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 ( Akkermansia muciniphila , referred to as A. muciniphila, Akkermansia muciniphila) is a new species of the Verrucomicrobiota, which was successfully isolated by researchers from the Microbiology Laboratory in Wageningen in 2004 (PMID: 15388697). Akkermansia muciniphila colonizes the gastrointestinal mucosa, can specifically degrade mucin, and is ubiquitous in the intestines of healthy adults and infants, accounting for 1% - 4% of the total intestinal microbiota (PMID: 18083887). As an important member of the intestinal flora, a large number of studies in recent years have shown that the abundance of Akkermansia muciniphila colonization is closely related to a variety of diseases, such as obesity, diabetes, hepatic steatosis, inflammation, and response to cancer immunotherapy. Therefore, Akkermansia muciniphila is considered to be one of the "next generation" probiotics, with important applications and commercial value in the future fields of food and medicine.

[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 different 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 relieve colitis symptoms by inhibiting macrophage infiltration and cytotoxic T lymphocytes (CTLs) activation, but also promote CTLs proliferation and activation by upregulating TNF-α expression and inhibiting PD-1 immune checkpoint molecules, 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 the expression level of BDNF in the hippocampus and inhibiting neuroinflammatory response (PMID: 34129964). These evidences indicate that Amuc_1100 protein is one of the important active substances that play a probiotic role in Akkermansia muciniphila against diseases, and also suggest that its content plays an important role in the beneficial effects of Akkermansia muciniphila.

[0004] Since Akkermansia muciniphila has many health benefits, current research or invention patents mainly focus on how to achieve large-scale production of Akkermansia muciniphila, including optimizing the fermentation production process by methods such as the composition of the culture medium, culture time, centrifugation conditions, and the addition of special derivatives. Although these optimizations are of great help in increasing the yield of Akkermansia muciniphila, due to the difficulty in quantitative detection of Amuc_1100 protein, there are no commercial antibodies or detection kits for Amuc_1100 protein on the market, and the use of mass spectrometry methods requires complex pre-treatment steps and high costs. Current research rarely focuses on the effects of these culture methods on the active component Amuc_1100 protein of Akkermansia muciniphila, nor on how to increase the yield of Amuc_1100 protein.

[0005] As an important effective component of Akkermansia muciniphila, the content of Amuc_1100 determines the efficacy of Akkermansia muciniphila in exerting probiotic effects to a certain extent. Although the optimization of the fermentation production process in the prior art can increase the yield of Akkermansia muciniphila, the higher the yield of Akkermansia muciniphila does not mean that the Amuc_1100 protein content in the fermentation broth is higher. Therefore, while increasing the yield of Akkermansia muciniphila, it is necessary to pay attention to the influence of the fermentation culture method on Amuc_1100.

[0006] Increasing the yield of Amuc_1100 protein plays an important role in the industrial production of Amuc_1100 and improving the probiotic effect of Akkermansia muciniphila. Therefore, it is of great significance to provide a fermentation process method 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 a specific type of peptone, 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, there is provided 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 ( Akkermansia muciniphila ).

[0009] The Akkermansia muciniphila in the present disclosure may be any Akkermansia muciniphila capable of producing Amuc_1100 protein. In some embodiments, the Akkermansia muciniphila may be any of the Akkermansia muciniphila described in Chinese patent application CN2024119901535. In some specific embodiments, the Akkermansia muciniphila is Akkermansia muciniphila ( Akkermansia muciniphila ) MNH19250 strain, deposited in Guangdong Microbiological Culture Collection Center (GDMCC), with the deposit number GDMCC NO: 63782 and the deposit date 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 ( Akkermansia muciniphila ), 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 ( Akkermansia muciniphila ), wherein the peptone comprises at least one of soy peptone, tryptone, wheat peptone or rice peptone.

[0013] In some embodiments, the method further comprises increasing the Amuc_1100 protein content in the fermentation broth by controlling the total bacterial count of the fermentation broth or preventing the total bacterial count of the fermentation broth from being too high. By controlling the total bacterial count of the fermentation broth or preventing the total bacterial count of 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 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 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 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.

[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 that are 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 equivalent to the magnesium sulfate content 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 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, 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 the present disclosure may be any Akkermansia muciniphila capable of producing Amuc_1100 protein. In some embodiments, the Akkermansia muciniphila may be any of the Akkermansia muciniphila in Chinese patent application CN2024119901535. In some specific embodiments, the Akkermansia muciniphila is Akkermansia muciniphila ( Akkermansia muciniphila ) MNH19250 strain, deposited in Guangdong Microbiological Culture Collection Center (GDMCC), with the deposit number GDMCC NO: 63782 and the deposit date 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 content of Amuc_1100 protein in the bacterial protein. In some embodiments, the quantitative detection includes: detecting the Amuc_1100 protein in the bacterial suspension by high performance liquid chromatography-mass spectrometry using a characteristic peptide segment of Amuc_1100, and the amino acid sequence of the characteristic peptide segment of Amuc_1100 is: AINSLVNK (SEQ ID NO: 1).

[0035] 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.

[0036] 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 a dried product thereof.

[0037] According to yet another aspect of the present disclosure, a composition is provided, comprising the aforementioned cell culture of the present disclosure.

[0038] 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-killed bacterium, preferably a pasteurized bacterium.

[0039] 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.

[0040] In some embodiments, the composition is any one of a medicine, a health product or a food.

[0041] 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.

[0042] In some embodiments, the composition is in the form of a liquid, foam, cream, spray, powder (eg, lyophilized powder), or gel.

[0043] 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.

[0044] In some embodiments, the composition is in the form of an oral dosage or an injection.

[0045] In some embodiments, the composition further comprises one or more pharmaceutically acceptable carriers, food carriers, excipients and / or adjuvants.

[0046] 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 agent.

[0047] 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.

[0048] In some embodiments, the composition further comprises one or more other active agents for preventing or treating metabolic diseases and / or tumors.

[0049] 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.

[0050] In some embodiments, suppressing appetite comprises reducing food intake and / or reducing appetite.

[0051] In some embodiments, the other active agent is selected from: a GLP-1 receptor agonist, a dual agonist of a GLP-1 receptor and a GCG receptor, a triple agonist of a GLP-1 receptor, a GIP receptor and a GCG receptor, an AMPK agonist or an active drug that promotes GLP-1 secretion.

[0052] 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.

[0053] In some embodiments, the other active agent may be an agent for preventing or treating tumors.

[0054] In some embodiments, the other active agent may be one or more of probiotics, prebiotics, or a combination thereof.

[0055] In some embodiments, the probiotics are selected from one or more of lactic acid bacteria, lactobacillus, lactococci, butyrate-producing bacteria, bifidobacteria, thermophilic Streptococcus, faecal Streptococcus, and mesenteric Leuconostoc.

[0056] 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, fructooligosaccharide (FOS), galacto-oligosaccharide (GOS), starch, cellulose, b-glucan, hemicellulose, lactulose, manno-oligosaccharide, manno-oligosaccharide (MOS), inulin rich in fructooligosaccharide, oligo-glucose, tagatose, trans-galacto-oligosaccharide, pectin, resistant starch, xylo-oligosaccharide (XOS) and any combination thereof.

[0057] In some embodiments, the composition can be formulated as a frozen composition, such as a frozen composition prepared by quick freezing and drying, or lyophilization, for storage and / or transportation.

[0058] In some embodiments, the composition is obtained by spray drying. In some embodiments, the composition is obtained by electrostatic spray drying.

[0059] 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 alive. 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.

[0060] 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.

[0061] In some embodiments, the composition is formulated for oral administration. In some embodiments, the composition is an enteric preparation. In some embodiments, the enteric preparation is a dosage form with an enteric coating. For example, the enteric preparation can be an enteric granule, an enteric tablet, or an enteric 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 capsule, or the capsule can be a microencapsulated capsule, or a microcapsule.

[0062] In some embodiments, the composition is a medicine, a health product or a food.

[0063] In some embodiments, the composition is in a dosage form suitable for infants, children or adults.

[0064] In some embodiments, the composition is in a parenteral dosage form.

[0065] 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 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.

[0066] According to another aspect of the present disclosure, 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 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.

[0067] 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 diseases, obesity and obesity-related diseases, cardiovascular diseases, cardiovascular and cerebrovascular diseases, diabetes, dyslipidemia, glucose intolerance, type I diabetes, type II diabetes, impaired glucose tolerance, insulin resistance, obesity, hyperglycemia, hyperinsulinemia, fatty liver, alcoholic fatty hepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglyceridemia, uremia, ketoacidosis, hypoglycemia, thrombotic diseases, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), atherosclerosis, and at least one of nephropathy.

[0068] In some embodiments, the liver and kidney diseases include liver diseases, liver function damage-related diseases, 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.

[0069] 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.

[0070] According to another aspect of the present disclosure, there is provided 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, reducing liver and / or kidney damage, 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.

[0071] 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.

[0072] 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 renal function.

[0073] In some embodiments, the metabolism-related disease is selected from at least one of obesity, metabolic syndrome, insulin deficiency or insulin resistance-related disorders, diabetes, glucose intolerance, abnormal lipid metabolism, hyperglycemia, dyslipidemia, irritable bowel syndrome, intestinal contraction disorders, hypercholesterolemia, and hyperlipidemia.

[0074] In some embodiments, the cardiovascular and cerebrovascular disease is selected from at least one of hypertension, atherosclerosis, coronary heart disease, and stroke.

[0075] In some embodiments, the intestinal barrier damage-related disease is selected from inflammatory bowel disease, Crohn's disease, and ulcerative colitis.

[0076] In some embodiments, the lipid reduction is selected from reducing visceral fat and / or local fat.

[0077] 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 hepatitis or non-alcoholic fatty liver; 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; lowering the blood lipid and / or cholesterol level of the subject; lowering the local fat, body fat rate and / or visceral fat of the subject; repairing the intestinal tissue mucosa of the subject; prevention, treatment and improvement of hypertension and atherosclerosis; regulating metabolism and immunity at the same time; repairing intestinal barrier damage while reducing abdominal fat; improving liver damage while losing weight; fighting inflammation while losing weight; fighting inflammation while lowering lipids; repairing intestinal barrier damage while lowering lipids; lowering serum AST and / or ALT while lowering lipids; improving liver damage while improving kidney damage; improving diabetes while improving liver damage; improving diabetes while improving kidney damage; improving atherosclerosis while improving liver damage; improving atherosclerosis while lowering lipids. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 The relationship between OD600 and bacterial count of Akkermansia muciniphila culture according to some embodiments of the present disclosure is shown.

[0079] Figure 2 Shown is an Amuc_1100 protein quantification standard curve according to some embodiments of the present disclosure.

[0080] Figure 3 The quantitative mass spectrometry peak diagram of the Amuc_1100 characteristic peptide of Akkermansia muciniphila cultured with different nitrogen sources according to some embodiments of the present disclosure is shown.

[0081] 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.

[0082] Deposit of strains The strain Akkermansia muciniphila ( Akkermansia muciniphila )MNH19250, deposited in Guangdong Microbiological Culture Collection Center (GDMCC), with the deposit number GDMCC No: 63782, the deposit date is 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, the proposed taxonomic name is Akkermansia muciniphila . DETAILED DESCRIPTION

[0083] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation to the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0084] 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.

[0085] Unless the context clearly dictates otherwise, the expressions "a", "an" and "an" as used herein 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.

[0086] 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).

[0087] Compositions or preparations disclosed herein can be used as pharmaceutical preparations, therapeutic compositions, dietary supplements, nutritional supplements, medical probiotics or medical foods. In some cases, the composition is applied in the form of a pharmaceutical preparation. In some cases, the composition is applied in the form of a nutritional supplement. In some cases, the composition is applied in the form of a dietary supplement. In some cases, the composition is applied in the form of a medical food. In some cases, the composition is applied in the form of a medical probiotic. In some cases, the composition (e.g., dietary supplements, nutritional supplements, medical probiotics or medical foods) can be orally administered, for example as a capsule, pill or tablet.

[0088] "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.

[0089] Compositions can be prepared using the Akkermansia muciniphila culture described herein, for example, by using a pharmaceutically acceptable excipient. The pharmaceutical composition comprises a pharmaceutically effective amount of the Akkermansia muciniphila culture, for example, the Akkermansia muciniphila culture with a deposit number of GDMCC NO: 63782. Similarly, the Akkermansia muciniphila culture with a deposit number of GDMCC NO: 63782 can also be prepared into a pharmaceutical composition, for example, by using a pharmaceutically acceptable excipient, which comprises a pharmaceutically effective amount of the Akkermansia muciniphila.

[0090] Suitable pharmaceutically acceptable excipients that may be used include, for example, carriers, excipients, diluents, lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners and flavors.

[0091] 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 bile infusion, by cardiac infusion, anteriorly, rectally, subcutaneously, sublingually, topically, intravaginally, transdermally, by ureteral or urethral routes.

[0092] Examples of dosage forms suitable for the composition 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.

[0093] In some embodiments, the composition is a sugar-coated tablet, a gel capsule, a gel, an emulsion, a tablet, a sheet capsule, a hydrogel, a nanofiber gel, an electrospun fiber, a food bar, a candy, a fermented milk, a fermented cheese, a chewing gum, a powder or a toothpaste, etc.

[0094] In some embodiments, administration can also be by inclusion in a subject's diet, such as inclusion in a functional food for humans or companion animals.

[0095] The compositions provided herein may include a pharmaceutically acceptable excipient, diluent or carrier. The pharmaceutically acceptable excipient, diluent or carrier is well known in the art.

[0096] In some embodiments, the Akkermansia muciniphila in the composition of the present disclosure is lyophilized. In some embodiments, the Akkermansia muciniphila in the composition of the present disclosure is spray-dried. In some embodiments, the Akkermansia muciniphila in the composition of the present disclosure is lyophilized or spray-dried and is alive. In some embodiments, the Akkermansia muciniphila in the composition of the present disclosure is lyophilized or spray-dried and can partially or completely colonize the intestine. In some embodiments, the lyophilized Akkermansia muciniphila is reconstituted prior to administration. In some embodiments, the reconstitution is performed using a diluent described herein.

[0097] 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.

[0098] In some embodiments, the composition is prepared by freeze-drying or spray-drying.

[0099] The compositions disclosed herein include pharmaceutical compositions, health products or foods.

[0100] The subject of the present disclosure may be a human or an animal, and the animal includes but is not limited to cattle, sheep, cats, dogs, horses, rabbits, monkeys, mice, rats, alpacas, camels, etc.

[0101] The pharmaceutical composition disclosed herein can be used to treat, prevent or alleviate metabolic diseases or diseases caused by metabolic disorders.

[0102] The pharmaceutical composition disclosed herein can be used to treat, prevent or alleviate tumors.

[0103] 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 fatty hepatitis, 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.

[0104] 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 reasons. It is characterized by absolute destruction of pancreatic islet function. It mostly occurs in children and adolescents. It must be treated with insulin to achieve satisfactory results, otherwise it will be life-threatening. Type 2 diabetes is a multifactorial syndrome characterized by abnormal carbohydrate / fat metabolism, usually including hyperglycemia, hypertension and abnormal cholesterol. Type 2 diabetes is caused by the inability of insulin to work effectively (low binding to receptors). Therefore, it is necessary not only to check fasting blood sugar, but also to observe blood sugar 2 hours after a meal, especially to do pancreatic islet function tests. There are two types of diabetes during pregnancy. One is that the patient has been diagnosed with diabetes before pregnancy, which is called "diabetes complicated with pregnancy"; the other is that the patient has normal glucose metabolism or potential glucose tolerance before pregnancy, and the diabetes that appears or is diagnosed during pregnancy, which is also called "gestational diabetes (GDM)". More than 80% of pregnant women with diabetes have GDM.

[0105] Four metabolic disease-related models, 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. The model mice are usually accompanied by metabolic diseases such as obesity, insulin resistance, hyperglycemia, hyperlipidemia, hypercholesterolemia, NAFLD / NASH, etc.

[0106] 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.

[0107] Liver function disease refers to abnormal liver function or liver function damage. Alanine aminotransferase (ALT) and / or aspartate aminotransferase (AST) are sensitive markers of liver function disease.

[0108] Non-alcoholic fatty liver disease (NAFLD) refers to the accumulation of excess fat in the liver in the form of triglycerides (TG) (fatty degeneration). Some patients with NAFLD have liver cell damage and inflammation (steatohepatitis) in addition to excess fat, namely non-alcoholic steatohepatitis (NASH). NASH is widely considered to be the liver manifestation of metabolic syndrome, such as type 2 diabetes, insulin resistance, central obesity, hyperlipidemia (low high-density lipoprotein cholesterol, high triglycerides) and hypertension.

[0109] Liver and kidney function diseases refer to functional acute renal failure that occurs in severe liver disease and decompensated cirrhosis. Due to insufficient effective circulating blood volume and decreased prostaglandins, hepatorenal syndrome may occur.

[0110] Obesity refers to a certain degree of obvious overweight and excessive fat layer. It is 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 accumulation of body fat due to excessive food intake or changes in body metabolism causes excessive weight gain and causes pathological, physiological changes or lurking in the human body. A body mass index of more than 25 is considered overweight, and more than 30 is considered obese. Obesity increases the risk of many physical and mental diseases. It is mainly related to metabolic syndrome, a combination of diseases including type 2 diabetes, hypertension, hypercholesterolemia and hypertriglyceridemia. In general, the effects of obesity on health can be divided into two categories: diseases that can be attributed to increased body fat (such as osteoarthritis, obstructive sleep apnea, etc.) and diseases that increase the number of fat cells (diabetes, dyslipidemia, cancer, cardiovascular disease, non-alcoholic fatty liver or non-alcoholic steatohepatitis, etc.). 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, atherosclerosis, 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.

[0111] 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.

[0112] The reagents used in the following examples are all commercially available or can be synthesized by known methods.

[0113] Example

[0114] 1. Materials and methods 1.1 Materials 1.1.1 Bacterial strains The strain used in this experiment is Akkermansia muciniphila MNH19250, which is deposited in Guangdong Microbiological Culture Collection Center. The deposited name is Akkermansia muciniphila MNH19250, the deposited number of the strain is GDMCC No: 63782, and the deposited date is June 21, 2024.

[0115] 1.1.2 Culture medium Liquid seed culture 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, 5.0 g / L anhydrous sodium acetate.

[0116] Basic culture 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.

[0117] Complete medium: Take the above basic medium as the basic component, add different types of peptone (refer to Table 1), the dosage is 30 g / L, and prepare complete medium with different types of peptone.

[0118]

[0119] 1.1.3 Strain activation and cultivation Take the MNH19250 glycerol cryopreserved tube, activate it with liquid seed culture medium in the anaerobic workstation for 24h-28h, and then subculture it in fresh liquid seed culture medium at 2% inoculation, and prepare seed liquid after culturing for 24h. Then inoculate the seed liquid into complete culture medium containing different nitrogen sources at 2.5%, culture it anaerobically at 37℃ for 48h, and collect the bacterial suspension for subsequent detection.

[0120] 1.1.4 Reagents The reagents used in the following examples are shown in Table 2.

[0121]

[0122] 1.1.5 Instruments and equipment The instruments and equipment used in the following examples are shown in Table 3.

[0123]

[0124] 1.2 Detection and analysis methods 1.2.1 Bacterial growth detection method 1) OD600 test: After the fermentation broth of the strain is diluted appropriately, the absorbance is within the range of 0.2-0.8, and the absorbance is measured at a wavelength of 600nm using a spectrophotometer. Turbidity OD600nm = OD600 after dilution × dilution multiple. Each sample is tested 3 times and the average value is taken.

[0125] 2) Counting plate: Hemocytometer determination. Take a clean hemocytometer, drop the diluted bacterial suspension into the small squares of the counting plate, let it stand for about 5 minutes, and then count. The calculation formula is: bacterial count (TFU / mL) = average number of bacterial cells in one square × 20000 × 1000 × dilution factor. Repeat the counting 3 times for each sample and take the average value.

[0126] 1.2.2 Amuc_1100 protein quantitative detection method Principle of quantitative detection: Use external standard, Amuc_1100 characteristic peptide, and mass spectrometry to quantitatively detect Amuc_1100 protein in Akkermansia muciniphila. That is, a standard substance (molecular weight 858.50434) of the characteristic peptide AINSLVNK (SEQ ID NO: 1) of Amuc_1100 protein with a purity of more than 98% is synthesized by chemical method, and a standard curve for characteristic peptide quantification is established by calculating the peptide signal using mass spectrometry, and finally the content of Amuc_1100 protein in Akkermansia muciniphila samples is detected by the standard curve.

[0127] The specific steps of the experiment are as follows: (1) Peptide standard stock solution Accurately weigh 5.00 mg of synthetic peptide (AINSLVNK standard), place 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.

[0128] (2) Drawing of standard curve The above peptide standard stock solution was diluted into a 10 mg / L standard working solution, and further diluted to obtain 500, 100, 50, 10, 5, and 1 μg / L standard curve working solutions. The characteristic peptide content in the peptide standard linear working dilution solution was then detected by triple quadrupole liquid chromatography-mass spectrometry, and a standard curve was drawn so that the standard curve could be used to quantify the characteristic peptide content in the real sample, and finally the Amuc_1100 protein content was converted.

[0129] (3) Preparation of protein peptides from bacterial samples Take an appropriate amount of sample (3.5-4ml), centrifuge, discard the supernatant, add 8M urea to the precipitate, ultrasonically break it, extract, centrifuge at 12000r / min for 20min, take the supernatant, and make up the supernatant volume to 500μL, and take a small amount of supernatant for protein concentration detection. Add acetone to the remaining supernatant, re-dissolve with 8M urea solution, add DTT (10 mM) to reduce at 37°C for 60min, then add IAA (50 mM), incubate at room temperature in the dark for 45min for alkylation, and finally dilute the urea concentration of the sample to less than 1M, add trypsin at a mass ratio of 1:50 (trypsin: protein), and digest at 37°C overnight. The peptide sample was desalted using WatersSEP-PAK C18, and finally dissolved with 100 μL 0.1% formic acid for subsequent mass spectrometry detection.

[0130] (4) Amuc_1100 protein quantitative detection The characteristic peptide standard was used to draw a standard curve, and then the triple quadrupole liquid chromatography-mass spectrometry was used to detect the characteristic peptide content in the sample. The Amuc_1100 protein content in the sample was calculated by the external standard method.

[0131] The HPLC-MS detection conditions are shown in Table 4 below:

[0132] The calculation formula of Amuc_1100 protein content in the sample is: 1) Amuc_1100 content of the test sample (ng / mL) = (CX-C0) × M1 × V / m / M2 / 1000 (Formula 1) m: liquid (volume of sample, in liters (L)); M1: protein molecular weight (target protein molecular weight), Amuc_1100 protein is 34213 Da; M2: molecular weight of peptide standard (characteristic peptide molecular weight), the peptide is 858.50434 Da; 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; C0: mass concentration of blank, in micrograms per liter (μg / L); CX: The mass concentration of the peptide to be tested, in micrograms per liter (μg / L).

[0133] Example 1: Fermentation of Akkermansia muciniphila in shake flasks Akkermansia muciniphila was cultured for 48 hours using 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.

[0134] The results are shown in Table 5. According to the results of OD600 determination, after 48 hours of culture, except for the trypsin and peptone groups whose OD600 exceeded 3, the OD600 of the other peptone groups of MNH19250 was between 2-3. 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 the Amuc_1100 content of bacteria, the bacterial count is used as the standard of measurement, which is more accurate than OD600.

[0135] According to Table 5, the median of the total bacterial count in the fermentation broth is 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.

[0136]

[0137] Example 2: Quantitative detection of Amuc_1100 protein To quantify the Amuc_1100 protein content in the sample, the 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. Then the established quantitative standard curve was used to detect the Amuc_1100 protein content in different samples.

[0138]

[0139] In Example 1, the bacterial suspension samples of 10 kinds of Akkermansia muciniphila cultured with different nitrogen sources were subjected to bacterial protein extraction and enzymatic hydrolysis to obtain peptides, and then the characteristic peptides of Amuc_1100 were detected and analyzed by 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, see the above formula 1 for the specific meaning), the content of Amuc_1100 protein in the sample was calculated, and then the effect of different nitrogen sources on the bacterial Amuc_1100 content was evaluated by combining the number of bacteria in the test sample.

[0140] The test results are shown in Table 7, where the median of Amuc_1100 content per milliliter of sample is 2.395 ng. The top four Amuc_1100 contents are 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 can significantly promote the increase of the functional protein Amuc_1100 level per unit volume of fermentation broth compared with peptones of other nitrogen sources, and the Amuc_1100 protein concentration of Akkermansia muciniphila fermentation broth cultured in the medium containing this nitrogen source is higher.

[0141] Every 10 10 Among the bacteria, the median of Amuc_1100 content was 3.61835 ng. The top four Amuc_1100 contents were soy peptone (9.8109 ng), wheat peptone (6.1857 ng), rice peptone (6.4008 ng), and tryptone (4.8539 ng), with contents exceeding 4 ng. Soy peptone had the highest content, close to 10 ng, indicating that the addition of soy peptone, wheat peptone, rice peptone, and tryptone could significantly promote the increase of the functional protein Amuc_1100 level in single cells compared with peptones of other nitrogen sources, and Akkermansia muciniphila cultured in a medium containing this nitrogen source may have more advantages in probiotic efficacy.

[0142]

[0143] 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 of 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 4It can be seen that after 48 hours of fermentation, the total bacterial count of the fermentation broth and the Amuc_1100 protein content in the fermentation broth showed a certain negative correlation. It is speculated that the higher the bacterial count of Akkermansia muciniphila, the more Amuc_1100 protein will be consumed or the synthesis of Amuc_1100 protein will be reduced, thereby reducing the Amuc_1100 protein content. Therefore, it is possible to consider increasing the Amuc_1100 protein yield by controlling the total bacterial count of the fermentation broth.

[0144] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. Application 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 ( Akkermansia muciniphila ).

2. Use of peptone or yeast extract powder in preparing a culture medium for improving the protein yield of Akkermansia muciniphila Amuc_1100, wherein the peptone comprises at least one of soy peptone, tryptone, wheat peptone or rice peptone.

3. A method for increasing the protein production of Akkermansia muciniphila Amuc_1100, the method comprising culturing Akkermansia muciniphila ( Akkermansia muciniphila ),in, The peptone includes at least one of soy peptone, tryptone, wheat peptone or rice peptone.

4. A method for increasing the yield of Amuc-1100 protein in a fermentation broth, the method comprising culturing Akkermansia muciniphila ( Akkermansia muciniphila ),in, The peptone includes at least one of soy peptone, tryptone, wheat peptone or rice peptone.

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 temperature of the culture is 35-40° C., and the time is 24-60 hours.

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, Tween 80, 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, 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.

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 Akkermansia muciniphila ( Akkermansia muciniphila ) MNH19250 strain, deposited in Guangdong Microbiological Culture Collection Center (GDMCC), with the deposit number GDMCC NO: 63782 and the deposit date June 21, 2024.

10. A cell culture obtained by the method according to any one of claims 3 to 9.

11. The cell culture according to claim 10, characterized in that 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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