Microorganisms belonging to ackermania genus

Abstract

The present invention is a microorganism belonging to the genus Ackermania, having a 16SrRNA gene comprising a nucleotide sequence or the like having more than 98.0% homology with respect to the nucleotide sequence depicted in SEQ ID NO: 3, and having NOD2 agonist activity, and a microorganism for activating NOD2, immunity and the like, which comprises the microorganism as an active ingredient. The strain has assimilation to D-mannitol, is negative in gram staining, is spherical to short-rod-shaped anaerobic bacteria, is negative in an oxidase test and is positive in a catalase test.

Description

Technical Field

[0001] The present invention relates to a novel microorganism belonging to the genus Akkermansia, and more specifically, to the microorganism, and a composition for activating NOD2 (nucleotide-binding oligomerization domain 2) and a composition for activating immunity using the microorganism as an effective ingredient. Background Art

[0002] In the human intestine, a variety of intestinal microorganisms gather and build a complex ecosystem. This ecosystem is also called the intestinal microbial flora, which affects various physiological functions of humans as hosts. Therefore, the types and diversity of microorganisms inhabiting the intestine are important in terms of human health and longevity.

[0003] As for microorganisms belonging to the genus Akkermansia, which is one type of intestinal microorganism, it is known that the abundance of Akkermansia in the intestine is effective for various symptoms, such as metabolic disorders such as obesity and type 2 diabetes, and inflammatory diseases such as ulcerative colitis and appendicitis. In addition, in recent years, the concept of the brain-gut-microbiota axis has been proposed, and it has been reported that the amount of Akkermansia is related to the personality (social ability) of the host (Non-Patent Document 1).

[0004] In addition, it is reported that in Amami Oshima (Japan), where many centenarians live (the centenarian rate is high), the intestinal flora of the elderly was analyzed and the ratio of Akkermansia and the like was higher than that of the elderly of the same age in other regions (non-patent document 2). Furthermore, it is also reported that the life span was extended after Akkermansia was administered to progeria model mice (non-patent document 3). These results suggest that the abundance of Akkermansia in the intestine has a good effect on the life span of the host.

[0005] As mentioned above, Akkermansia has attracted much attention in recent years because it affects the health and personality of the host and may even prolong life. However, there are only two species of microorganisms that are clearly identified as belonging to the genus Akkermansia (Akkermansia muchiniphila and Akkermansia glycaniphila).

[0006] Prior art literature

[0007] Non-patent literature

[0008] Non-patent literature 1: Katerina VA Johnson, Hum Microb J., “Gut microbiome composition and diversity are related to human personality traits”, March 15, 2020, doi: 10.1016 / j.humic.2019.100069.

[0009] Non-patent document 2: Hidetoshi Morita, Scientific Research Funding Project Research Result Report, "Indicators of Intestinal Bacteria in the Amami Islands' Centenarians' Longevity and Longevity Intestinal Science", June 23, 2021

[0010] Non-patent document 3: Clea Barcena et al., Nat Med., “Healthspan and lifespan extension by fecal microbiota transplantation into progeroid mice 1”, August 25, 2019, Vol. 8, pp. 1234-1242, doi: 10.1038 / s41591-019-0504-5. Summary of the invention

[0011] Problem that the invention aims to solve

[0012] The present invention aims to isolate a novel microorganism belonging to the genus Akkermansia and provide the microorganism. Another object of the present invention is to clarify the effect of the microorganism on a host and provide a composition for improving the health of the host based on the effect.

[0013] Solutions for solving problems

[0014] The present inventors have repeatedly conducted intensive research to achieve the above-mentioned purpose, and as a result, they have successfully detected the colony formation of microbial strains (WON2089, WON2090, and WON2093) belonging to the genus Akkermansia in the feces of three of the five subjects. In addition, the 16SrRNA (Ribonucleic Acid) sequences of these microbial strains were determined and compared, and the results showed that they all carried 16S rRNA genes containing the same sequence (sequence described in SEQ ID NO: 3).

[0015] Then, the 16S rRNA sequence of the above-mentioned microorganism was subjected to Blast+ analysis against the rRNA / ITS reference strain database of the National Center for Biotechnology Information (hereinafter referred to as NCBI). As a result, the homology with the known Akkermansia muciniphila (sequence recorded in sequence number: 1; Genbank accession number AY271254) was 98.0%, and the homology with the known Akkermansia sacchariphila was 94.0% (sequence recorded in sequence number: 2; Genbank accession number KT254068). Furthermore, the whole genome of WON2089 was sequenced, and based on the sequence, ANI (Average nucleotide identity) analysis was performed by DFAST to compare with two strains of the genus Akkermansia. As a result, the ANI value was about 82.5% compared with the known Akkermansia muciniphila, and the ANI value was about 77.4% compared with the known Akkermansia sacchariphila. 95% ANI is the critical value of the current bacterial species. Since it is lower than this value, it indicates that the isolated microbial strain may be a new species of Akkermansia.

[0016] In addition, in the results of the acid production test using API20A, it was observed that the microbial strains (WON2089, WON2090, and WON2093) all proliferated in D-glucose, lactose, and D-mannose, similarly to the two known Akkermansia species. However, unlike the two known Akkermansia species, the above-mentioned microbial strains were also observed to proliferate in D-mannitol.

[0017] Furthermore, when the ligand activity was analyzed, it was found that, while no NOD2 agonist activity was observed for the two known Akkermansia species, such activity was observed for all the microbial strains (WON2089, WON2090, and WON2093).

[0018] It should be noted that NOD2 is a pattern recognition receptor (PRR), and through the activation of this receptor, it is possible to detect invading pathogens or abnormal autologous cells and eliminate them (so-called innate immunity and acquired immunity).

[0019] In addition, the IgA (Immunoglobulin A) secretion promoting effect of WON2089 was evaluated and it was found that it has such an effect. Furthermore, it was found that the IgA secretion promoting effect is more than 2 times higher than that of the known Akkermansia muciniphila (standard strain Muc, strain ATCC BAA-835), thereby completing the present invention.

[0020] That is, the present invention provides the following aspects.

[0021] [1] A microorganism belonging to the genus Akkermansia,

[0022] It has a 16S rRNA gene comprising a nucleotide sequence having a homology of 98.0% or less to the nucleotide sequence described in SEQ ID NO: 1, a homology of 94.0% or less to the nucleotide sequence described in SEQ ID NO: 2, or a homology of more than 98.0% to the nucleotide sequence described in SEQ ID NO: 3, and

[0023] It has the following properties 1) to 4):

[0024] 1) Has NOD2 agonist activity;

[0025] 2) It has the ability to assimilate D-mannitol;

[0026] 3) Gram-negative, anaerobic bacteria with a spherical to short rod shape;

[0027] 4) Oxidase test was negative and catalase test was positive.

[0028] [2] A microorganism specified by deposit number NITE BP-03622, NITE BP-03623 or NITE BP-03624.

[0029] [3] The microorganism according to [1], which has an IgA secretion promoting effect that is more than twice that of the standard strain ATCC BAA-835 of Akkermansia muciniphila.

[0030] [4] A composition for activating immunity, comprising the microorganism according to any one of [1] to [3] as an active ingredient.

[0031] [5] A composition for activating NOD2, comprising the microorganism according to any one of [1] to [3] as an active ingredient.

[0032] [6] A composition for promoting the secretion of IgA, comprising the microorganism described in [2] or [3] as an active ingredient.

[0033] The present invention also relates to use of the microorganism described in any one of [1] to [3] for producing a composition for activating immunity, a composition for activating NOD2, or a composition for promoting secretion of IgA.

[0034] The present invention also relates to use of the microorganism described in any one of [1] to [3] for activating immunity, activating NOD2, or promoting secretion of IgA.

[0035] The present invention also relates to a microorganism according to any one of [1] to [3], which is used for activating immunity, activating NOD2, or promoting secretion of IgA.

[0036] The present invention also relates to a method for activating immunity, activating NOD2, or promoting IgA secretion, comprising administering an effective amount of the microorganism described in any one of [1] to [3] to a subject.

[0037] Effects of the Invention

[0038] According to the present invention, immunity can be activated through activation of NOD2 and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a phylogenetic tree created based on 16S rRNA sequences of the microorganism of the present invention (WON2089), known Akkermansia (Akkermansia muciniphila, Akkermansia sacchariphila), and representative strains belonging to the phylum Verrucomicrobia.

[0040] Figure 2 This is a graph showing the results of analyzing the NOD2 agonist activity against the microorganisms of the present invention (WON2089, WON2090, WON2093) and known Akkermansia (Akkermansia muciniphila, Akkermansia sacchariphila).

[0041] Figure 3 This is a graph showing the results of analyzing the secretion promoting effect of IgA on the microorganism of the present invention (WON2089) and a known Akkermansia (Akkermansia muciniphila (standard strain Muc, strain ATCC BAA-835)). DETAILED DESCRIPTION

[0042] (Novel microorganism belonging to the genus Akkermansia)

[0043] As shown in the examples described below, the present inventors have successfully isolated microbial strains (WON2089, WON2090, and WON2093) belonging to the genus Akkermansia from human feces. Moreover, it was found that these microorganisms belong to the same genus as the two known Akkermansia species (Akkermansia muciniphila and Akkermansia glycanophila), but belong to different species, and may be found to be new species. In addition, it was found that these novel microbial strains have different assimilability from known Akkermansia, and further have NOD2 agonist activation that has not been detected in known Akkermansia.

[0044] Therefore, the novel microorganism belonging to Akkermansia of the present invention (hereinafter, also simply referred to as "the microorganism of the present invention") relates to a microorganism having a 16S rRNA gene comprising a nucleotide sequence having an identity of 98.0% or less to the nucleotide sequence described in SEQ ID NO: 1, an identity of 94.0% or less to the nucleotide sequence described in SEQ ID NO: 2, or an identity of more than 98.0% to the nucleotide sequence described in SEQ ID NO: 3, and

[0045] It has the following properties 1) to 4).

[0046] 1) Has NOD2 agonist activity;

[0047] 2) It has the ability to assimilate D-mannitol;

[0048] 3) Gram-negative, anaerobic bacteria with a spherical to short rod shape;

[0049] 4) Oxidase test was negative and catalase test was positive.

[0050] Generally speaking, "microorganisms belonging to the genus Akkermansia" refer to anaerobic microorganisms (bacteria) belonging to the phylum Verrucomicrobia, class Verrucomicrobia, orders Verrucomicrobiales, family Verrucomicrobia, genus Akkermansia, and Gram-negative, and having a spherical to short rod shape. In addition, the microorganism can use mucin as a carbon source, a nitrogen source, and an energy source. Furthermore, it also has the properties of being negative in the oxidase test and positive in the catalase test. That is, the microorganism belonging to the genus Akkermansia is a microorganism that does not have cytochrome oxidase but has catalase. It should be noted that, as microorganisms belonging to the genus Akkermansia, before the present application was filed, two species were known: A. muciniphila (Akkermansia muciniphila) and A. glycaniphila (Akkermansia glycaniphila).

[0051] The microorganism of the present invention belongs to the genus Akkermansia, but is different from at least the above-mentioned two known Akkermansia species. That is, the following microorganisms: the homology of the nucleotide sequence of the 16S rRNA gene (16S rDNA) is 98.0% or less (for example, 97.0% or less, 96.0% or less, 95.0% or less, 94.0% or less, 93.0% or less, 92.0% or less, 91.0% or less, 90.0% or less, 89.0% or less, 88.0% or less, or 87.0% or less) with respect to the nucleotide sequence of the 16S rRNA gene of Akkermansia muciniphila (sequence described in SEQ ID NO: 1), or 94.0% or less (for example, 93.0% or less, 92.0% or less, 91.0% or less, 90.0% or less, 89.0% or less, 88.0% or less, or 87.0% or less) with respect to the nucleotide sequence of the 16S rRNA gene of Akkermansia saccharophyllova (sequence described in SEQ ID NO: 2). In addition, the microorganism of the present invention may be a microorganism having the following 16S rRNA gene, which comprises a nucleotide sequence having a homology of more than 98.0% (e.g., more than 98.5%, more than 98.7%, more than 99.0%, more than 99.5%, more than 99.7%, 100.0%) relative to the nucleotide sequence recorded in sequence number: 3 (nucleotide sequence of the 16S rRNA gene of WON2089, WON2090 and WON2093).

[0052] It should be noted that, in the present invention, "homology" may refer to similarity or identity. In addition, "homology" may particularly refer to identity. The homology of nucleotide sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool). For example, the homology of nucleotide sequences can be listed as the homology between sequences calculated using BLAST+ (National Center for Biotechnology Information) relative to the rRNA / ITS benchmark strain database of NCBI, and more specifically, the homology between sequences calculated using preset parameters (i.e., using the parameters initially set) for the BLAST+. In addition, in the present invention, homology is represented by the value obtained by rounding off the second decimal place of the numerical value thus calculated.

[0053] The microorganism of the present invention may be a microorganism that, in an ANI (average nucleotide identity) analysis, has a value less than 95% (e.g., less than 93%, less than 90%, less than 87%, less than 85%, less than 83%, less than 80%) compared to the whole genome sequence of a known Akkermansia (e.g., Akkermansia muciniphila). It should be noted that the ANI value can be obtained using DFAST and using preset parameters (i.e., using initially set parameters), for example, as shown in the embodiments described below. In addition, the GC (guanine-cytosine) content in the genomic DNA is preferably 56 to 57 mol% (especially 56.8 mol%).

[0054] In addition, as shown in the examples described later, the microorganism of the present invention also has properties that are different from the above-mentioned two known Akkermansia in terms of assimilation. That is, the microorganism of the present invention is characterized in that it has assimilation at least for D-mannitol (the property of being able to assimilate D-mannitol), and preferably does not have assimilation for maltose (having the property of being unable to assimilate maltose). In addition, the microorganism of the present invention may also have at least one of the following properties related to assimilation (for example, it may further have two or more properties, four or more properties, five or more properties, seven or more properties, ten or more properties, eleven or more properties, twelve or more properties, thirteen or more properties, or fourteen properties).

[0055] Properties related to assimilation:

[0056] The property of being able to assimilate D-glucose, the property of being able to assimilate lactose, the property of being able to assimilate D-mannose, the property of being unable to assimilate white sugar, the property of being unable to assimilate salicin, the property of being unable to assimilate D-xylose, the property of being unable to assimilate L-arabinose, the property of being unable to assimilate glycerol, the property of being unable to assimilate D-cellobiose, the property of being unable to assimilate D-melezitose, the property of being unable to assimilate D-raffinose, the property of being unable to assimilate D-sorbitol, the property of being unable to assimilate L-rhamnose, and the property of being unable to assimilate D-trehalose.

[0057] It should be noted that, in the present invention, "assimilability" refers specifically to sugar assimilation (the property of producing acid from sugar). In addition, the presence or absence of the assimilability can be determined by a known acid production test, which is shown in the examples described below, for example, using an API (Application Programming Interface) system (Apikenki) or the like.

[0058] In addition, as shown in the examples described below, the microorganism of the present invention also has different properties from the above two known Akkermansia in terms of agonist activity for pattern recognition receptors. That is, the microorganism of the present invention is also characterized by having NOD2 agonist activity that these known ones do not have. Furthermore, the microorganism of the present invention may have a TLR (Toll-like receptor) 2 agonist activity that is 1.5 times higher (preferably 1.7 times higher, more preferably 2 times higher) than that of known Akkermansia (e.g., Akkermansia muciniphila (strain ATCC BAA-835), Akkermansia sacchariphila (strain DSM100705)), and may also have a TLR4 agonist activity that is 1.2 times higher (preferably 1.3 times higher, more preferably 1.5 times higher).

[0059] It should be noted that in the present invention, "NOD2" is a protein also called nucleotide binding oligomerization domain 2, and if it is derived from humans, it is a protein comprising an amino acid sequence described in, for example, NCBI Reference Sequence (RefSeq) ID: NP_001280486, NP_071445, or NP_001357395. "TLR2" is a protein also called Toll-like receptor 2, and if it is derived from humans, it is a protein comprising an amino acid sequence described in, for example, RefSeq ID: NP_001305716, NP_001305718, NP_001305719, NP_001305720, NP_001305722, NP_001305724, NP_001305725, or NP_003255. “TLR4” is a protein also called Toll-like receptor 4, and when derived from human, is a protein comprising an amino acid sequence described in, for example, RefSeq ID: NP_003257, NP_612564, or NP_612567.

[0060] The above-mentioned "agonist activity" for pattern recognition receptors refers to the property of activating the receptor due to the recognition of the above-mentioned receptor (binding to it) by components derived from microorganisms. The agonist activity of the present invention includes not only the activation of the above-mentioned receptor itself (for example, multimerization), but also the activation of downstream signal transduction caused by the activation (for example, the binding of activated NOD2 with RICK as its downstream molecule, the activation of the IKK complex using RICK, the decomposition of IκB as an inhibitory molecule of NF-κB, the nuclear transfer of NF-κB released due to the decomposition, the transcriptional activation using the transfer, the inflammatory response caused by it, etc.). In addition, regarding the above-mentioned agonist activity, it can be quantitatively evaluated by those skilled in the art, for example, by using cells (reporter experimental system) introduced with pattern recognition receptor genes and reporter genes as shown in the examples described later. In addition, in the present invention, "having NOD2 agonist activity" can be appropriately judged by those skilled in the art according to the evaluation system used. For example, regarding the reporter experimental system shown in the examples described later, when the test microorganism (10 7 When the agonist activity value obtained by culturing cells introduced with the above-mentioned reporter gene in the presence of a positive control (for example, 10 μg / experimental system) is significantly higher than the agonist activity value obtained by culturing the cells in the absence of the microorganism (for example, only the culture medium), it can be judged that the tested microorganism has NOD2 agonist activity. It should be noted that the significance in this judgment can be evaluated by a technician in this field according to the number of samples (N number) as the object, etc., by appropriately selecting a known statistical method. For example, as shown in the examples described later, when the N number is 3 or more (for example, 4 or 5), when the P value obtained by performing a Student's t-test is less than 0.01, it can be judged that there is a significant difference. In addition, when the agonist activity value obtained by culturing cells introduced with the above-mentioned reporter gene in the presence of the positive control (for example, 10 μg / experimental system of muramyl dipeptide (MDP)) is compared, the agonist activity value obtained by culturing cells introduced with the above-mentioned reporter gene in the presence of the tested microorganism (10 7 When the agonist activity value obtained by culturing the cells in the presence of NOD2 / experimental system) is at least 1 / 4 (preferably 1 / 3 or more), the test microorganism can also be judged to have NOD2 agonist activity.

[0061] In addition, the microorganism of the present invention may have a higher IgA secretion promoting effect as shown in the embodiments described later. "IgA secretion" refers to the secretion of IgA (also known as IgA antibody, immunoglobulin A) from the mucosa (especially the intestinal mucosa in the present invention), and the "higher" promoting effect of the secretion means that the effect is more than 2 times (preferably more than 3 times, more preferably more than 4 times) compared with the known Akkermansia (e.g., Akkermansia muciniphila (strain ATCC BAA-835)). In addition, regarding the above-mentioned IgA secretion promoting effect, it can be quantitatively evaluated by a person skilled in the art, for example, by using a culture system containing Peyer's patches as shown in the embodiments described later, and immunologically detecting the amount of IgA secreted in the system (e.g., by ELISA (Enzyme Linked Immunosorbent Assay, enzyme-linked immunosorbent assay) method).

[0062] In addition, the microorganism of the present invention can also have the enzymatic activity shown in the embodiments described later. Specifically, it can have the activity of at least one enzyme selected from the group consisting of naphthol-AS-BI-phosphohydrolase, beta-galactosidase, beta-glucuronidase and N-acetyl-beta-glucosaminidase (for example, it can have the activity of 2, 3 or 4 enzymes). In addition, it can not have the activity of at least one enzyme selected from the group consisting of alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine allyl amidase, valine allyl amidase, cystine allyl amidase, trypsin, chymotrypsin, acid phosphatase, alpha-galactosidase, alpha-glucosidase, beta-glucosidase, alpha-mannosidase and alpha-fucosidase (for example, it can not have the activity of more than 5, more than 7, more than 10, more than 12 or 15 enzymes). As shown in the Examples described below, the presence or absence of the above-mentioned enzyme activity can be determined by a known enzyme activity test using APIZYM, for example.

[0063] In addition, the microorganism of the present invention may also have a bacterial fatty acid composition as shown in the examples described below. For example, it is preferred to contain 13:0anteiso which is not detected in the two known species of Akkermansia. In addition,

[0064] As a ratio, it is preferably 0.05% or more (for example, 0.06%, 0.07%, 0.08%, 0.1%). On the other hand, it is preferred that the Summed Feature 1 detected in the two known Akkermansia species is not contained (as a ratio, for example, less than 0.1%, less than 0.05%, less than 0.01%). In addition, the most important bacterial fatty acid is 15:0anteiso, and its ratio is preferably 47% or more (for example, more than 50%, more than 52%, more than 55%). In addition, it is preferred to contain C15:0 at a ratio of less than 10% (for example, less than 7%, less than 5%), preferably to contain C15:0iso 3OH at a ratio of more than 0.12% (for example, more than 0.15%, more than 0.2%), and preferably to contain Sum In Feature at a ratio of more than 0.25% (for example, more than 0.3%, more than 0.35%, more than 0.4%). 6, preferably contains C17:0iso at a ratio of 0.2% or more (for example, 0.25% or more, 0.3% or more), preferably contains C17:0anteiso at a ratio of 1% or more (for example, 1.2% or more, 1.5% or more, 1.7% or more, 1.8% or more), preferably contains C18:2w6,9c at a ratio of 0.5% or more, preferably contains C18:1w9c at a ratio of 4% or more (for example, 5% or more, 5.5% or more), preferably contains SumIn Feature 10 at a ratio of 0.4% or more, preferably contains C18:0 at a ratio of 0.8% or more (for example, 0.9% or more), preferably contains Sum In Feature at a ratio of 0.1% or more (for example, 0.15% or more, 0.2% or more) 11, or preferably contains C17:0anteiso 3OH at a ratio of 0.3% or more (e.g., 0.4% or more, 0.5% or more, 0.6% or more).

[0065] It should be noted that, as shown in the examples described below, the bacterial fatty acid composition can be analyzed, for example, according to the bacterial fatty acid composition analysis manual of the Sherlock Microbial Identification System. In addition, for the notation of fatty acids, refer to Table 8 described below.

[0066] In addition, the microorganism of the present invention is preferably shown as the cultivation conditions shown in the embodiments described below. For example, regarding the cultivation temperature, when cultured for 120 hours in a modified GAM (Gifu Anaerobic Medium) agar medium containing 0.1% mucin, it is preferably cultivated (proliferated) at 25 to 45°C, more preferably at 30 to 40°C, and further preferably at 30 to 37°C. Furthermore, under the same conditions, it is also ideal that it cannot be cultivated at below 20°C and / or above 50°C. That is, the optimal cultivation temperature is 30°C to 40°C, and it is ideal that no proliferation is confirmed at below 20°C and / or above 50°C. In addition, regarding the incubation pH, when incubated at 37°C for 120 hours in a modified GAM agar medium containing 0.1% mucin, it is preferably possible to incubate at a pH of 5.5 to 9.5, more preferably at a pH of 6.0 to 8.5, and even more preferably at a pH of 6.5 to 8.0. Furthermore, under the same conditions, it is also preferable that the cells cannot be incubated at a pH of 5.0 or less and / or a pH of 10.0 or more. That is, the optimal pH is 6.0 to 8.5, and it is preferable that no proliferation is confirmed at a pH of 5.0 or less and / or a pH of 10.0 or more.

[0067] The microorganism of the present invention can be appropriately prepared by a person skilled in the art based on a known method. For example, the microorganism can be prepared by culturing the digestive tract contents (feces, etc.) of humans, non-human mammals (e.g., even-toed ungulates such as pigs and cattle, rodents such as mice), poultry, reptiles, etc., under anaerobic conditions in a known medium for culturing Akkermansia (e.g., mucin medium, modified GAM medium containing mucin, and medium containing trypticase soy) as shown in the Examples described below.

[0068] In addition, as shown in the Examples described below, WON2089, WON2090 and WON2093 are particularly preferred embodiments of the microorganism of the present invention.

[0069] WON2089 was deposited on March 17, 2022 at the Patent Microorganism Depository of the Independent Administrative Institution for Product Evaluation and Research (now the National Institute for Technology Evaluation, Patent Microorganism Depository, Room 122, 2-5-8, Kazusa Kamata, Kisarazu-shi, Chiba Prefecture 292-0818, Japan) (identification mark: WON2089, deposit number: NITE BP-03622).

[0070] WON2090 was deposited in the Patent Microorganism Depository of the Institute of Technology for Product Evaluation, an independent administrative institution, on March 17, 2022 (identification mark: WON2090, deposit number: NITE BP-03623).

[0071] WON2093 was deposited in the Patent Microorganism Depository of the Institute of Technology for Product Evaluation, an independent administrative institution, on March 17, 2022 (identification mark: WON2093, deposit number: NITE BP-03624).

[0072] Therefore, these strains can be obtained by applying to the above-mentioned depository centers.

[0073] In addition, as long as the microorganism of the present invention belongs to Akkermansia and has at least NOD2 agonist activity, it can also be a microorganism obtained by breeding through mutation treatment, gene recombination, selection of natural variants, etc. The above-mentioned "variant strain" refers to: a person skilled in the art mutates a specific microorganism (strain) within a range that does not change its properties by a method well known to those skilled in the art; a person obtained by breeding by selecting natural variants, etc.; or a person skilled in the art can confirm that it is equivalent to it. In addition, the microorganism of the present invention is not limited to the strain itself (hereinafter also referred to as "deposited strain") that is deposited or registered in a prescribed institution, but also includes strains that are substantially equivalent to it (also referred to as "derivative strains" or "induced strains"). As the deposited strains of the present invention, for example, WON2089, WON2090 and WON2093 mentioned above can be listed. In addition, "strains substantially equivalent to the deposited strains" refer to strains that belong to Akkermansia and have at least NOD2 agonist activity. A strain substantially equivalent to the deposited strain can be, for example, a derivative strain with the deposited strain as the mother strain. Examples of the derivative strains include strains obtained by breeding from the deposited strains and strains naturally generated from the deposited strains.

[0074] The microorganism of the present invention may be a live bacterium or a dead bacterium, or both a live bacterium and a dead bacterium. As "dead bacterium", for example, a heat-treated body, a roasting-treated body, a boiling-treated body, a radiation (gamma ray, etc.) irradiation-treated body, an enzyme-treated body, a drug (antibiotics, etc.) treated body, a chemical substance (formalin, etc.) treated body, and an ultrasonic-treated body. In addition, the form of the microorganism of the present invention is not particularly limited, and may be any of a liquid or a solid, but from the perspective of easy handling during manufacture or storage, the form is preferably a dry form (e.g., a bacterial powder, etc.). The dried product can be prepared by the following method: drying the suspension obtained by dispersing the bacterium in a solvent (water, etc.). The drying method is not particularly limited, and for example, a spray drying method and a freeze drying method can be mentioned. The sterilization method is not particularly limited, and examples thereof include steam sterilization, UHT (Ultra Heat Treated) sterilization, air sterilization, pressurized sterilization, high-pressure steam sterilization, dry heat sterilization, circulating steam sterilization, electromagnetic wave sterilization, electron beam sterilization, high-frequency sterilization, radiation sterilization, ultraviolet sterilization, ethylene oxide gas sterilization, hydrogen peroxide plasma sterilization, chemical sterilization (alcohol sterilization, formalin fixation, electrolytic water treatment), etc. In addition, surfactant treatment, grinding, pulverization, enzyme treatment, fractionation treatment, etc. may be performed before or after sterilization treatment by heating or the like, or before or after drying treatment, depending on the circumstances, and the products obtained by these treatments may also be included in the composition of the present invention.

[0075] In addition, in the present invention, it can be provided not only in the form of the above-mentioned bacterial cells, but also in the form of the constituent substances of the microorganism, that is, the components of the microorganism itself (so-called bacterial components. For example, amino acids, peptides, nucleic acids, sugars, lipids, vitamins, hormones, and their complexes, as well as their complexes with phosphorus and sulfur metal elements, etc. that constitute the microorganism).

[0076] Furthermore, in the present invention, it can also be a culture of the microorganism of the present invention. The culture can be a culture containing the microorganism (including the culture solution, solid culture medium, etc. of the microorganism after proliferation), and the form of the culture is not particularly limited, and can be any of a liquid or a solid, but from the perspective of easy handling during manufacture or storage, the form is preferably a dry form (such as a culture dry product, etc.). It should be noted that the culture dry product, etc. can also be appropriately prepared by those skilled in the art using the above-mentioned drying method.

[0077] (Composition)

[0078] As shown in the examples described below, the microorganism of the present invention has at least NOD2 agonist activity. Therefore, the present invention provides a composition for activating NOD2 in a subject, which contains the microorganism of the present invention as an active ingredient.

[0079] Furthermore, the microorganism of the present invention may also have a high IgA secretion promoting effect. Therefore, the present invention provides a composition for promoting IgA secretion in a subject, comprising the microorganism of the present invention as an active ingredient.

[0080] In addition, NOD2 is a pattern recognition receptor, and by activating this receptor, it is possible to detect invading pathogens or abnormal autologous cells and eliminate them (natural immunity, acquired immunity). Therefore, the present invention provides a composition for activating natural immunity and / or acquired immunity in a subject, which contains the microorganism of the present invention as an active ingredient.

[0081] Furthermore, IgA secreted in mucosal tissues prevents the invasion of pathogens and the like on the surface of the tissues (so-called mucosal immunity). Therefore, the present invention provides a composition for activating mucosal immunity in a subject, which contains the microorganism of the present invention as an active ingredient.

[0082] In addition, as described above, the present invention provides a composition for activating immunity in a subject, comprising the microorganism of the present invention as an active ingredient.

[0083] The microorganism of the present invention contained as an effective ingredient in the above-mentioned composition can not only be the above-mentioned thalline (live bacteria, dead thalline), but also can be the form of its thalline component, culture. For example, as an effective ingredient for activating the composition of NOD2, the peptidoglycan (preferably the peptidoglycan with class MDP structure) derived from the microorganism of the present invention can be listed as a preferred one. As an effective ingredient for activating the composition of natural immunity and / or acquired immunity, in addition to the above-mentioned peptidoglycan, with regard to the viewpoint of activating TLR2 or TLR4, lipoteichoic acid, lipoprotein, lipoarabinomannan, yeast polysan, lipopolysaccharide (LPS), lipid A can also be listed as its preferred example. In addition, as an effective ingredient for promoting the secretion of IgA, Pam3CSK4, lipoteichoic acid, LPS can be listed as a preferred example.

[0084] The composition of the present invention can be the form of the reagent used in pharmaceutical composition (medicine, quasi-drug, etc.), food composition (food and drink, animal feed, etc.) or cell experiment, model animal experiment, etc. In addition, the composition of the present invention can be formulated by known pharmaceutics method according to the form of the microorganism of the present invention as active ingredient. For example, capsule, tablet, pill, liquid, powder, granule, fine granule, film-coated agent, round granule, buccal agent, sublingual agent, chewable agent, buccal tablet (buccal) agent, paste, syrup, suspension, elixir, emulsion, liniment, ointment, plaster, compress, percutaneous absorption type preparation, lotion, aspirator, aerosol, injection, suppository, etc., use in oral or parenteral mode, but with regard to non-invasive and the viewpoint that can be easily taken in, it is more desirable to be taken in by mouth.

[0085] In these formulations, can be with pharmacologically or as the carrier allowed by dietetics, specifically, can be with physiological saline, sterile water, culture medium, vegetable oil, solvent, excipient, base, emulsifying agent, suspending agent, surfactant, stabilizer, flavoring agent, aromatic agent, vehicle, preservative, binding agent, diluent, isotonic agent, analgesic, extender, disintegrating agent, buffer, coating agent, lubricant, coloring agent, sweetener, viscous agent, flavoring agent, solubilizing agent or other additives etc. suitable combination.

[0086] In addition, in these preparations, in view of the fact that the microorganism of the present invention is an intestinal microorganism, especially in preparations for the purpose of oral administration, it can be combined with a composition that can efficiently deliver the microorganism of the present invention to the intestinal tract. This composition that can be delivered to the intestinal tract is not particularly limited, and a known composition can be appropriately used, for example, a pH-sensitive composition, a composition that inhibits release to the intestinal tract (cellulose polymers, acrylic acid polymers and copolymers, ethylene acid polymers and copolymers, etc.), a bioadhesive composition that specifically adheres to the intestinal mucosa (for example, a polymer described in the specification of U.S. Patent No. 6368586), a composition containing a protease inhibitor, and a composition that is specifically decomposed by an enzyme in the intestinal tract.

[0087] When the composition of the present invention is used as a pharmaceutical composition, it can be used in combination with a known substance for activating immunity, etc. Examples of such known substances include probiotics such as lactic acid bacteria and bifidobacteria, and prebiotics.

[0088] When the composition of the present invention is used as a food composition, it can be, for example, a health food, a functional food, a food for specific health care, a nutritional functional food, a functional labeled food, a nutritional supplement, a patient's food, or an animal feed. It should be noted that functional foods are usually classified into four types based on their mechanism of action: probiotics, biogenics, prebiotics, and synbiotics. In the present invention, probiotics, biogenics, synbiotics, and prebiotics can be used.

[0089] The food and drink of the present invention can be taken in the form of various food and drink products in addition to the form of the composition as described above. As a specific example of food and drink products, it can be listed: liquid foods such as functional beverages, beverages, jelly-like beverages, refreshing drinks, milk beverages, tea beverages, alcoholic beverages, soups, etc.; fermented foods and fermented beverages such as yogurt and yogurt drinks; products containing oils such as edible oils, sauces, mayonnaise, margarine, etc.; carbohydrate-containing foods such as rice, noodles, bread, etc.; livestock processed foods such as ham and sausage; aquatic processed foods such as Japanese fish cakes, dried fish and shellfish, and pickled foods; vegetable processed foods such as pickles; semi-solid foods such as jelly; fermented foods such as miso; various snacks such as Western-style desserts, Japanese-style desserts, candies, chewing gums, soft candies, frozen desserts, ice cream, etc.; steamed products such as curry, sauced vegetables, and Chinese soups; fast food or microwave foods such as fast food soups and fast food miso juice, etc. Furthermore, healthy food and drink products prepared into powders, granules, tablets, capsules, liquids, pastes, or jelly can also be listed. It should be noted that the manufacturing of the food and drink product among the present invention can be implemented by manufacturing technology known in this technical field. In this food and drink product, also can add effective one or more compositions (for example, other intestinal microorganisms, effective composition of the proliferation of microorganism of the present invention in intestinal tract etc.) to immune activation etc.).In addition, also can make multifunctional food and drink product by with other compositions or other functional food combinations of the function except this activation etc.

[0090] It should be noted that the above-mentioned "other intestinal microorganisms" are not particularly limited, and examples thereof include probiotics such as lactic acid bacteria and bifidobacteria. Examples of the above-mentioned "ingredients effective for the proliferation of the microorganisms of the present invention in the intestines, etc." include, for example, N-acetyl D-glucosamine, N-acetyl D-galactosamine, and polyphenols.

[0091] In addition, in the food and drink composition proposed in the present invention, it is also possible to provide and sell the food and drink that indicates health-care use." indicating " behavior comprises all behaviors of informing the needer of the above-mentioned purposes, can be the person who will be attached to the composition itself for the statement that can directly identify the above-mentioned purposes or can think of, the statement that class pushes out the above-mentioned purposes etc., can also be the person who is attached to the container, packaging material or the accompanying document that the composition is housed. In addition, " indicating " can also be the advertisement etc. of the storage medium, electronic bulletin board or the Internet etc. such as leaflet, instruction manual, pop-up advertisement, catalogue, poster, books, DVD (Digital Versatile Disc, digitization versatile optical disc) etc., using the effective this content of the composition of the present invention as the relevant information of the composition of the present invention to indicate, the propagandist.

[0092] <Methods of activating immunity, etc.>

[0093] The present invention also provides a method for activating NOD2 in a subject, a method for promoting the secretion of IgA, or a method for activating immunity, wherein the method is characterized in that the subject is administered or ingested with the microorganism of the present invention, or a composition containing the microorganism as an active ingredient (the above-mentioned composition of the present invention). It should be noted that in the present invention, the "immunity" to be activated is as described above, and examples thereof include natural immunity, acquired immunity, and mucosal immunity. More specifically, examples thereof include: elimination of pathogens, induction of the production of antimicrobial peptides (such as α-defensins), promotion of differentiation of helper T cells, and control of intestinal bacteria.

[0094] The "subject" of the immune activation in the present invention is an animal including humans. As animals other than humans, there are no particular restrictions, and various livestock, poultry, pets, experimental animals, etc. can be used as objects. Specifically, pigs, cattle, horses, sheep, goats, chickens, wild ducks, ostriches, domestic ducks, dogs, cats, rabbits, hamsters, mice, rats, monkeys, etc. can be listed, but they are not limited to these. For example, people who feel that their immune function is reduced, the elderly, and those who want to activate their immune function can be listed. In addition, even if you have no special concerns about your immune function, you can also take it daily for the purpose of activating your immune function.

[0095] In addition, the present technology can be used for therapeutic purposes or non-therapeutic purposes. The concept of "non-therapeutic use" is as follows: it does not include medical behavior, that is, behavior that treats the human body through treatment. For example, health promotion, etc.

[0096] When administering or ingesting the microorganism of the present invention, or a composition containing the microorganism as an active ingredient (the above-mentioned composition of the present invention), the administration amount or ingestion amount is appropriately selected according to the age, weight, health status, type of composition (drugs, food and beverages, etc.), form of the active ingredient (e.g., live bacteria, dead bacteria, bacterial components, cultures), etc. of the subject.

[0097] The content or usage of the microorganisms (live or dead bacteria) of the present invention is not particularly limited, and in the above composition, it is 1×10 4 ~1×10 12 , preferably 1×10 6 ~1×10 12 , and preferably 1×10 8 ~1×10 12 It should be noted that the unit is CFU / g or CFU / mL, or cells / g or cells / mL. CFU stands for colony forming unit.

[0098] The daily dosage of the microorganisms (live or dead) of the present invention is not particularly limited, but is preferably 1×10 4 ~1×10 12 , or 1×10 6 ~1×10 12 , which can be 1×10 8 ~1×10 12 It should be noted that the unit is CFU / day or cells / day.

[0099] In addition, the combination of the upper limit and the lower limit of the above numerical range is only illustrative, and their combination can be appropriately changed. In addition, when the active ingredient is a component of a microorganism or a culture, for example, the component amount or the culture amount corresponding to the above-mentioned cell amount can be set.

[0100] It should be noted that the administration or intake amount of the composition (the above-mentioned composition of the present invention) as the microorganism of the present invention or comprising the microorganism as an active ingredient is as described above, but it can be administered or ingested once a day or divided into multiple times (e.g., 2 times, 3 times). In addition, the administration or intake period can also be suspended according to the state of immune function, but it is also possible to continuously administer or ingest without suspending. It should be noted that, regarding "continuously", it can be continuous every day, or it can be continuous at intervals of a certain time, but in terms of the effect, it is preferably continuously administered or ingested every day. The microorganism of the present invention or the composition comprising the microorganism as an active ingredient.

[0101] Example

[0102] The present invention will be described in more detail below based on examples, but the present invention is not limited to the following examples. In addition, in this example, novel microorganisms belonging to the genus Akkermansia were isolated using the following method, and the properties of these microorganisms were analyzed.

[0103] (Microbial Isolation Method)

[0104] At the Otsu Nutritional Products Research Institute of Otsu Pharmaceutical Co., Ltd. (Otsu City, Shiga Prefecture, Japan), stool was collected from 5 adults belonging to the same institute and transferred to an anaerobic chamber as quickly as possible. The stool was suspended in a physiological saline solution to which cysteine ​​was added in a concentration of 0.5 g / L, and after serial dilution, it was inoculated in a mucin medium (composition shown in Table 1 below) and liquid cultured in an anaerobic chamber at 37°C (maximum culture for 7 days).

[0105] [Table 1]

[0106]

[0107] After the culture solution in which the proliferation of microorganisms was confirmed was diluted continuously in the mucin medium, it was spread on the mucin medium agar culture plate and cultured anaerobically at 37°C. It should be noted that one culture plate was prepared for each subject's feces. Then, colonies were picked up from each culture plate as much as possible, inoculated into the mucin medium again, and cultured anaerobically. Then, glycerol was added to the culture solution in which the proliferation was observed so that the final concentration became 10%, and it was divided into 1 ml aliquots and stored at -80°C as a stock solution.

[0108] (Determination of the sequence of the 16S rRNA gene of microorganisms)

[0109] A portion of the culture solution obtained when preparing the above-mentioned stock solution was subjected to heat treatment at 98°C for 15 minutes to extract DNA. Using the DNA as a template, bacterial 16S rRNA PCR kit Fast (manufactured by Takara Bio) was used to amplify 800bp of the DNA region encoding a portion of 16S rRNA, and the sequence of the above region was determined by Sanger sequencing (ABIPRISM 3500xLGenetic Analyzer System). In addition, for the determined sequence, a 16S rRNA system analysis was performed using NCBI Blast (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and microbial strains classified as Akkermansia (WON2089, WON2090, WON2093) were selected. It should be noted that these three microbial strains were isolated from three different subjects.

[0110] In addition, Techno Suruga Lab Co., Ltd. (Shizuoka City, Shizuoka Prefecture, Japan) determined the approximate full-length sequences of 16S rRNA of these microbial strains by the following procedure.

[0111] The coding region of 16S rRNA was amplified by PCR (Polymerase Chain Reaction) using universal primers 9F and 1510R using the DNA extracted from the above-mentioned microbial strain as a template.

[0112] The obtained PCR products were purified and subjected to sequence analysis using primers 9F / 515F / 536R / 926R / 1099F / 1510R and ABIPRISM 3500xL Genetic Analyzer System to determine their sequences.

[0113] Furthermore, the full-length sequence thus obtained was subjected to Blast+ analysis against the rRNA / ITS reference strain database of NCBI, and the homology with known microbial strains belonging to the genus Akkermansia was calculated.

[0114] (Creation of phylogenetic tree)

[0115] Furthermore, 16S rRNA sequences of known microbial strains belonging to the genus Akkermansia (Akkermansia muciniphila, Akkermansia glycanophila) and representative strains belonging to the phylum Verrucomicrobia were obtained from the NCBI database, and a phylogenetic tree was created by the neighbor joining method (bootstrap 1000 times) using MEGA version 10.2.5.

[0116] (Genomic Analysis of Microorganisms)

[0117] The above-mentioned microbial strain (WON2089) was inoculated into a culture medium (containing tryptic soy broth (BD), 2 g / L of N-acetyl D-glucosamine (manufactured by Wako Pure Chemical Industries, Ltd.), 4 g / L of threonine (manufactured by Wako Pure Chemical Industries, Ltd.) and 0.05% cysteine ​​(manufactured by Wako Pure Chemical Industries, Ltd.)) and cultured at 37°C for 24 hours under anaerobic conditions.

[0118] DNA was extracted from the obtained culture using NucleoBond (registered trademark) HMW DNA (manufactured by Takara Bio). 2000 ng of the DNA was treated with Short Read Eliminator (manufactured by Circulomics) to remove low molecular weight DNA of approximately 10 Kbp or less. This low molecular weight removal operation was repeated twice to prepare genomic DNA.

[0119] A library for nanopore sequencing was prepared from the obtained genomic DNA (400 ng) using a ligation sequencing kit (manufactured by Nanopore), and then sequencing analysis (long read sequencing analysis) was performed using a MinION flow cell FLO-MIN106R9.41revD.

[0120] In addition, a library was prepared from the above genomic DNA (500 ng) using Illumina Nextera DNA Flex Library Prep Kit (manufactured by Illumina) according to the instructions, and PhiX was added to a final concentration of 15%, and 155 bp×2 sequence analysis (short read sequencing analysis) was performed using MiSeq.

[0121] Low-quality sequences were removed from the long reads and short reads thus obtained according to the following conditions.

[0122] Short read length is to remove those below Q30, and long read length is to remove those below Q10

[0123] Short read length is to remove less than 20bp, long read length is to remove less than 3,000bp

[0124] Remove remaining adapter sequences.

[0125] Then, the sequence data were collected by flye version 2.7 (https: / / www.nature.com / articles / s41587-019-0072-8) and unicycler version 0.4.8 (https: / / pubmed.ncbi.nlm.nih.gov / 28594827 / ) to obtain a circular sequence. Furthermore, when mismatches occurred in the circular sequence, corrections were made by Pilon version 1.23 (https: / / pubmed.ncbi.nlm.nih.gov / 25409509 / ) to obtain a final circular sequence.

[0126] In addition, the GC content was calculated from the circular sequence thus obtained. Furthermore, the ANI (average nucleotide identity) value was obtained by analysis using DFAST (https: / / pubmed.ncbi.nlm.nih.gov / 29106469 / ).

[0127] (Observation of cell morphology of microorganisms)

[0128] The above-mentioned microbial strains (WON2089, WON2090, WON2093) were inoculated on agar plates (containing modified GAM (manufactured by Nissui) and 0.1% mucin) and cultured at 37°C for 48 hours under anaerobic conditions. The formed colonies were scraped and smeared on a slide glass, and Gram staining was performed using fiber G "Nissui" (manufactured by Nissui Pharmaceutical). Then, the morphology was observed under an optical microscope.

[0129] (Assimilation)

[0130] The above-mentioned microbial strains (WON2089, WON2090, WON2093) were inoculated on agar plates (containing modified GAM and 0.1% mucin) and cultured at 37°C for 48 hours under anaerobic conditions. The formed colonies were scraped with a cotton swab and suspended in a culture medium provided with the anaerobic bacteria biochemical identification kit API20A (manufactured by Biomerieux Japan). The culture was performed under anaerobic conditions (N2:CO2:H2 / 90:5:5) for 48 hours to determine the acid production.

[0131] In addition, the colonies scraped with a cotton swab were suspended in sterile physiological saline, and then the enzyme activity was determined using an enzyme activity test kit APIZYM according to the instructions (http: / / products.sysmex-biomerieux.net / product / pdf / 25207E).

[0132] (Fatty acid determination)

[0133] The above-mentioned microbial strains (WON2089, WON2090, WON2093) were inoculated on agar plates (containing modified GAM and 0.1% mucin) and cultured at 37°C for 48 hours under anaerobic conditions. The formed colonies were scraped and freeze-dried. Then, the fatty acids were determined using the Sherlock Microbial Identification System (version 6.0), a bacterial fatty acid composition analysis system, according to its manual.

[0134] (Determination of optimal cultivation temperature and pH value)

[0135] The above-mentioned microbial strain (WON2089) was inoculated into a liquid culture medium (containing modified GAM and 0.1% mucin) and cultured under anaerobic conditions for 120 hours. During the culture process, the culture temperature was adjusted within the range of 10 to 50°C and the pH value was adjusted within the range of 4.5 to 10 to conduct the test.

[0136] (Ligand Analysis)

[0137] The above-mentioned microbial strains (WON2089, WON2090, WON2093) were inoculated into a culture medium (a culture medium containing trypticase soy (manufactured by BD), 2 g / L of N-acetyl D-glucosamine (manufactured by Wako Pure Chemical Industries, Ltd.), 4 g / L of threonine (manufactured by Wako Pure Chemical Industries, Ltd.), and 0.05% cysteine ​​(manufactured by Wako Pure Chemical Industries, Ltd.)), and cultured at 37°C for 24 hours under anaerobic conditions. The culture medium was removed and the cells were washed with Otsuka saline and centrifuged again at 10,000 × g for 5 minutes at room temperature to collect the cells. Then, the culture medium was removed and the cells were washed with Otsuka saline. The cells were centrifuged again at 10,000 × g for 5 minutes at room temperature to collect the cells. 7 The microorganisms were suspended in Otsuka physiological saline at a concentration of 1 microorganism / 20 μl and subjected to a heat treatment at 70° C. for 30 minutes. The number of microorganisms was measured by a fluorescent staining method.

[0138] On the other hand, the following human embryonic kidney cell (HEK) transformants were cultured in a culture medium (containing DMEM (manufactured by Thermofisher Scientific), 50 U / ml penicillin (manufactured by Thermofisher Scientific), 50 μg / ml streptomycin (manufactured by Thermofisher Scientific), HEK-Blue Selection (manufactured by Invivogen) and 10% FBS (manufactured by Thermofisher Scientific)) at 37°C and 5% CO2.

[0139] HEK-Blue hTLR2 (manufactured by Invivogen, hkbhtlr2),

[0140] HEK-Blue hTLR4 (manufactured by Invivogen, hkbhtlr4),

[0141] HEK-Blue hNOD2 (manufactured by Invivogen, hkbhnod2).

[0142] It should be noted that various pattern recognition receptor (PRR) genes (hTLR2, hTLR4, hNOD2) and reporter genes (SEAP) are stably integrated into these HEK transformants. In the above-mentioned transformants, in the presence of a PRR agonist, the expression of SEAP is promoted as the PRR is activated. Therefore, the color (absorbance) of the reagent (QuantiBlue Solution) that changes according to the expression can be used as an indicator to evaluate the PRR agonist activity of the above-mentioned microbial strains.

[0143] Then, the cells maintained in this manner were adjusted to 2.0×10 5 cells / ml, 180 μl was inoculated into each well of a 96-well culture plate. After cell inoculation, the 5 microorganisms / 20μl, 10 6 microorganisms / 20μl, 10 7 The cells of each microorganism / 20 μl were added to a 96-well culture plate at 20 μl per well and cultured for 24 hours. It should be noted that Pam3CSK4 (TLR2 agonist, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), LPS from Escherichia coli O111: B4 (TLR4 agonist, manufactured by Sigma), and MDP (NOD2 agonist, manufactured by Invivogen) were used as positive controls. 180 μl of QuantiBlue Solution (manufactured by Invivogen) was added to the obtained cell culture solution 20 μl, reacted at 37°C for 1 hour, and then the absorbance at 620 nm was measured.

[0144] (IgA assay)

[0145] First, Peyer's patch (PP) cells were prepared. Specifically, mice (BALB / c CrSlc / male, 9-13 weeks old) were first raised and maintained by free intake of MF solid feed and tap water. After the quarantined mice were euthanized by cervical dislocation, the small intestine was removed. After washing the outside of the intestine with physiological saline, the PP cut from the outside surface of the small intestine with ophthalmic scissors was placed in a 24-well culture plate supplemented with RPMI1640 and cooled in an ice bath. The cells were singulated using a stainless steel mesh and carefully washed with 5 mL of complete medium (+) (RPMI1640 medium supplemented with 50 μM mercaptoethanol, 100 U / mL penicillin, 100 μg / mL streptomycin, 15 mM-HEPES (pH 7.2), and 10% FBS). The cell suspension was filtered with a 40 μm cell strainer and centrifuged for 10 minutes at 4°C and 280×g. After centrifugation, the culture supernatant was removed by suction and resuspended in 5 mL of complete culture medium (+). The same operation was repeated twice, and the obtained precipitate was suspended in 1.0 mL of complete culture medium (+) to a cell concentration of 1.2 × 10 6 The cell suspension prepared in this way was inoculated into a 96-well culture plate at 200 μL per well and cultured at 37° C. and 5% CO 2 for about 2 hours until the PP cells fell to the bottom of the culture plate.

[0146] Then, the microbial strain (WON2089) was cultured using the method described in the above (ligand analysis), and the culture was heated to 10 7 Then, the killed bacteria solution thus obtained was added to PP cells at 20 μL each and cultured at 37°C and 5% CO2 for 6 days. After the culture, the culture plate was centrifuged at 1,000 rpm (180×g) for 10 minutes using a centrifuge (KUBOTA5200), and 150 μL of the supernatant was recovered in a microtube and stored frozen at -80°C until the IgA assay.

[0147] Then, IgA was measured using an IgA, Mouse, ELISA Quantitation Kit (manufactured by Bethyl Laboratories).

[0148] (result)

[0149] As described above, colony formation of microorganism strains (WON2089, WON2090, WON2093) belonging to the genus Akkermansia was detected in the feces of three of the five people. In addition, the 16S rRNA sequences of these microorganism strains were determined and compared, and the results showed that all carried 16S rRNA genes containing the same sequence.

[0150] The 16S rRNA sequences of the above microorganisms were subjected to Blast+ analysis with respect to the rRNA / ITS reference strain database of NCBI. The results are shown in Table 2 below. The homology with Akkermansia muciniphila was 98.0%, and the homology with Akkermansia glycanophila was 94.0%. In addition, as shown in the phylogenetic tree created based on the 16S rRNA sequence ( Figure 1 ) showed that the three isolated microbial strains belonged to the genus Akkermansia, but it was suggested that they might be different species.

[0151] [Table 2]

[0152] Registered name Strain name Accession number Homology rate (%) Akkermansia muciniphila Muc AY271254 98.0(1406 / 1435) Akkermansia glycaniphila Pyt KT254068 94.0(1355 / 1442) Persicirhabdus sediminis YM20-087 AB331886 86.5(1272 / 1471) Luteolibacter algae A5J-41-2 AB331893 86.0(1268 / 1475) Luteolibacter pohnpeiensis A4T-83 AB331895 85.8(1267 / 1476) Haloferula sargassicola MN1-1037 AB372856 86.4(1228 / 1421) Luteolibacter gellanilyticus CB-286403 LN833280 86.6(1212 / 1400) Oceaniferula marina N1E253 MN897724 85.5(1259 / 1472) Haloferula harenae YM23-227 AB372852 86.3(1217 / 1410) Haloferula rosea (Haloferula rosea) 06SJR1-1 AB372853 85.9(1216 / 1416) Haloferula luteola YC6886 FJ032193 86.2(1202 / 1395) Rubritalea halochordaticola MN1-1006 AB543683 85.1(1247 / 1465) Rubritalea sabuli (Rhodobacterium sabuli) YM29-052 AB353310 85.1(1247 / 1465) Rubritalea tangerina (Rubritalea tangerina) YM27-005 AB297806 85.1(1254 / 1473) Haloferula phyci (Haloferula phyci) AK18-024 AB372854 85.7(1213 / 1416) Haloferula helveola 05IJR53-1 AB372855 85.7(1212 / 1415) Haloferula chungangensis CAU1074 JN001489 85.6(1219 / 1424) Luteolibacter luojiensis CCTCC2010415 JN630810 85.7(1203 / 1403) Rubritalea spongiae YM21-132 AB297805 84.7((1248 / 1474) Rubritalea squalenifaciens HOact23 AB277853 84.99(1219 / 1436)

[0153] For WON2089, the whole genome sequencing analysis was determined, and based on the sequence, ANI analysis was performed using DFAST. Then, compared with two strains of Akkermansia muciniphila, the results are shown in Table 3 below, and the values ​​were about 82.5%, which is lower than the critical value of 95% ANI for current species. That is, it was shown that the isolated microbial strain was a new species of Akkermansia.

[0154] [Table 3]

[0155] Species name strain Accession number ANI Akkermansia muciniphila strain = DSM 22959 GCA_008000975.1 82.5 Akkermansia muciniphila strain=ATCC BAA-835 GCA_000020225.1 82.5 Akkermansia saccharophila strain=Pyt GCA_001683795.1 77.4

[0156] Based on the above-mentioned whole genome sequencing, the GC content was analyzed, and the result was 56.8 mol%. In addition, as shown in Table 4 below, the morphology of the microbial strains (WON2089, WON2090, WON2093) is spherical to short rod-shaped. Furthermore, all microbial strains are Gram-negative for Gram staining and oxidase reaction, but positive for catalase reaction. Furthermore, as shown in Table 5 below, the optimal cultivation temperature for all microbial strains is 30°C to 40°C, on the other hand, no proliferation is observed at 20°C or below or 50°C or above. In addition, the optimal pH value is 6.0 to 8.5, on the other hand, no proliferation is observed at pH values ​​below 5.0 or pH values ​​above 10.0. It should be noted that, regarding these GC contents, cell morphology, Gram staining, oxidase reaction, catalase reaction and cultivation conditions, they are the same properties as the known Akkermansia.

[0157] [Table 4]

[0158] Use agar medium: modified GAM + 0.1% mucin

[0159] Cultivation temperature 37℃

[0160] Cultivation time: 72 hours

[0161]

[0162] [Table 5]

[0163]

[0164] In the acid production test using API20A, as shown in Table 6 below, the microbial strains (WON2089, WON2090, WON2093) were observed to proliferate in D-glucose, lactose, and D-mannose, similar to the two known Akkermansia species (Ackermansia muciniphila (strain ATCC BAA-835) and Akkermansia sacchariphila (strain DSM100705)). On the other hand, unlike the two known Akkermansia species, the above-mentioned microbial strains were also observed to proliferate in D-mannitol. In addition, regarding the proliferation observed in Akkermansia sacchariphila in maltose, the above-mentioned microbial strains were not observed, similar to Akkermansia muciniphila.

[0165] [Table 6]

[0166] Acid production using API 20A

[0167]

[0168] As a result of the enzyme activity test using APIZYM, the enzyme activities shown in the following Table 7 were observed for WON2089.

[0169] [Table 7]

[0170] Utilizing the bacterial enzyme activity of APIZIM

[0171]

[0172] In addition, the fatty acids in the cells of WON2089 and two known species of Akkermansia (Akkermansia muciniphila and Akkermansia sacchariphila) were measured. As a result, the fatty acid compositions of the cells shown in Table 8 below were observed.

[0173] [Table 8]

[0174]

[0175] The ligand activity was analyzed and the results were as follows Figure 2 As shown, for the two known Akkermansia (Akkermansia muciniphila (strain ATCC BAA-835), Akkermansia saccharophyla (strain DSM100705)), NOD2 agonist activity was not observed, but the microbial strains (WON2089, WON2090, WON2093) all observed this activity. In addition, although not shown in the figure, TLR2 agonist activity and TLR4 agonist activity were observed for the two known species and the above-mentioned microbial strains. Regarding TLR2 agonist activity, the TLR2 agonist activity of the above-mentioned microbial strains was about 1.5 times higher than that of the two known species. In addition, regarding TLR4 agonist activity, the TLR4 agonist activity of the above-mentioned microbial strains was slightly higher than that of the two known species.

[0176] Furthermore, the IgA secretion promoting effect of WON2089 was evaluated. Figure 3 As shown, it was found that the compound has such an effect, and it was also found that the effect of promoting IgA secretion is more than two times higher than that of the known Akkermansia muciniphila (strain ATCC BAA-835).

[0177] Industrial Applicability

[0178] As described above, the microorganism belonging to the genus Akkermansia of the present invention is a novel microorganism that is different from the known Akkermansia at least in terms of assimilation of D-mannitol. In addition, the microorganism belonging to the genus Akkermansia of the present invention may have NOD2 agonist activity that is not found in the known microorganisms, and may also have a high IgA secretion promoting effect.

[0179] Therefore, the microorganism belonging to the genus Akkermansia of the present invention can activate host immunity, etc. through activation of NOD2, etc. Therefore, it can be used as a medicine, food, etc. having such an activating effect.

[0180] [Accession number]

[0181] (1) Identification mark: WON2089

[0182] (2) Accession number: NITE BP-03622

[0183] (3) Deposit date: March 17, 2022

[0184] (4) Depository: Patent Microorganism Depository of the Institute for Product Evaluation and Technology, an independent administrative institution

[0185] (1) Identification mark: WON2090

[0186] (2) Accession number: NITE BP-03623

[0187] (3) Deposit date: March 17, 2022

[0188] (4) Depository: Patent Microorganism Depository of the Institute for Product Evaluation and Technology, an independent administrative institution

[0189] (1) Identification mark: WON2093

[0190] (2) Accession number: NITE BP-03624

[0191] (3) Deposit date: March 17, 2022

[0192] (4) Depository: Patent Microorganism Depository of the Institute for Product Evaluation and Technology, an independent administrative institution

[0193]

[0194]

Claims

1. A microorganism belonging to the genus Akkermansia, It has a 16S rRNA gene comprising a nucleotide sequence having a homology of 98.0% or less to the nucleotide sequence described in SEQ ID NO: 1, a homology of 94.0% or less to the nucleotide sequence described in SEQ ID NO: 2, or a homology of more than 98.0% to the nucleotide sequence described in SEQ ID NO: 3, and It has the following properties 1) to 4): 1) Has NOD2 agonist activity; 2) It has the ability to assimilate D-mannitol; 3) Gram-negative, anaerobic bacteria with a spherical to short rod shape; 4) Oxidase test was negative and catalase test was positive.

2. A microorganism specified by deposit number NITE BP-03622, NITE BP-03623 or NITE BP-03624. 3 . The microorganism according to claim 1 , which has an IgA secretion promoting effect that is more than twice that of the standard strain ATCC BAA-835 of Akkermansia muciniphila.

4. A composition for activating immunity, comprising the microorganism according to any one of claims 1 to 3 as an active ingredient.

5. A composition for activating NOD2, comprising the microorganism according to any one of claims 1 to 3 as an active ingredient.

6. A composition for promoting the secretion of IgA, comprising the microorganism according to claim 2 or 3 as an active ingredient.

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