Method for evaluating test substance in intestinal flora

Abstract

The present disclosure provides a method for evaluating in vitro the effect of a test substance such as a food product or a drug candidate compound on the intestinal flora in a mammal, especially a human. Specifically, the present disclosure provides a method for evaluating a test substance in intestinal flora, wherein the method, etc. comprises the following steps: A) a step for culturing the intestinal flora in a culture medium for a period of time that is effective for reaching an original equilibrium state after starting culture by inoculating the intestinal flora into the culture medium; B) a step for adding the test substance to the culture medium containing the intestinal flora after passage of the abovementioned time; and C) a step for acquiring and evaluating evaluation items before and after the addition of the test substance.

Description

Methods for evaluating test substances in the intestinal microbiota

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

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

[0003] Patent No. 7051175

[0004] The present inventors have previously developed an apparatus for maintaining human intestinal bacterial species in a culture system using human feces as an inoculum source. After extensive research, the present inventors have discovered that a culture medium containing intestinal bacterial flora can be brought into a state of original equilibrium. Based on this finding, the present disclosure is as follows: [1] A method for evaluating a test substance in intestinal bacterial flora, comprising the following steps: A) culturing the intestinal bacterial flora in a medium for a time period effective for the intestinal bacterial flora to reach an original equilibrium state after the start of culture by inoculating the intestinal bacterial flora into the medium; B) adding the test substance to the medium containing the intestinal bacterial flora after the elapse of the time period; and C) obtaining and evaluating evaluation items before and after the addition of the test substance. [2] A method for evaluating a test substance in an intestinal bacterial flora, comprising the following steps: A) culturing the intestinal bacterial flora by inoculating it into a medium; B) adding the test substance to the medium containing the intestinal bacterial flora after the predetermined time has elapsed, wherein the predetermined time is determined based on a stabilization determination index; and C) obtaining and evaluating evaluation items before and after the addition of the test substance. [3] The method according to any one of the above items, wherein the test substance is added to the medium 6 hours or more after the start of culture. [4] The method according to any one of the above items, wherein the test substance is added to the medium 24 hours or more after the start of culture. [5] The method according to any one of the above items, wherein the evaluation item after the addition of the test substance is obtained while the original equilibrium state is maintained. [6] The method according to any one of the above items, wherein the evaluation item after the addition of the test substance is obtained within 96 hours after the start of culture. [7] The method according to any one of claims 1 to 5, wherein the evaluation item after addition of the test substance is obtained within 48 to 72 hours after the start of the culture. [8] The method according to any one of the above items, wherein the original equilibrium state includes equilibrating the microbiota diversity to the state of a pre-inoculation sample of the intestinal microbiota. [9] The method according to any one of the above items, wherein the original equilibrium state is determined based on structural analysis of the intestinal microbiota.

[10] The method according to any one of the above items, wherein the original equilibrium state is determined based on analysis of the 16S or genome of the intestinal microbiota.

[11] The method according to any one of the preceding items, wherein the structural analysis is a Pearson product-moment correlation coefficient.

[12] The method according to any one of the preceding items, wherein the original equilibrium state is determined by a Pearson product-moment correlation coefficient of 0.70 or higher.

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

[14] The method according to any one of the preceding items, wherein the original equilibrium state is determined from a time range during which a state in which the Pearson product-moment correlation coefficient of the bacterial flora structure before and after culture of the enterobacteria is maintained at 0.70 or higher for 24 hours or more.

[15] The method according to any one of the above items, wherein the medium contains mucin.

[16] The method according to any one of the above items, wherein the mucin is contained in the medium at a concentration of 0.4% or more.

[17] The method according to any one of the above items, wherein the intestinal microbiota is obtained from stool.

[18] The method according to any one of the above items, wherein the intestinal microbiota is obtained from human stool.

[19] The method according to any one of the above items, wherein the amount of the stool sample added is 0.1% or more and 1.25% or less of the medium.

[20] The method according to any one of the above items, wherein the method is carried out in at least one well of a multi-well plate.

[21] A method for producing an equilibrated intestinal microbiota sample by culturing a specimen containing intestinal microbiota in a medium, the method comprising culturing the specimen for a period of time sufficient for the culture, using a medium in which the Pearson product-moment correlation coefficient between the intestinal microbiota before culture and the intestinal microbiota during culture is 0.70 or more.

[22] The method according to any one of the above items, wherein the timing of addition of the test substance is determined based on a stabilization determination index.

[23] A method for evaluating a test substance in an intestinal bacterial flora, comprising the following steps: A) culturing the intestinal bacterial flora in a medium and adding the test substance, wherein the timing of adding the test substance is determined based on a stabilization determination index; and B) acquiring and evaluating evaluation items before and after adding the test substance.

[24] The method according to any one of the above items, wherein the medium is GAM medium or an improved GAM medium.

[25] The method according to any one of the above items, wherein the medium contains fucoidan and / or sodium lactate.

[26] The method according to any one of the above items, wherein the addition is performed 12 to 36 hours after the start of culture.

[27] The method according to any one of the above items, wherein the intestinal bacterial flora is provided in a frozen state.

[28] The method according to any one of the above items, wherein the intestinal bacterial flora is provided in a state obtained from the subject.

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

[0006] Based on the above, the present disclosure provides a method for evaluating a test substance, such as a food or a drug candidate compound, that enables the in vivo effect of the test substance on the intestinal environment to be reproduced in vitro. For example, the present disclosure provides a method for predicting the in vivo effect of the test substance on the intestinal environment by culturing a human intestinal microbiota in vitro, measuring changes in the microbiota structure or changes in metabolite concentrations, etc., and evaluating the test substance.

[0007] According to the method of the present disclosure, the effects of test substances such as foods and drug candidate compounds on the intestinal flora of mammals, particularly humans, can be more accurately evaluated.

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

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

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

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

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

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

[0014] 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).

[0015] 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 two bacterial flora. For example, original equilibrium can be achieved when the Pearson product-moment correlation coefficient between the intestinal bacterial flora before and after the start of culture is 0.50 or higher, or 0.60 or higher, usually 0.70 or higher, and preferably 0.80 or higher, or 0.85 or higher, 0.90 or higher, or 0.95 or higher. Furthermore, a state of original equilibrium is achieved when original equilibrium continues for a certain period of time. 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.

[0016] 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.

[0017] 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.

[0018] (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.

[0019] (Evaluation Method) In one aspect, the present disclosure provides a method for evaluating a test substance in an intestinal microbiota, comprising the following steps: A) culturing the intestinal microbiota in a medium for an effective time for the intestinal microbiota to reach a state of equilibrium 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; and C) acquiring and evaluating evaluation items before and after the addition of the test substance. While not wishing to be bound by theory, it is generally not expected that evaluation items will not be "measured" before addition. Since changes over time are often examined before and after the addition of the test substance, and there is considerable individual variation between samples, the Pearson product-moment correlation coefficient and Shannon's index may change from lot to lot due to small changes, it is preferable to evaluate a sample before the addition of the test substance.

[0020] In one embodiment, the same specimen was collected in advance, and it was demonstrated that the intestinal flora derived from the same specimen exhibited the same behavior, as shown by microwells, etc. Therefore, the timing of addition of the same specimen can be determined in advance, and this timing can be used to set up an evaluation system. In the present disclosure, it was found that the intestinal flora derived from the same specimen exhibited the same behavior. For example, as shown in the examples, when a 96-well plate was used, it was found that there were almost no error bars, demonstrating high reproducibility.

[0021] In one embodiment, the equilibration to the original state includes equilibrating the microbiota diversity to the state before inoculation of the intestinal microbiota. Without wishing to be bound by theory, this is because a sample is usually diluted and inoculated.

[0022] In one embodiment, the evaluation items after addition of the test substance of the present disclosure are obtained while the original equilibration state is maintained. By evaluating in the original equilibration state, it is possible to conduct a test under conditions close to those in the body, and to perform an evaluation equivalent to that in a real in vivo experiment.

[0023] In one embodiment, the evaluation items after addition of the test substance of the present disclosure are obtained while the original equilibrium is maintained after the start of culture, for example, within 120 hours or 96 hours, preferably within 48 to 72 hours, and are usually evaluated after at least 6 hours or 12 hours or more have passed, but are not limited to this.

[0024] In one embodiment, the test substance is added after 4 hours or more, preferably 6 hours or more, 8 hours or more, more preferably 12 hours or more, 18 hours or more, 24 hours or more, etc. have elapsed since the start of the culture, and the upper limit of the time may be within 90 hours, 84 hours, 78 hours, 72 hours, 66 hours, 60 hours, 54 hours, 48 ​​hours, etc.

[0025] In a preferred embodiment, the evaluation items after adding the test substance of the present disclosure can be measured after a certain period of time has elapsed since the addition of the test substance. Exemplary ranges include, but are not limited to, 6 hours to 90 hours, with lower limits of 4, 6, 8, 10, or 12 hours or more, and upper limits of 84, 96, 120, or 144 hours.

[0026] In another embodiment, the timing of addition of the test substance is determined based on a stabilization index. As used herein, the "stabilization index" is calculated as an index of the time required to achieve equilibrium, and is calculated based on the description herein. The timing of addition may be, for example, 6 to 90 hours or 12 to 36 hours after the start of culture.

[0027] In one embodiment, the present disclosure provides a method for determining whether the intestinal microbiota is in equilibrium and / or stabilized based on a structural analysis of the intestinal microbiota, such as quantitative PCR analysis, 16S amplicon analysis, or metagenomic analysis.

[0028] In another embodiment, the indicators for determining the original equilibrium state and / or stabilization of the present disclosure are determined based on quantitative PCR analysis, 16S amplicon analysis, metagenomic analysis, or the like of the intestinal microbiota.

[0029] In another embodiment, the structural analysis includes at least one selected from the Shannon index and the Pearson product-moment correlation coefficient of the intestinal microbiota. Preferably, the Pearson product-moment correlation coefficient is used for the assessment. Without wishing to be bound by theory, the Pearson product-moment correlation coefficient is an index that can evaluate both the degree of diversity and its relative ratio when evaluating a microbiota, and assessment using this coefficient is advantageous for determining whether the original equilibrium state has been properly achieved. In this specification, the original equilibrium state is normally determined to be appropriate when the Pearson product-moment correlation coefficient is, for example, 0.75 or higher. This coefficient can be set appropriately depending on the situation, and may be 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, etc.

[0030] In another embodiment, the present disclosure may typically include determining the timing of adding the test substance from a time range in which the Pearson product-moment correlation coefficient between the enterobacterial flora structure before and after culture is maintained at 0.70 or higher for 24 hours or longer. Preferably, the present disclosure may include determining the timing of adding the test substance from a time range in which the Pearson product-moment correlation coefficient between the enterobacterial flora structure before and after culture is maintained at 0.60 or higher, 0.65 or higher, 0.70 or higher, 0.75 or higher, 0.80 or higher, 0.85 or higher, 0.90 or higher, 0.95 or higher, typically 0.70 or higher, for 6 hours or longer, 12 hours or longer, 18 hours or longer, 24 hours or longer, 36 hours or longer, 48 hours or longer, and / or 90 hours or shorter, 84 hours or shorter, 78 hours or shorter, 72 hours or shorter, 66 hours or shorter, 60 hours or shorter, or 54 hours or shorter.

[0031] In one embodiment, the timing of adding the test substance in the present disclosure is determined by a method comprising the following steps A and B: (Step A) culturing a stool specimen containing the intestinal bacteria in a culture medium and obtaining time-course data in advance for at least one item selected from pH, short-chain fatty acid concentration, Shannon index of the bacterial flora, and Pearson product-moment correlation coefficient of the bacterial flora structure before and after culturing; and (Step B) determining in advance the timing of adding the test substance to the specimen containing the intestinal bacteria cultured in a culture medium from the time-course data obtained in Step A.

[0032] In a specific embodiment, the addition time is, but is not limited to, 6 to 24 hours or 12 to 36 hours after the start of culture.

[0033] In a specific embodiment, the present disclosure is carried out in at least one well of a multi-well plate.

[0034] In one embodiment, the indicator for determining the equilibrium state / stabilization is, for example, a state in which the Pearson correlation coefficient between the bacterial flora structure before and after the culture of the enterobacteria is 0.60 or more, 0.65 or more, 0.70 or more, 0.75 or more, 0.80 or more, 0.85 or more, 0.90 or more, 0.95 or more, typically 0.70 or more, is maintained for 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 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. This can be achieved by performing at least one of the following steps: determining the timing of adding the test substance from a time range in which the Shannon index after the culture in step A is maintained at 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, 95 or more, etc., when the Shannon index after the culture in step A is set to 100; and determining the timing of adding the test substance from a time range in which the state of initial pH ±1.5 (preferably ±1.0, or 0.5) in step A is maintained for, for example, 6 to 84 hours or more. The maintained time can be 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 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.

[0035] In one embodiment, the original equilibrated state in the present disclosure includes equilibrating the microbiota diversity to the state of the intestinal microbiota in a pre-inoculation sample. The importance of such equilibration was not previously known, and it was discovered for the first time by the present disclosure that it is important for appropriate evaluation of the evaluation items.

[0036] In one embodiment, the medium used in the present disclosure contains mucin, preferably at a concentration of 0.4% or more, or at any threshold, such as 0.2% or more, 0.3% or more, 0.5% or more, 0.6% or more, 0.7% or more, 0.8% or more, 0.9% or more, 1.0% or more, 1.5% or more, or 2.0% or more.

[0037] In one embodiment, the bacterial flora used in the present disclosure is preferably, but not limited to, obtained from feces. Preferably, the bacterial flora is obtained from human feces. To the inventors' knowledge, no system capable of reproducibly evaluating bacterial flora in humans has been provided, and the present disclosure is the first to successfully provide such a system.

[0038] In one embodiment, the amount of stool sample added to the culture medium used in the present disclosure may be 0.05% or more, preferably 0.1% or more, and / or 1.5% or less, 1.25% or less, 1% or less, etc.

[0039] In one embodiment, the present disclosure may be carried out in at least one well of a multi-well plate, such as a fermentor (jar type), 6-well, 12-well, 24-well, or 96-well plate.

[0040] (Method for "producing" a stable intestinal microbiota) In another aspect, the present disclosure provides a method for producing an equilibrated intestinal microbiota sample by culturing a specimen containing intestinal microbiota in a medium, the method comprising culturing the specimen for a period of time sufficient for culturing using a medium such that the Pearson product-moment correlation coefficient between the intestinal microbiota before culturing and the intestinal microbiota during culturing is a desired value of 0.60 or more, 0.65 or more, 0.70 or more, 0.50 or more, 0.80 or more, 0.85 or more, 0.90 or more, 0.95 or more, typically 0.70 or more. It is understood that in the method for "producing" a stable intestinal microbiota, any embodiment or combination thereof exemplified herein, for example, in (Evaluation method), can be used in this method.

[0041] (Time of Addition of Test Substance) In one embodiment, the improved evaluation method of the present disclosure is characterized in that the time of addition of the test substance to a sample containing intestinal microbiota is improved. More specifically, the present disclosure is characterized in that the test substance is added during the original equilibrium state. The addition time may be any time during the original equilibrium period, but from the standpoint of enabling a longer-term evaluation of the effect of the test substance on the intestinal microbiota after addition and evaluation efficiency, it is preferable to add the test substance at the beginning of the original equilibrium state. That is, after the start of culturing the Enterobacteria, the agent may be added 6 hours or more, 12 hours or more, 15 hours or more, 18 hours or more, 24 hours or more, 30 hours or more, 36 hours or more, 42 hours or more, 48 hours or more, and / or 96 hours or less, 90 hours or less, 84 hours or less, 78 hours or less, 72 hours or less, 66 hours or less, 60 hours or less, 54 hours or less, 48 ​​hours or less, for example, 6 to 90 hours, 6 to 24 hours, 12 to 48 hours, or 48 to 84 hours. In another embodiment, the agent may be added between 24 and 96 hours after the start of culturing, more preferably between 24 and 72 hours after the start of culturing. Alternatively, when the appropriate time for evaluation is between 72 and 96 hours after the start of culturing, particularly when 72 hours is advantageously used, it may be advantageous to add the agent between 6 and 24 hours after the start of culturing.

[0042] (Cultivation and Collection After Addition of Test Substance) In one embodiment, it may be advantageous to select the timing of sample acquisition for evaluation. After adding a test substance to a culture medium and further culturing, the culture medium is collected for evaluation. The sample may be collected at any time after adding the test substance to the culture medium, but it is preferably collected while the original equilibrium state is maintained. Specifically, the sample may be collected at any time after the addition of the test substance. For example, the sample may be 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 sampled 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. In one embodiment, it may be advantageous to obtain the sample for evaluation 24 to 96 hours, preferably 48 to 96 hours, after the start of culture.

[0043] (Culture Medium) The culture medium used in the present disclosure is not particularly limited as long as it is a medium in which enterobacteria can grow, and examples thereof include GAM medium, YCFA medium, modified YCFA medium, BBL medium, SOC medium, LB medium, etc. Among these, GAM medium, YCFA medium, and modified media thereof are 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.), YCFA medium, modified YCFA medium, etc. can be used. Two or more media selected from these media may be mixed in any ratio and used.

[0044] (Additional Components) In a preferred embodiment, various additional components can be added to the culture medium. For example, in an exemplary embodiment, fucoidan and / or sodium lactate may be added. Additional components added to the culture medium used in the present disclosure can also include high molecular weight glycoproteins. High molecular weight glycoproteins that can be used in the present disclosure include polypeptides comprising an amino acid sequence with a tandem repeat structure to which an O-linked glycan has been attached.

[0045] As used herein, a tandem repeat structure refers to a structure in which an amino acid sequence of one to several dozen amino acids in length is regularly repeated. Examples of O-linked sugar chains include, but are not limited to, O-mannose, O-N-acetylglucosamine, O-fucose, O-glucose, and O-galactose. In the high molecular weight glycoprotein of the present disclosure, GalNAc (N-acetylgalactomisan) 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.

[0046] 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.

[0047] The mucin may be of human or non-human origin, for example, porcine.

[0048] The amount of high-molecular-weight glycoprotein added to the medium is preferably 0.1% by mass or more, more preferably 0.2% by mass or more, and even more preferably 0.4% by mass or more, relative to the medium from the viewpoint of obtaining a pristine equilibrium state, and is preferably 4.0% by mass or less, more preferably 3.0% or less, and more preferably 2.0% or less, from the viewpoint of maintaining the bacterial flora structure.

[0049] In certain embodiments, additional components other than high molecular weight glycoproteins may be added to the medium, for example, carbon sources such as glucose, nitrogen sources such as ammonia, nutrient sources such as vitamins and inorganic salts, and scaffolds necessary for bacterial growth may be added as needed.

[0050] In the present disclosure, the medium may be sterilized, for example, by autoclaving, before culturing. Liquid culture is preferred, and the liquid culture is also referred to as a "culture medium." During culturing, the culture medium may be appropriately stirred.

[0051] (Intestinal Microbiota Used in the Present Disclosure and Sample Preparation) The intestinal microbiota used in the present disclosure may be obtained from a stool sample or from a sample other than stool. One or more specific intestinal bacteria may also be used. The sample may be from a human or a non-human animal, with human samples being preferred, and human stool being even more preferred. Furthermore, the stool sample may be obtained immediately after excretion from the intestine, may be frozen after collection, or may be collected from the intestine. After collection, the stool sample may be stored in a container such as an anaerobic culture swab until the start of culture. The collected stool may be mixed with phosphate buffer solution (PBS) to form a suspension. The phosphate buffer solution used to suspend the stool may also contain ascorbic acid or glycerin. The concentration of the stool in the stool suspension may be 0.01% (w / v) to 50% (w / v). From the viewpoint of bacterial flora diversity, the amount of stool sample to be inoculated into the culture medium is preferably 0.05% (w / v) or more in the culture solution, more preferably 0.1% (w / v) or more, and even more preferably 0.12% (w / v) or more. From the viewpoint of unintended bacterial growth, the amount is preferably 5.0% (w / v) or less, more preferably 2.5% (w / v) or less, and even more preferably 1.25% (w / v) or less. As used herein, "inoculation" refers to taking a fixed amount from a stool suspension and adding it to a culture medium.

[0052] (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. Furthermore, commercially available culture devices such as jar fermenters and shaker mixers can also be used.

[0053] (Culture Conditions - Atmosphere) In the present disclosure, the culture is carried out in an anaerobic environment. The anaerobic culture environment can be created by aerating an anaerobic gas into the culture medium. The anaerobic gas can be, for example, nitrogen, nitrogen and carbon dioxide, or nitrogen, carbon dioxide, and hydrogen. The anaerobic gas is aerated continuously or intermittently at a predetermined flow rate (e.g., 0.1 to 1.0 dL / min). Furthermore, since intestinal gas may contain, for example, nitrogen or carbon dioxide, the anaerobic gas is preferably a mixed gas consisting of nitrogen and carbon dioxide. Note that, in order to maintain highly anaerobic conditions, it is preferable to aerate the anaerobic gas continuously.

[0054] (Culture Conditions - pH) In the present disclosure, the pH of the culture medium at the start of culture is preferably 6.2 to 6.7, more preferably 6.2 to 6.5. By adjusting the pH to within the above range at the start of culture (for example, when the culture medium containing a fecal sample is placed in an anaerobic environment), it is possible to match the pH in the large intestine of a mammal corresponding to the feces used. It is sufficient that the pH of the culture medium at the start of culture is within the above range, and thereafter, the pH may be left as is without any particular adjustment, or the pH may be adjusted to within the above range using a pH adjuster as needed to prevent an extreme drop in pH.

[0055] (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 jar or flask, it is preferable to use a stirring blade or stirrer, or when using a multi-well plate, to agitate the culture solution during culture.

[0056] (Preparation and Amount of Test Substance Added) The test substance added in the present disclosure is preferably added in the range of 0.1 g to 50 g, more preferably 1 g to 20 g, per 1 L of culture solution. If the test substance is solid, it may be added after dissolving it in a solvent such as water.

[0057] (Acquisition and analysis of bacterial flora structure data) The collected culture solution is used to perform bacterial composition analysis and bacterial diversity analysis of the intestinal flora. Metagenomic analysis of the intestinal flora may be performed using 16sRNA gene sequences or whole genome sequences. For example, as in Reference Example 1 of JP 2021-153471 A, OTUs (Operational Taxonomic Units) that reach 97% similarity can be used to calculate the Shannon index and Pearson product-moment correlation coefficient. Details of the bacterial flora structure data analysis are described in the examples of this specification. Data such as the pH and short-chain fatty acid concentration of the collected culture solution can also be obtained as needed.

[0058] (When the timing of adding the test substance is determined in advance) The present disclosure may also include a step of determining the timing of adding the test substance in advance. The intestinal microbiota is cultured without adding the test substance, and the culture medium is periodically collected to obtain time-course data such as the Shannon index and Pearson product-moment correlation coefficient of the intestinal microbiota. From the obtained time-course data, the above-mentioned original equilibrium state can be determined, and the timing of adding the test substance can be determined.

[0059] 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.

[0060] 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.

[0061] Example 1 (Achieving Original Equilibrated Conditions (Jar Culture)) (Preparation of Fecal Suspension) Feces serving as an inoculum for intestinal bacterial flora were collected from healthy individuals on the day of culture. After collection, the fecal samples were stored in anaerobic culture swabs (212550 BD BBL Culture Swab; Becton, Tickinson & Company) and transported to the laboratory. To prepare the inoculum, 0.5 g of feces was 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 (Fujifilm Wako Pure Chemical Industries, Ltd.) to prepare a fecal suspension.

[0062] (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.

[0063] 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 (Able Co., Ltd., BJR-25NAIS-8M, hereinafter sometimes referred to as "jar") with a capacity of approximately 200 mL, and sterilized in an autoclave at 115°C for 15 minutes.

[0064] (Culture Conditions) After sterilization, anaerobic conditions were established in the culture vessel by aerating (15 mL / min) a mixed gas of nitrogen and carbon dioxide (N2:CO2 = 80:20 (volume ratio)) that had been sterilized by filtration through a 0.2 μm PTFE membrane (manufactured by Pall Corporation) for 1 hour at 37°C before culturing.

[0065] 500 μL of the fecal suspension was inoculated into a medium-containing vessel (0.125 g feces / 100 mL of culture solution), and anaerobic culture was initiated (culture time 0). During the culture, the medium was constantly bubbled with a sterilized, filtered mixed gas (N2:CO2 = 80:20 (volume ratio)) to maintain anaerobic conditions in the culture tank. The culture temperature was set to 37°C, and the incubation was performed with continuous stirring at approximately 300 rpm.

[0066] 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.

[0067] (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:

[0068] 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).

[0069] 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.

[0070] 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.

[0071] Shannon index and Pearson product-moment correlation coefficient were calculated using the QIIME software package.

[0072]

[0073] Table 1 shows the change in the Pearson product-moment correlation coefficient with the incubation time. After 6 hours of incubation, the Pearson product-moment correlation coefficient decreased to 0.74, but remained at 0.80 or higher between 24 and 96 hours of incubation.

[0074] From the above data on the Pearson product-moment correlation coefficient, the original equilibrium state can be determined to be in the range of 24 to 96 hours after the start of culture.

[0075] Example 2 (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; referred to as INU) as the test substance was added to the culture solution at 0.3% (w / v) 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.

[0076] Comparative Example 1 (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 to the culture medium at 0.3% (w / v) 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.

[0077] (Experimental Results) The percentage of Bifidobacterium bacteria present in the culture medium was compared for the culture medium of Example 1 (no inulin added), the culture medium of Example 2 to which inulin was added 24 hours after the start of culture, and the culture medium of Comparative Example 1 to which inulin was added at the start of culture. The results are shown in Table 2.

[0078]

[0079] The culture medium to which inulin, the test substance, was not added is referred to as "CUL," the culture medium to which inulin was added at the start of culture (0 hours) is referred to as "INU (0 h added)," and the culture medium to which inulin was added 24 hours after the start of culture is referred to as "INU (24 h added)."

[0080] Table 2 shows that in Comparative Example 1, in which inulin was added at the start of culture, the presence rate of Bifidobacterium was reduced at all culture times compared to the culture medium without inulin. On the other hand, in Example 2, in which inulin was added 24 hours after the start of culture, the presence rate of Bifidobacterium was improved at all culture times compared to Example 1, in which inulin was not added. The results showing that the presence rate of Bifidobacterium increased with the addition of inulin are consistent with the results of a study 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 could be reproduced in vitro by adding the test substance inulin after the original equilibrium state was reached.

[0081] Example 2A In Example 2, the timing of inulin addition was changed to 24 hours after the start of culture, and experiments were carried out at 6 hours, 12 hours, and 18 hours after the start of culture.

[0082] The results showed that the presence rate of Bifidobacterium was improved 48 and 72 hours after the start of culture, both 6, 12, and 18 hours after the start of culture, compared to Comparative Example 1, in which inulin was added at the start of culture. In particular, the improvement in the presence rate at 72 hours after the start of culture was remarkable, both 6, 12, and 18 hours after the start of culture.

[0083] Example 3 (Test substance: water-soluble indigestible dextrin added 24 hours after the start of culture) Cultivation and evaluation were carried out in the same manner as in Example 1, except that the test substance, water-soluble indigestible dextrin (Fibersol 2 (maltodextrin) manufactured by Matsutani Scientific; referred to as DEX), was added to the culture medium at 0.3% (w / v) 24 hours after the start of culture. The culture medium was sampled 48 hours, 72 hours, and 96 hours after the start of culture, and the abundance of bacteria of the genus Bifidobacterium and bacteria of the genus Faecalibacterium in each culture medium was determined. The results are shown in Tables 3 and 4.

[0084] Comparative Example 2 (Test Substance: Water-Soluble Indigestible Dextrin Added at the Start of Cultivation) Cultivation and evaluation were carried out in the same manner as in Example 1, except that the test substance, water-soluble indigestible dextrin (Fibersol 2 (maltodextrin), manufactured by Matsutani Scientific; referred to as DEX), was added to the culture medium at 0.3% (w / v) at the start of cultivation. The culture medium was sampled 48 hours, 72 hours, and 96 hours after the start of cultivation, and the abundance of bacteria of the genus Bacteroides and bacteria of the genus Faecalis bacterium in each culture medium was determined. The results are shown in Tables 3 and 4.

[0085]

[0086]

[0087] 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 (0 hours) is referred to as "DEX (0 h added)," and the culture solution to which dextrin was added 24 hours after the start of culture is referred to as "DEX (24 h added)."

[0088] Table 3 shows that in Comparative Example 2, in which dextrin was added at the start of culture, the abundance rate of bacteria of the genus Bacteroides was lower at all culture times compared to Example 1, in which no dextrin was added. On the other hand, in Example 3, in which dextrin was added 24 hours after the start of culture, the abundance rate of bacteria of the genus Bacteroides was higher at all culture times compared to the culture medium of Example 1, in which no dextrin was added.

[0089] Furthermore, Table 4 shows that in Comparative Example 2, in which dextrin was added at the start of culture, the abundance rate of bacteria of the genus Faecalibacterium was lower at all culture times compared to Example 1, in which no dextrin was added. On the other hand, in Example 3, in which dextrin was added 24 hours after the start of culture, the abundance rate of bacteria of the genus Faecalibacterium was higher at all culture times compared to the culture medium of Example 1, in which no dextrin was added.

[0090] These results are consistent with those of a study in which dextrin was administered to humans (Baer D.J. et al., J. Nutr, 144, 1014, pp. 1023-1029) and a study in which dextrin was administered to rats (Takagaki R. et al., Bioscience, Biotechnology, and Biochemistry, Vol. 84, Issue 4, 2020, pp. 824-831). This indicates that the addition of the test substance dextrin during the initial equilibration period can reproduce the in vivo test results in humans and rats in vitro.

[0091] Example 3A In Example 3, the timing of adding dextrin was changed to 24 hours after the start of culture, and experiments were carried out at 6 hours, 12 hours, and 18 hours after the start of culture.

[0092] The results showed that the abundance of bacteria of the genus Bacteroides was generally improved at 48 and 72 hours after the start of culture compared to Comparative Example 2, in which dextrin was added at the start of culture, at all times, 6 hours, 12 hours, and 18 hours after the start of culture. In particular, the improvement in the abundance at 72 hours after the start of culture was remarkable at all times, 6 hours, 12 hours, and 18 hours after the start of culture.

[0093] Example 4 (Multiwell Plate Culture) (Preparation of Fecal Suspension) Feces serving as an inoculum for intestinal bacterial flora were collected from healthy individuals on the day of culture. After collection, the fecal samples were stored in anaerobic culture swabs (212550 BD BBL Culture Swab; Becton, Tickinson & Company) and transported to the laboratory. To prepare the inoculum, 0.5 g of feces was suspended in 2 mL of 0.1 M phosphate buffer (PBS) buffer (pH 6.5, consisting of a 68.5:31.5 (molar ratio) mixture of 0.1 M NaH2PO4 and 0.1 M Na2HPO4) supplemented with 1.0% L-ascorbic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) to prepare a fecal suspension.

[0094] (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.

[0095] (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 agitated 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 set to 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 (the start of culture).

[0096] 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.

[0097] Example 5 (Test substance inulin added 24 hours after initiation of culture) Cultivation and evaluation were carried out in the same manner as in Example 4, except that inulin (derived from BENEO, OraftiGR, chicory; referred to as INU) as the test substance was added to the culture solution at 0.3% (w / v) 24 hours after initiation of culture. The culture solution was sampled 48 hours and 72 hours after initiation of culture, and the abundance rate of bacteria of the genus Bifidobacterium in each culture solution was determined.

[0098] Comparative Example 3 (Inulin Added at the Start of Culture) Cultivation and evaluation were carried out in the same manner as in Example 4, except that inulin (derived from BENEO, OraftiGR, chicory; referred to as INU) was added as the test substance to the culture medium at 0.3% (w / v) at the start of culture (0 hours into culture). The culture medium was sampled 48 hours and 72 hours after the start of culture, and the abundance of bacteria of the genus Bifidobacterium in each culture medium was determined.

[0099] The results of Examples 4 and 5 and Comparative Example 3 are shown in Table 5.

[0100]

[0101] In Table 5, it can be seen that in Comparative Example 3, in which inulin was added at the start of culture, the presence rate of Bifidobacterium was reduced at all culture times compared to Example 4, in which inulin was not added. On the other hand, in Example 5, in which inulin was added 24 hours after the start of culture, the presence rate of Bifidobacterium was improved at all culture times compared to Example 4, 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.

[0102] Example 6 (Test substance, water-soluble indigestible dextrin, added 24 hours after the start of culture (multi-well plate)) Cultivation and evaluation were carried out in the same manner as in Example 4, except that the test substance, water-soluble indigestible dextrin (Fibersol 2 (maltodextrin), manufactured by Matsutani Scientific; referred to as DEX), was added to the culture medium at 0.3% (w / v) 24 hours after the start of culture.

[0103] Comparative Example 4 (Water-soluble, indigestible dextrin added at the start of culture (multi-well plate)) Culture and evaluation were carried out in the same manner as in Example 4, except that water-soluble, indigestible dextrin (Fibersol 2 (maltodextrin), manufactured by Matsutani Scientific Co., Ltd.; referred to as DEX) was added as the test substance at 0.3% (w / v) to the culture medium at the start of culture.

[0104] The results of Examples 4 and 6 and Comparative Example 4 are shown in Table 6.

[0105]

[0106] 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 (0 hours) is referred to as "DEX (0 h added)," and the culture solution to which dextrin was added 24 hours after the start of culture is referred to as "DEX (24 h added)."

[0107] In Table 6, it can be seen that in Comparative Example 4, in which dextrin was added at the start of culture, the presence rate of Faecali- bacterium was reduced at any culture time compared to Example 4, in which dextrin was not added. On the other hand, in Example 6, in which dextrin was added 24 hours after the start of culture, the presence rate of Faecali- bacterium was increased at any culture time compared to the culture medium to which dextrin was not added. This result is consistent with the test results in which rats were given dextrin (Takagaki R et al., Bioscience, Biotechnology, and Biochemistry, Vol. 84, Issue 4, 2020, pp. 824-831). In other words, it was shown that by adding the test substance dextrin during the original equilibrium state, it is possible to reproduce the in vivo test results in vitro.

[0108] Example 7 (Original Equilibration State Up to 24 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. The results are shown in Table 7.

[0109]

[0110] Table 7 shows the change in the Pearson product-moment correlation coefficient with the incubation time. After 6 hours of incubation, the Pearson product-moment correlation coefficient decreased to 0.77, but remained above 0.8 between 12 and 24 hours of incubation. Therefore, it can be seen that the original equilibrium state can be achieved within 12 hours of incubation.

[0111] Example 8 (Preparation, storage and incubation of fecal suspensions) In this example, an experiment was conducted on the storage method of fecal samples as an example of starting conditions.

[0112] Fecal samples, which served as the inoculum source for the intestinal flora, were collected from healthy individuals and stored in anaerobic culture swabs (212550 BD BBL Culture Swab; manufactured by Becton, Tickinson and Company) in a 10% glycerol solution at −80°C. The samples were thawed and resuspended 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.) equivalent to 0.5 g of feces. ２ P.O. ４ and 0.1 M Na ２ HPO ４ A fecal suspension was prepared by adding a 68.5:31.5 (molar ratio) mixture of the above (a 68.5:31.5 mixture of the above) to a total volume of 2 mL.

[0113] Incubation with frozen samples yielded similar Pearson's cumulative coefficients up to 96 hours after incubation compared with incubation with fresh fecal samples.

[0114] Example 9 (Multiwell Plate Culture) In this example, culture media were examined in multiwell plate culture. (Preparation of Fecal Suspension) Feces serving as an inoculum for intestinal bacterial flora were collected from healthy individuals on the day of culture. After collection, the fecal samples were stored in anaerobic culture swabs (212550 BD BBL Culture Swab; manufactured by Becton, Tickinson and Company) and transported to the laboratory. To prepare the inoculum, 0.5 g of feces was suspended in 0.1 M phosphate buffer (PBS) buffer (pH 6.5, 0.1 M NaHCO3) supplemented with 1.0% L-ascorbic acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). ２ P.O. ４ and 0.1 M Na ２ HPO ４ A fecal suspension was prepared by suspending the feces in 2 mL of a 68.5:31.5 (molar ratio) mixture of

[0115] (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.

[0116] (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., may be referred to as a 96-well plate in this example). The multi-well plate was agitated 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 (the start of culture).

[0117] 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.

[0118] (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:

[0119] 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).

[0120] 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.

[0121] Example 10 (Study on GAM medium containing mucin) Cultivation and bacterial flora analysis were carried out in the same manner as in Example 9, 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.

[0122] Example 11 (Study on Modified GAM Medium) Cultivation and bacterial flora analysis were performed in the same manner as in Example 9, 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.

[0123] Example 12 (Study on modified GAM medium containing mucin) Cultivation and bacterial flora analysis were carried out in the same manner as in Example 11, except that in preparing the modified GAM medium, 8.0 g / L or 4.0 g / L of mucin (Sigma-Aldrich, derived from porcine stomach, Type III) was added to the modified GAM medium.

[0124] Example 13 (Study on YCFA medium) Culture and bacterial flora analysis were performed in the same manner as in Example 9, 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 volatile fatty acids per liter. The medium was prepared by mixing 3.1 ml (acetic acid 2.026 ml / L, propionic acid 0.715 ml / L, n-valeric acid 0.119 ml / L, isovaleric acid 0.119 ml / L, isovaleric acid 0.119 ml / L), vitamin mixture 1: 1 ml (biotin 1 mg / 100 ml, cyanocobalamin 1 mg / 100 ml, p-aminobenzoic acid 3 mg / 100 ml, folic acid 5 mg / 100 ml, pyridoxine 15 mg / 100 ml), vitamin mixture 2: 1 ml (thiamine 5 mg / 100 ml, riboflavin 5 mg / 100 ml) and 50 μL / L of antifoaming agent, and the pH was adjusted to 7.5 with a pH adjuster.

[0125] Example 14 (Study on YCFA medium containing mucin) Cultivation and bacterial flora analysis were carried out in the same manner as in Example 13, 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.

[0126] Example 15 (Modified YCFA Medium) Cultivation and bacterial flora analysis were performed in the same manner as in Example 9, 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.

[0127] Example 16 (Study on modified YCFA medium containing mucin) Cultivation and bacterial flora analysis were performed in the same manner as in Example 15, 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 the preparation of 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.

[0128] Table 6 shows the Pearson product-moment correlation coefficients after 72 hours of culture, relative to the results of Examples 9, 10, 11, and 12 in various media, which were set at 100, and the Pearson product-moment correlation coefficients of Examples 13, 14, 15, and 16 in mucin-supplemented media. The results in Table 8 clearly show that the addition of mucin was effective in maintaining the original equilibrium state in all media.

[0129]

[0130] Example 17 (Amount 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 9 and 10. Specific primers for Faecalibacterium duncaniae (Fd bacteria) and Blautia wexlerae (Bw bacteria) targeting the 16S rRNA gene of each bacteria were used to quantify the target bacterial genes from the extracted genomic DNA using a quantitative PCR device (Table 9).

[0131] The results in Table 9 show that when cultured in each medium without mucin, the abundance of the useful bacteria Fd and Bw decreased, whereas the addition of mucin to all media maintained Fd and Bw bacteria during culture, and their abundance was equal to or greater than that of the original sample (Fec), demonstrating its effectiveness in maintaining these useful bacteria.

[0132]

[0133] (Study of Mucin) Example 18 (Study of Medium Containing Porcine Type II Mucin) Cultivation and bacterial flora analysis were carried out in the same manner as in Example 9, 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.

[0134] Example 19 (Study on medium containing porcine mucin) Cultivation and bacterial flora analysis were carried out in the same manner as in Example 9, 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.

[0135] Example 20 (Study on medium containing skate mucin) Cultivation and bacterial flora analysis were carried out in the same manner as in Example 9, except that 8.0 g / L of mucin (derived from skate, manufactured by Marukyo Suisan Co., Ltd.) was added to GAM medium in the preparation of the medium. Relative occupancy rates were calculated from the classification data at the genus level for bacterial species attribution, and the Pearson product-moment correlation coefficient was calculated based on the relative occupancy rates.

[0136] Comparative Example 7 (Study on Mucin-Free Medium) In the preparation of the medium, the culture and bacterial flora analysis were carried out in the same procedures as in Example 9.

[0137] Table 10 shows the relative values ​​of the Pearson product-moment correlation coefficients for Examples 9, 18, 19, and 20 for the Pearson product-moment correlation coefficients after 72 hours of culture, with the result for Comparative Example 7 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.

[0138]

[0139] Example 21 (Administration of Culture Solution Preparation to Mice) The human intestinal flora culture solution prepared in Example 10 was administered to SPF mice, and they were subjected to a high-fat diet load to measure insulin sensitivity. The administration of the microorganism to mice and the efficacy evaluation test were performed according to the method described in Nature Communications (202) 13:4477. 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 solution prepared in Example 10 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.

[0140] (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. 2023-196981, filed with the Japan Patent Office on November 20, 2023, the contents of which are incorporated herein by reference in their entirety.

[0141] According to the method of the present disclosure, it is possible to evaluate in vitro the effects of test substances such as foods and candidate pharmaceutical compounds on the intestinal flora of mammals, particularly humans.

Claims

1. A method for evaluating a test substance in an intestinal bacterial flora, comprising the following steps: A) culturing the intestinal bacterial flora in a medium for a period of time effective for the intestinal bacterial flora to reach an original equilibrium state after the start of culture by inoculating the medium with the intestinal bacterial flora; B) adding the test substance to the medium containing the intestinal bacterial flora after the period of time has elapsed; and C) obtaining and evaluating evaluation items before and after the addition of the test substance.

2. A method for evaluating a test substance in an intestinal bacterial flora, comprising the following steps: A) culturing the intestinal bacterial flora by inoculating it into a culture medium; B) adding the test substance to the culture medium containing the intestinal bacterial flora after the specified time has elapsed, the specified time being determined based on a stabilization judgment index; and C) obtaining and evaluating evaluation items before and after the addition of the test substance.

3. The method according to claim 1 or 2, wherein the test substance is added to the medium at least 6 hours after the start of culture.

4. The method according to any one of claims 1 to 3, wherein the test substance is added to the medium after 24 hours or more have elapsed since the start of the culture.

5. A method according to any one of claims 1 to 4, wherein the evaluation items after addition of the test substance are obtained while the original equilibration condition is maintained.

6. The method according to any one of claims 1 to 5, wherein the evaluation item after addition of the test substance is obtained within 96 hours after the start of the culture.

7. A method according to any one of claims 1 to 6, wherein the evaluation item after addition of the test substance is obtained within 48 to 72 hours after the start of the culture.

8. The method of any one of claims 1 to 7, wherein the original equilibration condition comprises equilibrating the microbiota diversity to the state of a pre-inoculation sample of the intestinal microbiota.

9. The method according to any one of claims 1 to 8, wherein the original equilibrium state is determined based on a structural analysis of the intestinal flora.

10. The method of any one of claims 1 to 9, wherein the original equilibrium state is determined based on 16S or genomic analysis of the gut microbiota.

11. The method according to any one of claims 1 to 10, wherein the structural analysis is a Pearson product-moment correlation coefficient.

12. The method of any one of claims 1 to 11, wherein the original balanced state is determined by a Pearson product moment correlation coefficient of 0.70 or greater.

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

14. A method according to any one of claims 1 to 13, characterized in that the original equilibrium state is determined from a time range in which a state in which the Pearson product moment correlation coefficient between the bacterial flora structure before and after cultivation of the intestinal bacteria is 0.70 or more is maintained for 24 hours or more.

15. The method according to any one of claims 1 to 14, wherein the medium contains mucin.

16. The method according to claim 15, wherein the mucin is contained in the culture medium at a concentration of 0.4% or more.

17. The method according to any one of claims 1 to 16, wherein the intestinal flora is obtained from stool.

18. The method of any one of claims 1 to 17, wherein the intestinal flora is obtained from human stool.

19. The method according to any one of claims 1 to 18, wherein the amount of the stool sample added is 0.1% or more and 1.25% or less relative to the medium.

20. The method of any one of claims 1 to 19, which is carried out in at least one well of a multi-well plate.

21. A method for producing an equilibrated intestinal flora sample by culturing a specimen containing intestinal flora in a culture medium, the method comprising the step of culturing the specimen for a period of time sufficient for culturing using a culture medium in which the Pearson product moment correlation coefficient between the intestinal flora before culture and the intestinal flora during culture is 0.70 or greater.

22. The method according to any one of claims 1 to 21, wherein the intestinal microbiota is provided in a frozen state.

23. The method according to any one of claims 1 to 21, wherein the intestinal microbiota is provided in a state obtained from a subject.

Images