Application of Ackermann muciniphila in preparation of health care product composition for improving blood sugar level, composition and application thereof

Abstract

The invention relates to an application of Ackermann mucilaginosus in preparation of a health care product composition for improving the blood sugar level. 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 Ackermann muciniphile AM06 is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number of the Ackermann muciniphile AM06 is CGMCC No.22793; the Ackermann muciniphile AM02 is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number of the Ackermann muciniphile AM02 is CGMCC No.22794. In the application, blood sugar can be reduced, insulin resistance can be reduced, and islet inflammation can be reduced. The invention also relates to an application of the ackermann containing the mucophilic protein.

Description

[0001] Related Applications

[0002] This application is a divisional application of the Chinese patent application filed on June 8, 2022, with application number 2022106412230, and entitled "Application of Akkermansia muciniphila in the preparation of pharmaceutical compositions for the prevention and treatment of diabetes, compositions and their applications", the entire text of which is hereby incorporated by reference. Technical Field

[0003] The present invention relates to the technical field of health care products, in particular to the use of Akkermansia muciniphila in the preparation of a health care product composition for improving blood sugar levels, and also to the use of a composition containing Akkermansia muciniphila in the preparation of or as a health food for improving blood sugar levels. Background Art

[0004] With the development of society, the improvement of material living standards and the change of lifestyle, coupled with the sharp increase in the number of elderly people and the aggravation of the aging structure, the demand for improving blood sugar levels is increasing. Summary of the invention

[0005] Based on this, the invention objective of the present application includes providing the use of Akkermansia muciniphila and a composition containing the Akkermansia muciniphila in the preparation of a health care product composition / food for improving blood sugar level or liver fat accumulation, wherein the Akkermansia muciniphila is Akkermansia muciniphila AM06, Akkermansia muciniphila AM02 or a combination of the two; wherein,

[0006] The Akkermansia muciniphila AM06 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on June 28, 2021, with the deposit number of CGMCC No.22793;

[0007] The Akkermansia muciniphila AM02 was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on June 28, 2021, with the deposit number CGMCC No.22794.

[0008] In a first aspect of the present invention, there is provided a use of Akkermansia muciniphila in preparing a health care product composition for improving blood sugar levels, wherein the Akkermansia muciniphila is Akkermansia muciniphila AM06, Akkermansia muciniphila AM02 or a combination thereof; wherein,

[0009] The Akkermansia muciniphila AM06 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on June 28, 2021, with the deposit number of CGMCC No.22793;

[0010] The Akkermansia muciniphila AM02 was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on June 28, 2021, with the deposit number CGMCC No.22794.

[0011] In some embodiments, the Akkermansia muciniphila AM06 and the Akkermansia muciniphila AM02 are each independently live bacteria, inactivated bacteria, or a combination of live bacteria and inactivated bacteria.

[0012] In some embodiments, the manner of improving blood glucose levels comprises at least one of inducing secretion of GLP-1 and reducing insulin resistance.

[0013] In some embodiments, the liver fat accumulation is a reduction in liver steatosis score.

[0014] In some embodiments, the health care product composition includes the Akkermansia muciniphila and edible raw materials and auxiliary materials.

[0015] 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.

[0016] In a second aspect of the present invention, there is provided a use of a composition containing Akkermansia muciniphila in the preparation of or as a health food for improving blood sugar levels, wherein the Akkermansia muciniphila is as defined in the first aspect of the present invention.

[0017] In some embodiments, the composition comprising Akkermansia muciniphila further comprises a second active ingredient; and / or,

[0018] The composition containing Akkermansia muciniphila is a composite probiotic, and the composite probiotic also includes probiotics different from the Akkermansia muciniphila.

[0019] In a third aspect of the present invention, a composition containing Akkermansia muciniphila is provided, wherein the Akkermansia muciniphila is as defined in the first aspect of the present invention; further, the composition containing Akkermansia muciniphila further comprises a second active ingredient.

[0020] The inventors of the present application isolated strains AM06 (deposit number CGMCC No. 22793) and AM02 (deposit number CGMCC No. 22794). According to comprehensive analyses 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 tight junction proteins of intestinal cells, and the effect of inhibiting LPS-induced liver slice hepatitis), both strains belong to Akkermansia muciniphila, and are identified as new strains of Akkermansia muciniphila different from ATCC BAA-835 (standard strain) and other Akkermansia muciniphila.

[0021] The inventors have found that the above-isolated Akkermansia muciniphila AM06 and / or AM02 can be used to improve blood sugar levels. Therefore, the above-isolated Akkermansia muciniphila AM06 and / or AM02 can be used to prepare health care product compositions, and further can be used to prepare health care foods. The above-isolated Akkermansia muciniphila AM06 and / or AM02 can lower blood sugar, reduce insulin resistance, and reduce insulitis. Through experimental analysis, the applicant believes that the above-isolated Akkermansia muciniphila AM06 and / or AM02 can enhance the intestinal barrier and induce intestinal L cells to secrete GLP-1, thereby lowering blood sugar, reducing insulin resistance, and reducing insulitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0023] Figure 1 This is a colony characteristic diagram of Akkermansia muciniphila AM02 cultured in one embodiment of the present invention;

[0024] Figure 2 This is a colony characteristic diagram of Akkermansia muciniphila AM06 obtained in one embodiment of the present invention;

[0025] Figure 3 This is a microscopic observation picture of Akkermansia muciniphila AM02 cultured in one embodiment of the present invention after Gram staining;

[0026] Figure 4 This is a microscopic observation picture of Akkermansia muciniphila AM06 cultured in one embodiment of the present invention after Gram staining;

[0027] Figure 5This is a PCA analysis diagram of metabolites of several Akkermansia muciniphila culture supernatants in one embodiment of the present invention;

[0028] Figure 6 This is a fluorescence microscope image showing the effect of several muciniphilic Akkermansia on the reduction of tight junction protein ZO-1 expression induced by TNF-α and IFN-γ in Caco2 cells according to an embodiment of the present invention.

[0029] The Akkermansia muciniphila AM06 isolated in the present invention is classified and named Akkermansia muciniphila. It was deposited in the General Microbiology Center of China Culture Collection Administration on June 28, 2021, with the address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 22793; the strain was received and registered by the collection center on June 28, 2021, and was detected as a viable strain by the collection center on June 28, 2021.

[0030] The Akkermansia muciniphila AM02 isolated in the present invention is classified and named Akkermansia muciniphila, and was deposited in the General Microbiology Center of China Culture Collection Administration on June 28, 2021, with the address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 22794; the strain was received and registered by the collection center on June 28, 2021, and was detected as a viable strain by the collection center on June 28, 2021. DETAILED DESCRIPTION

[0031] The present invention will be further described in detail below in conjunction with the accompanying drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The purpose of providing these embodiments and examples is to make the understanding of the disclosure 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 embodiments and examples described herein. Those skilled in the art can make various changes or modifications without violating the connotation of the present invention, and the equivalent form obtained also falls within the protection scope of the present invention. In addition, in the following description, a large number of specific details are given in order to provide a more comprehensive understanding of the present invention. It should be understood that the present invention can be implemented without one or more of these details.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing implementation modes and embodiments and are not intended to limit the present invention.

[0033] the term

[0034] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0035] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of 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, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0036] In the present invention, "plurality", "multiple", "multiple times", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0037] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0038] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of the present invention, solve the technical problem of the present invention, and achieve the expected technical effect of the present invention.

[0039] Herein, "preferred", "better", "more preferred" and "suitable" are only used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute a limitation on the scope of protection of the present invention. If multiple "preferred" items appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "preferred" item is independent.

[0040] In the present invention, “further”, “furthermore”, “particularly”, etc. are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present invention.

[0041] In the present invention, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel solutions of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0042] In the present invention, in the "first aspect", "second aspect", "third aspect", "fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0043] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0044] In the present invention, when it comes to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e., the minimum value and the maximum value) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers in the numerical interval, including the two endpoint integers of the numerical range, and each integer between the two endpoints, is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all sub-ranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows for broadly including quantitative intervals such as percentage intervals, ratio intervals, and ratio intervals.

[0045] The temperature parameters in the present invention, if not specifically limited, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0046] 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.

[0047] In the present invention, when it comes to the unit of the data range, if there is a unit only after the right endpoint, it means that the units of the left endpoint and the right endpoint are the same. For example, 3-5h means that the units of the left endpoint "3" and the right endpoint "5" are both h (hours).

[0048] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as a reference separately. Unless they conflict with the invention purpose and / or technical solution of the present application, the cited documents involved in the present invention are cited with all contents and all purposes. When the present invention involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present invention involves cited documents, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement the present invention. It should be understood that when the content of the citation conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0049] In the present invention, "Akkermansia muciniphila" can be a living bacterium, or it can be an Akkermansia muciniphila that has been inactivated, genetically recombined, transformed or modified, attenuated, chemically treated, or physically treated and retains all or part of its original biological activity. It can also be a bacterial lysate, culture (such as supernatant) or a component extracted from the culture.

[0050] A first aspect of the present invention

[0051] In a first aspect of the present invention, there is provided a use of Akkermansia muciniphila in preparing a health care product composition for improving blood sugar levels, wherein the Akkermansia muciniphila is Akkermansia muciniphila AM06, Akkermansia muciniphila AM02 or a combination of the two.

[0052] The inventors of the present application isolated strains AM06 (deposit number CGMCC No. 22793) and AM02 (deposit number CGMCC No. 22794). According to comprehensive analyses 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 tight junction proteins of intestinal cells, and the effect of inhibiting LPS-induced liver slice hepatitis), both strains belong to Akkermansia muciniphila, and are identified as new strains of Akkermansia muciniphila different from ATCC BAA-835 (standard strain) and other Akkermansia muciniphila.

[0053] In the present invention, "Akermansia muciniphila" is consistent with the common definition in the art, including but not limited to Akkermansia muciniphila AM06 and AM02.

[0054] Akkermansia muciniphila (A. muciniphila) belongs to the phylum Verucomicrobia, family Akkermansiaceae, genus Akkermansia. A. muciniphila is a mucin-degrading bacterium. It is an anaerobic, non-motile, non-spore-forming, oval Gram-negative bacterium that grows at 20-40°C and pH 5.5-8.0 (optimum temperature 37°C, optimum pH 6.5). It is commonly found in the human intestine. Although it is an anaerobic bacterium, it can also tolerate a certain degree of oxygen. A. muciniphila can colonize in the mucus layer and colon by degrading and utilizing mucin as an energy source. Furthermore, it can also produce short-chain fatty acids (SCFA) through mucin degradation. The production of these short-chain fatty acids (including acetate, propionate and butyrate) plays an important role in human health.

[0055] In the present invention, “Akermansia muciniphila described in the present invention” specifically refers to Akkermansia muciniphila AM06, Akkermansia muciniphila AM02 or a combination of the two, which can be recorded as “AM06 and / or AM02”.

[0056] Akkermansia muciniphila AM06 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on June 28, 2021, with the deposit number CGMCC No. 22793. It can be isolated by the method of Example 1, and the strain can also be identified by one or more methods including but not limited to Examples 2 to 4.

[0057] In some embodiments, the colony culture characteristics of Akkermansia muciniphila AM06 include: round protrusions, neat edges, opaque, white, and unevenly sized colonies, and further, the colony size is about 0.08 to 2.2 mm.

[0058] In some embodiments, Akkermansia muciniphila AM06 is isolated from breast milk.

[0059] Akkermansia muciniphila AM02 was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on June 28, 2021, with the deposit number CGMCC No. 22794. It can be isolated by the method of Example 1, and the strain can also be identified by one or more methods including but not limited to Examples 2 to 4.

[0060] In some embodiments, the colony culture characteristics of Akkermansia muciniphila AM02 of CGMCC No. 22794 include: round protrusions, neat edges, opaque, white, and unevenly sized colonies, and further, the colony size is about 0.08 to 2.2 mm.

[0061] In some embodiments, the applicant also conducted an artificial gastric juice and artificial intestinal juice tolerance test. Among them, in the artificial gastric juice tolerance test, the number of live bacteria in different groups of 0.9wt% NaCl solution group, pH3 artificial gastric juice group and pH2 artificial gastric juice group was statistically analyzed after anaerobic incubation at 37±2℃ for 0h, 1.5h and 3h. The results showed that the artificial gastric juice tolerance of Akkermansia muciniphila strain was AM02>AM06>standard strain ATCC BAA-835. Among them, in the artificial intestinal juice tolerance test experiment, the number of live bacteria after anaerobic incubation at 37±2℃ for 0h, 4h and 8h was statistically analyzed, and the artificial intestinal juice tolerance was better than AM06>AM02>ATCC BAA-835.

[0062] In some in vitro experiments, Akkermansia muciniphila AM06 and AM02 were superior to the standard strain BAA-835 in terms of tolerance to artificial gastric juice and artificial intestinal juice, ability to inhibit inflammatory factors from destroying tight junction proteins of intestinal cells, and effect of inhibiting LPS-induced liver slice hepatitis.

[0063] Akkermansia muciniphila AM06 and AM02 can each independently be living bacteria, or inactivated Akkermansia muciniphila (i.e. inactivated bacteria, which can be fully or partially inactivated), or a bacterial lysate, culture (such as supernatant) or a component extracted from the culture.

[0064] In some embodiments, Akkermansia muciniphila AM06 and Akkermansia muciniphila AM02 are each independently live bacteria, inactivated bacteria, or a combination of live bacteria and inactivated bacteria. The inactivated bacteria may be inactivated bacteria with complete morphological structure, inactivated bacteria with incomplete morphological structure, or a combination thereof.

[0065] In some embodiments, Akkermansia muciniphila AM06 and Akkermansia muciniphila AM02 are each independently one or more of live bacteria, inactivated bacteria with intact morphological structures, and inactivated bacteria with incomplete morphological structures.

[0066] In some embodiments, the Akkermansia muciniphila of the present invention is selected from live bacteria of Akkermansia muciniphila.

[0067] In some embodiments, the Akkermansia muciniphila of the present invention is selected from inactivated Akkermansia muciniphila bacteria.

[0068] In the present invention, "morphologically intact" may include, but is not limited to, Akkermansia muciniphila that has been completely or partially inactivated through an inactivation treatment.

[0069] In the present invention, the situation of "incomplete morphological structure" may include but is not limited to bacterial lysate, culture (such as supernatant) or components extracted from the culture.

[0070] The inventors have found through a large number of experiments that the above-isolated Akkermansia muciniphila AM06 and / or AM02 can be used to improve blood sugar levels. Therefore, the above-isolated Akkermansia muciniphila AM06 and / or AM02 can be used to prepare health care product compositions, and further can be used to prepare health care foods. The above-isolated Akkermansia muciniphila AM06 and / or AM02 can lower blood sugar, reduce insulin resistance, and reduce insulitis. After experimental analysis, the applicant believes that the above-isolated Akkermansia muciniphila AM06 and / or AM02 can enhance the intestinal barrier and induce intestinal L cells to secrete GLP-1, thereby lowering blood sugar, reducing insulin resistance, and reducing insulitis.

[0071] For preparing health care product composition

[0072] In the present invention, "health care products" and "health food" have the same meaning and can be used interchangeably. In the present invention, "health care products" refer to foods with health care functions, which can regulate the physiological functions of animals (humans or other animals) and often provide beneficial effects. The scope of the beneficial effects of "health care products" in the body is not particularly limited, and can be systemic effects or only local effects.

[0073] In the present invention, "health care product composition" refers to a food composition with health care function. The health care product composition can be directly consumed as a health care product or as a dietary supplement.

[0074] In the present invention, "food" refers to a product that can be eaten directly. "Food composition" refers to a composition composed of edible substances. It should be understood that the food composition of the present invention may contain any other suitable edible substances in addition to the aforementioned Akkermansia muciniphila. In some embodiments, other edible substances can be selected from substances allowed to be added in food management regulations, and further, substances prohibited from being added in food management regulations are not included. If not specifically limited, food management regulations refer to the current regulations at the time of production.

[0075] In the present invention, "improving blood sugar level" means beneficially lowering blood sugar level, and "beneficial" means bringing about beneficial effects on health.

[0076] In some embodiments, the method for improving blood sugar level is not particularly limited. For example, in some embodiments, the method for improving blood sugar level includes at least one of inducing secretion of GLP-1 and reducing insulin resistance. In other embodiments, the method for improving blood sugar level is selected from one or more of inducing secretion of GLP-1 and reducing insulin resistance.

[0077] In some embodiments, a health care product composition comprises the Akkermansia muciniphila of the present invention and edible raw materials and auxiliary materials.

[0078] In the present invention, "edible raw materials and auxiliary materials" refer to edible raw materials, edible auxiliary materials or a combination of the two. Among them, "edible raw materials" refer to edible substances that can improve blood sugar levels alone, but are not AM06 and AM02; "edible auxiliary materials" refer to edible substances that do not have the function of improving blood sugar levels alone. It should be understood that "edible raw materials and auxiliary materials" can be selected from substances allowed to be added in the health care product management regulations, and further, do not contain substances prohibited from being added in the health care product management regulations. If not specifically limited, the health care product management regulations refer to the current regulations at the time of production.

[0079] In some embodiments, the food composition comprises the Akkermansia muciniphila (AM06 and / or AM02) of the present invention and edible excipients. In the present invention, food additives also belong to edible excipients. Examples of edible excipients include 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 excipients of the present invention can be used alone or in combination.

[0080] In some embodiments, the health care product composition consists of the Akkermansia muciniphila (AM06 and / or AM02) of the present invention and edible excipients.

[0081] In some embodiments, the health care product composition may further contain other health care ingredients, which are health care ingredients different from AM06 and AM02, and their health care functions may be to improve blood sugar levels or other health care functions.

[0082] In some embodiments, the health care product composition further contains other probiotics. Other probiotics can be selected from the modified bacteria of Akkermansia muciniphila (AM06 and / or AM02) described in the present invention and probiotics of other species. Probiotics of other species can include one or more of Bacteroides fragilis, Christensenella, Saccharomyces boulardii, Lactobacillus rhamnosus, Lactobacillus bifidum, and Enterococcus hilarii.

[0083] In some embodiments, the health care product composition further contains probiotics of other species.

[0084] Formulation type

[0085] In some embodiments, the health product composition is a health food.

[0086] In some embodiments, the dosage form of the health food is a pill, tablet, granule, capsule, solution, suspension or emulsion. Further, non-limiting examples include honey pills, water-honey pills, water pills, syrups, syrups, etc.

[0087] It should be understood that the health food contains a health-effective amount of Akkermansia muciniphila (AM06 and / or AM02) of the present invention. The "health-effective amount" herein refers to an amount that can improve blood sugar levels and can play a role in improving blood sugar levels when consumed in a normal dose of health food.

[0088] In the present invention, "subject" refers to a consumer who consumes the health care product composition.

[0089] In the present invention, "subject" is an animal, preferably a mammal, more preferably a human, and the subject includes but is not limited to the consumer of the health product. The subject in the present invention is preferably a mammal. 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, cattle, sheep, horses, humans, etc., preferably primates, more preferably humans.

[0090] In some embodiments, the subject is a mammal.

[0091] In some embodiments, the health care product composition is suitable for humans or other mammals. In the present invention, "other mammals" are not humans, and non-limiting examples of "other mammals" include cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, mice (such as rats, mice), pigs, cattle, sheep, horses, etc., and further include mice or rats; non-limiting examples of "other mammals" include primates.

[0092] In some embodiments, the subject is a human, mouse, or rat.

[0093] In some embodiments, the subject is a human or a mouse.

[0094] In the present invention, "patient" refers to an animal, preferably a mammal, such as a human, a mouse, or a rat.

[0095] Different subjects may result in different selection ranges of other ingredients in the aforementioned health care product composition.

[0096] Second aspect of the present invention

[0097] In a second aspect of the present invention, a composition containing Akkermansia muciniphila is provided for use in the preparation or as a health food for improving blood sugar levels. The "composition containing Akkermansia muciniphila" herein refers to a composition containing "Ackermansia muciniphila (AM06 and / or AM02) of the present invention". The definition of "Ackermansia muciniphila of the present invention" can refer to the first aspect of the present invention.

[0098] Furthermore, the Akkermansia muciniphila described in the present invention is Akkermansia muciniphila AM06 (with a deposit number of CGMCC No. 22793), Akkermansia muciniphila AM02 (with a deposit number of CGMCC No. 22794), or a combination of the two.

[0099] In some embodiments, the composition containing Akkermansia muciniphila is a nutraceutical composition.

[0100] In the second aspect of the present invention, the definitions of "composition", "health food", "improving blood sugar level" and "health 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.

[0101] The Akkermansia muciniphila described in the present invention may be a living bacterium, or an inactivated Akkermansia muciniphila (i.e., an inactivated bacterium, which may be fully or partially inactivated), or a lysate of the bacterium, a culture (such as a supernatant), or a component extracted from the culture.

[0102] In some embodiments, the composition comprising Akkermansia muciniphila further comprises a second active ingredient.

[0103] In the present invention, the "second active ingredient" refers to other active ingredients that have the effect of improving blood sugar levels, in addition to the Akkermansia muciniphila (AM06 and / or AM02) described in the present invention.

[0104] In some embodiments, the second active ingredient can be a modified bacterium of the 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, and the like. The modified Akkermansia muciniphila forms a new strain different from AM06 and AM02. The modified bacteria may still have the function of improving blood sugar levels, and may also be endowed with other new physiological functions.

[0105] In some embodiments, the composition comprising Akkermansia muciniphila does not include a pharmaceutically active ingredient.

[0106] In the present invention, "drug" includes any agent, compound, composition or mixture that provides pharmacological effects in vivo or in vitro, and often provides beneficial effects. The scope of the pharmacological effects of "drug" in vivo is not particularly limited, and it can be a systemic effect or only a local effect. The activity of the "drug" is not particularly limited, and it can be an active substance that can interact with other substances, or it can be an inert substance that does not interact.

[0107] In the present invention, "drug active ingredient" refers to a drug active ingredient having a preventive and therapeutic effect on diabetes.

[0108] In some embodiments, the composition containing Akkermansia muciniphila is a composite probiotic. In this case, the composition contains, in addition to the Akkermansia muciniphila (AM06 and / or AM02) described in the present invention, other probiotics different from the Akkermansia muciniphila described in the present invention.

[0109] In the present invention, the "composite probiotics" contain at least two probiotics. It should be understood that the "composite probiotics containing Akkermansia muciniphila" includes the Akkermansia muciniphila described in the present invention, and also includes other probiotics different from the Akkermansia muciniphila described in the present invention.

[0110] In some embodiments, other probiotics (different from the muciniphilic Akkermansia described in the present invention) may include modified bacteria of the muciniphilic Akkermansia described in the present invention and probiotics of other species. Probiotics of other species are probiotics different from muciniphilic Akkermansia, and may include but are not limited to Bacteroides fragilis, Christensen, Saccharomyces boulardii, Lactobacillus rhamnosus, Lactobacillus bifidum, Enterococcus hilarii, etc.

[0111] In some embodiments, the complex probiotic includes multiple species of Akkermansia muciniphila.

[0112] In some embodiments, the composite probiotic consists of at least one of Akkermansia muciniphila AM06 and AM02, and at least one of the modified bacteria of AM06 and the modified bacteria of AM02.

[0113] In some embodiments, the composite probiotics include the Akkermansia muciniphila described in the present invention, and also include probiotics of other species. The probiotics of other species are defined as above. It further includes modified bacteria of the Akkermansia muciniphila described in the present invention.

[0114] In the present invention, each component of the "composition containing Akkermansia muciniphila" can appear at any appropriate time. For example, it can be directly prepared as a mixture and opened for use, or it can be packaged separately and then prepared as a mixture when used, or it can be administered to a subject separately and appear at the local location in the body at the same time and exert a combined effect. The "composition containing Akkermansia muciniphila" that appears at any appropriate time is within the scope of protection of the present invention.

[0115] The third aspect of the present invention

[0116] In a third aspect of the present invention, a composition comprising Akkermansia muciniphila is provided;

[0117] The definition of "composition containing Akkermansia muciniphila" may refer to the second aspect of the present invention.

[0118] In some embodiments, the composition containing Akkermansia muciniphila of the present invention further comprises a second active ingredient. The definition of "second active ingredient" can refer to the second aspect of the present invention.

[0119] A fourth aspect of the present invention

[0120] In the fourth aspect of the present invention, a method for improving blood sugar level is also provided, comprising administering to a subject a health-care effective amount of the Akkermansia muciniphila of the present invention, or administering to a subject a health-care effective amount of a health-care composition containing the Akkermansia muciniphila of the present invention, or administering to a subject a health-care effective amount of a health-care food containing the Akkermansia muciniphila of the present invention.

[0121] In the fourth aspect of the present invention, the definitions of "Akkermansia muciniphila", "Akkermansia muciniphila described in the present invention", "composition", "improving blood sugar level", "health-care effective amount", "health-care composition" and "health-care food" can refer to the first aspect or the second aspect of the present invention.

[0122] Some specific embodiments are provided below.

[0123] The embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are preferably referred to the guidance provided in the present invention, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0124] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0125] In the following examples, 10-fold gradient dilution means that each gradient is diluted 10 times. The unit "day" can be expressed as "d".

[0126] Example 1. Isolation and identification of Akkermansia muciniphila

[0127] 1.1. Isolation and identification of strain AM02

[0128] Use a sterile sampling spoon to take soybean-sized feces (samples are from healthy adult men) into a 10mL centrifuge tube. After sampling, transfer the sample immediately to a 37℃ anaerobic workstation (85% N2, 10% H2, 5% CO2, volume percentage) and dilute the sample to 10 by 10-fold gradient dilution. -9 , take 1 mL of each dilution solution and inoculate it into 9 mL of basal medium with mucin as the sole carbon source, and culture it anaerobically for 7 days. -4 Dilution: 1 mL of culture medium was inoculated and diluted to 10 in a 1:10 gradient dilution. -6 , 100 μL of each dilution was spread on mucin agar medium, cultured anaerobically for 7 days, and a single colony was picked and inoculated into 2 mL of BHI broth (containing N-acetyl-D-glucosamine medium). The cultured bacterial solution was sequenced by 16s rRNA, and the 16S rRNA sequence was compared on the NCBI database. The isolated strain was identified as Akkermansia muciniphila, and the 16S rRNA sequencing results are as follows:

[0129]

[0130] The sequence was compared with the 16S rRNA sequence of ATCC BAA-835 at NCBI and the Per.Ident value was 99.43%.

[0131] 1.2. Isolation and identification of strain AM06

[0132] Freshly collected breast milk samples (from healthy adult women) were immediately injected into 5 mL anaerobic vials for storage. The samples were then transferred to a 37°C anaerobic workstation (85% N2, 10% H2, 5% CO2, volume percentage) and diluted to 10 by 10-fold gradient dilution. -6 , take 1 mL of each dilution solution and inoculate it into 9 mL of basal medium with mucin as the sole carbon source, and culture it anaerobically for about 1 month. -1 ~10 -4 Dilution: 1 mL of culture medium inoculated with 1:10 dilution to 10 -6 , 100 μL of each dilution was spread on mucin agar medium, cultured anaerobically for 7 days, and a single colony was picked and inoculated into 2 mL of BHI broth (containing N-acetyl-D-glucosamine medium). The cultured bacterial solution was sequenced and identified by 16S rRNA. The 16S rRNA sequence was compared with the NCBI database, and the result was identified as Akkermansia muciniphila. The sequencing results of 16S rRNA are as follows:

[0133]

[0134] The sequence was compared with the 16S rRNA sequence of ATCC BAA-835 at NCBI and the Per.Ident value was 99.22%.

[0135] 1.3. Strain deposit information

[0136] Akkermansia muciniphila AM02, whose classification name is Akkermansia muciniphila, was deposited in the General Microbiology Center of China Culture Collection Administration on June 28, 2021, with the address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.22794; the strain was received and registered by the collection center on June 28, 2021, and was tested as a viable strain by the collection center on June 28, 2021.

[0137] Akkermansia muciniphila AM06, whose classification name is Akkermansia muciniphila, was deposited in the General Microbiology Center of China Culture Collection Administration on June 28, 2021, with the address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.22793; the strain was received and registered by the collection center on June 28, 2021, and was tested as a viable strain by the collection center on June 28, 2021.

[0138] Example 2. Cultivation of Akkermansia muciniphila and preparation of inactivated bacteria

[0139] 2.1. Streak the Akkermansia muciniphila onto BHA plates and culture anaerobically for 3 days. Observe the colony morphology, staining properties, size, club shape, and distribution.

[0140] Colony characteristics: After 3 days of culture on the above medium, Akkermansia muciniphila AM02 and AM06 showed round, convex, neatly edged, opaque, white, and unevenly sized colonies, with a size of about 0.08 to 2.2 mm. Figure 1 (Akkermansia muciniphila AM02) and Figure 2 (Akermansia muciniphila AM06).

[0141] Microscopic morphology: Gram staining of Akkermansia muciniphila AM02 and AM06 showed that they were Gram-negative bacteria, oval in shape, arranged singly or in chains, as shown in Figure 3 (Akkermansia muciniphila AM02) and Figure 4 (Akermansia muciniphila AM06).

[0142] The colony characteristics of the standard strain ATCC BAA-835 are: round protrusions, neat edges, opaque, white, and unevenly sized colonies. Under the microscope, they are gram-negative bacteria, oval, single or in chain arrangement.

[0143] 2.2. A single colony was selected and inoculated into BHI broth for 48 hours (temperature was 37°C), and the obtained bacterial solution was centrifuged at a speed of 16000×g for 30 minutes, and the supernatant was removed and the precipitate was collected to obtain the Akkermansia muciniphila sludge. AM02, AM06, and ATCC BAA-835 Akkermansia muciniphila were obtained by culture.

[0144] 2.3. Preparation of inactivated bacteria: Take an appropriate amount of bacterial mud, add physiological saline at a ratio of bacterial mud: physiological saline (m:v) = 1:10, use a stirrer to stir at 1000rpm for 5-20min to evenly disperse the bacteria. Take 100mL 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 the magnetic stirrer, put in a sterile stirring bar, and insert a temperature electrode, set the speed to 300-500rpm, the temperature to 70℃, and heat for 30min to obtain inactivated bacteria of Akkermansia muciniphila.

[0145] Example 3. Non-targeted metabolic differential analysis of Akkermansia muciniphila culture supernatants

[0146] Sample preparation

[0147] After the culture of each Akkermansia muciniphila (AM02, AM06, ATCC BAA-835) in Example 2 was completed, 1 mL of the culture solution was centrifuged at 12000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm filter membrane, and the filtrate was taken as a sample to be tested for non-targeted metabolomics analysis. Five parallel samples to be tested were prepared for each strain.

[0148] 3.2 Experimental results

[0149] PCA is a data dimensionality reduction method, which is to reduce multiple variables to a set of new comprehensive variables, and then select the first few principal components that reflect the original variable information as much as possible, so as to achieve the purpose of dimensionality reduction. The PCA graph reflects the true distribution of samples, and is mainly used to observe the separation trend between sample groups and whether there are abnormal points. At the same time, it reflects the variability between and within groups from the original data.

[0150] The experimental results can be found in Figure 5 . Figure 5The PCA analysis of quality control samples (QC samples) and all samples was included. The QC samples were clustered together in the two principal component analysis diagrams, indicating that the instrument was stable during the test and the data collection had good repeatability. At the same time, the results also showed that the metabolites in the culture supernatant of AM06 were close to those in the culture supernatant of BAA-835, and the metabolites in the culture supernatants of AM02 and BAA-835 were quite different.

[0151] The results of the comparison of the number of differential metabolites between the strains can be seen in Table 1. It can be seen that compared with the standard strain BAA-835, the number of differential metabolites detected by positive ion (pos) mode and negative ion (neg) mode of AM02 was 205 and 135, respectively, and compared with the standard strain BAA-835, the number of differential metabolites detected by positive ion (pos) mode and negative ion (neg) mode of AM06 was 111 and 62, respectively.

[0152] Table 1. Statistics of differential metabolites

[0153]

[0154] Example 4. Efficacy verification of Akkermansia muciniphila

[0155] 4.1. Tolerance of Akkermansia muciniphila to artificial gastric fluid

[0156] 4.1.1. Experimental methods and grouping

[0157] Table 2. Experimental groups

[0158]

[0159] Table 3. Experimental methods

[0160]

[0161] “+” indicates that testing is required.

[0162] 4.1.1.1. Take one tube of Akkermansia muciniphila, remove the label, wipe and disinfect the outer surface of the glycerol cryopreservation tube with 75% (v / v) alcohol, vortex and oscillate to mix, and open. Pipette 100-500 μL of bacterial solution and inoculate into 10 mL / tube of BHI broth, shake well, prepare 3 tubes in total, and do not inoculate bacteria as a negative control, place at 37°C for anaerobic culture for 2-4 days to obtain the first-level seed solution.

[0163] Gram staining of the primary seed solution should show Gram-positive bacteria without spores or other bacteria.

[0164] 4.1.1.2. Take 10 mL of the above-mentioned first-level seed solution, centrifuge at 12000×g and 4°C for 10 min, discard the supernatant, add 1 mL of 0.9wt% NaCl solution and resuspend to prepare bacterial suspension for use.

[0165] 4.1.1.3. According to Table 2 and Table 3, add the bacterial suspension of AM06, AM02 and standard strains to 0.9wt% NaCl, pH3.0 and pH2.0 artificial gastric juice, mix well, dispense into 5mL / tube, place in an anaerobic glove box and incubate at 37℃ for 0h, 1.5h and 3h, then take out and test the bacterial concentration of each sample. Each experimental group is carried out in parallel 3 times.

[0166] 4.1.1.4. Determination of viable count:

[0167] Take the experimental sample and dilute it in a 10-fold series to different dilutions. Take 100 μL of the dilution and inoculate it on the BHA plate. Spread it evenly. Make 2 plates for each dilution. Generally, make 2 to 3 dilutions. At the same time, take 100 μL of the dilution on the BHA plate as a negative control. Place all the coated plates upright and culture them under anaerobic conditions for about 3 to 5 days. Observe the growth of the colonies on the plates and count them.

[0168] The number of viable bacteria was calculated based on the sum of the colony counts on the two plates using the following formula:

[0169] Viable bacteria count (CFU / mL) = sum of colony counts on 2 plates / 2 × 10 × final dilution

[0170] Survival rate calculation:

[0171]

[0172] 4.1.2 Experimental results

[0173] The experimental results can be seen in Table 4. The artificial gastric juice tolerance of different mucinotropic Akkermansia strains is AM02>AM06>standard strain BAA-835 from high to low.

[0174] Table 4 Statistical table of survival rate of Akkermansia muciniphila tolerance to artificial gastric fluid

[0175]

[0176] 4.2. Tolerance of Akkermansia muciniphila to artificial intestinal fluid

[0177] 4.2.1. Experimental methods and grouping

[0178] Table 5. Experimental groups

[0179]

[0180] 4.2.1.1. Preparation of primary seed solution

[0181] Take one tube of Akkermansia muciniphila and remove the label. Use 75% (v / v) alcohol to wipe and disinfect the outer surface of the glycerol cryopreservation tube. Vortex and oscillate to mix well and open. Pipette 100 μL of bacterial solution and inoculate into 10 mL / tube of BHI broth. Shake well and prepare 3 tubes in total. At the same time, do not inoculate bacteria as a negative control and place at 37°C for anaerobic culture for 2 to 4 days to obtain the first-level seed solution.

[0182] Gram staining of the primary seed solution should show Gram-positive bacteria without spores or other bacteria.

[0183] 4.2.1.2. Preparation of bacterial sludge

[0184] The above-mentioned first-level seed liquid was divided into 1.5 mL / tube, centrifuged at 12000×g for 10 min, and the supernatant was discarded to obtain bacterial sludge. 3 tubes of bacterial sludge were prepared for each of AM06, AM02 and standard strains.

[0185] 4.2.1.3. Evaluation of strain tolerance to artificial intestinal fluid

[0186] As shown in Tables 5 and 6, 1.5 mL of artificial intestinal fluid was added to each tube of the prepared bacterial sludge, mixed, and then each tube of solution was divided into 3 tubes at 0.5 mL / tube, and incubated anaerobically at 37°C for 0, 4, and 8 hours, respectively, and samples were taken to detect the number of viable bacteria. Three parallels were made for each group.

[0187] Table 6. Experimental methods

[0188]

[0189] “+” indicates that testing is required.

[0190] 4.2.1.4. Determination of viable count

[0191] Take the incubated samples separately, dilute them in 10-fold gradient series, take 100 μL of the dilution and inoculate them on the BHA plate, spread it evenly, make 2 plates for each dilution, usually make 2 to 3 dilutions, and take 100 μL of the dilution on the BHA plate as a negative control. Place all the coated plates upright and culture them under anaerobic conditions for about 3 to 5 days. Observe the growth of the colonies on the plates and count them.

[0192] Viable bacteria count (CFU / mL) = sum of colony counts on 2 plates / 2 × 10 × final dilution

[0193] Survival rate calculation:

[0194] Survival rate = number of viable bacteria at each time point / corresponding number of viable bacteria at 0h × 100%

[0195] 4.2.2 Experimental results

[0196] The results can be seen in Table 7. ATCC BAA-835, AM02, and AM06 strains have good tolerance to artificial intestinal fluid, and the tolerance is in the order of AM02>AM06>standard strain BAA-835.

[0197] Table 7. Survival rate statistics

[0198]

[0199] Effects of Akkermansia muciniphila on the expression of tight junction protein ZO-1 in Caco2 cells induced by TNF-α and IFN-γ

[0200] TNF-α: tumor necrosis factor-α; IFN-γ: interferon-γ.

[0201] 4.3.1. Experimental methods and grouping

[0202] Caco2 cells were inoculated into 96-well plates and cultured until the confluence was 80% to 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 to continue incubating with the cells for 24 h. The experimental grouping can be seen in Table 8, and 5 replicates were made 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.

[0203] Table 8. Experimental groups

[0204]

[0205] 4.3.2 Experimental results

[0206] The fluorescence intensity of the captured images was counted, and the results can be seen in Figure 6 and Table 9. Compared with the blank control group, the fluorescence intensity of the intercellular space in the inflammatory model group was significantly weakened after TNF-α and IFN-γ induction for 48 hours, indicating that the expression of ZO-1 protein was reduced and the tight junctions between cells were destroyed. Compared with the inflammatory model group, the fluorescence intensity of the treated group after intervention with AM02, AM06, and BAA-835 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).

[0207] Table 9. Fluorescence intensity statistics

[0208] serial number Group Fluorescence intensity A Blank control group 36.2647±5.8977** B Inflammation model group 25.6588±4.5303 C AM02 Group 41.7059±6.5664**aa D AM06 Group 40.2949±4.5437**aa E BAA-835 group 33.6688±4.13336**

[0209] 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.

[0210] Example 5. Akkermansia muciniphila induces GLP-1 secretion in NCI-H716 cells

[0211] 5.1. Experimental methods and grouping

[0212] NCI-H716 cells in the logarithmic growth phase were centrifuged and the supernatant was discarded. 0.02 g / L BSA solution was added to mix the cells and 3 × 10 6 The cells were inoculated with an inoculum of 100 cells / well into a 6-well plate. After culturing for 2 h, appropriate amounts of 0.02 g / L bovine serum albumin (BSA) solution, AM02, AM06, and BAA-835 were added to the cells according to the experimental groups in Table 10 and the cells were incubated for 36 h. The supernatant was collected and the GLP-1 content in the supernatant was detected by ELISA.

[0213] Table 10. Experimental groups

[0214] serial number Group Test substance A Blank control group / B AM02 Group <![CDATA[1×10 8 CFU / mL AM02]]> C AM06 Group <![CDATA[1×10 8 CFU / mL AM06]]> D BAA-835 group <![CDATA[1×10 8 CFU / mL BAA-835]]>

[0215] 5.2 Experimental results

[0216] The experimental results can be seen in Table 11. After adding AM02, AM06, and BAA-835, NCI-H716 cells were significantly induced to secrete a large amount of GLP-1 (p<0.01), and compared with BAA-835, the content of GLP-1 induced by AM02 and AM06 was significantly increased (p<0.05).

[0217] Table 11. GLP-1 content (pg / mL)

[0218] serial number Group GLP-1 content (pg / mL) A Blank control group 134.5±28.34 B AM02 Group 536.56±17.35**a C AM06 Group 573.45±23.21**a D BAA-835 group 475.46±34.14**

[0219] Note: ** indicates that compared with the model group, the difference is extremely significant p<0.01; a indicates that compared with the BAA-835 group, the difference is significant p<0.05.

[0220] Example 6

[0221] Experimental design and methods

[0222] Experimental animals: SPF grade db / db mice, male, 4-5 weeks old, 100; SPF grade C57BL / KsJ mice, male, 4-5 weeks old, 10. Breeding place during the experiment: SPF grade mouse room.

[0223] Experimental groups: The blank group consisted of C57BL / KsJ mice, 10 animals. The other experimental groups consisted of db / db mice, divided into: model group, positive group (semaglutide, 0.36 mg / kg, subcutaneous injection), AM06 low-dose group (10 6 CFU / mL), AM06 medium dose group (10 8 CFU / mL), AM06 high-dose group (10 10 CFU / mL), AM02 medium dose group (10 8 CFU / mL), ATCC BAA-835 medium dose group (10 8 CFU / mL), and the inactivated bacteria groups of AM06, AM02 and BAA-835 (the dosage of each group was 10 8 / mL), 10 animals per group. After a one-week adaptation period, each group began to be given the corresponding active ingredient. From the first day to the 42nd day of the experiment, each group of mice was given a normal diet, and each group was given the active ingredient once a day. On the 43rd day, the mice in each group were sampled.

[0224] Experimental design test:

[0225] On the 43rd day of the experiment, the mice were fasted for 12 hours and then euthanized with CO2. Blood samples were collected by cardiac puncture for the detection of blood glucose and insulin levels. The terminal body weight and liver weight were recorded. The liver tissue of the mice was taken, fixed with 4% paraformaldehyde, embedded in paraffin, sliced ​​at 5 μm, stained with HE, and the pathological changes of the liver were observed under a light microscope and the fatty degeneration was scored. The fatty degeneration scoring standard: 0 points means that the fat droplets in the hepatocytes are scattered, rare, and basically normal; 1 point means that the hepatocytes containing fat droplets do not exceed 1 / 4; 2 points means that the hepatocytes containing fat droplets do not exceed 1 / 2; 3 points means that the hepatocytes containing fat droplets do not exceed 3 / 4; 4 points means that the liver tissue is almost replaced by fat droplets.

[0226] 6.2 Experimental results

[0227] The results of each test index can be found in Table 12 and Table 13.

[0228] Table 12. Statistics of various test indicators (mean ± SD, n = 10)

[0229]

[0230] Table 13.

[0231]

[0232] In Table 12 and Table 13, Note 1: * indicates that compared with the model group, the difference is significant p<0.05, ** indicates that compared with the model group, the difference is extremely significant p<0.01; Note 2: a indicates that compared with the BAA-835 medium-dose group, the difference is significant p<0.05, aa indicates that compared with the BAA-835 medium-dose group, the difference is extremely significant p<0.01;

[0233] Note 3: b means that compared with the medium-dose group of BAA-835, the difference is significant p<0.05, bb means that compared with the medium-dose group of BAA-835, the difference is extremely significant p<0.01.

[0234] 6.2.1. Mouse body weight

[0235] As shown in Table 12, compared with the blank group, the body weight of mice in the model group increased significantly (p<0.01). Compared with the model group, the AM06 medium- and high-dose groups, the AM02 medium-dose group, the AM06 inactivated bacteria group, and the AM02 inactivated bacteria group could significantly reduce body weight (p<0.05), and the other groups given active ingredients had a trend of reducing the body weight of mice, but there was no significant difference. The body weight of the AM06 medium-dose group was significantly lower than that of the BAA-835 medium-dose group (p<0.05), and the body weight of the AM02 medium-dose group had a trend lower than that of the BAA-835 medium-dose group. The body weight of the AM02 and AM06 inactivated bacteria groups was significantly lower than that of the BAA-835 inactivated bacteria group. This shows that the live bacteria and inactivated bacteria of AM02 and AM06 are better than the live bacteria and inactivated bacteria of BAA-835 in reducing the weight gain of mice.

[0236] 6.2.2. Blood glucose, insulin and insulin resistance index in mice

[0237] As shown in Tables 12 and 13, compared with the blank group, the blood glucose level, insulin level and insulin resistance index of the mice in the model group were significantly increased (p<0.01), indicating that the mice in the model group had hyperglycemia and hyperinsulinemia, i.e., insulin resistance. Compared with the model group, each group given with active ingredients can reduce the blood glucose and insulin levels of animals to varying degrees, and significantly reduce the insulin resistance index (p<0.01). And at the same dose level, the insulin resistance index of the live or inactivated bacteria of AM06 and AM02 is significantly better than that of the live or inactivated bacteria of BAA-835. This shows that the live and inactivated bacteria of AM02 and AM06 are better than the live and inactivated bacteria of BAA-835 in reducing blood glucose and reducing insulin resistance.

[0238] 6.2.3. Mouse liver scoring

[0239] The livers of mice in each group were stained with HE, and the scores were calculated according to the degree of fatty degeneration of the sections. The results can be seen in Table 13. Compared with the blank control group, the liver fatty degeneration score of the model group increased significantly (p<0.01), indicating that the animals had fatty liver. Compared with the model group, each group given active ingredients could reduce the liver fatty degeneration score to varying degrees, and the low and medium dose groups of AM06 and AM02 could significantly reduce the liver fatty degeneration score. Low liver steatosis score (p<0.05). At the same dose level, the liver steatosis scores of live or inactivated bacteria of AM02 and AM06 tended to be lower than those of live or inactivated bacteria of BAA-835, indicating that the live and inactivated bacteria of AM02 and AM06 were slightly better than those of live and inactivated bacteria of BAA-835 in reducing liver fat accumulation.

[0240] In conclusion, Akkermansia muciniphila AM02 and AM06 can reduce body weight, lower fasting blood glucose and insulin levels, reduce insulin resistance and Fat accumulation in the liver , and the effects of each strain were AM06>AM02>BAA-835.

[0241] Example 7.

[0242] 7.1. Experimental animals: 80 SPF NOD mice, female, 4-5 weeks old; 10 SPF BALB / c mice, female, 4-5 weeks old. Breeding place during the experiment: SPF mouse room.

[0243] Experimental groups: The blank group consisted of BALB / c mice, 10 animals. The other experimental groups consisted of NOD mice, divided into: model group, positive group (glargine insulin, 50 IU / kg, subcutaneous injection), AM06 group (10 8 CFU / mL), AM02 group (10 8 CFU / mL), ATCCBAA-835 group (10 8 CFU / mL, AM06 inactivated bacteria group (10 8 / mL), AM02 inactivated bacteria group (10 8 / mL), ATCC BAA-835 inactivated bacteria group (10 8 / mL), 10 animals per group. After a one-week adaptation period, each group began to be given the corresponding active ingredient, and each group was given the active ingredient once a day for 24 weeks. On the 43rd day, samples were collected from each group of mice.

[0244] Experimental design

[0245] One drop of tail vein blood was collected weekly, and random blood glucose was measured with a rapid blood glucose meter. The incidence of blood glucose levels equal to or greater than 12.0 mmol / L was recorded in two consecutive readings.

[0246] After the experiment, the pancreatic tissue of the mice was obtained, fixed with paraformaldehyde, embedded in paraffin, sliced, stained with HE, and observed under a light microscope to score the insulitis. The severity of islet lymphocyte infiltration was scored according to the following standards: 0 points: no lymphocyte infiltration around the islets; 1 point: local lymphocyte infiltration around the islets; 2 points: partial lymphocyte infiltration in the islets, ranging from 1 / 3; 3 points: more lymphocyte infiltration in the islets, ranging from 1 / 3 to 1 / 2; 4 points: a large number of lymphocyte infiltration in the islets, ranging from >1 / 2, but not reaching 100%; 5 points: pan-insulitis.

[0247] 7.2 Experimental results

[0248] The test results can be seen in Table 14.

[0249] Table 14. Test results of each group of animal experiments

[0250]

[0251] Note 1: * 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; Note 2: a indicates that the difference is significant compared with the BAA-835 medium-dose group, p<0.05, aa indicates that the difference is extremely significant compared with the BAA-835 medium-dose group, p<0.01;

[0252] Note 3: b means that compared with the medium-dose group of BAA-835, the difference is significant p<0.05, bb means that compared with the medium-dose group of BAA-835, the difference is extremely significant p<0.01.

[0253] 7.2.1. Incidence of two consecutive readings equal to or greater than 12.0 mmol / L

[0254] As shown in Table 14, compared with the blank group, as the age of the mice increased, the incidence of two consecutive readings equal to or greater than 12.0mmol / L in the model group mice gradually increased, and the incidence of two consecutive readings equal to or greater than 12.0mmol / L reached 90% by the end of the experiment. Compared with the model group, each group given the active ingredient reduced and delayed the incidence of two consecutive readings equal to or greater than 12.0mmol / L to varying degrees. And the effects of live bacteria and inactivated bacteria of AM02 and AM06 were slightly better than those of live bacteria and inactivated bacteria of BAA-835.

[0255] 7.2.2. Pancreatic islet inflammatory score

[0256] As shown in Table 14, compared with the blank group, the pancreatic islet inflammatory score of the mice in the model group increased significantly (p<0.01), indicating that the mice in the model group had insulitis. Compared with the model group, each group given with active ingredients can reduce the pancreatic islet inflammatory score of animals to varying degrees, and the positive group, AM06 group and AM02 group significantly reduced the pancreatic islet inflammatory score (p<0.05). Among them, the pancreatic islet inflammatory score of the AM06 group and the AM02 group was significantly lower than that of the BAA-835 group, and the pancreatic islet inflammatory score of the AM06 and AM02 inactivated bacteria groups tended to be lower than that of the BAA-835 inactivated bacteria group. This shows that the live bacteria and inactivated bacteria of AM02 and AM06 are better than the live bacteria and inactivated bacteria of BAA-835 in reducing the pancreatic islet inflammatory score.

[0257] Example 8. Reducing postprandial blood sugar in mice

[0258] Experimental methods

[0259] Experimental animals: SPF-grade C57 / BL mice, male, 4-5 weeks old, 70 mice; breeding place during the experiment: SPF-grade mouse room.

[0260] Experimental grouping and modeling: 10 animals were selected as the blank group, which were fed with ordinary feed and no STZ (streptozotocin) injection during the experiment. The remaining animals were fed with high-fat feed for 4 weeks, and then injected with STZ 150mg / kg intraperitoneally. Then, the animals with random blood sugar greater than 16.7mmol / L were selected and randomly divided into the model group, AM06 group (10 9 CFU / animal), 10 animals per group. After grouping, the animals except the blank group continued to be fed with high-fat diet, and each group began to be given the corresponding active ingredient, once a day, for 3 weeks.

[0261] Blood sugar test: At the end of the experiment, all animals were fasted for 16 hours and their fasting blood sugar was tested. Then, the animals were given starch solution by gavage at a dose of 2g / kg and their blood sugar levels were tested 1 hour and 2 hours after the meal.

[0262] 8.2 Experimental results

[0263] The experimental results can be seen in Table 15. Compared with the blank group, the fasting blood glucose, blood glucose levels 1h and 2h after meal in the model group were significantly increased (p<0.01). Compared with the model group, the fasting blood glucose, blood glucose levels 1h and 2h after meal in the AM06 group were reduced to varying degrees (p<0.05 or p<0.01).

[0264] Table 15. Blood glucose test results of each group of animals

[0265]

[0266] Note 1: * 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;

[0267] The technical features of the above-mentioned embodiments and examples can be combined in any appropriate manner. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0268] The above embodiments only express several implementation methods of the present invention, which is convenient for understanding the technical solution of the present invention in detail, but it cannot be understood as limiting the scope of protection of the present invention. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present invention, several variations and improvements can be made, which all belong to the protection scope of the present invention. In addition, it should be understood that after reading the above teaching content 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 on the basis of the technical solution provided by the present invention through logical analysis, reasoning or limited experiments are all within the protection scope of the claims attached to the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the content of the attached claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. The use of Akkermansia muciniphila in the preparation of a health care product composition or food for improving blood sugar level or liver fat accumulation, characterized in that: The Akkermansia muciniphila is Akkermansia muciniphila AM06, Akkermansia muciniphila AM02 or a combination of the two; wherein, The Akkermansia muciniphila AM06 was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on June 28, 2021, with the deposit number of CGMCC No.22793; The Akkermansia muciniphila AM02 was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on June 28, 2021, 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 a live bacterium, an inactivated bacterium, or a combination of live and inactivated bacteria.

3. The use according to claim 1 or 2, characterized in that: The method for improving blood sugar level includes at least one of inducing secretion of GLP-1 and reducing insulin resistance.

4. The use according to claim 1 or 2, characterized in that: The health care product composition comprises the Akkermansia muciniphila and edible raw and auxiliary materials.

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

6. Use of a composition containing Akkermansia muciniphila in the preparation or as a health food for improving blood sugar level or liver fat accumulation, characterized in that: The Akkermansia muciniphila is as defined in claim 1 or 2.

7. The use according to claim 6, characterized in that The composition containing Akkermansia muciniphila is a health product composition.

8. The use according to claim 6 or 7, characterized in that The composition containing Akkermansia muciniphila further comprises a second active ingredient, and the second active ingredient does not include a pharmaceutically active ingredient.

9. The use according to claim 6 or 7, characterized in that The composition containing Akkermansia muciniphila is a composite probiotic, and the composite probiotic also includes probiotics different from the Akkermansia muciniphila.

10. The use according to claim 9, characterized in that The probiotic bacteria different from the Akkermansia muciniphila include one or more of Bacteroides fragilis, Christensenella, Saccharomyces boulardii, Lactobacillus rhamnosus, Lactobacillus bifidum and Enterococcus hilarii.

Images