Akkermansia muciniphila akk21 with blood glucose-lowering ability, and application, product and method thereof

Abstract

The application discloses a mucinophilic Akkermansia Akk21 with blood sugar reducing capacity, and application, products and methods thereof, and belongs to the technical field of microorganisms.The application provides a mucinophilic Akkermansia strain Akk21 with blood sugar reducing capacity, and the preservation number of the strain Akk21 is CCTCC NO:M 20242779.The application also provides a blood sugar reducing drug, a lead promoting function food, health products and a method for preventing and treating lead / arsenic pollution based on the strain Akk21.The mucinophilic Akkermansia Akk21 has excellent lead and arsenic tolerance and blood sugar reducing capacity, can effectively reduce the blood lipid level of a type 2 diabetes mouse, significantly reduces the inflammation of the mouse, and can significantly reduce the number of harmful intestinal flora by increasing the abundance of beneficial intestinal flora, and improve the species diversity and abundance of intestinal flora.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to Akkermansia muciniphila Akk21 with blood glucose lowering capacity and application, product and method thereof. BACKGROUND

[0002] Type II diabetes is a chronic metabolic disease caused by insufficient or abnormal insulin secretion in insulin-sensitive tissues, resulting in high blood glucose. The sustained high level of blood glucose often causes blood to become thick and flow poorly in the circulatory system, causing some small blood vessels to become congested, causing damage to multiple organs of the human body, and eventually leading to various complications such as kidney disease, myocardial disease, retinopathy. Among all diabetic patients, type II diabetes patients account for 95%. As a global epidemic, the number of diabetic patients is increasing, and the Lancet Online published a global disease burden study indicating that the number of diabetic patients will increase to 131 million in 2050. The global prevalence of diabetes is rising, and the prevention and control of type II diabetes remains a continuing challenge, and diabetes treatment targeting the gut microbiota has become an increasingly growing research area.

[0003] Probiotics are live microorganisms that provide various health benefits to the host, and the immunomodulatory effects of probiotics have attracted much research interest. Probiotics play an important role in maintaining intestinal flora balance, immune regulation, preventing gastrointestinal infections, and improving blood indicators. In addition, studies have shown that probiotics can stimulate the immune response of immunocompromised mice. Therefore, developing probiotic strains without side effects and capable of exerting immune stimulation can improve the 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 among strains within the same species. Therefore, it is necessary to characterize the effects of specific strains and determine their dosages to verify their immunomodulatory effects.

[0004] Akkermansia muciniphila is an anaerobic, gram-negative, non-motile, non-endospore ovoid intestinal bacterium found in human feces, and will become the next generation of probiotics with clinical application prospects as research continues.

[0005] Currently, there is no Akkermansia muciniphila that has both excellent blood glucose lowering effect and tolerance to lead / arsenic and other pollution elements. SUMMARY

[0006] In order to fill the above technical gaps in the prior art, the present application provides Akkermansia muciniphila Akk21 with blood glucose lowering capacity and application thereof.

[0007] The technical solutions of the present application are as follows:

[0008] Akkermansia muciniphila with blood glucose lowering ability, with a preservation number of CCTCC NO: M 20242779.

[0009] The application of Akkermansia muciniphila Akk21 with a preservation number of CCTCC NO: M 20242779 and with blood glucose lowering ability in preparing a blood glucose lowering auxiliary functional food, a blood lipid lowering auxiliary functional food, a lead excretion promoting functional food and a gut flora regulating functional food.

[0010] The blood glucose lowering auxiliary includes inhibiting alpha-glucosidase, improving the increase of diet and water intake, relieving weight loss, reducing blood glucose value, reducing serum insulin concentration, reducing oral glucose tolerance area, reducing insulin resistance index, reducing blood lipid level, reducing inflammation factor level, improving body immunity and regulating gut flora.

[0011] Preferably, the blood lipid lowering includes reducing the level of total cholesterol, total triglyceride, high-density lipoprotein and low-density lipoprotein in serum.

[0012] Preferably, the inflammation factor includes LPS, IL-6, TNF-alpha and IL-10.

[0013] Preferably, the gut flora regulating includes increasing the abundance of Blautia, Bifidobacterium, Ligilactobacillus, Akkermansia and unclassified_Muribaculaceae and reducing the abundance of Aerococcus and Allobaculum.

[0014] Preferably, the lead excretion promoting includes reducing the lead mass fraction of kidney.

[0015] A blood glucose lowering auxiliary functional food includes active ingredients; the active ingredients include Akkermansia muciniphila Akk21 with a preservation number of CCTCC NO: M 20242779 and with blood glucose lowering ability.

[0016] The blood glucose lowering auxiliary functional food further includes auxiliary materials.

[0017] The application discloses a functional food for assisting in reducing blood fat, which comprises an active ingredient; the active ingredient comprises Akkermansia muciniphila Akk21 with blood sugar reducing capacity with a preservation number of CCTCC NO: M 20242779.

[0018] The functional food for assisting in reducing blood fat further comprises a supplementary material.

[0019] The application discloses a functional food for promoting lead excretion, which comprises an active ingredient; the active ingredient comprises Akkermansia muciniphila Akk21 with blood sugar reducing capacity with a preservation number of CCTCC NO: M 20242779.

[0020] The functional food for promoting lead excretion further comprises a supplementary material.

[0021] The application discloses a functional food for regulating intestinal flora, which comprises an active ingredient; the active ingredient comprises Akkermansia muciniphila Akk21 with blood sugar reducing capacity with a preservation number of CCTCC NO: M 20242779.

[0022] The application has the following beneficial effects:

[0023] The application screens a strain of Akkermansia muciniphila Akk21 with excellent lead and arsenic tolerance and blood sugar reducing capacity from infant fecal samples, the strain has a tolerance to artificial gastric juice of 77.38%, a tolerance to bile salt solution of 78.84%, and a tolerance to lead and arsenic as high as 48.3% and 45.46% respectively. The Akk21 strain also has an alpha-glucosidase inhibiting capacity, and the inhibiting rate to alpha-glucosidase is 7.26%. Animal experiments prove that the Akk21 strain can effectively relieve the weight loss symptom of type 2 diabetes mice, effectively reduce the blood sugar value of the type 2 diabetes mice, and significantly improve and relieve the degree of insulin, and the Akk21 strain can effectively reduce the blood lipid level of the type 2 diabetes mice, significantly reduce the inflammation of the mice, and significantly reduce the number of harmful intestinal flora and improve the species diversity and abundance of intestinal flora by increasing the abundance of beneficial intestinal flora. The preservation information of the Akkermansia muciniphila Akk21 is as follows:

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

[0025] Classification name: Akkermansia muciniphila Akk21;

[0026] Depositary: China General Microbiological Culture Collection Center

[0027] Address of the depositary: Wuhan, China, Wuhan University

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

[0029] Figure 1 Figure for the influence of the mucinophilic Akkermansia Akk21 in experimental example 5 of the present application on the food and water intake of type Ⅱ diabetes mice.

[0030] Figure 2 Figure for the influence of the mucinophilic Akkermansia Akk21 in experimental example 5 of the present application on the body weight of type Ⅱ diabetes mice.

[0031] Figure 3 Figure for the influence of the mucinophilic Akkermansia Akk21 in experimental example 6 of the present application on the blood glucose level of type Ⅱ diabetes mice.

[0032] Figure 4 Figure for the influence of the mucinophilic Akkermansia Akk21 in experimental example 6 of the present application on the oral glucose tolerance of type Ⅱ diabetes mice.

[0033] Figure 5 Figure for the influence of the mucinophilic Akkermansia Akk21 in experimental example 6 of the present application on the serum insulin concentration and insulin resistance index of type Ⅱ diabetes mice.

[0034] Figure 6 Figure for the influence of the mucinophilic Akkermansia Akk21 in experimental example 8 of the present application on the inflammatory factors of type Ⅱ diabetes mice.

[0035] Figure 7 Figure for the Chao1 index and Shannon index of the intestinal microorganisms of mice in experimental example 9 of the present application.

[0036] Figure 8 Figure for the analysis of the β diversity of intestinal flora of mice in experimental example 9 of the present application

[0037] Figure 9 Figure for the way analysis of intestinal flora of mice in experimental example 9 of the present application

[0038] Figure 10 Figure for the cluster plot of the genus level abundance of intestinal flora of mice in experimental example 9 of the present application

[0039] Figure 11 Figure for the analysis of the genus level abundance of intestinal flora of mice in experimental example 9 of the present application

[0040] The labels in the figures are listed as follows:

[0041] Figures 1-8The group labels in the figures are as follows: NC is a normal group of mice, MC is a model group of mice, MET is a drug treatment group of mice, Akk21 is a probiotic group of mice.

[0042] Figure 3 The vertical coordinate FBG of the right graph refers to the fasting blood glucose value.

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

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

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

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

[0047] Source of biological material

[0048] First, the C57BL / 6j mice of the experimental examples 5-10 of the present application can be commercially available.

[0049] In the present application, "above" and "below" include the number.

[0050] In the present application, "Akkermansia muciniphila strain Akk21", "Akkermansia muciniphila Akk21", "Akk21", "Akk21 strain", "strain Akk21" all refer to: Akkermansia muciniphila strain Akk21 with the accession number CCTCC NO: M 20242779. Group 1 example, strain Akk21 of the present application

[0051] The present group of examples provides an Akkermansia muciniphila Akk21 with blood glucose lowering ability. All examples in the present group have the following common features: the accession number of the Akkermansia muciniphila Akk21 with blood glucose lowering ability is CCTCC NO: M20242779.

[0052] Any culture, propagation, fermentation, enrichment, production, preparation, use, inoculation, amplification, transformation, modification, alteration, sale, promise of sale, and / or combination of the strain of muciniphilic Akkermansia Akk21 with other probiotics, and / or antagonism of the strain of muciniphilic Akkermansia Akk21 against various pathogenic bacteria, and / or preparation of an antibacterial product, and / or preparation of a drug for improving constipation or regulating intestinal flora, and / or preparation of a health food with the functions of relieving constipation or regulating intestinal flora, which are preserved as CCTCC NO: M 20242779, fall within the protection scope of the present application.

[0053] The other probiotics include, but are not limited to, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus helveticus, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus salivarius, Saccharomyces, Torulopsis, Candida, Wickerham, Pichia, Cryptococcus, Starmerella, Rhodotorula, Schizosaccharomyces, Hansenula, Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium animalis, Bifidobacterium asteroides, Bifidobacterium bifidum, Bifidobacterium bovis, Bifidobacterium breve, Bifidobacterium dentium, Bifidobacterium infantis (i.e., Bifidobacterium longum subsp. infantis), Bifidobacterium lactis (i.e., Bifidobacterium animalis lactis subsp.), Bifidobacterium longum, Bifidobacterium pseudocatenulatum, and Bifidobacterium thermophilum and Bifidobacterium acidophilum.

[0054] The person skilled in the art can make routine selection or adjustment of the pharmaceutical excipients according to the actual production needs and in combination with the conventional technical means or basic common sense in the production process of the pharmaceutical field (for example, Encyclopedia of Pharmaceutical Technology, Pharmaceutical Preparation Technology, etc.), and then prepare products with different dosage forms, different storage conditions, and different shelf lives of the strain of muciniphilic Akkermansia Akk21, which is no technical obstacle for the person skilled in the art and can be and easily achieved.

[0055] Example 2, application of the strain Akk21 of the present application

[0056] The embodiments of the present application provide application of Akkermansia muciniphila Akk21 with blood glucose-lowering capacity with a preservation number of CCTCC NO: M 20242779 in preparation of a blood glucose-lowering functional food, a blood lipid-lowering functional food, a lead excretion promoting functional food and a gut flora regulating functional food.

[0057] In specific embodiments, the blood glucose-lowering assistance includes inhibition of alpha-glucosidase, improvement of increased food and water intake, relief of weight loss, reduction of blood glucose level, reduction of serum insulin concentration, reduction of oral glucose tolerance area, reduction of insulin resistance index, reduction of blood lipid level, reduction of inflammatory factor level, improvement of body immunity and regulation of gut flora.

[0058] Preferably, the blood lipid reduction includes reduction of levels of total cholesterol, total triglyceride, high-density lipoprotein and low-density lipoprotein in serum.

[0059] Preferably, the inflammatory factor includes LPS, IL-6, TNF-alpha and IL-10.

[0060] Preferably, the gut flora regulation includes increase of abundance of Blautia, Bifidobacterium, Ligilactobacillus, Akkermansia and unclassified_Muribaculaceae and decrease of abundance of Aerococcus and Allobaculum.

[0061] Preferably, the lead excretion promotion includes reduction of kidney lead mass fraction.

[0062] The third group of embodiments, the lead excretion promoting functional food of the present application

[0063] The embodiments of the present application provide a lead excretion promoting functional food. All the embodiments of the present application have the following common features: the lead excretion promoting functional food includes an active ingredient; the active ingredient includes Akkermansia muciniphila Akk21 with blood glucose-lowering capacity with a preservation number of CCTCC NO: M 20242779.

[0064] In further embodiments, the lead excretion promoting functional food further includes an auxiliary material.

[0065] In more specific embodiments, the excipient is selected from the group consisting of solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, tonicity adjusting agents, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adherents, integrating agents, penetration enhancers, pH adjusting agents, buffers, plasticizers, surfactants, antifoaming agents, defoaming agents, thickening agents, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retarders, and the like.

[0066] According to the content of the present application, in view of different requirements in practical production applications, in combination with conventional technical means in the field of pharmaceutical preparation (for example, Encyclopedia of Pharmaceutical Technology, Encyclopedia of Food and Food Production, Lactic Acid Bacteria and Fermented Food, Research and Application of Microbial Agent Technology, etc.), a person skilled in the art can select and adjust the above-mentioned excipients, and prepare different dosage forms of the mucinophilic Akkermansia muciniphila Akk21 with the preservation number of CCTCC NO: M 20242779, such as solid beverage, liquid beverage, powder, tablet, oral liquid, gel, plaster, spray, lotion, granule, etc.

[0067] In specific embodiments, the dosage form of the product is selected from one or more than one of powder, tablet, liquid, capsule.

[0068] Group 4 embodiments, the auxiliary blood glucose-lowering functional food of the present application

[0069] The embodiments of the present application provide an auxiliary blood glucose-lowering functional food. All the embodiments of the present application have the following common features: the auxiliary blood glucose-lowering functional food comprises: active ingredients; the active ingredients comprise a mucinophilic Akkermansia muciniphila Akk21 with blood glucose-lowering ability with the preservation number of CCTCC NO: M 20242779.

[0070] In further embodiments, the auxiliary blood glucose-lowering functional food further comprises: an excipient.

[0071] In more specific embodiments, the excipient is selected from the group consisting of solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, tonicity adjusting agents, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adherents, integrating agents, penetration enhancers, pH adjusting agents, buffers, plasticizers, surfactants, antifoaming agents, defoaming agents, thickening agents, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retarders, and the like.

[0072] According to the content of the present application, in order to meet different needs in practical production application, in combination 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 Food, Research and Application of Microbial Agent Technology, etc.), a person skilled in the art can select and adjust the above-mentioned excipients, and prepare different dosage forms of the mucinophilic Akkermansia muciniphila Akk21 with the preservation number of CCTCC NO: M 20242779, such as liquid beverage, solid beverage, powder, tablet, oral liquid, gel, patch, spray, lotion, granule, etc.

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

[0074] Group 5 embodiment, auxiliary hypolipidemic functional food of the present application

[0075] The auxiliary hypolipidemic functional food of the present application comprises: active ingredients; the active ingredients comprise a mucinophilic Akkermansia muciniphila Akk21 with hypolipidemic ability with the preservation number of CCTCC NO: M 20242779.

[0076] In a further embodiment, the auxiliary hypolipidemic functional food further comprises: excipients.

[0077] In a more specific embodiment, the excipients are selected from the group consisting of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavorings, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickening agents, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculants, filtration aids, release retardants, etc.

[0078] According to the content of the present application, in order to meet different needs in practical production application, in combination 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 Food, Research and Application of Microbial Agent Technology, etc.), a person skilled in the art can select and adjust the above-mentioned excipients, and prepare different dosage forms of the mucinophilic Akkermansia muciniphila Akk21 with the preservation number of CCTCC NO: M 20242779, such as liquid beverage, solid beverage, powder, tablet, oral liquid, gel, patch, spray, lotion, granule, etc.

[0079] In specific embodiments, the dosage form of the auxiliary hypolipidemic functional food is selected from one or more of a powder, a tablet, a liquid, and a capsule.

[0080] Example 6, the intestinal flora function regulating food of the present application

[0081] The present example provides an intestinal flora function regulating food. All examples of the present example have the following common features: the intestinal flora function regulating food comprises: an active ingredient; the active ingredient comprises a mucinophilic Akkermansia sp. Akk21 having a hypolipidemic ability with a preservation number of CCTCC NO: M 20242779.

[0082] In further embodiments, the intestinal flora function regulating food further comprises: an excipient.

[0083] In more specific embodiments, the excipient is selected from a solvent, a propellant, a solubilizer, a co-solvent, an emulsifier, a colorant, a binder, a disintegrant, a filler, a lubricant, a wetting agent, an osmotic pressure regulator, a stabilizer, a glidant, a flavoring agent, a preservative, a suspending agent, a coating material, an aromatic agent, an anti-adhesion agent, an integrating agent, a penetration enhancer, a pH regulator, a buffer, a plasticizer, a surfactant, a foaming agent, an antifoaming agent, a thickening agent, a complexing agent, a humectant, an absorbent, a diluent, a flocculating agent, a deflocculating agent, a filter aid, a release retardant, and the like.

[0084] According to the content of the present application, in view of different needs in actual production applications, combined with conventional technical means in the field of pharmaceutical preparation (for example, Encyclopedia of Pharmaceutical 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 adjust the above-mentioned excipients, and prepare different dosage forms of the mucinophilic Akkermansia sp. Akk21 with a preservation number of CCTCC NO: M 20242779, such as liquid beverages, solid beverages, powders, tablets, oral liquids, gels, patches, sprays, lotions, granules, and the like.

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

[0086] Experimental Example 1, Isolation and Screening and Identification of Mucinophilic Akkermansia sp.

[0087] (1) Isolation and Screening of Mucinophilic Akkermansia sp.

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

[0089] The frozen sample was thawed and diluted 10 times, and inoculated into the BHI + 0.2% mucin screening medium containing antibiotics at an inoculation amount of 2%, and incubated at 37°C under anaerobic conditions for 2 days. The fecal sample after enrichment was preliminarily screened and identified using the Akk-specific primer pair described in the article "Qiye Wang, Zhaobin Wang, Bohao Shang, Yilong Li, Fang Zhou, Xianglin Zeng, Zhonghua Liu, Huansheng Yang, Mingzhi Zhu, Teapolyphenols improve lipid deposition via modulation of gut microbiota in rats and Ningxiang pigs, Journal of Functional Foods, Volume 113, 2024, 106049, ISSN 1756-4646", and whether the Akk strain was contained in the enriched sample was preliminarily determined according to the presence or absence of the target band. The enrichment tube with positive PCR identification was selected for solid plate isolation culture.

[0090] The enrichment tube with positive identification result was diluted and separated, and 10 -1 to 10 -5 gradient plate coating was performed on the solid screening medium containing antibiotics and BHI + 0.2% mucin, and incubated at 37°C under anaerobic conditions for 5-7 days. The single colony suspected to be Akk in the antibiotic plate was selected, numbered, identified again using Akk-specific primers, and the positive strain was preserved.

[0091] Through the method, 29 positive enrichment tubes were obtained after preliminary screening and identification of 100 fecal samples enriched by antibiotic medium using Akk-specific primers. After dilution and separation of the positive enrichment tube, plate coating was performed on the solid screening medium containing antibiotics and BHI + 0.2% mucin, and the single colony suspected to be Akk growing in the antibiotic plate was selected. After identification using PCR-specific primers, 27 Akk strains were obtained, and the strains were numbered and preserved. The effective rate of isolating Akk strains from positive enrichment tubes by the method reached 93%, which greatly improved the isolation efficiency of Akk strains compared with ordinary strain isolation methods.

[0092] (2) Akk21 strain identification and preservation

[0093] The preserved target strain was liquidly expanded, the bacterial cells were collected, genomic DNA was extracted, and PCR amplification reaction was performed using general primers 27F and 1492R as described in Chinese invention patent 202210478937.4. The PCR amplification procedure was as follows: 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 extension at 72°C for 10 min. Subsequently, the content and purity of the PCR amplification product were detected, and after passing the detection, it was sent to Wuhan Jin Kai Rui Biological Engineering Co., Ltd. for sequencing. According to the sequencing results, homology comparison was performed using the BLAST tool in the NCBI database, and the identification standard was 16S rRNA gene sequence homology ≥ 99%. One strain had good gastric juice, bile salt, lead, and arsenic tolerance and sugar-lowering capacity, and the strain was identified as Akkermansia muciniphila, which was named Akk21 and preserved in the China Center for Type Culture Collection, with the following preservation information:

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

[0095] Classification name: Akkermansia muciniphila Akk21 Akkermansia muciniphila Akk21;

[0096] Preservation unit: China Center for Type Culture Collection;

[0097] Address of the preservation unit: Wuhan, Wuhan University, China;

[0098] Preservation date: December 10, 2024.

[0099] Experimental example 2, gastric juice and bile salt tolerance experiment

[0100] Simulated artificial gastric juice: 0.5% sodium chloride solution was prepared, and the pH was adjusted to 3.0 with 1 mol / L HCl. 0.3% pepsin was added, dissolved thoroughly, and then filtered with a 0.22 μm microporous filter to remove bacteria for standby use.

[0101] Bile salt solution: 0.2% sodium mercaptoacetate and 0.3% pig bile salt were weighed and added to BHI liquid medium. After thoroughly dissolving, it was sterilized in a high-pressure sterilization pot at 115°C for 20 min for standby use.

[0102] Akkermansia muciniphila Akk21 was cultured overnight at 37°C in BHI + 0.2% mucin liquid medium. The activated Akk21 bacterial liquid was centrifuged, the supernatant was discarded, the bacterial cells were collected, and the bacterial liquid concentration was adjusted to 10 8CFU / mL. 10 mL of the bacterial suspension was centrifuged, and the bacterial cells were collected and mixed with 10 mL of the prepared pH 3.0 simulated artificial gastric juice and bile salt, respectively, and incubated at 37°C. Meanwhile, the viable cell count of the 0 h and 3 h suspensions was detected to calculate the survival rate.

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

[0104]

[0105] In the formula, N0 represents the viable cell count (CFU / mL) of the strain at 0 h, and N t represents the viable cell count (CFU / mL) of the strain at 3 h.

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

[0107] Strain Artificial gastric juice (%) Cholic acid solution (%) Akkermansia muciniphila Akk21 77.38±5.80 78.84±5.71

[0108] The experimental results show that the survival rate of Akkermansia muciniphila Akk21 is 77.38% after 3 h of gastric juice digestion, and the survival rate of Akkermansia muciniphila Akk21 is 78.84% after 3 h of bile salt digestion, indicating that Akkermansia muciniphila Akk21 has strong tolerance to gastric acid and bile salt.

[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 transfer, the culture was incubated for 8 h, and then transferred for 3 generations. The culture was fermented in BML medium, 100 ug / mL lead concentration BML liquid medium, and 100 ug / mL arsenic concentration BML liquid medium for 15 h. The OD 600 value was determined. The tolerance rate was calculated according to the following formula:

[0111]

[0112] In the above formula, A: BML culture OD value; B: 100 ug / mL lead / arsenic concentration BML culture OD value. 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 show that the tolerance rate of strain Akk21 to 100 ug / mL lead concentration is 48.3%, and the tolerance rate to 100 ug / mL arsenic concentration is 45.46%.

[0116] Experimental Example 4, Inhibition of α-glucosidase by Akkermansia muciniphila

[0117] Akkermansia muciniphila was inoculated into liquid BML medium and cultured at 37°C overnight, subcultured twice and then cultured for 8 h, followed by subculturing for 14 h. The fermentation broth was centrifuged, the supernatant was discarded, and the bacteria were resuspended in physiological saline to a concentration of 10 8 CFU / mL. 150 μL of PBS solution was added to 75 μL of p-nitrophenol-α-D-glucopyranoside (PNPG) solution and 25 μL of the sample to be tested. The mixture was placed on ice for 3 min, 50 μL of α-glucosidase solution (0.2 U / mL) was added, and the mixture was incubated at 37°C for 15 min. 1 mL of 1 mol / L Na2CO3 was added as a reaction termination solution. The absorbance of the reaction solution was measured at 405 nm:

[0118]

[0119] A: sample + α-glucosidase;

[0120] B: sample without α-glucosidase;

[0121] C: without sample + α-glucosidase;

[0122] D: without sample without α-glucosidase

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

[0124] Strain Alpha-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 slow the increase in blood sugar by inhibiting α-glucosidase in the small intestinal epithelium, thereby producing a hypoglycemic effect.

[0126] Experimental Example 5, Effect of Akkermansia muciniphila on the food intake, water intake, and body weight of mice with type II diabetes

[0127] Male C57BL / 6j mice (5-6 weeks; 19±1 g) were housed in the animal room of Hubei Center for Disease Control and Prevention, and the experimental animal use license number was SCXK(E)2020-0019. The animal experiments were maintained at 20-22℃, humidity 40-60%, and circulate 12h light / 12h dark. Mice can freely eat and drink 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 2 hepatic glucose and lipid metabolism and gut microbiota in mice, Food Funct, 2019, mice were made into type 2 diabetes model. Mice were divided into 4 groups (n=10): normal group (NC group), model group (MC group), probiotic group (Akk21, 6×10 9 CFU / d), drug treatment group (MET, 0.2 g / kg / d). The normal group and the model group were given sterile normal saline by gavage, the probiotic group was given Akkermansia muciniphila Akk21 by gavage, once a day, for 35 consecutive days, and the drug treatment group was given metformin hydrochloride (MET) by gavage, once a day, for 35 consecutive days. The daily food intake, daily water intake, and weekly body weight of 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 are as shown in Figure 1 Gavage of Akk21 for five weeks effectively improved the increase in food intake and water intake of type 2 diabetic mice, and the mice gradually approached the healthy group. As Figure 2 The change in body weight of mice showed that the treatment of strains and drugs had a significant recovery effect on the weight loss symptoms of mice, and the body weight of mice in the strain treatment group showed a stable and small increase trend after two weeks of gavage.

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

[0130] Before treatment and during the five-week treatment, tail vein blood was taken every week to determine the fasting blood glucose value of mice. The fasting blood glucose (FBG) measurement results are as shown in Figure 3The oral glucose tolerance test was performed after five weeks of treatment. The mice were orally administered glucose, and the blood glucose levels were measured at 0 min, 15 min, 30 min, 60 min, 90 min, and 120 min after the oral administration of glucose. The area under the curve (AUC) of the oral glucose tolerance test was calculated. The serum insulin concentration was measured at the end of the experiment.

[0131] The oral glucose tolerance test was performed after five weeks of treatment. The mice were orally administered glucose, and the blood glucose levels were measured at 0 min, 15 min, 30 min, 60 min, 90 min, and 120 min after the oral administration of glucose. The area under the curve (AUC) of the oral glucose tolerance test was calculated. The serum insulin concentration was measured at the end of the experiment.

[0132] The oral glucose tolerance test was performed after five weeks of treatment. The mice were orally administered glucose, and the blood glucose levels were measured at 0 min, 15 min, 30 min, 60 min, 90 min, and 120 min after the oral administration of glucose. The area under the curve (AUC) of the oral glucose tolerance test was calculated. The serum insulin concentration was measured at the end of the experiment. Figure 3 The oral glucose tolerance test was performed after five weeks of treatment. The mice were orally administered glucose, and the blood glucose levels were measured at 0 min, 15 min, 30 min, 60 min, 90 min, and 120 min after the oral administration of glucose. The area under the curve (AUC) of the oral glucose tolerance test was calculated. The serum insulin concentration was measured at the end of the experiment.

[0133] The oral glucose tolerance test was performed after five weeks of treatment. The mice were orally administered glucose, and the blood glucose levels were measured at 0 min, 15 min, 30 min, 60 min, 90 min, and 120 min after the oral administration of glucose. The area under the curve (AUC) of the oral glucose tolerance test was calculated. The serum insulin concentration was measured at the end of the experiment. Figure 4 The oral glucose tolerance test was performed after five weeks of treatment. The mice were orally administered glucose, and the blood glucose levels were measured at 0 min, 15 min, 30 min, 60 min, 90 min, and 120 min after the oral administration of glucose. The area under the curve (AUC) of the oral glucose tolerance test was calculated. The serum insulin concentration was measured at the end of the experiment. Figure 4 The oral glucose tolerance test was performed after five weeks of treatment. The mice were orally administered glucose, and the blood glucose levels were measured at 0 min, 15 min, 30 min, 60 min, 90 min, and 120 min after the oral administration of glucose. The area under the curve (AUC) of the oral glucose tolerance test was calculated. The serum insulin concentration was measured at the end of the experiment. Figure 5It can be concluded that the serum insulin concentration of the mice treated for five weeks was significantly reduced, and reached a significant difference level compared with the model group, showing that it approached the healthy group without significant difference. Figure 5 b shows that the insulin level of the strain treatment group and the drug treatment group was significantly improved and relieved.

[0134] The above results show that the treatment of Akk21 strain can improve the glucose metabolism of mice, reduce the blood glucose level of mice, and control the development of mice.

[0135] Experimental Example 7, Effect of Akkermansia muciniphila on blood lipid levels of type Ⅱ diabetic mice

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

[0137] Table 4. Detection results of total cholesterol, total triglyceride, 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 ± 0.05 bc 2.536±0.31a 2.50±0.25a 1.266±0.13b MET group 0.829 ± 0.14 ab 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 can reduce the blood lipid level of type Ⅱ diabetic mice, and the effect is particularly significant in reducing the total triglyceride and low-density lipoprotein levels. According to the data in the table, the serum total triglyceride concentration of the Akk21 strain treatment group was significantly reduced and approached the healthy group (NC group) without 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 Ⅱ diabetic mice, and has good effect.

[0141] Experimental Example 8, Effect of Akkermansia muciniphila on inflammatory factors of type Ⅱ diabetic mice

[0142] Diabetes mellitus can cause systemic inflammation in the body, and the occurrence of chronic inflammatory diseases can lead to an increase in serum tumor necrosis factor 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 serum of the mice was determined by ELISA kit for inflammatory factors, including the concentration levels of LPS (lipopolysaccharide), IL-6, TNF-α, and IL-10. Figure 6a-c showed that compared with the model group (MC), 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), and slowed down the occurrence of inflammation in the body. Serum insulin level is an important indicator for diagnosing type 2 diabetes and an important monitoring indicator during treatment. During the onset process, the insulin concentration in the serum of patients will show an increase, such as Figure 6 d showed that Akk21 could significantly increase the concentration of serum interleukin-10 and approach the level of the healthy group without significant difference (p>0.05). This indicates that Akk21 can effectively reduce the systemic inflammation of type 2 diabetic mice and improve the body's immunity.

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

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

[0145] Alpha-diversity reflects the species richness and species diversity of the sample. Chao1 index and Shannon index are commonly used indicators for judging the abundance and diversity of intestinal microbial flora. As shown in Figure 7 As shown in, the abundance and diversity of the intestinal flora of type 2 diabetic mice were significantly lower than those of the healthy group (p<0.05). According to the Chao1 index analysis, Akk21 strain treatment 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, Akk21 strain significantly improved the diversity of the intestinal flora of type 2 diabetic mice and approached the healthy group without significant difference (p>0.05). The results showed that Akk21 strain could improve and improve the abundance and diversity of the intestinal flora of type 2 diabetic mice.

[0146] Beta-diversity is used to compare the similarity of species diversity between different samples. NMDS method is a commonly used analysis method, which can better reflect the nonlinear structure of ecological data. The closer the distance on the coordinate graph, the higher the similarity. As shown in Figure 8As shown, the intestinal flora of the model group, drug treatment group, and Akk21 treatment group were significantly different, indicating that the continuous treatment of drugs and probiotics effectively improved the intestinal health, and gradually showed differences between the model group and gradually approached the healthy group. Strains effectively improve the intestinal flora of type Ⅱ diabetes mice and tend 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 OTUs in the mouse intestinal flora that overlap or are not the same, the more common the number is, the higher the similarity between the two. As shown in Figure 9 As shown, the number of common OTUs in the Akk21 group and the NC group is 217, the number of common OTUs in the MET group and the NC group is 172, and the number of common OTUs in the MC group and the NC group is 182. The similarity between the Akk21 group and the NC group is higher through the number of common OTUs, indicating that the treatment of Akk21 makes the mouse state tend to be normal.

[0148] Metformin is a first-line drug for treating type Ⅱ diabetes, and its mechanism of action is constantly being revealed. Many studies have shown that metformin can affect type Ⅱ diabetes mice by changing the intestinal flora. As shown in Figure 10 The present application mainly compares the common response strains of the metformin group and the probiotic group at the genus level, and analyzes the influence of different strain changes on the blood glucose lowering effect. According to Figure 11 According to the results analysis, Blautia, Bifidobacterium, Ligilactobacillus, Akkermansia, unclassified_Muribaculaceae, etc. were significantly increased in the Akk21 treatment group (p<0.01), and the abundance of the treatment group was significantly higher than that of the drug treatment group at the level of Akkermansia. Aerococcus, Allobaculum, etc. in the intestinal flora of harmful bacteria in the Akk21 treatment group and the drug treatment group showed a very significant decrease (p<0.01).

[0149] The above results show that type Ⅱ diabetes mice have intestinal flora disorder, and the treatment of Akkermansia muciniphila Akk21 can significantly reduce the number of harmful bacteria in the intestine by increasing the abundance of beneficial bacteria in the intestine, improve the species diversity and abundance of intestinal flora, and thus effectively control and improve the blood glucose level of type Ⅱ diabetes mice.

[0150] Experimental Example 10, Remedy of Akkermansia muciniphila to heavy metal lead poisoning mice

[0151] Thirty 6-8 weeks old C57BL / 6 male mice were randomly divided into 3 groups after one week of environmental adaptation, each group of 10 mice, experimental grouping NC group (10), Pb group (10), Akk21+Pb group (10). Keep continuous light and allow free feeding and drinking water. Pb group and Akk21+Pb group freely drink lead acetate solution with a concentration of 300 mg / L instead of drinking water for 30 days. After the end of the experiment, the mice were sacrificed by carbon dioxide assisted with cervical dislocation, and the mouse kidney tissues were collected for index detection. The lead content in the mouse kidney was determined.

[0152] Table 5. Lead content in mouse kidney

[0153] Group Kidney 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, the lead content in the kidney of Akk21 strain intervention group was significantly lower than that of the model group. It shows that Akk21 strain has a certain alleviating effect on lead poisoning.

Claims

1. A type of Akkermansia myxophilus with hypoglycemic ability ( Akkermansia muciniphila Akk21, characterized in that, The preservation number of which is CCTCC NO: M 20242779.

2. The use of Akkermansia muciniphila Akk21 in claim 1 in the preparation of a food for lowering blood sugar function, a food for lowering blood lipid function, a food for promoting lead excretion function or a food for regulating intestinal flora function.

3. A food for specified health use having a function of assisting in reduction of blood glucose, characterized by comprising the food additive according to claim 1 or 2. Comprise: Active ingredients; the active ingredients comprise Akkermansia muciniphila Akk21 in claim 1.

4. The food for specified health use according to claim 3, characterized in that, Also comprise: Adjuvants.

5. A food for specified health use having a function of assisting reduction of blood lipids, characterized by comprising the composition according to any one of claims 1 to 4. Comprise: Active ingredients; the active ingredients comprise Akkermansia muciniphila Akk21 in claim 1.

6. The food for specified health use according to claim 5, characterized in that, Also comprise: Adjuvants.

7. A lead-removal promoting functional food, characterized by comprising, Comprise: Active ingredients; the active ingredients comprise Akkermansia muciniphila Akk21 in claim 1.

8. The food for promoting lead excretion according to claim 7, wherein Also comprise: Adjuvants.

9. A food for regulating intestinal flora function, characterized by comprising the composition according to any one of claims 1 to 8. Comprise: Active ingredients; the active ingredients comprise Akkermansia muciniphila Akk21 in claim 1. Also comprise: Adjuvants.

Images