CN118178478A

CN118178478A - AKKERMANSIA MUCINIPHILA and application of its product in preventing and treating diabetes

Info

Publication number: CN118178478A
Application number: CN202410442191.0A
Authority: CN
Inventors: 马新; 喻扬; 郁雪平; 赵欣
Original assignee: Thankcome Biotechnology Suzhou Co ltd
Current assignee: Thankcome Biotechnology Suzhou Co ltd
Priority date: 2024-04-12
Filing date: 2024-04-12
Publication date: 2024-06-14

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to AKKERMANSIA MUCINIPHILA and application of a product thereof in diabetes prevention and treatment. AKKERMANSIA MUCINIPHILA is AKK ONE, and the preservation number is CGMCC No.20954. The AKK ONE provided by the invention can effectively improve the blood sugar tolerance, relieve liver injury caused by diabetes, provide technical support for preventing or treating diabetes and improving lipid metabolism, and enlarge strain seed resources.

Description

AKKERMANSIA MUCINIPHILA and application of its product in preventing and treating diabetes

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to AKKERMANSIA MUCINIPHILA and application of a product thereof in diabetes prevention and treatment.

Background

Diabetes is a group of metabolic diseases characterized by chronic hyperglycemia due to multiple causes, due to defects in insulin secretion and/or utilization. Long-term hyperglycemic levels can cause multiple systemic lesions, leading to chronic progressive lesions, hypofunction, and failure in tissues and organs such as the eye, kidneys, nerves, heart, blood vessels, etc. Acute serious metabolic disorders such as diabetic ketoacidosis, hyperglycemic syndrome, etc. may be caused when the condition is serious or stressed.

Diabetes belongs to metabolic diseases, and is mainly characterized by excessive blood sugar level, and hyperglycemia can cause diabetic patients to have symptoms of polydipsia, polyphagia, diuresis and weight loss. Lipid metabolism is also an important aspect of human metabolism, and is mainly manifested by abnormal metabolism of cholesterol, triglycerides, and low-density lipoproteins. Cholesterol metabolic disorder is a major cause of diabetes, and hypercholesterolemia is a characteristic lesion of diabetes, and is mainly caused by excessive fat intake of patients, which leads to fatty liver, and excessive fat accumulation in the liver may prevent liver clearance, thereby causing elevated cholesterol levels. Triglyceride levels are also one of the etiologies of diabetes, and severe triglyceride can cause arteriosclerosis, increasing the risk of developing cardiovascular and cerebrovascular diseases. Low density lipoproteins can promote cholesterol synthesis in vivo, and cholesterol can be oxidized to low density lipoproteins in vivo. Therefore, the level of the low density lipoprotein is closely related to the onset of diabetes and is also a risk factor of cardiovascular and cerebrovascular diseases of diabetics. In summary, diabetes can lead to a number of metabolic abnormalities in the body, and lipid metabolism is one of these. Therefore, for diabetics, it is recommended to keep high dietary fiber diet, reduce fat intake, and help control blood sugar and blood lipid levels of diabetics.

The current drugs for treating diabetes are mainly western medicine preparations, including insulin preparations, sulfonylurea preparations, biguanide preparations, insulin resistance improvers, alpha-glucosidase inhibitors and the like. The medicines act on a certain link of blood sugar regulation and control to play a role in controlling blood sugar, and have the advantages of quick response, large side effect, single curative effect and easy resistance generation.

Chronic inflammation caused by endotoxin bleeding due to dysbacteriosis is one of important factors for development of metabolic diseases such as obesity and diabetes. Dysbacteriosis and unbalance can cause systemic chronic reaction inflammation, thereby causing islet beta cell injury and insulin resistance reduction, and simultaneously affecting the absorption of other cells in the body to sugar so as to convert energy, so that the human body is difficult to normally operate, and finally a series of complications are caused. Research shows that the probiotics can effectively inhibit the pre-diabetes from developing into type 2 diabetes, and the probiotics are helpful for reducing blood sugar concentration of human bodies, so that the symptoms of the pre-diabetes are inhibited.

Patent CN113330109a relates to the strain of akkermansia muciniphila (AKKERMANSIA MUCINIPHILA) SNUG 61027 (accession No. KCTC13530 BP) and its use. In particular, there is provided a composition for appetite control or prevention, improvement, alleviation or treatment of metabolic disorders, which comprises as an active ingredient B2UM07 protein, or a culture broth or the like thereof, or isolated therefrom, for appetite control or prevention, improvement, alleviation or treatment of metabolic disorders, which are impaired glucose tolerance, diabetes, arteriosclerosis, hyperlipidemia, hypercholesterolemia, fatty liver, cardiovascular disease or obesity, and a method of appetite control or prevention, improvement, alleviation or treatment of metabolic disorders using the composition.

Therefore, there is a great need to develop more safe and effective probiotics to meet the huge clinical demands.

The present invention is directed to autonomously developed microbial resources: AKKERMANSIA MUCINIPHILA (other patents have been submitted on the same day) with the preservation number of CGMCC No.20954, and the downstream application technology is developed.

After AKKERMANSIA MUCINIPHILA was obtained, the inventors performed various fields of application experiments including improvement of diabetes, anti-infection, improvement of respiratory diseases, regulation of lipid metabolism, improvement of enteritis, and the like, and obtained forward experimental results in some fields.

In view of the relevant regulations of patent law singleness, each indication is individually claimed where singleness is not present.

The present invention is directed to the protection of application techniques in improving diabetes.

Since the related evidences of microorganism identification, performance test and the like are published in the microorganism patents submitted on the same day, the early work of the inventor is quickly known, and the related information of AKKERMANSIA MUCINIPHILA is briefly disclosed in the present invention.

The conclusive information in this section is for facilitating the examination of the present invention and is not intended to limit the scope of the invention. The specific experimental process and experimental results are recorded in the microbial patents submitted on the same day as the invention.

1. AKKERMANSIA MUCINIPHILA is obtained by the separation and screening of the inventor.

2. AKKERMANSIA MUCINIPHILA preservation information: the preservation number is: CGMCC No.20954.

3. AKKERMANSIA MUCINIPHILA physiological and biochemical properties:

(1) Hydrophobicity: the hydrophobicity of the strain increased with time to 30% or more at 60 min.

(2) Self-aggregation: the self-aggregation of the strain increased with time, and was stable at 20 hours, and maintained at about 50%.

(3) Gastrointestinal fluid tolerance:

the survival rate of the strain in gastric juice is in a decreasing trend along with the time extension, and the survival rate is about 80% in 240 min;

The survival rate of the strain in intestinal juice is in a decreasing trend as a whole, and the survival rate is more than 80% at 240 min.

(4) Biofilm formation ability: weak nature.

(5) Bacterial back mutation assay: the bacterial back mutation test result shows that the strain is free from mutagenicity.

(6) Acute toxicity:

Mice did not die through acute gavage of AKK ONE at high, medium, and low doses. Daily weight change of mice in the test group and control physiological saline group;

During acute gastric lavage, there was no significant difference between daily changes in food intake of the mice tested and the mice in the control saline group;

During acute gastric lavage, there was no significant difference between daily blood glucose changes in the mice tested and the control saline group mice;

the blood indexes of the mice in the acute gastric lavage group and the control physiological saline group have no obvious difference;

The triglyceride levels of the mice in the test group and the mice in the control group have no significant difference in serum and liver;

The liver cholesterol levels of the mice with the high stomach-filling dose are extremely obviously different from those of the mice with the control group, and the other groups have no obvious difference in serum and liver;

the bile acid levels of the mice in the tested group and the mice in the control group are not significantly different in serum and liver;

the glucose levels of the mice in the tested group and the mice in the control group are not significantly different in serum and liver;

the total protein levels of the mice in the tested group and the mice in the control group are not significantly different in serum and liver;

the glutamic pyruvic transaminase enzyme activities of the tested mice and the mice of the control group are not significantly different in serum and liver;

the glutamic-oxaloacetic transaminase enzyme activities of the mice in the tested group and the mice in the control group have no significant difference in serum and liver;

The creatinine content of the mice in the test group and the creatinine content of the mice in the control group are not significantly different in serum;

the urea nitrogen concentration of the mice in the test group and the mice in the control group are not significantly different in serum;

The weight of main organs such as heart, liver, spleen, kidney, thymus, brain, testis, lung, stomach, intestine and the like of the tested mice and the mice of the control group are basically not obviously different;

the liver and kidney of the mice in the tested group and the mice in the control group have no obvious pathological damage.

(7) Sub-chronic toxicity test:

mice were perfused with high, medium, and low doses of AKK ONE over 90 consecutive days without mortality. No significant difference was observed between weekly weight changes in mice in the test group and the control saline group;

during the continuous gastric lavage period, no significant difference exists between the feed intake change of the tested mice and the mice in the control physiological saline group every week;

During continuous lavage, there was no significant difference between the blood glucose change in weekly medium dose lavage mice and control saline mice;

The blood indexes of the mice in the gastric lavage group and the control physiological saline group have no obvious difference;

The triglyceride levels of the mice in the test group and the mice in the control group have no significant difference in liver and serum;

The cholesterol levels of the mice in the test group and the mice in the control group have no significant difference in liver and serum;

The bile acid levels of the mice in the intragastric dosage and the mice in the control group have significant differences in the liver, while the other groups have no significant differences in serum and liver from the control group;

The glucose levels of the mice in the test group and the mice in the control group are not significantly different in liver and serum;

The total protein content of the mice in the tested group and the mice in the control group has no significant difference in liver and serum;

The serum of the mice with the dose in the stomach and the mice with the control group have obvious difference in glutamate pyruvate transaminase activity, and the other groups have no obvious difference in serum and liver;

the weight of main organs such as heart, liver, spleen, kidney, thymus, brain, testis, lung, pancreas, stomach and intestine of the tested mice and the mice of the control group are basically not significantly different;

the liver and the kidney of the mice in the tested group and the mice in the control group have no obvious pathological damage;

The blood sugar regulating ability of the tested mice and the mice of the control group is not significantly different.

Disclosure of Invention

In order to solve the problems, the invention provides a new AKKERMANSIA MUCINIPHILA which is named AKK ONE and has the preservation number of: CGMCC No.20954. The strain can be effectively used for preventing and treating diabetes and improving metabolic abnormality.

In one aspect, the invention provides the use of AKKERMANSIA MUCINIPHILA in the manufacture of a product for the prevention and/or treatment of diabetes; the preservation number of AKKERMANSIA MUCINIPHILA is CGMCC No.20954.

In particular, the product comprises AKKERMANSIA MUCINIPHILA fermentation broth, fermentation broth supernatant, fermentation broth precipitate, live bacteria, dead bacteria, lyophilized powder and/or cell lysate.

Further specifically, the fermentation broth refers to a liquid obtained by inoculating a strain into a culture medium and culturing for a period of time.

Further specifically, the fermentation broth supernatant refers to supernatant liquid of the fermentation broth after centrifugation; contains rich metabolites and a part of thallus fragments in the growth and propagation process of bacteria, and acid substances secreted by the bacteria and bacteriocins have antagonism and killing effects on harmful bacteria; amino acids after decomposing food by bacteria, and synthetic vitamins are in the culture solution, and also include enzymes secreted by bacteria useful for human body; and part of thallus components have immunity promoting effect on human body.

Further specifically, the fermentation liquid sediment refers to liquid sediment obtained by centrifugation, and the liquid sediment comprises free protein, residual thalli, broken cells and residues of culture matrixes, namely the protein and the matrixes in the cells.

Further specifically, the living bacteria are also called active bacteria group, can colonize and reproduce in intestinal tracts, and are beneficial to increasing the number of beneficial bacteria.

Further specifically, the dead bacteria are microorganisms which have lost viability, cannot grow and reproduce, and the probiotics are lost viability due to the production process.

Still more particularly, the methods of inactivation include, but are not limited to: any one or more of high temperature inactivation, radiation inactivation, or chemical inactivation;

The high-temperature inactivation can be dry heat inactivation or wet heat inactivation; the radiation inactivation may be optical radiation, which may be ultraviolet or infrared radiation, or ionizing radiation, which includes, but is not limited to, microwave radiation; the chemical inactivation includes, but is not limited to, gas sterilization using gaseous sterilizing agents such as ozone, formaldehyde, or liquid sterilization using liquid sterilizing agents.

More specifically, the freeze-dried powder is obtained by freeze-drying the culture solution. The freeze-dried powder also comprises a freeze-drying protective agent. Such lyoprotectants include, but are not limited to: pH buffers, fillers, sugars, nonionic surfactants, ligands, and the like. The pH buffer includes, but is not limited to, any one or more of Tris, amino acids or salts thereof, citric acid or salts thereof, acetic acid or salts thereof. The bulking agent includes, but is not limited to, any one or more of mannitol, glycine, bovine serum albumin. The saccharide may be a disaccharide such as any one or more of sucrose or trehalose. The nonionic surfactant includes, but is not limited to, tween, which may be tween-20, tween-60, tween-80, etc. The freeze-drying protective agent can also comprise an antioxidant and the like. The freeze-drying protective agent can also comprise albumin, polyethylene glycol and the like.

More specifically, the cell lysate may be obtained by lysing cells obtained by culturing the above culture. The cleavage may be physical or chemical. Such physical cleavage includes, but is not limited to: grinding, ultrasonic crushing, and the like. The chemical cleavage includes, but is not limited to, chemical reagent cleavage, enzymolysis, which may be hydrolase or oxidase. The lysis may also be the self-rupture of cells by increasing the intracellular pressure.

Specifically, the product is a drug, a food, a health product, a medical raw material, an industrial raw material, a food raw material or a health product raw material.

Further specifically, the product is a medicament, and the medicament also comprises pharmaceutically acceptable auxiliary materials; the pharmaceutically acceptable excipients include, but are not limited to: any one or more of excipients, stabilizers, diluents, binders, preservatives, lubricants, antioxidants.

Preferably, the pharmaceutically acceptable auxiliary material may be at least one selected from lactose, mannose, starch, acacia, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

In yet another aspect, the invention provides the use of AKKERMANSIA MUCINIPHILA in the manufacture of a product for ameliorating abnormal lipid metabolism.

Specifically, the preservation number of AKKERMANSIA MUCINIPHILA is CGMCC No.20954.

Further specifically, the product comprises AKKERMANSIA MUCINIPHILA fermentation broth, fermentation broth supernatant, fermentation broth precipitate, live bacteria, dead bacteria, lyophilized powder, and/or cell lysate.

Further specifically, the product is a medicament, and the medicament also comprises pharmaceutically acceptable auxiliary materials.

Further specifically, the medicine also comprises pharmaceutically acceptable auxiliary materials; the pharmaceutically acceptable excipients include, but are not limited to: any one or more of excipients, stabilizers, diluents, binders, preservatives, lubricants, antioxidants.

In yet another aspect, the present invention provides a method of preparing a medicament for preventing and/or treating diabetes.

Specifically, the method comprises culturing AKKERMANSIA MUCINIPHILA; the preservation number of AKKERMANSIA MUCINIPHILA is CGMCC No.20954.

Specifically, the medicine also comprises pharmaceutically acceptable auxiliary materials.

In yet another aspect, the present invention provides a method of preparing a medicament for ameliorating abnormal lipid metabolism.

The invention has the technical effects that:

(1) AKK ONE has the ability to improve blood glucose and glucose tolerance in T2DM mice.

(2) AKK ONE can effectively reduce the serum insulin content of T2DM mice.

(3) AKK ONE to alleviate HFD/STZ induced liver injury in mice.

(4) AKK ONE can improve the phenomenon of lipid increase in diabetic mice.

Preservation description:

strain name: AKK ONE;

Preservation number: CGMCC No.20954;

Classification naming: AKKERMANSIA MUCINIPHILA;

preservation date: 10 months and 26 days 2020;

preservation unit: china general microbiological culture Collection center (China Committee for culture Collection);

deposit unit address: the korean district North Star, beijing city, part No. 1, no. 3.

Drawings

FIG. 1 is a graph showing the change in blood glucose in the OGTT of mice.

FIG. 2 shows the effect of AKK ONE on insulin in T2DM mice.

FIG. 3 shows the effect of AKK ONE on serum AST and ALT of T2DM mice, wherein A is the AST content in the serum; b is ALT content in serum.

Fig. 4 is a histopathological section of mouse liver.

Fig. 5 is a histopathological section of the mouse pancreas.

FIG. 6 shows the expression level of mouse liver tissue gene.

Nouns and terminology:

Glutamate-aspartate aminotransferase (glutamic oxaloacetic transaminase, AST) is mainly distributed in cardiac muscle, and secondly in liver, kidney and skeletal muscle. The normal serum has very low glutamic-oxaloacetic transaminase content, but if the corresponding cells are lost, the cell membrane permeability is increased, and intracellular glutamic-oxaloacetic transaminase can be released into blood to cause the increase of the intracellular glutamic-oxaloacetic transaminase. Generally, the method is used for assisting in examining organ damage.

Glutamic-pyruvic transaminase (glutamic-pyruvic transaminase, ALT) is a transferase for representing liver function of a patient, and is alanine aminotransferase, and when the glutamic-pyruvic transaminase is increased, the damage of liver cells of the patient is prompted, and the liver function of the patient is prompted to be problematic.

Serum insulin (Seruminsulin, INS) is a protein hormone. Insulin is secreted in vivo by islet beta cells. Serum insulin is the only hormone in the body that reduces blood sugar, and is also the only hormone that promotes glycogen, fat, and protein synthesis at the same time.

Triglycoide (TG), also known as neutral fat, is a lipid formed by the reaction of glycerol molecules with fatty acids, and is one of the components of blood lipids, with the function of providing energy for cellular metabolism. Triglyceride measurement is an important item in blood lipid tests and is the sum of the triglycerides contained in the individual lipoproteins in plasma.

Total cholesterol (Total Cholesterol, TC) is the total sum of cholesterol contained in various lipoproteins in blood, and cholesterol is a main component of cell membranes and is also an important raw material for synthesizing physiologically active substances such as adrenocortical hormone, sex hormone, bile acid and vitamin D. The concentration of total cholesterol in human blood can be used as an index of lipid metabolism for evaluating the incidence risk of atherosclerosis and ischemic cardiovascular and cerebrovascular diseases.

High density lipoprotein cholesterol (HIGH DENSITY lipoprotein cholesterol, HDL-C) is an anti-atherosclerosis lipoprotein, which can transport cholesterol from extrahepatic tissues to the liver for metabolism, and is discharged from the body through bile, and the plasma content is inversely related to the risk of metabolic diseases.

Low density lipoprotein cholesterol (Low density lipoprotein cholesterol, LDL-C) is the major lipoprotein in fasting plasma, accounting for about 2/3 of plasma lipoproteins, and is the primary vehicle for transporting cholesterol to extrahepatic tissues. Its concentration is clearly positively correlated with the incidence of metabolic diseases.

PPAR-alpha is expressed in tissues with vigorous fatty acid metabolism, such as liver, heart, skeletal muscle and kidney, and is the most expressed PPAR subtype in human endothelial cells, which is involved in blood glucose and blood lipid metabolism, maintains energy metabolism balance and regulates inflammatory response.

CPT-1 is the first member of the CPT gene family and plays a key role in lipid metabolism. CPT-1 is located mainly in liver and muscle cells, and the liver controls how fat in food becomes energy.

C/EBP-alpha is a DNA binding protein with leucine zipper structure, and is used as eukaryotic cell transcription regulating factor to participate in the transcription regulation of liver cell, fat cell and other cell rich in the protein.

The SCD-1 gene encodes stearoyl-CoA desaturase, which converts saturated fatty acids to monounsaturated fatty acids.

HSL is mainly present in adipose tissue, adrenal gland, macrophage, placenta and ovary cells, the content of cardiac muscle and skeletal muscle is small, and the expression quantity of HSL gene has obvious inhibiting effect on TG deposition in adipose cells.

LPL is present in large amounts in the capillary endothelium of cardiac muscle, kidney, brain, skeletal muscle, adipose tissue, etc., with the highest content in adrenal glands and adipose tissue. LPL hydrolyzes TG carried and transported by VLDL, and loss and low activity of LPL can cause serious lipodystrophy in the body.

The SREBPs family regulates fat production, SREBP-1 mainly regulates the expression of FA synthesis genes and LDLR, while SREBP-2 preferentially controls the expression of cholesterol biosynthesis genes.

Expression regulation of the HMGCR gene can affect the accumulation of cholesterol in the body.

Detailed Description

The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

Example 1

The invention provides AKKERMANSIA MUCINIPHILA which is named AKK ONE and has the preservation number of CGMCC No.20954.

1 Experimental method

1.1 Preparation of the Experimental Strain heavy suspension

Inoculating the frozen strain into BHI liquid culture medium, culturing at 37deg.C for 15 hr, centrifuging at 4deg.C for 5min at 5000r/min, discarding supernatant to obtain bacterial mud, inoculating bacterial mud into BHI liquid culture medium according to 3% inoculum size, and activating at 37deg.C for 15 hr. And (3) a small amount of bacterial liquid is dipped by an inoculating loop, three-area lineation is carried out on the BHI solid culture medium, the culture is carried out for 48 hours at 37 ℃, single bacterial colony is selected and cultured in the BHI solid culture medium for 15 hours, and the bacterial suspension is obtained after continuous three generations of activation. The bacterial suspension was diluted and plated for counting, and the bacterial suspension concentration was adjusted to 10 ⁹ CFU/mL with sterilized PBS broth.

BHI medium: BHI (brain heart extract (Brain Heart Infusion), OXOID, cat No. CM 1032B)) +5% sheep blood.

Culture conditions: 8 layers of gauze are completely cultured anaerobically.

1.2 Grouping and administration of laboratory animals

40 Male C57BL/6J 6 week old mice (average body weight 21.1.+ -. 0.3 g) were purchased from Chongqing university animal experiment center. Mice were randomized into normal, model, metformin, AKK ONE groups (n=8) after 1 week of adaptive feeding. Normal mice were fed standard feed (Jiangsu province cooperative medical bioengineering Limited company, cat No. 1010001), model and experimental mice were fed 60% high fat feed (Jiangsu province cooperative medical bioengineering Limited company, cat No. XTHF), during which time all mice were free to drink water and the experiment was continued for 4 weeks. Beginning on week 5, 50 mg/kg.bw of STZ (Sigma-Aldrich, cat# V900890-1G) was dissolved in sodium citrate buffer, and mice from model and experimental groups were given continuous 5 days of intraperitoneal injection, while normal groups served as controls, with sodium citrate buffer only. Mice were assayed for fasting glycemia (FBG) (8 hours on a empty stomach) after 1 week, and if more than 11.1mmol/L, the mice were considered type 2 diabetes (T2 DM) mice. Subsequently, ONE group of mice in the experimental group was filled with fresh AKK ONE bacterial liquid (1X 10 ¹⁰ CFU/kg. Bw) daily, ONE group was filled with metformin (100 mg/kg. Bw; shandong Sibangde pharmaceutical Co., ltd., product number: H20060230) daily, and the other mice were filled with 10 mL/kg. Bw physiological saline daily, once daily for 4 weeks. During the experiment, the body weight of the mice was measured weekly. The food and water intake of the mice was recorded every 2d during the treatment period. On week 10, mice were fasted overnight and tested for FBG and Oral Glucose Tolerance Test (OGTT). At the end of the 10 th weekend experiment, the mice were fasted for 12h, blood samples were collected, centrifuged at 3000r/min for 10min at 4℃to separate serum, and transferred to-80℃for later use. Tissue samples (including liver, spleen, kidney) were rapidly processed, weighed, and parts of the tissue were fixed in 4% paraformaldehyde for 48h, and the remaining tissue was frozen with liquid nitrogen and then transferred to-80 ℃ for storage.

1.3 Oral Glucose Tolerance Test (OGTT)

Mice were OGTT, fasted for 12h, and were given free water at week 0. The body weight of the mice was measured before the experiment, blood glucose was measured (recorded as 0min blood glucose value), and then the stomach was irrigated with a 2 g/kg.bw glucose solution, and blood glucose levels were measured 1 time at 0min,30min,60min and 120min, and a blood glucose curve was drawn.

1.4 Determination of serum indicators

After the collected blood sample was placed in a refrigerator to prevent 30min from waiting for blood coagulation, it was centrifuged in a low temperature centrifuge at 4 ℃ under centrifugation conditions: 3000r/min, 10min, taking upper serum, and measuring the contents of AST (product number: C010-2-1), ALT (product number: C009-2-1), INS (product number: E-EL-M1382C), TC (product number: A111-1-1), TG (product number: A110-1-1), LDL-C (product number: A113-1-1) and HDL-C (product number: A113-1-1) in the mouse serum by adopting a Nanjing building blood lipid measuring kit method.

1.5 Liver and pancreas tissue HE pathology analysis

The liver and pancreas tissues of the mice were taken to a size of about 1cm ² and immersed in the tissue fixative for 24h for fixation. Then cleaning, waxing and slicing, finally staining by HE, and observing the morphological structure by a biological microscope.

1.6 Real-time fluorescent quantitative PCR

The liver tissue of mice in the experimental group is taken, genes are extracted, and the expression levels of PPAR-alpha, CPT-1, C/EBP-alpha, SCD-1, HSL, LPL, SREBP-1, SREBP-2 and HMGCR are measured. Total RNA extraction: the liver tissue of each group of mice was subjected to extraction of total RNA of cells according to the instructions of TRLzol kit (Invitrogen-cos, cat# 15596026), and the RNA quality was detected by agarose gel electrophoresis, and the concentration was measured by an enzyme-labeled instrument. Reverse transcription: the first strand of cDNA was synthesized according to the reverse transcription kit instructions. RT-PCR: and (3) carrying out RT-PCR amplification on the inverted cDNA by adopting a SYBR-Green quantitative PCR method. The amplification conditions were 95℃for 60s; continuing at 95 ℃ for 15s, and carrying out 40 cycles; the temperature is 55 ℃ for 30s; the temperature is 72 ℃ for 35s; the temperature is 95 ℃ for 30s; the temperature was 55℃for 35s. The experiment uses beta-actin gene as reference gene and adopts 2 ^-ΔΔCt relative quantitative method to calculate the expression condition of each group of genes. The primer sequences are shown in Table 1.

TABLE 1 primer sequences

1.7 Data analysis

Experimental data are expressed as mean ± Standard Deviation (SD). Statistical analysis and mapping was performed using GRAPH PAD PRISM 8.0.0 software.

2 Experimental results

2.1 Effect of AKK ONE on oral glucose tolerance in T2DM mice

The glucose change curve in the oral glucose tolerance (OGTT) test of mice is shown in fig. 1. After 30 minutes of glucose oral administration, the blood glucose levels reached the highest value in each group of mice. Subsequently, the blood glucose levels of the mice in each group showed a different degree of decrease trend over the period of 30 to 120 minutes. Wherein, compared with the normal group, the blood glucose level of the mice in the model group is rapidly increased after 30 minutes of glucose administration, and the higher blood glucose level is maintained within 30-120 minutes. After 120 minutes of glucose oral administration, the blood glucose level of the mice in the model group failed to return to the blood glucose level before glucose oral administration, suggesting that glucose tolerance of the diabetic mice was impaired. The blood glucose levels were significantly lower in the mice of the metformin and AKK ONE groups than in the model group within 30 minutes after glucose administration, and in particular slightly lower in the AKK ONE group at 120 minutes. The results show that AKK ONE has the ability to improve T2DM blood glucose and glucose tolerance.

2.2 Effect of AKK ONE on T2DM mouse insulin

As the results in fig. 2 show, the serum insulin levels of the model mice were significantly increased over the normal mice, indicating that islet cell dysfunction in diabetic mice resulted in hypersecretion of insulin. Serum insulin levels decreased after 4 weeks of probiotic AKK ONE and metformin dry prognosis.

2.3 Effect of AKK ONE on serum AST and ALT of T2DM mice

ALT and AST can be used as key markers when the liver is damaged. As shown in the results of fig. 3, the serum levels of ALT and AST were significantly increased in the mice of the model group compared to the normal group, indicating that long-term T2DM damage to the liver tissue of the mice was caused. While the AKK ONE and metformin prognosis levels were significantly reduced, but there was no significant difference between the groups, indicating that the strain can alleviate HFD/STZ-induced liver injury in mice.

2.4 Effect of AKK ONE on blood lipid in T2DM mice

The effect of AKK ONE on the lipid index of mice is shown in Table 2. The mice in the model group fed the high-fat feed had significantly higher TG, TC and LDL-C levels than the normal group, and significantly lower HDL-C levels than the normal group, indicating that diabetes causes lipid level disorders in the serum. The increase of blood lipid induced by high fat diet is significantly inhibited after gavage of AKK ONE and metformin. After the stomach is irrigated with AKK ONE and metformin, the contents of TG, LDL-C and TC in the serum of the diabetic mice are obviously reduced, which indicates that AKK ONE and metformin can improve the phenomenon of lipid increase of the diabetic mice.

TABLE 2 TG, TC, HDL-C and LDL-C levels in mouse serum

2.5AKK ONE histopathological analysis of mouse liver and pancreas

As shown in FIG. 4, the liver tissue of the normal group mice has no steatosis, no fat infiltration, abundant cytoplasm of liver cells, clear cell membranes, orderly arrangement, large and normal cell nuclei in the center and clear tissue structure. The liver cells of mice in the model group induced by high fat are swollen, obvious steatosis is visible, and most liver cells contain lipid droplets. Fat drops in hepatic cells are improved to different degrees after AKK ONE and metformin are dried, wherein the quantity and the density of fat drops in AKK ONE groups are smaller, and the fat degeneration in the liver is obviously improved. This suggests that AKK ONE improves liver lipid accumulation and prevents fat absorption in vivo.

As shown in fig. 5. The normal group mice have complete islet structure, full shape, abundant islet beta cells and even distribution. The mice in the model group are saturated in fat drops in pancreas, the islet volume is obviously reduced, and the number of islet beta cells is obviously reduced compared with that in the normal group. Compared with the model group, after the intervention of metformin and AKK ONE for 4 weeks, the islet atrophy of the diabetic mice can be obviously relieved, and the phenomenon of massive apoptosis of islet beta cells can be reduced. The above results indicate that metformin and AKK ONE can effectively improve pancreatic tissue damage caused by HFD and STZ, protecting islet morphology in diabetic mice.

2.6 Effect of AKK ONE on expression of T2DM mouse liver lipid metabolism Gene

As shown in FIG. 6, the expression intensities of C/EBP alpha, SCD-1, LPL, SREBP1 and HMGCR MRNA were the weakest in liver tissues of normal mice, and PPAR-alpha, CPT-1, HSL and SREBP2 were the strongest; whereas PPAR-alpha, CPT-1, HSL and SREBP2 were expressed the weakest in the diabetic model group mice and C/EBP alpha, SCD-1, LPL, SREBP1 and HMGCR were expressed the strongest. AKK ONE and metformin can down-regulate C/ebpα, SCD-1, lpl, SREBP1 and HMGCR expression and up-regulate PPAR- α, CPT-1, hsl and SREBP2 expression in liver tissue of diabetic mice. In conclusion, the probiotic AKK ONE can regulate the expression of lipid metabolism genes related to liver tissues of the diabetic mice.

Conclusion 3

The study establishes a T2DM mouse model through HFD and STZ induction, evaluates the blood glucose reducing effect of the probiotic AKK ONE by measuring indexes such as the OGTT and insulin of the mouse, and evaluates the effect of the probiotic AKK ONE on the viscera of the T2DM mouse by measuring the expression conditions of liver blood lipid level, AST and ALT level and liver lipid metabolism genes. AKK ONE significantly improved blood glucose levels in T2DM mice. Compared with the group D, after AKK ONE is interfered for 4 weeks, the oral glucose tolerance of the T2DM mice is gently hydrolyzed, the insulin level is improved, and the blood lipid level is also improved, which indicates that AKK ONE has the effects of obviously reducing blood sugar and regulating lipid metabolism.

Comparative example

With reference to the experimental procedure of example 1, a comparative example was set. The strain AKK ONE in example 1 was replaced with AKK PROBIO (preservation number CGMCC No. 20955). The relative expression level of the mouse liver lipid metabolism gene PPAR-alpha after AKK PROBIO days is detected, and the detection result is shown in Table 3.

TABLE 3 Table 3

	Differences from example 1	Relative expression level of mRNA of PPAR-alpha
			Comparative example	Replacement of AKK ONE administration with equivalent AKK PROBIO	1.52±0.21

The above results demonstrate that AKK ONE provided by the invention is significantly better than AKK PROBIO in up-regulating PPAR-alpha.

Claims

The application of 1.Akkermansia muciniphila in preparing the product for preventing and/or treating diabetes is characterized in that the preservation number of AKKERMANSIA MUCINIPHILA is CGMCC No.20954.
2. The use according to claim 1, wherein the product comprises AKKERMANSIA MUCINIPHILA fermentation broth, fermentation broth supernatant, fermentation broth pellet, live bacteria, dead bacteria, lyophilized powder and/or cell lysate.
3. The use according to claim 1 or 2, wherein the product is a medicament, a food, a health product, a pharmaceutical raw material, an industrial raw material, a food raw material or a health product raw material.
4. The use according to claim 3, wherein the product is a medicament, and wherein the medicament further comprises pharmaceutically acceptable excipients.
The application of 5.Akkermansia muciniphila in preparing the product for improving abnormal lipid metabolism is characterized in that the preservation number of AKKERMANSIA MUCINIPHILA is CGMCC No.20954.
6. The use according to claim 5, wherein the product comprises AKKERMANSIA MUCINIPHILA fermentation broth, fermentation broth supernatant, fermentation broth sediment, live bacteria, dead bacteria, lyophilized powder and/or cell lysate.
7. A medicament for preventing and/or treating diabetes mellitus, which comprises AKKERMANSIA MUCINIPHILA; the preservation number of AKKERMANSIA MUCINIPHILA is CGMCC No.20954.
8. A medicament for ameliorating abnormal lipid metabolism, said medicament comprising AKKERMANSIA MUCINIPHILA; the preservation number of AKKERMANSIA MUCINIPHILA is: CGMCC No.20954.
9. A method for preparing a medicament for preventing and/or treating diabetes mellitus, which comprises culturing AKKERMANSIA MUCINIPHILA; the preservation number of AKKERMANSIA MUCINIPHILA is CGMCC No.20954.
10. A method for preparing a medicament for ameliorating abnormal lipid metabolism, said method comprising culturing AKKERMANSIA MUCINIPHILA; the preservation number of AKKERMANSIA MUCINIPHILA is CGMCC No.20954.