Ackermania muciniphila Akk21 with hypoglycemic capacity, and application, product and method of ackermania muciniphila Akk21

Abstract

The invention relates to ackermania muciniphila Akk21 with hypoglycemic capacity and application, a product and a method thereof, and belongs to the technical field of microorganisms. The invention provides an ackermania muciniphila strain Akk21 with a blood sugar reducing capability. The preservation number of the ackermania muciniphila strain Akk21 is CCTCC (China Center for Type Culture Collection) NO: M 20242779. The invention also provides hypoglycemic drugs, functional food for promoting lead excretion, health care products and a method for preventing and treating lead / arsenic pollution based on the strain Akk21. The Ackermania muciniphila Akk21 has excellent lead and arsenic tolerance and hypoglycemic capacity, can effectively reduce the blood lipid level of mice with type II diabetes, has a remarkable reduction effect on inflammation of the whole body of the mice, and can remarkably reduce the number of harmful flora in the intestinal tract by increasing the abundance of effective flora in the intestinal tract, so that the content of the harmful flora in the intestinal tract is reduced, and the content of the harmful flora in the intestinal tract is reduced. The species diversity and abundance of intestinal flora are improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of microorganisms, and in particular relates to Akkermansia muciniphila Akk21 with the ability to lower blood sugar, and applications, products and methods thereof. Background Art

[0002] Type 2 diabetes is a chronic metabolic disease caused by insufficient or abnormal insulin secretion in insulin-sensitive tissues, which leads to elevated blood sugar. Sustained high levels of blood sugar often cause the blood in the circulatory system to become viscous and have poor fluidity, causing congestion in some small blood vessels, causing damage to multiple organs in the human body, and ultimately leading to complications such as nephropathy, cardiomyopathy, and retinopathy. Type 2 diabetes accounts for 95% of all diabetic patients. As a global epidemic, the number of diabetes patients continues to increase. The Global Burden of Disease Study published online in The Lancet indicates that the number of people with diabetes will increase to 1.31 billion in 2050. The global prevalence of diabetes is rising, and the prevention and control of type 2 diabetes remains an ongoing challenge. Diabetes treatment targeting the intestinal microbiota has become a growing research field.

[0003] Probiotics are live microorganisms that provide various health benefits to the host, and the immunomodulatory effects of probiotics in particular have attracted great research interest. Probiotics play an important role in maintaining the balance of intestinal flora, immunomodulation, preventing gastrointestinal infections, and improving blood indicators. In addition, studies have shown that probiotics can stimulate the immune response of immunocompromised mice. Therefore, the development of probiotic strains that have no side effects and can exert immunostimulatory effects can improve the impaired immune function of immunodeficient patients. Lactic acid bacteria, such as Lactobacillus acidophilus, are known as common probiotic species. However, their effects on the immune system vary greatly even between strains within the same species. Therefore, it is necessary to characterize the effects of specific strains and determine their doses to verify their immunomodulatory effects.

[0004] Akkermansia muciniphila is an anaerobic, Gram-negative, non-motile, non-endospore-forming oval intestinal bacterium that exists in human feces. With the continuous deepening of research, it will become the next generation of probiotics with promising clinical applications.

[0005] At present, there is no Akkermansia muciniphila in the art that has both excellent hypoglycemic effect and tolerance to polluting elements such as lead / arsenic. Summary of the invention

[0006] In order to fill the above technical gaps in the prior art, the present invention provides Akkermansia muciniphila Akk21 with blood sugar lowering ability and applications thereof.

[0007] The technical solution of the present invention is as follows:

[0008] The invention discloses Akkermansia muciniphila with the ability of lowering blood sugar, and its deposit number is CCTCC NO:M 20242779.

[0009] The invention discloses an Akkermansia muciniphila Akk21 with the ability to lower blood sugar and the preservation number of CCTCC NO: M 20242779, and its application in the preparation of functional foods for assisting in lowering blood sugar, assisting in lowering blood lipids, promoting lead excretion, and regulating intestinal flora.

[0010] The auxiliary blood sugar lowering includes: the auxiliary blood sugar lowering includes: inhibiting α-glucosidase, improving the increase of food intake and water intake, alleviating weight loss, reducing blood sugar value, reducing serum insulin concentration, reducing the area below the oral glucose tolerance line, reducing insulin resistance index, reducing blood lipid level, reducing inflammatory factor level, improving body immunity, and regulating intestinal flora;

[0011] Preferably, the lipid-lowering comprises: lowering the levels of total cholesterol, total triglycerides, high-density lipoprotein, and low-density lipoprotein in serum;

[0012] Preferably, the inflammatory factors include: LPS, IL-6, TNF-α, IL-10;

[0013] Preferably, the regulating intestinal flora includes: increasing the abundance of Blautia, Bifidobacterium, Ligilactobacillus, Akkermansia, and unclassified Muribacula, and reducing the abundance of Aerococcus and Allobaculum.

[0014] Preferably, said promoting lead excretion comprises: reducing the mass fraction of lead in the kidneys.

[0015] A blood sugar lowering functional food, comprising: an active ingredient; the active ingredient comprises Akkermansia muciniphila Akk21 with a blood sugar lowering ability and a preservation number of CCTCC NO: M 20242779.

[0016] The food with auxiliary blood sugar lowering function also includes auxiliary materials.

[0017] A functional food for assisting in lowering blood lipids, comprising: an active ingredient; the active ingredient comprises Akkermansia muciniphila Akk21 with a blood sugar lowering ability and a preservation number of CCTCC NO: M 20242779.

[0018] The functional food for assisting in lowering blood lipids also includes auxiliary materials.

[0019] A functional food for promoting lead excretion, comprising: an active ingredient; the active ingredient comprises Akkermansia muciniphila Akk21 with a preservation number of CCTCCNO: M 20242779 and the ability to lower blood sugar.

[0020] The functional food for promoting lead excretion also includes auxiliary materials.

[0021] A functional food for regulating intestinal flora, comprising: an active ingredient; the active ingredient comprises Akkermansia muciniphila Akk21 with a blood sugar lowering ability and a preservation number of CCTCC NO: M 20242779.

[0022] The beneficial effects of the present invention are as follows:

[0023] The present invention screens and obtains a strain of Akkermansia muciniphila Akk21 with excellent lead and arsenic tolerance and hypoglycemic ability from infant feces samples. The strain has an artificial gastric juice tolerance of 77.38%, a bile salt solution tolerance of 78.84%, and a lead and arsenic tolerance rate of 48.3% and 45.46%, respectively. The Akk21 strain also has the ability to inhibit α-glucosidase, and the inhibition rate of α-glucosidase is 7.26%. Animal experiments have shown that the Akk21 strain can effectively alleviate the weight loss symptoms of type II diabetic mice and effectively reduce the blood sugar level of type II diabetic mice and significantly improve and alleviate the insulin level. At the same time, the Akk21 strain can effectively reduce the blood lipid level of type II diabetic mice, significantly reduce the inflammation of the whole body of mice, and can significantly reduce the number of harmful intestinal bacteria by increasing the abundance of beneficial bacteria in the intestine, thereby improving the species diversity and abundance of intestinal bacteria. The deposit information of Akkermansia muciniphila Akk21 is as follows:

[0024] Deposit number: CCTCC NO:M 20242779;

[0025] Classification and nomenclature: Akkermansia muciniphila Akk21;

[0026] Depository: China Center for Type Culture Collection;

[0027] Address of depository: Wuhan University, Wuhan, China;

[0028] Date of preservation: December 10, 2024. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a graph showing the effect of Akkermansia muciniphila Akk21 of Experimental Example 5 of the present invention on the food intake and water intake of type 2 diabetic mice.

[0030] Figure 2 This is a graph showing the effect of Akkermansia muciniphila Akk21 of Experimental Example 5 of the present invention on the body weight of type 2 diabetic mice.

[0031] Figure 3 This is a graph showing the effect of Akkermansia muciniphila Akk21 of Experimental Example 6 of the present invention on the blood glucose level of type 2 diabetic mice.

[0032] Figure 4 This is a graph showing the effect of Akkermansia muciniphila Akk21 of Experimental Example 6 of the present invention on oral glucose tolerance in type 2 diabetic mice.

[0033] Figure 5 This is a graph showing the effect of Akkermansia muciniphila Akk21 on serum insulin concentration and insulin resistance index in type 2 diabetic mice in Experimental Example 6 of the present invention.

[0034] Figure 6 This is a graph showing the effect of Akkermansia muciniphila Akk21 of Experimental Example 8 of the present invention on inflammatory factors in type 2 diabetic mice.

[0035] Figure 7 This is the Chao1 index and Shannon index diagram of mouse intestinal microorganisms in Experimental Example 9 of the present invention.

[0036] Figure 8 This is an analysis diagram of the β diversity of the mouse intestinal flora in Experimental Example 9 of the present invention

[0037] Fig. 9 This is a Wayne analysis diagram of the mouse intestinal flora in Experimental Example 9 of the present invention

[0038] Fig.10 This is a cluster diagram of the abundance of the mouse intestinal flora at the genus level in Experimental Example 9 of the present invention.

[0039] Fig.11 This is a graph showing the abundance of the mouse intestinal flora at the genus level in Experimental Example 9 of the present invention.

[0040] The markings in the figure are listed as follows:

[0041] Figure 1-Figure 8The group numbers are as follows: NC is the normal group mice, MC is the model group mice, MET is the drug treatment group mice, and Akk21 is the probiotic group mice.

[0042] Figure 3 The vertical axis FBG in the right figure refers to the fasting blood glucose value.

[0043] Figure 4 The vertical axis OGTT AUC in the right graph refers to the value of the area under the curve (AUC) of the oral glucose tolerance test (OGTT).

[0044] Figure 5 The vertical axis HOMA-IR refers to the insulin resistance index.

[0045] Figure 8 The horizontal and vertical axes of NMDS1 refer to non-metric multidimensional scaling. DETAILED DESCRIPTION

[0046] The present invention is further described in detail below in conjunction with embodiments and experimental examples, but the embodiments of the present invention are not limited thereto, and the protection scope of the present invention is not limited thereto.

[0047] Sources of biological materials

[0048] 1. The C57BL / 6j mice of Experimental Examples 5-10 of the present invention are commercially available.

[0049] “Above” and “below” in this article include the original number.

[0050] The "Akkermansia muciniphila strain Akk21", "Akkermansia muciniphila Akk21", "Akk21", "Akk21 strain", "strain Akk21" herein all refer to the Akkermansia muciniphila strain Akk21 with a deposit number of CCTCC NO: M 20242779. Group 1 Example, strain Akk21 of the present invention

[0051] This group of embodiments provides an Akkermansia muciniphila Akk21 with the ability to lower blood sugar. All embodiments in this group have the following common features: the deposit number of the Akkermansia muciniphila Akk21 with the ability to lower blood sugar is CCTCC NO: M20242779.

[0052] Any act of culturing, propagating, fermenting, enriching, producing, preparing, using, inoculating, amplifying, transforming, modifying, transforming, selling, or promising to sell a strain of Akkermansia muciniphila Akk21 with a deposit number of CCTCC NO: M 20242779, and / or using a strain of Akkermansia muciniphila Akk21 with a deposit number of CCTCC NO: M 20242779 in combination with other probiotics, and / or using a strain of Akkermansia muciniphila Akk21 with a deposit number of CCTCC NO: M 20242779 to antagonize various pathogens, and / or preparing antibacterial products, and / or preparing drugs for improving constipation or regulating intestinal flora, and / or preparing health foods with laxative function or intestinal flora regulating function all fall within the protection scope of the present invention.

[0053] The other probiotics include but are not limited to: Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii lactic acid subsp., Lactobacillus helveticus, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus salivarius, Saccharomyces cerevisiae, Delb's spore-forming torula, Candida, Wickham's yeast, Pichia, Saccharomyces boulardii, Torulopsis spp., Saccharomyces cerevisiae, Rhodotorula, Schizosaccharomyces pombe , Saccharomyces cerevisiae, Bacillus thuringiensis, Bacillus laterosporus, Bacillus megaterium, Bacillus colloidal, Bacillus azotobacterium, Bacillus sphaericus, Clostridium butyricum, Bifidobacterium adolescentis, Bifidobacterium angularis, Bifidobacterium animalis, Bifidobacterium asteroides, Bifidobacterium bifidum, Bifidobacterium bovis, Bifidobacterium breve, Bifidobacterium dentate, Bifidobacterium infantis (i.e., Bifidobacterium longum infantis subsp. infantis), Bifidobacterium lactis (i.e., Bifidobacterium animalis subsp. cremoris), Bifidobacterium longum, Bifidobacterium pseudocatenulatum, as well as Bifidobacterium thermophilum and Bifidobacterium acidothermophilum.

[0054] Those skilled in the art can make routine selections or adjustments to pharmaceutical excipients according to actual production needs, combined with conventional technical means or basic common sense of pharmaceutical production processes (for example, "Encyclopedia of Preparation Technology", "Pharmaceutical Preparation Technology", etc.), and then prepare products with different dosage forms, different storage conditions, and different shelf lives from Akkermansia muciniphila Akk21 with a deposit number of CCTCC NO: M 20242779. This is possible and easy to do without technical barriers for those skilled in the art.

[0055] The second group of examples, application of strain Akk21 of the present invention

[0056] This group of examples provides the use of Akkermansia muciniphila Akk21 with the ability to lower blood sugar, which has a deposit number of CCTCC NO: M 20242779, in the preparation of functional foods that assist in lowering blood sugar, functional foods that assist in lowering blood lipids, functional foods that promote lead excretion, and functional foods that regulate intestinal flora.

[0057] In a specific embodiment, the auxiliary blood sugar reduction includes: inhibiting α-glucosidase, improving the increase of food intake and water intake, alleviating weight loss, reducing blood sugar levels, reducing serum insulin concentrations, reducing the area below the oral glucose tolerance line, reducing insulin resistance index, reducing blood lipid levels, reducing inflammatory factor levels, improving body immunity, and regulating intestinal flora;

[0058] Preferably, the lipid-lowering comprises: lowering the levels of total cholesterol, total triglycerides, high-density lipoprotein, and low-density lipoprotein in serum;

[0059] Preferably, the inflammatory factors include: LPS, IL-6, TNF-α, IL-10;

[0060] Preferably, the regulating intestinal flora includes: increasing the abundance of Blautia, Bifidobacterium, Ligilactobacillus, Akkermansia, and unclassified Muribacula, and reducing the abundance of Aerococcus and Allobaculum.

[0061] Preferably, said promoting lead excretion comprises: reducing the mass fraction of lead in the kidneys.

[0062] The third group of embodiments, the functional food for promoting lead removal of the present invention

[0063] This group of embodiments provides a functional food for promoting lead excretion. All embodiments in this group have the following common features: the functional food for promoting lead excretion comprises: an active ingredient; the active ingredient comprises Akkermansia muciniphila Akk21 with a blood sugar lowering ability and a preservation number of CCTCC NO: M20242779.

[0064] In a further embodiment, the functional food for promoting lead excretion further includes: auxiliary materials.

[0065] In a more specific embodiment, the excipient is selected from: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculating agents, filter aids, release retardants, etc.

[0066] According to the content of the present invention, for different needs in actual production and application, combined with conventional technical means in the field of drug preparation (for example, "Encyclopedia of Preparation Technology", "Encyclopedia of Food and Food Production", "Lactic Acid Bacteria and Fermented Foods", "Research and Application of Microbial Agent Technology", etc.), those skilled in the art can select and formulate the above-mentioned excipients, and prepare Akkermansia muciniphila Akk21 with a deposit number of CCTCC NO: M 20242779 into different dosage forms, such as solid beverages, liquid beverages, powders, tablets, oral liquids, gels, patches, sprays, lotions, granules, etc.

[0067] In a specific embodiment, the dosage form of the product is selected from: one or more of powder, tablet, liquid, and capsule.

[0068] The fourth group of embodiments, the blood sugar lowering functional food of the present invention

[0069] This group of embodiments provides a blood sugar lowering food. All embodiments in this group have the following common features: the blood sugar lowering food comprises: an active ingredient; the active ingredient comprises Akkermansia muciniphila Akk21 with a blood sugar lowering ability and a preservation number of CCTCCNO: M 20242779.

[0070] In a further embodiment, the blood sugar lowering auxiliary functional food further includes: auxiliary materials.

[0071] In a more specific embodiment, the excipient is selected from: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculating agents, filter aids, release retardants, etc.

[0072] According to the content of the present invention, for different needs in actual production and application, combined with conventional technical means in the field of drug preparation (for example, "Encyclopedia of Preparation Technology", "Encyclopedia of Food and Food Production", "Lactic Acid Bacteria and Fermented Foods", "Research and Application of Microbial Agent Technology", etc.), those skilled in the art can select and formulate the above-mentioned excipients, and prepare Akkermansia muciniphila Akk21 with a deposit number of CCTCC NO: M 20242779 into different dosage forms, such as liquid beverages, solid beverages, powders, tablets, oral liquids, gels, patches, sprays, lotions, granules, etc.

[0073] In a specific embodiment, the dosage form of the auxiliary blood sugar lowering functional food is selected from: one or more of powder, tablet, liquid, and capsule.

[0074] The fifth group of embodiments, the functional food for assisting in lowering blood lipids of the present invention

[0075] This group of embodiments provides a functional food for assisting in lowering blood lipids. All embodiments in this group have the following common features: the functional food for assisting in lowering blood lipids comprises: an active ingredient; the active ingredient comprises Akkermansia muciniphila Akk21 with a blood lipid lowering ability and a preservation number of CCTCCNO: M 20242779.

[0076] In a further embodiment, the functional food for assisting in lowering blood lipids further comprises: auxiliary materials.

[0077] In a more specific embodiment, the excipient is selected from: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculating agents, filter aids, release retardants, etc.

[0078] According to the content of the present invention, for different needs in actual production and application, combined with conventional technical means in the field of drug preparation (for example, "Encyclopedia of Preparation Technology", "Encyclopedia of Food and Food Production", "Lactic Acid Bacteria and Fermented Foods", "Research and Application of Microbial Agent Technology", etc.), those skilled in the art can select and formulate the above-mentioned excipients, and prepare Akkermansia muciniphila Akk21 with a deposit number of CCTCC NO: M 20242779 into different dosage forms, such as liquid beverages, solid beverages, powders, tablets, oral liquids, gels, patches, sprays, lotions, granules, etc.

[0079] In a specific embodiment, the dosage form of the functional food for assisting in lowering blood lipids is selected from: one or more of powder, tablet, liquid, and capsule.

[0080] The sixth group of embodiments, functional food for regulating intestinal flora of the present invention

[0081] This group of embodiments provides a functional food for regulating intestinal flora. All embodiments in this group have the following common features: the functional food for regulating intestinal flora comprises: an active ingredient; the active ingredient comprises Akkermansia muciniphila Akk21 with a lipid-lowering ability and a preservation number of CCTCC NO: M 20242779.

[0082] In a further embodiment, the functional food for regulating intestinal flora further includes: auxiliary materials.

[0083] In a more specific embodiment, the excipient is selected from: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculating agents, filter aids, release retardants, etc.

[0084] According to the content of the present invention, for different needs in actual production and application, combined with conventional technical means in the field of drug preparation (for example, "Encyclopedia of Preparation Technology", "Encyclopedia of Food and Food Production", "Lactic Acid Bacteria and Fermented Foods", "Research and Application of Microbial Agent Technology", etc.), those skilled in the art can select and formulate the above-mentioned excipients, and prepare Akkermansia muciniphila Akk21 with a deposit number of CCTCC NO: M 20242779 into different dosage forms, such as liquid beverages, solid beverages, powders, tablets, oral liquids, gels, patches, sprays, lotions, granules, etc.

[0085] In a specific embodiment, the dosage form of the functional food for regulating intestinal flora is selected from: one or more of powder, tablet, liquid, and capsule.

[0086] Experimental Example 1: Isolation, screening and identification of Akkermansia muciniphila

[0087] (1) Isolation and screening of Akkermansia muciniphila

[0088] 100 fecal samples from healthy infants in Hubei were collected, mixed evenly with 30% sterilized glycerol water, numbered and stored in a -80℃ refrigerator for later use. Vancomycin and streptomycin were selected as antibacterial agents and added to the basal culture medium (BHI + 0.2% mucin) at an addition amount of 0.5-10 μg / mL as the screening culture medium.

[0089] After thawing, the frozen samples were diluted 10 times and inoculated into the screening medium containing antibiotics and BHI + 0.2% mucin at a 2% inoculum, and cultured at 37°C for 2 days under anaerobic conditions. The Akk-specific primers described in the article "Qiye Wang, Zhaobin Wang, Bohao Shang, Yilong Li, Fang Zhou, Xianglin Zeng, Zhonghua Liu, Huansheng Yang, Mingzhi Zhu, Tea polyphenols improve lipid deposition via modulation of gut microbiota in rats and Ningxiang pigs, Journal of Functional Foods, Volume 113, 2024, 106049, ISSN 1756-4646" were used to preliminarily screen and identify the enriched fecal samples. The presence or absence of the target band can be used to preliminarily determine whether the enriched samples contain Akk strains, and the enriched tubes identified as positive by PCR are selected for separation and culture on solid plates.

[0090] Dilute and separate the enrichment tubes with positive identification results, and select 10 -1 Up to 10 -5 The gradient was plated on a solid screening medium containing antibiotics and BHI + 0.2% mucin, and cultured at 37°C under anaerobic conditions for 5-7 days. Single colonies suspected of Akk in the antibiotic plate were selected, numbered, identified again using Akk-specific primers, and the positive strains were preserved.

[0091] The present method uses Akk-specific primers to preliminarily screen and identify 100 stool samples enriched in antibiotic culture medium, and obtains 29 positive enrichment tubes. The positive enrichment tubes are diluted and separated, and then plated on a solid screening culture medium containing antibiotic BHI + 0.2% mucin, and single colonies suspected of Akk growing in the antibiotic plate are selected. After identification using PCR-specific primers, 27 Akk strains are obtained, and the strains are numbered and preserved. The efficiency of isolating Akk strains from positive enrichment tubes by this method reaches 93%, which greatly improves the isolation efficiency of Akk strains compared to ordinary strain isolation methods.

[0092] (2) Identification and preservation of Akk21 strain

[0093] The preserved target strain was cultured in liquid, the cells were collected, the genomic DNA was extracted, and the universal primers 27F and 1492R recorded in Chinese invention patent 202210478937.4 were used for PCR amplification reaction. The PCR amplification procedure was: 94°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 90 s, a total of 35 cycles, and finally extended at 72°C for 10 min. The content and purity of the PCR amplification product were then tested, and after passing the test, it was sent to Wuhan Jinkairui Bioengineering Co., Ltd. for sequencing. According to the sequencing results, the BLAST tool in the NCBI database was used for homology comparison, and the 16S rRNA gene sequence homology ≥ 99% was used as the identification standard. One of the strains had good tolerance to gastric juice, bile salts, lead, arsenic and glucose-lowering ability. It was identified as Akkermansia muciniphila and named Akk21. It was sent to China Center for Type Culture Collection for preservation. Its preservation information is as follows:

[0094] Deposit number: CCTCC NO:M 20242779;

[0095] Classification and nomenclature: Akkermansia muciniphila Akk21;

[0096] Depository: China Center for Type Culture Collection;

[0097] Address of depository: Wuhan University, Wuhan, China;

[0098] Date of preservation: December 10, 2024.

[0099] Experimental Example 2: Gastric Juice and Bile Salt Tolerance Experiment

[0100] Simulated artificial gastric juice: prepare 0.5% sodium chloride solution, adjust the pH to 3.0 with 1 mol / L HCl, add 0.3% pepsin, and filter with a 0.22 μm microporous filter membrane to sterilize after fully dissolving.

[0101] Bile salt solution: Weigh 0.2% sodium thioglycolate and 0.3% porcine bile salt and add them to BHI liquid culture medium. After fully dissolved, sterilize in an autoclave at 115°C for 20 min for later use.

[0102] Akkermansia muciniphila Akk21 was cultured in BHI + 0.2% mucin liquid medium at 37°C overnight. The activated Akk21 bacterial solution was centrifuged, the supernatant was discarded, the bacterial cells were collected, and the bacterial solution concentration was adjusted to 10 8CFU / mL. Take 10mL of bacterial suspension and centrifuge. After collecting the bacteria, add them into 10mL of prepared simulated gastric juice and bile salt at pH 3.0, mix them well, incubate them at 37℃, and take the suspension at 0h and 3h to detect the number of live bacteria and calculate the survival rate.

[0103] The survival rate of the strain was calculated according to the following formula:

[0104]

[0105] Where N0 represents the number of viable bacteria of the strain at 0h (CFU / mL), N t It indicates the number of viable bacteria of the strain at 3h (CFU / mL).

[0106] Table 1. Tolerance of Akkermansia muciniphila Akk21 to gastric juice and bile salts

[0107] strain Artificial gastric juice (%) Bile salt solution (%) Akkermansia muciniphila Akk21 77.38±5.80 78.84±5.71

[0108] The experimental results showed that after 3 hours of gastric juice digestion, the survival rate of Akkermansia muciniphila Akk21 was 77.38%, and after 3 hours of bile salt digestion, the survival rate of Akkermansia muciniphila Akk21 was 78.84%, indicating that Akkermansia muciniphila Akk21 has a strong ability to resist gastric acid and bile salts.

[0109] Experimental Example 3: Tolerance of Akkermansia muciniphila to lead and arsenic

[0110] Akkermansia muciniphila was inoculated into liquid BML medium and cultured at 37°C overnight. After the second generation, the culture was cultured for 8 hours. After the third generation, the culture was fermented in BML medium, BML liquid medium with a lead concentration of 100 ug / mL, and BML liquid medium with an arsenic concentration of 100 ug / mL for 15 hours. 600 The tolerance rate is calculated according to the following formula:

[0111]

[0112] In the above formula, A: BML culture OD value; B: BML culture OD value of 100ug / mL lead / arsenic concentration. The tolerance rate results are shown in Table 2 below:

[0113] Table 2. Tolerance of Akkermansia muciniphila Akk21 to lead and arsenic

[0114] strain Lead tolerance rate (%) Arsenic tolerance rate (%) Akkermansia muciniphila Akk21 48.30±0.078 45.46±0.102

[0115] The results showed that the tolerance rate of strain Akk21 to 100ug / mL lead concentration was 48.3%, and the tolerance rate to 100ug / mL arsenic concentration was 45.46%.

[0116] Experimental Example 4: Inhibitory ability of Akkermansia muciniphila on α-glucosidase

[0117] Akkermansia muciniphila was inoculated into liquid BML medium and cultured overnight at 37°C. After the second generation was transferred, it was cultured for 8 hours, and then the third generation was transferred for fermentation and cultured for 14 hours. The fermentation broth was centrifuged, the supernatant was discarded, and the culture was resuspended in physiological saline and the bacterial solution concentration was adjusted to 10 8 CFU / mL for later use. Add 75μL p-nitrophenol-α-D-pyranose glucoside (PNPG) solution and 25μL sample to be tested to 150μL PBS solution, place the mixture on ice for 3min, add 50μL α-glucosidase solution 0.2U / mL and react in a water bath at 37℃ for 15min, add 1mL 1mol / L Na2CO3 as the reaction stop solution, and measure the absorbance of the reaction solution at 405nm:

[0118]

[0119] A: add sample plus α-glucosidase;

[0120] B: adding sample without α-glucosidase;

[0121] C without sample plus α-glucosidase;

[0122] D No sample or α-glucosidase

[0123] Table 3. Inhibition rate of α-glucosidase by Akkermansia muciniphila Akk21

[0124] strain α-Glucosidase inhibition rate (%) Akkermansia muciniphila Akk21 7.26±0.01

[0125] As shown in Table 3 above, Akk21 has the ability to inhibit α-glucosidase. This indicates that the strain can inhibit the α-glucosidase of the small intestinal epithelium to slow down the increase of blood sugar, thereby producing a blood sugar lowering effect.

[0126] Experimental Example 5: Effects of Akkermansia muciniphila on food intake, water intake, and body weight in type 2 diabetic mice

[0127] Male C57BL / 6j mice (5 - 6 weeks old; 19 ± 1 g) were housed in the animal facility of Hubei Center for Disease Control and Prevention. The license number for the use of experimental animals was SCXK(E)2020 - 0019. The animal experiments were maintained at 20 - 22 °C, with a humidity of 40 - 60%, and a 12 h light / 12 h dark cycle. The mice had free access to food and water. After one week of adaptation, according to the reference F. Yan, N. Li, J. Shi, H. Li, Y. Yue, W. Jiao, N. Wang, Y. Song, G. Huo and B. Li, Lactobacillus acidophilus alleviates type 2 diabetes by regulating hepatic glucose and lipid metabolism and gut microbiota in mice, Food Funct, 2019, the mice were induced with type 2 diabetes. The mice were divided into 4 groups (n = 10): normal control group (NC group), model control group (MC group), probiotic group (Akk21, 6 × 10 9 CFU / d), and drug treatment group (MET, 0.2 g / kg / d). The NC group and the MC group were gavaged with sterile normal saline, the probiotic group was gavaged with Akkermansia muciniphila Akk21 once a day for 35 consecutive days, and the drug treatment group was gavaged with metformin hydrochloride (MET) once a day for 35 consecutive days. The daily food intake, daily water intake, and weekly body weight of the mice were monitored.

[0128] Since the typical symptoms of type 2 diabetic mice are "increased food intake, increased water intake, and weight loss", after five weeks of gavage treatment, the results were as Figure 1 shown. Gavage with Akk21 for five weeks effectively improved the increased food intake and water intake of type 2 diabetic mice. With the progress of gavage, it had a continuous and effective control on the mice, and the mouse state gradually approached that of the healthy group. From Figure 2 the change in the body weight value of the mice, it can be concluded that the treatment with the strain and the drug had an obvious restorative effect on the weight loss symptoms of the mice. The body weight value of the mice in the strain treatment group showed a stable and slightly increasing trend two weeks after gavage.

[0129] Experimental Example 6. Effect of Akkermansia muciniphila on glucose metabolism in type 2 diabetic mice

[0130] Before treatment and during the five - week treatment period, tail vein blood was collected weekly to measure the fasting blood glucose value of the mice. The measurement results of the fasting blood glucose value (FBG) were as Figure 3As shown. After five weeks of treatment, the oral glucose tolerance of mice was tested. Each group of mice was given oral glucose, and the blood glucose level was measured at 0min, 15min, 30min, 60min, 90min, and 120min after oral glucose tolerance test. The area below the oral glucose tolerance line in the oral glucose tolerance test was measured. At the end of the experiment, serum was taken to measure the serum insulin concentration of mice.

[0131] Experimental steps for oral glucose tolerance: The amount of glucose used in the mouse glucose tolerance experiment is 2g per kilogram of body weight (2g / kg). Prepare a glucose solution of appropriate concentration according to the experimental requirements, and use normal saline to prepare a 20% glucose solution. The mice were fasted for 16 hours, during which time the mice maintained normal drinking water; before starting the glucose tolerance experiment, weigh each mouse. Blood was collected from the tip of the mouse's tail, and the fasting blood sugar was measured with a blood glucose meter, and the measured value was considered the blood sugar value at 0min. Glucose was gavaged and the timing began from the moment it was completed. The blood sugar value of each mouse was measured at 15, 30, 60, 90 and 120min.

[0132] During the five-week treatment period, the blood sugar levels of mice gavaged with Akk21 strain were effectively controlled. At the end of the treatment, the blood sugar levels of mice were significantly different between 11.21mmol / L and 20.27mmol / L in the model group. Figure 3 It can be seen that the first week of intragastric administration of probiotics showed the effect of controlling and reducing the increase in blood sugar levels of mice, which was manifested by the blood sugar level dropping from 17.16mmol / L to 15.3mmol / L. As the intragastric administration continued, the blood sugar levels of mice fluctuated but showed an overall downward trend. This shows that the treatment of the strain effectively reduced the blood sugar levels of type 2 diabetic mice, and as the treatment continued, it played a good effect on stabilizing and reducing blood sugar levels.

[0133] At the end of the treatment, the mice were tested for oral glucose tolerance. Figure 4 It can be clearly seen that the strain treatment has a good effect on the recovery of blood glucose levels in mice within two hours and is close to that of the healthy group. The area under the curve (AUC) is calculated from the OGTT (oral glucose tolerance test). The area calculation better displays and analyzes the impaired glucose tolerance, such as Figure 4 It can be concluded that the glucose tolerance of mice treated with the strain for five weeks has been greatly restored. There is no significant difference in effect compared with drug treatment. The drug treatment and strain treatment groups have reached a significant difference level compared with the model group. And it shows a trend close to the healthy group. The insulin resistance index (HOMA-IR) is a value calculated by a formula based on serum insulin level and fasting blood sugar value. It is an indicator used to judge the degree of insulin resistance. Figure 5aIt can be concluded that after five weeks of treatment, the serum insulin concentration of mice was significantly reduced, reaching a significant difference level compared with the model group, and showed a similar trend to the healthy group with no significant difference. Figure 5 b shows that the insulin levels in the strain treatment group and the drug treatment group were significantly improved and alleviated.

[0134] The above results indicate that treatment with the Akk21 strain can improve and enhance the glucose metabolism ability of mice, thereby lowering their blood sugar levels and thus controlling the progression of the disease in mice.

[0135] Experimental Example 7: Effect of Akkermansia muciniphila on blood lipid levels in type 2 diabetic mice

[0136] The indicators of mouse serum were measured: triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C), as shown in Table 4:

[0137] Table 4. Results of total cholesterol, total triglycerides, high-density lipoprotein, and low-density lipoprotein in serum

[0138] Group TG (mmol / L) TC(mmol / L) HDL-C (mmol / L) LDL-C (mmol / L) Akk21 group 0.703±0.05bc 2.536±0.31a 2.50±0.25a 1.266±0.13b MET Group 0.829±0.14ab 2.486±0.17a 2.53±0.34a 1.518±0.10a MC Group 0.936±0.13a 2.655±0.16a 2.78±0.37a 1.684±0.16a NC Group 0.577±0.06c 1.649±0.25b 2.058±0.33b 0.698±0.21c

[0139] As shown in Table 4, the treatment of Akk21 strain has a role in reducing the blood lipid level of type 2 diabetic mice, and the effect of reducing total triglyceride and low-density lipoprotein levels is particularly significant. According to the data in the table, the serum total triglyceride concentration of the Akk21 strain treatment group was significantly reduced and close to the healthy group (NC group) with no significant difference (P>0.05), and the serum low-density lipoprotein level was significantly reduced compared with the model group (p<0.05).

[0140] The above shows that Akk21 strain can effectively reduce the blood lipid level of type 2 diabetic mice and has a very good effect.

[0141] Experimental Example 8: Effects of Akkermansia muciniphila on inflammatory factors in type 2 diabetic mice

[0142] The onset of diabetes will cause systemic inflammation in the body. The occurrence of chronic inflammatory diseases will lead to an increase in tumor necrosis factor in the patient's serum and promote the production of interleukin 6. At the end of the experiment, the whole blood of the mice was centrifuged to obtain serum, and the ELISA kit was used to measure the inflammatory factors in the mouse serum, including the concentration levels of LPS (lipopolysaccharide), IL-6, TNF-α, and IL-10. Figure 6As shown in Figure ac, compared with the model group (MC), oral administration of Akk21 strain significantly reduced the levels of lipopolysaccharide (LPS), interleukin-6 (IL-6) and tumor necrosis factor (TNF-α) in the serum of type 2 diabetic mice (p<0.05), slowing down the occurrence of inflammation in the body. Serum insulin level is usually an important indicator for diagnosing type 2 diabetes and is also an important monitoring indicator during treatment. During the onset of the disease, the insulin concentration in the patient's serum will show an increase in concentration, such as Figure 6 As shown in Figure d, oral administration of Akk21 can significantly increase the concentration of serum interleukin-10, and it is close to the level of the healthy group with no significant difference (p>0.05). This shows that oral administration of Akk21 can effectively reduce the systemic inflammation of type 2 diabetic mice and improve the body's immunity.

[0143] Experimental Example 9: Effect of Akkermansia muciniphila on intestinal flora of type 2 diabetic mice

[0144] After the experiment, the mice were killed, the abdomen was disinfected and dissected, and the cecal contents of each group of mice were taken for microbial diversity analysis. The 16S rDNA V3-V4 hypervariable regions of all bacteria in the samples were sequenced on the Mi Seq Illumina sequencing platform to determine the characteristics of the intestinal microbiota.

[0145] α-diversity reflects the species richness and diversity of the sample. Chao1 index and Shannon index are commonly used indicators for judging the abundance and diversity of intestinal microbial colonies. Figure 7 As shown in the figure, the abundance and diversity of intestinal flora in type 2 diabetic mice were significantly decreased compared with those in the healthy group (p<0.05). According to the Chao1 index analysis, Akk21 strain treatment increased and improved the abundance of intestinal flora and reached a significant level (p<0.01), and there was no significant difference compared with the drug treatment group (p>0.05). According to the Shannon index analysis, oral administration of Akk21 strain significantly increased the diversity of intestinal flora in type 2 diabetic mice, and it was close to the healthy group and there was no significant difference between the two (p>0.05). The results show that Akk21 strain can increase and improve the abundance and diversity of intestinal flora in type 2 diabetic mice.

[0146] β-diversity is used to compare the similarity of different samples in terms of species diversity. NMDS is a commonly used analysis method that can better reflect the nonlinear structure of ecological data. The closer the distance on the coordinate graph, the higher the similarity. Figure 8As shown, there are obvious differences in the intestinal flora of the model group, drug treatment group, and Akk21 treatment group, indicating that with the continuous treatment of drugs and probiotics, the intestinal health is effectively improved, and the differences between the model group and the model group gradually appear, and gradually approach the healthy group. This shows that the strain effectively improves the intestinal flora of type 2 diabetic mice and tends to be normal.

[0147] The Venn diagram is used to show the number of common or unique elements between groups, by showing the number of overlapping or different OTUs in the mouse intestinal flora. The more common numbers, the higher the similarity between the two. Fig. 9 As shown, the total number of OTUs between the Akk21 group and the NC group was 217, the total number of OTUs between the MET group and the NC group was 172, and the total number of OTUs between the MC group and the NC group was 182. The similarity between the Akk21 group and the NC group was higher through the number of shared OTUs, indicating that the treatment with Akk21 made the mice tend to be normal.

[0148] Metformin is a first-line drug for the treatment of type 2 diabetes, and its mechanism of action is constantly being revealed by research. Many studies have shown that metformin can have an effect on type 2 diabetic mice by changing the intestinal flora. Fig.10 The present invention mainly compares the co-response strains of the metformin group and the probiotic group at the genus level, and analyzes the influence of the changes of different strains on the hypoglycemic effect. Fig.11 Results showed that the genera of Blautia, Bifidobacterium, Ligilactobacillus, Akkermansia, and unclassified Muribacillus increased significantly in the Akk21 treatment group (p<0.01), and the abundance of Akkermansia was significantly higher than that of the drug treatment group. The harmful intestinal bacteria Aerococcus and Allobaculum decreased significantly in the Akk21 treatment group and the drug treatment group (p<0.01).

[0149] The above results indicate that type 2 diabetic mice have intestinal flora disorders, and treatment with Akkermansia muciniphila Akk21 can increase the abundance of beneficial flora in the intestine, significantly reduce the number of harmful intestinal flora, and improve the species diversity and abundance of intestinal flora, thereby effectively controlling and improving the blood sugar levels of type 2 diabetic mice.

[0150] Experimental Example 10: The alleviating effect of Akkermansia muciniphila on heavy metal lead poisoning in mice

[0151] Thirty clean-grade C57BL / 6 male mice aged 6 to 8 weeks were randomly divided into three groups after one week of environmental adaptation, with 10 mice in each group. The experimental groups were NC group (10 mice), Pb group (10 mice), and Akk21+Pb group (10 mice). Continuous light was maintained and food and water were allowed to be taken at will. The Pb group and Akk21+Pb group freely drank 300 mg / L lead acetate solution instead of drinking water for 30 days. After the experiment, the mice were killed by carbon dioxide-assisted cervical dislocation, and the mouse kidney tissues were collected for index detection. The lead mass fraction in the mouse kidney was determined.

[0152] Table 5. Results of lead content in mouse kidneys

[0153] Group Renal lead mass fraction (μg / g) Blank control group 0.29±0.13c Model Group 10.13±0.56a Akk21+ model group 7.64±0.83b

[0154] The results are shown in Table 5 below. The kidney lead mass fraction in the Akk21 strain intervention group was significantly lower than that in the model group, indicating that the Akk21 strain has a certain alleviating effect on lead poisoning.

Claims

1. An Akkermansia muciniphila Akk21 having the ability to lower blood sugar, characterized in that: Its deposit number is CCTCC NO:M 20242779.

2. The application of Akkermansia muciniphila Akk21 with the ability to lower blood sugar and the deposit number of CCTCC NO: M 20242779 in the preparation of functional foods that assist in lowering blood sugar, functional foods that assist in lowering blood lipids, functional foods that promote lead excretion, and functional foods that regulate intestinal flora.

3. The use of Akkermansia muciniphila Akk21 with hypoglycemic ability and the deposit number of CCTCC NO: M 20242779 according to claim 2 in the preparation of functional foods for assisting in hypoglycemic function, functional foods for assisting in lowering blood lipids, functional foods for promoting lead excretion, and functional foods for regulating intestinal flora, characterized in that: The auxiliary blood sugar lowering includes: inhibiting α-glucosidase, improving the increase of food intake and water intake, alleviating weight loss, reducing blood sugar levels, reducing serum insulin concentrations, reducing the area below the oral glucose tolerance line, reducing insulin resistance index, reducing blood lipid levels, reducing inflammatory factor levels, improving body immunity, and regulating intestinal flora; Preferably, the lipid-lowering comprises: lowering the levels of total cholesterol, total triglycerides, high-density lipoprotein, and low-density lipoprotein in serum; Preferably, the inflammatory factors include: LPS, IL-6, TNF-α, IL-10; Preferably, the regulating intestinal flora includes: increasing the abundance of Blautia, Bifidobacterium, Ligilactobacillus, Akkermansia, and unclassified Muribacula, and reducing the abundance of Aerococcus and Allobaculum. Preferably, said promoting lead excretion comprises: reducing the mass fraction of lead in the kidneys.

4. A functional food for assisting in lowering blood sugar, characterized in that: include: Active ingredient: The active ingredient includes Akkermansia muciniphila Akk21 with the deposit number of CCTCC NO: M 20242779 and the ability to lower blood sugar.

5. The blood sugar lowering functional food according to claim 4, characterized in that: Also includes: Auxiliary materials.

6. A functional food for assisting in lowering blood lipids, characterized in that: include: Active ingredient: The active ingredient includes Akkermansia muciniphila Akk21 with the deposit number of CCTCC NO: M 20242779 and the ability to lower blood sugar.

7. The functional food for assisting in lowering blood lipids according to claim 6, characterized in that: Also includes: Auxiliary materials.

8. A functional food for promoting lead excretion, characterized in that: include: Active ingredient: The active ingredient includes Akkermansia muciniphila Akk21 with the deposit number of CCTCC NO: M 20242779 and the ability to lower blood sugar.

9. The functional food for promoting lead excretion according to claim 8, characterized in that: Also includes: Auxiliary materials.

10. A functional food for regulating intestinal flora, characterized in that: include: Active ingredient: The active ingredient includes Akkermansia muciniphila Akk21 with the deposit number of CCTCC NO: M 20242779 and the ability to lower blood sugar.

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