Method for producing artificial intestinal microbiota

Abstract

To provide a method for producing an artificial intestinal microbiota including a step of culturing a solution containing cells in which two or more types of glycerol stocks including a microbiota cultured in a culture medium containing a medium additive are mixed and prepared.SOLUTION: Provided is a method for producing an artificial intestinal microbiota including a step of culturing a solution containing cells in which two or more types of glycerol stocks including a microbiota cultured in a culture medium containing a medium additive are mixed and prepared.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing an artificial intestinal microbiota. The production method of the present invention is characterized by comprising a step of culturing a solution containing cells (cell solution) prepared by mixing two or more types of glycerol stocks containing bacterial microbiota cultured in a medium containing a medium additive. The present invention also relates to an intestinal microbiota model, an intestinal microbiota improver, etc., which contain the artificial intestinal microbiota produced by the production method. [Background technology]

[0002] The human gut microbiota has a significant impact on host health and disease and is essential for the normal maturation of the host's immune system. Therefore, evaluation systems that simulate the human gut are important in the fields of medicine and healthcare. Representative systems include (i) in vivo fecal transplant mouse models, (ii) ex vivo organoid models, and (iii) in vitro fecal culture models (hereinafter sometimes referred to as gut simulators) (Non-Patent Document 1). Model (i) does not reflect the human gut microbiota and is therefore not reproducible due to the use of fresh human feces. Model (ii) makes it difficult to investigate the interaction between the human gut microbiota, including anaerobic bacteria, and organoids. Model (iii) is generally not reproducible due to the use of fresh human feces. Existing human gut evaluation systems struggle to simulate the human gut environment while achieving reproducibility, making them unsuitable for non-clinical trials in drug development, where reproducibility is required. Furthermore, fecal transplantation therapy requires the preparation of large quantities of fresh human feces on the same day, necessitating the establishment of a preservation method.

[0003] The above-mentioned gut simulators are divided into batch culture systems and continuous culture systems. However, to the best of the inventor's knowledge, no method has been developed to reproduce an artificial gut microbiota that is close to the standard human gut bacteria, regardless of whether a batch culture system or a continuous culture system is used (Patent Document 1 and Non-Patent Document 2). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2015 / 136916 [Non-patent literature]

[0005] [Non-Patent Document 1] Aguanno D, et al., Microbiome Res Rep. 1(3): 17, 2022 [Non-patent document 2] Bircher L, et al., mSystems. 5(1): e00521-19, 2020. Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, an objective of the present invention is to provide a method for producing an artificial intestinal microbiota. In particular, an objective of the present invention is to provide a method for producing an artificial intestinal microbiota using a preserved stock (glycerol stock) of fecal culture medium. Another objective of the present invention is to provide an intestinal microbiota model, an intestinal microbiota improver, etc., which include the artificial intestinal microbiota produced by the production method.

[0007] In investigating a method for producing an artificial intestinal microbiota, the present inventors considered the difficulty of preparing a large amount of fresh feces and therefore attempted to use fecal culture medium as an inoculum source for reproducing an artificial intestinal microbiota. They also considered using glycerol as a cryoprotectant for storing fecal culture medium, considering its cytotoxicity. Furthermore, when reproducing an artificial intestinal flora using a glycerol stock of culture medium, there was a risk that the main bacteria in fresh human stool (e.g., Faecalibacterium) might decrease, so we came up with the idea of ​​using two or more types of glycerol stock.

[0008] As a result of extensive research, the inventors surprisingly found that producing an artificial intestinal microbiota using two or more types of glycerol stocks can retain sufficient amounts of the major intestinal bacteria that make up the human intestinal microbiota, and can reproduce an artificial intestinal lumen microbiota that mimics the human intestinal microbiota as closely as possible. Based on these findings, the inventors conducted further research and have completed the present invention.

[0009] That is, the present invention is as follows. [1] A method for producing an artificial intestinal microbiota, comprising a step of culturing a solution containing cells prepared by mixing two or more types of glycerol stocks containing bacterial microbiota cultured in a medium containing a medium additive. [2] The method according to [1], wherein the medium additives contained in the glycerol stock preparation include one or more selected from the group consisting of prebiotics, dietary fiber, glycoproteins, and health food materials. [3] The method according to [1], wherein the medium additives included in the preparation of the glycerol stock include one or more selected from the group consisting of pectin, guar gum, fructooligosaccharide, xylan, and resistant starch. [4] The method according to [3], wherein one medium additive included in the preparation of a glycerol stock is guar gum, and at least one medium additive included in the preparation of a glycerol stock other than the glycerol stock is pectin, guar gum, fructooligosaccharide, and xylan. [5] The method according to [3], wherein at least one medium additive contained in the preparation of the glycerol stock other than the glycerol stock containing the bacterial flora cultured in the medium containing guar gum and the glycerol stock containing the bacterial flora cultured in the medium containing pectin, guar gum, fructooligosaccharide, and xylan is pectin. [6] The method according to any one of [1] to [5], wherein the artificial intestinal microbiota is an artificial human intestinal microbiota. [7] A gut microbiota model comprising an artificial gut microbiota produced by the method according to any one of [1] to [6]. [8] An agent for improving intestinal flora, comprising an artificial intestinal flora produced by the method according to any one of [1] to [6]. [9] A combination agent comprising the intestinal flora-improving agent according to [8] and an antibacterial agent. [Effects of the Invention]

[0010] The present invention makes it possible to reproduce an artificial intestinal lumen microbiota that retains sufficient amounts of the major intestinal bacteria that constitute the human intestinal microbiota and mimics the human intestinal microbiota as closely as possible. Furthermore, the artificial intestinal microbiota produced by the production method of the present invention retains sufficient amounts of the major intestinal bacteria that constitute the human intestinal microbiota, is an artificial intestinal lumen microbiota that mimics the human intestinal microbiota as closely as possible, and is also reproducible. Therefore, this artificial intestinal microbiota can be used to establish a model (evaluation system) for human intestinal microbiota research. Furthermore, the artificial intestinal microbiota in the glycerol stock mixture and the artificial intestinal microbiota produced therefrom retain sufficient amounts of the major intestinal bacteria that constitute the human intestinal microbiota and are an artificial intestinal lumen microbiota that mimics the human intestinal microbiota as closely as possible. Therefore, they can be used to improve the intestinal microbiota, for example, by transplantation. Furthermore, the artificial intestinal microbiota produced by the production method of the present invention can be transplanted to treat or prevent diseases that are candidates for fecal transplantation (Clostridium difficile infection, Crohn's disease, ulcerative colitis, nonspecific multiple small intestinal ulcers, intestinal Behçet's disease, small intestinal bacterial overgrowth (SIBO), drug-resistant irritable bowel syndrome, etc.). [Brief explanation of the drawings]

[0011] [Figure 1]Figure 1 shows the specific procedure for recreating an artificial gut microbiota in a gut simulator using glycerol stocks derived from human fecal cultures. For human subjects (F1), 100 μl of fresh fecal suspension was cultured for 48–52 hours in 100 ml of GAM medium containing 0.4% pectin (PE), 0.4% guar gum (GG), or 0.2% fructooligosaccharides (FOS) + 0.1% xylan (XL) + 0.2% PE + 0.1% GG. Glycerol stocks were then constructed from these fecal cultures. After storage, 250 μl of each of these glycerol stocks, either individually or in combination, was inoculated and cultured for 72 hours in 100 ml of GAM medium containing 0.2% FOS + 0.05% XL + 0.1% PE + 0.025% GG + 0.1% (resistant starch) RS. [Figure 2] Figure 2 shows the relative abundances of (a) family and (b) genus levels in the microbiota from human F1, as determined by 16S rRNA sequencing. Abbreviations: f (family), g (genus), control (no additives), GG (guar gum (0.4%)), PE (pectin (0.4%)), FXPG (0.2% fructooligosaccharides (FOS) + 0.1% xylan + 0.2% PE + 0.1% GG), FXPGR (0.2% FOS + 0.05% xylan + 0.1% PE + 0.025% GG + 0.1% resistant starch). [Figure 3] Figure 3 shows the ratios of short-chain fatty acids (SCFAs) produced by the recapitulated artificial gut microbiota of human subject F1. [Figure 4] Figure 4 shows a visual overview of the intestinal simulator for the artificial intestine and mucosal microbiota. Each vessel contains agitated autoclaved culture medium. The system was maintained at 37 °C and purged with a gas mixture. Each vessel contained five pieces of mucin agar, optionally fitted with a Teflon mesh. [Figure 5]Figure 5 shows the relative abundances of (a) the family level and (b) the genus level of the microbiota from human subject F2, as determined by 16S rRNA sequencing. Abbreviations: f (family), g (genus), control (no additives), GG (guar gum (0.2%)), PE (pectin (0.2%)), FXPG (0.14% fructooligosaccharides (FOS) + 0.07% xylan + 0.14% PE + 0.07% GG), FXPGR (0.36% FOS + 0.09% xylan + 0.18% PE + 0.045% GG + 0.18% resistant starch). [Figure 6] Figure 6 shows the relative abundances of (a) the family level and (b) the genus level of the microbiota from human subject F3, as determined by 16S rRNA sequencing. Abbreviations: f (family), g (genus), control (no additives), GG (guar gum (0.4%)), PE (pectin (0.4%)), FXPG (0.10% fructooligosaccharides (FOS) + 0.05% xylan + 0.10% PE + 0.05% GG), FXPGR (0.24% FOS + 0.06% xylan + 0.12% PE + 0.030% GG + 0.12% resistant starch). DETAILED DESCRIPTION OF THE INVENTION

[0012] 1. Method for producing the artificial intestinal microbiota of the present invention The present invention provides a method for producing an artificial intestinal microbiota (hereinafter sometimes referred to as the "production method of the present invention"), which comprises the step of culturing a solution containing cells (cell solution) prepared by mixing two or more types of glycerol stocks containing bacterial flora cultured in a medium containing medium additives.

[0013] The glycerol stock used in the present invention can be prepared using a fecal culture medium obtained by culturing a fecal sample collected from a subject in a medium containing a medium additive. Fecal samples can be collected by methods known per se (e.g., Sasaki et al. Sci Rep. 2021 Jul 16;11(1):14627. doi: 10.1038 / s41598-021-94210-8). Fecal samples may be cultured immediately after collection to prepare a glycerol stock without storage, or may be cultured after storage in a container such as an anaerobic culture swab. From the perspective of maintaining the subject's bacterial flora, it is preferable to start culturing within 24 hours after collection.

[0014] The glycerol stock used in the present invention may be prepared using a stool culture solution obtained by adding, for example, genome-editing specific bacteria contained in the stool to give them a desired function, and then culturing the bacteria in a medium containing a medium additive, separate from a stool sample collected from the subject.

[0015] The subject is not particularly limited, and examples thereof include humans and non-human mammals (e.g., monkeys, cows, horses, pigs, mice, rats, guinea pigs, hamsters, dogs, cats, rabbits, sheep, goats, etc.), preferably humans. In one embodiment, the subject is a healthy subject. In one embodiment, a "healthy subject" refers to a subject who is not suffering from at least one of the diseases for which fecal transplantation is performed (Clostridium difficile infection, Crohn's disease, ulcerative colitis, nonspecific multiple small intestinal ulcers, intestinal Behçet's disease, small intestinal bacterial overgrowth (SIBO), and drug-resistant irritable bowel syndrome). In another embodiment, a "healthy subject" refers to a subject for whom no abnormalities have been detected in a medical examination.

[0016] It is most preferable that the glycerol stock used in the present invention is capable of maintaining the subject's (fecal) bacterial flora, but in one embodiment, it is preferable that at least the major bacteria of the subject's bacterial flora be maintained. When comparing each of the major bacteria of the subject's bacterial flora (e.g., Bifidobacterium) with each of the bacteria in the glycerol stock (e.g., Bifidobacterium), it is more preferable that they are present in the glycerol stock at 10% or more (e.g., Bifidobacterium in glycerol stock / Bifidobacterium in subject's (fecal) bacterial flora × 100≧10%).

[0017] In one embodiment, the primary bacteria is a species of the genus Bifidobacterium, Bacteroides, Prevotella, Faecalibacterium, or Escherichia (particularly Escherichia-Shigella). Furthermore, it is preferable that minor bacteria are not significantly increased (for example, increased by 1% or more) compared to the bacterial flora of the subject. In one embodiment, the minor bacteria are Peptostreptococcus or Paraclostridium.

[0018] The medium additives are not particularly limited, but examples thereof include prebiotics, dietary fiber (preferably water-soluble dietary fiber), glycoproteins, health food materials, etc. As used herein, "prebiotics" generally refers to substances that are not broken down or absorbed in the upper digestive tract, serve as a selective nutrient source for beneficial bacteria that live symbiotically in the large intestine, promote their proliferation, improve and maintain a healthy balance of intestinal flora in the large intestine, and are useful for promoting and maintaining human health. Examples of prebiotics and dietary fibers include galactooligosaccharides, fructooligosaccharides, soybean oligosaccharides, lactoferrin oligosaccharides, xylooligosaccharides, isomaltose oligosaccharides, coffee bean mannooligosaccharides, raffinose, lactosucrose, 1-kestose, 4'-galactosyllactose, lactulose, cellobiose, isomaltose, lactitol, gluconic acid, polydextrose, guar gum (including guar gum hydrolysates), alginate, pectin, isomaltodextrin, resistant starch, barley β-glucan, inulin, indigestible dextrin, xylan, etc. In one embodiment, pectin, guar gum, fructooligosaccharides, xylan, and resistant starch are preferred. Examples of glycoproteins include lactoferrin, mucin, extensin, and arabinogalactan-protein (AGP). Examples of health food ingredients include green juice, Euglena, and barley leaf extract. In one aspect, the medium additive included in the preparation of a glycerol stock of the present invention is one or more selected from the group consisting of pectin, guar gum, fructooligosaccharide, xylan, and resistant starch.

[0019] The amounts of prebiotics, dietary fiber (preferably water-soluble dietary fiber), glycoproteins, and health food ingredients used in the preparation of the glycerol stock used in the present invention are 0.01 to 1.00% by weight. In one embodiment, the amounts of pectin are 0.10 to 0.40% by weight, guar gum is 0.025 to 0.40% by weight, fructooligosaccharides are 0.10 to 0.36% by weight, xylan is 0.050 to 0.10% by weight, and resistant starch is 0.10 to 0.18% by weight.

[0020] The medium for culturing a fecal sample is not particularly limited as long as it is a medium that can be used for anaerobic culture. The fecal sample may be prepared in advance as a suspension (e.g., suspended in 0.1 M phosphate buffer supplemented with 1% L-ascorbic acid), and 100 μl of the suspension may be added to 100 ml of medium. Examples of anaerobic culture media include Gifu University Anaerobic Medium (GAM medium), such as GAM bouillon and modified GAM bouillon (both manufactured by Nissui Pharmaceutical Co., Ltd.). Agar may also be added to the medium, and mucin (purified mucin), N-acetylneuraminic acid (NeuAc), etc. may be added to the agar as appropriate. The medium may be sterilized (e.g., autoclaved) before culturing. Liquid culture is preferred, and the culture solution may be appropriately stirred during culturing. The stirring speed is, for example, 100 to 400 rpm. Examples of medium additives contained in the medium for culturing a fecal sample include the above-mentioned prebiotics, dietary fiber (preferably water-soluble dietary fiber), glycoproteins, and health food materials. Typically, the amount of prebiotics, dietary fiber (preferably water-soluble dietary fiber), glycoproteins, and health food materials in the medium is 0.01 to 1.00 wt%. In one embodiment, the amounts of pectin are 0.10 to 0.40 wt%, guar gum are 0.025 to 0.40 wt%, fructooligosaccharides are 0.10 to 0.36 wt%, xylan is 0.050 to 0.10 wt%, and resistant starch are 0.10 to 0.18 wt%.

[0021] An anaerobic culture environment can be achieved by aerating an anaerobic gas into the medium. Examples of anaerobic gases include nitrogen, nitrogen and carbon dioxide, or nitrogen, carbon dioxide and hydrogen. The anaerobic gas may be continuously or intermittently aerated at a flow rate of, for example, 0.1 to 1.0 dl / min. The anaerobic gas is preferably a mixed gas consisting of nitrogen and carbon dioxide. It is also preferable to continuously aerate the anaerobic gas.

[0022] In the present invention, the pH of the culture medium for anaerobic culture is 6.2 to 6.7, preferably 6.2 to 6.5, at least at the start of culture. At the start of culture (for example, when the medium containing the fecal sample is placed in an anaerobic environment), the pH of the culture medium should be within the above range, and the pH may be adjusted to within the above range using a pH adjuster, if necessary. The pH adjuster may be appropriately selected from those known per se. After the start of culture, the pH is checked appropriately. During culture, it is not necessary to perform operations intended to maintain the pH within the above range (for example, pH adjustment by adding a pH adjuster or alkali, etc.).

[0023] The culture period can be set appropriately, for example, 24 to 100 hours, preferably 48 to 54 hours. The culture temperature is set to a temperature close to the body temperature of the subject, for example, 36°C to 38°C, preferably 37°C, when the subject is a human. The culture method is not particularly limited, but a single batch method is preferred.

[0024] The glycerol stock used in the present invention may be used immediately after preparation for the method for producing an artificial intestinal bacterial flora of the present invention, or may be frozen and stored until use.

[0025] In the present invention, "mixing two or more glycerol stocks containing bacterial flora cultured in media containing media additives" refers to mixing two or more glycerol stocks in which the media additives contained in at least one glycerol stock and the media additives contained in at least one other glycerol stock are not the same. Examples of media additives include prebiotics, dietary fiber (preferably water-soluble dietary fiber), glycoproteins, and health food ingredients, as described above. Furthermore, the media used for the two or more glycerol stocks in the present invention may be the same or different. In one embodiment, all of the media are Gifu University Formulated Anaerobic Medium (GAM Medium).

[0026] In one aspect, the medium additives included in the preparation of the glycerol stock used in the present invention include one or more selected from the group consisting of pectin, guar gum, fructooligosaccharide, xylan, and resistant starch.

[0027] In one embodiment, the medium additive included in the preparation of at least one glycerol stock is guar gum, and the medium additives included in at least one glycerol stock other than the glycerol stock are pectin, guar gum, fructooligosaccharides, and xylan.

[0028] In one aspect, pectin is a medium additive included in the preparation of at least one glycerol stock other than a glycerol stock containing a bacterial flora cultured in a medium containing guar gum and a glycerol stock containing a bacterial flora cultured in a medium containing pectin, guar gum, fructooligosaccharides, and xylan.

[0029] The medium contained in the culture solution prepared by mixing two or more glycerol stocks containing bacterial flora cultured in a medium containing a medium additive in the production method of the present invention is not particularly limited as long as it is a medium that can be used for anaerobic culture. In one embodiment, the medium contained in the culture solution is the same as that used when preparing the glycerol stock.

[0030] The culture method for the above culture medium may be the same as the method used to prepare the glycerol stock used in the present invention. The culture time can be appropriately set from the perspective of bacterial growth, but is typically, for example, 48 to 120 hours, and preferably 48 to 72 hours. It is also preferable to culture after adding medium additives (e.g., prebiotics, dietary fiber (preferably water-soluble dietary fiber), glycoproteins, health food materials, etc.) contained in the glycerol stock used in the production method of the present invention. Typically, the amount of prebiotics, dietary fiber (preferably water-soluble dietary fiber), glycoproteins, and health food materials in the culture medium is 0.01 to 1.00% by weight. In one embodiment, the pectin is 0.10 to 0.40% by weight, the guar gum is 0.025 to 0.40% by weight, the fructooligosaccharide is 0.10 to 0.36% by weight, the xylan is 0.050 to 0.10% by weight, and the resistant starch is 0.10 to 0.18% by weight.

[0031] The artificial intestinal microbiota obtained by the production method of the present invention typically maintains the major bacteria of the subject's (fecal) microbiota used to prepare the glycerol stock. Furthermore, when comparing the major bacteria of the subject's microbiota (e.g., Bifidobacterium) with the respective bacteria in the artificial intestinal microbiota (e.g., Bifidobacterium), it is preferable that the major bacteria in the subject's microbiota (e.g., Bifidobacterium) account for 10% or more of the total in the artificial intestinal microbiota (e.g., Bifidobacterium in the artificial intestinal microbiota / Bifidobacterium in the subject's (fecal) microbiota × 100≧10%).

[0032] In one embodiment, the primary bacteria is a species of the genus Bifidobacterium, Bacteroides, Prevotella, Faecalibacterium, or Escherichia (particularly Escherichia-Shigella).

[0033] Typically, the artificial intestinal microbiota obtained by the production method of the present invention maintains beneficial bacteria from the (fecal) microbiota of the subject used to prepare the glycerol stock. In one embodiment, the beneficial bacteria is at least one selected from the group consisting of Collinsella, Eubacterium halii group, Anaerostipes, Blautia, Dorea, Fusicatenibacter, Lachnospiraceae ND3007 group, Lachnospiraceae UCG-004, and Oscillospiraceae UCG-003.

[0034] Furthermore, in the artificial intestinal microbiota obtained by the production method of the present invention, the number of minor bacteria is typically not significantly increased (e.g., not less than 1%) compared to the (fecal) microbiota of the subject. Preferably, the number is not more than 0.6%. In one embodiment, the minor bacteria are Peptostreptococcus and Paraclostridium.

[0035] As described above, the artificial intestinal microbiota obtained by the production method of the present invention at least reflects the (fecal) microbiota of the subject used to prepare the glycerol stock, and is therefore suitable as an intestinal microbiota model. Furthermore, because this intestinal microbiota model reflects or maintains the intestinal microbiota of a living organism (e.g., a human), it is also suitable for screening drugs and prebiotics, including live bacterial preparations, intended to control the intestinal microbiota. The live bacteria contained in the live bacterial preparation may be prepared by genome editing. Furthermore, for example, the artificial intestinal microbiota obtained by the production method of the present invention is also suitable for screening drugs that ameliorate the change in the microbiota after administering a drug (e.g., a live bacterial preparation, an anticancer drug, an antibacterial agent, an anti-inflammatory agent, etc.) to the artificial intestinal microbiota obtained by the production method of the present invention.

[0036] For example, in the above screening, when the glycerol stock used to prepare the intestinal microbiota model of the present invention is prepared using feces from a subject with a disease caused by an imbalance in the intestinal microbiota, a test substance (e.g., a low molecular weight compound or live bacteria that have been genome-edited) is added to the intestinal microbiota model, and the model that results in a more normal balance of the intestinal microbiota is selected as a candidate substance for preventing or treating the disease caused by an imbalance in the intestinal microbiota, etc. Whether the balance of the intestinal microbiota has become more normal can be determined, for example, by comparing the intestinal microbiota of a healthy subject (typically, at least as a result of screening, a subject who is not suffering from the disease intended to be prevented or treated by the candidate substance, or a subject for whom no abnormalities were detected in a health check) before and after the addition of the test substance and determining whether the microbiota is closer to that of a healthy subject. In addition, the comparison was carried out using the above-mentioned major bacteria (Bifidobacterium, Bacteroides, Prevotella, Faecalibacterium, and Escherichia (particularly Escherichia-Shigella)), useful bacteria (Collinsella, Eubacterium halii group, Group), Anaerostipes, Blautia, Dorea, Fusicatenibacter, Lachnospiraceae ND3007 group, Lachnospiraceae UCG-004, and Oscillospiraceae UCG-003), as well as minor bacteria (Peptostreptococcus and Paraclostridium) may be appropriately combined depending on the purpose. Furthermore, the intestinal microbiota of a healthy subject may be the intestinal microbiota model of the present invention, or known data on the intestinal microbiota of a healthy subject may be used.

[0037] 2. Intestinal flora improving agent of the present invention The present invention also provides an agent for improving an intestinal microbiota, comprising an artificial intestinal microbiota obtained by the production method of the present invention. As described above, the artificial intestinal microbiota obtained by the production method of the present invention reflects or maintains the (fecal) microbiota of the subject used to prepare the glycerol stock. Therefore, if the subject is healthy, administering the agent can make the intestinal microbiota of the subject more similar to that of a healthy subject. For example, the agent for improving an intestinal microbiota of the present invention is also suitable as a therapeutic or preventive agent for diseases targeted by fecal transplantation (FMT) (e.g., Clostridium difficile infection, Crohn's disease, ulcerative colitis, nonspecific multiple small intestinal ulcers, intestinal Behçet's disease, small intestinal bacterial overgrowth (SIBO), and drug-resistant irritable bowel syndrome). Furthermore, the artificial intestinal microbiota obtained by the production method of the present invention is capable of effectively producing butyrate, making it suitable as a therapeutic or preventive agent for diseases targeted by fecal transplantation (FMT) from this perspective.

[0038] Furthermore, when attempting to improve the intestinal flora, it may be useful to treat the target intestinal flora with an antibacterial agent in advance. Therefore, the present invention also provides a combination of the intestinal flora-improving agent of the present invention and an antibacterial agent. In the combination of the present invention, the respective agents may be administered simultaneously via the same or different administration routes, or may be administered sequentially via the same or different administration routes. When administered sequentially, the order is not particularly limited.

[0039] The antibacterial agent is not particularly limited as long as it can exert the desired effect, and examples thereof include ampicillin, bacampicillin, amoxicillin, pivmecillinam, amoxicillin, sultamicillin, piperacillin, aspoxilin, benzylpenicillin, cloxacillin, oxacillin, carbenicillin, cephalocrilla, cefroxadine, cefadroxil, cefixime, cefteram pivoxil, cefuroxime axetil, cefpodoxime proxetil, cefotiam hexetil, cefdinir, ceftibuten, cefditoren pivoxil, cefcapene pivoxil, cefazolin, cephalosporin ... Phosphate, cefozopran, cefmetazole, cefotiam, cefsulodin, cefoperazone, cefotaxime, cefmenoxime, ceftriaxone, ceftazisim, cefodisim, cefpirome, cefepime, faropenem, imipenem, panipenem, meropenem, biapenem, doripenem, aztreonam, vancomycin, teicoplanin, fosmicin, polymyxin B sulfate, colistin sulfate, gramicidin S, amphotericin B, levofloxacin, ofloxacin, norfloxacin, enoxacin, ciprofloxacin, lomefloxacin, tosuf Loxacin, sparfloxacin, gatifloxacin, prulifloxacin, moxifloxacin, pazufloxacin, rifampicin, dibekacin, tobramycin, amikacin, isepamicin, micronomycin, streptomycin, kanamycin, gentamicin, erythromycin, rokitamycin, josamycin, roxthromycin, clarithromycin, azithromycin, telithromycin, doxycycline, minocycline, chloramphenicol, lincomycin, clindamycin, trimethoprim, clavulanic acid, sulva ctam, tazobactam, sulfamethoxazole, salazopyrin, isoniazid, rifampicin, pyrazinamide, ethambutol, griseofulvin, amphotericin B, 5-fluorocytosine, fluconazole, miconazole, itraconazole, acyclovir, ganciclovir, foscavir, idoxuridine, amantadine, interferon gamma, ribapirin, lamipudin, metronidazole, tinidazole, fluconazole, mebendazole, pyrantel pamoate, diethylcarbamazine, praziquantel, albendazole, ivermectin,Examples of antihistamines include quinupristin, dalfopristin, linezolid, spectinomycin, netilmicin, sisomycin, lincosamin, ramoplanin, telithromycin, nystatin, fusidic acid, chlorhexidine, and polyhexanid.

[0040] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples in any way. [Example]

[0041] Example 1: Preparation of glycerol stocks (1) Collection of fecal samples Fecal samples were collected from three healthy Japanese volunteers according to the procedures outlined in a previous study (Sasaki et al. Sci Rep. 2021 Jul 16;11(1):14627. doi: 10.1038 / s41598-021-94210-8). All participants provided written informed consent prior to specimen collection. Each fecal sample was stored in an anaerobic culture swab (Becton Dickinson, New Jersey, USA) and utilized within 24 hours. This study was approved by the Institutional Review Board of Biopalette Co., Ltd. (study code BE-2021-02). All methods employed in this study conformed to the principles of the Declaration of Helsinki.

[0042] (2) Preparation of culture medium and mucin agar The autoclaved culture medium consisted primarily of Gifu University Formulated Anaerobic Medium (GAM) (59 g) (Product No. 05422, Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) and antifoaming agent PE-M (50 μl) (Wako, Osaka, Japan). Depending on the requirements, xylan (XL) (0.050–0.10 g, Product No. 38500, Serva, Germany), citrus pectin (PE) (0.10–0.40 g, Wako), and / or guar gum (GG) (0.025–0.40 g, Wako) were added before autoclaving (115 °C, 15 min). After autoclaving, filter-sterilized fructooligosaccharide (FOS) solution (0.10–0.36 g, Wako) and / or tapioca-derived resistant starch (RS) (0.10–0.18 g, Nippon Garlic Co., Ltd.) were added as needed. These additives were used alone or in combination (FXPG: FOS + XL + PE + GG, or FXPGR: FXPG + RS).

[0043] Purified mucin obtained from commercially available porcine gastric mucin (type II, Sigma-Aldrich, St. Louis, MO, USA) was used to prepare mucin agar according to a previously described method (Glenister DA et al. Microb Ecol Health Dis. 1988;1:31-38). Mucin agar was made by boiling dH2O containing 2.5–5% purified mucin, 1% agar, and 0, 0.25, or 0.5% N-acetylneuraminic acid (NeuAc) (Nacalai Tesque, Kyoto, Japan). Each piece of mucin agar measured 7 mm x 7 mm x 5 mm.

[0044] (3) Operation of an intestinal simulator for artificial intestinal microflora An artificial gut microbiota (Sasaki et al. Sci Rep. 2021 Jul 16;11(1):14627. doi: 10.1038 / s41598-021-94210-8) was established using a small-scale multichannel fermenter (Bio Jr. 8; ABLE, Tokyo, Japan). Fecal samples from swabs were suspended in 0.1 M phosphate buffer (pH 6.5) supplemented with 1% L-ascorbic acid (Wako). The culture medium was stirred at 300 rpm and purged with a filter-sterilized N2-CO2 mixture (80:20 v / v) at 37 °C for 1 h. Then, 1 ml of fecal suspension was inoculated and cultured for 48–54 h. The remaining fecal suspension was stored at -20 °C until use. The interior of each vessel was stirred and controlled at 37 °C, and anaerobic conditions were maintained by purging with a filter-sterilized gas mixture during the culture. The method for reproducing an artificial intestinal microflora will be described later.

[0045] As needed, five pieces of mucin agar-containing Teflon mesh were immersed in each container containing culture medium (Figure 4). The Teflon mesh was attached to the container with stainless steel. After incubation, the culture medium and mucin agar were aliquoted, and the culture medium was stored at -20°C until use. Short-chain fatty acids (SCFAs) (acetate, propionate, butyrate) and lactate were measured using high-performance liquid chromatography (Shimadzu Corporation, Kyoto, Japan) equipped with an Aminex HPX-87H column (300 × 7.8 mm; Bio-Rad, Hercules, CA, USA).

[0046] Example 2: Production of an artificial gut microbiota (1) Reproduction of artificial intestinal microbiota through cryopreservation The fecal microbiota cultured as described above was collected after incubation and concentrated to a final concentration of 5x by centrifugation at 8000 × g for 10 minutes. The resulting mixture was transferred to an anaerobic chamber (10% CO2, 3% H2, and 87% N2; Coy Laboratories, Grass Lake, MI, USA), after which the supernatant was removed. Cryopreservation was performed according to the FMT process (Keller JJ et al., United European Gastroenterol J. 2021 Mar;9(2):229-247). The concentrated microbiota was suspended in saline (0.8% NaCl, Nacalai Tesque) containing a final concentration of 16% glycerol (Nacalai Tesque) and stored at -80°C until use. After storage, the cryopreserved microbiota was transferred back to the anaerobic chamber and thawed on ice. 2.5 ml / L of the cryopreserved microbiota was inoculated into the intestinal simulator, and incubation was initiated for 72 hours. The procedure was the same as described above.

[0047] (2) Bacterial 16S rRNA profiling Genomic DNA extraction was performed from various samples, including 200 μl of fecal suspension, culture broth, glycerol stock, and 200 mg of mucin agar, according to a method described in a previous study (Tourlousse DM et al., Microbiome. 2021;9:95). For genomic DNA extraction, the ISOSPIN Fecal DNA Kit (Nippon Gene, Tokyo, Japan) was used, following the manufacturer's recommended procedure. Subsequently, sequencing, read processing, and analysis were performed according to published procedures (Tourlousse DM et al., Microbiome. 2021;9:95).

[0048] (3) Quantitative real-time PCR Quantitative real-time PCR assays were performed using LightCycle 96 (Roche Diagnostics, Rotkreuz, Switzerland) and FastStart Essential DNA Green Master Mix (Roche).

[0049] (4) Data availability The raw sequencing data generated in this study have been deposited in the DDBJ, EMBL, and Genbank databases ( http: / / getentry.ddbj.nig.ac.jp ) under BioProject PRJDB17564 with accession numbers DRR530900–DRR530932.

[0050] Results (Examples 1 and 2) (1) Development of a new method for reproducing artificial gut microbiota We attempted to recreate an artificial gut microbiota by starting the process with human fecal cultures via glycerol stocks (Figure 1). The following genera have been identified as common in the guts of diverse human populations, including Japanese individuals (Frioux C et al., Microb Ecol Health Dis. 1988;1:31-38; Takagi T et al., Microorganisms. 2022;10:664).

[0051] To preserve common genera in the artificial gut microbiota, prebiotics such as PE, GG, FOS, XL, and RS were used as additives in both fecal culture and post-storage culture (Portincasa et al., Int J Mol Sci. 2022;23:1105). Specifically, to reproduce an ideal artificial gut microbiota, three glycerol stocks of PE, GG, and FXPG (FOS + XL + PE + GG) were mixed in equal amounts and used as inoculum for post-storage culture. For comparison, a control group was used without additives in either fecal culture or post-storage culture.

[0052] 16S rRNA gene sequencing revealed the characteristics (diversity estimates) of these samples from human subjects (F1) (Table 1).

[0053] [Table 1]

[0054] For the fecal-derived artificial gut microbiota, the additives (PE, GG, and FXPG) showed a Shannon diversity index similar to that of the control, but the abundance of amplicon sequence variants (ASVs) decreased. On the other hand, when glycerol stocks of PE, GG, and FXPG were combined, the alpha diversity, expressed as both ASV and Shannon diversity, was comparable to or greater than that of the glycerol-based artificial gut microbiota control. The Bray-Curtis dissimilarity index (BCU) with the original feces also decreased. Similarly, for both human subjects F2 and F3, mixing the glycerol stocks increased both the ASV and Shannon index and decreased the Bray-Curtis dissimilarity index of the fecal-derived samples compared to the glycerol-based artificial gut microbiota control (Tables 2 and 3). Sample characteristics (diversity estimates) for human subjects F2 and F3 are as follows (Tables 2 and 3).

[0055] [Table 2]

[0056] [Table 3]

[0057] (2) Examination of artificial gut microbiota at the phylum and genus level Common genera such as Bifidobacteria, Bacteroides, Prevotella, Faecalibacterium, and Escherichia should be retained in the reconstructed artificial gut microbiota (Figure 2). As expected, the artificial gut microbiota reconstructed using a combination of glycerol stocks (PE, GG, and FXPG) retained at least 10% of the common genera compared to the original F1 fecal microbiota (Table 4). However, the control without prebiotics showed a decrease in the genera Prevotella and Faecalibacterium. A comparison of the metagenomic 16S sequencing results for the fecal and gut simulator luminal microbiota of human subjects (F1) with previous reports is shown in Table 4.

[0058] [Table 4]

[0059] In Table 4, the numbers in parentheses indicate the relative proportion to the original feces. Abbreviations: KUHIMM, Kobe University human intestinal microbiota model; M-SHIME, mucosal simulator of human intestinal microbial ecosystem.

[0060] Combining the three glycerol stocks maintained the Bacteroides ratio and the Bacteroides genus. In contrast, in the artificial gut microbiota derived from a single glycerol stock containing additives (FXPG or GG), these ratios were lower than those in the original feces. Minor Peptostreptococcaceae and Peptostreptococcus species were not detected in the artificial microbiota derived from the glycerol stocks containing additives, but they were increased in the control.

[0061] Interestingly, the three glycerol-stocked artificial microbiota contained at least 10% of other commensal bacteria, including Collinsella, Eubacterium halii group, Anaerostipes, Blautia, Dorea, Fusicatenibacter, Lachnospiraceae ND3007 group, Lachnospiraceae UCG-004, and Oscillospiraceae UCG-003, compared with the original fecal microbiota. However, one commensal genus, Ruminococcus, was reduced in the glycerol-stocked artificial microbiota.

[0062] The artificial gut microbiota combined with glycerol stocks showed a higher butyrate ratio (26.4-26.9%) and butyrate production (54.2-54.6 mM) among short-chain fatty acids compared to the control (19.7% and 49.1 mM, respectively) (Figure 3). Similar results were obtained for human subjects F2 and F3 (Figures 5 and 6 and Tables 5 and 6). Unexpectedly, Clostridium sensu stricto 1 was increased in the artificial microbiota mediated by the three glycerol stocks in F2 and F3. Metagenomic 16S sequencing results for the fecal and gut simulator luminal microbiota of human subjects (F2) and (F3) are shown in Tables 5 and 6.

[0063] [Table 5]

[0064] [Table 6]

[0065] In Tables 5 and 6, the numbers in parentheses indicate the relative proportion to the original feces.

[0066] As can be seen from the results above, we have developed a method for preserving and regenerating an artificial gut microbiota that includes common genera and other commensal genera while suppressing minor genera. This method is achieved by using glycerol, a food-grade cryoprotectant. To evaluate the usefulness of the cryopreserved artificial gut microbiota, simulations were performed using a gut simulator. When preserved via glycerol stocks, cryopreservation was observed to reduce the abundance and diversity of ASVs in the artificial gut microbiota compared to the original feces. However, this adverse effect was found to be mitigated by combining multiple glycerol stocks made using various media additives. The ASV counts (166-190) of the artificial gut microbiota regenerated via glycerol stocks using a combination of various media additives were found to be comparable to the values ​​reported in Japanese human feces (median: approximately 150 or 200) (Kameoka S et al., BMC Genomics. 2021;22:1-10). Thus, for example, a combination of three glycerol stocks (PE, GG, and FXPG) may be applied in FMT therapy.

[0067] Ideally, the composition of the reconstructed artificial gut microbiota would be similar to that of the human fecal microbiota. However, previous studies have shown that Peptostreptococcaceae predominates over Bacteroidaceae, and a decrease in Faecalibacterium, a major bacterium in the human fecal microbiota, has been observed after cryopreservation of in vitro models (Bircher L et al., mSystems. 2020;5:e00521-19 and Bircher L et al., Microb Biotechnol. 2018;11:163-175).

[0068] As can be seen from the examples, the artificial gut microbiota recreated using the glycerol stock of the present invention successfully retained common genera (including Bacteroides and Faecalibacterium) and other commensal genera, and successfully suppressed minor genera (Peptostreptococcus and Paraclostridium, both of the Peptostreptococcaceae family). These genera ratios were comparable to previously reported human values ​​(Frioux C et al., Cell Host Microbe. 2023;31:1111-1125.e6 and Takagi T et al., Microorganisms. 2022;10:664) and conventional intestinal simulator model values ​​(Deyaert S et al., Front Microbiol. 2023;13:1054061, Sasak K et al., Sci Rep. 2021;11:1-8, and Van den Abbeele P et al., ISME J. 2013;7:949-61) (Table 4). Furthermore, the higher butyrate production achieved by the glycerol stock combination of the present invention supports better retention of butyrate-producing bacteria. These bacteria have been reported to be sensitive to environmental conditions in reconstructed artificial gut microbiota (Bircher L et al., Microb Biotechnol. 2018;11:163-175.).

[0069] Considering that these common genera and other commensal genera are present at more than 0.1% in the artificial gut microbiota, the question arises as to whether they can be maintained in the human intestine. 8 It has been reported that when lactic acid bacteria were ingested for 10 days, they were detected in a rectal biopsy on the 11th day (Derrien M et al., Trends Microbiol. 2015;23:354-366.). In this regard, the artificial intestinal microbiota of the present invention contains approximately 10 11 cells, which is the number found in feces (10 per gram). 11This is comparable to the number of cells in the human intestinal flora (Derrien M et al., Trends Microbiol. 2015;23:354-366). Therefore, it is predicted that the artificial intestinal flora of the present invention will be sufficiently effective when administered at a dose of, for example, 1 ml per day.

[0070] In conclusion, we successfully recreated an artificial gut microbiota comparable to the original fecal microbiota by combining a glycerol stock with a prebiotic additive in the luminal compartment of a gut simulator.

[0071] The present invention is useful because it allows for the reproduction of an artificial intestinal lumen bacterial flora that retains sufficient amounts of the major intestinal bacteria that constitute the human intestinal microbiota and mimics the human intestinal microbiota as closely as possible. Furthermore, the artificial intestinal microbiota produced by the production method of the present invention retains sufficient amounts of the major intestinal bacteria that constitute the human intestinal microbiota, is an artificial intestinal lumen bacterial flora that mimics the human intestinal microbiota as closely as possible, and is also reproducible, making it useful because it allows for the establishment of a model (evaluation system) for research on the human intestinal microbiota. Furthermore, the artificial intestinal microbiota produced by the production method of the present invention retains sufficient amounts of the major intestinal bacteria that constitute the human intestinal microbiota and is an artificial intestinal lumen bacterial flora that mimics the human intestinal microbiota as closely as possible, making it useful because it allows for the improvement of the intestinal microbiota, for example, by transplantation. Furthermore, the artificial intestinal microbiota produced by the production method of the present invention is useful because, by transplantation, it can be used to treat or prevent diseases that are targets of fecal transplantation (Clostridium difficile infection, Crohn's disease, ulcerative colitis, nonspecific multiple small intestinal ulcers, intestinal Behçet's disease, small intestinal bacterial overgrowth (SIBO), drug-resistant irritable bowel syndrome, etc.).

Claims

1. A method for producing an artificial intestinal microbiota, comprising a step of culturing a solution containing cells prepared by mixing two or more types of glycerol stocks containing bacterial flora cultured in a medium containing a medium additive.

2. The method according to claim 1, wherein the medium additives included in the preparation of the glycerol stock include one or more selected from the group consisting of prebiotics, dietary fiber, glycoproteins, and health food ingredients.

3. 2. The method of claim 1, wherein the medium additives included in the preparation of the glycerol stock include one or more selected from the group consisting of pectin, guar gum, fructooligosaccharides, xylan, and resistant starch.

4. The method of claim 3, wherein one medium additive included in the preparation of a glycerol stock is guar gum, and at least one medium additive included in the preparation of a glycerol stock other than the glycerol stock is pectin, guar gum, fructooligosaccharide, and xylan.

5. 4. The method of claim 3, wherein at least one medium additive contained in the preparation of the glycerol stock other than the glycerol stock containing the bacterial flora cultured in the medium containing guar gum and the glycerol stock containing the bacterial flora cultured in the medium containing pectin, guar gum, fructooligosaccharide, and xylan is pectin.

6. The method according to any one of claims 1 to 5, wherein the artificial intestinal microbiota is an artificial human intestinal microbiota.

7. An intestinal microbiota model comprising an artificial intestinal microbiota produced by the method according to any one of claims 1 to 6.

8. An agent for improving intestinal flora, comprising an artificial intestinal flora produced by the method according to any one of claims 1 to 6.

9. A combination comprising the intestinal flora-improving agent according to claim 8 and an antibacterial agent.

Images