Novel microbial compositions and methods of use thereof

Abstract

The invention provides a novel Akkermansia muciniphila (Akkermansia muciniphila) strain and a preparation method of the novel Akkermansia muciniphila strain. Compositions comprising the strains are also provided. Also provided are methods comprising administering the compositions to a subject in need thereof. In some embodiments, methods of treating a metabolic disorder in a subject are provided, such methods comprising administering to the subject an amount of a composition of the present disclosure effective to treat the metabolic disorder in the subject. Also provided are methods comprising culturing the novel strains.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 320,000, filed on March 15, 2022, which is incorporated herein by reference in its entirety. Background Art

[0003] Akkermansia is a gram-negative, strictly anaerobic, non-motile, non-spore-forming, oval-shaped bacterium. Due to the strict anaerobic requirements of Akkermansia, it can be difficult to culture and formulate compositions comprising the bacterium. In addition, Akkermansia is traditionally cultured under anaerobic conditions on a medium containing gastrosin.

[0004] Provided herein is a novel strain of Akkermansia that grows in vegetable culture medium under anaerobic conditions and does not contain mucin. Summary of the invention

[0005] The present disclosure provides bacteria of the Akkermansia muciniphila strain as deposited at the American Type Culture Collection (ATCC) under ATCC Accession No. PTA- 126838. The bacteria include a nucleic acid encoding a polypeptide including the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, or both.

[0006] The present disclosure provides a composition comprising bacteria of the strain Akkermansia muciniphila deposited at the American Type Culture Collection (ATCC) under ATCC Accession No. PTA- 126838. As described herein, the composition comprises a nucleic acid encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, or both.

[0007] Also presented herein is a composition for treating a metabolic disorder in a subject, wherein the composition comprises a bacterium such as the strain Akkermansia muciniphila deposited with the American Type Culture Collection (ATCC) under ATCC Accession No. PTA- 126838 in an amount effective to treat the metabolic disorder. In one embodiment, the metabolic disorder is selected from a metabolic disorder selected from the group consisting of or consisting essentially of: an insulin resistance-based disorder, an insulin sensitivity-based disorder, type 1 diabetes, type 2 diabetes, and obesity.

[0008] In certain embodiments, the composition further comprises one or more additional microorganisms having a 16S rRNA sequence comprising at least 97% identity over the entire length of the 16S rRNA sequence of a microorganism selected from the group consisting of Anaerostipes caccae, Anaerobutyricumhallii, Bacteroides finegoldii, Bacteroides ovatus, Bacteroides stercoris, Eubactrium hallii, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, Blautia hydrogenotrophica, Blautia producta, Butyrivibrio fibrinolyticus, fibrisolvens), Clostridium acetobutylicum, Clostridium aminophilum, Clostridium beijerinckii, Clostridium butyricum, Clostridium colinum, Clostridium indolis, Clostridium innocuum, Clostridium orbiscindens, Enterococcus faecium, Eubacterium rectale, Faecalibacterium prausnitzii, Fibrobacter succinogenes, Oscillospira guilliermondii, Roseburia cecicola, Roseburia glucovorans inulinivorans), Ruminococcus flavefaciens, Ruminococcus gnavus, Ruminococcus ovaleobeum), Streptococcus cremoris, Streptococcus faecium, Streptococcus infantis, Streptococcus mutans, Streptococcus thermophilus, Anaerofustis stercorihominis, Anaerostipes hadrus, Anaerotruncus colihominis, Clostridium sporogenes, Clostridium tetani, Coprococcus eutactus, Eubacterium cylindroides, Eubacterium dolichum, Eubacterium ventriosum, Roseburia faeccis, Roseburia hominis hominis, Roseburia intestinalis, Collinsella aerofaciens, Coprococcus comes, Eubacterium limosum, and Ruminococcus faecis, and any combination thereof.

[0009] In certain embodiments, the composition further comprises one or more additional microorganisms selected from the group consisting of: a butyrate co-producing bacterium, a Clostridium butyricum, a Clostridium beijerinckii, a Clostridium butyricum, and any combination thereof.

[0010] In certain embodiments, the composition includes at least 10Λ5 AFU / g of each of the one or more additional microorganisms.

[0011] In further embodiments, the composition comprises at least one preservative and / or enteric coating. In some embodiments, the composition comprises a prebiotic selected from the group consisting of inulin, green banana, ganoderma lucidum, cassava starch, oats, pectin, potato or its extract, complex carbohydrates, complex sugars, resistant dextrins, resistant starch, amino acids, peptides, nutritional compounds, biotin, polydextrose, fructooligosaccharides (FOS), galacto-oligosaccharides (GOS), starch, lignin, psyllium, chitin, chitosan, gum (e.g., guar gum), high amylose corn starch (HAS), cellulose, b-glucan, hemicellulose, lactulose, manno-oligosaccharides, manno-oligosaccharides (MOS), oligofructose-enriched inulin, oligofructose, oligoglucose, tagatose, trans-galacto-oligosaccharides, pectin, resistant starch, xylo-oligosaccharides (XOS) and any combination thereof. In some preferred embodiments, the prebiotic used is inulin.

[0012] In some embodiments, compositions are presented herein that include lyophilized bacteria. In some embodiments, compositions presented herein include lyophilized bacteria such as the strain Akkermansia muciniphila deposited with the American Type Culture Collection (ATCC) under ATCC Accession No. PTA-126838.

[0013] In some embodiments, the compositions presented herein include viable bacteria. In some embodiments, the compositions presented herein include viable bacteria such as the strain Akkermansia muciniphila deposited with the American Type Culture Collection (ATCC) under ATCC Accession No. PTA-126838.

[0014] In some embodiments, the compositions presented herein include non-viable bacteria. In some embodiments, the compositions presented herein include non-viable bacteria such as the strain Akkermansia muciniphila deposited with the American Type Culture Collection (ATCC) under ATCC Accession No. PTA-126838.

[0015] In some embodiments, the composition is formulated as a pharmaceutical formulation. In some embodiments, the composition is formulated as a nutritional supplement. In some embodiments, the composition is formulated as a dietary supplement. In some embodiments, the composition is formulated as a medical food.

[0016] In some embodiments, described herein is a dairy-free composition. In some embodiments, the composition does not include animal products.

[0017] In some embodiments, the composition is in the form of a pill, capsule, lozenge, food bar, or gummy ball.

[0018] Also provided herein is a method of treating a metabolic disorder in a subject, the method comprising administering to the subject a composition comprising a bacterium such as a strain of Akkermansia muciniphila deposited with the American Type Culture Collection (ATCC) under ATCC Accession No. PTA- 126838. The composition may further comprise a co-producing butyric acid bacterium, Clostridium butyricum, Clostridium beijerinckii, Clostridium butyricum, and any combination thereof.

[0019] The metabolic disorder mentioned in the treatment method is selected from disorders based on insulin resistance, disorders based on insulin sensitivity, type 1 diabetes, type 2 diabetes and obesity, and intestinal related disorders.

[0020] Provided herein are methods of culturing an Akkermansia muciniphila strain as deposited with the American Type Culture Collection (ATCC) under ATCC Accession No. PTA- 126838 under anaerobic growth conditions in / on a growth medium. The growth medium used herein is VEG medium. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : Depicted is a comparison of growth phases between Akkermansia muciniphila strains Amuc-OG and Amuc-i09.

[0022] Figure 2 : Depicted are growth curves comparing three independent replicates each between Akkermansia muciniphila (OG) and Amuc-i09.

[0023] Figure 3 : Depicts a comparison of doubling time, pH consumption, and glucose consumption between Amuc-i09 and Amuc-OG during the growth phase. DETAILED DESCRIPTION

[0024] Before describing the present invention, it should be understood that the present invention is not limited to the specific embodiments described, as these embodiments can certainly vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to be restrictive, as the scope of the present invention will only be limited by the appended claims.

[0025] Where a range of values ​​is provided, it is understood that each intermediate value between the upper and lower limits of the range to the tenth unit of the lower limit (unless the context clearly indicates otherwise) is also specifically disclosed. Each smaller range between any stated value or intermediate value in the stated range and any other stated value or intermediate value in the stated range is included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded in the stated range, and each range that includes either, does not include both, or includes both limits in the smaller range is also included in the present invention, subject to any specifically excluded limits in the stated range. Where the stated range includes one or both of the limits, ranges that exclude either or both of those included limits are also included in the present invention.

[0026] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. Although any method and material similar or equivalent to those described herein can be used to practice or test the present invention, some potential and exemplary methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in conjunction with the cited publications. It should be understood that in the event of a conflict, the present disclosure replaces any disclosure of the incorporated publication.

[0027] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0028] Strain preservation

[0029] Under the terms of the Budapest Treaty, the following biological materials have been deposited with the American Type Culture Collection Patent depository - P0071744 at 10801 University Boulevard Manassas, Virginia 20110 and has been assigned the following accession number: PTA-126838.

[0030] Name / Nomenclature Patent deposit number Patent deposit number Akkermansia muciniphila WB-STR-i09 PTA-126838

[0031] A. Bacteria

[0032] New strains of anaerobic bacteria are described herein. In some embodiments, the bacteria as described herein are by The strain deposited under deposit number PTA-126838 (referred to herein as Amuc-i09).

[0033] Provided herein is a novel strain of Akkermansia muciniphila. The strain disclosed herein is deposited with the American Type Culture Collection (ATCC) under ATCC Accession No. PTA-126838. by The bacterial genome of the Akkermansia muciniphila strain deposited under accession number PTA-126838 encodes a polypeptide comprising SEQ ID NO: 1 and a polypeptide comprising SEQ ID NO: 2:

[0034] Sequence information

[0035] SEQ ID NO:1

[0036] MFLPCGGCRRAFLYPPGLFSGV

[0037] SEQ ID NO:2

[0038] MNQLLSLESLRAYMIILQGEHHPKNKVAPARQTVTARNPRTMISFRYNTENNTVDAAKTGRDDTN

[0039] B. Other bacteria

[0040] Embodiments of the compositions described herein include, but are not limited to, compositions comprising Amuc-i09 alone or in combination with one or more other bacteria. Examples of bacteria that can be combined with Amuc-i09 in the composition include, but are not limited to: Clostridium butyricum, Clostridium beijerinckii, anaerobic Corynebacterium faecalis, butyric acid co-producing bacteria, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, Butyrivibrio fibrinolyticus, Clostridium acetobutylicum, Clostridium aminophilum, Clostridium beijerinckii, Clostridium butyricum, Clostridium intestinale, Clostridium indole, Clostridium flavone-lyticum, Enterococcus faecalis, Eubacterium rectum, Faecalibacterium prausnitzii, succinimidobacterium, Oscillating Spirillum gibbsii, Roseburia caecum, Roseburia glucovorans, Ruminococcus xanthotrophicus, Ruminococcus activus, Ruminococcus ovale, Streptococcus cremoris, Streptococcus faecalis, Streptococcus infantis, Streptococcus mutans, Streptococcus thermophilus, fecal anaerobic rod-shaped bacteria, butyrate-producing bacteria, human colon anaerobic rod-shaped bacteria, Clostridium sporogenes, Clostridium tetani, regular coccus, Eubacterium columnarum, Eubacterium elongatum, Eubacterium convexum, Roseburia faecum, Roseburia hominis, Roseburia intestinalis, and any combination thereof.

[0041] In certain embodiments, the composition comprising Amuc-i09 further comprises a bacterium of a genus selected from the group consisting of Clostridium, Eubacterium, Bifidobacterium, Anaerobic Corynebacterium, Coprococcus, Bacteroides, Blautia, Ruminococcus, Faecalibacterium, Oscillatory Spirillum, Streptococcus, and Roseburia.

[0042] In one embodiment, the composition includes Amuc-i09 and one or more additional microorganisms selected from the group consisting of: butyric acid co-producing bacteria DSM 3353, Akkermansia muciniphila ATCC BAA83, Clostridium butyricum ATCC 1939, Clostridium beijerinckii ATCC 5174, and any combination thereof.

[0043] In one embodiment, the composition includes Amuc-i09 and one or more additional bacterial strains selected from the group consisting of: butyric acid co-producing bacteria, Akkermansia muciniphila, Clostridium butyricum, Clostridium beijerinckii, and any combination thereof.

[0044] In one embodiment, the additional strains in the composition are anaerobic bacteria. In another embodiment, the additional bacteria are non-viable. In still other embodiments, the additional bacteria are freeze-dried. In other embodiments, the additional bacteria are freeze-dried and viable.

[0045] In another embodiment, the present disclosure provides a composition comprising Amuc-i09 and at least one additional microorganism having a 16S rRNA sequence comprising at least about 85%, 87%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity over the entire length of the 16S rRNA sequence of a microorganism selected from the group consisting of Clostridium butyricum, Clostridium beijerinckii, anaerobic Corynebacterium faecalis, butyric acid co-producing bacteria, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, Butyrivibrio fibrinolyticus, Clostridium acetobutylicum, Clostridium aminophilum, Clostridium beijerinckii, Clostridium butyricum, Clostridium indolizum, Clostridium flavoneolyticum, Enterococcus faecalis, Eubacterium rectum , Faecalibacterium prausnitzii, succinimidyl filamentous bacteria, Oscillating Spirillum gibbsii, Roseburia caecum, Roseburia glucovorans, Ruminococcus xanthophylla, Ruminococcus activus, Ruminococcus ovale, Streptococcus cremoris, Streptococcus faecalis, Streptococcus infantis, Streptococcus mutans, Streptococcus thermophilus, fecal anaerobic rod-shaped bacteria, butyrate-producing bacteria, human colon anaerobic rod-shaped bacteria, Clostridium sporogenes, Clostridium tetani, regular coccus, Eubacterium columnarum, Eubacterium elongatum, Eubacterium convexum, Roseburia faecalis, Roseburia hominis, Roseburia intestinalis, and any combination thereof.

[0046] In another embodiment, described herein is a bacterium that expresses a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1, a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, or both. In some embodiments, such a bacterium is a strain of Akkermansia muciniphila bacteria.

[0047] C. Characteristics of bacteria

[0048] In an embodiment, the novel strain of Akkermansia muciniphila, Amuc-i09, as disclosed herein, exhibits unique phenotypic properties, such as improved viability (measured in CFU per gram), faster and / or improved growth characteristics of Amuc-i09 compared to other Akkermansia strains, such as Amuc-OG.

[0049] The growth characteristics of frozen or lyophilized bacterial strains are measured and compared with the basic strains. Such basic strains can be basic strains or any other starting materials preserved in a certification depository. In a specific embodiment, the growth characteristics of the lyophilized bacterial strains of Amuc-i09 are measured and compared with the characteristics of Amuc-OG.

[0050] a. Vitality

[0051] Viability as defined herein refers to the measure of living cells. Specifically, viability is defined as the ability to reproduce by binary fission under controlled conditions. Viability is measured in colony forming units (CFU) and is measured by culturing microorganisms and counting only viable cells. Another unit used is the active fluorescent unit (AFU), which measures viability based on flow cytometric results, thereby correlating cell wall integrity with living cells.

[0052] Flow cytometry (BD Accuri TM C6) Viability measurement. Such flow cytometry measurements involve staining a mixture of cells and detecting the fluorescence of each cell as it flows through. Staining of a mixture of cells can be performed using any of a number of stains that preferentially stain cells, particularly live cells and dead cells, or only dead cells. Examples of such stains include, but are not limited to, thiazole orange, propidium iodide, 7-AAD, DAPI, SYTO 9, such as Zombie Aqua TM 、ZombieGreen TM 、Zombie NIR TM , Zombie Red TM 、Zombie Violet TM 、Zombie UV TM and Zombie Yellow TMIn one embodiment, the dye used in the process of the present disclosure is a combination of thiazole orange and propidium iodide.

[0053] As disclosed herein, Amuc-i09 exhibits higher activity per gram compared to the wild-type variant Amuc-OG. For example, as shown in the Examples, 4g of dry weight Amuc-i09 has 8.72x 10^11CFU, while 21.3g of dry weight Amuc-OG only has 8.29x 10^11CFU. Calculated per gram, Amuc-i09 has 2.18x 10^11CFU / g of microorganisms, while Amuc-OG has only 0.38x 10^11CFU / g of Amuc-OG microorganisms. This also shows that there are more live bacteria per gram of Amuc-i09 than per gram of Amuc-OG strains.

[0054] In some embodiments, Amuc-i09 is in a viable, lyophilized state. In some embodiments, the bacteria of the present disclosure are non-viable.

[0055] In further embodiments, one or more bacteria in any of the compositions described herein are in a lyophilized state. In addition, one or more bacteria in any of the compositions described herein are viable or non-viable.

[0056] In some embodiments, the bacteria of the present disclosure are present at about 1×10 3 Up to 1×10 14 The concentration of active fluorescent units (AFU) / g is present, including any number within the stated range, and in other embodiments, the concentration is about 1×10 9 Up to 1×10 13 AFU / g, about 1×10 5 Up to 1×10 7 AFU / g or about 1×10 8 Up to 1×10 9 AFU / g, including any number within the stated range.

[0057] In certain embodiments, the compositions of the present disclosure include at least 10^5CFU per gram of each strain. In other embodiments, the compositions include at least 10^6CFU / g, or at least 10^7CFU / g, or at least 10^8CFU / g, or at least 10^9CFU / g, or at least 10^10CFU / g, or at least 10^11CFU / g or at least 10^12CFU / g of at least one strain in the composition. In other embodiments, when there is more than one strain, the vitality of each strain (measured in CFU or AFU) can be different from other strains in the composition.

[0058] b. Faster and / or improved growth characteristics

[0059] As disclosed herein, Amuc-i09 has faster growth characteristics compared to the wild-type strain. Growth is measured as OD or optical density of the medium in which the microorganism is cultured.

[0060] As used herein, the term "faster growth" refers to where the doubling time of a particular organism is less than that of another organism, other things being equal.

[0061] In some embodiments, Amuc-i09 has improved growth characteristics compared to wild-type strain Amuc-OG. As used herein, the term "improved" refers to a quantitative improvement in the number of viable cells after a certain time point in the growth phase of bacteria. Specifically, it is shown herein that, in the case where both bacteria are under the same conditions, at a time point after the same time, such as 9 hours or 12 hours later, the number of viable cells of Amuc-i09 (measured in CFU / g) is more than the number of viable cells of Amuc-OG (measured in CFU / g).

[0062] As in Example 1 and Figure 3 As described in , Amuc-i09 is characterized by a faster doubling time compared to Amuc-OG during the exponential growth phase.

[0063] c. Improved granulation properties

[0064] In some embodiments, as disclosed herein, Amuc-i09 has improved pelleting properties compared to the pelleting properties of Amuc-OG.

[0065] As used herein, "pelleting properties" refers to the percentage of organisms in liquid culture present in the recoverable cell pellet after centrifugation. All other things being equal, an organism with "improved pelleting properties" will have a greater ratio of organisms present in the pellet:supernatant during centrifugation than another precipitated organism's pellet to supernatant (pellet:supernatant) ratio. In the examples described herein, Amuc-i09 was shown to have a greater pellet:supernatant ratio when cultured and precipitated under the same conditions compared to Amuc-OG.

[0066] In certain embodiments, Amuc-i09 exhibits "improved pelleting characteristics" by exhibiting a (visually) clearer supernatant than the supernatant of the pelleted Amuc-OG organism, all other things being equal.

[0067] Specifically, as described in Example 2 and shown in Table 2, Amuc-i09 exhibited improved pelleting properties compared to Amuc-OG.

[0068] D. How to use

[0069] In certain embodiments, the present disclosure provides methods of treating a disorder. Such a disorder may be an intestinal-based disorder or any other disorder based on an imbalance of a bacterial population in the intestinal tract. By way of non-limiting example, such a population may be a population of Akkermansia muciniphila in the intestinal tract.

[0070] In certain embodiments, the present disclosure provides methods of increasing the Akkermansia muciniphila population in the small intestine.

[0071] In further embodiments, the compositions disclosed herein can be used to treat any one or more of conditions such as irritable bowel syndrome, inflammatory bowel disease, gastric ulcer, pouchitis, Helicobacter pylori infection, diarrhea, type 1 diabetes, type 2 diabetes, and / or obesity.

[0072] In embodiments, the compositions described herein may include a pharmaceutically acceptable carrier suitable for oral administration to a mammal, for example, as a powdered food supplement, various granular formulations, or liquid formulations. In some embodiments, the composition is in the form of a pill, capsule, lozenge, food bar, and / or soft candy ball. In some embodiments, the composition is in the form of a suppository or injection for delivery to a mammal.

[0073] In some embodiments, the compositions described herein can be used as a nutritional supplement or dietary supplement for a mammal. In some cases, the mammal can be a human.

[0074] E. Preparation method

[0075] Embodiments include methods of producing bacteria with improved growth characteristics and improved pelleting characteristics. Such bacteria can be obtained by a method involving isolating the bacteria from a biological sample, inoculating and culturing it in a suitable culture medium under suitable anaerobic conditions, precipitating with a cryoprotectant, freezing, lyophilizing, and resuspending in a culture medium.

[0076] In some embodiments, the bacteria are grown in any suitable culture medium; some non-limiting examples include PYG, RCM, GYT veg, BHI, nutrient medium, minimal medium, selective medium, and differential medium. In a specific embodiment, the culture medium is a vegetable-based culture medium. As used herein, "vegetable-based culture medium" refers to a growth medium that is substantially free of any animal-based or milk-based ingredients or derivatives. In some embodiments, the vegetable-based culture medium is a meat-free culture medium that does not contain any animal-derived ingredients. In some embodiments, the vegetable-based culture medium is a liquid "VEG medium" whose components are shown in Table 1:

[0077]

[0078]

[0079] Table 1: Composition of VEG medium

[0080] Cryoprotectants that can be used in the methods of this description include, but are not limited to, lactate, trehalose, glycerol, DMSO, propylene glycol, 2-methyl-2,4-pentanediol, formamide, glycerophospholipids, proline, sorbitol, diethylene glycol, sucrose, glucose, and polymers such as polyvinyl alcohol, PEG, hydroxyethyl starch, skim milk, tapioca starch, polyvinyl polypropylene, inulin, methylcellulose, sodium alginate, gum arabic, propylene glycol or xylitol, and all possible combinations thereof. In an embodiment, a cryoprotectant is added to the bacteria before freezing.

[0081] In certain embodiments, the bacterial pellet is resuspended in a cryoprotectant. In some embodiments, the cryoprotectant used is sucrose.

[0082] The cryoprotected bacteria were then resuspended in VEG medium and centrifuged to obtain a pellet.When comparing the bacterial pellets of Amuc-OG and Amuc-i09, it was surprisingly found that Amuc-i09 had improved pelleting properties compared to Amuc-OG.

[0083] After resuspension, the cryo-suspended bacterial pellet is frozen. For example, the pellet can be rapidly frozen in liquid nitrogen. Other freezing techniques that can be employed by one of ordinary skill in the art are contemplated as part of this disclosure - including cryogenic freezing (e.g., in ethanol and dry ice or carbon dioxide), mechanical freezing, or rapid freezing.

[0084] In certain embodiments, the mixture of cells and cryoprotectants is freeze-dried. Such freeze drying may require 12-48 hours. Freeze drying can be performed under vacuum and at temperatures below freezing. Non-limiting examples of freeze drying conditions are 0.008 millibars and a temperature of -84°C. Other freeze drying conditions that can be changed include the surface area of ​​the product exposed to sublimation, controlling the pressure and temperature of the freeze drying chamber, and changing the heat applied to the product during the sublimation process throughout the primary drying stage and during the desorption process throughout the secondary drying stage.

[0085] In a specific embodiment, the bacterial pellet is lyophilized in a LabConco FreeZone 2.5 Plus lyophilizer at -84°C and a vacuum pressure of 0.008 to 0.1 mbar for 8-24 hours.

[0086] Then the lyophilized or frozen bacteria are recovered. In an embodiment, the bacteria are recovered by placing the bacteria in a culture medium and incubating under growth conditions. In a specific embodiment, the bacteria are recovered to a ratio of about 0.01 mg / mL by placing the lyophilized cells in a plant culture medium (as defined above) and growing the culture for 12-20 hours at a temperature suitable for bacterial growth. This process screens cells that can recover rapidly after freezing or lyophilization. The process can then be repeated by another cycle to further select a bacterial colony.

[0087] At the end of each cycle, the bacteria are reconstituted, frozen or lyophilized, and grown in culture under appropriate conditions. Comparative studies are performed to determine differences in growth characteristics between the novel Amuc-i09 strain and Amuc-OG.

[0088] As described herein, the novel Amuc-i09 strain has improved growth characteristics compared to Amuc-OG. Improved growth characteristics are defined as those achieved by Figure 1 To characterize the faster growth phase, the growth characteristics of Amuc-i09 ("i09-3" is the third replicate of Amuc-i09 grown in the medium as described above) were compared relative to Amuc-OG when grown in the same medium as described above.

[0089] Obviously, in Figure 1 Among them, i09-3 (Amuc-i09 batch 3) grew to a higher OD value within 9 h, while OG-2 (Amuc-OG batch 2) did not reach the same high OD value even after 12 h.

[0090] In certain embodiments, a single colony of bacterium or a sample of bacterium recovers and grows in vegetable culture medium as above. The number of cycles that selected colony may experience can be at least 5 cycles, at least 10 cycles, at least 15 cycles, at least 20 cycles, at least 25 cycles, at least 30 cycles, at least 35 cycles, at least 40 cycles, at least 45 cycles, at least 50 cycles, at least 55 cycles, at least 60 cycles, at least 65 cycles, at least 70 cycles, at least 75 cycles, at least 80 cycles, at least 85 cycles, at least 90 cycles, at least 95 cycles, at least 100 cycles, at least 105 cycles, at least 110 cycles, at least 115 cycles, at least 120 cycles, at least 125 cycles, at least 130 cycles, at least 135 cycles, at least 140 cycles, at least 145 cycles or at least 150 cycles. Each cycle may bring one or more challenges to bacterium. Examples of such challenges include, but are not limited to, changes in growth conditions, nutrient medium components, temperature, pressure, oxygen conditions of the isolation, freezing and / or lyophilization and / or recovery processes of the microorganisms as described herein.

[0091] In one embodiment, freeze-dried powders of each of the Amuc-i09 strain and the Amuc-OG strain were recovered on agar plates. From a single colony of each of the strains, three replicates were cultured in a vegetable medium as described herein. Data showing the growth phase of each of these replicates are shown in Table 1. Figure 2 as shown in .

[0092] F. Composition

[0093] Provided herein are compositions comprising one or more bacteria of the present disclosure. Such compositions can be administered to a subject. In certain embodiments, such compositions can be administered as a therapeutic agent or as a dietary supplement. As described herein, one or more bacteria of the present disclosure can be used to prepare a pharmaceutical formulation comprising an effective amount of bacteria for treating a subject. The microorganism can be in any formulation known in the art. Some non-limiting examples of such formulations include topical, capsule, pill, enema, liquid, injection formulation, etc. In certain embodiments, one or more strains disclosed herein can be included in food or beverage products, cosmetics, or nutritional supplements.

[0094] In some embodiments, the subject is a mammal. As used herein, mammal refers to any mammal, including but not limited to humans, mice, cats, rats, dogs, sheep, monkeys, goats, rabbits, hamsters, horses, cows or pigs. In a preferred embodiment, the mammal is a human.

[0095] The composition may comprise one or more active ingredients. Active ingredients include, but are not limited to, active ingredients selected from the group consisting of antibiotics, prebiotics, probiotics, sugars (e.g., as baits that limit the binding of specific bacteria / viruses to the intestinal wall), bacteriophages, microorganisms, etc.

[0096] In some embodiments, the composition includes a prebiotic. Prebiotics can be used as a combination or as a single ingredient. In some embodiments, the prebiotic can be a starch source, mucin, fructooligosaccharide, pectin, gum, glucan, xylan, arabinogalactan, seaweed polysaccharide or a derivative thereof. Some examples of such sources are inulin, green banana, ganoderma lucidum, tapioca starch, oats, pectin, raw or cooked potatoes, corn, rice, rice bran, cereals, porcine or human mucin sources, L-threonine derivatives, chicory root, agave, artichoke, dandelion, lemon peel, apple peel, berries, guar gum, xanthan gum, locust chia seeds, barley, sorghum, corn, larch, gum arabic, seaweed, dulse or any combination or derivative thereof. In some embodiments, the prebiotic is inulin. Inulin is used as an energy source for the microbial composition.

[0097] In some embodiments, the prebiotic is selected from the group consisting of inulin, green banana, ganoderma lucidum, tapioca starch, oats, pectin, potato or its extracts, complex carbohydrates, complex sugars, resistant dextrins, resistant starch, amino acids, peptides, nutritional compounds, biotin, polydextrose, fructooligosaccharides (FOS), galacto-oligosaccharides (GOS), starch, lignin, psyllium, chitin, chitosan, gum (e.g., guar gum), high amylose corn starch (HAS), cellulose, b-glucan, hemicellulose, lactulose, manno-oligosaccharides, manno-oligosaccharides (MOS), oligofructose-enriched inulin, oligofructose, oligoglucose, tagatose, trans-galacto-oligosaccharides, pectin, resistant starch, xylo-oligosaccharides (XOS), and any combination thereof.

[0098] In some other embodiments, olive oil polyphenols, including hydroxytyrosol and its derivatives, such as oleuropein complex and tyrosol ("phenol alcohols"), are used as prebiotics.

[0099] The composition can be administered by a suitable method to deliver it to any part of the gastrointestinal tract of a subject, including the oral cavity, mouth, esophagus, stomach, duodenum, small intestinal region (including duodenum, jejunum, ileum) and large intestinal region (including cecum, colon, rectum and anal canal). In some embodiments, the composition is formulated for delivery to the ileum and / or colon region of the gastrointestinal tract.

[0100] In some embodiments, for example, the administration of the composition is carried out orally by capsules, pills, powders, tablets, gels or liquids, designed to release the composition in the gastrointestinal tract. In some embodiments, for example, the administration of the composition is carried out by injection for compositions comprising butyrate, propionate, acetate and short-chain fatty acids. In some embodiments, the administration of the composition is carried out by application to the skin, such as an ointment, liquid or patch. In some embodiments, the administration of the composition is carried out by suppositories and / or by enemas. In some embodiments, a combination of routes of administration is used.

[0101] The microbial composition can be formulated as a dietary supplement. The microbial composition can be combined with a vitamin supplement. The microbial composition can be formulated into a chewable form, such as a probiotic soft candy. The microbial composition can be incorporated into a certain form of food and / or beverage. Non-limiting examples of foods and beverages into which the microbial composition can be incorporated include, for example, bars, milk shakes, fruit juices, infant formulas, beverages, frozen foods, fermented foods, and cultured dairy products, such as yogurt, yogurt drinks, cheese, acidophilus beverages, and kefir.

[0102] The compositions of the present disclosure can be administered as part of a fecal transplant procedure. The compositions can be administered to a subject via a tube, such as a nasogastric tube, a nasojejunal tube, a nasoduodenal tube, an oral gastric tube, an oral jejunal tube, or an oral duodenal tube. The compositions can be administered to a subject via colonoscopy, endoscopy, sigmoidoscopy, and / or enema.

[0103] In some embodiments, the microbial composition is formulated so that one or more microorganisms can replicate once delivered to a target habitat (e.g., intestinal tract). In a non-limiting example, the microbial composition is formulated into a pill. The shelf life of such a pill can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. In another non-limiting example, the microbial composition is formulated so that the microorganisms can reproduce once they enter the intestinal tract.

[0104] In certain embodiments, Amuc-i09 can be formulated into a pill together with other additives, and the pill can be administered orally to an individual so that the pill will not disintegrate before arriving at the intestinal tract of the individual. In certain embodiments, other ingredients can also be added to extend the shelf life of the microbial composition. In certain embodiments, Amuc-i09 can be formulated in a manner that enables it to survive in a non-natural environment. For example, the microorganism originating in the intestinal tract may not survive in an oxygen-rich environment. In order to overcome this limitation, microorganisms can be formulated to reduce or eliminate exposure to oxygen. Other strategies for improving the shelf life of microorganisms can also include other microorganisms (for example, one or more strains in the composition improve the survival rate of one or more of the other strains in the composition).

[0105] In some embodiments, Amuc-i09 can be formulated with polyphenols. In some embodiments, the polyphenols can be a combination of one or more polyphenols. Commonly used polyphenols are flavonoids, which include flavonoids, flavonols, flavanols, flavanones, isoflavones, proanthocyanidins, and anthocyanidins. Flavonoids that are particularly abundant in food are catechins (tea, fruit), hesperidin (citrus fruits), cyanidins (red fruits and berries), daidzein (soybeans), proanthocyanidins (apples, grapes, cocoa), and quercetin (onions, tea, apples). Phenolic acids include caffeic acid, while lignans are polyphenols derived from phenylalanine found in flaxseed and other grains. In one embodiment, tannic acid extracted from papaya is used.

[0106] In some embodiments, Amuc-i09 is lyophilized.

[0107] In some embodiments, Amuc-i09 is lyophilized and formulated into a composition as provided herein. Such compositions can be formulated into powders, tablets, enteric-coated capsules (e.g., for delivery to the ileum / colon) or pills that can be administered to a subject by any suitable route. In some embodiments, the lyophilized formulation can be mixed with saline or other solutions before administration.

[0108] In some embodiments, wherein the composition comprises Amuc-i09, wherein the bacteria are fully viable (comprising 100% live cell population), partially viable (comprising <50% live cell population), or largely nonviable (comprising >50% dead cell population).

[0109] In some embodiments, a composition comprising Amuc-i09 and Amuc-OG is provided. In some embodiments, the composition comprises at least 50% live Amuc-i09 bacteria. In some embodiments, the composition comprises at least 50% dead Amuc-i09. In some embodiments, the composition comprises Amuc-i09-derived extracellular vesicles.

[0110] In some embodiments, the microbial composition is formulated for oral administration, for example, as an enteric-coated capsule or pill for delivery of the contents of the formulation to the ileum and / or colon region of a subject.

[0111] I. Capsule ingredients

[0112] In some embodiments, the microbial composition is formulated for oral administration. In some embodiments, the microbial composition is formulated into enteric coated pills or capsules for oral administration. In some embodiments, the microbial composition is formulated for delivery of microorganisms to the ileum region of a subject. In some embodiments, the microbial composition is formulated for delivery of microorganisms to the colon region (e.g., upper colon and / or lower colon) of a subject. In some embodiments, the microbial composition is formulated for delivery of microorganisms to the ileum and colon regions of a subject.

[0113] In one embodiment, the composition comprising Amuc-i09 is administered as a capsule for oral delivery.

[0114] In one embodiment, the composition comprising the combination of Amuc-i09 and Amuc-OG is administered as a capsule for oral delivery.

[0115] In one embodiment, the composition comprising a combination of Amuc-i09 and another anaerobic bacteria is administered as an oral formulation.

[0116] In one embodiment, the size of the capsule is No. 0, but the capsule size can be any size suitable for use to a subject. In addition, in the embodiment, the capsule has an enteric coating. The enteric coating can protect the content of the oral formulation from the influence of gastric acid, and provides delivery to the ileum and / or upper colon region. The non-limiting examples of enteric coatings include pH-sensitive polymers (e.g., acrylic resin FS30D), methyl acrylate-methacrylic acid copolymers, cellulose acetate succinate, hydroxypropyl methyl-cellulose phthalate, hydroxypropyl methyl-cellulose succinate ethyl ester (e.g., hydroxypropyl methylcellulose succinate ethyl ester), polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymers, shellac, cellulose acetate trimellitate, sodium alginate, zein, other polymers, fatty acids, waxes, shellac, plastics and plant fibers. In certain embodiments, the enteric coating is formed by a pH-sensitive polymer. In certain embodiments, the enteric coating is formed by acrylic resin FS30D.

[0117] The enteric coating can be designed to dissolve at any suitable pH. In some embodiments, the enteric coating is designed to dissolve at a pH greater than about pH 6.5 to about pH 7.0. In some embodiments, the enteric coating is designed to dissolve at a pH greater than about pH 6.5. In some embodiments, the enteric coating is designed to dissolve at a pH greater than about pH 7.0. The enteric coating can be designed to dissolve at a pH greater than: 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4 or 7.5 pH units.

[0118] In some embodiments, administration of the formulations of the present disclosure may precede, for example, colon cleansing methods such as colon irrigation / hydrotherapy, enemas, administration of laxatives, dietary supplements, dietary fiber, enzymes, and magnesium or its derivatives.

[0119] The formulations provided herein can include adding one or more medicaments to increase the stability and / or survival rate of the microbial formulations in therapeutic agents or cosmetics. The limiting examples of stabilizers include genetic factors, glycerol, ascorbic acid, skim milk, lactose, tween, alginate, xanthan gum, carrageenan, mannitol, palm oil and poly-L-lysine (POPL).

[0120] II. Microbial Formulations and Compositions in Microbial Consortia

[0121] The compositions disclosed herein may include a single microbial species or a combination of microorganisms. In one embodiment, the formulation includes only Amuc-i09. In another embodiment, the formulation comprises Amuc-i09 and at least one microorganism selected from the group consisting of anaerobic Corynebacterium faecalis, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, Butyrivibrio fibrinolyticus, Clostridium acetobutylicum, Clostridium aminophilum, Clostridium beijerinckii, Clostridium butyricum, Clostridium intestinale, Clostridium indolizum, Clostridium flavone-lyticus, Enterococcus faecalis, Eubacterium hallii, Eubacterium rectum, Faecalis prausnitzii, Mylobacterium succinicigenes, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus caucasicus, Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus lactis, lactis, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Oscillatory Spirochete Gibbsii, Roseburia caeca, Roseburia glucovorans, Ruminococcus xanthophytes, Ruminococcus activus, Ruminococcus ovale, Streptococcus cremoris, Streptococcus faecalis, Streptococcus infantis, Streptococcus mutans, Streptococcus thermophilus, anaerobic rod-shaped bacteria of feces, butyrate-producing bacteria, anaerobic rod-shaped bacteria of human colon, Clostridium sporogenes, Clostridium tetani, Coccus regularus, Eubacterium columnaris, Eubacterium elongatum, Eubacterium convexum, Roseburia faecalis, Roseburia hominis, Roseburia intestinalis, and any combination thereof.

[0122] In another embodiment, the composition as disclosed herein includes one or more recombinant microorganisms or genetically modified microorganisms. In other embodiments, one or more microorganisms are unmodified or recombinant. In some embodiments, the composition includes a microorganism that can be regulated, for example, a microorganism including an operator or promoter to control microbial growth. As disclosed herein, microorganisms can be produced, grown or modified using any suitable method (including recombinant methods).

[0123] In one embodiment, the bacterial population that can be administered as part of the composition disclosed herein can include any one or more of the species selected from the group consisting of Amuc-i09, Amuc-OG, Akkermansia muciniphila, Anaerobic Corynebacterium faecalis, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, Butyrivibrio fibrinolyticus, Clostridium acetobutylicum, Clostridium aminophilum, Clostridium beijerinckii, Clostridium butyricum, Clostridium intestinale, Clostridium indolizum, Clostridium flavoneolyticus, Enterococcus faecalis, Eubacterium hallii, Eubacterium rectum, Faecalibacterium prausnitzii, Succinic acid-producing filamentous bacillus, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus bulgaricus any other strain of the invention, including but not limited to Lactobacillus, Lactobacillus casei, Lactobacillus caucasus, Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus lactis, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Oscillatory Spirochete Gibbsii, Roseburia caecum, Roseburia glucovorans, Ruminococcus xanthogenes, Ruminococcus activus, Ruminococcus ovale, Streptococcus cremoris, Streptococcus faecalis, Streptococcus infantis, Streptococcus mutans, Streptococcus thermophilus, anaerobic rod-shaped bacteria of feces, butyrate-producing bacteria, anaerobic rod-shaped bacteria of the human colon, Clostridium sporogenes, Clostridium tetani, regular coccus, Eubacterium columnarum, Eubacterium elongatum, Eubacterium convexum, Roseburia faecum, Roseburia hominis, Roseburia intestinalis, and any combination thereof.

[0124] In some embodiments, a therapeutic composition for treating a metabolic disorder is provided, the therapeutic composition comprising a purified microbial population consisting of bacteria, wherein the purified microbial population has at least about 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity with the 16S rRNA and / or 23S rRNA of a microorganism selected from the group consisting of Amuc-i09, Eubacterium hallii DSM 3353, Akkermansia muciniphila ATCC BAA83, Clostridium butyricum ATCC 1939, Clostridium beijerinckii ATCC 5174, Akkermansia muciniphila, Anaerobic Corynebacterium faecalis, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, Butyrivibrio fibrinolyticus, Clostridium acetobutylicum, Clostridium aminophilum, Clostridium beijerinckii, Clostridium butyricum, Clostridium intestinale, Clostridium indolizum, Clostridium flavone-soluble, Enterococcus faecalis, Eubacterium hallii, Eubacterium rectum, Faecalis prausnitzii, Succinic acid-producing filamentous bacillus, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus caucasus, Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus lactis Bacteria, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Oscillatory Spirillum gibbsii, Roseburia caecum, Roseburia glucovorans, Ruminococcus xanthotrophus, Ruminococcus activus, Ruminococcus ovale, Streptococcus cremoris, Streptococcus faecalis, Streptococcus infantis, Streptococcus mutans, Streptococcus thermophilus, fecal anaerobic rod-shaped bacteria, butyrate-producing bacteria, human colon anaerobic rod-shaped bacteria, Clostridium sporogenes, Clostridium tetani, regular coccus, Eubacterium columnarum, Eubacterium elongatum, Eubacterium convexum, Roseburia faecum, Roseburia hominis, Roseburia intestinalis, and any combination thereof.

[0125] In some embodiments, the metabolic disorder may be selected from an insulin resistance based disorder, an insulin sensitivity based disorder, type 1 diabetes, type 2 diabetes, and obesity.

[0126] In some embodiments, a therapeutic composition for treating type 2 diabetes or obesity is provided, the therapeutic composition comprising a purified microbial population consisting of bacteria, wherein the purified microbial population has at least about 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity with the 16S rRNA and / or 23S rRNA of a microorganism selected from the group consisting of Amuc-i09, Eubacterium hallii DSM 3353, Akkermansia muciniphila ATCC BAA83, Clostridium butyricum ATCC 1939, Clostridium beijerinckii ATCC5174, Akkermansia muciniphila, Anaerobic Corynebacterium faecalis, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, Butyrivibrio fibrinolyticus, Clostridium acetobutylicum, Clostridium aminophilum, Clostridium beijerinckii, Clostridium butyricum, Clostridium intestinale, Clostridium indolizum, Clostridium flavone-lyticum, Enterococcus faecalis, Eubacterium hallii, Eubacterium rectum, Faecalibacterium prausnitzii, Succinic acid-producing filamentous bacillus, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus caucasus, Lactobacillus fermentum, Lactobacillus helveticus Bacteria, Lactobacillus lactis, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Oscillatory Spirillum gibbsii, Roseburia caecum, Roseburia glucovorans, Ruminococcus xanthophyllus, Ruminococcus activus, Ruminococcus ovale, Streptococcus cremoris, Streptococcus faecalis, Streptococcus infantis, Streptococcus mutans, Streptococcus thermophilus, fecal anaerobic rod-shaped bacteria, butyrate-producing bacteria, human colon anaerobic rod-shaped bacteria, Clostridium sporogenes, Clostridium tetani, regular coccus, Eubacterium columnarum, Eubacterium elongatum, Eubacterium convexum, Roseburia faecum, Roseburia hominis, Roseburia intestinalis, and any combination thereof.

[0127] In some embodiments, a therapeutic composition for treating type 2 diabetes or obesity is provided, the therapeutic composition comprising a purified microbial population comprising bacteria, wherein the purified microbial population has at least about 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity with the 16S rRNA and / or 23S rRNA of Amuc-i09.

[0128] In some embodiments, a therapeutic composition for treating type 2 diabetes or obesity is provided, the therapeutic composition comprising a purified microbial population comprising bacteria, wherein the purified microbial population has at least about 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity with: 16S rRNA and / or 23S rRNA of Amuc-OG.

[0129] In some embodiments, a therapeutic composition for treating a neurological disease or a behavioral disorder is provided, the therapeutic composition comprising a purified microbial population consisting of bacteria, wherein the purified microbial population has at least about 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity with the 16S rRNA and / or 23S rRNA of a microorganism selected from the group consisting of Amuc-i09, Eubacterium hallii DSM 3353, Akkermansia muciniphila ATCC BAA83, Clostridium butyricum ATCC 1939, Clostridium beijerinckii ATCC 5174, Akkermansia muciniphila, Anaerobic Corynebacterium faecalis, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, Butyrivibrio fibrinolyticus, Clostridium acetobutylicum, Clostridium aminophilum, Clostridium beijerinckii, Clostridium butyricum, Clostridium intestinale, Clostridium indolizum, Clostridium flavone-soluble, Enterococcus faecalis, Eubacterium hallii, Eubacterium rectum, Faecalis prausnitzii, Succinic acid-producing filamentous bacillus, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus caucasus, Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus lactis Bacteria, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Oscillatory Spirillum gibbsii, Roseburia caecum, Roseburia glucovorans, Ruminococcus xanthotrophus, Ruminococcus activus, Ruminococcus ovale, Streptococcus cremoris, Streptococcus faecalis, Streptococcus infantis, Streptococcus mutans, Streptococcus thermophilus, fecal anaerobic rod-shaped bacteria, butyrate-producing bacteria, human colon anaerobic rod-shaped bacteria, Clostridium sporogenes, Clostridium tetani, regular coccus, Eubacterium columnarum, Eubacterium elongatum, Eubacterium convexum, Roseburia faecum, Roseburia hominis, Roseburia intestinalis, and any combination thereof.

[0130] In some embodiments, the neurological disease or behavioral disorder is selected from gut-brain axis disorders, anxiety, or hyperalgesia. In some embodiments, the compositions disclosed herein modulate neurotransmitter levels in the brain of a subject.

[0131] In some embodiments, a therapeutic composition for treating a neurological disease or a behavioral disorder is provided, the therapeutic composition comprising a purified microbial population consisting of Amuc-i09.

[0132] In some embodiments, a therapeutic composition for treating a gut-related disorder is provided, the therapeutic composition comprising a purified microbial population consisting of Amuc-i09. In some embodiments, the gut-related disorder is selected from constipation, bloating, intestinal damage-related disorders, diarrhea, gastritis, intestinal inflammation, Crohn's disease, irritable bowel syndrome, inflammatory bowel disease or disorders.

[0133] In some embodiments, the therapeutic combination includes Akkermansia muciniphila, Bifidobacterium adolescentis, Bifidobacterium infantis, Clostridium beijerinckii, Clostridium butyricum, and Amuc-i09.

[0134] In some embodiments, the therapeutic combination includes Akkermansia muciniphila and Eubacterium hallii or a butyrate co-producing bacterium.

[0135] In some embodiments, the therapeutic combination includes Amuc-i09 and Eubacterium hallii or a butyrate co-producing bacterium.

[0136] In some embodiments, the therapeutic combination includes Amuc-i09 and any one of Bifidobacterium adolescentis, Bifidobacterium infantis, Clostridium beijerinckii, and Clostridium butyricum.

[0137] The therapeutic composition may include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 45, or at least 50, or at least 75, or at least 100 types of bacteria. The therapeutic composition may include at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 28, at most 29, at most 30, at most 31, at most 32, at most 33, at most 34, at most 35, at most 36, at most 37, at most 38, at most 39, at most 40, at most 45, or at most 50, or at most 75, or at most 100 types of bacteria.

[0138] In some embodiments, combining one or more microorganisms in a therapeutic composition or combination can provide a synergistic effect when administered to an individual.

[0139] For example, administration of a first microorganism can be beneficial to the subject, and administration of a second microorganism can be beneficial to the subject, but when both microorganisms are administered to the subject together, the benefit is greater than the benefit of administering either microorganism alone.

[0140] Different types of microorganisms in the therapeutic composition can be present in the same amount or in different amounts. For example, the ratio of the two bacteria in the therapeutic composition can be about 1:1, 1:2, 1:5, 1:10, 1:25, 1:50, 1:100, 1:1000, 1:10,000 or 1:100,000.

[0141] III. Compositions including probiotics

[0142] In one embodiment, a composition comprising a single microorganism as a probiotic is disclosed. In another embodiment, a composition comprising a consortium of at least two different bacteria is disclosed. In addition to the probiotic, the composition may include a prebiotic, an antibiotic, or a combination of active agents as described herein.

[0143] In some embodiments, the composition is administered before, during, and / or after treatment with an antimicrobial agent such as an antibiotic. For example, the composition may be administered at least about 1 hour, 2 hours, 5 hours, 12 hours, 1 day, 3 days, 1 week, 2 weeks, 1 month, 6 months, or 1 year before and / or after treatment with an antibiotic. The formulation may be administered up to 1 hour, 2 hours, 5 hours, 12 hours, 1 day, 3 days, 1 week, 2 weeks, 1 month, 6 months, or 1 year before and / or after treatment with an antibiotic.

[0144] In some embodiments, the compositions as disclosed herein are administered during, after, and / or before treatment with an antidiabetic drug such as insulin or a drug such as metformin. For example, the formulations may be administered at least about 1 hour, 2 hours, 5 hours, 12 hours, 1 day, 3 days, 1 week, 2 weeks, 1 month, 6 months, or 1 year before and / or after treatment with an antidiabetic drug.

[0145] In some embodiments, the composition is administered before, during, and / or after food is ingested by the subject. In some embodiments, the composition is administered together with food ingested by the subject. In some embodiments, the composition is administered together with (e.g., simultaneously with) food ingestion.

[0146] In some embodiments, the composition is administered before food is ingested by the subject. In some embodiments, the composition is more effective or more potent for treating metabolic disorders when administered before food is ingested. For example, the composition may be administered about 1 minute, about 2 minutes, about 3 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, or about 1 day before food is ingested by the subject.

[0147] In some embodiments, the composition is administered after the subject ingests food. In some embodiments, the composition is more effective or more potent for treating metabolic disorders when administered after ingesting food. For example, the composition may be administered at least about 1 minute, 2 minutes, 3 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 5 hours, 10 hours, 12 hours, or 1 day after ingesting food by the subject. For example, the formulation may be administered at most about 1 minute, 2 minutes, 3 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 5 hours, 10 hours, 12 hours, or 1 day after ingesting food by the subject.

[0148] The compositions provided herein include those suitable for oral (including buccal and sublingual), intranasal, topical, transdermal, transdermal patch, pulmonary, vaginal, rectal, suppository, mucosal, systemic, or parenteral (including intramuscular, intraarterial, intrathecal, intradermal, intraperitoneal, subcutaneous, and intravenous) administration or in a form suitable for administration by nebulization, inhalation, or insufflation.

[0149] In some embodiments, the composition may be a therapeutic composition, a pharmaceutical composition, or a dietary supplement composition.

[0150] As disclosed herein, therapeutic compositions may include carriers and excipients (including but not limited to buffers, carbohydrates, lipids, mannitol, proteins, polypeptides or amino acids (such as glycine), antioxidants, antibacterial agents, chelating agents, suspending agents, thickeners and / or preservatives), metals (e.g., iron, calcium), salts, vitamins, minerals, water, oils (including oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc.), saline solutions, aqueous glucose and glycerol solutions, flavoring agents, coloring agents, anti-adhesive agents and other acceptable additives, adjuvants or binders, other pharmaceutically acceptable auxiliary substances as required by the general physiological conditions, such as pH buffers, tonicity adjusters, emulsifiers, wetting agents, etc. Examples of excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc.

[0151] Non-limiting examples of pharmaceutically acceptable excipients suitable for use in the present disclosure include granulating agents, binders, lubricants, disintegrants, sweeteners, glidants, antiadherents, antistatic agents, surfactants, antioxidants, gums, coating agents, colorants, flavoring agents, dispersion enhancers, disintegrants, coating agents, plasticizers, preservatives, suspending agents, emulsifiers, plant cellulose materials and spheronizing agents, and any combination thereof.

[0152] Non-limiting examples of pharmaceutically acceptable excipients can be found in, e.g., Remington: The Science and Practice of Pharmacy, 19th ed. (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 1975; Liberman, H. A. and Lachman, L., eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed. (Lippincott Williams & Wilkins 1999), each of which is incorporated by reference in its entirety.

[0153] The therapeutic composition may be substantially free of preservatives. In some applications, the composition may contain at least one preservative.

[0154] The therapeutic composition can be encapsulated in a suitable vehicle, such as liposomes, microspheres or microparticles. Microspheres formed from polymers or proteins can be customized to enter the blood directly through the gastrointestinal tract. Alternatively, the compound can be combined with microspheres or complexes of microspheres and can be implanted to slowly release over a period of days to months.

[0155] The therapeutic composition can be formulated into a sterile solution or suspension. The therapeutic composition can be sterilized by conventional techniques, or can be aseptically filtered. The resulting aqueous solution can be packaged for use as is or lyophilized. The lyophilized preparation of the microbial composition can be packaged in a suitable form for oral administration, such as a capsule or a pill. In one embodiment, the capsule can include an enteric polymer that does not disintegrate until the capsule leaves the stomach of the subject.

[0156] In one embodiment, the capsule ingredients may include methylcellulose, hydroxypropylmethylcellulose phthalate, propylene glycol, and derivatives of such polymers.

[0157] The composition can be applied topically and can be formulated into various compositions for topical application, such as solutions, suspensions, lotions, gels, pastes, medicated sticks, balms, creams and ointments. Such pharmaceutical compositions can contain solubilizers, stabilizers, tonicity regulators, buffers and preservatives.

[0158] The compositions may also be formulated in rectal compositions such as enemas, rectal gels, rectal foams, rectal aerosols, suppositories, jelly suppositories or retention enemas containing traditional suppository bases such as cocoa butter or other glycerides, as well as synthetic polymers such as polyvinylpyrrolidone, PEG, etc. In suppository forms of compositions, a low melting wax such as a mixture of fatty acid glycerides, optionally in combination with cocoa butter may be employed.

[0159] When practicing the therapeutic methods or purposes provided herein, the microbial composition as herein described of therapeutically effective dose is applied to the experimenter suffering from disease to be treated or symptom in the form of pharmaceutical composition. In certain embodiments, the experimenter is a mammal, such as people. The therapeutically effective dose can be greatly different according to the severity of the disease, the age of the experimenter and relative health status, the effectiveness of the formulation and other factors. For example, the experimenter can be a person, an elderly person, an adult, a teenager, a prepubertal child, a child, a toddler, a baby or a newborn.

[0160] A subject may be a patient. A subject may be an individual participating in a clinical study. A subject may be an experimental animal, such as a mammal. A subject may be a human.

[0161] Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers including excipients and adjuvants that assist in processing the microorganism into a pharmaceutically usable formulation. The formulation can be modified according to the selected route of administration. The pharmaceutical compositions described herein can be prepared in a conventional manner, such as by conventional mixing, dissolving, granulating, vitrifying, spray drying, freeze drying, grinding, encapsulating, capturing, emulsifying or compression processes.

[0162] In some embodiments, the pharmaceutical composition is prepared in a dry form, for example by spray drying or lyophilization. In some embodiments, the formulation is prepared as a liquid capsule to maintain the liquid state of the microorganism.

[0163] The compositions provided herein can be stored at any suitable temperature. For example, the formulation can be stored in a freezer at a temperature of about -80 ° C, about -20 ° C, about -4 ° C or about 4 ° C. For example, the storage temperature can be about 0 ° C, about 1 ° C, about 2 ° C, about 3 ° C, about 4 ° C, about 5 ° C, about 6 ° C, about 7 ° C, about 8 ° C, about 9 ° C, about 10 ° C, about 12 ° C, about 14 ° C, about 16 ° C, about 20 ° C, about 22 ° C or about 25 ° C. In some embodiments, the storage temperature is about 2 ° C to about 8 ° C. Storing the microbial composition at low temperatures, such as about 2 ° C to about 8 ° C, can maintain the vitality of the microorganism and improve the efficiency of the composition, for example, when present in a liquid or gel formulation. Storing at a freezing temperature below 0 ° C and using a cryoprotectant can further extend stability.

[0164] The pH of the composition can range from about 3 to about 12. For example, the pH of the composition can be from about 3 to about 4, from about 4 to about 5, from about 5 to about 6, from about 6 to about 7, from about 7 to about 8, from about 8 to about 9, from about 9 to about 10, from about 10 to about 11, or from about 11 to about 12 pH units.

[0165] For example, the pH of the composition can be about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12 pH units. For example, the pH of the composition can be at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 pH units. For example, the pH of the composition can be at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, or at most 12 pH units. If the pH exceeds the range designed by the formulator, the pH can be adjusted by using enough pharmaceutically acceptable acids and bases. In some embodiments, the pH of the composition is about 4 to about 6.

[0166] Pharmaceutical compositions containing microorganisms as herein described can be used for preventive and / or therapeutic treatment. In therapeutic applications, the composition can be enough to cure or at least partially prevent the symptom of a disease or condition or cure, recover, improve or alleviate the amount of the condition to be used to a subject who has suffered from the disease or condition. Microbial compositions can also be used to reduce the development, infection or deterioration of the condition. The effective amount for this purpose can depend on the severity and course of disease of the disease or condition, previous therapy, the health status of the subject, body weight and the response of the drug and the judgment of the attending physician and change.

[0167] The multiple therapeutic agents may be administered in any order or simultaneously. If used simultaneously, the multiple therapeutic agents may be provided in a single, unified form or in multiple forms, such as multiple individual pills. The composition may be packaged together or separately in a single package or multiple packages. One or all of the therapeutic agents may be administered in multiple doses. If not taken simultaneously, the time between multiple doses may vary up to about one month.

[0168] Compositions as described herein can be applied before, during or after a disease or condition occurs, and the time of applying the composition can vary. For example, the microbial composition can be used as a prophylactic, and can be continuously applied to a subject with a condition or disease tendency to reduce the possibility of a disease or condition occurring. The microbial composition can be applied to a subject as soon as possible during or after the onset of symptoms. The application of the microbial composition can start within the first 48 hours of symptom onset, within the first 24 hours of symptom onset, within the first 6 hours of symptom onset, or within 3 hours of symptom onset. Any formulation as described herein can be used to initially apply by any practical approach as described herein (such as by any approach as described herein). The microbial composition can be applied as soon as possible after detecting or suspecting a disease or condition onset, and the time length required for the treatment of the disease can be continued, such as about 1 month to about 3 months. The treatment length of each subject can be different.

[0169] In one aspect, the composition can be administered in conjunction with another therapy, such as immunotherapy, chemotherapy, radiation therapy, anti-inflammatory agents, antiviral agents, antimicrobial agents, and antifungal agents.

[0170] On the other hand, the composition can be packaged into a test kit. In certain embodiments, the test kit includes written instructions for the use / purpose of the composition. For example, the written material can be a label. The written material can suggest the method of application of the condition. These instructions provide the best guidance for the subject and the guiding doctor so that the best clinical effect can be obtained from the use of the therapy. The written material can be a label. In certain embodiments, the label can be approved by a regulatory agency, such as the U.S. Food and Drug Administration (FDA), the European Medicines Agency (EMA) or other regulatory agencies.

[0171] G. Treatment methods

[0172] The present disclosure provides methods for treating subjects. Subjects may suffer from or be susceptible to symptoms associated with diseases caused by an imbalance of intestinal microbiota. Changing the composition of the microbiota in such subjects can have expected health outcomes. The compositions of the present disclosure can be administered as treatments and / or dietary supplements for treating health conditions. Treatments designed to change the host microbiota can alleviate patient symptoms, prevent disease and or treat disease or health conditions. For example, changing the intestinal microbiota can reduce the risk of health conditions such as metabolic disorders, neurological diseases or behavioral disorders.

[0173] Therefore, the present disclosure provides a method for restoring a microbial habitat of a subject to a healthy state. The method may include correction and / or adjustment of the microbiome, including, for example, supplementation of natural microorganisms, removal of pathogenic microorganisms, administration of prebiotics and growth factors required for the survival of the microbiome. In some embodiments, the method further includes administration of an antimicrobial agent, such as an antibiotic.

[0174] Based on the microbiome profile, the present disclosure provides a universal treatment recommendation method for a subject and a treatment recommendation method for a specific subject. The treatment method may include one of the following steps: determining a first ratio of a subject-specific microbiome profile level to a second microbiome profile level in a biological sample obtained from at least one subject, detecting the presence or absence of a disease in the subject based on the determination result, and recommending at least one universal or subject-specific treatment to the subject to improve disease symptoms.

[0175] Diseases that can be treated include diseases in which changes in the intestinal microbiota are beneficial to the subject suffering from the disease. Specifically, the intestinal microbiota of the subject may lack a population of at least one live, active intestinal bacteria selected from the group consisting of Akkermansia muciniphila, Clostridium butyricum, Clostridium beijerinckii, Anaerobic Corynebacterium faecalis, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, Butyrivibrio fibrinolyticus, Clostridium acetobutylicum, Clostridium aminophilum, Clostridium beijerinckii, Clostridium butyricum, Clostridium intestinale, Clostridium indolizole, Clostridium flavoneolyticus, Enterococcus faecalis, Eubacterium rectum, Eubacterium hallii, and Eubacterium prausnitzii. Faecalibacterium, succinimidyl bacteria, Oscillating Spikelet, Roseburia caecum, Roseburia glucovorans, Ruminococcus xanthotrophica, Ruminococcus activus, Ruminococcus ovale, Streptococcus cremoris, Streptococcus faecalis, Streptococcus infantis, Streptococcus mutans, Streptococcus thermophilus, fecal anaerobic rod-shaped bacteria, butyrate-producing bacteria, human colon anaerobic rod-shaped bacteria, Clostridium sporogenes, Clostridium tetani, regular coccus, Eubacterium columnarum, Eubacterium elongatum, Eubacterium convexum, Roseburia faecum, Roseburia hominis, Roseburia intestinalis, and any combination thereof.

[0176] In one embodiment, the intestinal microbe at a lower level in a subject suffering from or susceptible to a condition as described above is Akkermansia muciniphila. In another embodiment, the intestinal microbe at a lower level in a subject suffering from or susceptible to a condition as described above is Eubacterium hallii. In another embodiment, the intestinal microbe at a lower level in a subject suffering from or susceptible to a condition as described above belongs to the genera Clostridium, Eubacterium, Bifidobacterium, Anaerobic Corynebacterium, Coprococcus, Bacteroides, Blautia, Ruminococcus, Faecalibacterium, Oscillatory Spirillum, Streptococcus, and Roseburia.

[0177] In another embodiment, the level of more than one intestinal microorganism in the subject is lower than the level of the intestinal microorganism in a normal subject. Specifically, the microorganism can be at least one or more of the following: Akkermansia muciniphila, Clostridium butyricum, Clostridium beijerinckii, anaerobic Corynebacterium faecalis, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, Butyrivibrio fibrinolyticus, Clostridium acetobutylicum, Clostridium aminophilum, Clostridium beijerinckii, Clostridium butyricum, Clostridium intestinalis, Clostridium indoliferum, Clostridium flavone-lyticum, Enterococcus faecalis, Eubacterium rectum, Eubacterium hallii, Faecalibacterium prausnitzii, succinic acid-producing filamentous rods Bacteria, Oscillatory Spikeleti, Roseburia caecalis, Roseburia glucovorans, Ruminococcus xanthotrophus, Ruminococcus activus, Ruminococcus ovale, Streptococcus cremoris, Streptococcus faecalis, Streptococcus infantis, Streptococcus mutans, Streptococcus thermophilus, fecal anaerobic rod-shaped bacteria, butyrate-producing bacteria, human colon anaerobic rod-shaped bacteria, Clostridium sporogenes, Clostridium tetani, Coccus regularus, Eubacterium columnarum, Eubacterium elongatum, Eubacterium convexum, Roseburia faecalis, Roseburia hominis, Roseburia intestinalis, and any combination thereof.

[0178] Measuring the microbiota of a subject can show that a microbiota lacking various strains of microorganisms can result in health conditions and / or disease states. Restoring one or more of the lacking strains (e.g., by bacterial strains such as Akkermansia muciniphila) can result in a change in health conditions. Some non-limiting examples include altering the intestinal microbiota to increase the host's ability to obtain energy, improve insulin sensitivity and / or decrease appetite, reduce anxiety, reduce pain sensation, reduce behavioral problems such as anxiety, regularize bowel movements, reduce bloating, and reduce constipation.

[0179] For example, some non-limiting health conditions that may be affected by an imbalance in the microbiome include type 2 diabetes (T2DM), premature birth, chronic fatigue syndrome, skin diseases such as acne, allergies, autism, asthma, depression, hypertension, metabolic syndrome, obesity, lactose intolerance, oral thrush, ulcerative colitis, drug metabolism, vaginitis, atopic dermatitis, psoriasis, type 1 diabetes (T1DM), multiple sclerosis, neurological diseases such as Parkinson's disease, Clostridium difficile infection, heart disease, diabetic foot ulcers, bacteremia, pediatric colic, cancer, cystic fibrosis, multiple sclerosis, urinary tract infection, radiation enteropathy, drug metabolism, dental caries, and bad breath.

[0180] Measuring the microbiome of a subject can be performed by analyzing a body fluid sample or performing a microbial analysis on the feces of the subject. Candidate strains can be found in scientific literature and research. These candidate strains can be used as parameters to be measured from the microbiome analysis of the subject. By comparing the ideal level of each microorganism in a healthy human body and the level of the microorganism in the subject, a conclusion about the level of the microorganism in the intestinal tract of the subject can be drawn. Therefore, the composition as disclosed herein can be administered to supplement and restore the colony of the microorganism of the intestinal tract as determined by microbial analysis that is lacking.

[0181] I. Administration

[0182] The appropriate amount of therapeutic composition to be administered, the number of treatments, and the unit dosage may vary depending on the subject and / or the subject's disease state.

[0183] The pharmaceutical compositions described herein can be in unit dosage form for single administration of precise dosage. In unit dosage form, the formulation can be divided into unit doses containing an appropriate amount of one or more microbial compositions. The unit dose can be in a packaged form containing a discrete number of formulations. Unrestricted examples are liquids in vials or ampoules. Aqueous suspension compositions can be packaged in single-dose non-resealed containers. The composition can be in multi-dose form. For example, a multi-dose resealed container can be used in combination with a preservative. The formulation for parenteral injection can be presented in unit dosage form, for example in an ampoule, or in a multi-dose container containing a preservative.

[0184] The dosage may be in the form of a solid, semisolid or liquid composition. Non-limiting examples of dosage forms include feeds, foods, pills, lozenges, liquids, elixirs, aerosols, inhalants, sprays, powders, tablets, pills, capsules, gels, gel tablets, nanosuspensions, nanoparticles, microgels, suppositories, lozenges, aqueous or oily suspensions, ointments, patches, lotions, dentifrices, emulsions, creams, drops, dispersible powders or granules, emulsions in hard or soft capsules, syrups, botanicals, nutraceuticals, dietary supplements, and any combination thereof.

[0185] Microorganisms can be present in the pharmaceutical composition at any suitable concentration. For example, the concentration of the microorganism can be about 10^1 to about 10^18 CFU or AFU per gram of culture medium. For example, the concentration of the microorganism can be at least 10^1, at least 10^2, at least 10^3, at least 10^4, at least 10^5, at least 10^6, at least 10^7, at least 10^8, at least 10^9, at least 10^10, at least 10^11, at least 10^12, at least 10^13, at least 10^14, at least 10^15, at least 10^16, at least 10^17 or at least 10^18 CFU or AFU per gram. For example, the concentration of microorganisms can be at most 10^1, at most 10^2, at most 10^3, at most 10^4, at most 10^5, at most 10^6, at most 10^7, at most 10^8, at most 10^9, at most 10^10, at most 10^11, at most 10^12, at most 10^13, at most 10^14, at most 10^15, at most 10^16, at most 10^17 or at most 10^18 CFU or AFU per gram. In some embodiments, the concentration of microorganisms is about 10^8 CFU to about 10^9 CFU or AFU per gram. In some embodiments, the concentration of microorganisms is about 10^8 CFU or AFU per gram. In some embodiments, the concentration of microorganisms is about 10^9 CFU or AFU per gram.

[0186] As described herein, pharmaceutical compositions can be formulated with any suitable therapeutically effective concentration of prebiotics. For example, the therapeutically effective concentration of prebiotics can be at least about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 10 mg / ml, about 15 mg / ml, about 20 mg / ml, about 25 mg / ml, about 30 mg / ml, about 35 mg / ml, about 40 mg / ml, about 45 mg / ml, about 50 mg / ml, about 55 mg / ml, about 60 mg / ml, about 65 mg / ml, about 70 mg / ml, about 75 mg / ml, about 80 mg / ml, about 85 mg / ml, about 90 mg / ml, about 95 mg / ml, about 100 mg / ml, about 110 mg / ml, about 125 mg / ml, about 130 mg / ml, about 140 mg / ml, or about 150 mg / ml. For example, the therapeutically effective concentration of a prebiotic can be up to about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 10 mg / ml, about 15 mg / ml, about 20 mg / ml, about 25 mg / ml, about 30 mg / ml, about 35 mg / ml, about 40 mg / ml, about 45 mg / ml, about 50 mg / ml, about 55 mg / ml, about 60 mg / ml, about 65 mg / ml, about 70 mg / ml, about 75 mg / ml, about 80 mg / ml, about 85 mg / ml, about 90 mg / ml, about 95 mg / ml, about 100 mg / ml, about 110 mg / ml, about 125 mg / ml, about 130 mg / ml, about 140 mg / ml, or about 150 mg / ml. For example, the therapeutically effective concentration of prebiotics can be about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 10 mg / ml, about 15 mg / ml, about 20 mg / ml, about 25 mg / ml, about 30 mg / ml, about 35 mg / ml, about 40 mg / ml, about 45 mg / ml, about 50 mg / ml, about 55 mg / ml, about 60 mg / ml, about 65 mg / ml, about 70 mg / ml, about 75 mg / ml, about 80 mg / ml, about 85 mg / ml, about 90 mg / ml, about 95 mg / ml, about 100 mg / ml, about 110 mg / ml, about 125 mg / ml, about 130 mg / ml, about 140 mg / ml, or about 150 mg / ml. In some embodiments, the concentration of prebiotics in the pharmaceutical composition is about 70 mg / ml. In some embodiments, the prebiotic is inulin.

[0187] As in the present disclosure, pharmaceutical compositions can be administered, for example, 1, 2, 3, 4, 5 or more times per day. In one embodiment, for example, pharmaceutical compositions can be administered every day, every other day, three times per week, twice per week, once per week or with other appropriate intervals to treat a condition.

[0188] Although the foregoing invention has been described in some detail for the purpose of clarity and understanding, it will be apparent to those skilled in the art from reading this disclosure that various changes in form and detail may be made without departing from the true scope of the invention. For example, all of the compositions and methods described above may be used in different combinations. All publications, patents, patent applications, and / or other documents cited in this application are incorporated by reference in their entirety for all purposes, to the same extent as if each individual publication, patent, patent application, and / or other document were individually and separately indicated to be incorporated by reference for all purposes.

[0189] H. Examples

[0190] Example 1: Improved growth phase of Amuc-i09 relative to Amuc-OG

[0191] Objective: The aim of the study was to determine the growth differences between AMUC_i09 and AMUC_OG.

[0192] Methods: AMUC colonies were grown on VEG agar plates for 5 days until a single colony was identified. A single colony was inoculated into duplicate 8 ml tubes containing VEG medium at 37°C with shaking at 180 rpm. The optical density at 600 nm (OD600) was measured at different time intervals. Figure 3 As shown in , in terms of exponential growth, Amuc-i09 is characterized by a faster doubling time compared to Amuc-OG. During the exponential growth phase, there were no differences in pH and glucose consumption between the two strains.

[0193] Example 2: Improved granulation properties of Amuc-i09

[0194] Purpose: The purpose of this experiment was to determine the physiological differences between Amuc-i09 and Amuc-OG.

[0195] Method: Amuc-i09 and Amuc-OG colonies were grown on VEG agar plates for 5 days until a single colony was identified. A single colony was inoculated into two replicate 8ml test tubes containing VEG culture medium shaken at 180rpm at 37C. The optical density (OD600) at 600nm was measured at different time intervals throughout the growth phase of the 8ml test tube. When OD600 reached 1.0, 8ml culture fluid was used to inoculate 100ml bottles. It was 1.0 to grow the 100ml bottle to OD600, and then it was subcultured into 500ml bottles. It was 1.0 to grow the 500ml bottle to OD600, and it was centrifuged at 7,500rpm for 20 minutes. Compared with Amuc-OG, Amuc-i09 cells show improved granulation characteristics. As shown in Table 2, this makes the dry weight after the product is lyophilized different.

[0196] Table 2: Differences in dry weight of sediments

[0197] strains Cryoprotectant Dry weight (g) Total CFU CFU / g Amuc-i09 10% sucrose 4 8.72E+11 2.18E+11 Amuc-OG 10% sucrose 21.3 8.29E+11 3.89E++10

[0198] Example 3: Treatment of metabolic disorders with compositions comprising Amuc-i09

[0199] Objective: To treat metabolic disorders in humans using a composition comprising Amuc-i09.

[0200] Method: Treating an individual with a metabolic disorder with a composition comprising AMuc-i09. The composition comprises Amuc-i09 in a range of about 10^9 CFU to 10^10 CFU and inulin.

[0201] The expected delivery form of the oral composition is an enteric (e.g., pH sensitive polymer Eudragit FS30D) pill, which can resist gastric acid and be delivered to the ileum / upper colon region of the subject. The enteric coating is designed to dissolve at a pH greater than about 6.5-7. In certain embodiments, the oral composition is administered as a liquid capsule. The subject administers the composition twice a day before food intake (e.g., 1 hour before meals) for fourteen consecutive days. In some cases, the composition is administered simultaneously with food intake.

[0202] The microbial composition changes the microbial habitat of the subject's intestine to be the same as the microbial habitat of a healthy subject. The subject loses weight. The composition treats a metabolic condition in the subject, e.g., obesity, insulin insensitivity, T2DM, and / or T1DM.

[0203] Example 4: Sequencing Amuc-i09

[0204] Purpose: To sequence Amuc-i09 and determine the unique genomic sequence

[0205] Methods: Amuc-i09 was sequenced using PacBio RSII and assembled into a contig that appears to be a complete chromosome displayed through repeats so that the ends overlap.

[0206] After assembly, a circular contig was obtained that included the entire genome. A database of all publicly available Akkermansia muciniphila proteins was created by downloading all proteins assigned to Akkermansia muciniphila from reference sequences. A mutual blast search of the predicted Amuc-i09 protein sequence against this database in combination with the genome of AMUC-OG showed a total of 2 unique genes (diamond blastp, maximum e-value of 1e-03), SEQ ID NO:1 and SEQ ID NO:2. Of these genes, 0 genes were associated with genes of organisms outside the Akkermansia clade (blastp against reference sequences).

[0207] Therefore, the foregoing only shows the principle of the present disclosure.It should be understood that those skilled in the art can design various arrangements, although not explicitly described or shown herein, the various arrangements embody the principle of the present invention and are included in the spirit and scope of the present invention.In addition, all examples and conditional language narrated herein are mainly intended to help readers understand the principle of the present invention and the concept contributed by the inventor to the development of the art, and should be interpreted as but not limited to the examples and conditions of this specific narration.In addition, the statements narrating the principles, aspects and embodiments of the present invention and its specific examples herein are intended to cover both its structural equivalents and its functional equivalents.In addition, this equivalent is intended to include both currently known equivalents and equivalents developed in the future, that is, any element of the developed performance of the same function, regardless of the structure.Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein.

Claims

1. A bacterium such as the strain Akkermansia muciniphila deposited with the American Type Culture Collection (ATCC) under ATCC Accession No. PTA-126838.

2. A bacterium of the strain Akkermansia muciniphila, wherein the bacterium comprises one or more nucleic acids encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1, a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2, or both.

3. A bacterium of the strain Akkermansia muciniphila, wherein the bacterium comprises a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 and a polypeptide comprising the amino acid sequence shown in SEQ ID NO:

2.

4. A bacterium that expresses a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1, a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2, or both.

5. A composition comprising the bacterium according to any one of claims 1 to 4.

6. A composition for treating a metabolic disorder in a subject, the composition comprising the bacterium according to any one of claims 1 to 4 in an amount effective to treat the metabolic disorder.

7. The composition of claim 5 or 6, further comprising one or more additional microorganisms having a 16S rRNA sequence comprising at least 97% identity over the entire length of the 16S rRNA sequence of a microorganism selected from the group consisting of Anaerostipes caccae, Anaerobutyricum hallii, Bacteroides finegoldii, Bacteroides ovatus, Bacteroides stercoris, Eubactrium hallii, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, Blautia hydrogenotrophica, Blautia producta, Butyrivibrio fibrinolyticus, fibrisolvens), Clostridium acetobutylicum, Clostridium aminophilum, Clostridium beijerinckii, Clostridium butyricum, Clostridium colinum, Clostridium indolis, Clostridium innocuum, Clostridium orbiscindens, Enterococcus faecium, Eubacterium rectale, Faecalibacterium prausnitzii, Fibrobacter succinogenes, Oscillospira guilliermondii, Roseburiacecicola, Roseburia inulinivorans), Ruminococcus flavefaciens, Ruminococcus activegnavus), Ruminococcusobeum, Streptococcus cremoris, Streptococcus faecium, Streptococcus infantis, Streptococcus mutans, Streptococcus thermophilus, Anaerofustis stercorihominis, Anaerostipes hadrus, Anaerotruncus colihominis, Clostridium sporogenes, Clostridium tetani, Coprococcus eutactus, Eubacterium cylindroides, Eubacterium dolichum, Eubacterium ventriosum, Roseburia faeccis, Roseburia hominis, Roseburia intestinalis, Collinsella aerofaciens, Coprococcus comes, Eubacterium limosum, and Ruminococcus faecis, and any combination thereof.

8. The composition of claim 5 or 6, further comprising at least one additional microorganism selected from the group consisting of: co-producing butyrate bacteria, Clostridium butyricum, Clostridium beijerinckii, Clostridium butyricum, and any combination thereof.

9. The composition of any one of claims 5 to 8, wherein the composition comprises at least 10^5 AFU / g of each of the one or more additional microorganisms.

10. The composition according to any one of claims 5 to 8, wherein the composition further comprises at least one preservative.

11. The composition according to any one of claims 5 to 8, wherein the composition further comprises an enteric coating.

12. The composition according to any one of claims 5 to 8, wherein the composition further comprises a prebiotic.

13. The composition of claim 12, wherein the prebiotic is selected from the group consisting of inulin, green banana, ganoderma lucidum, tapioca starch, oats, pectin, potato or extracts thereof, complex carbohydrates, complex sugars, resistant dextrins, resistant starch, amino acids, peptides, nutritional compounds, biotin, polydextrose, fructooligosaccharides (FOS), galacto-oligosaccharides (GOS), starch, lignin, psyllium, chitin, chitosan, gums (e.g., guar gum), high amylose corn starch (HAS), cellulose, b-glucan, hemicellulose, lactulose, mannooligosaccharides (MOS), fructooligosaccharide-enriched inulin, fructooligosaccharides, oligoglucose, tagatose, trans-galacto-oligosaccharides, pectin, resistant starch, xylo-oligosaccharides (XOS), and any combination thereof.

14. The composition of claim 12, wherein the prebiotic is inulin.

15. The composition according to any one of claims 5 to 14, wherein the bacteria are freeze-dried.

16. The composition of claim 7 or 8, wherein the one or more additional microorganisms are freeze-dried.

17. A composition according to any one of claims 5 to 16, wherein the bacteria is viable.

18. The composition of any one of claims 5 to 16, wherein the bacteria is non-viable.

19. The composition according to any one of claims 5 to 18, wherein the composition is formulated as a pharmaceutical formulation.

20. The composition of any one of claims 5 to 18, wherein the composition is formulated as a nutritional supplement.

21. The composition of any one of claims 5 to 18, wherein the composition is formulated as a dietary supplement.

22. The composition according to any one of claims 5 to 18, wherein the composition is formulated as a medical food.

23. A composition according to any one of claims 5 to 22, wherein the composition is dairy-free.

24. The composition of any one of claims 5 to 22, wherein the composition comprises substantially no animal products.

25. The composition according to any one of claims 5 to 22, wherein the composition is in the form of a pill, capsule, lozenge, food bar or gummy ball.

26. A method comprising administering to a subject in need thereof a composition according to any one of claims 5 to 25.

27. A method of treating a metabolic disorder in a subject, the method comprising: The composition of any one of claims 5 to 25 is administered to the subject in an amount effective to treat the metabolic disorder in the subject.

28. The method of claim 27, wherein at least one dose of the composition is administered to the subject per day.

29. The method of claim 27 or 28, wherein a single dose of at least 1 x 10^8 CFU of the Akkermansia muciniphila strain is administered to the subject.

30. The method of any one of claims 27 to 29, wherein the metabolic disorder is selected from the group consisting of or consisting essentially of: insulin resistance based disorders, gut-related disorders, insulin sensitivity based disorders, type 1 diabetes, type 2 diabetes, and obesity.

31. A method comprising cultivating the bacterium according to any one of claims 1 to 4 under anaerobic conditions in / on a growth medium.

32. The method of claim 31 , wherein the growth medium is a vegetable based medium.

33. The method of claim 32, wherein the vegetable based medium is VEG medium.

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