Application of Akkermansia muciniphila and its products in regulating lipid metabolism

Abstract

The present invention belongs to the field of microbial technology, and specifically relates to the application of Akkermansia muciniphila and its products in regulating lipid metabolism. The Akkermansia muciniphila is named AKK ONE, and the deposit number is CGMCC No.20954. AKK ONE can improve the blood lipid level of hypercholesterolemia mice and effectively improve liver damage. AKK ONE can regulate the abundance of intestinal flora, increase the content of SCFAs, and improve the BAs structure in the intestine of mice to improve hypercholesterolemia caused by a high cholesterol diet. The present invention provides new insights into the relationship between probiotics and intestinal flora and metabolites, and proves that AKK ONE has potential application value in improving hypercholesterolemia.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to application of Akkermansia muciniphila and products thereof in regulating lipid metabolism. Background Art

[0002] Cholesterol has the functions of synthesizing bile acid, forming cell membranes, and synthesizing hormones. When it is severely elevated, it will block blood vessels, thereby inducing coronary heart disease, cerebral infarction, hypertension, myocardial infarction and other diseases. Hypercholesterolemia refers to the increase in cholesterol levels in the blood, which is a key risk factor for various metabolic diseases such as atherosclerotic cardiovascular disease (ASCVD) and obesity. At present, the types of clinical drugs are very limited, and only a part of people have good effects when taking them, but long-term use has side effects. The cholesterol-lowering effect of probiotics has achieved certain results in clinical trials, reducing the risk of cardiovascular and other diseases.

[0003] Akkermansia muciniphila (A. muciniphila) is a mucin-degrading bacterium that inhabits the intestines of humans and many other animals. AKK can effectively improve symptoms such as obesity, type I / II diabetes, hyperuricemia, hepatic steatosis, intestinal inflammation, and cognitive impairment in mice. For example, A. muciniphila ATCC BAA-835 can effectively improve hepatic steatosis, inflammation, and liver damage in mice fed a high-fat, high-cholesterol diet model, effectively improve mitochondrial oxidation and bile acid metabolism in the gut-liver axis, improve oxidative stress-induced intestinal cell apoptosis, and effectively improve the composition of intestinal microorganisms. Another study showed that A. muciniphila could induce the expression of uncoupling protein 1 in brown adipose tissue of mice fed a high-fat diet, thereby promoting thermogenesis and inducing systemic glucagon-like peptide-1 secretion to improve glucose homeostasis in mice. In addition, A. muciniphila can also regulate host functions through functional components such as bile acids (BAs), membrane proteins, and short-chain fatty acids (SCFAs). A large number of studies have shown that the levels of BAs and SCFAs in the host are positively correlated with the abundance of A. muciniphila. In a sleep deprived mouse model, intestinal flora disorders stimulated microglia to over-phagocytose hippocampal synaptic proteins, leading to cognitive impairment. After supplementing A. muciniphila, the microglial phagocytosis function was regulated by increasing the production of SCFAs, thereby improving the cognitive impairment of mice. These studies show that A. muciniphila, as an important genus of intestinal bacteria, plays an important role in host health. However, the functionality of different strains is different. Both A. muciniphila 139 and A. muciniphila ATCC BAA-835 strains have anti-inflammatory effects on chronic colitis, but the anti-inflammatory effect of A. muciniphila ATCC BAA-835 is stronger than that of strain 139. The former significantly reduced the spleen weight and colon inflammation index of mice with chronic colitis, while A. muciniphila 139 did not significantly reduce it.

[0004] Therefore, developing more effective probiotics is an effective way to address high cholesterol-related diseases.

[0005] The present invention is the development of downstream application technology carried out by the inventor for the independently developed microbial resources: Akkermansia muciniphila with the deposit number of CGMCC No.20954 (other patents have been submitted on the same day).

[0006] After obtaining Akkermansia muciniphila, the inventors conducted application experiments in multiple fields including improving diabetes, anti-infection, improving respiratory diseases, regulating lipid metabolism, improving enteritis, etc., and obtained positive experimental results in some fields.

[0007] Taking into account the relevant provisions of the Patent Law on unity, protection is requested separately for each indication that does not have unity.

[0008] The present invention is directed to the protection of the application technology in regulating lipid metabolism.

[0009] Since relevant evidence such as microbial identification and performance testing was published in the microbial patent submitted on the same day, in order to facilitate the review of the present invention and quickly understand the inventor's previous work, the relevant information of Akkermansiamuciniphila is briefly disclosed in the content of the present invention.

[0010] The conclusive information in this section is for the convenience of the review of the present invention, but is not intended to limit the scope of protection of the present invention. The specific experimental process and experimental results are recorded in the microbiological patent filed on the same day as the present invention.

[0011] 1. Akkermansia muciniphila was obtained by separation and screening by the present inventors.

[0012] 2. The deposit information of Akkermansia muciniphila: The deposit number is: CGMCC No.20954.

[0013] 3. Physiological and biochemical properties of Akkermansia muciniphila:

[0014] (1) Hydrophobicity: The hydrophobicity of the strain increases with time, reaching more than 30% at 60 minutes. (2) Self-aggregation: The self-aggregation of the strain increases with time, tends to be stable at 20 hours, and remains at about 50%. (3) Gastrointestinal fluid tolerance: The survival rate of the strain in gastric fluid shows a downward trend with time, and the survival rate is about 80% at 240 minutes; the survival rate of the strain in intestinal fluid shows an overall downward trend, and the survival rate is above 80% at 240 minutes. (4) Biofilm formation ability: weak. (5) Bacterial reverse mutation test: The results of the bacterial reverse mutation test show that the strain is not mutagenic. (6) Acute toxicity: non-toxic. (7) Subchronic toxicity test: no toxic side effects. Summary of the invention

[0015] In order to solve the above problems, the present invention provides a new strain of Akkermansia muciniphila, named AKK ONE, with a deposit number of CGMCC No. 20954. AKK ONE can improve the blood lipid level of hypercholesterolemia mice, thereby improving hypercholesterolemia caused by a high cholesterol diet.

[0016] In one aspect, the present invention provides the use of Akkermansia muciniphila in preparing a product for regulating lipid metabolism, wherein the deposit number of the Akkermansia muciniphila is CGMCC No.20954.

[0017] Specifically, the product comprises fermentation broth, fermentation broth supernatant, fermentation broth precipitate, live bacteria, dead bacteria, lyophilized powder and / or cell lysate of Akkermansia muciniphila.

[0018] More specifically, the fermentation liquid refers to the liquid obtained by inoculating the strain into the culture medium and culturing it for a period of time. The fermentation liquid supernatant refers to the clarified liquid on the upper layer of the fermentation liquid after centrifugation; it contains abundant metabolites and part of the bacterial fragments during the bacterial growth and reproduction process; the acidic substances and bacteriocins secreted by the bacteria have antagonistic and killing effects on harmful bacteria; the amino acids after the bacteria decompose the food, as well as the synthesized vitamins are all in the culture liquid, and also include enzymes secreted by the bacteria that are useful to the human body; and some of the bacterial components also have an immune-promoting effect on the human body. The fermentation liquid precipitate refers to the liquid precipitate obtained by centrifugation, including free protein, residual bacteria, broken cells, and residues of the culture matrix, mainly protein and intracellular matrix. The live bacteria, also known as active bacterial flora, can be colonized and multiplied in the intestine, which is conducive to increasing the number of beneficial bacteria. The dead bacteria are microorganisms that have lost their vitality and cannot grow and reproduce, and the probiotics lose their vitality due to the production process. Preferably, the inactivation method includes but is not limited to: any one or more of high temperature inactivation, radiation inactivation or chemical inactivation. The freeze-dried powder is obtained by freeze-drying the aforementioned culture liquid. The freeze-dried powder generally includes a freeze-dried protective agent. The freeze-dried protective agent includes, but is not limited to, pH buffer, filler, sugar, non-ionic surfactant, ligand, etc. The pH buffer includes, but is not limited to, any one or more of Tris, amino acids or their salts, citric acid or its salts, acetic acid or its salts. The filler includes, but is not limited to, any one or more of mannitol, glycine, and bovine serum albumin. The sugar may be a disaccharide, such as any one or more of sucrose or trehalose. The non-ionic surfactant includes, but is not limited to, Tween, and the Tween may be Tween-20, Tween-60, Tween-80, etc. The freeze-dried protective agent may also include antioxidants, etc. The freeze-dried protective agent may also specifically include albumin, polyethylene glycol, etc. The cell lysis solution may be obtained by lysing the bacteria obtained by the aforementioned culture. The lysis may be physical lysis or chemical lysis. The physical lysis includes, but is not limited to, grinding, ultrasonic crushing, etc. The chemical lysis includes, but is not limited to, chemical reagent lysis and enzymatic hydrolysis, and the enzymatic hydrolysis may be a hydrolase or an oxidase. The lysis may also be achieved by increasing the intracellular pressure to cause the cells to rupture.

[0019] Specifically, the product is a medicine, food, health care product, pharmaceutical raw material, industrial raw material, food raw material or health care product raw material. More specifically, the product is a medicine, and the medicine also includes a pharmaceutically acceptable excipient. More specifically, the pharmaceutically acceptable excipient includes, but is not limited to, any one or more of an excipient, a stabilizer, a diluent, a binder, a preservative, a lubricant, and an antioxidant. Preferably, the pharmaceutically acceptable excipient can be selected from at least one of lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0020] In another aspect, the present invention provides the use of Akkermansia muciniphila in the preparation of products for preventing and / or treating hypercholesterolemia, wherein the Akkermansia muciniphila has a deposit number of CGMCC No. 20954. Specifically, the product comprises fermentation broth, fermentation broth supernatant, fermentation broth precipitate, live bacteria, dead bacteria, lyophilized powder and / or cell lysate of Akkermansia muciniphila. Specifically, the product is a medicine, a food, a health product, a pharmaceutical raw material, an industrial raw material, a food raw material or a health product raw material. More specifically, the product is a medicine, and the medicine also includes pharmaceutically acceptable excipients.

[0021] In another aspect, the present invention provides a pharmaceutical composition, comprising Akkermansia muciniphila; the Akkermansia muciniphila has a deposit number of CGMCC No. 20954. Specifically, the bacterial concentration of the Akkermansia muciniphila is 1×10 8 CFU / mL×10 12 CFU / mL or 1×10 8 CFU / g×10 12 CFU / g. Preferably, the bacterial concentration of Akkermansia muciniphila is 1×10 9 CFU / mL×10 11 CFU / mL or 1×10 9 CFU / g×10 11 CFU / g. Specifically, the pharmaceutical composition is used to regulate lipid metabolism. Specifically, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0022] In another aspect, the present invention provides a method for preparing the aforementioned pharmaceutical composition, the method comprising culturing Akkermansia muciniphila; the deposit number of Akkermansia muciniphila is CGMCC No. 20954. Specifically, the culture medium comprises BHI culture medium.

[0023] The technical effects achieved by the present invention are as follows: AKK ONE can improve the blood lipid levels of hypercholesterolemia mice, AKK ONE can significantly reduce the total TC and LDL-C content in the serum and liver of hypercholesterolemia mice, increase the HDL-C content, improve liver damage, reduce serum ALT, AST and TBA levels, and improve fasting blood glucose levels and glucose tolerance. Further analysis of intestinal flora and metabolites showed that AKK ONE significantly increased the abundance of Faecalibaculum, Oscillospira, Bacteroides, and Akkermansia in the intestines of mice, increased the content of SCFAs including acetic acid, isobutyric acid, and valeric acid, and regulated the composition of the BAs pool in the intestine, especially secondary BAs. In short, AKK ONE improves hypercholesterolemia caused by a high-cholesterol diet by regulating the abundance of intestinal flora, especially increasing the SCFAs-producing flora, thereby increasing the SCFAs content and enhancing the conversion of cholesterol to BAs.

[0024] Storage Instructions:

[0025] Strain name: AKK ONE; Deposit number: CGMCC No.20954; Classification name: Akkermansiamuciniphila; Deposit date: October 26, 2020; Deposit unit: General Microbiology Center of China Microbiological Culture Collection Administration; Deposit unit address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the effect of AKK ONE on the feeding habits of mice.

[0027] Figure 2 The figure shows the effect of AKK ONE on the blood lipid level of mice, where A is TG in serum (mmol / L); B is total TC in serum (mmol / L).

[0028] Figure 3 The figure shows the effect of AKK ONE on blood lipid levels in mice, where A is LDL-C (mmol / L) in serum; B is HDL-C (mmol / L) in serum.

[0029] Figure 4The figure shows the effect of AKK ONE on blood lipid levels in mice, where A is TBA (μmol / L) in serum; B is ALT (U / L) in serum; and C is AST (U / L) in serum.

[0030] Figure 5 The effect of AKK ONE on blood glucose homeostasis in mice, where A is the fasting blood glucose level (mM); B is the glucose content (mM); C is the area under the curve of glucose tolerance (mM 120min).

[0031] Figure 6 The figure shows the effect of AKK ONE on the liver lipid level of mice, where A is the total TC in the liver (mmol / g); B is the total TG in the liver (mmol / g); C is the LDL-C in the liver (mmol / g); and D is the HDL-C in the serum (mmol / g).

[0032] Figure 7 The effect of AKK ONE on mouse liver degeneration, where A is the color of mouse liver; B is the pathological section of mouse liver tissue.

[0033] Figure 8 The effect of AKK ONE on the intestinal flora of mice, where A is the result of PCoA analysis of diversity; B is the Venn diagram made from the ASV / OTU abundance table.

[0034] Fig. 9 The effects of AKK ONE on the intestinal flora of mice, where A is the composition analysis at the phylum level; B is the composition analysis at the genus level.

[0035] Fig.10 This is the effect of AKK ONE on the intestinal flora of mice, where A and B are composition analyses at the genus level.

[0036] Fig.11 The figure shows the effect of AKK ONE on short-chain fatty acids in the intestine of mice, where A is total short-chain fatty acids (μg / g); B is acetic acid (μg / g); C is propionic acid (μg / g); D is butyric acid (μg / g); E is isobutyric acid (μg / g); F is valeric acid (μg / g); G is isovaleric acid (μg / g); and H is hexanoic acid (μg / g).

[0037] Fig.12 Effect of AKK ONE on intestinal bile acid pool in mice.

[0038] Fig.13 This is the effect of AKK ONE on the bile acid content in mice.

[0039] Fig.14 The effect of AKK ONE on the primary bile acid content in mice.

[0040] Fig.15 The effect of AKK ONE on the secondary bile acid content in mice.

[0041] Fig.16 The effect of AKK ONE on the free bile acid content in mice.

[0042] Fig.17 The effect of AKK ONE on the content of conjugated bile acids in mice.

[0043] Fig.18 The effect of AKK ONE on the content of conjugated bile acids in mice.

[0044] Fig.19 This is the correlation analysis between AKK ONE's intestinal microorganisms and physical and chemical indicators and metabolites, where A is the correlation analysis between intestinal microorganisms and physical and chemical indicators; B is the correlation analysis between intestinal microorganisms and metabolites.

[0045] Note: The * in the figure indicates the comparison among ND, Sim, AKK ONE and HCD groups, and P<0.05 and 0.0001 are marked as * and ****, respectively. DETAILED DESCRIPTION

[0046] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0047] Example 1

[0048] 1.1 Materials and Instruments

[0049] Ordinary maintenance feed for mice, Beijing Huafukang Biotechnology Co., Ltd.; high cholesterol feed for mice (ASHF4), Deci Biotechnology Co., Ltd.; simvastatin, Shandong Lukang Pharmaceutical Group Saite Co., Ltd.; blood glucose meter and blood glucose test strips, Sino Biosensor Co., Ltd.; total cholesterol (total TC) determination kit (A111-1-1), triglyceride (TG) determination kit (A110-1-1), low-density lipoprotein (LDL-C) determination kit (A113-1-1), high-density lipoprotein (HDL-C) determination kit (A112-1-1), total bile acid (TBA) determination kit (E003-2-1), aspartate aminotransferase (AST) test kit (C010-2-1), alanine aminotransferase (ALT) test kit (C009-1-1), Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.

[0050] FA1004B electronic balance, Shanghai Youke Instrument Co., Ltd.; SPARK 10M microplate reader, Tecan, Switzerland; Centrifuge 5424R desktop centrifuge, Eppendorf AG, Germany.

[0051] 1.2 Experimental methods

[0052] 1.2.1 Strains and culture conditions

[0053] Akkermansia muciniphila ONE (AKK ONE) was isolated from the feces of healthy adults and is currently deposited in the China General Microbiological Culture Collection Center with the deposit number CGMCC No. 20954. AKK ONE was cultured in BHI broth medium and placed in an anaerobic incubator (80% N2, 15% CO2, 5% H2) at 37°C for 48-72h.

[0054] 1.2.2 Animal experiments

[0055] Animal experiments were approved by the Animal Ethics Committee of Jilin Agricultural University (experimental ethics number: 20230831001) and were conducted in accordance with the international animal experiment ethics. 40 male C57BL / 6 mice aged 5 weeks (Beijing Huafukang Biotechnology Co., Ltd. (SCXK (Beijing) 2019-0008) were raised in an SPF-grade, room temperature (25℃±1℃), 12 / 12h light-dark cycle environment. After one week of adaptive culture in the animal room, the 40 mice were divided into 4 groups, 10 mice in each group, namely the normal group (ND), high cholesterol group (HCD), simvastatin group (Sim), and AKK ONE group. The normal group was fed with maintenance feed, and the other three groups were fed with high cholesterol feed. At the same time, the Sim group and AKK ONE group were gavaged with simvastatin solution (3.8 mg / kg BW) and AKK ONE bacterial solution (1.1×10 10 CFU / mL), the control group and the high cholesterol group were gavaged with the same amount of 0.9% NaCl, once a day, for 4 consecutive weeks, and the food intake and weight changes of the mice were recorded during the experiment. The day before the end of the experiment, the feces of the mice were collected under sterile conditions, and the mice were fasted overnight before being killed. Serum, liver, kidney, spleen, epididymal white adipose tissue, inguinal white adipose tissue, brown adipose tissue, cecal contents, etc. were collected and stored in a -80℃ refrigerator for use.

[0056] 1.2.3 Oral glucose tolerance test

[0057] One day after the experiment, the oral glucose tolerance of mice in each group was measured. The mice were fasted overnight before the measurement. The fasting blood glucose level of mice was measured before oral glucose administration, and the tail vein blood glucose content was measured at 15, 30, 60, and 120 minutes after oral glucose administration (2g / kg BW). The area under the curve of blood glucose concentration and time was calculated.

[0058] 1.2.4 Determination of organ index

[0059] The collected liver, kidney, spleen, epididymal white adipose tissue, inguinal white adipose tissue, and brown adipose tissue were weighed, and the ratio to the body weight of the mice on the last day was the organ index (%).

[0060] 1.2.5 Determination of serum and liver physicochemical indicators

[0061] The collected serum was used to measure the levels of total TC, TG, LDL-C, HDL-C, TBA, AST, and ALT in mouse serum according to the kit. The liver was accurately weighed, mixed with anhydrous ethanol at a ratio of 1:9, homogenized, and the supernatant was taken to measure the levels of total TC, TG, LDL-C, and HDL-C in mouse liver according to the kit.

[0062] 1.2.6 Oil Red O staining of liver tissue

[0063] The liver tissue was embedded and frozen, fixed with tissue fixative for 15 minutes, washed and dried. The slices were added with oil red dye and covered in light-proof staining for 8-10 minutes. Take out the slices, stay for 3 seconds, and then immerse them in two cylinders of 60% isopropanol for differentiation, 3 seconds and 5 seconds respectively. And then immerse them in 2 cylinders of pure water for 10 seconds each. Take out the slices and stay for 3 seconds, then immerse them in hematoxylin staining solution for 3-5 minutes, and then immerse them in 3 cylinders of pure water for 5 seconds, 10 seconds, and 30 seconds respectively. Differentiation solution for 2-8 seconds, and then immerse them in 2 cylinders of distilled water for 10 seconds. Then, blueing solution was used for 1 second, and the slices were gently immersed in 2 cylinders of tap water for 5 seconds and 10 seconds respectively, and the staining effect was examined under a microscope. Then, the slices were sealed with glycerol gelatin sealing agent, and the images were collected under a microscope.

[0064] 1.2.7 16S rRNA sequencing analysis

[0065] Weigh an appropriate amount of fecal sample, extract total RNA, and quantify DNA using Nanodrop. The quality of DNA extraction was tested by 1.2% agarose gel electrophoresis. Primers (standard bacteria 16S V3V4, 338F (SEQ ID NO.1): ACTCCTACGGGAGGCAGCA; 806R (SEQ ID NO.2): GGACTACHVGGGTWTCTAAT) were used and sample-specific Barcode sequences (Table 1) were added to perform PCR amplification of the rRNA gene variable region (single or multiple consecutive sequences) or specific gene fragments, and the amplified products were purified and recovered using magnetic beads. The PCR amplification and recovery products were subjected to fluorescence quantification. Sequencing was performed according to the fluorescence quantification results. The sequencing library was prepared using Illumina's TruSeq Nano DNA LT Library Prep Kit, and the library was quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, Cat. No.: P7589) on the Promega QuantiFluor fluorescence quantification system. The original data from high-throughput sequencing are screened according to sequence quality, and the original sequences that pass the quality screening are annotated and evaluated according to the index and Barcode information.

[0066] Table 1 Sample-specific Barcode sequences

[0067]

[0068]

[0069] 1.2.8 Determination of SCFAs

[0070] Working standard solutions of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid were prepared. The internal standard (4-methylvaleric acid) was prepared to 375 μg / mL with ether. 200 μL of a series of working standard solutions of 6 short-chain fatty acids, 100 μL of 15% phosphoric acid, 20 μL of a series of working standard solutions of hexanoic acid, 20 μL of the internal standard, and 260 μL of ether were mixed to prepare ten standard curve points (0.02, 0.1, 0.5, 2, 10, 25, 50, 100, 250, 500 μg / mL).

[0071] Take an appropriate amount of intestinal content homogenate, centrifuge and take 200μL supernatant, add 100μL 15% phosphoric acid, add 20μL internal standard solution (375μg / mL) and 280μL ether solution to homogenize for 1min, centrifuge and take the supernatant for testing. Chromatographic machine conditions: Thermo Trace 1310 gas phase system, chromatographic column is Agilent HP-INNOWAX capillary column (30m×0.25mm ID×0.25μm); split injection, injection volume is 1μL, split ratio is 10:1. Injection port temperature is 250℃; ion source temperature is 300℃; transfer line temperature is 250℃. The program temperature starts at 90℃; then it is heated to 120℃ at 10℃ / min; then it is heated to 150℃ at 5℃ / min; finally it is heated to 250℃ at 25℃ / min and maintained for 2min. The carrier gas was helium with a carrier gas flow rate of 1.0 mL / min. Mass spectrometry conditions: Thermo ISQ LT mass spectrometer, electron impact ionization (EI) source, SIM scanning mode, electron energy 70 eV. The short-chain fatty acid content of the sample was calculated based on the standard curve of 7 short-chain fatty acids.

[0072] 1.2.9 Determination of BAs

[0073] Prepare bile acid standards and dilute ten standard curve points with 30% methanol. Take an appropriate amount of intestinal contents and add 400 μL of methanol. Centrifuge after shaking to obtain the supernatant. Filter the supernatant through a 0.2 μm filter and dilute 50 times before testing on the chromatography machine. BEH C18 chromatographic column (2.1×100mm, 1.7μm), injection volume of 5μL, column temperature of 40℃, mobile phase A of 0.01% formic acid water, mobile phase B of acetonitrile. Gradient elution conditions were 0-4min, 25% mobile phase B; 4-9min, 25-30% mobile phase B; 9-14min, 30-36% mobile phase B; 14-18min, 36-38% mobile phase B; 18-24min, 38-50% mobile phase B; 24-32min, 50-75% mobile phase B; 32-33min, 75-90% mobile phase B; 33-35.5min, 90-25% mobile phase B. Flow rate 0.25mL / min. Mass spectrometry conditions: electrospray ionization (ESI) source, negative ionization mode. The ion source temperature was 500°C, the ion source voltage was -4500 V, the collision gas was 6 psi, the curtain gas was 30 psi, and the nebulizer gas and auxiliary gas were both 50 psi. Multiple reaction monitoring was used for scanning.

[0074] 1.3 Statistical analysis

[0075] All data results were expressed as mean ± standard deviation and were statistically analyzed using Graphpad prism 8.0. The t-test was used to analyze the significance of the difference between the two groups of samples. The spearman algorithm was used for correlation analysis. P < 0.05 indicated that the difference was significant.

[0076] 1.4 Results and Analysis

[0077] 1.4.1 Effect of AKK ONE on blood lipid levels in hypercholesterolemia mice

[0078] During the experiment, the mice's food intake and body weight changes were as follows: Figure 1 As shown. Only the food intake of the ND group was significantly different from that of the HCD group, indicating that the food intake of mice in each experimental group was not significantly different from that of the HCD group. The organ index is used to evaluate the relative size of mouse organs. The ratio of organs to body weight of normal mice is relatively constant. As can be seen from Table 2, a high cholesterol diet will significantly increase the liver index of mice. After oral administration of AKK ONE, the liver index of mice was significantly reduced, while other organ indices did not change significantly.

[0079] The effect of AKK ONE on blood lipid levels in mice was detected. Compared with the ND group, the high cholesterol diet significantly increased the total TC content in the serum of mice, while it was significantly decreased after oral administration of AKK ONE ( Figure 2 Similarly, a high cholesterol diet resulted in a significant increase in serum LDL-C levels and a significant decrease in HDL-C levels, whereas after oral administration of AKK ONE, serum LDL-C levels significantly decreased by 20.41% and HDL-C levels significantly increased by 82.17% ( Figure 3 ). However, a high cholesterol diet caused a decrease in serum TG levels, but there was no significant change after oral administration of AKK ONE ( Figure 2 A in the figure). Serum ALT, AST and TBA are important indicators of liver damage. AKK ONE can significantly reduce liver damage caused by a high cholesterol diet. Compared with the HCD group, serum TBA content decreased by 24.37% ( Figure 4 A), ALT and AST decreased to 47.48U / L and 19.75U / L respectively (B and C in Figure 4). The above results show that AKKONE can effectively improve the blood lipid level of hypercholesterolemia mice.

[0080] Table 2 Mouse organ index

[0081]

[0082]

[0083] Note: Data are expressed as mean ± standard deviation (n=8-10), * indicates the comparison among ND, Sim, AKK ONE and HCD groups, P<0.05, 0.0001 are marked with *, ****, respectively.

[0084] 1.4.2 Effect of AKK ONE on blood glucose homeostasis in hypercholesterolemic mice

[0085] High cholesterol diet disrupts glucose levels in mice Figure 5 As shown in A, the fasting blood glucose level of mice in the HCD group was significantly higher than that in the ND group. After oral administration of AKK ONE, the fasting blood glucose level of mice was reduced and was similar to that of the ND group. The oral glucose tolerance test is used to determine the body's ability to regulate glucose homeostasis. After the HCD group mice ingested the glucose solution, the blood glucose level rose rapidly and then slowly dropped to the initial state ( Figure 5 B), while after oral administration of AKK ONE, the AUC values ​​corresponding to the glucose tolerance of mice decreased by 22.26% and 16.80% ( Figure 5 C), indicating that AKK ONE can improve the fasting blood glucose level and oral glucose tolerance of hypercholesterolemia mice and improve blood glucose homeostasis in mice.

[0086] Effects of AKK ONE on liver lipid levels and liver degeneration in hypercholesterolemic mice

[0087] As an important organ for lipid metabolism, the liver is closely related to blood lipid levels, and the homeostasis of liver lipid metabolism is crucial to maintaining normal liver metabolism. Figure 6 As shown in A, B, and C, the high cholesterol diet led to a significant increase in the levels of total TC, TG, and LDL-C in the liver of mice. After oral administration of AKK ONE, the levels of these three lipids were significantly reduced. At the same time, the intake of AKK ONE also significantly increased the level of HDL-C in the liver of mice, even higher than the level of HDL-C in the liver of mice in the normal group ( Figure 6 The liver of mice fed a high cholesterol diet was grayish yellow and enlarged, which was consistent with the liver index in Table 1. After oral administration of AKK ONE, the grayish yellow color of the liver decreased and its color was close to the reddish brown color of the liver in the ND group ( Figure 7 A in the figure). Next, the liver tissue pathological sections of the mice in the AKK ONE group were observed. Oil red O staining observed ( Figure 7 In B), the liver lipid droplets of mice fed a high cholesterol diet were stained red by Oil Red O and were obviously accumulated in flakes, but the liver lipid droplets of the AKK ONE group were significantly reduced and the fat volume was reduced. The above results show that AKK ONE can improve the liver degeneration of mice induced by a high cholesterol diet and improve the lipid level of the mouse liver.

[0088] 1.4.4 Effect of AKK ONE on intestinal flora of hypercholesterolemia mice

[0089] Effects of AKK ONE on intestinal flora in hypercholesterolemia mice Figure 8 The PCoA analysis showed that the sample point distances of the intestinal flora of mice on a high cholesterol diet were quite different from those of mice on a normal diet, indicating that the two communities were different. After oral administration of AKK ONE, the community structure of the mice in the AKK ONE group was somewhat different from that in the HCD group ( Figure 8 A). Furthermore, the Venn diagram made using the ASV / OTU abundance table showed that the number of ASV / OTUs in the ND group was the highest, and the number of ASV / OTUs in the HCD group was the lowest. In addition, the number of ASV / OTUs in the AKK ONE group was 235 more than that in the HCD group, an increase of 32.32% ( Figure 8 B) Specifically, the composition analysis of each group at the phylum level is as follows Fig. 9 As shown in A, Firmicutes, Verrucomicrobiota, Bacteroidota, and Actinobacteriota were the most abundant phyla in all groups. Compared with the ND group, the HCD group significantly reduced the relative abundance of Bacteroidota, while after oral administration of AKK ONE, the abundance of Firmicutes, Verrucomicrobiota, and Bacteroidota in the intestine of AKK ONE mice increased compared with the HCD group. Fig. 9 B and Fig.10 ), it was found from the heat map that the microbiota structure of the ND, HCD, and AKK ONE groups was clearly divided into three parts, indicating changes in the microbiota structure of mice. Compared with the HCD group, AKK ONE significantly increased the abundance of Faecalibaculum, Bacteroides, Akkermansia, and Oscillospira. The abundance of Enterococcus, Erysipelatoclostridium, and Lachnoclostridium in the HCD group was significantly higher than that in the ND group, and the intake of AKK ONE significantly reduced the abundance of these genera. The above results show that AKK ONE can improve the intestinal flora imbalance caused by a high cholesterol diet in mice and improve the composition and abundance of intestinal flora.

[0090] 1.4.5 Effects of AKK ONE on intestinal SCFAs and bile acid pools in hypercholesterolemic mice

[0091] SCFAs are the main metabolites of intestinal microorganisms. The effect of AKK ONE on intestinal SCFAs in hypercholesterolemia mice is as follows: Fig.11 Compared with mice on a normal diet, high cholesterol diet led to a significant decrease in the total SCFAs content in the intestine of mice ( Fig.11 A in the figure, while after oral administration of AKK ONE, the total SCFAs content increased significantly. Similarly, a high cholesterol diet led to an increase in intestinal acetic acid ( Fig.11 B), propionic acid ( Fig.11 C), isobutyric acid ( Fig.11 E in), valeric acid ( Fig.11 F in), isovaleric acid ( Fig.11 G in), hexanoic acid ( Fig.11 The content of H) in the glutathione decreased significantly, and the intake of AKK ONE significantly improved the content of acetic acid, isobutyric acid, and valeric acid, while the content of propionic acid and butyric acid ( Fig.11 D) Although the isovaleric acid content showed an upward trend after taking AKK ONE, it was not significant.

[0092] The effect of AKK ONE on the intestinal bile acid pool in hypercholesterolemic mice was further investigated by bile acid targeted metabolomics. Fig.12 It can be seen that the high-cholesterol diet significantly improved the bile acid metabolism composition of the mouse intestine. Compared with the control group, the total bile acid content of the mice on the high-cholesterol diet increased significantly, about three times that of the control group. After taking AKK ONE, the total bile acid content of the mice decreased significantly ( Fig.13 Similarly, the levels of total primary bile acid and total secondary bile acid were significantly increased in mice fed a high cholesterol diet ( Fig.13 ), after oral administration of AKK ONE, the levels of primary bile acids chenodeoxycholic acid (CDCA), cholic acid (CA), glycocholic acid (GCA), and taurocholic acid (TCA) in the intestine of mice decreased significantly ( Fig.14 ), the contents of secondary bile acids taurohyodeoxycholic acid + tauroursodeoxycholic acid (THDCA + TUDCA), taurodeoxycholic acid (TDCA), tauro-α-muricholic acid (T-α-MCA), and tauro-β-muricholic acid (T-β-MCA) decreased significantly, while compared with the control group, the contents of secondary bile acids alloLCA, lithocholic acid (LCA), and hyodeoxycholic acid (HDCA) in the intestine of mice fed a high cholesterol diet decreased significantly, but there was no significant change after oral administration of AKKONE ( Fig.15 ).

[0093] Further analysis of bile acid structure revealed that a high cholesterol diet also significantly increased the levels of total free bile acid and total conjugated bile acid in the intestines of mice, but after oral administration of AKK ONE, total conjugated bile acid decreased significantly, and total free bile acid showed a downward trend but was not significant ( Fig.13 Specifically, compared with mice fed a high cholesterol diet, the levels of free bile acids isolithocholic acid (isoLCA), 7-keto lithocholic acid (7-ketoLCA), CDCA, and CA were significantly reduced after AKK ONE intake, while other free bile acids did not change significantly ( Fig.16 ). Similarly, the intake of AKK ONE significantly changed the content of conjugated bile acids, and the content of GCA, THDCA+TUDCA, TDCA, and taurocholic acid (TCA) all decreased significantly ( Fig.17 ), and the content of bile acids conjugated to glycine and taurine also decreased significantly ( Fig.18 ). In conclusion, the above results indicate that AKK ONE changes the metabolic structure of intestinal SCFAs and bile acid pools in hypercholesterolemia mice, thereby alleviating hypercholesterolemia.

[0094] 1.4.6 Correlation analysis between AKK ONE intestinal microorganisms and physicochemical indicators and metabolites

[0095] The Spearman algorithm was used to analyze the correlation between AKK ONE intestinal microorganisms and physical and chemical indicators and metabolites. The results are shown in Fig.19. Bacteroides and Muribaculaceae were significantly negatively correlated with liver index, serum TC, LDL-C, TBA, ALT, AST, liver TC, LDL-C, and significantly positively correlated with serum HDL-C, while Enterococcus, Erysipelatoclostridium, and Lachnoclostridium were significantly positively correlated with liver index, serum TC, LDL-C, TBA, ALT, AST, liver TC, LDL-C, and negatively correlated with serum HDL-C and liver HDL-C. In addition, Akkermansia and Oscillospira were significantly positively correlated with liver HDL-C. Total SCFAs, acetic acid, propionic acid, and caproic acid were significantly positively correlated with Bacteroides and Muribaculaceae, and significantly negatively correlated with Enterococcus, Lachnoclostridium, and Faecalibaculum. Isobutyric acid was significantly negatively correlated and positively correlated with Enterococcus and Oscillospira, respectively. Bacteroides and Muribaculaceae were significantly negatively correlated with the contents of BAs except T-β-MCA, while Enterococcus, Erysipelatoclostridium and Lachnoclostridium were significantly positively correlated with all BAs. In addition, Faecalibaculum was significantly positively correlated with CA, GCA, TDCA and total BAs, and Oscillospira was significantly negatively correlated with T-α-MCA and T-β-MCA.

[0096] Comparative Example

[0097] The AKK ONE in the AKK ONE group in Example 1 was replaced with A. muciniphila ATCC BAA-835 (Shanghai Fuxiang Biotechnology Co., Ltd., XF1.1346) and AKK PROBIO (deposit number CGMCC No. 20955), and the level of HDL-C in mouse liver was measured according to the method of Example 1. The test results are shown in Table 2.

[0098] Table 2

[0099]

[0100] The above results show that AKK ONE of the present invention can significantly increase the level of HDL-C, and the degree of increase is significantly better than A. muciniphila ATCC BAA-835 and AKK PROBIO.

Claims

1. Akkermansia muciniphila Akkermansia muciniphila ) for use in the preparation of a medicament for preventing and / or treating hypercholesterolemia, characterized in that: The Akkermansia muciniphila ( Akkermansia muciniphila )’s deposit number is CGMCC No. 20954.

2. The use according to claim 1, characterized in that: The drug contains Akkermansia muciniphila ( Akkermansia muciniphila ) of the fermentation broth, fermentation broth precipitate and / or lyophilized powder.

3. The use according to claim 1 or 2, characterized in that: The medicine also includes pharmaceutically acceptable excipients.

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