Methods for improving amuc_1100 protein production

Abstract

The present disclosure provides a method for improving the yield of Amuc_1100 protein, the method comprising culturing Akkermansia muciniphila in a culture medium containing proteose peptone or yeast extract powder, wherein the proteose peptone comprises at least one of soy peptone, tryptone, wheat peptone or rice peptone. The method of the present disclosure can significantly improve the yield of the active ingredient Amuc_1100 protein of Akkermansia muciniphila.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for increasing the yield of Amuc_1100 protein. Background Technology

[0002] Akkermansia muciniphila (A. muciniphila, Akk) is a new species of a new genus in the phylum Verrucomicrobiota, successfully isolated in 2004 by researchers at the Microbiology Laboratory in Wageningen (PMID: 15388697). Akkermansia muciniphila colonizes the mucosa of the digestive gastrointestinal tract, specifically degrades mucin, and is ubiquitous in the intestines of healthy adults and infants, accounting for 1%–4% of the total gut microbiota (PMID: 18083887). As an important member of the gut microbiota, numerous studies in recent years have shown that the abundance of Akkermansia muciniphila colonization is closely related to various diseases, such as obesity, diabetes, hepatic steatosis, inflammation, and response to cancer immunotherapy. Therefore, Akkermansia muciniphila is considered one of the "next-generation" probiotics, with significant applications and commercial value in the food and pharmaceutical fields.

[0003] Amuc_1100, a key functional protein of the outer membrane of Akkermansia muciniphila, has been shown to be a specific effector molecule mediated by this bacterium in regulating host health. Multiple studies have revealed that this protein has significant biological activity in different disease models: In the field of metabolic diseases, Amuc_1100 effectively regulates metabolic disorders in obese and diabetic animal models 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 alleviate colitis symptoms by inhibiting macrophage infiltration and cytotoxic T lymphocyte (CTL) activation, and it can also inhibit colitis-related carcinogenesis by upregulating TNF-α expression, inhibiting PD-1 immune checkpoint molecules, and promoting CTL proliferation and activation (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). This evidence suggests that Amuc_1100 protein is one of the important active substances in Akkermansia myxophilus that exerts a beneficial effect on disease, and also indicates that its content plays an important role in the beneficial effects of Akkermansia myxophilus.

[0004] Because *Ackermania myxophilus* offers numerous health benefits, current research and patents primarily focus on achieving large-scale production. This includes optimizing fermentation processes by modifying culture medium composition, culture time, centrifugation conditions, and adding specific derivatives. While these optimizations significantly contribute to increasing *Ackermania myxophilus* yield, the quantitative detection of Amuc_1100 protein remains challenging. Commercially available antibodies or detection kits for Amuc_1100 are unavailable, and mass spectrometry methods require complex pretreatment steps and are costly. Current research has largely ignored the impact of these culture methods on the active component of *Ackermania myxophilus*, Amuc_1100 protein, and has also neglected methods to increase its yield.

[0005] Amuc_1100, as an important active component of Akkermansia myxophilus, significantly influences the probiotic effects of the bacteria. While optimizing fermentation processes can increase Akkermansia myxophilus yield, a higher yield does not necessarily indicate a higher Amuc_1100 protein content in the fermentation broth. Therefore, while improving Akkermansia myxophilus yield, it is crucial to consider the impact of fermentation methods on Amuc_1100 levels.

[0006] Increasing the yield of Amuc_1100 protein is crucial for the industrial production of Amuc_1100 and enhancing the probiotic effects of Akkermansia myxophilus. Therefore, providing a fermentation process for producing Akkermansia myxophilus and significantly increasing the yield of Amuc_1100 protein is of great significance. Summary of the Invention

[0007] This disclosure improves the yield of Amuc_1100 protein, the active ingredient of Akkermansia muciniphila, by optimizing the nitrogen source in the culture medium. It was found that yeast extract or specific types of peptone, such as soybean 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 this disclosure, the use of peptone or yeast extract in increasing the yield of Amuc_1100 protein is provided, said peptone including at least one of soybean peptone, trypsin, wheat peptone or rice peptone, said Amuc_1100 protein being derived from Akkermansia muciniphila.

[0009] The Akkermansia muciniphila in this disclosure can be any Akkermansia muciniphila capable of producing the Amuc_1100 protein. In some embodiments, the Akkermansia muciniphila can be any Akkermansia muciniphila strain listed in Chinese patent application CN2024119901535. In some specific embodiments, the Akkermansia muciniphila is strain MNH19250, deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) with accession number GDMCC NO: 63782 and deposit date of June 21, 2024.

[0010] Another aspect of this disclosure provides the use of peptone or yeast extract in the preparation of a culture medium for increasing the protein yield of Akkermansia amuc_1100, said peptone comprising at least one of soybean peptone, trypsinone, wheat peptone, or rice peptone.

[0011] According to another aspect of this disclosure, a method for increasing the protein yield of Akkermansia muciniphila Amuc_1100 is provided, the method comprising culturing Akkermansia muciniphila in a culture medium containing peptone, wherein the peptone comprises at least one of soybean peptone, trypsin peptone, wheat peptone, or rice peptone.

[0012] According to another aspect of this disclosure, a method for increasing the yield of Amuc_1100 protein in fermentation broth is provided, the method comprising culturing Akkermansia muciniphila in a culture medium containing peptone or yeast extract, wherein the peptone includes at least one selected from soybean peptone, trypsin, wheat peptone, or rice peptone.

[0013] In some embodiments, the Amuc_1100 protein content in the fermentation broth is increased by controlling the total bacterial count in the fermentation broth or preventing the total bacterial count from becoming too high. By controlling the total bacterial count in the fermentation broth or preventing the total bacterial count from becoming 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 peptone content 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 between them.

[0015] In some embodiments, the culture medium further includes 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 peptone content 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 between them.

[0018] In some embodiments, the content of glucose monohydrate in the culture medium may 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 between them, 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 between them.

[0020] In some embodiments, the content of L-cysteine ​​hydrochloride monohydrate in the culture medium may 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 between them, or other forms of L-cysteine ​​hydrochloride equivalent to the L-cysteine ​​hydrochloride content 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 between them.

[0022] In some embodiments, the content of magnesium sulfate heptahydrate in the culture medium may 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 between them, or other forms of magnesium sulfate equivalent to the magnesium sulfate content in magnesium sulfate heptahydrate.

[0023] In some embodiments, the content of ferrous sulfate heptahydrate in the culture medium may 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 between them, or other forms of ferrous sulfate equivalent to the ferrous sulfate content in ferrous sulfate heptahydrate.

[0024] In some embodiments, the manganese sulfate content 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 between them.

[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 between them.

[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 between them.

[0027] In some embodiments, the potassium citrate content 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 between them.

[0028] In some embodiments, the sodium acetate content 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 between them.

[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 in this disclosure can be any Akkermansia muciniphila capable of producing the Amuc_1100 protein. In some embodiments, the Akkermansia muciniphila can be any Akkermansia muciniphila strain listed in Chinese patent application CN2024119901535. In some specific embodiments, the Akkermansia muciniphila is strain MNH19250, deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) with accession number GDMCC NO: 63782 and deposit date of June 21, 2024.

[0031] In some embodiments, the culture is carried out under anaerobic conditions at a temperature of 35-40°C for 24-60 hours.

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

[0033] In some implementations, the incubation time is 24h, 28h, 32h, 36h, 40h, 44h, 48h, 52h, 56h, 60h, or any value between them.

[0034] In some embodiments, the method further includes extracting bacterial proteins from a cultured suspension of Akkermansia obliterans; and optionally, quantitatively detecting the Amuc_1100 protein content in the bacterial proteins.

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

[0036] According to another aspect of this disclosure, a cell culture is provided, said cell culture being obtained by the methods described above in this 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 another aspect of this disclosure, a composition is provided comprising the cell cultures described above.

[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, a heat-inactivated bacterium, preferably pasteurized.

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

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

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

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

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

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

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

[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 carriers, excipients, diluents, lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners, and flavorings.

[0048] In some embodiments, the composition comprises one or more of the following: buffers (e.g., sodium bicarbonate, infant formula or sterile human milk or other agents that allow bacteria to survive and grow (e.g., to survive in the acidic environment of the stomach and to grow in the intestinal environment)), lyophilization protectants, preservatives, stabilizers, binders, compactors, lubricants, dispersants, disintegrants, antioxidants, flavoring agents, sweeteners and colorants).

[0049] In some embodiments, the composition further comprises one or more other active agents for the prevention or treatment of 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, and (e) treating tumors.

[0051] In some embodiments, the appetite suppression includes reducing food intake and / or reducing appetite.

[0052] In some embodiments, the other active agent is selected from: GLP-1 receptor agonists, dual agonists of GLP-1 receptor and GCG receptor, triple agonists of GLP-1 receptor, GIP receptor and GCG receptor, AMPK agonists or active drugs that promote GLP-1 secretion.

[0053] In some embodiments, the other active agents are selected from: metformin, sulfonylureas, chloroanisole, thiazolidinediones, DPP-4 inhibitors, GLP-1 receptor agonists, SGLT2 inhibitors, insulin, pioglitazone, rosiglitazone, pentoxifylline, omega-3 fatty acids, statins, ezetimibe, ursodeoxycholic acid, smegglutide, liraglutide, exenatide, and benaglutide.

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

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

[0056] In some embodiments, the probiotics are selected from one or more of lactic acid bacteria, lactobacilli, lactococci, butyric acid bacteria, bifidobacteria, thermophilic streptococci, fecal streptococci, and mesenteric streptococci.

[0057] In some embodiments, the prebiotic is selected from 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, nutrient compounds, biotin, polydextrose, fructooligosaccharides (FOS), galactooligosaccharides (GOS), starch, cellulose, β-glucan, hemicellulose, lactulose, mannooligosaccharides, mannooligosaccharides (MOS), inulin rich in fructooligosaccharides, oligodextrose, tagatose, trans-galactooligosaccharides, pectin, resistant starch, xylooligosaccharides (XOS), and any combination thereof.

[0058] In some embodiments, the composition may be formulated into a frozen composition, such as a frozen composition prepared by quick-freezing and drying, or freeze-drying, for storage and / or transport.

[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 the diluent described herein.

[0061] In some embodiments, the composition may 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 with an enteric coating. For example, the enteric-coated formulation may be enteric-coated granules, enteric-coated tablets, or enteric-coated capsules. 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 may be a microencapsulated capsule or microcapsule.

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

[0064] In some embodiments, the composition is an infant-specific dosage form, a pediatric-specific dosage form, or an adult-specific dosage form.

[0065] In some embodiments, the composition is a gastrointestinal or non-gastrointestinal dosage form.

[0066] According to another aspect of this disclosure, the use of the Amuc_1100 protein obtained by the methods described in this disclosure, the cell cultures described in this disclosure, or the compositions described in this disclosure in the preparation of medicaments, health products, or foods for the prevention and / or treatment of inflammatory diseases, liver and kidney diseases, cardiovascular and cerebrovascular diseases, metabolic-related diseases, lipid-lowering drugs, intestinal barrier damage-related diseases, immune-related diseases, neuropsychiatric diseases, and tumors is provided.

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

[0068] In some embodiments, the metabolic disease is a metabolic disorder, metabolic disturbance, or a disease caused by a metabolic disturbance, including but not limited to: liver diseases, obesity and obesity-related diseases, cardiovascular diseases, cerebrovascular diseases, 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 diseases, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), atherosclerosis, and at least one of the following: kidney disease.

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

[0070] According to another aspect of this disclosure, the Amuc_1100 protein obtained by the methods described in this disclosure, the cell cultures described in this disclosure, or the compositions described in this disclosure are provided for use in the preparation of medicaments, health products, or foods 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.

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

[0072] In some embodiments, the inflammatory disease is selected from at least one of bronchitis, cervicitis, conjunctivitis, esophagealitis, myocarditis, 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, liver fibrosis, cirrhosis, alcoholic liver disease, drug-induced liver injury, kidney injury, and abnormal kidney 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 diseases are selected from at least one of hypertension, arteriosclerosis, coronary heart disease, and stroke.

[0076] In some embodiments, the intestinal barrier damage-related diseases are selected from inflammatory bowel disease, Crohn's disease, and ulcerative colitis.

[0077] In some embodiments, the lipid-lowering is selected from reducing visceral fat and / or localized 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 reduction of liver damage, non-alcoholic steatohepatitis, or non-alcoholic fatty liver disease; prevention, treatment, improvement, or reduction of kidney damage or abnormal kidney function; promotion of type I interferon IFNβ expression and enhancement of immunity; prevention, treatment, improvement, or reduction of cancer; prevention, treatment, improvement, or reduction of diabetes; reduction of blood lipids and / or cholesterol levels in the subject; reduction of local fat, body fat percentage, and / or visceral fat in the subject; repair of intestinal mucosa in the subject; prevention, treatment, or improvement of hypertension and atherosclerosis; regulation of metabolism and immunity; reduction of abdominal fat and repair of intestinal barrier damage; improvement of liver damage and weight loss; anti-inflammatory effects during weight loss; anti-inflammatory effects during lipid reduction; repair of intestinal barrier damage during lipid reduction; reduction of serum AST and / or ALT and lipid reduction; improvement of liver damage and kidney damage; improvement of diabetes and liver damage; improvement of diabetes and kidney damage; improvement of atherosclerosis and liver damage; improvement of atherosclerosis and lipid reduction. Attached Figure Description

[0079] Figure 1 The relationship between OD600 and bacterial count of Akkermansia muciniphila culture is shown in some embodiments of this disclosure.

[0080] Figure 2 The Amuc_1100 protein quantification standard curves according to some embodiments of this disclosure are shown.

[0081] Figure 3 The images show quantitative mass spectrometry peaks of Amuc_1100 characteristic peptides cultured with different nitrogen sources according to some embodiments of the present disclosure.

[0082] Figure 4 The graph shows the relationship between the total number of bacteria in the fermentation broth and the content of Amuc_1100 protein in the fermentation broth when Akkermansia myxophilus is cultured using different nitrogen sources. Preservation of strains

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

[0084] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been 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 pertains. For purposes 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 indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0087] As used in this article, the term "prebiotic" can be a general term referring to chemical substances and / or components that can affect the growth and / or activity of microorganisms in a host (e.g., that can allow specific changes in the composition and / or activity of the microbiome).

[0088] The compositions or formulations disclosed herein can be administered as pharmaceutical preparations, therapeutic compositions, dietary supplements, nutritional supplements, medical probiotics, or medical foods. In some cases, the compositions are administered as pharmaceutical preparations. In some cases, the compositions are administered as nutritional supplements. In some cases, the compositions are administered as dietary supplements. In some cases, the compositions are administered as medical foods. In some cases, the compositions are administered as medical probiotics. In some cases, the compositions (e.g., dietary supplements, nutritional supplements, medical probiotics, or medical foods) can be administered orally, for example, as capsules, pills, or tablets.

[0089] As used herein, “supernatant” or “supernatant” means the culture supernatant of a bacterial strain according to the present disclosure, optionally containing compounds and / or cell debris of the strain, and / or metabolites and / or molecules secreted by the strain.

[0090] Compositions can be prepared using the Akkermansia myxophilus cultures described herein, for example, by using pharmaceutically acceptable excipients. The pharmaceutical composition comprises a pharmaceutically effective amount of the Akkermansia myxophilus culture, such as the Akkermansia myxophilus culture with accession number GDMCC NO: 63782. Similarly, the Akkermansia myxophilus culture with accession number GDMCC NO: 63782 can also be prepared into a pharmaceutical composition, for example, by using pharmaceutically acceptable excipients, comprising a pharmaceutically effective amount of the Akkermansia myxophilus.

[0091] Suitable pharmaceutically acceptable excipients include, for example, carriers, excipients, diluents, lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners, and flavorings.

[0092] The compositions described herein can be formulated in any form suitable for enhancing the abundance of Akkermansia myxophilus in a subject. The compositions can be administered orally (e.g., via oral gavage), intramuscularly, by inhalation, intracranially, intralymphaticly, intraocularly, intraperitoneally, intrapleurally, intrathecally, intratracheally, intrauterinely, intravascularly, intravenously, intravesically, intranasally, intragastrically, intra-intestinally, via bile infusion, intracardiac infusion, via preanal, rectal, subcutaneous, sublingual, local, intravaginal, percutaneously, via ureter, or via the urethra.

[0093] Examples of dosage forms to which the compositions described herein are applicable include, but are not limited to, tablets, aerosols, chewing 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 implementations, administration may also be carried out by including the subject in their diet, for example, in a functional food intended for humans or companion animals.

[0096] The compositions provided herein may contain pharmaceutically acceptable excipients, diluents, or carriers. Such pharmaceutically acceptable excipients, diluents, or carriers are well known in the art.

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

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

[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 food products.

[0101] The subject of this 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 compositions disclosed herein can be used to treat, prevent, or alleviate metabolic diseases or diseases caused by metabolic disorders.

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

[0104] In some embodiments, the metabolic diseases, metabolic disorders, or diseases caused by metabolic disorders include, but are not limited to, at least one of the following: liver diseases, obesity and obesity-related diseases, cardiovascular diseases, diabetes, dyslipidemia, cardiovascular and cerebrovascular diseases, 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 diseases, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), atherosclerosis, and kidney disease.

[0105] Diabetes includes type 1 diabetes (T1D), type 2 diabetes (T2D), and gestational diabetes mellitus (GDM). Type 1 diabetes is caused by autoimmune damage or idiopathic factors, characterized by absolute destruction of pancreatic function. It mostly occurs in children and adolescents and requires insulin treatment to achieve satisfactory results; otherwise, it can be life-threatening. Type 2 diabetes is a multifactorial syndrome characterized by abnormal carbohydrate / lipid metabolism, typically including hyperglycemia, hypertension, and abnormal cholesterol. Type 2 diabetes is caused by the ineffective function of insulin (low binding to receptors), therefore, not only fasting blood glucose but also 2-hour postprandial blood glucose should be monitored, and pancreatic function tests should be performed in particular. There are two types of diabetes during pregnancy: one is diabetes diagnosed before pregnancy, called "diabetes mellitus complicated by pregnancy"; the other is diabetes that appears or is diagnosed during pregnancy but occurs during pregnancy, also known as "gestational diabetes mellitus (GDM)," which accounts for more than 80% of diabetes cases in pregnant women.

[0106] Four types of metabolic disease-related models—high-fat diet (HFD) induced mouse obesity model, high-fat, high-glucose, and high-cholesterol induced mouse NASH model, high-fat diet combined with streptozotocin (HFD-STZ) induced mouse type II diabetes model, and leptin receptor gene defective mouse model (db / db)—are commonly used mouse models of metabolic diseases. These model mice are usually accompanied by metabolic diseases such as obesity, insulin resistance, hyperglycemia, hyperlipidemia, hypercholesterolemia, and NAFLD / NASH.

[0107] Insulin resistance refers to a decrease in the efficiency of insulin in promoting glucose uptake and utilization due to various reasons. The body compensates by secreting excessive insulin, resulting in hyperinsulinemia to maintain stable blood glucose levels. Insulin resistance can easily lead to metabolic syndrome and type II diabetes.

[0108] Liver dysfunction is characterized by abnormal or impaired liver function. Alanine aminotransferase (ALT) and / or aspartate aminotransferase (AST) are sensitive markers of liver dysfunction.

[0109] Nonalcoholic fatty liver disease (NAFLD) refers to the excessive accumulation of fat in the liver as triglycerides (TG) (steatodegeneration). Some NAFLD patients also have hepatocellular damage and inflammation in addition to excessive fat (steatohepatitis), a condition known as nonalcoholic steatohepatitis (NASH). NASH is widely considered a liver manifestation of metabolic syndromes such as type 2 diabetes, insulin resistance, central obesity, hyperlipidemia (low high-density lipoprotein cholesterol, high triglycerides), and hypertension.

[0110] Liver and kidney dysfunction refers to functional acute renal failure that occurs in severe liver disease. In decompensated cirrhosis, liver and kidney syndrome can occur due to insufficient effective circulating blood volume, reduced prostaglandins, and other reasons.

[0111] Obesity refers to a significant degree of overweight and excessive fat accumulation, a state caused by excessive accumulation of body fat, especially triglycerides. It is an abnormal or excessive accumulation of fat that poses a risk to health. Excessive food intake or changes in metabolism lead to excessive fat accumulation, resulting in excessive weight gain and causing or inducing pathological and physiological changes in the body. 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 a combination of metabolic syndrome, including type 2 diabetes, hypertension, hypercholesterolemia, and hypertriglyceridemia. In general, the health effects of obesity fall into two main categories: diseases attributable to increased body fat (such as osteoarthritis, obstructive sleep apnea, etc.) and diseases with an increased number of fat cells (diabetes, dyslipidemia, cancer, cardiovascular disease, non-alcoholic fatty liver disease, or non-alcoholic steatohepatitis, etc.). The term "obesity-related diseases" can be selected from the following diseases: overeating, binge eating, bulimia, hypertension, diabetes, elevated plasma insulin levels, insulin resistance, hyperlipidemia, metabolic syndrome, insulin resistance syndrome, obesity-related gastroesophageal reflux disease, arteriosclerosis, hypercholesterolemia, hyperuricemia, lower back pain, cardiac hypertrophy and left ventricular hypertrophy, lipid metabolism disorders, non-alcoholic steatohepatitis, cardiovascular disease, and polycystic ovary syndrome, as well as those with these obesity-related diseases who wish to lose weight.

[0112] Obesity-related diseases in this disclosure include at least one of the following: 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 all commercially available or can be synthesized by known methods.

[0114] Example

[0115] 1. Materials and Methods

[0116] 1.1 Materials

[0117] 1.1.1 Microbial strains

[0118] The strain used in this experiment was Akkermansia muciniphila MNH19250, which was deposited at the Guangdong Provincial Microbial Culture Collection Center under the name Akkermansia muciniphila MNH19250, with accession number GDMCC No: 63782 and deposit date of June 21, 2024.

[0119] 1.1.2 Culture medium

[0120] Liquid seed culture medium: 15.0 g / L yeast peptone, 15.0 g / L yeast extract, 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.

[0121] The basal culture medium formula is as follows: 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.

[0122] Complete culture medium: Based on the above-mentioned basic culture medium, different types of peptone (refer to Table 1) are added, all at a dosage of 30 g / L, to prepare complete culture media of different types of peptone.

[0123] Table 1. Complete culture media with different candidate nitrogen sources

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

[0125] 1.1.3 Strain activation and culture

[0126] Take MNH19250 glycerol cryovials and activate them in an anaerobic workstation using liquid seed culture medium for 24-28 hours. Then, subculture them into fresh liquid seed culture medium at a 2% inoculum rate and culture for 24 hours to prepare seed culture. Next, inoculate the seed culture at 2.5% onto complete culture media containing different nitrogen sources and anaerobic culture at 37°C for 48 hours. Collect the bacterial suspension for subsequent analysis.

[0127] 1.1.4 Reagents

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

[0129] Table 2

[0130] reagents level Manufacturer hydrochloric acid Analytical Pure Sinopharm Chemical Reagent Co., Ltd. acetone Analytical Pure Sinopharm Chemical Reagent Co., Ltd. Urea Analytical Pure Aladdin Acetonitrile Chromatographic purity Fisher Scientific methanol Chromatographic purity Sigma Aldrich Formic acid Mass spectrometry pure Macklin trypsin Mass spectrometry pure Fisher Scientific water Grade I water Fisher Chemical Iodoacetamide (IAA) Analytical Pure Sigma Aldrich Dithiothreitol (DTT) Analytical Pure Sigma Aldrich Tris base Analytical Pure Solarbio Yeast peptone food grade Angel Yeast Co., Ltd. Yeast extract food grade Angel Yeast Co., Ltd. Soy peptone food grade Angel Yeast Co., Ltd. trypsin food grade Beijing Hongrun Baoshun Technology Co., Ltd. wheat peptone food grade Shanghai Hanni Biotechnology Co., Ltd. Beef Powder food grade Guangdong Huankai Microbial Technology Co., Ltd. Pea peptone food grade Angel Yeast Co., Ltd. Cucumber 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 embodiments are shown in Table 3 below.

[0133] Table 3

[0134]

[0135] 1.2 Detection and Analysis Methods

[0136] 1.2.1 Bacterial growth detection methods

[0137] 1) OD600 detection: After appropriate dilution of the bacterial fermentation broth to achieve an absorbance range of 0.2–0.8, the absorbance was measured at 600 nm using a spectrophotometer. Turbidity OD600nm = OD600 after dilution × dilution factor. Each sample was measured three times, and the average value was taken.

[0138] 2) Hemocytometer Counting: A clean hemocytometer is used. A bacterial suspension diluted to a certain extent is added to the small squares of the counting chamber. After standing for approximately 5 minutes, the cells are counted. The calculation formula is: Bacterial count (TFU / mL) = Average number of cells per square × 20000 × 1000 × Dilution factor. Each sample is counted three times, and the average value is taken.

[0139] 1.2.2 Amuc_1100 Protein Quantitative Detection Method

[0140] The principle of quantitative detection: Using an external standard, mass spectrometry is used to quantitatively detect the Amuc_1100 protein in *Ackermania pseudomallei* via the characteristic peptide of Amuc_1100. Specifically, a standard (molecular weight 858.50434) of the characteristic peptide AINSLVNK (SEQ ID NO: 1) of Amuc_1100 protein with a purity of over 98% is synthesized chemically. A standard curve for the quantitative detection of the characteristic peptide is established by calculating the peptide signal using mass spectrometry. Finally, the content of Amuc_1100 protein in *Ackermania pseudomallei* samples is detected using the standard curve.

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

[0142] (1) Stock solution of polypeptide standard

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

[0144] (2) Plotting the standard curve

[0145] The aforementioned peptide standard stock solution was diluted to a 10 mg / L standard working solution, and further diluted to obtain standard curve working solutions of 500, 100, 50, 10, 5, and 1 μg / L. The content of characteristic peptides in the linearly diluted peptide standard solutions was then detected using a triple quadrupole liquid chromatography-mass spectrometry (LC-MS) system to plot standard curves. These standard curves were then used to quantify the content of characteristic peptides in real samples, and finally, the content of Amuc_1100 protein was calculated.

[0146] (3) Preparation of bacterial sample protein peptides

[0147] Take an appropriate amount of sample (3.5-4 ml), centrifuge, discard the supernatant, add 8 M urea to the precipitate, sonicate to disrupt, extract, centrifuge at 12000 r / min for 20 min, collect the supernatant, and make up the supernatant volume to 500 μL. Take a small amount of supernatant for protein concentration detection. Precipitate the remaining supernatant with acetone, redissolve with 8 M urea solution, add DTT (10 mM) and reduce at 37°C for 60 min, then add IAA (50 mM) and incubate at room temperature in the dark for 45 min for alkylation. Finally, dilute the urea concentration of the sample to below 1 M, add trypsin at a mass ratio of 1:50 (trypsin:protein), and incubate overnight at 37°C. Desalt the peptide sample using a WatersSEP-PAK C18, and finally dissolve in 100 μL of 0.1% formic acid for subsequent mass spectrometry detection.

[0148] (4) Amuc_1100 protein quantification detection

[0149] A standard curve was plotted using characteristic peptide standards, and then the content of characteristic peptides in the samples was detected using a triple quadrupole liquid chromatography-mass spectrometry (LC-MS) system. The Amuc_1100 protein content in the samples was calculated using the external standard method.

[0150] The detection conditions for high performance liquid chromatography-mass spectrometry are shown in Table 4 below:

[0151] Table 4

[0152]

[0153] Formula for calculating Amuc_1100 protein content in a sample:

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

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

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

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

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

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

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

[0161] Example 1: Fermentation and shake-flask culture of Akkermansia myxophilus

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

[0163] The results are shown in Table 5. According to the OD600 measurements, after 48 h of culture, the OD600 of MNH19250 was between 2 and 3 for the tryptone and cucurbitacin groups, except for the tryptone and cucurbitacin groups which exceeded 3. OD600 usually reflects bacterial concentration and is used to evaluate bacterial growth. However, because the metabolites secreted by *Akkermansia obliterans* can affect absorbance, the measured absorbance value may not accurately reflect the bacterial count, thus affecting the subsequent accurate evaluation of Amuc_1100 protein content in the bacteria. Therefore, this embodiment further utilizes a counting chamber to directly count the bacteria under a microscope to determine the total bacterial count in the culture. The 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 The curves fitting OD600 to the corresponding bacterial count, R 2 =0.0924, indicating no linear relationship between the two. Therefore, in subsequent evaluations of bacterial Amuc_1100 content, bacterial count is a more accurate measure than OD600.

[0164] According to Table 5, the median total bacterial count in the fermentation broth is 6.835 × 10⁻⁶. 9 Cells / mL. Tryptone, pea peptone, casein peptone, yeast peptone, and yeast extract can significantly promote the increase of the total number of Akkermansia muciniphila in the fermentation broth.

[0165] Table 5. Concentration and number of MNH19250 cells after 48 h of culture with 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 Amuc_1100 characteristic peptide standards via external standard method. (Table 6 and...) Figure 2 The results showed that a linear equation was fitted based on the peptide concentration (ng / ml) and the response of the peptide detected by mass spectrometry, with the formula: y = 389.827848 × x - 234.964403, R0 2 =0.99811738. Then, the Amuc_1100 protein content in different samples was detected using the established quantitative standard curve.

[0169] Table 6 Results of Amuc_1100 Protein Quantitative Standard Curve Detection

[0170]

[0171] In Example 1, bacterial suspensions of *Ackermania pseudomallei* cultured with 10 different nitrogen sources were used. Bacterial proteins were extracted and enzymatically digested to obtain peptides. Then, high-performance liquid chromatography-tandem high-resolution mass spectrometry (HPLC-MS / MS) was used to detect and analyze the characteristic peptides of Amuc_1100. The quantitative mass spectrometry peak diagrams of the characteristic peptides for each sample are shown below. Figure 3 Then, based on the formula of the established Amuc_1100 protein characteristic peptide quantitative standard curve (y = 389.827848 × x - 234.964403) and the formula for calculating the Amuc_1100 protein content in the sample (Amuc_1100 content in the test sample (ng / mL) = (CX-C0) × M1 × V / m / M2 / 1000, see Formula 1 above for specific meaning), the Amuc_1100 protein content in the sample was calculated. Then, by combining the detection of the number of bacteria in the 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 sources of Amuc_1100 were soybean peptone (4.474 ng), tryptone (3.898 ng), wheat peptone (3.235 ng), and yeast extract (2.828 ng). This indicates that the addition of soybean peptone, tryptone, wheat peptone, and yeast extract significantly increased the level of the functional protein Amuc_1100 per unit volume of fermentation broth compared to other nitrogen sources. The Amuc_1100 protein concentration was even higher in the fermentation broth of *Ackermania pseudomallei* cultured in a medium containing this nitrogen source.

[0173] Every 10 10 Among the bacteria, the median Amuc_1100 content was 3.61835 ng. The four sources with the highest Amuc_1100 content were soybean peptone (9.8109 ng), wheat peptone (6.1857 ng), rice peptone (6.4008 ng), and trypsin (4.8539 ng), all exceeding 4 ng. Soybean peptone had the highest content, approaching 10 ng, indicating that the addition of soybean peptone, wheat peptone, rice peptone, and trypsin significantly promoted the increase of the functional protein Amuc_1100 level in individual cells compared to other nitrogen sources. Therefore, Akkermansia myxophilus cultured in a medium containing this nitrogen source may have a greater advantage in probiotic efficacy.

[0174] Table 7 Amuc_1100 protein quantification results

[0175]

[0176] By plotting the bacterial counts after 48 h of culture in Table 5 with the Amuc_1100 content of the fermentation broth samples in Table 7, the relationship between the total bacterial count and the Amuc_1100 protein content in the fermentation broth can be obtained. Figure 4 The graph shows the relationship between the total bacterial count and the Amuc_1100 protein content in the fermentation broth. From... Figure 4 It can be seen that after 48 hours of fermentation, there is a certain negative correlation between the total bacterial count and the Amuc_1100 protein content in the fermentation broth. It is speculated that a higher count of *Ackermania pseudomallei* will consume or reduce the synthesis of Amuc_1100 protein, thus lowering its content. Therefore, controlling the total bacterial count in the fermentation broth can be considered to increase the yield of Amuc_1100 protein.

[0177] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. The application of peptone in promoting the yield of the functional protein Amuc_1100 in single cells of Akkermansia muciniphila, wherein the peptone includes at least one of soybean peptone, trypsin peptone, wheat peptone, or rice peptone, and the Amuc_1100 protein is derived from Akkermansia muciniphila. The promotion of Amuc_1100 yield per single cell of Akkermansia muciniphila is achieved by controlling the total bacterial count in the fermentation broth while simultaneously increasing the Amuc_1100 protein yield. The control of the total bacterial count in the fermentation broth is defined as an order of magnitude of 10 when Akkermansia muciniphila has been cultured for 48 h. 9 per mL.

2. The application of peptone in the preparation of a culture medium that promotes the production of the functional protein Amuc_1100 per single cell of *Ackermania myxophilus*, wherein the peptone includes at least one of soybean peptone, trypsin peptone, wheat peptone, or rice peptone, and the promotion of the production of the functional protein Amuc_1100 per single cell of *Ackermania myxophilus* is achieved by increasing the production of Amuc_1100 protein while controlling the total bacterial count in the fermentation broth. The control of the total bacterial count in the fermentation broth is defined as an order of magnitude of 10 when *Ackermania myxophilus* has been cultured for 48 h. 9 per mL.

3. A method for promoting the production of the functional protein Amuc_1100 in single cells of Akkermansia muciniphila, the method comprising culturing Akkermansia muciniphila in a medium containing peptone, wherein, The peptone includes at least one of soybean peptone, trypsin peptone, wheat peptone, or rice peptone. The method for promoting the production of the functional protein Amuc_1100 per single cell of *Ackermania myxophilus* is to increase the Amuc_1100 protein production while controlling the total bacterial count in the fermentation broth. The control of the total bacterial count in the fermentation broth is defined as an order of magnitude of 10 when *Ackermania myxophilus* has been cultured for 48 h. 9 per mL.

4. A method for increasing the yield of the functional protein Amuc_1100 in a unit volume of Akkermansia muciniphila fermentation broth, the method comprising culturing Akkermansia muciniphila in a medium containing peptone or yeast extract, wherein, The peptone includes at least one of soybean peptone, tryptone, and wheat peptone. The method for increasing the yield of the functional protein Amuc_1100 per unit volume of Akkermansia myxophilia ferment broth is to increase the Amuc_1100 protein yield while controlling the total bacterial count in the ferment broth. Controlling the total bacterial count in the ferment broth means that after culturing Akkermansia myxophilia for 48 hours, the total bacterial count in the ferment broth is on the order of 10. 9 per mL.

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

6. The method according to claim 3 or 4, characterized in that, The culture medium also includes 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 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.

8. The method according to claim 7, characterized in that, 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, 1.5-3 g / L dipotassium hydrogen phosphate, 3-5 g / L potassium citrate, and 4-6 g / L sodium acetate.

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

10. A cell culture of Akkermansia myxophilus, said cell culture being obtained by the method of any one of claims 3-9, wherein the cell culture is prepared in batches of 10... 10 The content of the functional protein Amuc_1100 in each cell exceeded 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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