Akkermansia muciniphila for increasing glucagon-like peptide-1, its extract, and its uses

Abstract

The present invention provides a composition that has the effect of increasing glucagon-like peptide-1. [Solution] Provided are Akkermansia muciniphila containing LWHK0001, LWHK0002, LWHK0004, LWHK0005, LWHK0006, or LWHK0008. Furthermore, a use is provided for increasing glucagon-like peptide-1 by Akkermansia muciniphila containing LWHK0001, LWHK0002, LWHK0003, LWHK0004, LWHK0005, LWHK0006, or LWHK0008. Furthermore, an Akkermansia muciniphila extract having the effect of increasing glucagon-like peptide-1 is provided. Another embodiment provides a composition comprising the Akkermansia muciniphila and a pharmaceutically or food-acceptable carrier.

Description

[Technical Field]

[0001] This disclosure relates to Akkermansia muciniphila, its extracts and uses, and more particularly to Akkermansia muciniphila for glucagon-like peptide-1 elevation, its fractions, extracts and uses. [Background technology]

[0002] Since 1975, the global prevalence of obesity has nearly doubled, primarily due to unhealthy eating habits. Because obesity is associated with an increased risk of many chronic diseases, the global spread of obesity has become a significant public health issue worldwide. While numerous conventional weight-loss drugs exist, none are effective for obese individuals. Because the causes of obesity are diverse, some obese individuals may have a glucagon-like peptide-1 (GLP-1) deficiency. [Overview of the Initiative] [Problems that the invention aims to solve]

[0003] Therefore, conventional techniques need improvement in how to increase glucagon-like peptide-1 levels. [Means for solving the problem]

[0004] One embodiment of the present disclosure provides an Akkermansia muciniphila (AKK) selected from the group consisting of Akkermansia muciniphila LWHK0001 (BCRC911246), Akkermansia muciniphila LWHK0002 (BCRC911247), Akkermansia muciniphila LWHK0004 (BCRC911248), Akkermansia muciniphila LWHK0005 (BCRC911249), Akkermansia muciniphila LWHK0006 (BCRC911250), and Akkermansia muciniphila LWHK0008 (NITE BP-04475).

[0005] Another embodiment of the present disclosure provides a composition comprising the Akkermansia muciniphila and a pharmaceutically or food acceptable carrier.

[0006] In some embodiments, the carrier is selected from the group consisting of microcrystalline cellulose, trehalose, maltodextrin, rice flour, magnesium stearate, inositol, dextrose, sucrose, and any combination thereof.

[0007] Another embodiment of the present disclosure provides a composition for increasing glucagon-like peptide-1 comprising Akkermansia muciniphila selected from the group consisting of Akkermansia muciniphila LWHK0001, LWHK0002, LWHK0003 (BCRC911209), LWHK0004, LWHK0005, LWHK0006, and LWHK0008.

[0008] In some embodiments, the Akkermansia muciniphila is a live bacterium.

[0009] In some embodiments, the composition is a food composition or a pharmaceutical composition.

[0010] In some embodiments, the composition is administered to an individual orally or parenterally.

[0011] Another embodiment of the present disclosure provides an Akkermansia muciniphila culture supernatant prepared by a method comprising: providing a bacterial culture solution comprising Akkermansia muciniphila selected from the group consisting of LWHK0001, LWHK0002, LWHK0003, LWHK0004, LWHK0005, LWHK0006, and LWHK0008; and centrifuging the bacterial culture solution to obtain a culture supernatant containing the secretions of Akkermansia muciniphila, the metabolites of Akkermansia muciniphila, or a combination thereof.

[0012] In some embodiments, Akkermansia muciniphila is LWHK0008, and a fraction with a molecular weight exceeding 3 kDa, a fraction with a molecular weight between 3 kDa and 10 kDa, a fraction with a molecular weight between 3 kDa and 50 kDa, a fraction with a molecular weight between 50 kDa and 100 kDa, or a fraction with a molecular weight exceeding 100 kDa is separated from the culture supernatant.

[0013] Another embodiment of the present disclosure provides a composition for increasing glucagon-like peptide-1 comprising the Akkermansia muciniphila culture supernatant.

[0014] In some embodiments, the composition further comprises a pharmaceutically or food-acceptable carrier.

[0015] Another embodiment of the present disclosure provides a method for preparing an Akkermansia muciniphila extract, comprising the steps of: providing the Akkermansia muciniphila culture supernatant wherein Akkermansia muciniphila is LWHK0003; extracting a part of the culture supernatant with ethyl acetate to obtain an ethyl acetate aqueous layer and an ethyl acetate organic layer; drying the ethyl acetate aqueous layer and the ethyl acetate organic layer respectively to obtain an ethyl acetate aqueous layer extract and an ethyl acetate organic layer extract; extracting the remaining part of the culture supernatant with normal hexane to obtain a normal hexane aqueous layer and a normal hexane organic layer; drying the normal hexane aqueous layer and the normal hexane organic layer respectively to obtain a normal hexane aqueous layer extract and a normal hexane organic layer extract.

[0016] Another embodiment of the present disclosure provides a composition for increasing glucagon-like peptide-1 comprising the Akkermansia muciniphila extract comprising an ethyl acetate aqueous layer extract, an ethyl acetate organic layer extract, or a normal hexane organic layer extract.

[0017] In some embodiments, the composition further comprises a pharmaceutically or food-acceptable carrier.

[0018] In some embodiments, the composition is a food composition or a pharmaceutical composition.

[0019] In some embodiments, the composition is administered to an individual orally or via a non-enteral route. The various aspects of this disclosure will be most easily understood by reading the following detailed description in conjunction with the attached drawings. It should be noted that, in accordance with industry standard operating procedures, the various feature structures are not necessarily drawn to scale. For practical purposes and for clarity, the dimensions of the various feature structures may be arbitrarily enlarged or reduced. To make the above and other purposes, features, advantages, and embodiments of this disclosure clearer and easier to understand, the attached drawings are described below. [Brief explanation of the drawing]

[0020] [Figure 1] This document shows the differences in GLP-1 secretion in the blood of mice after glucose ingestion for different AKK strains of several embodiments of this disclosure. One-way ANOVA, * indicates p<0.05, ** indicates p<0.01. [Figure 2] The differences in GLP-1 secretion after treating NCI-H716 cells with different AKK culture supernatants of several embodiments of this disclosure are shown. t-tests are performed, with * indicating p<0.05 and ** indicating p<0.01. [Figure 3] The differences in GLP-1 secretion after treating NCI-H716 cells with LWHK0003 culture supernatant extracts of several embodiments of this disclosure are shown. t-tests are used, * indicates p<0.05, ** indicates p<0.01. Con: Equivolence of dimethyl sulfoxide (DMSO) is used as the negative control, and CA7S: Cholic acid-7-sulfate is used as the positive control. [Figure 4] The differences in GLP-1 secretion after treating NCI-H716 cells with LWHK0008 culture supernatant of several embodiments of this disclosure are shown. t-test, ** indicates p<0.01. [Figure 5]The LWHK0008 culture supernatants of several embodiments of this disclosure were separated into different fractions by molecular weight separation, and the differences in GLP-1 secretion after treatment with NCI-H716 cells are shown. t-test, ** indicates p<0.01. BHI: Brain Heart Infusion culture medium. [Figure 6] This document shows the differences in plasma GLP-1 secretion in mice administered via enteral feeding with fractions of the LWHK0008 strain with molecular weights ranging from 3 kDa to 10 kDa according to several embodiments of this disclosure. t-tests are used, with * indicating p<0.05. [Modes for carrying out the invention]

[0021] To make the description of this disclosure more detailed and complete, embodiments and specific examples of this disclosure are described below, but these are not the only forms of carrying out or utilizing the specific examples of this disclosure. Each of the embodiments disclosed below may be combined or substituted for one another as useful, and other embodiments may be added to one embodiment without further description or explanation. The following description elaborates on many specific details in order to allow the reader to fully understand the following embodiments. However, embodiments of this disclosure may be carried out without such specific details.

[0022] Furthermore, spatial relative terms such as "down" and "up" are used for convenience to describe the relative relationship between one element or feature and another in the drawings. These spatial relative terms are intended to include different orientations in which the device is used or operated, in addition to the orientation shown in the drawings. The device may be installed in yet another orientation (e.g., rotated 90 degrees or in another orientation), and the spatial relative expressions used herein shall be interpreted accordingly.

[0023] In this specification, unless otherwise specified, the articles “one” and “the aforementioned” may refer to singular or plural. Furthermore, it should be understood that the terms “including,” “equipped,” “possessing,” and similar terms used herein specify the described features, areas, integers, processes, operations, elements, and / or components, but do not exclude one or more other described or additional features, areas, integers, processes, operations, elements, components, and / or groups thereof.

[0024] Furthermore, where numerical values ​​or ranges of numerical values ​​are described by terms such as “approximately,” “approximately,” and similar terms, such terms are intended to include numerical values ​​within a reasonable range that takes into account variations that a person skilled in the art would understand to be inherently occurring in the manufacturing process. For example, based on known manufacturing tolerances related to manufacturing features (characteristics related to numerical values), numerical values ​​or ranges of numerical values ​​include a reasonable range that includes the stated numerical value, e.g., a range within + / - 10% of the stated numerical value. Notwithstanding the foregoing, numerical values ​​and / or letters may be repeatedly referenced in various examples in this disclosure. This repetition is for conciseness and clarity and does not imply any relationship between the various embodiments and / or configurations discussed in themselves.

[0025] Because the causes of obesity are diverse, some obese subjects may have GLP-1 deficiency. Supplementing with GLP-1 can slow down intestinal peristalsis, reduce the rate of digestion, decrease the rate of energy intake, and achieve an ideal effect for weight loss.

[0026] In some embodiments of the present disclosure, Akkermansia muciniphila selected from the group consisting of Akkermansia muciniphila LWHK0001 (BCRC911246), Akkermansia muciniphila LWHK0002 (BCRC911247), Akkermansia muciniphila LWHK0004 (BCRC911248), Akkermansia muciniphila LWHK0005 (BCRC911249), Akkermansia muciniphila LWHK0006 (BCRC911250), and Akkermansia muciniphila LWHK0008 (NITE BP - 04475) is provided.

[0027] In some embodiments, the probiotics are live bacteria.

[0028] In some embodiments, the content of the probiotics is 1×10 8 CFU / g to 1×10 11 CFU / g, for example, 1×10 8 CFU / g, 2×10 8 CFU / g, 3×10 8 CFU / g, 4×10 8 CFU / g, 5×10 8 CFU / g, 6×10 8 CFU / g, 7×10 8 CFU / g, 8×10 8 CFU / g, 9×10 8 CFU / g, 1×10 9 CFU / g, 2×10 9 CFU / g, 3×10 9 CFU / g, 4×10 9 CFU / g, 5×10 9 CFU / g, 6×10 9 CFU / g, 7×10 9 CFU / g, 8×10 9 CFU / g, 9×10 9 CFU / g, 1×10 10 CFU / g, 2×10 10 CFU / g, 3×10 10 CFU / g, 4×10 10 CFU / g, 5×10 10 CFU / g, 6×1010 CFU / g, 7 x 10 10 CFU / g, 8 x 10 10 CFU / g, 9 x 10 10 CFU / g, 1 x 10⁻⁶ 11 CFU / g, or any value between any two of these values.

[0029] In some embodiments, the composition is applied to obese individuals. In some examples, obesity is caused by a group selected from diet, type 2 diabetes, hyperglycemia, glucose intolerance, dyslipidemia, insulin resistance, hyperinsulinemia, fatty liver, cardiovascular disease, stroke, cancer, and combinations thereof.

[0030] In some embodiments, the composition is administered to the individual orally or via a non-enteral route. In some embodiments, the composition is prepared as an oral dosage form selected from the group consisting of solutions, suspensions, emulsions, powders, tablets, pills, syrups, lozenges, tablets, chewable gums, and capsules, and administered to the individual.

[0031] In some embodiments, pharmaceutically acceptable carriers include water, alcohols, glycols, preserving agents, antioxidants, solvents, emulsifiers, suspending agents, decomposers, binding agents, excipients, stabilizing agents, chelating agents, diluents, gelling agents, preservatives, lubricants, absorption enhancers, active agents, humectants, odor absorbers, fragrances, pH adjusting agents, occlusive agents, emollients, thickeners, and solubilizing agents. This includes, but is not limited to, agents, penetration enhancers, anti-irritants, colorants, propellants, surfactants, and other similar or other carriers applicable to this disclosure.

[0032] In some embodiments, the composition may be a food composition. For example, it may be added to edible materials in the form of a food additive to prepare a food product for human or animal consumption. Food compositions include, but are not limited to, general foods, health foods, beverages, dietary supplements, dairy products, or animal feed. In the example of an oral dosage form, the composition may further selectively contain pharmaceutically and food-acceptable carriers, excipients, and / or additives. In other examples, the dosage form of the synbiotic may include, but is not limited to, powders, tablets, granules, suppositories, microcapsules, ampoules, liquid sprays, or suppositories.

[0033] In some embodiments, the composition is administered orally or via a non-enteral route.

[0034] In some embodiments, the culture supernatant of Ackermansia muciniphila is prepared by suspending a certain amount of AKK strain in BHI culture medium to create a culture medium containing the bacteria, and culturing it for 32 hours. In some examples, a certain amount of AKK strain is 1 × 10 5 CFU / mL ~ 1 x 10 8 This is CFU / mL, for example, 1 × 10⁻⁶ 5 CFU / mL, 1 x 10 6 CFU / mL, 5×10 6 CFU / mL, 1 x 10 7 CFU / mL, 5×10 7 CFU / mL, 1 x 10 8 The value includes, but is not limited to, CFU / mL, or any value between any two of these values. Next, the culture medium containing the bacteria is centrifuged to obtain a bacterial precipitate and a culture supernatant. In some examples, the purpose of centrifugation is to preliminarily isolate and remove the bacterial strain and retain secretions and metabolites produced during the strain culture process; therefore, conventional centrifugation speeds and times can be selected to achieve this effect. Centrifugation conditions include, but are not limited to, 10000 × g for 30 minutes. Next, the culture supernatant can be selectively filtered through a 0.22 μm filter membrane to remove any remaining bacterial strain. In some examples, the culture supernatant after centrifugation may contain no bacterial strain or only a small amount; if a small amount of bacterial strain is present, it can be further filtered using the filter membrane to remove any remaining bacterial strain.

[0035] In some embodiments, the Akkermansia muciniphila extract was obtained by extracting a portion of the culture supernatant described above with ethyl acetate (EA) to obtain an aqueous ethyl acetate layer (EA_H2O) and an organic ethyl acetate layer (EA_EA). In some examples, the volume percentage of ethyl acetate included, but was not limited to, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99.5%, or any value between any two of these values. The substances in the aqueous ethyl acetate layer and the organic ethyl acetate layer extracted with different volume percentages of ethyl acetate were of almost the same type, the only difference being that the higher the volume percentage of ethyl acetate, the larger the volume of the organic ethyl acetate layer and the more extractable nonpolar metabolites there were (the type remained the same), and the smaller the volume (water content) of the aqueous ethyl acetate layer and the fewer extractable polar metabolites there were (the type remained the same), and vice versa. The remainder of the culture supernatant is extracted with n-hexane (Hex) to obtain a n-hexane aqueous layer (Hex_H2O) and a n-hexane organic layer (Hex_Hex). In some examples, the volume percentage of n-hexane includes, but is not limited to, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99.5%, or any value between any two of these values. The substances in the n-hexane aqueous layer and the n-hexane organic layer extracted with different volume percentages of n-hexane are of almost the same type, the only difference being that a higher volume percentage of n-hexane results in a larger volume of the n-hexane organic layer and more extractable nonpolar metabolites (without changing the type), and a smaller volume (water content) of the n-hexane aqueous layer and fewer extractable polar metabolites (without changing the type), and vice versa.

[0036] One embodiment of the present disclosure further provides an application for using Ackermansia muciniphila, Ackermansia muciniphila extract, Ackermansia muciniphila culture supernatant, or a fraction of Ackermansia muciniphila culture supernatant in the production of a composition for the prevention or treatment of glucagon-like peptide-1 secretion deficiency, wherein Ackermansia muciniphila is selected from the group consisting of Ackermansia muciniphila LWHK0001, LWHK0002, LWHK0003, LWHK0004, LWHK0005, LWHK0006 and LWHK0008, and Ackermansia muciniphila culture supernatant is Ackermansia muciniphila The Ackermansia muciniphila extract contains secretions of Ackermansia muciniphila, metabolites of Ackermansia muciniphila, or a combination thereof. The Ackermansia muciniphila extract contains an ethyl acetate aqueous layer extract, an ethyl acetate organic layer extract, or a n-hexane organic layer extract. The Ackermansia muciniphila culture supernatant contains fractions with a molecular weight greater than 3 kDa, fractions with a molecular weight between 3 kDa and 10 kDa, fractions with a molecular weight between 3 kDa and 50 kDa, fractions with a molecular weight between 50 kDa and 100 kDa, or fractions with a molecular weight greater than 100 kDa.

[0037] One embodiment of the present disclosure further provides an application for using Ackermansia muciniphila, Ackermansia muciniphila extract, Ackermansia muciniphila culture supernatant, or a fraction of Ackermansia muciniphila culture supernatant in the production of a composition for the prevention or treatment of glucagon-like peptide-1 deficiency, wherein Ackermansia muciniphila is selected from the group consisting of Ackermansia muciniphila LWHK0001, LWHK0002, LWHK0003, LWHK0004, LWHK0005, LWHK0006 and LWHK0008, and Ackermansia muciniphila culture supernatant is Ackermansia The Akkermansia muciniphila extract comprises the secretions of Akkermansia muciniphila, metabolites of Akkermansia muciniphila, or a combination thereof, wherein the Akkermansia muciniphila extract comprises the ethyl acetate aqueous layer extract and the ethyl acetate organic layer extract, or the n-hexane organic layer extract, and the Akkermansia muciniphila culture supernatant comprises a fraction with a molecular weight greater than 3 kDa, a fraction with a molecular weight between 3 kDa and 10 kDa, a fraction with a molecular weight between 3 kDa and 50 kDa, a fraction with a molecular weight between 50 kDa and 100 kDa, or a fraction with a molecular weight greater than 100 kDa.

[0038] One embodiment of the present disclosure further provides an application for using Ackermansia muciniphila, Ackermansia muciniphila extract, Ackermansia muciniphila culture supernatant, or a fraction of Ackermansia muciniphila culture supernatant in the production of a composition for the prevention or treatment of glucagon-like peptide-1 deficiency, wherein Ackermansia muciniphila is selected from the group consisting of Ackermansia muciniphila LWHK0001, LWHK0002, LWHK0003, LWHK0004, LWHK0005, LWHK0006 and LWHK0008, and Ackermansia muciniphila culture supernatant is Ackermansia The Akkermansia muciniphila extract comprises the secretions of Akkermansia muciniphila, metabolites of Akkermansia muciniphila, or a combination thereof, wherein the Akkermansia muciniphila extract comprises the ethyl acetate aqueous layer extract and the ethyl acetate organic layer extract, or the n-hexane organic layer extract, and the Akkermansia muciniphila culture supernatant comprises a fraction with a molecular weight greater than 3 kDa, a fraction with a molecular weight between 3 kDa and 10 kDa, a fraction with a molecular weight between 3 kDa and 50 kDa, a fraction with a molecular weight between 50 kDa and 100 kDa, or a fraction with a molecular weight greater than 100 kDa.

[0039] Several examples and experimental examples are listed below to illustrate the compositions of this disclosure in more detail, but these are for illustrative purposes only and do not limit this disclosure. The scope of protection of this disclosure is defined by the following claims. [Examples]

[0040] The following lists several examples and experimental examples to illustrate in more detail the glucagon-like peptide-1-enhancing Akkermansia muciniphila, its culture supernatant, extract, and its uses as disclosed herein, but these are for illustrative purposes only and do not limit the disclosure. The scope of protection of this disclosure is as defined by the subsequent claims.

[0041] For clarity, features and elements that are known in the field and are not essential for understanding the described principles are omitted.

[0042] Example 1: Identification of bacterial strains

[0043] The Akkermansia muciniphila LWHK0001, 0002, 0003, 0004, 0005, and 0006 strains disclosed herein have been deposited with the Bioresource Collection and Research Center (BCRC), a foundation of the Food Industry Development Institute in Taiwan, located at No. 331, Food Road, Hsinchu City 300, Taiwan. The Akkermansia muciniphila LWHK0008 strain has been deposited with the International Patent Organism Depositary (IPOD) of the National Institute of Technology and Evaluation (NITE, located at Room 120, 2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture, Japan, 292-0818). Detailed information is shown in Tables 1 and 2 below.

[0044] [Table 1]

[0045] The Ackermansia muciniphila strains LWHK0001, 0002, 0003, 0004, 0005, 0006, and 0008 disclosed herein were obligate anaerobic bacteria isolated from the feces of healthy humans in Taiwan. The strains were oval in shape, with a diameter of approximately 0.4–0.6 micrometers and a length of approximately 0.6–1 micrometer. The Ackermansia muciniphila strains disclosed herein were cultured in Brain Heart Infusion (BHI broth) under an oxygen-free environment at 37°C. The Ackermansia muciniphila strains LWHK0001, 0002, 0003, 0004, 0005, 0006, and 0008 are classified under SEQ ID It has a common sequence shown in NO:8, located between approximately 2234kbp and 2237kbp in the LWHK0001 genomic DNA, between approximately 2643kbp and 2646kbp in the LWHK0002 genomic DNA, between approximately 603kbp and 600kbp in the LWHK0003 genomic DNA, between approximately 658kbp and 655kbp in the LWHK0004 genomic DNA, between approximately 2338kbp and 2341kbp in the LWHK0005 genomic DNA, between approximately 2318kbp and 23210kbp in the LWHK0006 genomic DNA, and between approximately 2232kbp and 2235kbp in the LWHK0008 genomic DNA. Furthermore, this common sequence is not present in the standard strain of Ackermansia muciniphila, ATCC BAA-835. Analysis of this common sequence using an online system (Open reading frame finding https: / / www.ncbi.nlm.nih.gov / orffinder / ) predicted the presence of 20 open reading frames (ORFs). Of these, ORF 11 had 838 amino acids (SEQ ID NO: 9), and homology searches showed no agreement with any known genes. ORF 12 had 997 amino acids (SEQ ID NO: 10), and homology searches suggested that it was a gene encoding a protein containing the β-N-acetylglucosaminidase (β-NAGase) domain.β-NAGase is a type of glycosidase that plays a role in cleaving the bond between β-N-acetylglucosamine (β-GlcNAc, β-NAG) and other sugar residues. Proteins containing this functional domain are mainly involved in polysaccharide metabolism, protein modification regulation, cell wall degradation, or extracellular matrix (ECM) remodeling.

[0046] Whole-genome identification was performed on the Ackermansia muciniphila strains LWHK0001, 0002, 0003, 0004, 0005, 0006, and 0008 described herein, with the standard strain ATCC BAA-835 used as the reference. The identification items were ANIb (average nucleotide identity on BLAST, calculated based on BLAST+), ANIm (calculated based on MUMmer), Pearson product-moment correlation analysis (Pearson correlation coefficient (PCC), statistical analysis of tetranucleotide usage patterns), and alignment (Aligned, alignment based on the total nucleotide count of ATCC BAA-835, 2,664,102 bp).

[0047] [Table 2]

[0048] As can be seen from Table 2 above, based on the analysis of whole-genome mean base identity (ANI) with the standard strain ATCC BAA-835, differences existed between the genomes. LWHK0001, LWHK0002, LWHK0003, LWHK0004, LWHK0005, LWHK0006, and LWHK0008 were all shown to be novel isolates.

[0049] Example 2: Differences in blood GLP-1 secretion after glucose intake in mice using different AKK strains.

[0050] In this experiment, germ-free C57BL / 6 male mice aged 8-12 weeks were randomly assigned to three experimental groups and one control group. At the start of the experiment, each experimental group received a different strain of Akkermansia muciniphila (AKK) via tube feeding: BAA-835 (deposited with ATCC and freely available), LWHK0001, and LWHK0003 live bacteria. At the start of the experiment, each group of germ-free mice received a different strain via tube feeding, with a dose of 4 × 10⁶ bacteria per dose. 8 The strain was CFU (container-fed unit) suspended in phosphate-buffered saline (PBS) containing 2.5% glycerol. The control group received the same volume of PBS containing 2.5% glycerol via tube. One week later, mouse feces were collected, smears were prepared, and Gram staining was performed to confirm that the strain had colonized the mouse intestinal tract and that there was no contamination by other bacteria. Two weeks after the administration of live bacteria via tube, an efficacy test for GLP-1 secretion was conducted.

[0051] In mice or humans, intestinal L cells are stimulated to secrete GLP-1 into the bloodstream after ingesting carbohydrates, fats, and proteins. To compare the differences in the GLP-1 secretion-promoting effects of different AKK strains in mice, blood GLP-1 concentrations were measured after administering glucose solution to mice. The experimental procedure involved fasting mice for 5 hours, followed by intravenous administration of glucose solution at a dose of 2 g / kg BW. Blood samples were collected 10 minutes later, and a dipeptidyl peptidase-4 (DPP4) inhibitor was added to prevent GLP-1 degradation. After separating plasma from the blood samples, the GLP-1 content in the samples was analyzed using an ELISA kit.

[0052] As shown in Figure 1, diagram F1, after different AKK strains were colonized in mice for two weeks, the differences in blood GLP-1 secretion after glucose intake were examined in each group of mice. The blood GLP-1 concentration was 32.49 pg / mL in the control group, 42.55 pg / mL in the BAA-835 group, 50.96 pg / mL in the LWHK0001 group, and 50.95 pg / mL in the LWHK0003 group. Statistical analysis revealed that mice colonized with the LWHK0001 and LWHK0003 strains had significantly elevated blood GLP-1 levels compared to the control and BAA-835 groups. Based on the experimental results above, all live AKK BAA-835, LWHK0001, and LWHK0003 bacteria had a promoting effect on GLP-1 secretion in mice, with AKK LWHK0001 and LWHK0003 showing particularly superior effects compared to AKK BAA-835.

[0053] Example 3: Differences in GLP-1 secretion after treating NCI-H716 cells with different AKK culture supernatants.

[0054] A method for preparing AKK culture supernatant is 1 × 10 6 AKK strains with a CFU / mL concentration were suspended in BHI culture medium to obtain a bacterial culture medium, which was incubated for 32 hours. Next, the bacterial culture medium was centrifuged at 10000×g, 4°C, for 30 minutes to obtain a bacterial precipitate and culture supernatant. The culture supernatant was then filtered through a 0.22 μm filter membrane to remove any remaining bacterial strains. The culture supernatant was then concentrated fivefold using a centrifuge tube (x5 concentrated). This five-fold concentrated AKK culture supernatant was used in subsequent cell experiments.

[0055] The cell experiment procedure involved using the NCI-H716 cell line as a cell model (this cell line was collected from ascites fluid of colorectal adenocarcinoma and is currently a human-derived model used in in vitro studies of GLP-1 regulation). The NCI-H716 cell line was cultured in RPMI 1640 culture medium containing 10% fetal bovine serum. Next, the cells were seeded into Matrigel-coated 96-well plates, with a cell count of 6 × 10⁶. 4 Cells were divided into wells. Next, the cells were cultured in a Matrigel-coated 96-well plate for 2 days before the experiment. Next, the cell culture medium was removed and the cells were washed once with Hank's balanced salt solution (HBSS). Next, the HBSS was removed and 180 μL of HBSS containing 0.2% BSA was added to the cells and treated for 2 hours. Next, 20 μL of 5-fold concentrated AKK culture supernatant was added to the experimental group and the same volume of HBSS was added to the control group and treated for 2 hours. Next, the cell culture medium was collected and Diprotin A (final concentration 10 μM) was added (Note: Diprotin A is a DPP4 inhibitor that inhibits the DPP4 enzyme to prevent GLP-1 from being degraded by DPP4). Finally, the GLP-1 concentrations in the cell culture medium of the different groups were analyzed using an ELISA kit.

[0056] As shown in Figure 2, F2, after treating different groups of NCI-H716 cells with the culture supernatant or HBSS of AKK BAA-835, LWHK0001, LWHK0002, LWHK0003, LWHK0004, LWHK0005, and LWHK0006 for 2 hours, the GLP-1 concentration in the control group was 1.69 pg / mL, the GLP-1 concentration in the BAA-835 group was 3.63 pg / mL, and the GLP-1 concentration in the LWHK0001 group was 3.63 pg / mL. The GLP-1 concentration was 7.16 pg / mL. The GLP-1 concentration in the LWHK0002 group was 12.12 pg / mL, in the LWHK0003 group it was 30.10 pg / mL, in the LWHK0004 group it was 24.37 pg / mL, in the LWHK0005 group it was 10.24 pg / mL, and in the LWHK0006 group it was 10.41 pg / mL. Statistical analysis showed that the inducing effect of the culture supernatants of AKK LWHK0001, LWHK0002, LWHK0003, LWHK0004, LWHK0005, and LWHK0006 on GLP-1 secretion from NCI-H716 cells was significantly higher than that of the culture supernatant of AKK BAA-835. The experimental results above show that the culture supernatants of AKK BAA-835, LWHK0001, LWHK0002, LWHK0003, LWHK0004, LWHK0005, and LWHK0006 all have a promoting effect on GLP-1 secretion, and that the effects of AKK LWHK0001, LWHK0002, LWHK0003, LWHK0004, LWHK0005, and LWHK0006 are superior to those of AKK BAA-835.

[0057] Example 4: Differences in GLP-1 secretion after treating cells with different parts of the LWHK0003 culture supernatant extract.

[0058] The method for preparing the culture supernatant extract of AKK LWHK0003 is as follows: 1 × 10 6AKK strains with a CFU / mL concentration were suspended in BHI culture medium to obtain a bacterial culture solution, which was incubated for 32 hours. Next, the bacterial culture solution was centrifuged at 10000×g, 4°C, for 30 minutes to obtain a bacterial precipitate and a culture supernatant. The culture supernatant was then filtered through a 0.22 μm filter membrane to remove any remaining bacterial strains. A portion of the culture supernatant was then extracted with 99.5% ethyl acetate (EA) to obtain an aqueous ethyl acetate layer (EA_H2O) and an organic ethyl acetate layer (EA_EA). The remainder of the culture supernatant was extracted with 95% n-hexane (Hex) to obtain an aqueous n-hexane layer (Hex_H2O) and an organic n-hexane layer (Hex_Hex). Next, the ethyl acetate aqueous layer (EA_H2O) and the ethyl acetate organic layer (EA_EA), and the n-hexane aqueous layer (Hex_H2O) and the n-hexane organic layer (Hex_Hex) were dried to obtain extracts from the ethyl acetate aqueous layer, the ethyl acetate organic layer, the n-hexane aqueous layer, and the n-hexane organic layer, respectively. The BHI culture medium without cultured bacteria was also extracted using the above method and dried. After drying, 20 mg of the sample was redissolved in 0.5 mL of DMSO and used for cell experiments.

[0059] The cell experiment procedure involved using the NCI-H716 cell line as a cell model and culturing it in RPMI 1640 culture medium containing 10% FBS. Next, the cells were seeded into a Matrigel-coated 96-well plate, with a cell count of 6 × 10⁶. 4 Cells were divided into wells. Next, the cells were cultured in a Matrigel-coated 96-well plate for 2 days before the experiment. Next, the cell culture medium was removed and the cells were washed once with HBSS. Next, the HBSS was removed and 200 μL of HBSS containing 0.2% BSA was added to the cells and treated for 2 hours. Next, the HBSS containing 0.2% BSA was removed. Next, 0.2% BSA HBSS containing the extract was added. The control group was given the same volume of 0.2% BSA HBSS containing DMSO as the extract and treated for 2 hours. Next, the cell culture medium was collected and Diprotin A (final concentration 10 μM) was added. Next, the GLP-1 concentration in the cell culture medium of each group was analyzed using an ELISA kit.

[0060] As shown in Figure 3, Diagram F3, after treating different groups of NCI-H716 cells with different AKK LWHK0003 culture supernatant extracts for 2 hours, the GLP-1 concentration in the control group was 1.69 pg / mL, the GLP-1 concentration of 1 μM CA7S was 18.13 pg / mL, the GLP-1 concentration of EA_EA extract from BHI culture medium without cultured bacteria was 6.80 pg / mL, the GLP-1 concentration of EA_EA extract from AKK LWHK0003 culture supernatant was 8.69 pg / mL, the GLP-1 concentration of EA_H2O extract from BHI culture medium without cultured bacteria was 4.65 pg / mL, the GLP-1 concentration of EA_H2O extract from AKK LWHK0003 culture supernatant was 6.78 pg / mL, and the GLP-1 concentration of Hex_Hex extract from BHI culture medium without cultured bacteria was 4.30 pg / mL. The GLP-1 concentration of the Hex_Hex extract from the LWHK0003 culture supernatant was 8.60 pg / mL, the GLP-1 concentration of the Hex_H2O extract from the BHI culture medium without cultured bacteria was 7.51 pg / mL, and the GLP-1 concentration of the Hex_H2O extract from the AKK LWHK0003 culture supernatant was 6.48 pg / mL. Statistical analysis showed that the GLP-1 secretion induction effect of the EA_H2O extract from the AKK LWHK0003 culture supernatant on NCI-H716 cells was significantly higher than that of the EA_H2O extract from the BHI culture medium without cultured bacteria, and the GLP-1 secretion induction effect of the Hex_Hex extract from the AKK LWHK0003 culture supernatant on NCI-H716 cells was significantly higher than that of the Hex_Hex extract from the BHI culture medium without cultured bacteria.

[0061] Example 5

[0062] The preparation method for the AKK LWHK0008 culture supernatant was the same as in Example 3, but the difference was that the bacterial strain used was AKK LWHK0008. This 5-fold concentrated AKK LWHK0008 culture supernatant was used for the experimental group. The BHI culture medium without bacterial culture was concentrated 5-fold in a centrifuge tube and then used for the control group.

[0063] The cell experiment procedure involved using the NCI-H716 cell line as a cell model and culturing it in RPMI 1640 culture medium containing 10% FBS. Next, the cells were seeded into Matrigel-coated 96-well plates, with a cell count of 6 × 10⁶. 4 Cells were divided into wells. Next, the cells were cultured in a Matrigel-coated 96-well plate for 2 days before the experiment. Next, the cell culture medium was removed and the cells were washed once with HBSS. Next, the HBSS was removed and 180 μL of HBSS containing 0.2% BSA was added to the cells and treated for 2 hours. Next, 20 μL of 5-fold concentrated AKK LWHK0008 culture supernatant was added to the experimental group, and the same volume of 5-fold concentrated uncultured BHI culture medium was added to the control group and treated for 2 hours. Next, the cell culture medium was collected and Diprotin A (final concentration 10 μM) was added. (Note: Diprotin A is a DPP4 inhibitor that inhibits the DPP4 enzyme to prevent GLP-1 from being degraded by DPP4). Finally, the GLP-1 concentrations in the cell culture medium of the different groups were analyzed using an ELISA kit.

[0064] As shown in Figure 4, diagram F4, after treating different groups of NCI-H716 cells for 2 hours with either AKK LWHK0008 culture supernatant or BHI culture medium without cultured bacteria, the GLP-1 concentration in the control group was 6.27 pg / mL, and the GLP-1 concentration in the LWHK0008 group was 15.61 pg / mL. Statistical analysis showed that the GLP-1 secretion induction effect of the LWHK0008 culture medium was significantly higher than that of the control group. These results indicate that AKK LWHK0008 generates a product in the culture medium that promotes GLP-1 secretion after culturing.

[0065] Example 6

[0066] A method for preparing different molecular weight fractions of the AKK LWHK0008 culture supernatant is as follows: 6AKK LWHK0008 at CFU / mL was cultured in BHI culture medium to obtain a bacterial culture medium, which was incubated for 32 hours. Next, the bacterial culture medium was centrifuged at 10000×g, 4°C, for 30 minutes to obtain a bacterial precipitate and bacterial culture supernatant. Next, the bacterial culture supernatant was filtered through a 0.22 μm filter membrane to remove any remaining bacterial strains. Next, the filtered culture supernatant was added to a 3 kDa molecular weight cut-off (MWCO) centrifugation tube, and centrifugation was performed to obtain concentrates of <3 kDa and >3 kDa. Next, some of the >3 kDa concentrate was added to a 50 kDa MWCO centrifugation tube, and centrifugation was performed to obtain concentrates of 3-50 kDa and >50 kDa. Next, the >50 kDa concentrate was added to a 100 kDa MWCO centrifugation tube, and centrifugation was performed to obtain concentrates of 50-100 kDa and >100 kDa. Ultimately, the >3kDa, 3-50kDa, 50-100kDa, and >100kDa concentrates mentioned above were used in subsequent cell experiments. BHI cultures without cultured bacteria were isolated using the same method and used as control samples.

[0067] The cell experiment procedure involved using the NCI-H716 cell line as a cell model and culturing it in RPMI 1640 culture medium containing 10% FBS. Next, the cells were seeded into Matrigel-coated 96-well plates, with a cell count of 6 × 10⁶. 4 Cells were divided into wells. Next, the cells were cultured in a Matrigel-coated 96-well plate for 2 days before the experiment. Next, the cell culture medium was removed and the cells were washed once with HBSS. Next, the HBSS was removed and 180 μL of HBSS containing 0.2% BSA was added to the cells and treated for 2 hours. Next, 20 μL of the culture supernatant fraction was added to the experimental group and the same volume of the non-cultured BHI culture fraction was added to the control group and treated for 2 hours. Next, the cell culture medium was collected and Diprotin A (final concentration 10 μM) was added (Note: Diprotin A is a DPP4 inhibitor that inhibits the DPP4 enzyme to prevent GLP-1 from being degraded by DPP4). Finally, the GLP-1 concentrations in the cell culture medium of the different groups were analyzed using an ELISA kit.

[0068] As shown in Figure 5, Diagram F5, NCI-H716 cells from different groups were treated for 2 hours with fractions of either AKK LWHK0008 culture medium or BHI culture medium without cultured bacteria. In the >3kDa fraction, the GLP-1 concentration in the control group was 6.31 pg / mL, and in the LWHK0008 group it was 9.15 pg / mL. In the 3-50kDa fraction, the GLP-1 concentration in the control group was 8.95 pg / mL, and in the LWHK0008 group it was 9.69 pg / mL. In the 50-100kDa fraction, the GLP-1 concentration in the control group was 9.44 pg / mL, and in the LWHK0008 group it was 11.47 pg / mL. In the >100kDa fractions, the GLP-1 concentration in the control group was 7.17 pg / mL, while the GLP-1 concentration in the LWHK0008 group was 11.25 pg / mL. Statistical analysis revealed that the >3kDa and >100kDa fractions of the LWHK0008 culture supernatant had a significantly higher inducing effect on GLP-1 secretion from NCI-H716 cells compared to the control group. Furthermore, the 50-100kDa fraction of the LWHK0008 culture supernatant tended to have a higher inducing effect on GLP-1 secretion from NCI-H716 cells compared to the control group. These results indicate that AKK LWHK0008 generates a product that promotes GLP-1 secretion in the culture medium after culturing. The molecular weight ranges of the active substances are >3kDa, 3-50kDa, 50-100kDa, and >100kDa, and they generate products in the culture medium that promote GLP-1 secretion.

[0069] Example 7

[0070] A method for preparing the 3-10 kDa fraction of the AKK LWHK0008 culture supernatant is as follows: 1 × 10 6CFU / mL AKK LWHK0008 was cultured in BHI culture medium to obtain a bacterial-containing culture medium, which was incubated for 32 hours. Next, the bacterial-containing culture medium was centrifuged at 10000×g, 4°C, for 30 minutes to obtain a bacterial-containing precipitate and culture supernatant. Next, the culture supernatant was filtered through a 0.22 μm filter membrane to remove any remaining bacterial strains. Next, the filtered culture supernatant was added to a 3 kDa molecular weight cut-off (MWCO) centrifugation tube, and centrifugation was performed to obtain concentrates of <3 kDa and >3 kDa. Next, the >3 kDa concentrate was added to a 10 kDa MWCO centrifugation tube, and centrifugation was performed to obtain concentrates of 3-10 kDa and >10 kDa. Finally, the 3-10 kDa concentrate was used in subsequent animal experiments. BHI culture medium without cultured bacteria was isolated in the same manner and used as a control sample.

[0071] The experimental procedure involved using 8-week-old C57BL / 6 male mice, randomly dividing them into one experimental group and one control group, with 8 mice in each group. On the day of the experiment, the mice were fasted for 4 hours, after which 200 μL of the sample was administered via tube to each mouse. Five hours after tube administration, blood was collected, and a DPP4 inhibitor was added to prevent GLP-1 degradation. After separating the plasma from the blood samples, the GLP-1 content in the samples was analyzed using an ELISA kit.

[0072] As shown in Figure 6, diagram F6, mice that had been fasted for 4 hours were administered a 3-10 kDa fraction of BHI culture medium without cultured bacteria and a culture supernatant containing AKK LWHK0008. Five hours later, the blood GLP-1 concentration in the control group was 8.32 pg / mL, and in the LWHK0008 group it was 9.95 pg / mL. Statistical analysis revealed that the LWHK0008 group of mice had significantly elevated blood GLP-1 levels compared to the control group. These experimental results indicate that the 3-10 kDa fraction of the LWHK0008 culture supernatant has a promoting effect on GLP-1 secretion.

[0073] While the present disclosure is disclosed in embodiments as described above, these embodiments are not intended to limit the present disclosure. Any person skilled in the art could make various changes and modifications without departing from the spirit and scope of the present disclosure, and the scope of protection of the present disclosure should be based on the claims appended thereto.

[0074] [Explanation of symbols] F1, F2, F3, F4, F5, F6: Drawings

[0075] [Biological material deposit] Domestic deposit information (listed in the order of depositary institution, date, and number) Depository: Center for Biological Resource Conservation and Research, Taiwan Food Industry Development Research Institute Deposit date: November 21, 2024 Deposit number:BCRC911246 Domestic deposit information (listed in the order of depositing country, institution, date, and number) Depository: Center for Biological Resource Conservation and Research, Taiwan Food Industry Development Research Institute Deposit date: November 21, 2024 Deposit number: BCRC911247 Domestic deposit information (listed in the order of depositing country, institution, date, and number) Depository: Center for Biological Resource Conservation and Research, Taiwan Food Industry Development Research Institute Deposit date: November 21, 2023 Deposit number: BCRC911209 Domestic deposit information (listed in the order of depositing country, institution, date, and number) Depository: Center for Biological Resource Conservation and Research, Taiwan Food Industry Development Research Institute Deposit date: November 21, 2024 Deposit number:BCRC911248 Domestic deposit information (listed in the order of depositing country, institution, date, and number) Depository: Center for Biological Resource Conservation and Research, Taiwan Food Industry Development Research Institute Deposit date: November 21, 2024 Deposit number: BCRC911249 Domestic deposit information (listed in the order of depositing country, institution, date, and number) Depository: Center for Biological Resource Conservation and Research, Taiwan Food Industry Development Research Institute Deposit date: November 21, 2024 Deposit number: BCRC911250 Information on overseas deposits (listed in the order of depositing country, institution, date, and number) Depository: National Institute of Technology and Evaluation (NITE) Deposit date: October 22, 2025 Deposit number: NITE BP-04475

Claims

1. Ackermansia muciniphylla LWHK0001 (BCRC911246), Ackermansia muciniphylla LWHK0002 (BCRC911247), Ackermansia muciniphylla LWHK0004 (BCRC911248), Ackermansia muciniphylla LWHK0005 (BCRC911249), Ackermansia muciniphylla LWHK0006 (BCRC911250), and Akkermansia muciniphylla selected from the group consisting of Akkermansia muciniphylla LWHK0008 (NITE BP-04475).

2. Ackermansia muciniphylla as described in claim 1, A pharmaceutical or food-acceptable carrier, A composition containing the following:

3. The composition according to claim 2, wherein the carrier is selected from the group consisting of microcrystalline cellulose, trehalose, maltodextrin, rice flour, magnesium stearate, inositol, dextrose, sucrose, and any combination thereof.

4. A glucagon-like peptide-1 boosting composition comprising Akkermansia muciniphila selected from the group consisting of LWHK0001, LWHK0002, LWHK0003 (BCRC911209), LWHK0004, LWHK0005, LWHK0006, and LWHK0008.

5. The composition according to claim 4, wherein the Ackermansia muciniphila is a live fungus.

6. The composition according to claim 4, which is a food composition or a pharmaceutical composition.

7. The composition according to claim 4, administered to an individual by oral or non-enteral route.

8. A step of providing a culture medium containing Ackermansia muciniphila selected from the group consisting of LWHK0001, LWHK0002, LWHK0003, LWHK0004, LWHK0005, LWHK0006 and LWHK0008, A step of centrifuging the culture solution containing the bacteria to obtain a culture supernatant containing the secretions of Ackermansia muciniphila, metabolites of Ackermansia muciniphila, or a combination thereof, A conditioned medium of Ackermansia muciniphila prepared by a method including [a specific method].

9. The Akkermansia muciniphila culture supernatant according to claim 8, wherein the Akkermansia muciniphila is LWHK0008, and a fraction having a molecular weight greater than 3 kDa, a fraction having a molecular weight between 3 kDa and 10 kDa, a fraction having a molecular weight between 3 kDa and 50 kDa, a fraction having a molecular weight between 50 kDa and 100 kDa, or a fraction having a molecular weight greater than 100 kDa is separated from the culture supernatant.

10. A composition for increasing glucagon-like peptide-1, comprising the culture supernatant of Ackermansia muciniphila as described in claim 8.

11. The composition according to claim 10, further comprising a pharmaceutically or food-acceptably approved carrier.

12. A composition for increasing glucagon-like peptide-1, comprising the culture supernatant of Ackermansia muciniphila as described in claim 9.

13. The composition according to claim 12, further comprising a pharmaceutically or food-acceptably approved carrier.

14. A step of providing the culture supernatant of Ackermansia muciniphila according to claim 8, wherein the Ackermansia muciniphila is LWHK0003, A step of extracting a portion of the culture supernatant with ethyl acetate to obtain an aqueous ethyl acetate layer and an aqueous ethyl acetate layer, The process involves drying the aqueous ethyl acetate layer and the organic ethyl acetate layer, respectively, to obtain an extract of the aqueous ethyl acetate layer and an extract of the organic ethyl acetate layer, respectively. The remaining portion of the culture supernatant is extracted with n-hexane to obtain a n-hexane aqueous layer and a n-hexane organic layer. The process involves drying the n-hexane aqueous layer and the n-hexane organic layer, respectively, to obtain a n-hexane aqueous layer extract and a n-hexane organic layer extract. Akkermansia muciniphila extract prepared by a method including [the specified method].

15. A glucagon-like peptide-1 increasing composition comprising the Akkermansia muciniphila extract according to claim 14, which comprises the ethyl acetate aqueous layer extract, the ethyl acetate organic layer extract, or the n-hexane organic layer extract.

16. The composition according to claim 15, further comprising a pharmaceutically or food-acceptably approved carrier.

17. The composition according to claim 15, which is a food composition or a pharmaceutical composition.

18. The composition according to claim 15, administered to an individual by oral or non-enteral route.

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