Method for improving yield of Amuc1100 protein

Abstract

The present disclosure provides a method for increasing the yield of Amuciniphila, the method comprising culturing Akkermansia muciniphila with a culture medium containing peptone or yeast extract powder, the peptone comprising at least one of soy peptone, tryptone, wheat peptone, or rice peptone, and the method comprising: culturing Akkermansia muciniphila with a culture medium containing yeast extract powder; according to the method disclosed by the invention, the yield of the active ingredient Amuc1100 protein of Ackermania muciniphila can be obviously improved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for increasing the yield of Amuc_1100 protein. Background Art

[0002] Akkermansia muciniphila (A. muciniphila, or Akkermansia) is a new genus and species in the Verrucomicrobiota phylum. It was successfully isolated in 2004 by researchers at the Wageningen Microbiology Laboratory (PMID: 15388697). Akkermansia muciniphila colonizes the gastrointestinal mucosa and specifically degrades mucin. It is ubiquitous in the intestines of healthy adults and infants, comprising 1% to 4% of the total intestinal microbiota (PMID: 18083887). As a crucial member of the intestinal microbiota, numerous studies in recent years have shown that the abundance of A. muciniphila colonization is closely associated with various diseases, such as obesity, diabetes, hepatic steatosis, inflammation, and response to cancer immunotherapy. Therefore, A. muciniphila is considered a "next-generation" probiotic with significant potential applications and commercial value in food, medicine, and other fields.

[0003] Amuc_1100, as a key functional protein in the outer membrane of Akkermansia muciniphila, has been proven to be a specific effector molecule that mediates host health regulation by the bacterium. Multiple studies have revealed that this protein has significant biological activity in various disease models: in the field of metabolic diseases, Amuc_1100 effectively regulates metabolic disorders in obese and diabetic model animals by activating the TLR2 signaling pathway, significantly improving insulin sensitivity (PMID: 27892954); in intestinal inflammation and tumor models, its mechanism of action exhibits bidirectional regulatory characteristics - it can not only alleviate colitis symptoms by inhibiting macrophage infiltration and cytotoxic T lymphocyte (CTLs) activation, but also promote CTLs proliferation and activation by upregulating TNF-α expression and inhibiting the PD-1 immune checkpoint molecule, thereby inhibiting the progression of colitis-related carcinogenesis (PMID: 32169907); in neuropsychiatric disease models, this protein can reverse chronic stress-induced depressive-like behavior, and its neuroprotective effect is closely related to regulating BDNF expression levels in the hippocampus and inhibiting neuroinflammatory responses (PMID: 34129964). These evidences indicate that Amuc_1100 protein is one of the important active substances that Akkermansia muciniphila exerts a probiotic effect on diseases, and also suggest that its content plays an important role in the beneficial effects of Akkermansia muciniphila.

[0004] Because Akkermansia muciniphila has numerous health benefits, current research and patent applications primarily focus on achieving large-scale production of Akkermansia muciniphila, including optimizing fermentation processes through factors such as culture medium composition, culture time, centrifugation conditions, and the addition of specialized derivatives. While these optimizations significantly contribute to increasing Akkermansia muciniphila production, quantitative detection of the Amuc_1100 protein presents challenges. There are no commercially available antibodies or detection kits for Amuc_1100, and mass spectrometry methods require complex pretreatment steps and high costs. Consequently, current research has focused little on the effects of these culture methods on Amuc_1100, the active component of Akkermansia muciniphila, or on how to increase its production.

[0005] Amuc_1100, a key active component of Akkermansia muciniphila, determines its probiotic efficacy to a certain extent. While existing technologies for optimizing fermentation production processes can increase Akkermansia muciniphila yield, a higher Akkermansia muciniphila yield does not necessarily translate into a higher Amuc_1100 protein content in the fermentation broth. Therefore, while improving Akkermansia muciniphila yield, it is important to consider the impact of fermentation methods on Amuc_1100.

[0006] Increasing the yield of Amuc_1100 protein is important for the industrial production of Amuc_1100 and enhancing the probiotic effects of Akkermansia muciniphila. Therefore, it is of great significance to provide a fermentation process for producing Akkermansia muciniphila and significantly increasing the yield of Amuc_1100 protein. Summary of the Invention

[0007] The present invention improves the yield of Amuc_1100 protein, an active ingredient of Akkermansia muciniphila, by optimizing the nitrogen source of the culture medium. It is found that yeast extract powder or specific types of peptones such as soy peptone, tryptone, wheat peptone or rice peptone can significantly increase the yield of Amuc_1100 protein, which is of great significance for the large-scale production and industrial application of Amuc_1100 protein.

[0008] According to a first aspect of the present disclosure, provided is a use of peptone or yeast extract powder in increasing the yield of Amuc_1100 protein, wherein the peptone comprises at least one of soy peptone, tryptone, wheat peptone or rice peptone, and the Amuc_1100 protein is derived from Akkermansia muciniphila.

[0009] The Akkermansia muciniphila herein can be any Akkermansia muciniphila capable of producing the Amuc_1100 protein. In some embodiments, the Akkermansia muciniphila can be any of the Akkermansia muciniphila disclosed in Chinese patent application CN2024119901535. In some specific embodiments, the Akkermansia muciniphila is Akkermansia muciniphila strain MNH19250, deposited with the Guangdong Provincial Microbiological Culture Collection (GDMCC) under the GDMCC NO: 63782 and a deposit date of June 21, 2024.

[0010] Another aspect of the present disclosure provides the use of peptone or yeast extract powder in preparing a culture medium for improving the protein production of Akkermansia muciniphila Amuc_1100, wherein the peptone includes at least one of soy peptone, tryptone, wheat peptone or rice peptone.

[0011] According to another aspect of the present disclosure, a method for increasing the protein production of Akkermansia muciniphila Amuc_1100 is provided, the method comprising culturing Akkermansia muciniphila using a culture medium containing peptone, wherein the peptone comprises at least one of soy peptone, tryptone, wheat peptone or rice peptone.

[0012] According to another aspect of the present disclosure, a method for increasing the yield of Amuc_1100 protein in a fermentation broth is provided, the method comprising culturing Akkermansia muciniphila using a culture medium containing peptone or yeast extract powder, wherein the peptone comprises at least one of soy peptone, tryptone, wheat peptone, or rice peptone.

[0013] In some embodiments, the method further includes increasing the Amuc_1100 protein content in the fermentation broth by controlling the total bacterial count in the fermentation broth or preventing the total bacterial count in the fermentation broth from being too high. By controlling the total bacterial count in the fermentation broth or preventing the total bacterial count in the fermentation broth from being too high, the consumption of Amuc_1100 protein in the fermentation broth can be reduced, or the expression of Amuc_1100 protein can be promoted, thereby increasing the Amuc_1100 protein content in the fermentation broth.

[0014] In some embodiments, the content of the peptone in the culture medium is 25-40 g / L, for example, 25 g / L, 26 g / L, 28 g / L, 30 g / L, 32 g / L, 35 g / L, 38 g / L, 40 g / L or any value therebetween.

[0015] In some embodiments, the culture medium further comprises at least one of glucose, N-acetyl-D-glucosamine, L-cysteine ​​hydrochloride, L-threonine, magnesium sulfate, ferrous sulfate, manganese sulfate, Tween 80, dipotassium hydrogen phosphate, potassium citrate, and sodium acetate.

[0016] In some embodiments, the culture medium comprises 25-40 g / L peptone, 2-10 g / L glucose monohydrate, 5-20 g / L N-acetyl-D-glucosamine, 0.5-2 g / L L-cysteine ​​hydrochloride monohydrate, 1-5 g / L L-threonine, 0.05-0.2 g / L magnesium sulfate heptahydrate, 0.01-0.1 g / L ferrous sulfate heptahydrate, 0.02-0.1 g / L manganese sulfate, 0.5-2 g / L Tween 80, 1-5 g / L dipotassium hydrogen phosphate, 2-8 g / L potassium citrate, and 2-8 g / L sodium acetate.

[0017] In some embodiments, the content of peptone in the culture medium can be 25 g / L, 26 g / L, 28 g / L, 30 g / L, 32 g / L, 35 g / L, 38 g / L, 40 g / L or any value therebetween.

[0018] In some embodiments, the content of glucose monohydrate in the culture medium can be 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L or any value therebetween, or other forms of glucose equivalent to the glucose content in glucose monohydrate.

[0019] In some embodiments, the content of N-acetyl-D-glucosamine in the culture medium can be 5 g / L, 7.5 g / L, 10 g / L, 12.5 g / L, 15 g / L, 17.5 g / L, 20 g / L or any value therebetween.

[0020] In some embodiments, the content of L-cysteine ​​hydrochloride monohydrate in the culture medium can be 0.5 g / L, 0.75 g / L, 1 g / L, 1.25 g / L, 1.5 g / L, 1.75 g / L, 2 g / L, or any value therebetween, or other forms of L-cysteine ​​hydrochloride equivalent to the content of L-cysteine ​​hydrochloride in L-cysteine ​​hydrochloride monohydrate.

[0021] In some embodiments, the content of L-threonine in the culture medium can be 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, 5 g / L or any value therebetween.

[0022] In some embodiments, the content of magnesium sulfate heptahydrate in the culture medium can be 0.05 g / L, 0.075 g / L, 0.1 g / L, 0.125 g / L, 0.15 g / L, 0.175 g / L, 0.2 g / L, or any value therebetween, or other forms of magnesium sulfate with a content equivalent to that of magnesium sulfate in magnesium sulfate heptahydrate.

[0023] In some embodiments, the content of ferrous sulfate heptahydrate in the culture medium can be 0.01 g / L, 0.02 g / L, 0.03 g / L, 0.04 g / L, 0.05 g / L, 0.06 g / L, 0.07 g / L, 0.08 g / L, 0.09 g / L, 0.1 g / L, or any value therebetween, or other forms of ferrous sulfate equivalent to the ferrous sulfate content in ferrous sulfate heptahydrate.

[0024] In some embodiments, the content of manganese sulfate in the culture medium can be 0.02 g / L, 0.03 g / L, 0.04 g / L, 0.05 g / L, 0.06 g / L, 0.07 g / L, 0.08 g / L, 0.09 g / L, 0.1 g / L or any value therebetween.

[0025] In some embodiments, the content of Tween 80 in the culture medium can be 0.5 g / L, 0.75 g / L, 1 g / L, 1.25 g / L, 1.5 g / L, 1.75 g / L, 2 g / L or any value therebetween.

[0026] In some embodiments, the content of dipotassium hydrogen phosphate in the culture medium can be 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, 5 g / L or any value therebetween.

[0027] In some embodiments, the content of potassium citrate in the culture medium can be 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L or any value therebetween.

[0028] In some embodiments, the content of sodium acetate in the culture medium can be 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L or any value therebetween.

[0029] In some embodiments, the culture medium comprises 28-35 g / L peptone, 4-6 g / L glucose monohydrate, 8-15 g / L N-acetyl-D-glucosamine, 0.8-1.5 g / L L-cysteine ​​hydrochloride monohydrate, 1-3 g / L L-threonine, 0.08-0.15 g / L magnesium sulfate heptahydrate, 0.02-0.08 g / L ferrous sulfate heptahydrate, 0.04-0.08 g / L manganese sulfate, 0.8-1.5 g / L Tween 80, 1.5-3 g / L dipotassium hydrogen phosphate, 3-5 g / L potassium citrate, and 4-6 g / L sodium acetate.

[0030] The Akkermansia muciniphila herein can be any Akkermansia muciniphila capable of producing the Amuc_1100 protein. In some embodiments, the Akkermansia muciniphila can be any of the Akkermansia muciniphila disclosed in Chinese patent application CN2024119901535. In some specific embodiments, the Akkermansia muciniphila is Akkermansia muciniphila strain MNH19250, deposited with the Guangdong Provincial Microbiological Culture Collection (GDMCC) under the GDMCC NO: 63782 and a deposit date of June 21, 2024.

[0031] In some embodiments, the culturing is performed under anaerobic conditions, the culturing temperature is 35-40° C., and the culturing time is 24-60 h.

[0032] In some embodiments, the culture temperature can be 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, or any value therebetween.

[0033] In some embodiments, the culturing time is 24 h, 28 h, 32 h, 36 h, 40 h, 44 h, 48 h, 52 h, 56 h, 60 h or any value therebetween.

[0034] In some embodiments, the method further comprises extracting bacterial protein from the cultured Akkermansia muciniphila bacterial suspension; and optionally, quantitatively detecting the Amuc_1100 protein content in the bacterial protein.

[0035] In some embodiments, the quantitative detection includes: detecting the Amuc_1100 protein in the bacterial suspension using a characteristic peptide segment of Amuc_1100 using high performance liquid chromatography-mass spectrometry, and the amino acid sequence of the characteristic peptide segment of Amuc_1100 is: AINSLVNK (SEQ ID NO: 1).

[0036] According to yet another aspect of the present disclosure, a cell culture is provided, wherein the cell culture is obtained by the aforementioned method of the present disclosure.

[0037] In some embodiments, the cell culture is selected from any one of the following: fermentation broth, supernatant of fermentation broth, inactivated product of fermentation broth, or dried product thereof.

[0038] According to yet another aspect of the present disclosure, a composition is provided, comprising the aforementioned cell culture of the present disclosure.

[0039] In some embodiments, the Akkermansia muciniphila in the composition is an attenuated bacterium, a killed bacterium, a freeze-dried bacterium, or an irradiated bacterium, for example, it can be a heat-inactivated bacterium, preferably pasteurized.

[0040] In some embodiments, the culture is selected from any one of the following: a fermentation culture, a supernatant of a fermentation culture, or a dried product thereof.

[0041] In some embodiments, the composition is any one of a medicine, a health product or a food.

[0042] In some embodiments, the health product or food is any one of a nutritional composition, a food, a candy, a food bar, a food additive, a drink additive, and a dietary supplement.

[0043] In some embodiments, the composition is in the form of a liquid, foam, cream, spray, powder (eg, lyophilized powder), or gel.

[0044] In some embodiments, the composition is in the form of a powder, microencapsulated powder, capsule, tablet, lozenge, granule, oral solution, suspension, emulsion, liquid preparation, sustained-release preparation, nanoformulation, or microencapsulated capsule.

[0045] In some embodiments, the composition is in the form of an oral dosage or an injection.

[0046] In some embodiments, the composition further comprises one or more pharmaceutically acceptable carriers, food carriers, excipients and / or adjuvants.

[0047] The pharmaceutically acceptable excipients are well known to those skilled in the art. In some embodiments, the excipients may be at least one selected from a carrier, an excipient, a diluent, a lubricant, a wetting agent, an emulsifier, a suspension stabilizer, a preservative, a sweetener, and a flavoring.

[0048] In some embodiments, the composition comprises one or more of a buffer (e.g., sodium bicarbonate, infant formula or sterilized human milk or other agents that allow bacteria to survive and grow (e.g., survive in the acidic environment of the stomach and grow in the intestinal environment)), a lyoprotectant, a preservative, a stabilizer, a binder, a compacting agent, a lubricant, a dispersion enhancer, a disintegrant, an antioxidant, a flavoring agent, a sweetener, and a coloring agent.

[0049] In some embodiments, the composition further comprises one or more other active agents for preventing or treating metabolic diseases and / or tumors.

[0050] In some embodiments, the other active agent has at least one of the following functions: (a) suppressing appetite, (b) preventing metabolic diseases, (c) treating metabolic diseases, (d) preventing tumors, or (e) treating tumors.

[0051] In some embodiments, suppressing appetite comprises reducing food intake and / or reducing appetite.

[0052] In some embodiments, the other active agent is selected from: a GLP-1 receptor agonist, a dual agonist of the GLP-1 receptor and the GCG receptor, a triple agonist of the GLP-1 receptor, the GIP receptor and the GCG receptor, an AMPK agonist or an active drug that promotes GLP-1 secretion.

[0053] In some embodiments, the other active agent is selected from the group consisting of metformin, sulfonylureas, meglitinide, thiazolidinediones, DPP-4 inhibitors, GLP-1 receptor agonists, SGLT2 inhibitors, insulin, pioglitazone, rosiglitazone, pentoxifylline, omega-3 fatty acids, statins, ezetimibe, ursodeoxycholic acid, semaglutide, liraglutide, exenatide, and benaglutide.

[0054] In some embodiments, the other active agent may be an agent for preventing or treating tumors.

[0055] In some embodiments, the other active agent may be one or more of probiotics, prebiotics, or a combination thereof.

[0056] In some embodiments, the probiotics are selected from one or more of lactic acid bacteria, lactobacilli, lactococci, butyrate-producing bacteria, bifidobacteria, thermophilic Streptococcus, faecal Streptococcus, and mesenteric Leuconostoc.

[0057] In some embodiments, the prebiotic is selected from the group consisting of inulin, mulberry leaf extract, berberine, Ganoderma lucidum, green coffee bean extract, oats, pectin, potato or its extract, citrus polyphenols, Ceylon cinnamon, chromium, ergothioneine, astaxanthin, quercetin, curcumin, proanthocyanidins, resistant dextrin, yeast β-glucan, ginseng or its extract, nutritional compounds, biotin, polydextrose, fructooligosaccharides (FOS), galacto-oligosaccharides (GOS), starch, cellulose, β-glucan, hemicellulose, lactulose, manno-oligosaccharides, manno-oligosaccharides (MOS), inulin rich in fructooligosaccharides, oligo-glucose, tagatose, trans-galacto-oligosaccharides, pectin, resistant starch, xylo-oligosaccharides (XOS), and any combination thereof.

[0058] In some embodiments, the composition can be formulated as a frozen composition, for example, by quick freezing and drying, or lyophilization, for storage and / or transportation.

[0059] In some embodiments, the composition is obtained by spray drying. In some embodiments, the composition is obtained by electrostatic spray drying.

[0060] In some embodiments, the strain in the composition is freeze-dried or spray-dried. In some embodiments, the strain in the composition is electrostatically spray-dried. In some embodiments, the strain in the composition is freeze-dried or spray-dried and is viable. In some embodiments, the strain in the composition is freeze-dried or spray-dried and is capable of partially or completely colonizing the intestine. In some embodiments, the strain is reconstituted prior to administration. In some cases, the reconstitution is performed using a diluent as described herein.

[0061] In some embodiments, the composition can be administered alone or in combination with a carrier such as a pharmaceutically acceptable carrier or a biocompatible scaffold.

[0062] In some embodiments, the composition is formulated for oral administration. In some embodiments, the composition is an enteric-coated formulation. In some embodiments, the enteric-coated formulation is a dosage form having an enteric coating. For example, the enteric-coated formulation can be an enteric granule, an enteric-coated tablet, or an enteric-coated capsule. In some embodiments, the composition is a capsule. In some embodiments, the capsule is a hard capsule or a soft capsule; or the capsule is a sustained-release capsule, a controlled-release capsule, or an enteric-coated capsule, or the capsule can be a microencapsulated capsule or a microcapsule.

[0063] In some embodiments, the composition is a medicine, a health product, or a food.

[0064] In some embodiments, the composition is in an infant-suitable dosage form, a child-suitable dosage form, or an adult-suitable dosage form.

[0065] In some embodiments, the composition is in a parenteral or enteral dosage form.

[0066] According to another aspect of the present disclosure, provided is the use of the Amuc_1100 protein obtained by the method described herein, the cell culture described herein, or the composition described herein in the preparation of drugs, health products, or foods for preventing and / or treating inflammatory diseases, liver and kidney diseases, cardiovascular and cerebrovascular diseases, metabolic-related diseases, lipid-lowering, intestinal barrier damage-related diseases, immune-related diseases, neuropsychiatric diseases, and tumors.

[0067] According to another aspect of the present disclosure, provided is a method for preventing and / or treating inflammatory diseases, liver and kidney diseases, cardiovascular and cerebrovascular diseases, metabolism-related diseases, lipid-lowering, intestinal barrier damage-related diseases, immune-related diseases, neuropsychiatric diseases, and tumors, the method comprising administering to a subject in need thereof an effective amount of the Amuc_1100 protein obtained by the method described in the present disclosure, the cell culture described in the present disclosure, or the composition described in the present disclosure.

[0068] In some embodiments, the metabolic disease is a metabolic disease, a metabolic disorder, or a disease caused by a metabolic disorder, including but not limited to: liver disease, obesity and obesity-related diseases, cardiovascular disease, cardiovascular and cerebrovascular disease, diabetes, dyslipidemia, glucose intolerance, type I diabetes, type II diabetes, impaired glucose tolerance, insulin resistance, obesity, hyperglycemia, hyperinsulinemia, fatty liver, alcoholic steatohepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglyceridemia, uremia, ketoacidosis, hypoglycemia, thrombotic disease, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), atherosclerosis, and at least one of kidney disease.

[0069] In some embodiments, the liver and kidney diseases include liver diseases, diseases related to liver damage, kidney diseases such as kidney damage and abnormal kidney function. Liver diseases include, but are not limited to, at least one of fatty liver, NAFLD / NASH, abnormal liver function, extrahepatic cholestasis, hepatitis, liver damage, intrahepatic cholestasis, liver fibrosis, cirrhosis, and liver cell damage.

[0070] According to another aspect of the present disclosure, provided is the use of the Amuc_1100 protein obtained by the method described in the present disclosure, the cell culture described in the present disclosure, or the composition described in the present disclosure in the preparation of a medicine, health product, or food for affecting or regulating immune signal transduction, affecting intestinal barrier function, affecting or regulating fasting glucose homeostasis, affecting or regulating cholesterol homeostasis, triglyceride homeostasis, repairing liver damage, kidney damage, promoting local adipose tissue metabolism, and reducing liver and / or kidney damage.

[0071] According to another aspect of the present disclosure, a method for influencing or regulating immune signaling, influencing intestinal barrier function, influencing or regulating fasting glucose homeostasis, influencing or regulating cholesterol homeostasis, triglyceride homeostasis, repairing liver damage, kidney damage, promoting local adipose tissue metabolism, and reducing liver and / or kidney damage is provided, the method comprising administering to a subject in need thereof an effective amount of the Amuc_1100 protein obtained by the method described in the present disclosure, the cell culture described in the present disclosure, or the composition described in the present disclosure.

[0072] In some embodiments, the inflammatory disease is selected from at least one of bronchitis, cervicitis, conjunctival inflammation, esophageal inflammation, myocardial inflammation, rectal inflammation, scleral inflammation, gum inflammation, bone inflammation, lung inflammation, respiratory tract inflammation, inflammatory skin disease, fibrotic disease, and encephalitis.

[0073] In some embodiments, the liver and kidney diseases are selected from at least one of non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, liver fibrosis, cirrhosis, alcoholic liver disease, drug-induced liver injury, kidney injury, and abnormal renal function.

[0074] In some embodiments, the metabolic-related disease is selected from at least one of obesity, metabolic syndrome, insulin deficiency or insulin resistance-related disorders, diabetes, glucose intolerance, lipid metabolism disorders, hyperglycemia, dyslipidemia, irritable bowel syndrome, intestinal contraction disorders, hypercholesterolemia, and hyperlipidemia.

[0075] In some embodiments, the cardiovascular and cerebrovascular disease is selected from at least one of hypertension, atherosclerosis, coronary heart disease, and stroke.

[0076] In some embodiments, the disease associated with intestinal barrier damage is selected from inflammatory bowel disease, Crohn's disease, and ulcerative colitis.

[0077] In some embodiments, the lipid reduction is selected from the group consisting of reducing visceral fat and / or local fat.

[0078] In some embodiments, the cell culture or the composition can treat, prevent, alleviate or improve at least one of the following: weight loss or weight control; prevention, treatment, improvement or alleviation of liver damage, non-alcoholic fatty liver disease or non-alcoholic fatty liver disease; prevention, treatment, improvement or alleviation of kidney damage and abnormal renal function; promotion of type I interferon IFNβ expression and improvement of immunity; prevention, treatment, improvement or alleviation of cancer; prevention, treatment, improvement or alleviation of diabetes; reduction of blood lipid and / or cholesterol levels in the subject; reduction of local fat, body fat percentage and / or visceral fat in the subject; repair of intestinal tissue mucosa in the subject; prevention, treatment and improvement of hypertension and atherosclerosis; regulation of metabolism and immunity at the same time; repair of intestinal barrier damage while reducing abdominal fat; improvement of liver damage while losing weight; anti-inflammatory while losing weight; anti-inflammatory while lowering lipids; repair of intestinal barrier damage while lowering lipids; reduction of serum AST and / or ALT while lowering lipids; improvement of liver damage while improving kidney damage; improvement of diabetes while improving liver damage; improvement of diabetes while improving kidney damage; improvement of atherosclerosis while improving liver damage; improvement of atherosclerosis while improving atherosclerosis and lowering lipids. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 The relationship between OD600 and bacterial count of Akkermansia muciniphila culture according to some embodiments of the present disclosure is shown.

[0080] Figure 2 Shown is an Amuc_1100 protein quantification standard curve according to some embodiments of the present disclosure.

[0081] Figure 3 Shown is a quantitative mass spectrometry peak diagram of the Amuc_1100 characteristic peptide when Akkermansia muciniphila is cultured using different nitrogen sources according to some embodiments of the present disclosure.

[0082] Figure 4 A graph showing the relationship between the total bacterial count in the fermentation broth and the Amuc_1100 protein content in the culture broth of Akkermansia muciniphila cultured using different nitrogen sources. Strain preservation

[0083] The strain Akkermansia muciniphila MNH19250 was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) with the deposit number GDMCC No: 63782 and the deposit date on June 21, 2024. The address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences. The deposit name is Akkermansia muciniphila MNH19250, and the proposed taxonomic name is Akkermansia muciniphila. DETAILED DESCRIPTION

[0084] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and examples. The specific embodiments described herein are intended only to illustrate the present invention and are not intended to limit the present invention in any way. In addition, descriptions of known structures and techniques are omitted in the following description to avoid unnecessary confusion about the concepts of the present disclosure. Such structures and techniques are also described in many publications.

[0085] Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly used in the field to which this disclosure belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0086] Unless the context clearly dictates otherwise, as used herein, the expressions "a" and "an" include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0087] As used herein, the term "prebiotic" may be a general term referring to chemicals and / or ingredients that can affect the growth and / or activity of microorganisms in a host (e.g., can allow for specific changes in the composition and / or activity of a microbiome).

[0088] Compositions or preparations of the present disclosure can be used as pharmaceutical preparations, therapeutic compositions, dietary supplements, nutritional supplements, medical probiotics or medical food. In some cases, the composition is used in the form of a pharmaceutical preparation. In some cases, the composition is used in the form of a nutritional supplement. In some cases, the composition is used in the form of a dietary supplement. In some cases, the composition is used in the form of a medical food. In some cases, the composition is used in the form of a medical probiotic. In some cases, the composition (for example, dietary supplements, nutritional supplements, medical probiotics or medical food) can be orally administered, for example, as a capsule, pill or tablet.

[0089] "Supernatant" or "supernatant" within the meaning herein refers to the culture supernatant of the bacterial strain according to the present disclosure, optionally comprising compounds and / or cell debris of said strain, and / or metabolites and / or molecules secreted by said strain.

[0090] Compositions can be prepared using the Akkermansia muciniphila cultures described herein, for example, using pharmaceutically acceptable excipients. The pharmaceutical compositions contain a pharmaceutically effective amount of the Akkermansia muciniphila culture, such as the Akkermansia muciniphila culture deposited with GDMCC NO: 63782. Similarly, the Akkermansia muciniphila culture deposited with GDMCC NO: 63782 can also be prepared into pharmaceutical compositions, for example, using pharmaceutically acceptable excipients, containing a pharmaceutically effective amount of the Akkermansia muciniphila.

[0091] Suitable pharmaceutically acceptable excipients that may be used include, for example, carriers, excipients, diluents, lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners, and flavors.

[0092] The compositions herein can be formulated into any form suitable for enhancing the abundance of Akkermansia muciniphila in a subject. The compositions can be administered orally (e.g., by oral gavage), intramuscularly, by inhalation, intracranially, intralymphatically, intraocularly, intraperitoneally, intrapleurally, intrathecally, intratracheally, intrauterinely, intravascularly, intravenously, intravesically, intranasally, intragastrointestinally, by biliary infusion, by cardiac infusion, preanally, rectally, subcutaneously, sublingually, topically, intravaginally, transdermally, or by ureteral or urethral administration.

[0093] Examples of dosage forms suitable for the compositions herein include, but are not limited to, tablets, aerosols, chewable sticks, capsules, capsules containing coated granules, capsules containing sustained-release granules, capsules containing sustained-release granules, and concentrates.

[0094] In some embodiments, the composition is a sugar-coated tablet, gel capsule, gel, emulsion, tablet, sheet capsule, hydrogel, nanofiber gel, electrospun fiber, food bar, candy, fermented milk, fermented cheese, chewing gum, powder or toothpaste, etc.

[0095] In some embodiments, administration can also be by inclusion in a subject's diet, such as in a functional food for humans or companion animals.

[0096] The compositions provided herein may comprise a pharmaceutically acceptable excipient, diluent or carrier. Such pharmaceutically acceptable excipients, diluents or carriers are well known in the art.

[0097] In some embodiments, the Akkermansia muciniphila in the compositions of the present disclosure is lyophilized. In some embodiments, the Akkermansia muciniphila in the compositions of the present disclosure is spray-dried. In some embodiments, the Akkermansia muciniphila in the compositions of the present disclosure is lyophilized or spray-dried and is viable. In some embodiments, the Akkermansia muciniphila in the compositions of the present disclosure is lyophilized or spray-dried and is capable of partially or completely colonizing the intestine. In some embodiments, the lyophilized Akkermansia muciniphila is reconstituted prior to administration. In some embodiments, the reconstitution is performed using a diluent as described herein.

[0098] In some embodiments, the compositions of the present disclosure are administered orally. Oral administration may involve swallowing, thereby allowing the composition to enter the gastrointestinal tract, and / or administration through the mouth, tongue, or sublingually.

[0099] In some embodiments, the composition is prepared by freeze-drying or spray-drying.

[0100] The compositions disclosed herein include pharmaceutical compositions, health products or foods.

[0101] The subject of the present disclosure may be a human or an animal, including but not limited to cattle, sheep, cats, dogs, horses, rabbits, monkeys, mice, rats, alpacas, camels, etc.

[0102] The pharmaceutical composition disclosed herein can be used to treat, prevent or alleviate metabolic diseases or diseases caused by metabolic disorders.

[0103] The pharmaceutical composition disclosed herein can be used to treat, prevent or alleviate tumors.

[0104] In some embodiments, the metabolic disease, metabolic disorder, or disease caused by metabolic disorder includes but is not limited to: liver disease, obesity and obesity-related diseases, cardiovascular disease, diabetes, dyslipidemia, cardiovascular and cerebrovascular disease, glucose intolerance, atherosclerosis, coronary heart disease or hypertension, type I diabetes, type II diabetes, impaired glucose tolerance, insulin resistance, obesity, hyperglycemia, hyperinsulinemia, fatty liver, alcoholic steatohepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglyceridemia, uremia, ketoacidosis, hypoglycemia, thrombotic disease, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), atherosclerosis, and at least one of kidney disease.

[0105] Diabetes includes type 1 diabetes (T1D), type 2 diabetes (T2D), and gestational diabetes mellitus (GDM). Type 1 diabetes, caused by autoimmune or idiopathic factors, is characterized by the complete destruction of pancreatic islet function. It primarily occurs in children and adolescents and requires insulin therapy for satisfactory results; otherwise, it can be life-threatening. Type 2 diabetes is a multifactorial syndrome characterized by abnormal carbohydrate / fat metabolism, typically including hyperglycemia, hypertension, and abnormal cholesterol levels. Type 2 diabetes is caused by ineffective insulin action (low insulin receptor binding), so it is important to monitor not only fasting blood sugar levels but also two hours after meals, with particular emphasis on pancreatic islet function tests. There are two types of diabetes during pregnancy: one is diagnosed with diabetes before pregnancy, known as "diabetes complicated by pregnancy"; the other is normal glucose metabolism or potential impaired glucose tolerance before pregnancy, which develops or is diagnosed during pregnancy. This is also known as "gestational diabetes mellitus (GDM)." Over 80% of pregnant women with diabetes have GDM.

[0106] Four metabolic disease-related models, namely the high-fat diet (HFD)-induced mouse obesity model, the high-fat, high-sugar, and high-cholesterol-induced mouse NASH model, the high-fat diet combined with streptozotocin (HFD-STZ)-induced mouse type II diabetes model, and the leptin receptor gene-deficient mouse model (db / db), are all commonly used metabolic disease mouse models. Model mice usually have metabolic diseases such as obesity, insulin resistance, hyperglycemia, hyperlipidemia, hypercholesterolemia, NAFLD / NASH, etc.

[0107] Insulin resistance refers to the decrease in the efficiency of insulin in promoting glucose uptake and utilization due to various reasons. The body compensatory secretion of excessive insulin produces hyperinsulinemia to maintain blood sugar stability. Insulin resistance can easily lead to metabolic syndrome and type 2 diabetes.

[0108] Liver disease is defined as abnormal liver function or liver damage. Alanine aminotransferase (ALT) and / or aspartate aminotransferase (AST) are sensitive markers of liver disease.

[0109] Non-alcoholic fatty liver disease (NAFLD) refers to the accumulation of excess fat in the form of triglycerides (TG) in the liver (steatosis). Some patients with NAFLD also experience liver cell damage and inflammation (steatohepatitis) in addition to excess fat, a condition known as non-alcoholic steatohepatitis (NASH). NASH is widely considered the hepatic manifestation of metabolic syndrome, which includes type 2 diabetes, insulin resistance, central obesity, hyperlipidemia (low high-density lipoprotein cholesterol, high triglycerides), and hypertension.

[0110] Liver and kidney function diseases refer to functional acute renal failure that occurs in severe liver disease and decompensated cirrhosis, which can cause hepatorenal syndrome due to insufficient effective circulating blood volume and decreased prostaglandins.

[0111] Obesity refers to a certain degree of significant overweight and an excessive fat layer. It is a condition caused by excessive accumulation of body fat, particularly triglycerides. It is an abnormal or excessive accumulation of fat that poses a health risk. Excessive body fat accumulation, caused by excessive food intake or altered metabolism, leads to excessive weight gain and can cause pathological or physiological changes or potential health problems. A body mass index (BMI) over 25 is considered overweight, and over 30 is considered obese. Obesity increases the risk of many physical and mental illnesses. It is primarily associated with metabolic syndrome, a combination of conditions including type 2 diabetes, hypertension, hypercholesterolemia, and hypertriglyceridemia. Generally speaking, the health effects of obesity fall into two main categories: conditions attributable to increased body fat (such as osteoarthritis and obstructive sleep apnea) and conditions attributed to an increased number of fat cells (such as diabetes, dyslipidemia, cancer, cardiovascular disease, non-alcoholic fatty liver disease or non-alcoholic steatohepatitis). The "obesity-related diseases" can be selected from the following diseases: overeating, binge eating, bulimia, hypertension, diabetes, elevated plasma insulin concentration, insulin resistance, hyperlipidemia, metabolic syndrome, insulin resistance syndrome, obesity-related gastroesophageal reflux disease, arteriosclerosis, hypercholesterolemia, hyperuricemia, low back pain, cardiac hypertrophy and left ventricular hypertrophy, lipodystrophy, non-alcoholic steatohepatitis, cardiovascular disease, and polycystic ovary syndrome, as well as subjects with these obesity-related diseases including those who wish to lose weight.

[0112] The obesity-related diseases in the present disclosure include at least one of the following diseases: obesity, metabolic syndrome, cardiovascular disease, hyperlipidemia, hypercholesterolemia, hypertension, insulin resistance syndrome, obesity-related gastroesophageal reflux disease, and steatohepatitis.

[0113] The reagents used in the following examples are either commercially available or can be synthesized by known methods.

[0114] Example

[0115] 1. Materials and Methods

[0116] 1.1 Materials

[0117] 1.1.1 Bacterial strains

[0118] The strain used in this experiment is Akkermansia muciniphila MNH19250, which is deposited in Guangdong Provincial Microbiological Culture Collection Center. The deposit name is Akkermansia muciniphila MNH19250, the strain deposit number is GDMCC No: 63782, and the deposit date is June 21, 2024.

[0119] 1.1.2 Culture medium

[0120] Liquid seed medium: 15.0 g / L yeast peptone, 15.0 g / L yeast extract powder, 5.0 g / L edible glucose monohydrate, 10 g / L N-acetyl-D-glucosamine, 1.0 g / L L-cysteine ​​hydrochloride monohydrate, 2.0 g / L L-threonine, 0.1 g / L magnesium sulfate heptahydrate, 0.05 g / L ferrous sulfate heptahydrate, 0.05 g / L manganese sulfate, 1.0 g / L Tween 80, 2.0 g / L anhydrous dipotassium hydrogen phosphate, 4.0 g / L potassium citrate, and 5.0 g / L anhydrous sodium acetate.

[0121] Basal medium formula: 5.0 g / L edible glucose monohydrate, 10 g / L N-acetyl-D-glucosamine, 1.0 g / L L-cysteine ​​hydrochloride monohydrate, 2.0 g / L L-threonine, 0.1 g / L magnesium sulfate heptahydrate, 0.05 g / L ferrous sulfate heptahydrate, 0.05 g / L manganese sulfate, 1.0 g / L Tween 80, 2.0 g / L anhydrous dipotassium hydrogen phosphate, 4.0 g / L potassium citrate, 5.0 g / L anhydrous sodium acetate.

[0122] Complete culture medium: Using the above basal culture medium as the basic component, add different types of peptone (see Table 1) at a dosage of 30 g / L to prepare complete culture medium with different types of peptone.

[0123] Table 1 Complete culture medium supplemented with different candidate nitrogen sources

[0124] Complete medium nitrogen source 1 Basic medium + 30 g / L yeast peptone 2 Basic culture medium + 30 g / L yeast extract powder 3 Basal medium + 30 g / L soy peptone 4 Basal medium + 30 g / L tryptone 5 Basal medium + 30 g / L wheat peptone 6 Basic culture medium + 30 g / L beef extract powder 7 Basal medium + 30 g / L pea peptone 8 Basal medium + 30 g / L citrulline peptone 9 Basal medium + 30 g / L casein peptone 10 Basal medium + 30 g / L rice peptone

[0125] 1.1.3 Strain activation and cultivation

[0126] Take a glycerol cryovial of MNH19250 and activate it in liquid seed medium in an anaerobic workstation for 24-28 hours. Then, inoculate it with fresh liquid seed medium at a 2% inoculum and incubate it for 24 hours to prepare a seed solution. Then, inoculate the seed solution at 2.5% into complete medium containing different nitrogen sources and incubate anaerobically at 37°C for 48 hours. Collect the bacterial suspension for subsequent testing.

[0127] 1.1.4 Reagents

[0128] The reagents used in the following examples are shown in Table 2.

[0129] Table 2

[0130] Reagents level Manufacturer hydrochloric acid analytically pure Sinopharm Chemical Reagent Co., Ltd. acetone analytically pure Sinopharm Chemical Reagent Co., Ltd. urea analytically pure Aladdin Acetonitrile Chromatographically pure Fisher Scientific Methanol Chromatographically pure Sigma Aldrich Formic acid Mass spectrometry pure Macklin Trypsin Mass spectrometry pure Fisher Scientific water First-class water Fisher Chemical Iodoacetamide (IAA) analytically pure Sigma Aldrich Dithiothreitol (DTT) analytically pure Sigma Aldrich Tris base analytically pure Solarbio Yeast peptone Food grade Angel Yeast Co., Ltd. Yeast extract powder Food grade Angel Yeast Co., Ltd. Soybean Peptone Food grade Angel Yeast Co., Ltd. Tryptone Food grade Beijing Hongrun Baoshun Technology Co., Ltd. Wheat peptone Food grade Shanghai Hanni Biotechnology Co., Ltd. Beef dip powder Food grade Guangdong Huankai Microbiology Technology Co., Ltd. Pea Peptone Food grade Angel Yeast Co., Ltd. Melon Peptone Food grade Shanghai Hanni Biotechnology Co., Ltd. Casein peptone Food grade Shanghai Hanni Biotechnology Co., Ltd. Rice peptone Food grade Shanghai Hanni Biotechnology Co., Ltd.

[0131] 1.1.5 Instruments and equipment

[0132] The instruments and equipment used in the following examples are shown in Table 3.

[0133] Table 3

[0134]

[0135] 1.2 Detection and analysis methods

[0136] 1.2.1 Bacterial growth detection method

[0137] 1) OD600 Assay: Dilute the fermentation broth to an absorbance between 0.2 and 0.8. Measure the absorbance at 600 nm using a spectrophotometer. OD600nm = OD600 × dilution factor. Perform three dilutions per sample and take the average value.

[0138] 2) Counting plate: Hemocytometer measurement. Take a clean hemocytometer and drop a diluted bacterial suspension onto the small squares of the counting plate. Let it sit for approximately 5 minutes before counting. The calculation formula is: TFU / mL = average number of cells per square × 20,000 × 1,000 × dilution factor. Repeat the count three times for each sample and take the average value.

[0139] 1.2.2 Amuc_1100 protein quantitative detection method

[0140] Principle of quantitative detection: Quantitative detection of Amuc_1100 protein in Akkermansia muciniphila is performed using an external standard and a characteristic Amuc_1100 peptide using mass spectrometry. Specifically, a standard (molecular weight 858.50434) of the characteristic Amuc_1100 peptide, AINSLVNK (SEQ ID NO: 1), with a purity exceeding 98% was chemically synthesized. A standard curve for the characteristic peptide was established by calculating the peptide signal using mass spectrometry. Finally, the Amuc_1100 protein content in Akkermansia muciniphila samples was determined using this standard curve.

[0141] The specific steps of the experiment are as follows:

[0142] (1) Peptide standard stock solution

[0143] Accurately weigh 5.00 mg of synthetic peptide (AINSLVNK standard) and place it in a 5 mL volumetric flask. Dissolve it in 0.1% formic acid aqueous solution and dilute to the mark. Shake well to prepare a stock solution with a concentration of 1 mg / mL.

[0144] (2) Drawing of standard curve

[0145] The peptide standard stock solution was diluted to a 10 mg / L standard working solution, and further diluted to create 500, 100, 50, 10, 5, and 1 μg / L standard curve working solutions. The characteristic peptide content in the peptide standard linear working dilutions was then measured using a triple quadrupole liquid chromatography-mass spectrometry instrument. A standard curve was then constructed to quantify the characteristic peptide content in real samples and ultimately convert it into the Amuc_1100 protein content.

[0146] (3) Preparation of protein peptides from bacterial samples

[0147] An appropriate amount of sample (3.5-4 ml) was centrifuged and the supernatant discarded. 8 M urea was added to the pellet, ultrasonically disrupted, and extracted. The pellet was centrifuged at 12,000 rpm for 20 min. The supernatant was removed and the volume was made up to 500 μL. A small amount of the supernatant was used for protein concentration analysis. The remaining supernatant was precipitated with acetone, reconstituted with 8 M urea solution, and reduced with DTT (10 mM) at 37°C for 60 min. IAA (50 mM) was then added and incubated at room temperature in the dark for 45 min for alkylation. Finally, the sample was diluted to a urea concentration below 1 M, and trypsin was added at a 1:50 mass ratio (trypsin:protein) for digestion at 37°C overnight. Peptide samples were desalted using Waters SEP-PAK C18 and dissolved in 100 μL of 0.1% formic acid for subsequent mass spectrometry analysis.

[0148] (4) Amuc_1100 protein quantitative detection

[0149] A standard curve was drawn using characteristic peptide standards, and the characteristic peptide content in the samples was detected by triple quadrupole liquid chromatography-mass spectrometry. The Amuc_1100 protein content in the samples was calculated using the external standard method.

[0150] The HPLC-MS detection conditions are shown in Table 4 below:

[0151] Table 4

[0152]

[0153] The calculation formula of Amuc_1100 protein content in the sample is:

[0154] 1) Amuc_1100 content of the test sample (ng / mL) = (CX-C0) × M1 × V / m / M2 / 1000 (Formula 1)

[0155] m: liquid (volume of sample, in liters (L));

[0156] M1: protein molecular weight (target protein molecular weight), Amuc_1100 protein is 34213 Da;

[0157] M2: molecular mass of peptide standard (characteristic peptide molecular weight), the peptide is 858.50434 Da;

[0158] V: Final volume of the sample solution, in milliliters (mL), which refers to the final volume of the desalted lyophilized peptide, i.e. 0.1 mL;

[0159] C0: mass concentration of blank, in micrograms per liter (μg / L);

[0160] CX: The peptide concentration of the test substance, expressed in micrograms per liter (μg / L).

[0161] Example 1: Fermentation of Akkermansia muciniphila in shake flasks

[0162] Akkermansia muciniphila was cultured for 48 hours using prepared complete culture medium containing different nitrogen sources to explore the effects of different nitrogen sources on the growth of Akkermansia muciniphila, including bacterial concentration (OD value) and bacterial count.

[0163] The results are shown in Table 5. According to the results of OD600 determination, after 48 h of culture, except for the trypsin and citrulline peptone groups whose OD600 exceeded 3, the OD600 of the other peptone groups of MNH19250 were between 2 and 3. OD600 usually reflects the concentration of bacteria and is used to evaluate the growth of bacteria. However, since the metabolites secreted by Akkermansia muciniphila will affect the absorbance, the measured absorbance value may not truly reflect the number of bacteria, thereby affecting the subsequent accurate evaluation of the Amuc_1100 protein content in the bacteria. Therefore, this embodiment further utilizes the counting plate counting method to directly count the number of bacteria under a microscope to determine the number of bacteria in the culture set. The detection results of the total bacterial count are shown in Table 5. By analyzing the relationship between OD600 and the corresponding bacterial count, the results show that, Figure 1 , OD600 and the corresponding bacterial count fitting curve, R 2 =0.0924, indicating that there is no linear relationship between the two. Therefore, in the subsequent evaluation of bacterial Amuc_1100 content, the bacterial count is used as the standard of measurement, which is more accurate than OD600.

[0164] According to Table 5, the median of the total bacterial count in the fermentation broth was 6.835×10 9 Tryptone, pea peptone, casein peptone, yeast peptone and yeast extract powder can significantly promote the increase of the total number of Akkermansia muciniphila in the fermentation broth.

[0165] Table 5 Concentration and bacterial count of MNH19250 after 48 h of culture on different nitrogen sources

[0166]

[0167] Example 2: Quantitative detection of Amuc_1100 protein

[0168] To quantify the Amuc_1100 protein content in the sample, a quantitative standard curve was first established using the Amuc_1100 characteristic peptide standard by external standard method. Figure 2 The results showed that the linear equation was fitted based on the peptide concentration (ng / ml) and the response (Responses) of the peptide detected by mass spectrometry, and the formula was: y = 389.827848 × x - 234.964403, R 2 =0.99811738. The established quantitative standard curve was then used to detect the Amuc_1100 protein content in different samples.

[0169] Table 6 Amuc_1100 protein quantification standard curve detection results

[0170]

[0171] In Example 1, the bacterial suspension samples of 10 strains of Akkermansia muciniphila cultured with different nitrogen sources were subjected to bacterial protein extraction and enzymatic hydrolysis to obtain peptides. The characteristic peptides of Amuc_1100 were then detected and analyzed using high-performance liquid chromatography-tandem high-resolution mass spectrometry. The quantitative peaks of the characteristic peptide mass spectra of each sample are shown in FIG. Figure 3 Then, according to the established formula of the quantitative standard curve of the characteristic peptide of Amuc_1100 protein (y = 389.827848 × x - 234.964403) and the calculation formula of the Amuc_1100 protein content of the sample (Amuc_1100 content of the test sample (ng / mL) = (CX-C0) × M1 × V / m / M2 / 1000, for the specific meaning, see the above formula 1), the content of Amuc_1100 protein in the sample was calculated. Then, by combining the number of bacteria in the test sample, the effect of different nitrogen sources on the bacterial Amuc_1100 content was evaluated.

[0172] The test results are shown in Table 7. The median Amuc_1100 content per milliliter of sample was 2.395 ng. The top four Amuc_1100 content extracts were soy peptone (4.474 ng), tryptone (3.898 ng), wheat peptone (3.235 ng), and yeast extract powder (2.828 ng). This indicates that the addition of soy peptone, tryptone, wheat peptone, and yeast extract powder significantly increased the level of the functional protein Amuc_1100 per unit volume of fermentation broth compared to peptones containing other nitrogen sources. Fermentation broths of Akkermansia muciniphila cultured in medium containing these nitrogen sources exhibited higher Amuc_1100 protein concentrations.

[0173] Every 10 10 Among the individual strains, the median Amuc_1100 content was 3.61835 ng. The top four Amuc_1100 strains were soy peptone (9.8109 ng), wheat peptone (6.1857 ng), rice peptone (6.4008 ng), and tryptone (4.8539 ng), with levels exceeding 4 ng. Soy peptone had the highest content, approaching 10 ng. This indicates that the addition of soy peptone, wheat peptone, rice peptone, and tryptone significantly increased the level of the functional protein Amuc_1100 in individual cells compared to peptones derived from other nitrogen sources. This suggests that Akkermansia muciniphila cultured in media containing this nitrogen source may have a more beneficial effect on probiotics.

[0174] Table 7 Amuc_1100 protein quantification results

[0175]

[0176] By plotting the bacterial counts after 48 h of culture in Table 5 and the Amuc_1100 content of the fermentation broth samples in Table 7, we can obtain the relationship between the total bacterial counts and the Amuc_1100 protein content in the fermentation broth. Figure 4 The relationship between the total bacterial count in the fermentation broth and the Amuc_1100 protein content in the fermentation broth is shown. Figure 4 As can be seen, after 48 hours of fermentation, there was a negative correlation between the total bacterial count in the fermentation broth and the Amuc_1100 protein content. It is speculated that a higher Akkermansia muciniphila bacterial count may consume Amuc_1100 protein or reduce its synthesis, thereby reducing Amuc_1100 protein content. Therefore, it is possible to increase Amuc_1100 protein production by controlling the total bacterial count in the fermentation broth.

[0177] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. Any technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. Use of peptone or yeast extract powder in promoting the production of the functional protein Amuc_1100 in individual cells of Akkermansia muciniphila, wherein the peptone comprises at least one of soy peptone, tryptone, wheat peptone, or rice peptone, and the Amuc_1100 protein is derived from Akkermansia muciniphila. The production of the functional protein Amuc_1100 per individual cell of Akkermansia muciniphila is promoted by controlling the total bacterial count in the fermentation broth while increasing the Amuc_1100 protein yield.

2. Use of peptone or yeast extract powder in preparing a culture medium for promoting the production of the functional protein Amuc_1100 per single cell of Akkermansia muciniphila, wherein the peptone comprises at least one of soy peptone, tryptone, wheat peptone, or rice peptone. The promotion of the production of the functional protein Amuc_1100 per single cell of Akkermansia muciniphila is achieved by increasing the Amuc_1100 protein yield while controlling the total bacterial count in the fermentation broth.

3. A method for promoting the production of a functional protein Amuc_1100 in a single cell of Akkermansia muciniphila, the method comprising culturing Akkermansia muciniphila in a medium containing peptone, wherein: The peptone includes at least one of soy peptone, tryptone, wheat peptone or rice peptone. The method of promoting the yield of the functional protein Amuc_1100 in each single cell of Akkermansia muciniphila is to increase the yield of Amuc_1100 protein while controlling the total bacterial count in the fermentation liquid.

4. A method for increasing the yield of functional protein Amuc_1100 per unit volume of fermentation broth of Akkermansia muciniphila, the method comprising culturing Akkermansia muciniphila in a culture medium containing peptone or yeast extract, wherein: The peptone includes at least one of soy peptone, tryptone, wheat peptone, or rice peptone. The method of promoting the yield of the functional protein Amuc_1100 per unit volume of Akkermansia muciniphila fermentation broth is to increase the yield of Amuc_1100 protein while controlling the total bacterial count in the fermentation broth.

5. The method according to claim 3 or 4, characterized in that The content of peptone in the culture medium is 25-40 g / L; and / or the culture is carried out under anaerobic conditions, the culture temperature is 35-40° C., and the culture time is 24-60 h.

6. The method according to claim 3 or 4, characterized in that The culture medium further comprises at least one of glucose, N-acetyl-D-glucosamine, L-cysteine ​​hydrochloride, L-threonine, magnesium sulfate, ferrous sulfate, manganese sulfate, dipotassium hydrogen phosphate, potassium citrate, and sodium acetate.

7. The method according to claim 3 or 4, characterized in that The culture medium includes 25-40 g / L peptone, 2-10 g / L glucose monohydrate, 5-20 g / L N-acetyl-D-glucosamine, 0.5-2 g / L L-cysteine ​​hydrochloride monohydrate, 1-5 g / L L-threonine, 0.05-0.2 g / L magnesium sulfate heptahydrate, 0.01-0.1 g / L ferrous sulfate heptahydrate, 0.02-0.1 g / L manganese sulfate, 0.5-2 g / L Tween 80, 1-5 g / L dipotassium hydrogen phosphate, 2-8 g / L potassium citrate, and 2-8 g / L sodium acetate.

8. The method according to claim 7, characterized in that The culture medium includes 28-35 g / L peptone, 4-6 g / L glucose monohydrate, 8-15 g / L N-acetyl-D-glucosamine, 0.8-1.5 g / L L-cysteine ​​hydrochloride monohydrate, 1-3 g / L L-threonine, 0.08-0.15 g / L magnesium sulfate heptahydrate, 0.02-0.08 g / L ferrous sulfate heptahydrate, 0.04-0.08 g / L manganese sulfate, 1.5-3 g / L dipotassium hydrogen phosphate, 3-5 g / L potassium citrate, and 4-6 g / L sodium acetate.

9. The use according to claim 1 or 2 or the method according to claim 3 or 4, characterized in that The Akkermansia muciniphila is the Akkermansia muciniphila MNH19250 strain, which is deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) with a deposit number of GDMCC NO: 63782 and a deposit date of June 21, 2024.

10. A cell culture of Akkermansia muciniphila, obtained by the method according to any one of claims 3 to 9, wherein the content of the functional protein Amuc_1100 in each single cell of the cell culture exceeds 4 ng; The cell culture is selected from any one of the following: fermentation broth, supernatant of fermentation broth, inactivated product of fermentation broth, or dried product thereof.

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