Application of Ackermann muciniphila in preparation of health care product composition for reducing body weight and application of composition

Abstract

The invention relates to an application of Ackermann mucilaginosus in preparation of a health care product composition for reducing body weight. The Ackermann mucilaginosus is Ackermann mucilaginosus AM06, Ackermann mucilaginosus AM02 or a combination of the Ackermann mucilaginosus AM06 and the Ackermann mucilaginosus AM02. Wherein the preservation number of the ackermann myxotroph AM06 is CGMCC (China General Microbiological Culture Collection Center) No.22793, and the preservation number of the ackermann myxotroph AM02 is CGMCC No.22794. The ackermann myxotroph AM06 and the In the application, the lipoprotein lipase LPL activity and / or the lipoliver enzyme HL activity in serum can be further improved by inducing and inhibiting lipid accumulation in fat cells, and the CCAAT / enhancer binding protein alpha and / or peroxisome proliferator activated receptor gamma can be further reduced, so that the body weight is effectively reduced. The invention also relates to an application of a composition containing the Ackermann muciniphila.

Description

[0001] Related Application

[0002] This application is a divisional application of the Chinese patent application with the application number CN2022106424558 and the title "Application of Akkermansia muciniphila in the Preparation of Pharmaceutical Compositions for the Prevention and Treatment of Obesity, Compositions and Their Applications", filed on June 8, 2022, the full text of which is hereby incorporated by reference in its entirety. Technical Field

[0003] The present invention relates to the technical field of health products, and particularly relates to the application of Akkermansia muciniphila in the preparation of health product compositions for reducing body weight, and also relates to the application of compositions containing Akkermansia muciniphila in the preparation or as health foods for reducing body weight, and also relates to compositions containing Akkermansia muciniphila. Background Art

[0004] Obesity is a chronic metabolic disease caused by the interaction of multiple factors such as environment, diet, and genetics, characterized by excessive accumulation of body fat, abnormal fat distribution, and weight gain. It is mainly manifested by an increase in the number of adipocytes, dysregulation of body fat distribution, and local fat deposition. Obese patients are often accompanied by social and psychological factors such as low self-esteem, anxiety, and depression. The occurrence and development of diseases such as hypertension, diabetes, coronary heart disease, and atherosclerosis are also closely related to obesity and its complications. In addition, the number of people dying from obesity and its secondary chronic metabolic diseases is increasing globally.

[0005] Obesity can be classified into simple obesity, secondary obesity, and drug-induced obesity according to its etiology and clinical manifestations. Among them, simple obesity is relatively common clinically, with no obvious cause and relatively slow weight gain; secondary obesity is caused by diseases, endocrine disorders, or metabolic disorders; drug-induced obesity is caused by the use of drugs.

[0006] Currently, obesity is mainly treated by methods such as adjusting the feeding method, increasing physical activity, and drug intervention. The main treatment methods are as follows:

[0007] (1) Lifestyle and behavioral therapy. Restricting calorie intake and increasing calorie consumption are the most commonly used methods and the primary choice for preventing and treating overweight / obesity. It mainly improves human obesity by improving physical activity (PA), reasonably arranging diet, and changing environmental factors. Behavioral therapy advocates a low-calorie, low-fat, high-fiber diet, corrects bad living habits, and increases exercise. This method is simple and easy to implement, but has high requirements for patient compliance, slow onset, and unstable treatment effects.

[0008] (2) Drug treatment. When the body mass index (BMI) exceeds 27 kg / m 2 or 30 kg / m 2When accompanied by obesity complications, drug-assisted treatment is required. The main targets of drugs with weight loss effects are to suppress appetite in the central nervous system and inhibit lipid absorption in the gastrointestinal tract. Available drugs include phentermine, orlistat, phentermine / topiramate, lorcaserin, and bupropion / naltrexone, etc. Currently, the obesity treatment drug approved by the National Medical Products Administration in China is orlistat. However, long-term use of the drug can cause adverse reactions and drug resistance.

[0009] (3) Surgical treatment. Common surgical procedures for surgical treatment include laparoscopic sleeve gastrectomy (LSG), Roux-en-Y gastric bypass (RYGB), laparoscopic adjustable gastric banding (LAGB), and biliopancreatic diversion (BPD). It is applicable to patients with severe obesity (BMI between 30 and 35 kg / m 2 ²), who have failed to lose weight and are accompanied by serious complications, and these complications may be improved by weight loss. Compared with intensive lifestyle intervention and drug treatment, metabolic surgery can more effectively reduce and maintain the weight after weight loss for a long time, and at the same time can effectively improve blood glucose, blood lipids, blood pressure, etc. However, surgical operations usually cost a lot, and as an invasive treatment method, it has greater risks and side effects, and even has a certain risk of death. This method also has a negative impact on the mental health of patients.

[0010] (4) Other means. With the progress of technology and the development of the economy, there have been some relatively novel treatment methods in recent years, such as targeted therapy, gene-directed therapy, etc. However, the above weight loss methods have certain risks and uncertainties, and there are not enough clinical trials, and their safety needs to be further verified.

[0011] At present, it is still necessary to further develop health products that can be used to reduce weight. Summary of the Invention

[0012] Based on this, the invention objects of the present application include providing the application of Akkermansia muciniphila in the preparation of a health product composition for reducing weight, and also providing the application of a composition containing Akkermansia muciniphila in the preparation or as a health food for reducing weight, and also providing a composition containing Akkermansia muciniphila; the Akkermansia muciniphila is Akkermansia muciniphila AM06 (deposit number: CGMCC No. 22793), Akkermansia muciniphila AM02 (deposit number: CGMCC No. 22794) or a combination of both. The Akkermansia muciniphila of the present invention can inhibit lipid accumulation in adipocytes by induction. Further, it can also improve the levels of low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, thyroglobulin and / or total cholesterol in serum. Furthermore, it can also regulate the activities of alanine aminotransferase and / or aspartate aminotransferase.

[0013] In the first aspect of the present invention, there is provided the use of Akkermansia muciniphila in the preparation of a health care product composition for reducing body weight, wherein the Akkermansia muciniphila is Akkermansia muciniphila AM06, Akkermansia muciniphila AM02 or a combination of both; wherein,

[0014] The Akkermansia muciniphila AM06 was deposited with the China General Microbiological Culture Collection Center on June 28, 2021, and the deposit number is CGMCC No. 22793;

[0015] The Akkermansia muciniphila AM02 was deposited with the China General Microbiological Culture Collection Center on June 28, 2021, and the deposit number is CGMCC No. 22794.

[0016] In some embodiments, the Akkermansia muciniphila AM06 and the Akkermansia muciniphila AM02 are each independently live bacteria, inactivated bacteria or a combination thereof.

[0017] In some embodiments, the health care product composition comprises the Akkermansia muciniphila and edible raw and auxiliary materials.

[0018] In some embodiments, the health care product composition is a health food, and its dosage form is pills, tablets, granules, capsules, solutions, suspensions or emulsions.

[0019] In the second aspect of the present invention, there is provided the use of a composition containing Akkermansia muciniphila in the preparation or as a health food for reducing body weight, wherein the Akkermansia muciniphila is as defined in the first aspect of the present invention.

[0020] In some embodiments, the composition containing Akkermansia muciniphila further comprises a second active ingredient.

[0021] In some embodiments, the composition containing Akkermansia muciniphila is a compound probiotic, and the compound probiotic further comprises a probiotic different from the Akkermansia muciniphila;

[0022] Preferably, the probiotic different from the Akkermansia muciniphila includes one or more of Bacteroides fragilis, Saccharomyces boulardii, Christensenella, Enterococcus hirae, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus gasseri, Lactobacillus plantarum, Lactobacillus curvatus and Bifidobacterium.

[0023] In the third aspect of the present invention, there is provided a composition containing Akkermansia muciniphila, which comprises the Akkermansia muciniphila defined in the first aspect of the present invention and a second active ingredient.

[0024] The inventors of the present application isolated the strains AM06 (deposit number CGMCC No. 22793) and AM02 (deposit number CGMCC No. 22794). Through comprehensive analysis such as 16S rRNA analysis, morphological analysis, metabolite component analysis, and efficacy analysis (such as tolerance to artificial gastric juice and artificial intestinal juice, the ability to inhibit inflammatory factors from destroying the tight junction proteins of intestinal cells, and the effect of inhibiting LPS-induced hepatitis in liver slices), both of them belong to Akkermansia muciniphila, and are identified as new strains of Akkermansia muciniphila different from ATCC BAA-835 (standard strain) and others.

[0025] The inventors found that the isolated Akkermansia muciniphila AM06 and / or AM02 can be used to reduce body weight. Therefore, the isolated Akkermansia muciniphila AM06 and / or AM02 can be used to prepare a health care product composition, and further can be used to prepare a health food. The isolated Akkermansia muciniphila AM06 and / or AM02 can effectively reduce body weight by inducing the inhibition of lipid accumulation in adipocytes, further improving the activity of LPL (lipoprotein lipase) and / or HL (hepatic lipase) in serum, and further down-regulating CCAAT / enhancer-binding protein α and / or peroxisome proliferator-activated receptor γ. Brief Description of the Drawings

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present application and more comprehensively understand the present application and its beneficial effects, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative efforts.

[0027] Figure 1 It is a colony characteristic diagram of Akkermansia muciniphila AM02 cultured in an embodiment of the present invention;

[0028] Figure 2 It is a colony characteristic diagram of Akkermansia muciniphila AM06 obtained in an embodiment of the present invention;

[0029] Figure 3 It is a microscopic observation diagram of Akkermansia muciniphila AM02 cultured in an embodiment of the present invention after Gram staining;

[0030] Figure 4 It is a microscopic observation diagram of Akkermansia muciniphila AM06 cultured in an embodiment of the present invention after Gram staining;

[0031] Figure 5PCA analysis chart of metabolites in the culture supernatant of several Akkermansia muciniphila strains in an embodiment of the present invention;

[0032] Figure 6 Fluorescence microscopy images showing the effect of several Akkermansia muciniphila strains on the decreased expression of tight junction protein ZO-1 induced by TNF-α and IFN-γ in Caco2 cells in an embodiment of the present invention.

[0033] The Akkermansia muciniphila strain AM06 isolated in the present invention, classified and named Akkermansia muciniphila, was deposited on June 28, 2021 at the General Microbiology Center of the China Microbial Culture Collection Center, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 22793; the strain was received and registered by the deposit center on June 28, 2021, and was detected as a viable strain by the deposit center on June 28, 2021.

[0034] The Akkermansia muciniphila strain AM02 isolated in the present invention, classified and named Akkermansia muciniphila, was deposited on June 28, 2021 at the General Microbiology Center of the China Microbial Culture Collection Center, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 22794; the strain was received and registered by the deposit center on June 28, 2021, and was detected as a viable strain by the deposit center on June 28, 2021. Detailed implementation manners

[0035] The present invention will be further described in detail below in conjunction with the accompanying drawings, implementation manners and examples. It should be understood that these implementation manners and examples are only used to illustrate the present invention and not to limit the scope of the present invention. The purpose of providing these implementation manners and examples is to make the understanding of the disclosed content of the present invention more thorough and comprehensive. It should also be understood that the present invention can be implemented in many different forms and is not limited to the implementation manners and examples described herein. Those skilled in the art can make various changes or modifications without departing from the connotation of the present invention, and the equivalent forms obtained also fall within the protection scope of the present invention. In addition, in the following description, a large number of specific details are given to provide a more thorough understanding of the present invention. It should be understood that the present invention can be implemented without one or more of these details.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the description of the present invention herein are only for the purpose of describing the implementation manners and examples and are not intended to limit the present invention.

[0037] Term

[0038] Unless otherwise specified or there are contradictions, the terms or phrases used in this text have the following meanings:

[0039] As used herein, the selection scope of the terms "and / or", "or / and", and "and / or" includes any one of two or more related listed items, and also includes any and all combinations of the related listed items. The said any and all combinations include combinations of any two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, this technical solution undoubtedly includes the technical solution connected by "logical AND", and also undoubtedly includes the technical solution connected by "logical OR". For example, "A and / or B" includes three parallel solutions: A, B, and A + B. Another example is the technical solution of "A, and / or, B, and / or, C, and / or, D", which includes any one of A, B, C, and D (that is, the technical solution connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, it includes combinations of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution connected by "logical AND").

[0040] In the present invention, when it comes to "multiple", "multiple types", "multiple times", etc., without special limitations, it means greater than 2 or equal to 2 in quantity. For example, "one or more types" means one type or greater than or equal to two types.

[0041] As used herein, "its combination", "any combination thereof", "any combination mode thereof", etc. include all suitable combination modes of any two or any two or more items in the listed items.

[0042] In this text, the "suitable" in "suitable combination mode", "suitable mode", "any suitable mode", etc. is subject to being able to implement the technical solution of the present invention, solve the technical problems of the present invention, and achieve the expected technical effects of the present invention.

[0043] In this text, "preferred", "better", "more preferable", "it is advisable" are only used to describe the implementation modes or embodiments with better effects. It should be understood that they do not constitute a limitation on the protection scope of the present invention. If there are multiple "preferred" in a technical solution, without special instructions and without contradictions or mutual restrictions, each "preferred" is independent.

[0044] In the present invention, "further", "even further", "especially", etc. are used for descriptive purposes, indicating differences in content, but should not be understood as a limitation on the protection scope of the present invention.

[0045] In the present invention, "optionally", "optional", and "option" mean that it is either present or absent, that is, it refers to any one of two alternative scenarios of "present" or "absent". If "optional" appears multiple times in a technical solution, without special instructions, and without contradictions or mutual constraints, each "optional" is independent of each other.

[0046] In the present invention, in "the first aspect", "the second aspect", "the third aspect", etc., the terms "first", "second", "third", etc. are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or quantity, nor can they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", etc. only serve the purpose of non-exhaustive enumerative description and should be understood not to constitute a closed limitation on quantity.

[0047] In the present invention, among the technical features described in an open-ended manner, it includes a closed technical solution composed of the listed features, as well as an open technical solution containing the listed features.

[0048] In the present invention, regarding a numerical interval (i.e., a numerical range), without special instructions, the distribution of the optional numerical values within this numerical interval is considered continuous, and it includes the two numerical endpoints of this numerical interval (i.e., the minimum value and the maximum value), as well as each numerical value between these two numerical endpoints. Without special instructions, when the numerical interval only refers to the integers within this numerical interval, it includes the two endpoint integers of this numerical range, as well as each integer between the two endpoints, which is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be combined. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein. The "numerical value" in this numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. The "numerical interval" is allowed to broadly include quantitative intervals such as percentage intervals, ratio intervals, and ratio value intervals.

[0049] For the temperature parameter in the present invention, without special limitations, it allows both constant temperature treatment and fluctuations within a certain temperature range. It should be understood that the so-called constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0050] In the present invention, the term "room temperature" generally refers to 4°C to 35°C, preferably 20°C ± 5°C. In some embodiments of the present invention, room temperature refers to 20°C to 30°C.

[0051] In the present invention, for units related to data ranges, if a unit is only attached after the right endpoint, it indicates that the units of the left and right endpoints are the same. For example, 3~5h means that the units of both the left endpoint "3" and the right endpoint "5" are h (hours).

[0052] All documents mentioned in the present invention are cited herein for reference as if each document was cited individually for reference. Unless it conflicts with the invention purpose and / or technical solution of the present application, the cited documents related to the present invention are cited in their entirety and for all purposes. When referring to cited documents in the present invention, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When referring to cited documents in the present invention, examples and preferred ways of the relevant technical features cited can also be incorporated as references into the present application, but only to the extent that the present invention can be implemented. It should be understood that when the cited content conflicts with the description in the present application, the present application shall prevail or be amended adaptively according to the description in the present application.

[0053] Obesity is a group of heterogeneous diseases with a relatively long occurrence process. The main reason lies in the long-term excessive energy intake and reduced metabolic level of the body, which subsequently leads to a decreased ability to utilize fat and causes fat accumulation in the body. In recent years, people have conducted in-depth research on obesity, and obesity is considered to be closely related to genetics, diet, living environment, etc. The main causes and pathogenesis of obesity include:

[0054] (1) Genetics and genes. Some studies have shown that 20%~40% of obesity cases are related to genetic factors. In particular, simple obesity has a high correlation with genetics. Although the genetic basis is unclear, obesity has an obvious family aggregation tendency. Currently, genes such as UCP-2 gene, RBP4 gene, UCP gene, Hedeghog gene, KNA-PK gene, CRTC3 gene, and SRC gene have been found to be related to obesity. However, most cases of obesity are caused by the combined action of multiple genes and environmental factors, rather than single genes.

[0055] (2) Environmental and social factors. Overeating, changes in diet structure, reduced physical activity, and the choices of diet and lifestyle affected by policies, cultural traditions, news media, and science and education publicity will all affect the body's consumption and energy conversion.

[0056] (3) Adiposity-related factors. As an inert substance for storing fat, adipose tissue can secrete various adipokines and chemokines, including leptin, adiponectin, resistin, retinol-binding protein 4 (RBP4), tumor necrosis factor (TNF-α), interleukins (IL-1, IL-6, etc.), monocyte chemoattractant protein-1, etc. These factors play important roles in the processes of energy metabolism and the homeostatic regulation of the immune system. Among them, leptin can regulate feeding behavior, and the lack of leptin may lead to overeating, obesity, insulin resistance, etc.; adiponectin (APN) is a cytokine specifically expressed in adipocytes and has the function of promoting the catabolism of lipid substances and reducing the content of triglycerides in tissues.

[0057] The first aspect of the present invention

[0058] In the first aspect of the present invention, there is provided the use of Akkermansia muciniphila in the preparation of a health product composition for reducing body weight, wherein the Akkermansia muciniphila is Akkermansia muciniphila AM06, Akkermansia muciniphila AM02 or a combination of both.

[0059] In the present invention, "Akkermansia muciniphila" is consistent with the general definition in the art and includes, but is not limited to, Akkermansia muciniphila AM06 and AM02.

[0060] In the present invention, "Akkermansia muciniphila" can be live bacteria, or Akkermansia muciniphila that has been inactivated, genetically recombined, modified or decorated, attenuated, chemically treated, physically treated and still retains all or part of its original biological activity, and can also be the lysate, culture (such as supernatant) of the bacteria or the components extracted from the supernatant culture.

[0061] The inventors of the present application have isolated strains AM06 (deposit number CGMCC No. 22793) and AM02 (deposit number CGMCC No. 22794). Through comprehensive analysis such as 16S rRNA analysis, morphological analysis, metabolite component analysis, efficacy analysis (such as tolerance to artificial gastric juice and artificial intestinal juice, the ability to inhibit inflammatory factors from destroying the tight junction proteins of intestinal cells, and the effect of inhibiting LPS-induced hepatitis in liver slices), both belong to Akkermansia muciniphila and are identified as new strains different from Akkermansia muciniphila such as ATCC BAA-835 (standard strain).

[0062] Akkermansia muciniphila belongs to the phylum Verucomicrobia, family Akkermansiaceae, and genus Akkermansia. Akkermansia muciniphila is a mucin-degrading bacterium, an anaerobic, non-motile, sporeless, oval-shaped Gram-negative bacterium, suitable for growth at 20-40 °C and pH 5.5-8.0 (optimum temperature 37 °C, optimum pH 6.5). It is commonly present in the human intestine. Although it is an anaerobe, it can also tolerate a certain degree of oxygen. Akkermansia muciniphila can colonize in the mucus layer and colon by degrading and utilizing mucin. Further, it can also produce short-chain fatty acids (SCFAs) through mucin degradation. The production of these short-chain fatty acids (including acetate, propionate, and butyrate) plays an important role in human health.

[0063] In the present invention, "the Akkermansia muciniphila described in the present invention" specifically refers to Akkermansia muciniphila AM06, Akkermansia muciniphila AM02, or a combination of both, which can be denoted as "AM06 and / or AM02".

[0064] Akkermansia muciniphila AM06 was deposited with the China General Microbiological Culture Collection Center on June 28, 2021, with the deposit number CGMCC No. 22793. It can be isolated by the method of Example 1 and can also be identified by one or more methods including but not limited to Examples 2 to 4.

[0065] In some embodiments, the colony culture characteristics of Akkermansia muciniphila AM06 include: colonies that are round and raised, with neat edges, opaque, white, and of uneven size. Further, the colony size is about 0.08-2.2 mm.

[0066] In some embodiments, Akkermansia muciniphila AM06 is isolated from breast milk.

[0067] Akkermansia muciniphila AM02 was deposited with the China General Microbiological Culture Collection Center on June 28, 2021, with the deposit number CGMCC No. 22794. It can be isolated by the method of Example 1 and can also be identified by one or more methods including but not limited to Examples 2 to 4.

[0068] In some embodiments, the colony culture characteristics of Akkermansia muciniphila AM02 of CGMCC No. 22794 include: colonies that are round and raised, with neat edges, opaque, white, and of uneven size. Further, the colony size is about 0.08-2.2 mm.

[0069] In some embodiments, the applicant also conducted an investigation on the tolerance of artificial gastric juice and artificial intestinal juice. Among them, in the investigation of the tolerance of artificial gastric juice, the viable bacteria counts of different groups including the 0.9 wt% NaCl solution group, the artificial gastric juice group with pH 3, and the artificial gastric juice group with pH 2 were statistically analyzed after anaerobic incubation at 37 ± 2 °C for 0 h, 1.5 h, and 3 h. The results showed that the tolerance of Akkermansia muciniphila strains to artificial gastric juice was in the order of AM02 > AM06 > the standard strain ATCC BAA - 835. Among them, in the experiment of investigating the tolerance of artificial intestinal juice, the viable bacteria counts were statistically analyzed after anaerobic incubation at 37 ± 2 °C for 0 h, 4 h, and 8 h. The tolerance to artificial intestinal juice was AM06 > AM02 > ATCC BAA - 835.

[0070] In some in vitro experiments, the tolerance of Akkermansia muciniphila AM06 and AM02 to artificial gastric juice and artificial intestinal juice, the ability to inhibit the destruction of tight junction proteins of intestinal cells by inflammatory factors, and the effect of inhibiting LPS - induced hepatitis in liver slices were all superior to the standard strain BAA - 835.

[0071] Akkermansia muciniphila AM06 and AM02 can each independently be viable bacteria, or Akkermansia muciniphila that has been inactivated (i.e., inactivated bacteria, which can be completely or partially inactivated), or lysates of the bacteria, cultures (such as supernatants), or components extracted from the above cultures.

[0072] In some embodiments, Akkermansia muciniphila AM06 and Akkermansia muciniphila AM02 each independently are viable bacteria, inactivated bacteria, or a combination thereof (i.e., a combination of viable bacteria and inactivated bacteria). The inactivated bacteria can be inactivated bacteria with a complete morphological structure, inactivated bacteria with an incomplete morphological structure, or a combination thereof.

[0073] In some embodiments, Akkermansia muciniphila AM06 and Akkermansia muciniphila AM02 each independently are one or more of viable bacteria, inactivated bacteria with a complete morphological structure, and inactivated bacteria with an incomplete morphological structure.

[0074] In some embodiments, the Akkermansia muciniphila described in the present invention is selected from viable Akkermansia muciniphila bacteria.

[0075] In some embodiments, the Akkermansia muciniphila described in the present invention is selected from inactivated Akkermansia muciniphila bacteria.

[0076] In the present invention, "complete morphological structure" can include, but is not limited to, Akkermansia muciniphila that has been completely or partially inactivated through inactivation treatment.

[0077] In the present invention, the situation of "incomplete morphological structure" may include, but is not limited to, lysates of bacterial cells, cultures (such as supernatants), or components extracted from the above cultures, etc.

[0078] Through a large number of experiments, the inventors found that the above-mentioned isolated Akkermansia muciniphila AM06 and / or AM02 can be used to reduce body weight. The above-mentioned isolated Akkermansia muciniphila AM06 and / or AM02 can be used to prepare a health care product composition, and further can be used to prepare a health food. The above-mentioned isolated Akkermansia muciniphila AM06 and / or AM02 can effectively reduce body weight by inducing the inhibition of lipid accumulation in adipocytes, further improving the activity of LPL (lipoprotein lipase) and / or HL (hepatic lipase) in serum, and further down-regulating CCAAT / enhancer-binding protein α and / or peroxisome proliferator-activated receptor γ.

[0079] In the present invention, the "composition" can be a combination of multiple substances. Further, it can be used in combination or be a mixture formed by combination.

[0080] In some embodiments, after administering Akkermansia muciniphila AM06 or Akkermansia muciniphila AM02 to a subject (such as a human, a rat, or a mouse), improvements are observed in one or more of the following aspects: body weight, weight gain, body length, Lee's index, total fat mass, fat body ratio, serum lipid biochemical indices (including TC, TG, LDL-C, and HDL-C), serum leptin content, serum adiponectin content, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities. In some embodiments, the body weight and Lee's index are decreased. Further, the decreasing amplitude is AM06≈AM02>BAA-835. In some embodiments, the total fat mass and the fat body ratio both decrease to varying degrees. Further, the decreasing amplitude is AM06≈AM02>BAA-835. In some embodiments, the contents of serum TC, TG, and LDL-C decrease to varying degrees, while the content of HDL-C increases. Further, the change amplitude satisfies AM06≈AM02>BAA-835. In some embodiments, the leptin level increases and the adiponectin level decreases. Further, the change amplitude satisfies AM06≈AM02>BAA-835. See Examples 5 to 7 for reference.

[0081] For preparing a health care product composition

[0082] In the present invention, "health care product" and "health food" have the same meaning and can be used interchangeably. In the present invention, a "health care product" refers to a food with health care functions, which can regulate the physiological functions of an animal body (human or other animals), and usually provides beneficial effects. There is no particular limitation on the range of beneficial effects produced by the "health care product" in the body, which can be a systemic effect or only a local effect.

[0083] In the present invention, a "health care product composition" refers to a food composition with health care functions. The health care product composition can be directly consumed as a health care product or consumed as a dietary additive.

[0084] In the present invention, "food" refers to a product that can be directly consumed. A "food composition" refers to a composition composed of edible substances. It should be understood that the food composition in the present invention may contain, in addition to the aforementioned Akkermansia muciniphila, any other suitable edible substances. In some embodiments, the other edible substances can be selected from substances permitted to be added in food management regulations, and further, do not contain substances prohibited from being added in food management regulations. Unless otherwise specified, the food management regulations refer to the current regulations during production.

[0085] In the present invention, "weight loss in the health care product composition" means beneficial weight loss, that is, "weight loss" brings beneficial effects to health.

[0086] In some embodiments, the health care product composition includes the Akkermansia muciniphila described in the present invention and edible raw and auxiliary materials.

[0087] In the present invention, "edible raw and auxiliary materials" refer to edible raw materials, edible auxiliary materials, or a combination of both. Among them, "edible raw materials" refer to edible substances that can individually provide health care functions, but not AM06 and AM02; "edible auxiliary materials" refer to edible substances that do not have the function of individually providing health care functions. It should be understood that the "edible raw and auxiliary materials" can be selected from substances permitted to be added in health care product management regulations, and further, do not contain substances prohibited from being added in health care product management regulations. Unless otherwise specified, the health care product management regulations refer to the current regulations during production.

[0088] In some embodiments, the food composition includes the Akkermansia muciniphila (AM06 and / or AM02) described in the present invention and edible auxiliary materials. In the present invention, food additives also belong to edible auxiliary materials. Examples of edible auxiliary materials include edible sugar, fructose, honey, glucose, starch, vitamins, beneficial trace elements and medium elements (such as calcium powder), soybean powder, mung bean powder, maltodextrin, milk powder, vegetable juice, fruit juice, spices, flavors, etc. The edible auxiliary materials of the present invention can be used singly or in combination.

[0089] In some embodiments, the health care product composition is composed of Akkermansia muciniphila (AM06 and / or AM02) as described in the present invention and edible excipients.

[0090] In some embodiments, the health care product composition may further contain other health care components, which are health care components different from AM06 and AM02, and their health care functions may be beneficially reducing body weight or other health care functions.

[0091] In some embodiments, the health care product composition further contains other probiotics. The other probiotics may be selected from the modified strains of Akkermansia muciniphila (AM06 and / or AM02) as described in the present invention and probiotics of other species. The probiotics of other species may include one or more of lactic acid bacteria, streptococci, Clostridium perfringens, staphylococci, and bifidobacteria.

[0092] In some embodiments, the health care product composition further contains probiotics of other species.

[0093] Formulation type

[0094] In some embodiments, the health care product composition is a health food.

[0095] In some embodiments, the dosage form of the health food is pills, tablets, granules, capsules, solutions, suspensions or emulsions. Further, non-limiting examples include honey pills, water-honey pills, water pills, syrups, syrup preparations, etc.

[0096] It should be understood that the health food contains a health-effective amount of Akkermansia muciniphila (AM06 and / or AM02) as described in the present invention. The "health-effective amount" herein refers to the amount that can beneficially reduce body weight and can play a role in beneficially reducing body weight at the normal dosage of the health food.

[0097] In the present invention, a "subject" refers to a consumer who consumes the health care product composition.

[0098] In the present invention, a "subject" is an animal, preferably a mammal, more preferably a human. The subjects include but are not limited to consumers of health care products. The subjects in the present invention are preferably mammals. The term "mammal" mainly refers to warm-blooded vertebrate mammals, including but not limited to: cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, mice (such as rats, mice), pigs, cows, sheep, horses, humans, etc., preferably primates, more preferably humans.

[0099] In some embodiments, the health care product composition is applicable to humans or other mammals. In the present invention, "other mammals" do not include humans. Non-limiting examples of "other mammals" include cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, rats (such as rats, mice), pigs, cows, sheep, horses, etc., and further include mice or rats; non-limiting examples of "other mammals" also include primates.

[0100] In some embodiments, the subject is a mammal.

[0101] In some embodiments, the subject is a human, a rat or a mouse.

[0102] Differences in the subjects may lead to different selection ranges of other components in the aforementioned health care product composition.

[0103] The second aspect of the present invention

[0104] In the second aspect of the present invention, there is provided the use of a composition containing Akkermansia muciniphila in the preparation of or as a health food for reducing body weight. The "composition containing Akkermansia muciniphila" herein refers to a composition containing "Akkermansia muciniphila (AM06 and / or AM02) as described in the present invention". The definition of "Akkermansia muciniphila as described in the present invention" can refer to the first aspect of the present invention.

[0105] Furthermore, the Akkermansia muciniphila as described in the present invention is Akkermansia muciniphila AM06 (deposit number: CGMCC No. 22793), Akkermansia muciniphila AM02 (deposit number: CGMCC No. 22794) or a combination of both.

[0106] In some embodiments, the composition containing Akkermansia muciniphila is a health care product composition.

[0107] In the second aspect of the present invention, the definitions of "composition", "health food", "reducing body weight", and "health care product composition" can refer to the first aspect of the present invention. The definition of "health food" can also refer to the fourth aspect of the present invention.

[0108] The Akkermansia muciniphila as described in the present invention can be viable bacteria, or inactivated Akkermansia muciniphila (i.e., inactivated bacteria, which can be completely or partially inactivated), or a lysate of the bacteria, a culture (such as a supernatant) or a component extracted from the culture supernatant.

[0109] In some embodiments, the composition containing Akkermansia muciniphila further includes a second active ingredient.

[0110] In the present invention, the "second active ingredient" refers to an active ingredient other than Akkermansia muciniphila (AM06 and / or AM02) described in the present invention, which has the beneficial effect of reducing body weight.

[0111] In some embodiments, the second active ingredient may be a modified strain of Akkermansia muciniphila (AM06 and / or AM02) described in the present invention. The modification methods include but are not limited to gene editing, chemical treatment, physical treatment, etc. The modified Akkermansia muciniphila forms a new strain different from AM06 and AM02. This modified strain may still have the function of beneficially reducing body weight and may also be endowed with other new physiological functions.

[0112] In some embodiments, the composition containing Akkermansia muciniphila does not include a pharmaceutically active ingredient.

[0113] In the present invention, the "pharmaceutically active ingredient" refers to a pharmaceutically active ingredient having the effect of preventing and treating obesity.

[0114] In the present invention, a "drug" includes any medicament, compound, composition or mixture that provides a pharmacological effect in vivo or in vitro, and usually provides a beneficial effect. The range of the pharmacological effect produced by the "drug" in vivo is not particularly limited and can be a systemic effect or only a local effect. The activity of the "drug" is not particularly limited and can be an active substance that can interact with other substances or an inert substance that does not interact.

[0115] In some embodiments, the composition containing Akkermansia muciniphila is a compound probiotic. At this time, in addition to containing Akkermansia muciniphila (AM06 and / or AM02) described in the present invention, the composition also contains other probiotics different from Akkermansia muciniphila described in the present invention.

[0116] In the present invention, a "compound probiotic" contains at least two probiotics. It should be understood that the "compound probiotic containing Akkermansia muciniphila" includes Akkermansia muciniphila described in the present invention and also includes other probiotics different from Akkermansia muciniphila described in the present invention.

[0117] In some embodiments, other probiotics (different from Akkermansia muciniphila described in the present invention) may include modified strains of Akkermansia muciniphila described in the present invention and probiotics of other species. Probiotics of other species, which are different from Akkermansia muciniphila, may include, but are not limited to, one or more of Bacteroides fragilis, Christensenella, Saccharomyces boulardii, lactic acid bacteria, Enterococcus lactis, Streptococcus, Clostridium perfringens, Staphylococcus, Bifidobacterium, etc. These probiotics of other species can antagonize pathogenic microorganisms, compete for adhesion to the intestinal mucosa, affect the regulation of the gastrointestinal immune system, or affect the gene expression of obese patients. Studies have shown that when used alone or in combination, lactic acid bacteria, Bifidobacterium, and Pediococcus pentosaceus in adults can significantly reduce body weight, BMI, waist circumference, and fat mass.

[0118] In some embodiments, the composite probiotics include multiple strains of Akkermansia muciniphila.

[0119] In some embodiments, the composite probiotics are composed of at least one of Akkermansia muciniphila AM06 and AM02, and at least one of the modified strains of AM06 and the modified strains of AM02.

[0120] In some embodiments, the composite probiotics include Akkermansia muciniphila described in the present invention and also include probiotics of other species. The definition of probiotics of other species is as above. Further, it also includes the modified strains of Akkermansia muciniphila described in the present invention.

[0121] In the present invention, the components in the "composition containing Akkermansia muciniphila" can appear at any suitable time. For example, it can be directly formulated into a ready-to-use mixture, or it can be separately packaged and then formulated into a mixture during use, or it can be separately administered to the subject and appear simultaneously at a local position in the body and exert a combined effect. The "composition containing Akkermansia muciniphila" that appears at any suitable time is within the protection scope of the present invention.

[0122] The third aspect of the present invention

[0123] In the third aspect of the present invention, there is provided a composition containing Akkermansia muciniphila;

[0124] Among them, the definition of the "composition containing Akkermansia muciniphila" can refer to the second aspect of the present invention.

[0125] In some embodiments, the composition containing Akkermansia muciniphila in this aspect further includes a second active ingredient. The definition of the "second active ingredient" can refer to the second aspect of the present invention.

[0126] In some embodiments, the composition containing Akkermansia muciniphila of this aspect comprises the Akkermansia muciniphila described in the present invention and a second active ingredient. The definition of "second active ingredient" can refer to the second aspect of the present invention.

[0127] The fourth aspect of the present invention

[0128] In the fourth aspect of the present invention, a method for beneficially reducing body weight is also provided, which includes administering a health-effective amount of the Akkermansia muciniphila described in the present invention to a subject, or administering a health-care composition containing the Akkermansia muciniphila described in the present invention to a subject, or administering a health-care food containing the Akkermansia muciniphila described in the present invention to a subject.

[0129] In the fourth aspect of the present invention, the definitions of "Akkermansia muciniphila", "the Akkermansia muciniphila described in the present invention", "composition", "beneficially reducing body weight", "health-effective amount", "health-care composition", and "health-care food" can refer to the first aspect or the second aspect of the present invention.

[0130] Some specific embodiments are provided below.

[0131] The embodiments of the present invention will be described in detail below in conjunction with the examples. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. For the experimental methods without specific conditions noted in the following examples, the guidance given in the present invention is preferably referred to, and it can also be carried out according to the experimental manuals or conventional conditions in the art, or according to the conditions recommended by the manufacturers, or referring to the experimental methods known in the art.

[0132] In the following specific examples, for the measurement parameters of raw material components, if not otherwise specified, there may be slight deviations within the weighing accuracy range. For the temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0133] In the following examples, a 10-fold serial dilution means a dilution method in which each dilution is 10-fold. The unit "day" can be expressed as "d".

[0134] Example 1. Isolation and identification of Akkermansia muciniphila

[0135] 1.1. Isolation and identification of strain AM02

[0136] Using a sterile sampling spoon, take feces the size of a soybean (the sample is from an adult healthy male) into a 10 mL centrifuge tube. After sampling, immediately transfer the sample to an anaerobic workstation at 37 °C (85% N 2 、10% H 2 、5% CO 2, volume percentage), dilute the sample to 10 in a 10-fold gradient dilution -9 , take 1 mL of each dilution solution and inoculate it into 9 mL of a basal medium with mucin as the sole carbon source, and anaerobically culture for 7 days. Take 10 -4 The culture solution inoculated at the dilution factor, and dilute the culture solution to 10 in a 1:10 gradient dilution method -6 , respectively take 100 μL of each dilution and spread it on the mucin agar medium, anaerobically culture for 7 days, pick a single colony and inoculate it into 2 mL of BHI broth (medium containing N-acetyl-D-glucosamine). Perform 16s rRNA sequencing on the cultured bacterial solution, and perform sequence alignment of the 16S rRNA sequence on the NCBI database. The isolated strain was identified as Akkermansia muciniphila, and the 16S rRNA sequencing results are shown below (SEQ ID No.1):

[0137]

[0138] The alignment result of this sequence with the 16S rRNA sequence of ATCC BAA-835 on NCBI shows that the Per.Ident value is 99.43%.

[0139] 1.2. Isolation and identification of strain AM06

[0140] The freshly collected breast milk samples (from adult healthy women) were immediately injected into 5 mL anaerobic vials for preservation, and then the samples were transferred to an anaerobic workstation at 37°C (85% N 2 、10% H 2 、5% CO 2 , by volume percentage), and the samples were diluted to 10 -6 in 10-fold serial dilutions. 1 mL of each dilution solution was inoculated into 9 mL of a basal medium with mucin as the sole carbon source and anaerobically cultured for about 1 month. 1 mL of the culture broth inoculated at a dilution of 10 -1 ~10 -4 was diluted to 10 -6 in a 1:10 dilution, and 100 μL of each dilution was spread on mucin agar medium and anaerobically cultured for 7 days. Single colonies were picked and inoculated into 2 mL of BHI broth (containing N-acetyl-D-glucosamine medium). The cultured bacterial solution was identified by 16S rRNA sequencing. The 16S rRNA sequence was aligned with the NCBI database, and the result was identified as Akkermansia muciniphila. The sequencing result of 16S rRNA is as follows (SEQ ID No.2):

[0141]

[0142] The alignment result of this sequence with the 16S rRNA sequence of ATCC BAA-835 on NCBI shows that the Per.Ident value is 99.22%.

[0143] 1.3. Strain preservation information

[0144] Akkermansia muciniphila AM02, with the taxonomic name Akkermansia muciniphila, was deposited on June 28, 2021, at the General Microbiology Center of the China National Center for Biotechnology Development, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 22794; the strain was received and registered by the deposit center on June 28, 2021, and was detected as a viable strain by the deposit center on June 28, 2021.

[0145] Akkermansia muciniphila AM06, with the taxonomic name Akkermansia muciniphila, was deposited on June 28, 2021, at the General Microbiology Center of the China National Center for Biotechnology Development, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 22793; the strain was received and registered by the deposit center on June 28, 2021, and was detected as a viable strain by the deposit center on June 28, 2021.

[0146] Example 2. Cultivation and preparation of heat-inactivated Akkermansia muciniphila

[0147] 2.1. Streak-inoculate the Akkermansia muciniphila strain on a BHA plate and incubate anaerobically for 3 days. Observe the colony morphological characteristics, staining properties, size, coccobacillus shape, and distribution.

[0148] Colony characteristics: After culturing Akkermansia muciniphila AM02 and AM06 on the above medium for 3 days, both showed circular raised, neat-edged, opaque, white, and uneven-sized colonies, with a colony size of approximately 0.08 - 2.2 mm. See Figure 1 (Akkermansia muciniphila AM02) and Figure 2 (Akkermansia muciniphila AM06).

[0149] Morphology under the microscope: Gram staining microscopy of Akkermansia muciniphila AM02 and AM06 showed Gram-negative bacteria, oval-shaped, arranged singly or in chains. See Figure 3 (Akkermansia muciniphila AM02) and Figure 4 (Akkermansia muciniphila AM06).

[0150] The colony characteristics of the standard strain ATCC BAA-835 are: round and raised colonies with neat edges, opaque, white, and of uneven size. Under the microscope, the morphology is Gram-negative bacteria, oval, arranged singly or in chains.

[0151] 2.2. Select a single colony and inoculate it into BHI broth for culturing for 48 hours (temperature is 37°C). The obtained bacterial solution is centrifuged and precipitated at a rotation speed of 16,000×g for 30 minutes. Remove the supernatant and collect the precipitate to obtain the Akkermansia muciniphila bacterial sludge. The Akkermansia muciniphila strains AM02, AM06, and ATCC BAA-835 are cultured respectively.

[0152] 2.3. Preparation of inactivated bacteria: Take an appropriate amount of bacterial sludge, add physiological saline according to the ratio of bacterial sludge: physiological saline (m:v) = 1:10, and use a stirrer to stir at 1000 rpm for 5 - 20 minutes to evenly disperse the bacterial cells. Take 100 mL of the dispersed bacterial solution and place it in a sterile three-necked round-bottom flask (try to ensure that the bacterial solution does not adhere to the inner wall of the flask). Place the three-necked round-bottom flask on the heating plate of a magnetic stirrer, put in a sterile magnetic stir bar, and insert a temperature electrode. Set the rotation speed at 300 - 500 rpm and the temperature at 70°C, and heat for 30 minutes to obtain the inactivated Akkermansia muciniphila bacteria.

[0153] Example 3. Non-targeted metabolomics difference analysis of the culture supernatant of Akkermansia muciniphila

[0154] 3.1. Sample preparation

[0155] Take the culture supernatants of each Akkermansia muciniphila (AM02, AM06, ATCC BAA-835) after the culture in Example 2. After taking 1 mL of each bacterial solution and centrifuging it at 12,000 rpm for 5 minutes, the supernatant is filtered through a 0.22 μm filter membrane, and the filtrate is taken as the sample to be tested for non-targeted metabolomics analysis. Five parallel samples to be tested are prepared for each strain.

[0156] 3.2. Experimental results

[0157] PCA is a data dimensionality reduction method, that is, reducing multiple variables to a set of new comprehensive variables, and then selecting the first few principal components that can reflect as much information of the original variables as possible to achieve the purpose of dimensionality reduction. The PCA graph reflects the true distribution of the samples, mainly used to observe the separation trend between sample groups and whether there are abnormal points, and at the same time reflects the variability between groups and within groups from the original data.

[0158] The experimental results can be seen in Figure 5 . Figure 5It includes the PCA analysis of quality control samples (QC samples) and all samples. Each QC sample clustered together in the two principal component analysis plots, indicating that the instrument was stable during the detection and the repeatability of the collected data was good. At the same time, the results also showed that the metabolites in the culture supernatant of AM06 were closer to those in the culture supernatant of BAA-835, while there were greater differences in the culture supernatant metabolites between AM02 and BAA-835.

[0159] The comparison results of the number of differential metabolites among different strains can be seen in Table 1. It can be seen that compared with the standard strain BAA-835, the numbers of differential metabolites detected in the positive ion (pos) mode and the negative ion (neg) mode for AM02 were 205 and 135 respectively, and for AM06 were 111 and 62 respectively.

[0160] Table 1. Statistical table of differential metabolites

[0161]

[0162] Example 4. Efficacy verification of Akkermansia muciniphila

[0163] 4.1. Tolerance of Akkermansia muciniphila to artificial gastric juice

[0164] 4.1.1. Experimental method and grouping

[0165] Table 2. Experimental grouping

[0166]

[0167] Table 3. Experimental method

[0168]

[0169] “+” indicates that detection is required.

[0170] 4.1.1.1. Take 1 tube of Akkermansia muciniphila strain, remove the label, wipe and disinfect the outer surface of the glycerol cryopreservation tube with 75% (v / v) alcohol, vortex and mix well, then open it. Pipette 100 - 500 μL of the bacterial solution and inoculate it into 10 mL / tube of BHI broth, shake well. Prepare 3 tubes in total, and at the same time, use the non-inoculated one as the negative control. Incubate at 37°C anaerobically for 2 - 4 days to obtain the primary seed solution.

[0171] The primary seed solution is subjected to Gram staining and microscopic examination, and it should be G - bacilli, without spores and without contaminants.

[0172] 4.1.1.2. Take 10 mL of the above-mentioned primary seed liquid, centrifuge it at 12,000×g for 10 min at 4°C, discard the supernatant, add 1 mL of 0.9 wt% NaCl solution to resuspend, and prepare bacterial solutions respectively for standby.

[0173] 4.1.1.3. According to Tables 2 and 3, add the bacterial solutions of AM06, AM02 and the standard strain into artificial gastric juices with 0.9 wt% NaCl, pH 3.0 and pH 2.0 respectively, mix well, dispense into 5 mL / tube, take out and detect the bacterial concentration of each sample after incubating at 37°C for 0 h, 1.5 h and 3 h in an anaerobic glove box. Do 3 parallels for each experimental group.

[0174] 4.1.1.4. Determination of viable bacteria count:

[0175] Take the experimental samples, dilute them in a 10-fold serial dilution to different dilution degrees, take 100 μL of the dilution and inoculate it onto BHA plates, spread it evenly. Do 2 plates for each dilution degree, generally do 2 - 3 dilution degrees. At the same time, take 100 μL of the dilution and place it on a BHA plate as a negative control. All the spread plates are placed upright and cultured under anaerobic conditions for about 3 - 5 days. Observe the colony growth on the plates and count.

[0176] Calculate the viable bacteria count according to the sum of the colony counts on 2 plates using the following formula:

[0177] Viable bacteria count (CFU / mL) = sum of the colony counts on 2 plates / 2 × 10 × final dilution degree

[0178] Calculation of survival rate:

[0179]

[0180] 4.1.2. Experimental results

[0181] The experimental results can be seen in Table 4. The tolerance of different Akkermansia muciniphila strains to artificial gastric juice from high to low is AM02 > AM06 > standard strain BAA - 835.

[0182] Table 4. Statistical table of survival rate results of the tolerance of Akkermansia muciniphila to artificial gastric juice

[0183]

[0184] 4.2. Tolerance of Akkermansia muciniphila to artificial intestinal juice

[0185] 4.2.1. Experimental methods and grouping

[0186] Table 5. Experimental grouping

[0187]

[0188] 4.2.1.1. Preparation of primary seed liquid

[0189] Take 1 tube of Akkermansia muciniphila strain, remove the label, wipe and disinfect the outer surface of the glycerol cryopreservation tube with 75% (v / v) alcohol, vortex and mix well, then open it. Pipette 100 μL of the bacterial liquid and inoculate it into BHI broth at 10 mL / tube, shake well. A total of 3 tubes are prepared, and at the same time, a non-inoculated negative control is set up. Incubate anaerobically at 37 °C for 2 - 4 days to obtain the primary seed liquid.

[0190] The primary seed liquid is subjected to Gram staining and microscopic examination, and it should be Gram-negative bacilli, without spores and without contaminants.

[0191] 4.2.1.2. Preparation of bacterial sludge

[0192] Aliquot the above-mentioned primary seed liquid into tubes at 1.5 mL / tube, centrifuge at 12000×g for 10 min, discard the supernatant to obtain the bacterial sludge. Prepare 3 tubes of bacterial sludge for each of AM06, AM02 and the standard strain.

[0193] 4.2.1.3. Evaluation of the tolerance of the strain to artificial intestinal fluid

[0194] As shown in Table 5 and Table 6, add 1.5 mL of artificial intestinal fluid to each tube of the prepared bacterial sludge, mix well, then aliquot each tube of the solution at 0.5 mL / branch, prepare 3 tubes, incubate anaerobically at 37 °C for 0, 4 h and 8 h respectively, and take samples to detect the viable cell count. Each group is done in 3 parallels.

[0195] Table 6. Experimental method

[0196]

[0197] “+” indicates that detection is required.

[0198] 4.2.1.4. Determination of viable cell count

[0199] Take the incubated samples respectively, after serial dilution at 10-fold gradient, take 100 μL of the diluted solution and inoculate it onto BHA plates, spread evenly. A total of 2 plates are made for each dilution degree, generally 2 - 3 dilution degrees are done. At the same time, take 100 μL of the diluted solution onto BHA plates as the negative control. All spread plates are placed upright and cultured under anaerobic conditions for about 3 - 5 days, observe the colony growth on the plates and count.

[0200] Viable cell count (CFU / mL) = sum of the colony counts of 2 plates / 2 × 10 × final dilution factor

[0201] Calculation of survival rate:

[0202] Survival rate = viable cell count at each time point / viable cell count at 0 h × 100%

[0203] 4.2.2. Experimental results

[0204] The results can be seen in Table 7. Strains ATCC BAA-835, AM02, and AM06 have good tolerance to artificial intestinal fluid, and the tolerance from high to low is AM02 > AM06 > standard strain BAA-835 in turn.

[0205] Table 7. Statistical table of survival rate

[0206]

[0207] 4.3. Effects of Akkermansia muciniphila on the expression of tight junction protein ZO-1 in Caco2 cells induced by TNF-α and IFN-γ

[0208] 4.3.1. Experimental methods and grouping

[0209] Caco2 cells were inoculated into 96-well plates and cultured until the confluence reached 80% - 90%. After inducing Caco2 cells with 100 ng / mL TNF-α + 100 ng / mL IFN-γ for 24 h, AM02, AM06, and BAA-835 were added respectively and incubated with the cells for another 24 h. The experimental grouping can be seen in Table 8, and 5 replicate wells were set for each group. Immunofluorescence was used to observe the effects of BAA-835, AM06, and AM02 on the expression of tight junction protein ZO-1 in Caco2 cells induced by TNF-α and IFN-γ.

[0210] Table 8. Experimental grouping

[0211]

[0212]

[0213] 4.3.2. Experimental results

[0214] The fluorescence intensity of the taken pictures was statistically analyzed, and the results can be seen in Figure 6 and Table 9. Compared with the blank control group, after inducing with TNF-α and IFN-γ for 48 h, the fluorescence intensity of the cell gap in the inflammation model group was significantly weakened, indicating a decrease in the expression of ZO-1 protein and the disruption of tight junctions between cells. Compared with the inflammation model group, after intervention with AM02, AM06, and BAA-835, the fluorescence intensity of the treatment group of cells increased significantly (p < 0.05), and the ability of AM02 and AM06 to inhibit the reduction of ZO-1 protein in Caco2 cells induced by inflammatory factors was significantly better than that of BAA-825 (p < 0.05).

[0215] Table 9. Statistical table of fluorescence intensity

[0216] Number Group Fluorescence intensity A Blank control group 36.2647±5.8977** B Inflammatory model group 25.6588±4.5303 C AM02 group 41.7059±6.5664**aa D AM06 group 42.2949±4.5437**aa E BAA-835 group 33.6688±4.13336**

[0217] Note: ** indicates that compared with the model group, the difference is extremely significant p < 0.01; aa indicates that compared with the BAA-835 group, the difference is extremely significant p < 0.01.

[0218] Example 5.

[0219] Experimental design and methods

[0220] After SD rats were adaptively fed with the basic diet for 3 days, they were randomly divided into 2 groups: a normal group (10 rats) and a modeling group (120 rats), with half males and half females in each group. The normal group was fed with the basic diet, and the modeling group was fed with the nutritional obesity modeling diet. The rats were weighed once a week for 10 weeks. When the obesity degree was greater than 20%, it was considered obese, indicating that the obesity model was successfully established.

[0221] After the model was established, 90 rats with successful model were selected and randomly divided into 9 groups, with 10 rats in each group. The groups were: high-fat group, positive group (orlistat, 0.044 g / kg / d), AM06 high-dose live bacteria group (10 10 CFU / mL AM06), AM06 medium-dose live bacteria group (10 8 CFU / mL AM06), AM06 low-dose group (10 6 CFU / mL AM06), AM02 high-dose live bacteria group (10 10 CFU / mL AM02), AM02 medium-dose live bacteria group (10 8 CFU / mL AM02), AM02 low-dose live bacteria group (10 6 CFU / mLAM02) and ATCC BAA-835(10 10 CFU / mL) live bacteria group. Meanwhile, the above normal group was used as a blank control group. The following experimental groups were given active ingredients in the morning and evening: high-fat group, positive group, AM06 high-dose live bacteria group, AM06 medium-dose live bacteria group, AM06 low-dose group, AM02 high-dose live bacteria group, AM02 medium-dose live bacteria group, AM02 low-dose live bacteria group and ATCC BAA-835 live bacteria group; in addition, the blank group and the model group were given normal saline. Akkermansia muciniphila was prepared by the method of Example 1.

[0222] After 6 weeks, the rats in each group were weighed, their body lengths were measured, and Lee's index [Lee's index = body weight 1 / 3 (g)×10 3 / Body length (cm)], as much as possible, remove all the fat around the kidneys and in the pelvic cavity, wash away the blood stains with physiological saline, and dry it with filter paper. Weigh the wet weight of the fat and calculate the fat body ratio [Fat body ratio = total fat mass / body weight × 100%]. Take blood from the femoral artery and detect the contents of serum triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C).

[0223] 5.2. Experimental results

[0224] 5.2.1. Body weight, body length and Lee's index of rats

[0225] The detection results of the body weight, body length and Lee's index of rats can be seen in Table 10. Compared with the blank group, the body weight and Lee's index of the rats in the model group increased significantly; compared with the model group, the body weight and Lee's index of the experimental groups given active ingredients decreased to some extent. Among them, the descending range was AM06 > AM02 > positive group > BAA-835 group.

[0226] Table 10. Effects of Akkermansia muciniphila on body weight, body length and Lee's index of rats (mean±SD, n = 10)

[0227]

[0228]

[0229] Note: * indicates significant difference compared with the model group, p < 0.05.

[0230] 5.2.2. Total fat mass and fat body ratio

[0231] The detection results of the total fat mass and fat body ratio can be seen in Table 11. Compared with the blank group, the total fat mass and fat body ratio of the rats in the model group increased significantly; compared with the rats in the model group, the total fat mass and fat body ratio of Akkermansia muciniphila decreased to varying degrees. Among them, the descending range was AM06 > AM02 > positive group > BAA-835 group.

[0232] Table 11. Effects of Akkermansia muciniphila on total fat mass and fat body ratio of rats (mean±SD, n = 10)

[0233]

[0234] Note: * indicates significant difference compared with the model group, p < 0.05; ** indicates extremely significant difference compared with the model group, p < 0.01.

[0235] 5.2.3. Serum lipid biochemical indicators (TC, TG, LDL-C, HDL-C)

[0236] According to the kit instructions, the contents of low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), thyroglobulin (TG), and total cholesterol (TC) in rats were detected. The results are shown in Table 12. Compared with the blank group, the contents of serum TC, TG, and LDL-C in the model group rats were significantly increased, while the content of HDL-C was significantly decreased. Compared with the model group, the contents of serum TC, TG, and LDL-C in rats treated with intragastric administration of Akkermansia muciniphila decreased to varying degrees, while the content of HDL-C increased. The change range was AM06 > AM02 > positive group > BAA-835 group.

[0237] In summary, Akkermansia muciniphila can effectively improve obesity in rats, and the effect is AM06 > AM02 > positive group > BAA-835 group.

[0238] Table 12. Effects of Akkermansia muciniphila on various serum biochemical indexes in rats (mean±SD, n = 10)

[0239]

[0240] Note: * indicates significant difference compared with the model group, p < 0.05.

[0241] Example 6.

[0242] 6.1. Experimental design and methods

[0243] Experimental grouping and administration of active ingredients: 100 male 4-week-old C57BL / 6N mice. After 1 week of adaptive feeding, they were randomly divided into 10 groups, with 10 mice in each group. The groups were: blank control group, high-fat group, positive group (orlistat, 0.044 g / kg / d), high-dose group of heat-killed AM06 (10 11 CFU / mL AM06), medium-dose group of heat-killed AM06 (10 9 CFU / mL AM06), low-dose group of heat-killed AM06 (10 7 CFU / mL AM06), high-dose group of heat-killed AM02 (10 11 CFU / mL AM02), medium-dose group of heat-killed AM02 (10 9 CFU / mL AM02), low-dose group of heat-killed AM02 (10 7 CFU / mL AM02), and ATCC BAA-835 (10 11CFU / mL) group. The blank control group was fed with a basic feed, and the other groups were fed with a nutritional obesity modeling feed, and the other feeding conditions were the same. Obesity is considered when the obesity degree is greater than 20%, indicating that the obesity model was successfully established. The blank group and the model group were given normal saline, and the experimental group given the active ingredient was given the active ingredient in the morning and evening at the corresponding dose of the active ingredient. Muciniphila Akkermansia was prepared by the method of Example 1.

[0244] After 8 weeks of feeding, the mice were weighed on an empty stomach. After fasting for 12 hours, the mice were anesthetized with ether and blood was collected by decapitation. The serum levels of TC, TG, LDL-C, HDL-C, blood glucose and leptin were measured according to the instructions of the relevant kits. The liver and epididymal adipose tissue were removed, washed with saline, wiped dry and weighed.

[0245] 6.2 Experimental results

[0246] 6.2.1. Weight gain and fat mass in mice

[0247] As shown in Table 13, the body weight and fat weight of mice in the blank group were significantly lower than those in the model group; compared with the model group, the body weight and fat weight of mice in the experimental groups given active ingredients decreased to varying degrees, with the decrease in AM06>AM02>positive group>BAA-835 group.

[0248] Table 13. Effects of Akkermansia muciniphila on body weight and fat weight of mice (mean ± SD, n = 10)

[0249]

[0250] Note: * indicates that the difference is significant compared with the model group, p<0.05; ** indicates that the difference is extremely significant compared with the model group, p<0.01.

[0251] 6.2.2. Serum factors (TC, TG, LDL-C and HDL-C)

[0252] According to the kit instructions, the levels of low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), thyroglobulin (TG) and total cholesterol (TC) in the serum of mice were tested. The results are shown in Table 14. Compared with the blank group, the levels of TC, TG and LDL-C in the serum of the mice in the model group increased significantly, and the level of HDL-C decreased significantly. Compared with the model group, the levels of TC, TG and LDL-C in the serum of mice treated with inactivated Akkermansia muciniphila by oral gavage decreased to varying degrees, while the level of HDL-C increased to a certain extent, and the range of change was AM06>AM02>positive group>BAA-835 group.

[0253] Table 14. Effects of Akkermansia muciniphila on serum factors in mice (mean ± SD, n = 10)

[0254]

[0255] Note: * indicates significant difference compared with the model group, p < 0.05; ** indicates extremely significant difference compared with the model group, p < 0.01.

[0256] 6.2.3. Leptin and adiponectin

[0257] The contents of leptin and adiponectin in serum were measured, and the experimental results are shown in Table 15. Compared with the blank group, the leptin level of the model group mice increased significantly, while the adiponectin level decreased significantly; compared with the model group, the leptin content of each experimental group given active ingredients decreased to varying degrees, and the adiponectin content increased, among which the change range was AM06 > AM02 > positive group > BAA-835 group.

[0258] In summary, heat-inactivated Akkermansia muciniphila has an obvious inhibitory effect on the occurrence and development of obesity in rats.

[0259] Table 15. Effects of Akkermansia muciniphila on leptin and adiponectin in mice (mean ± SD, n = 10)

[0260]

[0261] Note: * indicates significant difference compared with the model group, p < 0.05; ** indicates extremely significant difference compared with the model group, p < 0.01.

[0262] Example 7. Regulatory effect of Akkermansia muciniphila lysate on obesity in mice

[0263] 7.1. Experimental design and methods

[0264] Six-week-old SPF-grade C57BL / 6J male mice were adaptively fed a high-fat diet for 7 days and then randomly divided into 2 groups: a normal group (10 mice) and a model group (50 mice). The normal group was fed a low-fat diet, and the model group was fed a high-fat diet and randomly divided into 5 groups, with 10 mice in each group. The body weights of the mice in each group were measured. Experimental grouping: blank control group (fed a low-fat diet), high-fat group (fed a high-fat diet), positive group (orlistat, 0.044 g / kg / d), AM06 lysate group, AM02 lysate group, and ATCC BAA-835 lysate group. During this period, each experimental group given active ingredients was intragastrically administered the active ingredients, and the blank group and the model group were given normal saline.

[0265] After 8 weeks, all mice were fasted overnight, weighed, and then sacrificed by cervical dislocation. Blood samples were collected. The mice were dissected, and the weights of the liver, heart, kidney, spleen, epididymal fat, peritoneal fat, and perirenal fat of the mice were measured.

[0266] Experimental Results

[0267] 7.2. Experimental Results

[0268] 7.2.1. Mouse Weight Gain and Fat Weight

[0269] As shown in Table 16, the weight gain and fat weight of mice in the model group were significantly higher than those in the blank group; compared with the model group, the weight gain and fat weight of mice in each experimental group given the active ingredient decreased to some extent, among which the AM06 and AM02 lysate groups were significantly lower than the model group, and the degree of decrease among groups was AM06 > AM02 > positive group > BAA-835 group.

[0270] Table 16. Effects of Akkermansia muciniphila on Mouse Body Weight and Fat Weight (mean±SD, n = 10)

[0271]

[0272] Note: * indicates significant difference compared with the model group, p < 0.05; ** indicates extremely significant difference compared with the model group, p < 0.01.

[0273] 7.2.2. Concentrations of TC, TG, HDL-C and LDL-C

[0274] As shown in Table 17, the contents of TC, TG and LDL-C in the serum of mice in the model group were significantly higher than those in the blank group, while HDL-C was significantly lower than that in the blank group; compared with the model group, the levels of TC, TG and LDL-C in the serum of mice in each experimental group given the active ingredient decreased to varying degrees, while HDL-C increased to some extent. Among them, the AM06 and AM02 lysate groups were significantly different from the model group, and the change range among different groups was AM06 > AM02 > positive group > BAA-835 group.

[0275] Table 17. Effects of Akkermansia muciniphila on Mouse Body Weight and Fat Weight (mean±SD, n = 10)

[0276]

[0277] Note: * indicates significant difference compared with the model group, p < 0.05; ** indicates extremely significant difference compared with the model group, p < 0.01.

[0278] 7.2.3. Activities of Serum Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST)

[0279] As shown in Table 18, compared with the blank group, the activities of AST and ALT in the model group increased significantly; compared with the model group, the activities of AST and ALT in each experimental group given the active ingredient decreased to some extent, and the degree of decrease was AM06 > AM02 > positive group > BAA-835 group.

[0280] In summary, Akkermansia muciniphila lysate has the effect of improving obesity.

[0281] Table 18. Effects of Akkermansia muciniphila lysate on body weight and fat weight of mice (mean±SD, n = 10)

[0282]

[0283] Note: * indicates significant difference compared with the model group, p < 0.05.

[0284] Example 8. Experiment on improving obesity in mice

[0285] 8.1. Experimental design and methods

[0286] After 4-week-old SPF-grade C57BL / 6J mice were acclimated to the environment for 7 days, they were randomly divided into 2 groups: a normal group (10 mice) and a model group (100 mice). The normal group was fed a low-fat diet, and the model group was fed a high-fat diet and randomly divided into 10 groups, with 10 mice in each group. The body weights of the mice in each group were measured. Experimental grouping: blank control group (fed a low-fat diet), high-fat group (fed a high-fat diet), AM06 live bacteria group, AM02 live bacteria group, AM06 inactivated bacteria group, AM02 inactivated bacteria group. During this period, the experimental groups given the active ingredient (high-fat group, AM06 live bacteria group, AM02 live bacteria group, AM06 inactivated bacteria group, and AM02 inactivated bacteria group) were given the active ingredient by gavage, and the blank group and the model group were given an equal amount of normal saline. The treatments involving Akkermansia muciniphila (live bacteria group and inactivated bacteria group) had a concentration of 10 11 CFU / mL.

[0287] After 21 weeks, all the mice were fasted overnight, weighed, and then sacrificed by cervical dislocation. Blood samples were collected. The mice were dissected, and liver samples were collected to measure the TG and TC contents.

[0288] 8.2. Experimental results

[0289] 8.2.1. Weight gain and liver weight of mice

[0290] As shown in Table 19, the weight gain and fat weight of the mice in the model group were significantly higher than those in the blank group; compared with the model group, the weight gain and fat weight of the experimental groups given the active ingredient decreased to varying degrees.

[0291] Table 19. Effects of Akkermansia muciniphila on weight gain and liver weight of mice (mean±SD, n = 10)

[0292] Group Weight gain (g) Liver weight (g) Blank group 7.33±0.18* 0.82±0.05** Model group 15.02±0.21 1.39±0.07 AM06 viable bacteria 9.28±0.14* 1.05±0.05 AM02 viable bacteria 9.94±0.31* 1.18±0.02 Inactivated AM06 10.05±0.07 1.10±0.05 Inactivated AM02 10.94±0.17 1.21±0.01

[0293] Note: * indicates significant difference compared with the model group, p < 0.05; ** indicates extremely significant difference compared with the model group, p < 0.01.

[0294] 8.2.2. TC and TG Concentrations

[0295] As shown in Table 20, the contents of TC and TG in the serum of mice in the model group were significantly higher than those in the blank group. Compared with the model group, the levels of TC and TG in the serum of experimental groups given active ingredients decreased to varying degrees.

[0296] Table 20. Effects of Akkermansia muciniphila on Plasma TC and TG Concentrations in Mice (mean ± SD, n = 10)

[0297] Group TC TG Blank group 2.81±0.19* 1.64±0.16* Model group 4.33±0.23 2.56±0.20 AM06 viable bacteria 3.09±0.13 1.77±0.06 AM02 viable bacteria 3.31±0.15 1.89±0.04 Inactivated AM06 3.20±0.21 1.85±0.03* Inactivated AM02 3.36±0.25 1.98±0.09

[0298] Note: * indicates significant difference compared with the model group, p < 0.05.

[0299] The technical features of the above embodiments and examples can be combined in any suitable way. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered to be within the scope described in this specification.

[0300] The above examples only represent several embodiments of the present invention, which are convenient for understanding the technical solutions of the present invention specifically and in detail, but should not be construed as limiting the protection scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and the equivalent forms obtained also fall within the protection scope of the present invention. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments based on the technical solutions provided by the present invention are all within the protection scope of the appended claims of the present invention. Therefore, the protection scope of this invention patent should be determined by the content of the appended claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. Use of Akkermansia muciniphila in the preparation of a health care product composition, food composition or food for reducing body weight, characterized in that, the Akkermansia muciniphila is Akkermansia muciniphila AM06, Akkermansia muciniphila AM02 or a combination of both; wherein, the Akkermansia muciniphila AM06 was deposited on June 28, 2021 at the General Microbiology Center of the China National Culture Collection Center, with the deposit number CGMCC No. 22793; the Akkermansia muciniphila AM02 was deposited on June 28, 2021 at the General Microbiology Center of the China National Culture Collection Center, with the deposit number CGMCC No. 22794.

2. The use according to claim 1, characterized in that, the Akkermansia muciniphila AM06 and the Akkermansia muciniphila AM02 are each independently viable bacteria, inactivated bacteria or a combination thereof.

3. The use according to claim 1 or 2, characterized in that, the Akkermansia muciniphila AM06 or Akkermansia muciniphila AM02 can improve one or more of the following aspects of the subject: body weight, weight gain, body length, Lee's index, total fat mass, fat mass ratio, serum lipid biochemical indices, serum leptin content, serum adiponectin content, serum alanine aminotransferase and aspartate aminotransferase activities; the serum lipid biochemical indices include TC, TG, LDL-C and HDL-C.

4. The use according to claim 1 or 2, characterized in that, the Akkermansia muciniphila effectively reduces body weight by inducing inhibition of lipid accumulation in adipocytes, improving lipoprotein lipase activity and / or hepatic lipase activity in serum, and down-regulating CCAAT / enhancer-binding protein α and / or peroxisome proliferator-activated receptor γ.

5. The use according to claim 1 or 2, characterized in that, the health care product composition includes the Akkermansia muciniphila and edible excipients; the food composition includes the Akkermansia muciniphila and edible adjuncts.

6. The use according to claim 4, characterized in that, the health care product composition is a health food, and its dosage form is pills, tablets, granules, capsules, solutions, suspensions or emulsions.

7. Use of a composition containing Akkermansia muciniphila in the preparation or as a health food for reducing body weight, characterized in that, the Akkermansia muciniphila is as defined in claim 1 or 2.

8. The application according to claim 7, wherein, the composition containing Akkermansia muciniphila is a health care product composition; optionally, the health care product composition further contains other probiotics, and the other probiotics are selected from the modified bacteria of Akkermansia muciniphila and probiotics of other species, and the probiotics of other species include one or more of lactic acid bacteria, streptococcus, Clostridium perfringens, staphylococcus, and bifidobacterium.

9. The application according to claim 7 or 8, wherein, the composition containing Akkermansia muciniphila further includes a second active ingredient, and the second active ingredient does not include a drug active ingredient.

10. The application according to claim 7, wherein, the composition containing Akkermansia muciniphila is a compound probiotic, and the compound probiotic further includes a probiotic different from Akkermansia muciniphila; preferably, the probiotic different from Akkermansia muciniphila includes one or more of Bacteroides fragilis, Saccharomyces boulardii, Christensenella, Enterococcus hirae, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus gasseri, Lactobacillus plantarum, Lactobacillus curvatus, and bifidobacterium.

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