A lipopolysaccharide derived from akkermansia muciniphila akk-101 and use thereof

Abstract

The present application relates to the field of microorganisms, in particular to a lipopolysaccharide derived from Akkermansia muciniphila Akk-101 and application thereof. Akkermansia muciniphila It is found that the lipopolysaccharide extracted from Akkermansia muciniphila (Akk-101) has a significantly better metabolic improvement effect on diet-induced obese mice, and has a more prominent performance in reducing whole body fat (especially visceral fat), reducing blood sugar, regulating insulin level and improving liver function indicators, and can more significantly reduce the level of plasma endotoxin (LPS) and pro-inflammatory factor level, and has a stronger anti-inflammatory effect. At the same time, the extraction efficiency of the lipopolysaccharide in the present application is higher, and it does not need complex steps such as freeze-drying and gel chromatography, and can obtain higher yield of ALPS with simpler operation, which is more suitable for industrial production requirements.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and more specifically, to a lipopolysaccharide derived from Akk-101 of Akkermansia myxophilus and its applications. Background Technology

[0002] In recent decades, numerous experimental and clinical studies have clearly revealed a causal relationship between gut microbiota dysbiosis and metabolic diseases. Current evidence suggests that supplementing with specific gut commensal bacteria, which are reduced in abundance in patients with obesity, diabetes, and cardiovascular disease, can help prevent and alleviate these metabolic conditions. Among them, *Akkermansia myxophilus* (… Akkermansia mucinophile Akkermansia myxophilus (Akk.) has attracted considerable attention due to its high abundance in healthy individuals and its significant therapeutic potential for various metabolic diseases. Early cohort studies have shown a negative correlation between its abundance and diseases such as obesity, diabetes, atherosclerosis, and colitis. Animal model studies have further confirmed that administration of live or pasteurized Akkermansia myxophilus effectively reduces obesity and atherosclerosis, improves insulin resistance, and enhances the efficacy of anti-PD-1 immunotherapy. Importantly, its benefits for overweight and obese individuals have been validated in a clinical trial.

[0003] Lipopolysaccharides (LPS) are important components of the outer membrane of Gram-negative bacteria. LPS not only mediates direct interactions between gut bacteria and intestinal immune cells but also influences systemic immune responses by entering the circulation. The chemical properties of LPS significantly affect its effectiveness in the immune response. LPS derived from gut microbiota have different physiological effects. For example, LPS from… Bacteroides dorei LPS can enhance immune tolerance, derived from B. vulgaris LPS reduced the response to subsequent LPS stimulation and alleviated intestinal inflammation in mice, derived from Rhodobacter sphaeroides LPS improves insulin sensitivity, derived from Parabacteroides goldsteinii LPS can alleviate chronic obstructive pulmonary disease.

[0004] In the paper published by Sun et al. in iMeta in 2025 Akkermansia muciniphila ‐derivedhypoacylated rough‐type lipopolysaccharides alleviate diet‐induced obesityvia activation of TLR4 IL-23 The article "IL-22 immune axis" and CN120137065A focus on lipopolysaccharides (ALPS) derived from Akkermansia muciniphila strain HW07. Through a series of experiments, their chemical characteristics and anti-obesity functions were clarified: In terms of chemical structure, ALPS was identified as a low-acylation (mainly tetraacylated), mono / diphosphorylated, rough LPS (without O-polysaccharide chains) by SDS-PAGE, HPLC-ESI-MS², and other techniques. The lipid A mainly contains 3-hydroxypentadecanoic acid (C15:0(3-OH)) and 3-hydroxytetrazoic acid (C13:0(3-OH)), and the oligosaccharide chains include 7 types from OS1 to OS7. Regarding the components and anti-obesity mechanism, it was confirmed that ALPS works by activating the TLR4-IL-23-IL-22 immune axis, that is, by upregulating IL-23 expression in dependence of TLR4, thereby inducing intestinal type 3 innate lymphocytes (ILC3s) to secrete IL-22, ultimately achieving weight suppression, intestinal barrier repair and microbiota regulation. In terms of safety and efficacy, there was no death in mice treated with ALPS at a dose of 10 mg / kg (the mortality rate in the E. coli LPS treatment group was 40%), and it could reduce the weight of diet-induced obese (DIO) mice and improve blood lipids (plasma triglycerides decreased by 55.9% and total cholesterol decreased by 44.8%).

[0005] However, it has the following drawbacks that limit its industrial application and effect optimization: First, the yield is low, with the ALPS yield of this strain being only 54.6±3.1mg / g (based on bacterial dry weight), and there is still room for further improvement; second, the metabolic improvement effect needs to be improved, and the reduction of visceral fat is limited; third, it cannot quickly and clearly identify the acylation characteristics of lipid A, and cannot quickly and effectively distinguish between low-acylated LPS and high-inflammatory LPS. Summary of the Invention

[0006] This invention first provides a lipopolysaccharide, which is derived from Akkermansia muciniphila (…). Akkermansia mucinophile Akk-101 bacterial cell extract after culture; wherein, the Akkermansia myxophilus ( Akkermansia muciniphila Akk-101 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.40786.

[0007] The present invention also provides the use of a drug containing the lipopolysaccharide in the prevention or treatment of diseases, wherein the diseases include one or more of the following: obesity, fatty liver, lipid metabolism disorders, glucose metabolism disorders, metabolic syndrome, and obesity-related inflammation.

[0008] The present invention also provides the application of the lipopolysaccharide in the preparation of a drug, wherein the drug is used to prevent or treat one or more of the following diseases: obesity, fatty liver, lipid metabolism disorders, glucose metabolism disorders, metabolic syndrome, and obesity-related inflammation.

[0009] The present invention also provides a medicine containing the aforementioned lipopolysaccharide.

[0010] The present invention also provides a functional food or health product containing the aforementioned lipopolysaccharide.

[0011] This invention discovers that, extracted from Akkermansia myxophilus (… Akkermansia muciniphila The lipopolysaccharide (LPS) of Akk-101 showed significantly better metabolic improvement effects in diet-induced obese mice, demonstrating outstanding performance in reducing systemic fat (especially visceral fat), lowering blood glucose, regulating insulin levels, and improving liver function indicators. Furthermore, it significantly reduced plasma endotoxin (LPS) and pro-inflammatory factor levels, exhibiting a stronger anti-inflammatory effect. Simultaneously, the extraction efficiency of the LPS in this invention is higher, eliminating the need for complex freeze-drying and gel chromatography steps, allowing for a simpler operation to obtain higher yields of ALPS, making it more suitable for industrial production needs. Attached Figure Description

[0012] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0013] Figure 1 This shows the weight changes of mice at different time points after ALPS treatment in this embodiment of the invention. Detailed Implementation

[0014] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the invention. Those skilled in the art can make various modifications and variations to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment can be used in another embodiment to produce further embodiments.

[0015] Unless otherwise stated, all terms used to disclose this invention (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Further guidance is provided below for a better understanding of the teachings of this invention. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0016] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected using at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B.

[0017] The terms “containing,” “comprising,” and “including” as used in this invention are synonyms and are inclusive or open-ended, and do not exclude additional, uncited members, elements, or method steps.

[0018] In this invention, the numerical range represented by endpoints includes all numerical values ​​and fractions contained within that range, as well as the endpoints mentioned.

[0019] The numerical values ​​involved in this invention include fluctuations within a certain range. For example, fluctuations are allowed within a corresponding precision range. For instance, 1% can fluctuate within ±0.05%. For larger values ​​or values ​​that do not require overly precise control, even greater fluctuations are permitted. For example, 75% can fluctuate within ranges of ±1%, ±2%, ±5%, etc.

[0020] In this invention, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity greater than or equal to 2.

[0021] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0022] In this invention, terms such as "preferred," "better," "more suitable," and "ideal" are merely descriptions of more effective implementation methods or embodiments, and should be understood not to limit the scope of protection of this invention.

[0023] In this invention, "optionally," "optionally," "optionally," "optionally," "optionally," and "optional" mean that they are optional, that is, they are selected from either "with" or "without." If multiple "optional" or "optional" terms appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, then each "optional" or "optional" term is independent.

[0024] In this invention, "ALPS" refers to a lipopolysaccharide extract derived from the cultured cells of Akk-101 Akmya fasciata.

[0025] In this invention, metabolic syndrome refers to a group of symptoms centered on metabolic disorders. Its core logic is that multiple metabolic abnormalities (obesity, abnormal glucose metabolism, abnormal lipid metabolism, abnormal blood pressure, etc.) coexist and are interconnected, synergistically increasing the risk of chronic diseases.

[0026] In this invention, obesity-associated inflammation, also known as obesity-induced chronic low-grade inflammation, refers to a persistent, low-intensity systemic inflammatory response caused by excessive accumulation of adipose tissue (especially visceral fat) in the body. It is a key pathological bridge between obesity and metabolic syndromes (such as type 2 diabetes, hyperlipidemia, and non-alcoholic fatty liver disease).

[0027] The applicant of this invention isolated a strain of Akkermansia mycotoxin with excellent efficacy from fecal samples of centenarians (age ≥ 90 years) in Hainan. Akkermansia muciniphila Akk-101, this strain not only exhibits superior acid-base tolerance and colonization ability compared to the standard strain, but also more effectively inhibits lipid accumulation in liver cells, reduces fat deposition in nematodes, and demonstrates weight loss and improved glucose and lipid metabolism. This strain was deposited on September 13, 2023, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), and is classified as *Ackermania myxophilus*. Akkermansia mucinophile The accession number is CGMCC NO.40786. This strain has also been disclosed in the applicant's previous patent applications CN202311622616.8 and CN202511062459.9. In subsequent research, this invention further discovered that the strain extracted from *Ackermania pseudomallei* (…) Akkermansia muciniphila The lipopolysaccharide of Akk-101 has shown excellent improvement effects on various indicators related to metabolic syndrome and is expected to be used in related drugs.

[0028] Based on the above findings, the present invention first provides a lipopolysaccharide (ALPS) from Akkermansia myxophilus (…). Akkermansia muciniphila Akk-101 bacterial cell extract after culture; wherein, the Akkermansia myxophilus ( Akkermansia muciniphila Akk-101 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.40786.

[0029] In practice, those skilled in the art can use common sense, known extraction methods, and / or commercially available kits to extract *Akermansia myxophilus* (… Akkermansia muciniphila Lipopolysaccharide was extracted from Akk-101. In some embodiments, it was obtained from Akk-101. Akkermansia muciniphila The method for extracting lipopolysaccharides (ALPS) from Akk-101 includes the following steps: (1) mixing Akkermansia myxophilus culture medium with proteinase K solution and incubating at 50±5℃ to degrade bacterial proteins; (2) centrifuging to remove the supernatant and collecting the bacterial precipitate; (3) resuspending the precipitate with lysis buffer, adding chloroform and incubating at room temperature; (4) centrifuging and collecting the supernatant containing ALPS, adding purification buffer and incubating at -20±2℃ to precipitate ALPS; (5) centrifuging to remove the supernatant, washing the precipitate and drying to obtain ALPS. Based on the strain of the present invention and the above method, a higher yield of ALPS can be efficiently extracted from Akkermansia myxophilus Akk-101.

[0030] Those skilled in the art can identify other operational details and specific parameters involved in the lipopolysaccharide extraction method by combining common sense with the instructions of the commercially available reagent kits used.

[0031] In some specific embodiments, the concentration of the proteinase K solution in step (1) is 20~40 mg / mL. For example, it can be 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, or 40 mg / mL, etc.

[0032] In some specific embodiments, the incubation temperature in step (1) can be 45°C, 48°C, 49°C, 50°C, 51°C, 52°C, or 55°C, etc.

[0033] In some specific implementations, the incubation time in step (1) can be 40 to 50 minutes. For example, it can be 40 minutes, 43 minutes, 44 minutes, 45 minutes, 46 minutes, 48 ​​minutes, or 50 minutes, etc.

[0034] In some specific implementations, the incubation temperature in step (3) is 23°C, 24°C, 25°C, 26°C, or 27°C, etc.

[0035] In some specific embodiments, the volume ratio of chloroform to lysis buffer in step (3) is 1:3 to 5. For example, it can be 1:3, 1:3.5, 1:4, 1:4.5, or 1:5, etc.

[0036] In some specific implementations, the incubation time in step (3) can be 25 to 40 minutes. For example, it can be 25 minutes, 28 minutes, 30 minutes, 32 minutes, 35 minutes, 37 minutes, or 40 minutes, etc.

[0037] In some specific embodiments, the centrifugation conditions in the method are 12,000 to 15,000 rpm. For example, it can be 12,000 rpm, 13,000 rpm, 14,000 rpm, or 15,000 rpm, etc.

[0038] In some specific embodiments, the incubation temperature in step (4) is -18℃, -19℃, -20℃, -21℃, or -22℃, etc.

[0039] In some specific embodiments, the precipitate is washed with a 70±5% ethanol solution in step (5).

[0040] This invention also provides the use of a drug containing the aforementioned lipopolysaccharide in the prevention or treatment of diseases, wherein the diseases include one or more of the following: obesity, fatty liver, lipid metabolism disorders, glucose metabolism disorders, metabolic syndrome, and obesity-related inflammation. That is, this method also provides a method for preventing or treating diseases, comprising: administering a drug containing the aforementioned lipopolysaccharide to a subject. The following further limitations on the application also apply to this method.

[0041] The present invention also provides the application of the lipopolysaccharide in the preparation of a drug, wherein the drug is used to prevent or treat one or more of the following diseases: obesity, fatty liver, lipid metabolism disorders, glucose metabolism disorders, metabolic syndrome, and obesity-related inflammation.

[0042] In some embodiments, the drug prevents or treats obesity by one or more of the following methods: (a1) inhibiting weight gain; (a2) reducing subcutaneous fat; (a3) ​​reducing visceral fat.

[0043] In some embodiments, the fatty liver includes non-alcoholic fatty liver disease.

[0044] In some embodiments, the drug prevents or treats fatty liver by one or more of the following methods: (b1) reducing one or more of aspartate aminotransferase and alanine aminotransferase in plasma; (b2) reducing one or more of liver index and liver collagen volume fraction.

[0045] In some embodiments, the lipid metabolism abnormality includes hyperlipidemia.

[0046] In some embodiments, the drug prevents or treats lipid metabolism disorders by one or more of the following methods: (c1) reducing one or more of triglycerides, total cholesterol, low-density lipoprotein cholesterol, and free fatty acids in plasma; (c2) reducing one or more of triglycerides, total cholesterol, and low-density lipoprotein cholesterol in the liver.

[0047] In this invention, abnormal glucose metabolism is defined as a state of imbalance in blood glucose regulation. In some embodiments, the abnormal glucose metabolism includes diabetes (especially type 2 diabetes) or simple insulin resistance without progression to diabetes.

[0048] In some embodiments, the drug prevents or treats abnormal glucose metabolism by one or more of the following methods: (d1) lowering blood glucose levels; (d2) lowering insulin levels; and (d3) increasing insulin sensitivity index.

[0049] In some embodiments, the drug prevents or treats obesity-related inflammation by one or more of the following methods: (e1) reducing the level of one or more of lipopolysaccharide, TNF-α, IL-1β, IL-6, and MCP-1 in plasma; (e2) increasing the level of IL-10 in plasma; and (e3) reducing the TNF-α / IL-10 ratio in plasma.

[0050] In some embodiments, the drug is an oral formulation (such as capsules or tablets) or an injectable formulation (such as an intraperitoneal injection or an intravenous injection).

[0051] In specific implementations, the subjects of the application described in this invention can be mammals, including primates (such as humans, non-human primates such as monkeys and chimpanzees), and non-primates (such as cattle, pigs, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, or mice). In some embodiments, the subjects are preferably primates, and more preferably humans.

[0052] The present invention also provides a medicine containing the aforementioned lipopolysaccharide.

[0053] In some specific embodiments, the drug also includes pharmaceutically acceptable excipients, the selection of which needs to be adapted to the physicochemical properties (such as hydrophilicity and stability) of lipopolysaccharides (ALPS) and the drug dosage form requirements, so as to ensure the safety, efficacy and stability of the drug. For example, if the drug is an injectable dosage form (such as intraperitoneal injection or intravenous injection), excipients may include isotonic adjusters (such as sodium chloride or glucose), pH adjusters (such as phosphate buffer or citrate buffer), and stabilizers (such as mannitol or sucrose). Isotonic adjusters maintain the osmotic pressure of the drug solution consistent with that of body fluids, avoiding stimulation of the injection site tissue. pH adjusters control the pH of the drug solution within the physiological range of 7.0-7.4, reducing damage to the vascular endothelium. Stabilizers inhibit the aggregation or degradation of ALPS during storage. If the drug is an oral dosage form (such as capsules or tablets), excipients may include fillers (such as lactose or microcrystalline cellulose), binders (such as hydroxypropyl methylcellulose), disintegrants (such as crospovidone), and coating materials (such as enteric-coated acrylic resin). Fillers and binders ensure the formability of the formulation, disintegrants promote rapid disintegration and release of the drug in the gastrointestinal tract, and enteric coatings protect ALPS from the strong acidic environment in the stomach, allowing it to dissolve and be absorbed after reaching the intestines, thus improving bioavailability.

[0054] In some specific embodiments, the drug may also contain other components with metabolic-improving activities to achieve a synergistic effect. For example, it may be combined with an insulin sensitizer (such as metformin analogues), which can work with ALPS to improve insulin resistance—ALPS reduces metabolic disorder triggers by repairing the intestinal barrier and regulating inflammatory balance, while insulin sensitizers directly enhance cellular sensitivity to insulin; the combination of the two can more effectively lower blood sugar. Alternatively, it may be combined with lipid-lowering components (such as phytosterols); while ALPS lowers LDL cholesterol, phytosterols can inhibit intestinal cholesterol absorption, further optimizing blood lipid indicators. In addition, intestinal mucosal protectants (such as glutamine) may be added to synergistically enhance intestinal barrier function with ALPS, reduce endotoxin entry into the bloodstream, alleviate obesity-related inflammation from the source, and comprehensively improve the intervention effect on metabolic syndrome.

[0055] The present invention also provides a functional food or health product containing the aforementioned lipopolysaccharide.

[0056] In some specific embodiments, the functional foods or health products also include food-grade acceptable matrix components and functional excipients, ensuring both palatability, stability, and ease of consumption, while also synergistically working with ALPS to regulate metabolism. The matrix components can be selected based on the product form: for solid forms (such as compressed candies, meal replacement bars, and probiotic compound powders), they can be combined with cereal powders (such as oat flour and quinoa flour), dietary fiber (such as inulin and fructooligosaccharides), and natural sweeteners (such as erythritol and steviol glycosides). Cereal powders provide basic nutrition and a chewy texture, dietary fiber promotes intestinal motility and improves gut microbiota structure, and natural sweeteners enhance palatability while avoiding blood sugar fluctuations caused by added sucrose. For liquid forms (such as oral liquids, fermented milk, and plant-based beverages), purified water, skim milk powder, and fruit and vegetable juices (such as apple juice and blueberry juice) can be selected as the matrix. Skim milk powder provides high-quality protein without increasing fat burden, while fruit and vegetable juices impart natural flavor and vitamins to the product, catering to popular drinking habits.

[0057] In some specific embodiments, the functional foods or health products may also contain other natural active ingredients with synergistic metabolic effects to further enhance the product's auxiliary effects on improving obesity and metabolic disorders. For example, they may be combined with L-carnitine (which promotes fatty acid β-oxidation); or with green tea extract; or with B vitamins (such as vitamins B6 and B12), which, as coenzymes for energy metabolism, can improve the body's energy conversion efficiency under the regulation of ALPS and prevent excess energy accumulation caused by metabolic disorders.

[0058] In some specific implementations, the dosage form of the functional food or health product can be designed to suit daily consumption scenarios. For example, portable powder is suitable for business travelers, fermented milk is suitable as a breakfast or snack, and compressed candy can be used as a daily snack to replace high-sugar and high-fat foods.

[0059] In practice, those skilled in the art can combine the above-mentioned implementation methods with common sense to obtain more embodiments of the present invention.

[0060] The embodiments of the present invention will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this invention, or follow experimental manuals or conventional conditions in the art, or other experimental methods known in the art, or follow the conditions recommended by the manufacturer.

[0061] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0062] 1 Experimental Design

[0063] 1.1 Experimental mice Eight-week-old male C57BL / 6J mice with diet-induced obesity (DIO) were selected and housed under specific pathogen-free (SPF) conditions with equal light and dark periods. They were fed a standard diet (13.5% calories from fat; HFK; 2032) and a high-fat diet (HFD; 60 kcal% fat; New Brunswick; D12492).

[0064] 1.2 Extraction of ALPS from Akkermansia myxophila ALPS from Akk-101 bacterial culture medium of Akkermansia myxophilus were extracted using a lipopolysaccharide extraction kit (iNtRON biotechnology, Cat. No. 17141). The specific steps are as follows: (1) After thoroughly mixing 4.5 mL of the cultured bacterial solution (1.0≤OD600≤1.2) with 5 μL of proteinase K solution (concentration of 30 mg / mL), incubate at 50℃ for 45 minutes; (2) Centrifuge at 13000 rpm for 10 minutes at room temperature and discard the supernatant; (3) Add 1 mL of lysis buffer to the centrifuge tube, repeatedly aspirate and resuspend the bacterial pellet using a pipette, and vortex vigorously until the cell clumps completely disappear, so that the bacterial cells are fully and evenly distributed in the lysis buffer. (4) Add 250 μL of chloroform to the centrifuge tube and vortex vigorously for 10-20 seconds to mix thoroughly; (5) Incubate at room temperature for 5 minutes; (6) Centrifuge at 13000 rpm and 4℃ for 15 minutes, and transfer 450 μL of supernatant to a new 1.5 mL centrifuge tube; Note: When aspirating the supernatant, maintain a slow and uniform aspiration rate, and avoid aspirating the lower sediment.

[0065] (7) Add 850 μL of purification buffer to the supernatant after transfer, and invert the centrifuge tube 8-10 times to mix the liquid in the tube thoroughly. (8) Place the centrifuge tubes in a -20°C freezer and incubate for half an hour; (9) Remove the centrifuge tube from the -20℃ freezer and centrifuge at 13000 rpm and 4℃ for 20 minutes, then discard the supernatant; (10) Add 1 mL of 70% ethanol solution to the centrifuge tube and invert the centrifuge tube 2-3 times to mix well; (11) Centrifuge at 13000 rpm and 4℃ for 5 minutes, discard the supernatant and dry the centrifuge tube thoroughly, and save the precipitate ALPS in the tube for later use.

[0066] 1.3 Component Identification of ALPS (1) Fatty acid composition analysis: ALPS were subjected to acid methanololysis reaction, and gas chromatography-mass spectrometry was used to analyze the types and relative contents of fatty acids to determine the acylation type.

[0067] (2) Monosaccharide composition analysis: After hydrolyzing ALPS, the monosaccharide composition was detected by high efficiency anion exchange chromatography to determine its polysaccharide structural characteristics.

[0068] (3) Isolation and identification of lipid A: The purified ALPS were subjected to mild acid hydrolysis to release lipid A from the polysaccharide domains. Lipid A was obtained by organic phase extraction. Subsequently, liquid chromatography-mass spectrometry and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry were used to analyze the molecular weight distribution, acylation number and phosphorylation state of lipid A.

[0069] (4) Analysis of oligosaccharide domains: ALPS were deacylated to obtain oligosaccharide domains. The linkage mode and configuration characteristics of the sugar residues were confirmed by monosaccharide linkage analysis and nuclear magnetic resonance spectroscopy. This step is used to clarify the core oligosaccharide structure of ALPS and improve the accuracy of structural identification.

[0070] (5) Auxiliary verification and quality control: Infrared spectroscopy was used to verify the overall structural features of ALPS.

[0071] 1.4 Determination of ALPS content The ALPS content of Akk-101 from Akkermansia myxophilus was determined using a general lipopolysaccharide (LPS) ELISA kit (Catalog No: RDR-LPS-Ge, Reddot Biotech). The procedure was performed according to the kit instructions.

[0072] (1) Preparation of standard: Dissolve the standard in 1.0 mL of standard diluent, let stand at room temperature for 10 minutes, and gently shake to mix (avoid generating bubbles). At this point, the concentration of the standard stock solution is 200 ng / mL. Prepare 7 test tubes, add 0.2 mL of standard diluent to each tube, and then perform serial dilution as shown in Table 1 below. After each transfer, mix thoroughly before proceeding to the next step.

[0073] Table 1 Gradient Dilution Series

[0074] (2) Determine the wells for standard diluents, blanks, and samples. Prepare 7 wells for standards and 1 well for blanks. Add 50 μL of standard diluents, blanks, and samples of each concentration to the corresponding wells. Then immediately add 50 μL of detection solution A to each well. Gently shake the microplate using a microplate shaker. Cover with the sealing film and incubate at 37°C for 1 hour.

[0075] (3) Remove the solution from the wells, add 350 μL of 1× washing buffer to each well, pipette to mix and wash, and let stand for 1–2 minutes. Completely remove all residual liquid from the wells, and gently tap the plate on absorbent paper. Wash a total of 3 times. After the last wash, remove the residual washing solution by aspirating or pouring, invert the plate and gently tap it on absorbent paper.

[0076] (4) Add 100 μL of detection solution B to each well. Cover with sealing film and incubate at 37°C for 1 hour.

[0077] (5) Repeat the washing process in step (3) for a total of 5 washes.

[0078] (6) Add 90 μL of substrate solution to each well. Cover with a new sealing film and incubate at 37°C in the dark for 15–25 minutes (not exceeding 30 minutes). The solution will turn blue after adding the substrate solution.

[0079] (7) Add 50 μL of stop solution to each well. The solution will turn yellow after addition. Mix the liquid by gently tapping the side of the plate. If the color change is uneven, gently tap the plate to ensure thorough mixing.

[0080] (8) Remove water droplets and fingerprints from the bottom of the plate and confirm that there are no air bubbles on the surface of the liquid in the wells. Immediately use an ELISA reader to perform the detection at a wavelength of 450 nm.

[0081] (9) Use the absorbance of the standard to plot the standard curve and calculate the concentration of the sample.

[0082] 1.5 Metabolic Syndrome Detection Twenty mice fed a high-fat diet (HFD) for 8 weeks were randomly divided into two groups (n=10 per group). Mice were intraperitoneally injected every two days with either 0.2 mg / kg ALPS (ALPS treatment group) or an equal volume (0.1 mL) of saline (model group). Body weight was measured and recorded weekly, and plasma and liver-related indicators were measured after 56 days. After euthanasia, subcutaneous fat, epididymal fat, perirenal fat, and mesenteric fat were removed (avoiding the introduction of surrounding tissues). Visible fluid was gently aspirated, and the mice were weighed immediately.

[0083] 1.6 Mouse lethality test C57BL / 6J mice fed with standard diet were randomly divided into three groups: ELPS treatment group, ALPS treatment group, and saline group (n=10 in each group). Mice were intraperitoneally injected with a single dose of LPS (ELPS, 10 mg / kg, Sigma, L2880) derived from E. coli O55:B5, ALPS (10 mg / kg), or an equal volume of saline, respectively. Mice survival was observed, and they were sacrificed 24 hours after administration. Plasma parameters were collected and analyzed.

[0084] 1.7 Anti-metabolic syndrome effect C57BL / 6J mice fed a high-fat diet were intraperitoneally injected with ALPS (ALPS treatment group, n = 10) or ELPS (ELPS treatment group, n = 10) at a dose of 0.2 mg / kg every two days, or injected with an equal volume (0.1 mL) of physiological saline (model group). After 8 weeks, body weight was measured and plasma-related indicators were detected.

[0085] 2. Experimental Results 2.1 ALPS production Based on ALPS production, the average ALPS content in Akk-101 of Akkermansia myxophilus was 66.31 mg / g.

[0086] 2.2 Component identification results of ALPS The purified ALPS samples were tested and found to have residual nucleic acid levels below the detection limit and residual protein levels below 0.5% (w / w). No obvious protein characteristic peaks were detected in the infrared spectrum. This indicates that the obtained sample is high-purity ALPS, meeting the requirements for subsequent structural identification. Compositional analysis can quickly distinguish the differences between ALPS and conventional inflammatory LPS in terms of fatty acid and monosaccharide composition. The ALPS of this invention exhibits low acylation characteristics in lipid A, with a lower number of major acyl groups than traditional highly inflammatory LPS, thus providing a structural basis for subsequent safety and functional applications.

[0087] Fatty acid composition analysis revealed that the main fatty acid components of ALPS were β-hydroxy fatty acids with a carbon chain length of 14-16, and no long-chain fatty acids with a carbon chain length of 18 or more were detected, suggesting a low degree of lipid A acylation. Monosaccharide analysis of ALPS after hydrolysis showed that it contained glucose, galactose, glucosamine, heptose, and 3-deoxy-D-manno-2-octylpyranuronic acid (Kdo), while rhamnose was not detected. This monosaccharide composition is consistent with the core oligosaccharide characteristics of typical Gram-negative bacteria LPS.

[0088] The structure of lipid A was identified by ESI-MS / MS in negative ion mode. The main molecular ion peaks of lipid A were distributed within the following range: the main peak molecular weight was concentrated in the theoretical molecular weight range of tetraacylated and pentaacylated lipid A; the characteristic peak of hexaacylated lipid A was not detected or was only trace. MALDI-TOF MS verification showed that the analytical results were consistent with ESI-MS / MS, with the main signal concentrated in the molecular weight range corresponding to low-acylated lipid A, and the peak distribution was relatively concentrated with low heterogeneity. The results demonstrate that lipid A in ALPS is predominantly low-acylated, significantly different from highly inflammatory lipopolysaccharides.

[0089] Oligosaccharide domain analysis and GC-MS analysis of the methylated derivatives showed that the polysaccharide structure contained multiple 1→4 and 1→6 linked sugar residues, and no highly branched complex O-antigen structure was detected. Nuclear magnetic resonance (NMR) analysis revealed the presence of multiple characteristic sugar residue signals, with well-defined glycosidic bond configurations, distinguishable between α / β configurations. These results corroborated the analysis of monosaccharide composition and linkage patterns. The oligosaccharide structure of ALPS exhibits relatively good stability.

[0090] Multiple infrared spectroscopy analyses confirmed the stable presence of the following characteristic absorption peaks: phosphate group characteristic peak, amide and ester bond characteristic peaks, and glycosyl backbone characteristic peaks. This validates the feasibility of the ALPS component identification method.

[0091] The above test results confirm that the ALPS of the present invention exhibits significant low acylation characteristics in chemical composition and structural features. These structural features enable it to maintain the basic functions of lipopolysaccharide while having lower potential irritation, making it suitable for further application as a functional ingredient.

[0092] 2.3 Results of body weight and other parameters in mice fed a high-fat diet with ALPS treatment Table 2 Changes in mouse body weight

[0093] Table 3 Metabolic indicators at the experimental endpoint

[0094] In Tables 2 and 3: * p < 0.05, ** p < 0.01, *** p < 0.001. ↓ indicates a decrease, and ↑ indicates an increase.

[0095] Based on Tables 2-3 and Figure 1The results showed that ALPS treatment significantly reduced high-fat diet-induced weight gain and related metabolic disorders. After 56 days, the amount of fat in subcutaneous, epididymal, perirenal, and mesenteric adipose tissues decreased by 43.59%, 53.85%, 44.93%, and 47.06%, respectively. Plasma insulin levels decreased by 37.43%. Plasma triglyceride (TG), total cholesterol (TC), free fatty acids (FFA), and low-density lipoprotein cholesterol (LDL) levels also decreased significantly by 28.33%, 39.23%, 43.75%, and 47.62%, respectively. In addition, ALPS treatment alleviated obesity-related liver dysfunction, manifested as decreased plasma aspartate aminotransferase (AST), alanine aminotransferase (ALT), and hepatic TGF-β1, as well as improvements in liver index and liver collagen volume fraction (CVF). The levels of triglycerides (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL) in the liver were also significantly reduced by 52.78%, 44.12%, and 40.00%, respectively.

[0096] ALPS treatment significantly reduced plasma lipopolysaccharide (LPS, 43.39%), TNF-α (44.04%), IL-6 (51.34%), IL-1β (49.74%), MCP-1 (45.41%) levels and the TNF-α / IL-10 ratio (63.64%), while increasing IL-10 levels (47.97%) and improving the insulin sensitivity index (ISI, 39.68%).

[0097] 2.4 Results of ALPS treatment in mice 24 hours Table 4

[0098] * p<0.05, for the saline group.

[0099] The safety of ALPS in mice was evaluated. As shown in Table 4, a single injection of 10 mg / kg resulted in a mortality rate of nearly 40% in the ELPS-treated group, while no deaths occurred in the ALPS-treated group at the same dose. Furthermore, ALPS induced a weaker inflammatory response compared to ELPS.

[0100] 2.5 ALPS has a better anti-obesity effect than ELPS Table 5

[0101] The anti-obesity effects of ALPS and ELPS were compared at the same dose of 0.2 mg / kg. Table 5 shows that ALPS and ELPS were similarly effective in reducing weight gain, but ALPS was more effective than ELPS in lowering blood glucose, alleviating hyperlipidemia (plasma triglycerides TG, total cholesterol TC, and low-density lipoprotein cholesterol LDL), and reducing the level of the inflammatory cytokine IL-6. The anti-obesity effects and favorable safety profile of ALPS support its application in alleviating metabolic disorders.

[0102] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of protection of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A lipopolysaccharide, which is derived from Akkermansia muciniphila (… Akkermansia muciniphila Akk-101 bacterial cell extract after culture; among which, The Ackermansia muciniphila ( Akkermansia muciniphila Akk-101 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.40786.

2. The use of the lipopolysaccharide according to claim 1 in the preparation of a drug, wherein, The drug is used to prevent or treat one or more of the following conditions: obesity, fatty liver, dyslipidemia, dysglucose metabolism, metabolic syndrome, and obesity-related inflammation.

3. The application according to claim 2, wherein, The drug prevents or treats obesity through one or more of the following methods: (a1) Inhibits weight gain; (a2) Reduce subcutaneous fat; (a3) Reduce visceral fat.

4. The application according to claim 2, wherein, The fatty liver includes non-alcoholic fatty liver disease; Optionally, the drug prevents or treats fatty liver by one or more of the following methods: (b1) Reduce one or more of aspartate aminotransferase and alanine aminotransferase in plasma; (b2) Reduce one or more of the liver index and liver collagen volume fraction.

5. The application according to claim 2, wherein, The lipid metabolism abnormalities include hyperlipidemia; Optionally, the drug prevents or treats lipid metabolism disorders through one or more of the following methods: (c1) Reduces one or more of the following in plasma: triglycerides, total cholesterol, low-density lipoprotein cholesterol, and free fatty acids; (c2) Reduces one or more of the following in the liver: triglycerides, total cholesterol, and low-density lipoprotein cholesterol.

6. The application according to claim 2, wherein, The glucose metabolism abnormalities include diabetes mellitus or simple insulin resistance that has not progressed to diabetes. Optionally, the drug prevents or treats abnormal glucose metabolism through one or more of the following methods: (d1) Lowers blood sugar levels; (d2) Lower insulin levels; (d3) Improves insulin sensitivity index.

7. The application according to claim 2, wherein, The drug prevents or treats obesity-related inflammation through one or more of the following mechanisms: (e1) Reduces the levels of one or more of the following in plasma: lipopolysaccharide, TNF-α, IL-1β, IL-6, and MCP-1; (e2) Increases plasma IL-10 levels; (e3) Reduces the TNF-α / IL-10 ratio in plasma.

8. The application according to claim 2, wherein, The drug is an oral or injectable formulation.

9. A drug comprising the lipopolysaccharide of claim 1.

10. A functional food or health product containing the lipopolysaccharide as described in claim 1.

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