used from losing its original equilibrium state after achieving it (also referred to as a deviation or decline from the original equilibrium state). Such a substance is advantageous in cases where evaluation of a candidate substance requires long-term evaluation and requires maintaining the original equilibrium state for a long period of time, since it can prevent the original equilibrium state from being lost after it has been achieved.

[0033] (Culture medium for original state equilibration) In one embodiment, the culture medium of the present disclosure may be a culture medium for original state equilibration of intestinal bacterial flora, comprising a high molecular weight glycoprotein and a culture medium component.

[0034] In this embodiment, the medium used in the present disclosure is for evaluating test substances in the gut microbiota.

[0035] In a preferred embodiment, the macromolecular protein comprises a mucin.

[0036] In a preferred embodiment, mucin is contained at a concentration (w / v%) relative to the medium components 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, or 1.0 w / v% or less.

[0037] 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, LB medium, etc. Among these, GAM medium is preferred, and for example, GAM agar medium, modified GAM agar medium, GAM semi-solid high-layer medium, GAM bouillon, and modified GAM bouillon (all manufactured by Nippon Pharmaceutical Co., Ltd.) can be used. Two or more types of medium selected from these media may be mixed in any ratio and used.

[0038] In one embodiment, the medium components used in the present disclosure are components constituting GAM medium, which may be present in an amount of 50 to 200%, for example, 75 to 150%, 80 to 120%, etc.

[0039] In one embodiment, the medium of the present disclosure may contain peptone / hemin and vitamin K. While not wishing to be bound by theory, the presence of these components may enhance the growth of many anaerobic bacteria. Furthermore, since some bacteria require these components, it is believed that the inclusion of these components is advantageous. Therefore, the present disclosure provides a composition for promoting and maintaining equilibrium when culturing intestinal bacterial flora in a medium, comprising peptone / hemin / vitamin K and a medium containing peptone / hemin / vitamin K or a modified version thereof. Modified versions of GAM medium can be easily prepared by those skilled in the art. Examples of such modified versions include those described at https: / / axel.as-1.co.jp / asone / d / 65-9404-61 / , for example, Acudia ＴＭ Examples include, but are not limited to, modified GAM broth.

[0040] The present disclosure provides a composition for promoting and maintaining equilibrium when culturing intestinal bacterial flora in a medium, comprising GAM medium or a variant thereof.

[0041] In one embodiment, the GAM medium may be advantageously semi-solid. A semi-solid medium 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 intestinal bacterial flora in a medium.

[0042] 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 to 5.0% and sodium lactate may be contained at 1.0 to 50 mM.

[0043] In another embodiment, in the present disclosure, the amount of the sample added to the culture medium is 0.05% or more, alternatively 0.1% or more, and 1.5 w / v% or less, alternatively 1.25 w / v% or less.

[0044] In one embodiment, any mucin may be used, although secretory mucins derived from the gastrointestinal tract (MUC2, 5AC, 5B, 6, 7, etc.) may be advantageous. The fermentation substrate for gastrointestinal and oral bacteria may be classified according to known classifications in the art, but is not limited thereto. When culturing intestinal bacterial flora, any mucin may be used as long as it also contains a composition for promoting and maintaining equilibrium by referring to this specification. Without wishing to be bound by theory, mucins include secretory mucins produced by epithelial cells and membrane-bound mucins that have hydrophobic transmembrane domains and exist bound to the cell membrane. Mucin core proteins are collectively referred to as MUCs and are numbered in the order of their discovery. The genes encoding these core proteins are known to be of at least 23 types in 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.

[0045] (Substances for promoting and / or maintaining original equilibrium) In this aspect, the present disclosure provides a composition for promoting and maintaining original equilibrium when culturing intestinal bacterial flora in a medium, the composition comprising a high molecular weight glycoprotein.

[0046] In one aspect, the present disclosure provides a substance for promoting or maintaining equilibrium in a natural state. Typically, the present disclosure uses a high molecular weight glycoprotein as the substance for promoting equilibrium in a natural state.

[0047] When a high molecular weight glycoprotein is used as a substance for promoting state equilibration in the present disclosure, the effect of promoting and / or maintaining state equilibration can be achieved by adding the high molecular weight glycoprotein to the medium used. In this case, the high molecular weight glycoprotein can function as a substance for promoting state equilibration and can also have the function of maintaining state equilibration. Exemplary high molecular weight glycoproteins of the present disclosure include polypeptides comprising an amino acid sequence having a tandem repeat structure to which an O-linked glycan has been attached.

[0048] A tandem repeat structure refers to a structure in which an amino acid sequence of one to several dozen amino acids in length is regularly repeated. Examples of O-linked sugar chains include, but are not limited to, O-mannose, O-N-acetylglucosamine, O-fucose, O-glucose, and O-galactose. In the high molecular weight glycoprotein of the present disclosure, GalNAc (N-acetylgalactomisan) of the sugar chain is bound to a hydroxyl group of serine or threonine in the polypeptide via an O-glycosidic bond. The molecular weight of the high molecular weight glycoprotein of the present disclosure is from about 500,000 to about 20 million, more preferably from about 1 million to about 10 million.

[0049] Examples of high molecular weight glycoproteins include secretory mucins and membrane-bound mucins. Examples of secretory mucins include MUC2, MUC5AC, MUC5B, MUC6, and MUC7, while examples of membrane-bound mucins include MUC1, MUC3, MUC4, MUC12, MUC13, MUC16, MUC17, MUC20, and MUC21. Other mucins that can be used include MUC8, 9, 10, 11, 14, 15, 18, and 19. Of these, secretory mucins such as MUC2, MUC5AC, MUC5B, MUC6, and MUC7 are preferably used.

[0050] The mucin may be of human or non-human origin, for example, porcine.

[0051] 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, with the aim of promoting and maintaining the original equilibrium. From the viewpoint of maintaining the bacterial flora structure, the amount is preferably 2.0 w / v% or 1.9 w / v% or less, and more preferably 0.8 w / v% or less.

[0052] In this embodiment, the medium used in the present disclosure is not particularly limited as long as it allows the growth of enterobacteria and does not inhibit the return to normal equilibrium, and examples thereof include GAM medium, YCFA medium, modified YCFA medium, BBL medium, SOC medium, and LB medium. Among these, GAM medium is preferred, and for example, GAM agar medium, modified GAM agar medium, GAM semi-solid high-layer medium, GAM bouillon, and modified GAM bouillon (all manufactured by Nippon Pharmaceutical Co., Ltd.) can be used. Two or more media selected from these media may be mixed in any ratio.

[0053] In another embodiment, the present disclosure uses GAM medium as the original equilibrating material.

[0054] In one embodiment, the GAM medium may be, for example, GAM agar medium, modified GAM agar medium, GAM semi-solid high-layer medium, GAM bouillon, and modified GAM bouillon (all manufactured by Nippon Pharmaceutical Co., Ltd.), but is not limited thereto as long as it has the effect of promoting and / or maintaining the original equilibrium.

[0055] In a preferred embodiment, the present disclosure can utilize a composition comprising at least a high molecular weight glycoprotein and a GAM medium, each in combination with any of the specific aspects described elsewhere herein.

[0056] Exemplary high molecular weight glycoproteins are as described in (Substances Promoting State Equilibration), but examples that can be used include mucin derived from pig stomach and mucin derived from skate, but are not limited to these.

[0057] 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 colony structure, they are contained at 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.

[0058] 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 scaffold necessary for bacterial growth. Additionally, pH adjusters, surfactants, thickeners, dispersants, preservatives, etc. may also be added.

[0059] By using the present composition for culturing a bacterial flora or by adding the present composition to a culture medium, the original equilibrium is promoted and maintained.

[0060] (Additional Components of Medium) If necessary, a carbon source such as glucose, a nitrogen source such as ammonia, a source of nutrients such as vitamins and inorganic salts, and a scaffold necessary for cell growth may be added to the medium.

[0061] In one embodiment, the medium may be sterilized, for example, by autoclaving, before culturing. Liquid culture is preferred, and the liquid culture is also referred to as a "culture medium." During culturing, the culture medium may be stirred as appropriate.

[0062] (Intestinal Microbiota Used in the Present Disclosure and Sample Preparation) The intestinal microbiota used in the present disclosure may be obtained from a stool sample or from a sample other than stool. One or more specific intestinal bacteria may also be used. The sample may be from a human or a non-human animal, with human samples being preferred, and human stool being even more preferred. Furthermore, the stool sample may be collected immediately after excretion from the intestine, frozen after collection, or collected from the intestine. After collection, the stool sample may be stored in a container such as an anaerobic culture swab until the start of culture. The collected stool may be mixed with phosphate buffer solution (PBS) to form a suspension. The phosphate buffer solution used to suspend the stool may also contain ascorbic acid or glycerin. The concentration of the stool in the stool suspension may be 0.01 w / v% to 50 w / v%, or 0.05 to 2.5 g / m, or 0.1 to 1.0 g / ml, for example, 0.25 g / ml.

[0063] 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, and preferably 0.05 to 1.5 w / v%, based on the culture medium from the viewpoint of bacterial flora diversity, and is more preferably 1.5 w / v% or less, 1.25 w / v% or less, from the viewpoint of inhibiting the growth of various bacteria. As used herein, "inoculation" refers to taking a fixed amount from a stool suspension and adding it to a culture medium.

[0064] (Culture Vessels and Apparatus Used in the Present Disclosure) The culture vessels used in the present disclosure may be flasks, commercially available culture vessels, multi-well plates, or the like. Multi-well plates are preferably used to increase the throughput of evaluation. The shape of each well of the multi-well plate may be approximately hemispherical, approximately rectangular, or approximately cylindrical, and the bottom may be flat or round. When using a multi-well plate, the volume per well is preferably 5 mL or less, more preferably 3 mL or less, and even more preferably 2 mL or less. Furthermore, the volume per well is preferably 0.1 mL or more, more preferably 0.2 mL or more, and even more preferably 0.3 mL or more.

[0065] Furthermore, commercially available culture devices such as jar fermenters and shaking mixers can be used.

[0066] (Culture Conditions - Atmosphere) In the present disclosure, the culture is carried out in an anaerobic environment. The anaerobic culture environment can be created by aerating an anaerobic gas into the culture medium. The anaerobic gas is, for example, nitrogen, nitrogen and carbon dioxide, or nitrogen, carbon dioxide, and hydrogen. The anaerobic gas is aerated continuously or intermittently at a predetermined flow rate (e.g., 0.1 to 1.0 dL / min), 0.1 to 1.0 dL / min, for example, 0.15 dL / min. Furthermore, 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. Note that, in order to maintain highly anaerobic conditions, it is preferable to aerate the anaerobic gas continuously.

[0067] (Culture Conditions—pH) In the present disclosure, the pH of the culture medium at the start of culture is typically 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 culture (for example, when the culture medium containing a fecal sample is placed in an anaerobic environment), the pH can be adapted to the pH in the large intestine of a mammal corresponding to the feces used. It is sufficient that the pH of the culture medium at the start of culture is within the above range, and thereafter, the pH may be left as is without any particular adjustment, or the pH may be adjusted to within the above range using a pH adjuster as needed to prevent an extreme drop in pH.

[0068] (Culture conditions - temperature, agitation) The culture temperature is preferably set to a temperature close to the body temperature of the mammal corresponding to the feces used, in order to mimic the environment in the large intestine of the mammal. For example, when human feces is used, the culture temperature is 36°C to 38°C, preferably 36°C to 37°C, since this is a temperature close to that of a healthy human. The culture method is not particularly limited, but a single batch method is preferred. Furthermore, when the culture vessel is a flask or the like, it is preferable to agitate the culture solution during culture using an agitator or stirrer, or when using a multi-well plate, it is preferable to agitate the culture solution using a shaker.

[0069] (Preparation of Test Substance and Amount Added) The intestinal microbiota sample according to the present disclosure can be used to test the effect of a test substance on the intestinal microbiota. 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 solid, it may be dissolved in a solvent such as water and added.

[0070] (Timing of Addition of Test Substance) The test substance may be added at any time while the intestinal bacterial flora is in a state of original equilibrium, but is preferably added at an early stage of the original equilibrium state, from the viewpoint of enabling a longer-term evaluation of the effect of the test substance on the intestinal bacterial flora after addition and of evaluation efficiency. That is, the test substance may be added at least 6 hours, at least 12 hours, at least 15 hours, at least 18 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 42 hours, at least 48 hours, and / or at most 96 hours, at most 90 hours, at most 84 hours, at most 78 hours, at most 72 hours, at most 66 hours, at most 60 hours, at most 54 hours, at most 48 hours, e.g., 6-90 hours, 12-48 hours, or 48-84 hours, or in another embodiment, between 24 and 96 hours, most preferably between 24 and 72 hours, after the start of culture.

[0071] (Cultivation and Collection After Addition of Test Substance) In one embodiment, it may be advantageous to select the timing of sample acquisition for evaluation. After adding a test substance to a culture medium and further culturing, the culture medium is collected for evaluation. The culture medium may be collected at any time after adding the test substance to the culture medium, but is preferably collected while the original equilibrium state is maintained. Specifically, the culture medium is collected between 6 and 120 hours after the start of culturing the intestinal bacterial flora, and this time may be at least 6 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 36 hours, at least 48 hours, and / or at most 120 hours, at most 114 hours, at most 108 hours, at most 102 hours, at most 96 hours, at most 90 hours, at most 84 hours, at most 78 hours, at most 72 hours, at most 66 hours, at most 60 hours, at most 54 hours, or at most 48 hours. The timing at which the culture medium is collected for evaluation is 6 hours or more, 12 hours or more, 18 hours or more, 24 hours or more, 36 hours or more, 48 hours or more, and / or 126 hours or less, 120 hours or less, 114 hours or less, 108 hours or less, 102 hours or less, 96 hours or less, 90 hours or less, 84 hours or less, 78 hours or less, 72 hours or less, 66 hours or less, 60 hours or less, 54 hours or less, or 48 hours or less after the test substance is added to the culture medium.

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

[0073] (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 bacterial flora is cultured without adding the test substance, the culture medium is periodically collected, and time-course data such as the Shannon index and Pearson product-moment correlation coefficient of the intestinal bacterial flora is obtained. From the obtained time-course data, the above-mentioned original equilibrium state can be determined, and the timing of adding the test substance can be determined.

[0074] (Bacterial Preparation) 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 genera such as Bifidobacterium (genus Bifidibacterium), Lactobacillus (genus Lactobacillus), Faecalis (genus Faecalis), Blautia, Akkermansia, Roseburia, Ruminococcus, Bacteroides, Enterococcus, and Clostridium, and in particular Faecalis 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. The fact that Faecalibacterium duncaniae (Fd bacteria) and Blautia wexlerae (Bw bacteria) are useful bacteria has been described in, for example, Fd bacteria: FEMS Microbiology Reviews, 2023, 47, 1-18, and Bw bacteria: Nature Communications | (2022) 13: 4477, but no technology has been provided that allows them to be maintained as bacterial preparations.

[0075] Therefore, it was unexpected that these useful bacteria could be maintained using high molecular weight glycoproteins, as disclosed in the present invention. In particular, the effect of mucin is particularly remarkable.

[0076] The composition of the present disclosure may be provided as a technique (method, medium, etc.) for producing a bacterial preparation. A "bacterial preparation" herein refers to a composition containing bacteria, preferably a pharmaceutical composition. The bacterial preparation may be composed of a bacterial suspension. A "bacterial suspension" herein includes medically and / or pharmaceutically acceptable bacteria and a medically and / or pharmaceutically acceptable drug. The type of bacteria is not particularly limited as long as it is medically and / or pharmaceutically acceptable, but it is advantageous to use bacteria contained in the intestinal flora, particularly useful bacteria.

[0077] In one aspect of the present disclosure, the present disclosure provides a method for producing a bacterial preparation enriched in beneficial bacteria, comprising: A) culturing an intestinal microbiota in a medium containing a high-molecular-weight glycoprotein; B) collecting the grown intestinal microbiota; and C) optionally washing the intestinal microbiota, and then adding the intestinal microbiota to an administration vehicle (e.g., an administration liquid, physiological saline, or a suspension) to produce a bacterial preparation. The bacterial preparation produced by this production method maintains or grows beneficial bacteria (this is referred to as being enriched). Any of the high-molecular-weight glycoproteins and beneficial bacteria described herein can be used. As used herein, "enriched in beneficial bacteria" refers to the ratio or amount, preferably the ratio, of beneficial bacteria present in the starting intestinal microbiota in the target composition, such as a bacterial preparation, being maintained or increased. Typically, when producing a bacterial preparation, the total amount of bacteria contained in the starting intestinal microbiota increases, so the amount of beneficial bacteria is usually increased.

[0078] 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 beneficial bacteria is maintained or increased, and in this respect, the ratio or amount of beneficial bacteria is maintained or increased, or increased, compared to when the preparation is simply made from intestinal bacterial flora, and therefore, it can be said that a new bacterial preparation is provided.

[0079] The administration vehicle used in the present disclosure may be any vehicle used in pharmaceuticals or food and beverages (e.g., any liquid or solid excipient, etc.), including, but not limited to, physiological saline.

[0080] In one embodiment, the high molecular weight glycoprotein is preferably a mucin.

[0081] (Bacterial preparations (probiotics) can be used not only in medicines but also in foods, supplements, and food and beverage additives.

[0082] In one embodiment, oral compositions in the present disclosure specifically include, but are not limited to, foods (including foods in the narrow sense, beverages, etc.), additives for foods and beverages, supplements, feed, feed additives, pharmaceuticals (including quasi-drugs), and the like.

[0083] 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-promoting foods (foods for specified health uses, foods with nutrient functions, foods with functional claims, etc.), health supplements, nutritional supplements, etc. The form of the food can be selected appropriately, such as solid, liquid, or paste. In a broad sense, food may include beverages, but in a narrow sense, it may be intended to be a concept excluding beverages.

[0084] The bacterial preparation of the present disclosure may be provided as a beverage. In the present disclosure, "beverage" includes soft drinks, dairy drinks, alcoholic drinks, etc. The term "food and beverage" may be used as a combined concept of "food and beverage."

[0085] The bacterial preparations disclosed herein may be provided as supplements. In the present disclosure, the term "supplement" may be in any form, including, but not limited to, tablets, granules, powders, sugar-coated tablets, capsules, syrups, suspensions, liquids, emulsions, etc. Furthermore, the bacterial preparations may be provided as enteric-coated preparations, which are coated with a coating that exhibits different 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 function in the intestine.

[0086] As used herein, the bacterial formulation of the present disclosure may be provided as feed. In the present disclosure, "feed" includes feed for livestock or pets, and the form thereof may be selected appropriately, such as solid, liquid, or paste.

[0087] The bacterial preparations disclosed herein may be provided as additives for foods and beverages. In the present disclosure, "additives for foods and beverages" can be used as additives for foods and beverages. Furthermore, "feed additives" can be used as additives for livestock or pet feed. These may be in any form, such as tablets, granules, powders, sugar-coated tablets, capsules, syrups, suspensions, liquids, emulsions, etc.

[0088] The bacterial formulations disclosed herein may be provided as pharmaceuticals. 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 formulations such as liquids and suspensions may be provided in a freeze-dried and storable state, and may be dissolved in a buffer solution containing water or saline solution to an appropriate concentration before use. Solid dosage forms such as tablets may be coated as needed (e.g., sugar-coated tablets, gelatin-encapsulated tablets, enteric-coated tablets, etc.), or may be formulated into controlled-release formulations such as sustained-release, delayed-release, or immediate-release formulations using known techniques. Furthermore, enteric-coated formulations may be coated with coatings that differ in solubility at different pH levels to protect lactic acid-producing bacteria and butyric acid-producing bacteria from gastric acid and bile acid and allow them to act in the intestine.

[0089] Diseases to which the bacterial preparation 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 diseases, stroke, cerebral infarction, intracerebral hematoma, cerebrovascular plegia, 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 a disease selected from diseases caused by aging and / or inflammation, articular cartilage defect, meniscus injury, osteochondrosis elastosis, avascular necrosis, knee osteoarthritis, 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, psychiatric diseases (dementia, etc.), and cancer.

[0090] The bacterial formulation of the present disclosure is not particularly limited as long as it can maintain the bacteria in a pharmaceutically usable form and is administrable to animals such as humans. The medically and / or pharmaceutically acceptable agent may be mixed with the bacterial pharmaceutical formulation during the manufacturing process or during administration to a patient or subject. Examples of the medically and / or pharmaceutically acceptable agent include, but are not limited to, saline, electrolyte solution, Ringer's solution, hyperalimentation, glucose solution, water for injection, amino acid electrolytes, etc. Furthermore, the medically and / or pharmaceutically acceptable agent may optionally contain salts, vitamins, amino acids, polysaccharides, dimethyl sulfoxide, buffers, albumin, culture media, cell cryoprotectants, etc. It may also contain pharmaceutically active ingredients such as immunosuppressants, antibiotics, albumin preparations, vitamin preparations, and anti-inflammatory agents. In one embodiment, for example, administering an antibiotic and then killing or reducing the subject's native intestinal bacteria before administering the bacterial preparation of the present disclosure is particularly advantageous when the subject has an abnormal intestinal flora. For example, patients with inflammatory bowel disease (IBD), cancer, etc. are often known to have disrupted intestinal flora, and disruption of the intestinal flora has also been reported in other diseases. Therefore, such a treatment method may be advantageous for any of the diseases described herein or other diseases. The period required to kill or reduce intestinal bacteria may vary depending on the subject's condition and the antibiotic. Therefore, the state of the intestinal flora may be observed before administering the bacterial preparation of the present disclosure, or an appropriate period may be allowed to elapse depending on the condition before administering the bacterial preparation of the present disclosure. This period may be shortened or extended as appropriate, and may be, but is not limited to, about 3 days, 1 week, 2 weeks, 3 weeks, or 1 month.

[0091] Examples of 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.

[0092] 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, and the like.

[0093] Examples of 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 naturally occurring amino acids such as L-cysteine.

[0094] Examples of 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, heparin sulfate, keratosulfate, chondroitin sulfate, dermatan sulfate, rhamnan sulfate, alginic acid, and salts thereof.

[0095] The medium is not particularly limited as long as it is composed of components that allow bacteria to survive and are suitable for administration to animals such as humans, and examples include GAM medium, YCFA medium, modified YCFA medium, BBL medium, SOC medium, and LB medium. Among these, GAM medium is preferred, and media such as GAM agar medium, modified GAM agar medium, GAM semi-solid high-layer medium, GAM bouillon, and modified GAM bouillon (all manufactured by Nippon Pharmaceutical Co., Ltd.) can be used. For oral administration to humans, BSA-free media or media free of animal-derived components such as meat extract can be used. Alternatively, microorganisms (bacteria) can be cultured using the media exemplified above, and the collected bacteria can be 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 used for administration to humans.

[0096] 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.

[0097] Antibiotics that can be used in the present disclosure include, for example, sulfa drugs, penicillin, phenethicillin, methicillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, nafcillin, ampicillin, amoxicillin, cyclacillin, carbenicillin, ticarcillin, piperacillin, azlocillin, mexlocillin, mecillinam, andinocillin, cephalosporin and its derivatives, oxolinic acid, amifloxacin, temafloxacin, nalidixic acid, piromidic acid, ciprofloxacin, cinoxacin, norfloxacin, perfloxacin, rozaxacin, ofloxacin, enoxacin, and pipemid. Acids that can be used include, but are not limited to, penicillin, sulbactam, clavulic acid, β-bromopenicillanic acid, β-chloropenicillanic acid, 6-acetylmethylene-penicillanic acid, cefoxazole, sultampicillin, formaldehyde foudate esters of adinocillin and sulbactam, tazobactam, aztreonam, sulfazetine, isosulfazetine, norcadicine, m-carboxyphenyl, methyl phenylacetamidophosphonate, chlortetracycline, oxytetracycline, tetracycline, demeclocycline, doxycycline, methacycline, and minocycline.

[0098] Anti-inflammatory agents that can 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.

[0099] The bacterial formulations of the present disclosure may also contain various additives to increase storage stability, isotonicity, absorption, and / or viscosity, such as emulsifiers, dispersing agents, buffers, preservatives, humectants, antioxidants, chelating agents, thickeners, gelling agents, pH adjusters, etc.

[0100] Examples of thickening agents that can be used in the present disclosure include, but are not limited to, hydroxyethyl starch, dextran, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, etc. The concentration of the thickening agent will depend on the thickening agent selected, but can be set arbitrarily within a range that is safe when administered to a patient or subject and achieves the desired viscosity.

[0101] The viscosity of the bacterial suspension is not particularly limited, but if the viscosity is too high, rectal administration cannot be performed appropriately. For example, when measured using a rotational viscometer TV-20 (manufactured by Toki Sangyo Co., Ltd.) 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.

[0102] 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 impose a physical burden on the patient or test subject. On the other hand, if the bacterial concentration is too low, it may take a long time to administer an amount of bacteria sufficient to obtain a therapeutic effect, which may lead to a deterioration in the quality of the bacterial preparation during that time, or may lead to a burden on the patient or test subject, such as prolonged confinement. Therefore, the preferred bacterial concentration is set appropriately taking these factors into consideration, and is not particularly limited, but may be, for example, 1 x 10 ４ cells / mL or more, 2 x 10 ４ cells / mL or more, 3 x 10 ４ cells / mL or more, 4 x 10 ４ cells / mL or more, 5 x 10４ 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 and aboveであり、また、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 ７cells / 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 x 10 ６ cells / mL or less, 3×10 ６ cells / mL or less, 2.5×10 ６ cells / mL or less, 2×10 ６ In addition to the above, the amount can be appropriately determined depending on the administration form, the purpose of use, and the age, weight, symptoms, etc. of the patient or subject.

[0103] The administration method (e.g., administration rate, characteristics of the tubing used in combination, etc.) in which the bacterial preparation of the present disclosure is used may be described on the label, package insert, or instruction manual attached to the cell preparation.

[0104] 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 a subject (patient or subject) for internal administration is not particularly limited, but can be, for example, rectal administration.

[0105] The container used to provide the bacterial preparation of the present disclosure is not particularly limited, as 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 directly for the administration method described below. From the standpoint of hygiene and ease of operation, it is preferable to use the container used to provide the bacterial preparation directly for the administration method described below. In this case, the provided bacterial preparation may be administered as is, or if frozen, may be thawed and then administered, or may be diluted with physiological saline, Ringer's solution, or the like before administration, as necessary.

[0106] All references cited in this disclosure, including scientific literature, patents, patent applications, and the like, are incorporated by reference in their entirety into this disclosure to the same extent as if each were specifically set forth.

[0107] The present disclosure has been described above by showing preferred embodiments for ease of understanding. Hereinafter, the present disclosure will be described based on examples. However, the above description and the following examples are provided for illustrative purposes only and are not intended to limit the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments or examples specifically described herein, but is limited only by the scope of the claims.

[0108] Example 1 (Achieving Original Equilibrated Conditions (Jar Culture of 0.8 g of Mucin)) (Preparation of Fecal Suspension) Feces serving as an inoculum for intestinal bacterial flora were collected from healthy individuals on the day of culture. After collection, the fecal samples were stored in anaerobic culture swabs (212550 BD BBL Culture Swab; manufactured by Becton, Tickinson and Company) and transported to the laboratory. To prepare the inoculum, 0.5 g of feces was added to a total of 2 mL of 0.1 M phosphate buffer (PBS) buffer (pH 6.5, consisting of a 68.5:31.5 (molar ratio) mixture of 0.1 M NaH2PO4 and 0.1 M Na2HPO4) supplemented with 1.0% L-ascorbic acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), to prepare a fecal suspension.

[0109] (Preparation of medium) A medium was prepared by mixing 59 g / L of Gifu University-prescribed anaerobic medium (GAM medium [Code 05422] (manufactured by Nissui Pharmaceutical Co., Ltd.), 8.0 g / L of mucin (manufactured by Sigma-Aldrich, derived from porcine stomach, Type III), and 50 μL / L of 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 the mixture was sterilized in an autoclave at 115° C. for 15 minutes.

[0110] (Culture Conditions) After sterilization, a nitrogen and carbon dioxide mixed gas (N ) that had been sterilized by filtration through a 0.2 μm PTFE membrane (manufactured by Pall Corporation) was introduced at 37° C. for 1 hour before culturing. ２ :CO ２ Anaerobic conditions were created in the culture vessel by aeration (15 mL / min) of a mixture of 100% ethanol and 100% ethanol (aqueous phase) in a volume ratio of 80:20.

[0111] 500 μL of the fecal suspension was inoculated into a medium-containing vessel (0.125 g feces per 100 mL of culture solution), and anaerobic culture was initiated (culture time 0). During the culture, a filtered and sterilized mixed gas (N ２ :CO ２ The medium was constantly bubbled at a ratio of 80:20 (volume ratio) to maintain an anaerobic state in the culture tank. The culture temperature was set at 37°C, and the incubation was performed with continuous stirring at approximately 300 rpm.

[0112] The culture medium was collected 6, 24, 30, 48, 72, and 96 hours after the start of culture, and subjected to bacterial flora analysis. The culture medium was collected using a syringe without opening the culture vessel, without introducing air.

[0113] (Bacterial Flora Analysis) Genomic DNA of the bacterial flora was extracted from the culture medium collected at various times before and after the start of cultivation. The V3-V4 region of the bacterial 16S rRNA gene was amplified from the extracted genomic DNA and sequenced using a next-generation sequencer to perform bacterial diversity analysis and bacterial composition analysis. The procedure is as follows:

[0114] The bacterial 16S rRNA gene was amplified using the primer pair S-D-Bact-0341-b-S-17 (SEQ ID NO: 1) and S-D-Bact-0785-a-A-21 (SEQ ID NO: 2) with extracted genomic DNA as a template. An Illumina adapter overhang nucleotide sequence (Illumina, Inc.) was added to the gene-specific sequence. PCR cycling reactions were performed according to the manufacturer's instructions. The confirmed amplicons were purified using AMPure XP DNA purification beads (Beckman Coulter, Inc.) and eluted in 25 μl of 10 mM Tris (pH 8.5). The amplicons were quantified on an Agilent Bioanalyzer 2100 DNA 1000 chip (Agilent Technologies, Inc.) and pooled at equimolar concentrations. The 16S rRNA gene product (together with an internal control (PhiX control V3; Illumina)) was subjected to paired-end sequencing using a MiSeq sequencer (Illumina) with a 600-cycle MiSeq reagent kit (Illumina).

[0115] Paired-end reads with a Q score of 20 or more obtained by extracting the PhiX sequence using Basespace Sequence Hub (https: / / basespace.illumina.com / ) were combined using QIIME 2 version 2022.2 and quality control and correction were performed in the DADA2 pipeline, after which the OTU was inferred. The obtained OTU was used to estimate alpha diversity and calculate the Shannon index. In addition, the obtained OTU was classified using a naive Bayes classifier trained on the Greengenes 13_8 99% OTU full-length sequence database, and bacterial species assignment was performed. Using Excel (Microsoft Japan Co., Ltd.), the relative occupancy rate was calculated from the genus-level classification data of bacterial species attribution, and the Pearson product-moment correlation coefficient was calculated based on the relative occupancy rate.

[0116] Real-time PCR was performed using a QuantStudio® 3 Real-Time PCR System (Thermo Fisher Scientific). Amplification was performed using a primer set targeting all enterobacteria, as described in Takagi, R. et al., PLoS One 11, e0160533 (2016). Total bacterial counts were calculated from a calibration curve prepared from known concentrations of E. coli.

[0117] Shannon index and Pearson product-moment correlation coefficient were calculated using the QIIME software package.

[0118] Comparative Example 1 (without mucin addition) Cultivation and analysis of the bacterial flora were carried out in the same manner as in Example 1, except that 8.0 g / L of mucin (Sigma-Aldrich, derived from porcine stomach, Type III) was not added in the preparation of the medium.

[0119] Table 1 shows the change in Pearson product-moment correlation coefficients with culture time in Example 1 and Comparative Example 1.

[0120]

[0121] It can be seen that in Example 1 according to the present invention, the Pearson product-moment correlation coefficient remained at 0.8 or higher from 24 to 96 hours of culture. On the other hand, in Comparative Example 1, the Pearson product-moment correlation coefficient was 0.8 or higher at 24 and 30 hours of culture, but fell below 0.8 after 48 hours of culture. A comparison between Example 1 and Comparative Example 1 revealed that the mucin added to the medium was effective in maintaining the original equilibrium state.

[0122] Example 2 (Original Equilibrated 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.

[0123] Comparative Example 2 (without mucin addition) 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.

[0124] The results of Example 2 and Comparative Example 2 are shown in Table 2.

[0125]

[0126] In Example 2, the Pearson product-moment correlation coefficient was 0.80 or higher after 15 hours of culture, whereas in Comparative Example 2, the Pearson product-moment correlation coefficient reached 0.80 or higher after 18 hours of culture. This indicates that the mucin added to the medium was effective in promoting the return to equilibrium.

[0127] 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 feces 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.

[0128] Comparative Example 3 Cultivation and analysis of bacterial flora were carried out in the same manner as in Example 1, except that in the preparation of the medium in Example 3, 4.0 g / L of mucin (Sigma-Aldrich, derived from porcine stomach, Type III) was not added.

[0129] The results of the bacterial flora analysis in Example 3 and Comparative Example 3 are shown in Table 3.

[0130]

[0131] Table 3 shows the relative values ​​of the Pearson product-moment correlation coefficients for Example 3 after 72 hours of culture, with the value for Comparative Example 3 set at 100. Even under different conditions of the inoculation amount of fecal suspension and the amount of mucin added, a high Pearson product-moment correlation coefficient was obtained according to the present invention.

[0132] Reference Example 1 (Test substance inulin added 24 hours after initiation of culture) Cultivation and evaluation were carried out in the same manner as in Example 1, except that inulin (derived from BENEO, OraftiGR, chicory; denoted as INU) was added as the test substance at 0.3 w / v % to the culture solution 24 hours after initiation of culture. The culture solution was sampled 48 hours, 72 hours, and 96 hours after initiation of culture, and the abundance rate of bacteria of the genus Bifidobacterium in each culture solution was determined.

[0133] 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 (derived from BENEO, OraftiGR, chicory; denoted as INU) was added as the test substance at 0.3 w / v % to the culture medium at the start of culture (0 hours into culture). The culture medium was sampled 48 hours, 72 hours, and 96 hours after the start of culture, and the abundance rate of bacteria of the genus Bifidobacterium in each culture medium was determined.

[0134] (Experimental Results) The percentage of Bifidobacterium bacteria present 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.

[0135]

[0136] The culture solution to which the test substance inulin was not added is referred to as "CUL," the culture solution to which inulin was added at the start of culture is referred to as "INU (added 0 h)," and the culture solution to which inulin was added 24 hours after the start of culture is referred to as "INU (added 24 h)."

[0137] 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 reduced 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 increased by the addition of inulin is consistent with the test results in which humans ingested inulin (Daniel So, et al., Am. J. Clin. Nutr 2018(107), 965-983). In other words, it was shown that the evaluation results of the human intestinal flora can be reproduced in vitro by adding the test substance inulin after the original equilibrium state was reached.

[0138] Reference Example 3 (Multiwell Plate Culture, No Test Substance Added) (Preparation of Fecal Suspension) Feces serving as an inoculum for intestinal bacterial flora were collected from healthy individuals on the day of culture. After collection, the fecal samples were stored in anaerobic culture swabs (212550 BD BBL Culture Swab; Becton, Tickinson & Company) and transported to the laboratory. To prepare the inoculum, 0.5 g of feces was suspended in 2 mL of 0.1 M phosphate buffer (PBS) buffer (pH 6.5, consisting of a 68.5:31.5 (molar ratio) mixture of 0.1 M NaH2PO4 and 0.1 M Na2HPO4) supplemented with 1.0% L-ascorbic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) to prepare a fecal suspension.

[0139] (Preparation of medium) A medium was prepared by mixing 59 g / L of Gifu University-prescribed anaerobic medium (GAM medium [Code 05422] (manufactured by Nissui Pharmaceutical Co., Ltd.), 8.0 g / L of mucin (manufactured by Sigma-Aldrich, derived from porcine stomach, Type III), and 50 μL / L of an antifoaming agent. The pH was adjusted to 6.5 with 0.1 M phosphate buffer, and the medium was sterilized in an autoclave at 115° C. for 15 minutes.

[0140] (Culture Conditions) 1.0 mL of medium was dispensed into the required number of wells in a clean bench. A 96-well multi-well plate (model number 82.1972.002, 2.2 mL capacity, manufactured by Sarstedt Co., Ltd.) was used (hereinafter sometimes referred to as 96-well). The multi-well plate was stirred at approximately 500 rpm using a 96-well shaking incubator (manufactured by Biosan, model number TS-DW) installed in an anaerobic chamber. The culture temperature was 37°C. Culture was initiated by inoculating 50 μL / well of fecal suspension (12.5 mg / mL feces per culture solution), and this was designated as culture time 0.

[0141] 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.

[0142] Reference Example 4 (Test substance, water-soluble indigestible dextrin, added 24 hours after the start of culture (multi-well plate)) Culture 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 culture.

[0143] Reference Example 5 (Addition of water-soluble, indigestible dextrin at the start of culture (multi-well plate)) Culture and evaluation were carried out in the same manner as in Reference Example 3, except that 0.3 w / v % of water-soluble, indigestible dextrin (Fibersol 2 (maltodextrin), manufactured by Matsutani Scientific Co., Ltd.; referred to as DEX) was added to the culture medium as the test substance at the start of culture.

[0144] The results of Reference Examples 3, 4 and 5 are shown in Table 5.

[0145]

[0146] The culture solution to which dextrin was not added is referred to as "CUL," the culture solution to which dextrin was added at the start of culture is referred to as "DEX (added 0 h)," and the culture solution to which dextrin was added 24 hours after the start of culture is referred to as "DEX (added 24 h)."

[0147] In Table 5, in Reference Example 5, in which dextrin was added at the start of culture, it can be seen that the presence rate of Faecalibacterium was reduced at any culture time 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, it can be seen that the presence rate of Faecalibacterium was increased at any culture time 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 the test results obtained in vivo can be reproduced in vitro by adding the test substance dextrin during the original equilibration state.

[0148] Example 4 (Culture on a Multiwell Plate) This example shows culture on a multiwell plate, and in particular, the medium was examined.

[0149] (Preparation of fecal suspension) Feces serving as an inoculum of the intestinal bacterial flora were collected from healthy individuals on the day of culture. After collection, the fecal samples were stored in anaerobic culture swabs (212550 BD BBL Culture Swab; manufactured by Becton, Tickinson and Company) and transported to the laboratory. To prepare the inoculum, 0.5 g of feces was dissolved in 0.1 M phosphate buffer (PBS) buffer (pH 6.5, 0.1 M NaHCO3) containing 1.0% L-ascorbic acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). ２ P.O. ４and 0.1 M Na ２ HPO ４ A fecal suspension was prepared by suspending the feces in 2 mL of a 68.5:31.5 (molar ratio) mixture of

[0150] (Preparation of medium) A medium was prepared by mixing 59 g / L of Gifu University-prescribed anaerobic medium (GAM medium [Code 05422] (manufactured by Nissui Pharmaceutical Co., Ltd.), 8.0 g / L of mucin (manufactured by Sigma-Aldrich, derived from porcine stomach, Type III), and 50 μL / L of an antifoaming agent. The pH was adjusted to 6.5 with 0.1 M phosphate buffer, and the medium was sterilized in an autoclave at 115° C. for 15 minutes.

[0151] (Culture Conditions) 1.0 mL of medium was dispensed into the required number of wells in a clean bench. A 96-well multi-well plate (model number 82.1972.002, 2.2 mL capacity, manufactured by Sarstedt Co., Ltd.) was used (hereinafter sometimes referred to as 96-well). The multi-well plate was stirred at approximately 500 rpm using a 96-well shaking incubator (manufactured by Biosan, model number TS-DW) installed in an anaerobic chamber. The culture temperature was 37°C. Culture was initiated by inoculating 50 μL / well of fecal suspension (12.5 mg / mL feces per culture solution), and this was designated as culture time 0.

[0152] The culture medium was collected 6, 24, 30, 48, 72, and 96 hours after the start of culture, and subjected to bacterial flora analysis. The culture medium was collected without opening the anaerobic chamber.

[0153] (Bacterial Flora Analysis) Genomic DNA of the bacterial flora was extracted from the culture medium collected at various times before and after the start of cultivation. The V3-V4 region of the bacterial 16S rRNA gene was amplified from the extracted genomic DNA and sequenced using a next-generation sequencer to perform bacterial diversity analysis and bacterial composition analysis. The procedure is as follows:

[0154] The bacterial 16S rRNA gene was amplified using the primer pair S-D-Bact-0341-b-S-17 (SEQ ID NO: 1) and S-D-Bact-0785-a-A-21 (SEQ ID NO: 2) with extracted genomic DNA as a template. An Illumina adapter overhang nucleotide sequence (Illumina, Inc.) was added to the gene-specific sequence. PCR cycling reactions were performed according to the manufacturer's instructions. The confirmed amplicons were purified using AMPure XP DNA purification beads (Beckman Coulter, Inc.) and eluted in 25 μl of 10 mM Tris (pH 8.5). The amplicons were quantified on an Agilent Bioanalyzer 2100 DNA 1000 chip (Agilent Technologies, Inc.) and pooled at equimolar concentrations. The 16S rRNA gene product (together with an internal control (PhiX control V3; Illumina)) was subjected to paired-end sequencing using a MiSeq sequencer (Illumina) with a 600-cycle MiSeq reagent kit (Illumina).

[0155] Paired-end reads with a Q score of 20 or more obtained by extracting the PhiX sequence using Basespace Sequence Hub (https: / / basespace.illumina.com / ) were combined using QIIME 2 version 2022.2 and quality control and correction were performed in the DADA2 pipeline, after which the OTU was inferred. The obtained OTU was used to estimate alpha diversity and calculate the Shannon index. In addition, the obtained OTU was classified using a naive Bayes classifier trained on the Greengenes 13_8 99% OTU full-length sequence database, and bacterial species assignment was performed. Using Excel (Microsoft Japan Co., Ltd.), the relative occupancy rate was calculated from the genus-level classification data of bacterial species attribution, and the Pearson product-moment correlation coefficient was calculated based on the relative occupancy rate.

[0156] Example 5 (Study on GAM medium containing mucin) Cultivation and bacterial flora analysis were carried out 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.

[0157] Example 6 (Study on 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 41.7 g / L of modified Gifu University prescribed anaerobic medium (modified GAM medium [Code 05433] (manufactured by Nissui Pharmaceutical Co., Ltd.) and 50 μL / L of an antifoaming agent.

[0158] Example 7 (Study on modified GAM medium containing mucin) In preparing the modified GAM medium, culture and bacterial flora analysis were carried out in the same manner as in Example 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.

[0159] Example 8 (Study on YCFA medium) Culture and bacterial flora analysis were carried out in the same manner as in Example 4, except that YCFA medium was used instead of GAM medium for medium preparation. YCFA medium contained 10.0 g / L of casein hydrolysate, 2.5 g / L of yeast extract, 4.0 g / L of sodium bicarbonate, 2.0 g / L of glucose, 2.0 g / L of maltose, 2.0 g / L of cellobiose, 1.0 g / L of L-cysteine ​​HCl, 0.001 g / L of resazurin, 0.45 g / L of dipotassium hydrogen phosphate, 0.45 g / L of potassium dihydrogen phosphate, 0.9 g / L of ammonium sulfate, 0.9 g / L of sodium chloride, 0.09 g / L of magnesium sulfate, 0.09 g / L of calcium chloride, and 0.01 g / L of hemin, and contained 3.1 ml of volatile fatty acids (acetic acid) per liter. The medium was prepared by mixing 2.026 ml / L of ATP, 0.715 ml / L of propionic acid, 0.119 ml / L of n-valeric acid, 0.119 ml / L of isovaleric acid, 0.119 ml / L of isovaleric acid), 1 ml of vitamin mixture 1 (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), 1 ml of vitamin mixture 2 (thiamine 5 mg / 100 ml, riboflavin 5 mg / 100 ml) and 50 μL / L of antifoaming agent, and the pH was adjusted to 7.5 with a pH adjuster.

[0160] Example 9 (Study on YCFA medium containing mucin) Cultivation and bacterial flora analysis were carried out 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.

[0161] 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 of casein hydrolysate, 2.5 g / L of yeast extract, 5.0 g / L of glucose, 0.045 g / L of magnesium sulfate, 0.09 g / L of calcium chloride, 0.45 g / L of dipotassium hydrogen phosphate, 0.45 g / L of potassium dihydrogen phosphate, 0.9 g / L of sodium chloride, 0.001 g / L of resazurin, 1.0 g / L of L-cysteine ​​HCl, 4.0 g / L of sodium bicarbonate, and 0.01 g / L of hemin, and contained 2.7 ml of volatile fatty acids (2.026 ml / L of acetic acid, 0.026 ml / L of 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.

[0162] Example 11 (Study on modified YCFA medium containing mucin) Cultivation and bacterial flora analysis were performed in the same manner as in Example 9, except that 8.0 g / L or 4.0 g / L of mucin (Sigma-Aldrich, derived from porcine stomach, Type III) was added to the modified YCFA medium in preparing the modified YCFA medium. Relative occupancy rates were calculated from genus-level classification data of bacterial species attribution, and Pearson product-moment correlation coefficients were calculated based on the relative occupancy rates.

[0163] Table 6 shows the relative values ​​of the Pearson product-moment correlation coefficients after 72 hours of culture for Examples 4, 5, 6, and 7 in various media, with the results set at 100, and the Pearson product-moment correlation coefficients for Examples 8, 9, 10, and 11 in mucin-supplemented media. The results in Table 6 clearly demonstrate that the addition of mucin is effective in maintaining the original equilibrium state in all media.

[0164]

[0165] Example 12 (Abundance of Useful Bacteria: Effect of Mucin Addition) Genomic DNA of bacteria in the bacterial flora was extracted from the culture medium collected before the start of culture and 72 hours after the start of culture in Examples 4 and 5. Using specific primers targeting the 16S rRNA genes of Faecalibacterium duncaniae (Fd bacteria) and Blautia wexlerae (Bw bacteria), the target bacterial genes were quantified using a quantitative PCR device (Table 7). The results in Table 7 show that when cultured in each medium without mucin, the abundance of the useful bacteria Fd and Bw decreased, whereas the addition of mucin to all media maintained Fd and Bw bacteria during culture, and their abundance was equal to or greater than that of the original sample (Fec), demonstrating that mucin addition was effective in maintaining these useful bacteria.

[0166]

[0167] (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 preparing the medium, 8.0 g / L of mucin (Sigma-Aldrich, derived from porcine stomach, Type II) was added to the GAM medium.

[0168] Example 14 (Study on medium containing porcine mucin) Cultivation and bacterial flora analysis were carried out in the same manner as in Example 4, except that in preparing the medium, 8.0 g / L of mucin (produced by Fujifilm Wako Pure Chemical Industries, Ltd., derived from porcine stomach) was added to the GAM medium.

[0169] Example 15 (Study on medium containing skate mucin) 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 GAM medium in the preparation of the medium. Relative occupancy rates were calculated from genus-level classification data of bacterial species attribution, and Pearson product-moment correlation coefficients were calculated based on the relative occupancy rates.

[0170] Comparative Example 4 (Study on Mucin-Free Medium) In preparing the medium, the same procedures as in Example 4 were used to culture and analyze the bacterial flora.

[0171] Table 8 shows the relative values ​​of the Pearson product-moment correlation coefficients for Examples 1, 2, 3, and 4 after 72 hours of culture, with the result for Comparative Example 4 set at 100. The results in Table 8 clearly show that the addition of all mucins of different origins was effective in maintaining the original equilibrium state.

[0172]

[0173] Example 16 (Administration of culture preparation to mice) The human intestinal flora culture prepared in Example 5 was administered to SPF mice, and insulin sensitivity was assessed by subjecting them to a high-fat diet. The administration of the microorganism to mice and the efficacy evaluation test were carried out by the method described in Nature Communications (202) 13:4477. Specifically, SPF mice (6 weeks old) were fed a high-fat diet (AIN-93G, Oriental Yeast Co., Ltd.) for 10 weeks, and the human intestinal flora culture prepared in Example 5 was administered at a concentration of 5 x 10 ９ CFU are orally administered three times a week, and the weight of each individual is measured. Eight weeks after administration of the human intestinal flora culture medium, serum is collected, and HOMA-IR and insulin concentrations are measured, and an IPGTT test is also performed.

[0174] (Note) As described above, the present disclosure has been illustrated using preferred embodiments of the present disclosure, but the present disclosure should not be construed as being limited to these embodiments. It is understood that the scope of the present disclosure should be interpreted solely by the scope of the claims. It is understood that a person skilled in the art can implement an equivalent scope based on the description of the present disclosure and common general technical knowledge from the description of specific preferred embodiments of the present disclosure. It is understood that the contents of patents, patent applications, and literature cited in this specification are incorporated by reference into this specification as if the contents themselves were specifically set forth in this specification. This application claims priority to Japanese Patent Application No. 2023-196982, filed with the Japan Patent Office on November 20, 2023, the contents of which are incorporated herein by reference in their entirety.

[0175] According to the present disclosure, it is possible to provide a composition that enables in vitro evaluation of the effect of a test substance, such as a food or a drug candidate compound, on the intestinal flora of mammals, particularly humans.

Claims

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 of claim 1, wherein the high molecular weight protein comprises a mucin.

3. The composition according to claim 2, wherein the mucin is contained in the culture medium at a concentration of 0.4 w / v % or more.

4. The composition according to any one of claims 1 to 3, wherein the medium is GAM medium, YCFA medium or a modified version thereof.

5. A composition according to any one of claims 1 to 4, wherein the equilibration is performed for the purpose of evaluating a test substance in the intestinal bacterial flora.

6. The composition according to any one of claims 1 to 5, wherein the composition is for maintaining the original equilibrium.

7. The composition according to any one of claims 1 to 6, which is intended to inhibit the medium from losing its equilibrium after it has achieved equilibrium.

8. A medium for equilibrating intestinal flora, comprising high molecular weight glycoproteins and medium components.

9. The original equilibrated medium of claim 8, wherein the medium is for evaluating a test substance in intestinal flora.

10. The medium according to claim 8 or 9, wherein the high molecular weight protein comprises mucin.

11. 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 relative to the medium components.

12. The medium according to any one of claims 8 to 11, wherein the medium components are 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 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).

15. The composition according to claim 13 or 14, wherein the high molecular weight glycoprotein is a mucin.

16. A composition according to any one of claims 13 to 15 for use in the manufacture of a bacterial preparation.

17. A method for producing a bacterial preparation enriched with beneficial bacteria, comprising: 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 to obtain a bacterial preparation.

18. The method of claim 17, wherein the high molecular weight glycoprotein is a mucin.

19. A bacterial preparation produced by the method of claim 17.

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