WO2023208223A1 - 药物组合物、巨球型菌及其应用 - Google Patents

药物组合物、巨球型菌及其应用 Download PDF

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WO2023208223A1
WO2023208223A1 PCT/CN2023/091789 CN2023091789W WO2023208223A1 WO 2023208223 A1 WO2023208223 A1 WO 2023208223A1 CN 2023091789 W CN2023091789 W CN 2023091789W WO 2023208223 A1 WO2023208223 A1 WO 2023208223A1
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megasphaera
diabetes
bacterial strain
strain
diseases
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PCT/CN2023/091789
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English (en)
French (fr)
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贤一博
蒋先芝
张东亚
赵国振
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慕恩（广州）生物科技有限公司
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Priority to CN202380017662.7A priority Critical patent/CN118891356A/zh
Publication of WO2023208223A1 publication Critical patent/WO2023208223A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the invention relates to the field of biomedicine, and in particular to a pharmaceutical composition, Macrococcus bacteria and their applications.
  • the gut microbiome composed of the collective genome and functional material from trillions of bacteria, archaea, fungi, viruses, and other microeukaryotic colonizers, has been widely recognized as a “second brain” in regulating human health and disease development. and perform multiple functions in clinical practice.
  • the intestine is the largest microecological environment in the human body and participates in many important physiological processes such as nutrient absorption, energy metabolism, tissue and organ development, immune defense, and endocrine regulation. There are a large number of symbiotic microorganisms in the human intestine, and the total amount of genetic information they carry is 50-100 times that of the human genome, which is the "gut microbiome".
  • the intestinal microbiome is the largest and most direct external environment of the human body and plays an important role in maintaining human health. Increasing evidence is revealing the protective interactions of specific gut microbes, products released through microbial metabolic activity, and the host in manipulating host immunity, metabolism, and cancer development. With the rapid development of molecular biology, genomics, bioinformatics analysis technology, high-throughput sequencing technology and microbial culture technology, the impact and role of intestinal flora on intestinal and extraintestinal diseases has become increasingly clear. Microbiome-based therapies, many different bacterial species can affect the human digestive system, circulatory system, and nervous system, and are closely related to various human diseases, including cancer, infection, gastrointestinal inflammation, autoimmunity, metabolic disorders, central nervous system and mental illness. Studying the relationship between intestinal flora and human health and disease is not only an important scientific research, but also has important significance and value in clinical diagnosis, treatment, and even transformation.
  • intestinal microbiome in nutritional disorders, metabolic abnormalities and complex diseases (such as obesity, diabetes, inflammatory bowel disease and metabolism, etc.), but the types of microorganisms currently studied only involve specific extremes. Few species.
  • intestinal microorganisms are usually concentrated in the field of probiotics such as Bifidobacterium, Bacteroides, and Lactobacillus, while other intestinal microorganisms have yet to be developed to form live bacterial drugs for metabolic diseases. treatment or prevention.
  • Megacoccus mainly exists in the intestines of humans or animals. It is a strictly anaerobic microorganism and has extremely high requirements for nutrition and culture environment, and has a long growth cycle. In addition, when verifying drug efficacy, bacteria of this genus are difficult to verify through in vitro cell experiments because they are anaerobic; during in vivo animal experiments, bacteria of this genus are difficult to culture and highly anaerobic. Maintain a stable viable bacterial count and suffer from insufficient reproducibility. These technical difficulties related to both in vivo and in vitro have limited the discovery and application of new strains of the genus Megacoccus.
  • Microbial SCFAs have been fully studied by existing technologies for the treatment or prevention of metabolic diseases such as obesity and diabetes.
  • the prior art "Gut microbial metabolites in obesity, NAFLD and T2DM" shows that metabolites produced by carbohydrate fermentation related to weight control include acetic acid, propionic acid, butyric acid, and succinic acid; acetate and butyrate are also used. Demonstrated induction of satiety through central mechanisms and increased thermogenesis in adipose tissue and liver, as well as induction of adipose tissue browning and leptin secretion.
  • GLP1 glucagon-like peptide 1
  • PYY peptide YY
  • a 2017 mouse study showed that long-term oral administration of butyrate prevented diet-induced obesity, NAFLD progression, and insulin resistance. These effects are primarily related to a reduction in food intake, through butyrate-induced inhibition of the activity of neuropeptide Y-expressing orexigenic neurons in the hypothalamus, and a reduction in neural activity in the brainstem nucleus of the solitary tract and the dorsal vagal complex.
  • intraperitoneal injection of acetic acid, propionic acid, and butyric acid has been shown to inhibit energy intake in mice through a mechanism related to vagal afferent stimulation, which propionate and butyrate prevent by inducing intestinal gluconeogenesis (IGN). Obesity and insulin resistance.
  • One aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising bacteria of the genus Megasphaera
  • the identity of the 16S rRNA sequence of the bacteria of the genus Megacoccus and SEQ is ⁇ 95%;
  • the SEQ includes at least one of SEQ ID No: 1, SEQ ID No: 2 and SEQ ID No: 3.
  • composition can be used to suppress appetite, prevent, treat and/or alleviate metabolic diseases
  • the suppressing appetite includes: at least one of reducing food intake and reducing appetite;
  • the metabolic disease is at least one of liver disease, obesity, cardiovascular disease, cardiovascular and cerebrovascular disease, hyperlipidemia, diabetes, and impaired glucose tolerance;
  • the liver disease may be at least one of fatty liver, NAFLD, NASH, reduced liver weight, and abnormal liver function;
  • the liver diseases include those caused by high-fat diet, those caused by high-cholesterol diet, those caused by high-sugar diet, those caused by high-fat and high-cholesterol, those caused by high-fat and high-sugar and/or those caused by high-fat, high-cholesterol and high-sugar. caused;
  • the obesity disease can be obesity caused by high-fat diet, obesity caused by high cholesterol, obesity caused by high-sugar diet, obesity caused by high-fat and high-cholesterol, obesity caused by high-fat and high-sugar, high-fat Obesity caused by high cholesterol and high sugar, obesity in NAFLD/NASH patients;
  • the cardiovascular disease or cardiovascular and cerebrovascular disease may be atherosclerosis and/or cardiovascular disease in NAFLD/NASH patients and/or cardiovascular and cerebrovascular disease in NAFLD/NASH patients and/or hypercholesterolemia. ;
  • the liver diseases include liver diseases or diseases of abnormal liver function caused by at least one of a high-fat diet, a high-cholesterol diet, and a high-sugar diet; further, include those caused by a high-fat diet, a high-cholesterol diet, Caused by a high-sugar diet, caused by high-fat and high-cholesterol, caused by high-fat and high-sugar and/or caused by high-fat, high-cholesterol and high-sugar Liver diseases or abnormal liver function;
  • the obesity disease includes obesity caused by at least one of a high-fat diet, a high-cholesterol diet, and a high-sugar diet; further, includes obesity caused by a high-fat diet, obesity caused by high cholesterol, and a high-sugar diet.
  • the cardiovascular disease or cardiovascular and cerebrovascular disease may be atherosclerosis and/or cardiovascular disease in NAFLD/NASH patients and/or cardiovascular and cerebrovascular disease in NAFLD/NASH patients and/or hypercholesterolemia. ;
  • the diabetes includes diabetes caused by obesity, type II diabetes, and diabetes in NAFLD/NASH patients;
  • the diabetes includes diabetes caused by at least one of a high-fat diet, a high-cholesterol diet, and a high-sugar diet; further, includes diabetes caused by a high-fat diet, diabetes caused by high cholesterol, and diabetes caused by a high-sugar diet.
  • Diabetes diabetes caused by high fat and high cholesterol, diabetes caused by high fat and high sugar, diabetes caused by high fat, high cholesterol and high sugar, diabetes in patients with NAFLD/NASH.
  • the metabolic disease is type I diabetes, type II diabetes, gestational diabetes, impaired glucose tolerance, insulin resistance, weight control, overweight, glycemic control, prediabetes, obesity, hyperglycemia, hyperinsulinemia, Fatty liver, alcoholic steatohepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglyceridemia, uremia, ketoacidosis, hypoglycemia, thrombotic diseases, At least one of dyslipidemia, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), atherosclerosis, nephropathy, diabetic neuropathy, diabetic retinopathy, skin disease, dyspepsia, or edema kind.
  • NAFLD non-alcoholic fatty liver disease
  • NASH non-alcoholic steatohepatitis
  • atherosclerosis nephropathy, diabetic neuropathy, diabetic retinopathy
  • skin disease dyspepsia
  • the pharmaceutical composition also includes a second active substance or a combination drug, and the second active substance or combination drug includes a GLP-1 receptor agonist, a GLP-1 receptor and a GCG receptor dual agonist, GLP- 1 receptor, GIP receptor and GCG receptor triple agonist, AMPK agonist or active drug that promotes GLP-1 secretion, DPP-4 receptor inhibitor, PPAR receptor agonist, PPAR ⁇ receptor agonist, PPAR ⁇ receptor Body agonist, PPAR ⁇ receptor agonist, PPAR ⁇ / ⁇ receptor dual agonist, PPAR ⁇ / ⁇ receptor dual agonist, PPAR ⁇ / ⁇ / ⁇ receptor triple agonist, CRTC, PGC-1 ⁇ , SREBP, FXR, FGF21 , ASK1, THR- ⁇ , LXR, NF- ⁇ B, SNDRI, MC4R, PNLIP, MOA, DRI and other mechanisms/targets, at least one active drug;
  • the GLP-1 receptor agonist includes any one of the following: semaglutide, liraglutide, exenatide or beinaglutide;
  • the AMPK agonist or active drug that promotes GLP-1 secretion includes Metformin, Semaglutide and/or liraglutide;
  • the pathogenesis of some diseases or conditions is characterized by reduced microbiota stability.
  • diseases and conditions are IBS, IBD, diabetes (eg type 2 diabetes), allergic diseases, autoimmune diseases and metabolic diseases/conditions.
  • diabetes eg type 2 diabetes
  • allergic diseases e.g., allergic diseases
  • autoimmune diseases e.g., allergic rhinitis
  • metabolic diseases/conditions e.g., allergic rhinitis, rhepatocytes, and digestive tracts.
  • the bacterial strains of the present invention can also treat or prevent disease by modulating the stability of the microbiota.
  • Another aspect of the present invention also relates to the application of the pharmaceutical composition or the bacterial strain or the Megalococcus bacteria in the preparation of drugs for the treatment and/or prevention of diseases;
  • the disease includes at least one of tumors, infectious diseases, metabolic diseases, immune diseases, inflammatory diseases or neurological diseases;
  • the metabolic disease includes: at least one of liver disease, obesity, cardiovascular disease, cardiovascular and cerebrovascular disease, hyperlipidemia, diabetes, and impaired glucose tolerance;
  • the liver disease may be at least one of fatty liver, NAFLD, NASH, reduced liver weight, and abnormal liver function;
  • the liver diseases include those caused by high-fat diet, those caused by high-cholesterol diet, those caused by high-sugar diet, those caused by high-fat and high-cholesterol, those caused by high-fat and high-sugar and/or those caused by high-fat, high-cholesterol and high-sugar. caused;
  • the obesity disease can be obesity caused by high-fat diet, obesity caused by high cholesterol, obesity caused by high-sugar diet, obesity caused by high-fat and high-cholesterol, obesity caused by high-fat and high-sugar, high-fat Obesity caused by high cholesterol and high sugar, obesity in NAFLD/NASH patients;
  • the cardiovascular disease or cardiovascular and cerebrovascular disease may be atherosclerosis and/or cardiovascular disease in NAFLD/NASH patients and/or cardiovascular and cerebrovascular disease in NAFLD/NASH patients and/or hypercholesterolemia. ;
  • the diabetes includes diabetes caused by obesity, type II diabetes, and diabetes in NAFLD/NASH patients;
  • the diabetes can be selected from diabetes caused by high-fat diet, diabetes caused by high cholesterol, diabetes caused by high-sugar diet, diabetes caused by high-fat and high-cholesterol, diabetes caused by high-fat and high-sugar, high-fat and high-cholesterol Diabetes caused by high sugar, diabetes in NAFLD/NASH patients.
  • the identity of the 16S rRNA sequence of the bacteria of the genus Megalococcus and SEQ is ⁇ 95%;
  • the SEQ includes at least one of SEQ ID No: 1, SEQ ID No: 2 and SEQ ID No: 3.
  • the present invention isolates and screens out a number of anaerobic strains belonging to the genus Megasphaera from the human intestine, which are natural strains (non-cloned and processed strains); through identification, it is determined that they belong to new strains of the genus Megasphaera. species; multiple strains of these strains can express EC2.8.3.9 enzymes, and the integrity of the butyrate-producing pathway reaches at least 70%. They can effectively produce butyric acid and other SCFAs, and effectively prevent and treat metabolic diseases.
  • the present invention utilizes the similar commonality of Megasphaera bacteria in producing butyric acid, and further conducts candidate drug screening of strains through gene and butyric acid pathway analysis to improve the screening efficiency.
  • the screened strains can be effectively used for metabolic diseases after verification. Prevention and treatment; the selected bacterial strains can inhibit the activity of histone acetylase by regulating short-chain fatty acids/short-chain fatty acid salts to prevent and treat metabolic diseases;
  • the microbial preparation provided by the present invention contains the megacoccus bacterium or the metabolite of the megacoccus bacterium and has the effect of preventing and treating metabolic diseases.
  • the medicines for preventing and/or treating metabolic diseases provided by the present invention include the megacoccus bacteria isolated and screened from the human intestinal tract or the metabolites of the megacoccus bacteria, and can be used for the treatment of diseases. It also has lower side effects.
  • the pharmaceutical composition provided by the present invention can achieve better therapeutic effects on metabolic diseases by combining the drug containing Macrococcus with other drugs for treating metabolic diseases.
  • the pharmaceutical composition provided by the invention can regulate at least one short-chain fatty acid or lactate and is used for the treatment of metabolic diseases.
  • Figure 1 shows the colony morphology of strains MNH 05026, MNH22004 and MNH27256 cultured on anaerobic blood plates for 48 hours;
  • Figure 2 shows the microscopic morphology of strains MNH 05026, MNH22004 and MNH27256;
  • Figure 3 shows the Gram staining microscopic morphology of strains MNH 05026, MNH22004 and MNH27256;
  • Figure 4 shows the stained microscopic morphology of spores of strains MNH 05026, MNH22004 and MNH27256;
  • Figure 5 shows the results of different concentrations of NaCl tolerance of strains MNH 05026, MNH22004 and MNH27256;
  • Figure 6 shows the results of different pH tolerances of strains MNH 05026, MNH22004 and MNH27256;
  • Figure 7 shows the results of different concentrations of bile salt tolerance of strains MNH 05026, MNH22004 and MNH27256;
  • Figure 8 shows the results of culturing strain MNH 05026 in medium API 20
  • Figure 9A is the 16S rRNA gene phylogenetic tree of strain MNH 05026;
  • Figure 9B is the 16S rRNA gene phylogenetic tree of strain MNH 22004;
  • Figure 9C is the 16S rRNA gene phylogenetic tree of strain MNH 27256;
  • Figure 10 shows the effect of strains MNH05026 and MNH22004 on the liver weight of obese mice induced by high-fat diet
  • Figure 11 is a graph showing the effect of strain MNH05026 on the serum alanine aminotransferase (ALT) content of high-fat diet-induced obese mice;
  • Figure 12 shows the effects of strains MNH05026 and MNH27256 on fasting blood glucose (FGB) in high-fat diet-induced obese mice.
  • Figure 13 shows the effect of MNH05026 combined with Metformin on the oral glucose tolerance (OGTT) of type II diabetic mice
  • Figure 13 (A) shows the effect of MNH05026 combined with Metformin on the oral glucose tolerance (OGTT) of type II diabetic mice ( OGTT)
  • Figure 13 (B) shows the area under the oral glucose tolerance curve results of experimental mice in each treatment group;
  • Figure 14 shows the results of the effects of MNH05026 and MNH27256 combined with Metformin on fasting blood glucose in type II diabetic mice;
  • Figure 15 is a graph showing changes in fasting blood glucose of type II diabetic mice using MNH27256 alone and in combination with Metformin;
  • Figure 16 shows the weight gain of strains MNH05026, MNH27256 and MNH22004 on high-fat diet-induced obese mice
  • Figure 17 shows the body weight changes of strains MNH05026, MNH22004, and MNH27256 respectively combined with Semaglutide in NAFLD/NASH mice induced by a high-fat, high-sugar, high-cholesterol diet;
  • Figure 18 is a graph showing the effects of MNH05026, MNH22004 and MNH27256 combined with Semaglutide on liver weight in mice with NAFLD/NASH induced by a high-fat, high-sugar, high-cholesterol diet;
  • Figure 19 is a graph showing the effects of MNH05026, MNH22004 and MNH27256 combined with Semaglutide on the oral glucose tolerance of the NAFLD/NASH mouse model induced by a high-fat, high-sugar, high-cholesterol diet;
  • Figure 19 (A) shows the effects of MNH05026, MNH22004, MNH27256 and Semaglutide. The effect of combined use on the oral glucose tolerance Day30 of the NAFLD/NASH mouse model induced by a high-fat, high-sugar, and high-cholesterol diet.
  • Figure 19 shows the effect of the combination of MNH05026 and Semaglutide on the NAFLD/NASH mice model induced by a high-fat, high-sugar, and high-cholesterol diet. Effects of Day 55 on oral glucose tolerance in the mouse model.
  • C) and (D) in Figure 19 are the area under the oral glucose tolerance curve results corresponding to (A) and (B) in Figure 19.
  • E) in Figure 19 are MNH05026 and Fasting blood glucose values on Day 30 of the oral glucose tolerance of the NAFLD/NASH mouse model induced by a high-fat, high-sugar, high-cholesterol diet when MNH22004 and MNH27256 were combined with Semaglutide;
  • Figure 20 shows the effects of MNH05026, MNH22004 and MNH27256 in combination with Semaglutide on the subcutaneous fat pad (subcutaneous fat pad), inguinal fat pad (Inguinal fat pad) and brown fat weight of the NAFLD/NASH mouse model induced by a high-fat, high-sugar and high-cholesterol diet. (brown adipose tissue) and epididymis fat weight (epididymis fat pad);
  • Figure 21 shows the effects of MNH05026 combined with Semaglutide on the effects of high-fat, high-sugar, and high-cholesterol diet. Effects of triglycerides (TG), total cholesterol (CHO), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) in the NAFLD/NASH mouse model;
  • TG triglycerides
  • CHO total cholesterol
  • HDL high-density lipoprotein
  • LDL low-density lipoprotein
  • Figure 22 shows the effect of MNH05026, MNH22004 and MNH27256 in combination with Semaglutide on liver steatosis (Steatosis), liver lobular inflammation (Lobular inflammation) and liver ballooning (Ballooning) in NAFLD/NASH mice induced by a high-fat, high-sugar and high-cholesterol diet. And the impact map of liver NAS score and fibrosis;
  • Figure 23 is a diagram showing the inhibitory effects of MNH22004 and MNH05026 on sirtuin activity.
  • bacterial species can be classified and identified through traditional classification methods and molecular biology methods.
  • Traditional classification methods include, for example, cell morphology observation, Gram staining, flagellar staining, various metabolic experiments, etc.
  • Molecular biology methods include ribosomal RNA sequence determination, determination methods based on whole-genome sequencing, etc.
  • 16S rRNA is a ribosomal RNA of prokaryotes.
  • the 16S rRNA gene consists of a variable region and a conserved region.
  • the conserved region is common to all bacteria, while the variable region differs to varying degrees among different bacteria.
  • Identity between the sequences of two nucleic acid molecules can be determined using known computer algorithms, such as the "FASTA” program, the GCG package, BLASTN or FASTA.
  • a commercially or publicly available program may also be, for example, the DNAStar "MegAlign” program.
  • Average nucleotide identity (ANI) of bacterial genomes refers to the similarity of homologous genes between two bacterial genomes. ANI values can be calculated by methods such as BLAST. In the field of bacterial taxonomy, it is generally believed that the ANI value needs to reach more than 95% to be considered to belong to the same species (Jain C, Rodriguez-R L M, Phillippy A M, et al. High throughput ANI analysis of 90K prokaryotic genomes reveal clear speciesboundaries[J].Nature Communications,2018,9(1):5114.).
  • ANI value calculation tools such as local computing software Jspecies (/jspecies) and Schmidtees (/documentation.html), online calculation tools ANI caculator (http:/enveomics.gatech.edu/), EzGenome ( /ezgenome/ani) and ANItools.
  • an isolated strain belongs to a new species of the genus Megasphaera discovered by the inventor. For example, when the average nucleotide identity ANI value with strains with deposit numbers GDMCC No: 62001, GDMCC No: 62000, GDMCC No: 61999 is at least 95%, such as 95.5%, 96%, 96.5%, 97%, When 97.5%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%, it can be determined that they belong to the same strain.
  • compositions may be prepared using the Megasphaera strains described herein, for example by using pharmaceutically acceptable excipients.
  • the pharmaceutical composition contains a pharmaceutically effective amount of Megasphaera bacteria of the bacterial species, for example, strains with deposit numbers GDMCC No: 62001, GDMCC No: 62000, and GDMCC No: 61999.
  • the Megasphaera strains to which the strains deposited as GDMCC No: 62001, GDMCC No: 62000, and GDMCC No: 61999 belong can also prepare pharmaceutical compositions, for example, by using pharmaceutically acceptable excipients, which contain a pharmaceutically effective amount.
  • Suitable pharmaceutically acceptable excipients may be used, such as carriers, excipients, diluents, lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners and flavors.
  • pharmaceutically acceptable excipients are lactose, glucose, sucrose, sorbitol, mannose, starch, corn starch, trehalose, fructose, vitamin C sodium salt, L-cysteine hydrochloride, skim milk powder, Sodium alginate, calcium chloride, sodium carboxymethylcellulose, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl fiber One or more of hydroxybenzoate, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.
  • Supernatant within the meaning of the present invention means a culture supernatant of a bacterial strain according to the invention, optionally containing compounds and/or cell fragments of said strain, and/or metabolites secreted by said strain and/or molecules.
  • the pharmaceutical composition provided by the present invention may contain pharmaceutically acceptable excipients, diluents or carriers.
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art.
  • suitable carriers include lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol or sorbitol, and the like.
  • suitable diluents include ethanol, glycerol and water.
  • the choice of pharmaceutical carrier, excipient, or diluent can be based on the intended route of administration and standard pharmaceutical practice.
  • the pharmaceutical composition may contain as or in addition to a carrier, excipient or diluent any suitable binder, lubricant, suspending agent, coating agent or solubilizing agent and the like.
  • binders examples include starch, gelatin, natural sugars and natural or synthetic gums.
  • natural sugars such as glucose, anhydrous lactose, free-flowing lactose, beta-lactose or corn sweeteners.
  • Natural or synthetic gums such as acacia, tragacanth, sodium alginate, carboxymethyl cellulose or polyethylene glycol.
  • suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate or sodium chloride, and the like.
  • Preservatives, stabilizers, dyes and even flavoring agents can be provided in pharmaceutical compositions. Examples of preservatives include sodium benzoate, sorbic acid or parabens.
  • Antioxidants and suspending agents may also be used. Antioxidants and suspending agents may also be used.
  • Judgment criteria When the similarity between the 16S rRNA gene sequences of two strains is less than 97% to 98.65%, they can be judged to belong to different species.
  • nucleic acid sequences of two nucleic acid molecules can be determined as percent identity using known computer algorithms, such as the "FASTA” program, using default parameters, for example in Pearson et al. (Other programs include the GCG package (Devereux, J., et al., Nucleic Acids Research 12(I):387(1984)), BLASTP, BLASTN, FASTA).
  • FASTA Pearson et al.
  • GCG Pearson et al.
  • BLASTP BLASTP
  • BLASTN BLASTN
  • FASTA Pearson et al.
  • the BLAST function of the NCBI database other commercially or publicly available programs include the DNAStar "MegAlign" program.
  • Insulin resistance refers to the decrease in the efficiency of insulin in promoting glucose uptake and utilization due to various reasons. The body compensatoryly secretes too much insulin to produce hyperinsulinemia to maintain blood sugar stability. Insulin resistance can easily lead to metabolic syndrome and type 2 diabetes. .
  • This patent applies four metabolic disease-related models: mouse obesity model induced by high-fat diet (HFD), mouse NASH model induced by high-fat, high-fructose and high-cholesterol, high-fat diet combined with streptozotocin (Streptozotocin) ( HFD-STZ) induced mouse type 2 diabetes model, leptin receptor gene deficient mouse model (db/db), these 4 types
  • HFD-STZ high-fat diet
  • HFD-STZ streptozotocin
  • db/db leptin receptor gene deficient mouse model
  • the models are commonly used mouse models of metabolic diseases. Model mice are usually accompanied by metabolic diseases such as obesity, insulin resistance, hyperglycemia, hyperlipidemia, high cholesterol, NAFLD/NASH and other metabolic diseases.
  • ALT and AST are sensitive markers of liver cell damage.
  • ALT and AST are sensitive markers of liver cell damage.
  • ALT and AST are sensitive markers of liver cell damage.
  • ALT and AST are sensitive markers of liver cell damage.
  • Liver and kidney disease refers to functional acute renal failure that occurs in severe liver disease, decompensated cirrhosis, and hepatorenal syndrome due to insufficient effective circulating blood volume and reduced prostaglandins.
  • the present invention focuses on related research on liver-related diseases, mainly NAFLD/NASH.
  • Non-alcoholic fatty liver disease refers to the accumulation of excess fat in the form of triglycerides (steatosis) in the liver. Some NAFLD patients also have liver cell damage and inflammation in addition to excess fat. (steatohepatitis), that is, NASH, NASH is widely considered to be the hepatic manifestation of metabolic syndrome, such as type 2 diabetes, insulin resistance, central obesity, hyperlipidemia (low high-density lipoprotein cholesterol, high triglyceridemia disease) and hypertension.
  • metabolic syndrome such as type 2 diabetes, insulin resistance, central obesity, hyperlipidemia (low high-density lipoprotein cholesterol, high triglyceridemia disease) and hypertension.
  • TG mainly participates in energy metabolism in the human body and produces heat energy. Excessive TG content in the blood can cause blood viscosity, causing lipids to deposit on the blood vessel walls, gradually forming small plaques, which is atherosclerosis. Increased LDL-C is a major and independent risk factor for the occurrence and development of atherosclerosis; the increased level of LDL-C is also an indicator of coronary heart disease. Because HDL-C can transport cholesterol in the blood vessel wall to the liver for catabolism (ie, reverse cholesterol transport), it can reduce the deposition of cholesterol in the blood vessel wall and play an anti-atherosclerotic role.
  • Type 1 diabetes is caused by autoimmune damage or idiopathic reasons. It is characterized by the 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 abnormalities in carbohydrate/fat metabolism, often including hyperglycemia, hypertension, and cholesterol abnormalities. Type 2 diabetes is caused by the inability of insulin to function effectively (less binding to receptors). Therefore, it is necessary to check not only fasting blood sugar, but also 2-hour postprandial blood sugar, and in particular, pancreatic islet function testing.
  • diabetes There are two types of diabetes during pregnancy. One is diabetes diagnosed before pregnancy, which is called “diabetes combined with pregnancy”; the other is diabetes that has normal glucose metabolism before pregnancy or has potential impaired glucose tolerance and only appears or is diagnosed during pregnancy. , also known as “gestational diabetes mellitus (GDM)", more than 80% of pregnant women with diabetes have GDM.
  • GDM gestational diabetes mellitus
  • Oral glucose tolerance is used to measure pancreatic beta cell function and the body's ability to regulate blood sugar. It is currently a recognized diagnostic indicator for the diagnosis of diabetes. When glucose metabolism is disordered, blood sugar rises sharply after oral administration of a certain amount of glucose, or the rise is not obvious, but cannot be reduced to the fasting level or the original level within a short period of time. This is abnormal glucose tolerance or impaired glucose tolerance; abnormal glucose tolerance indicates The body's ability to metabolize glucose is reduced, which is common in type 2 diabetes and obesity.
  • HOMA-IR is an index used to evaluate an individual's insulin resistance level. It has been widely used in clinical evaluation of insulin sensitivity in patients with diabetes. Its calculation method is: fasting blood glucose level (FPG, mmol/L) ⁇ fasting insulin level (FINS, ⁇ U/mL)/22.5.
  • FPG fasting blood glucose level
  • FINS fasting insulin level
  • HOMA-IR index of a normal individual is 1. As the level of insulin resistance increases, the HOMA-IR index will be higher than 1.
  • GLP-1 glucagon-like peptide-1
  • the inventor found that Christensenii bacteria can increase the secretion level of glucagon-like peptide-1 (GLP-1), thereby Regulate the body's blood sugar balance, improve the body's glucose tolerance, further improve the body's insulin sensitivity and leptin sensitivity, thereby preventing and/or treating diabetes and/or hyperlipidemia.
  • Lipopolysaccharide also known as intracellular toxin, is a phospholipid that constitutes the outer cell wall of Gram-negative bacteria.
  • lipopolysaccharides protect these bacteria from breakdown by bile salts secreted by the gallbladder. Normally, lipopolysaccharide is kept out of the bloodstream by tight junctions in the intestinal wall cells. If lipopolysaccharide enters the bloodstream, it will induce a strong inflammatory response in animals. So lipopolysaccharide levels in the blood can reflect inflammation levels.
  • Resistin is a hormone or adipokine secreted by adipose tissue and is related to obesity and insulin resistance.
  • resistin has been characterized as a hormone expressed and secreted by immune cells, particularly macrophages, and has been implicated in many inflammatory responses, including adipose tissue inflammation due to macrophage infiltration. Resistin may play an important role in the development and progression of obesity and insulin resistance through resistin-induced inflammation. Resistin has also been linked to other chronic diseases, such as cardiovascular disease and cancer, and has been proposed as an important biomarker for metabolism-related diseases in many studies.
  • Obesity refers to a certain degree of obvious overweight and excessive fat layer. It is a state caused by excessive accumulation of fat in the body, especially triglycerides. It is an abnormal or excessive accumulation of fat that poses a risk to health. Excessive accumulation of fat in the body due to excessive food intake or changes in body metabolism leads to excessive weight gain and causes pathological, physiological changes or latent changes in the human body. A body mass index above 25 is considered overweight, and a body mass index above 30 is considered obese. Obesity increases the risk of many physical and mental illnesses. It is primarily associated with metabolic syndrome, a combination of conditions including type 2 diabetes, hypertension, hypercholesterolemia, and hypertriglyceridemia.
  • the health effects of obesity fall into two broad categories: diseases attributable to increased body fat (such as osteoarthritis, obstructive sleep apnea, etc.) and diseases that increase the number of fat cells (diabetes, dyslipidemia, etc.) , cancer, cardiovascular disease, non-alcoholic fatty liver disease or non-alcoholic steatohepatitis, etc.).
  • diseases attributable to increased body fat such as osteoarthritis, obstructive sleep apnea, etc.
  • diseases that increase the number of fat cells diabetes, dyslipidemia, etc.
  • cancer cardiovascular disease
  • non-alcoholic fatty liver disease or non-alcoholic steatohepatitis etc.
  • Glucagon-like peptide-1 (GLP-1) is a hormone mainly produced by intestinal L cells and is an incretin.
  • GLP-1RA Glucagon-like peptide-1 receptor agonist
  • GLP-1RA Glucagon-like peptide-1 receptor agonist
  • Leptin is a hormone secreted by adipose tissue, and its content in serum is proportional to the size of animal adipose tissue. Leptin acts on receptors (Leptin Receptor) located in the central nervous system to regulate biological behavior and metabolism. When an animal loses body fat or is in a low-energy state (such as hunger), the leptin content in the serum will decrease significantly, thereby stimulating the animal's foraging behavior and reducing its own energy consumption. Conversely, when an organism's body fat increases, leptin levels in the serum increase, which inhibits eating and accelerates metabolism. Leptin regulates the body's energy balance and body weight through such a negative feedback mechanism.
  • leptin resistance As fat mass continues to grow, leptin continues to be secreted. Long-term stimulation of large amounts of leptin makes the brain no longer sensitive to leptin, which is medically called "leptin resistance.”
  • Diseases related to liver function impairment include at least one of the following diseases: fatty liver disease, non-alcoholic fatty liver disease, Non-alcoholic steatohepatitis, liver fibrosis, cirrhosis and liver cancer.
  • Obesity-related diseases include at least one of the following diseases: obesity, metabolic syndrome, cardiovascular disease, hyperlipidemia, hypercholesterolemia, hypertension, insulin resistance syndrome, and obesity-related gastroesophageal reflux disease and steatohepatitis.
  • Diabetes-related diseases include at least one of the following diseases: type II diabetes, insulin resistance syndrome, glucose intolerance, hyperlipidemia, complications of diabetic nephropathy, diabetic neuropathy, diabetic eye disease, cardiovascular disease, Diabetic foot.
  • the above-mentioned "obesity-related diseases” may be selected from the following diseases: overeating, binge eating, bulimia, hypertension, diabetes, elevated plasma insulin concentration, insulin resistance, hyperlipidemia, Metabolic syndrome, insulin resistance syndrome, obesity-related gastroesophageal reflux disease, arteriosclerosis, hypercholesterolemia, hyperuricemia, low back pain, cardiac hypertrophy and left ventricular hypertrophy, lipodystrophy, non-alcoholic Steatohepatitis, cardiovascular disease, and polycystic ovary syndrome, as well as subjects with these obesity-related conditions including those who wish to lose weight.
  • diseases including those who wish to lose weight.
  • the second active substance is a therapeutic agent for other metabolic diseases, such as GLP-1 receptor agonist, GLP-1 receptor and GCG receptor dual agonist, GLP-1 receptor, GIP receptor and GCG receptor triple agonist, AMPK agonists or active drugs that promote GLP-1 secretion; may also include metformin, sulfonylureas, meglitinide, thiazolidinediones, DPP-4 inhibitors, GLP-1 receptor agonists, SGLT2 inhibitors agent, insulin, pioglitazone, rosiglitazone, pentoxifylline, omega-3-fatty acids, statins, ezetimibe, or ursodeoxycholic acid, semaglutide, liraglutide, exenatide, or beinaglutide;
  • GLP-1 receptor agonist GLP-1 receptor and GCG receptor dual agonist
  • GLP-1 receptor GIP receptor and GCG receptor triple agonist
  • the method further comprises administering a prebiotic to the subject.
  • the prebiotic is fructooligosaccharide, galactooligosaccharide, transgalactooligosaccharide, xylo-oligosaccharide, chito-oligosaccharide, soy oligosaccharide, gentio-oligosaccharide, isomaltooligosaccharide, Mannose oligosaccharides, maltose oligosaccharides, mannose oligosaccharides, lactulose, lactose sucrose, palatinose, sugar sucrose, guar gum, gum arabic, tagatose, amylose, amylopectin, pectin , xylan or cyclodextrin.
  • administering generally refers to the route by which a composition (eg, a pharmaceutical composition) is administered to a subject.
  • routes of administration include oral, rectal, topical, inhalation (nasal), or injection.
  • Injection administration includes intravenous (IV), intramuscular (IM), intratumoral (IT) and subcutaneous (SC) administration.
  • the pharmaceutical compositions described herein may be administered in any form by any effective route, including, but not limited to, intratumoral, oral, parenteral, enteral, intravenous, intraperitoneal, topical, transdermal (e.g., using any standard patch ), intradermal, intraocular, (intranasal, topical, non-oral, e.g.
  • the pharmaceutical compositions described herein are administered orally, rectally, intratumorally, topically, intravesically, by injection into or near a draining lymph node, intravenously, by inhalation or aerosol, or subcutaneously.
  • the pharmaceutical compositions described herein are administered orally, intratumorally, or intravenously.
  • the term “increase” or “enhancement” or “improvement” refers to a change such that the post-treatment difference is at least 10%, 20%, 30%, 40%, 50%, 60%, as the case may be, compared to the pre-treatment state. 70%, 80%, 90%, 2x, 4x, 10x, 100x, 103x , 104x , 105x , 106x and/or 107x . Properties that may be increased include the number of immune cells, bacterial cells, stromal cells, myeloid-derived suppressor cells, fibroblasts, metabolites; levels of cytokines; or other physical parameters (such as tumor size).
  • reduction or “decrease” or “decrease” refers to a change such that the post-treatment difference is at least 10%, 20%, 30%, 40%, 50%, 60%, as the case may be, compared to the pre-treatment state. 70%, 80%, 90%, 1/100, 1/1000, 1/10,000, 1/100,000, 1/1,000,000 or undetectable. Characteristics that may be reduced include the number of immune cells, bacterial cells, stromal cells, myeloid-derived suppressor cells, fibroblasts, metabolites; levels of cytokines; or their other physical parameters (such as ear thickness (e.g., in DTH animal models) or tumor size).
  • Insulin resistance has its common meaning in the art. Insulin resistance is a physiological condition in which the natural hormone insulin becomes less effective at lowering blood sugar. The resulting increase in blood sugar may push blood sugar levels outside the normal range and lead to adverse health effects such as metabolic syndrome, dyslipidemia, and subsequent type 2 diabetes.
  • insulin resistance-related complications and “insulin resistance-related disorders” include, but are not limited to, metabolic syndrome, dyslipidemia, and type 2 diabetes, as well as insulin resistance in endocrine diseases (e.g., obese subjects with 2 type 1 diabetes), type 1 diabetes, Cushing's disease, and lipodystrophy syndrome).
  • metabolic disease refers to a group of conditions that occur together and increase the risk of heart disease, stroke, and type II diabetes. These conditions include increased blood pressure, high blood sugar, excess fat, obesity, and abnormal cholesterol or triglyceride levels.
  • the metabolic disease is type II diabetes, impaired glucose tolerance, insulin resistance, weight control, overweight, glycemic control, prediabetes, obesity, hyperglycemia, hyperinsulinemia, fatty liver disease, non-alcoholic fat Hepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglyceridemia, ketoacidosis, hypoglycemia, thrombotic diseases, dyslipidemia, non-alcoholic fatty liver disease (NAFLD) or related diseases.
  • the associated disease is cardiovascular disease, atherosclerosis, nephropathy, nephropathy,
  • metabolite refers to a compound, composition, molecule, ion, cofactor, catalyst or nutrient from any cellular or microbial metabolic reaction that is used as a substrate or as a product.
  • a “strain” refers to a member of a bacterial species that has genetic characteristics such that it can be distinguished from closely related members of the same bacterial species.
  • the genetic characteristic may be the absence of all or part of at least one gene, the absence of all or part of at least one regulatory region (e.g., promoter, terminator, riboswitch, ribosome binding site), absence (at least one natural "cure” of the plasmid), the presence of at least one recombinant gene, the presence of at least one mutated gene, the presence of at least one foreign gene (a gene from another species), at least one mutated regulatory region (e.g., promoter subon, terminator, riboswitch, ribosome binding site), the presence of at least one non-natural plasmid, the presence of at least one antibiotic resistance cassette, or a combination thereof.
  • Genetic characteristics between different strains can be identified by PCR amplification, optionally followed by DNA sequencing of the genomic region of interest or the entire genome. In cases where one strain (compared to another strain of the same species) acquires or loses antibiotic resistance or acquires or loses biosynthetic ability (e.g. auxotrophic strains), this can be achieved by selection or counter-selection using antibiotics Differentiate strains or nutrients/metabolites.
  • Supernatant within the meaning of the present invention means a culture supernatant of a bacterial strain according to the invention, optionally containing compounds and/or cell fragments of said strain, and/or metabolites secreted by said strain and/or molecules.
  • subject refers to any mammal.
  • a subject or patient described as “in need” is a person in need of treatment (or prevention) of a disease.
  • Mammals i.e., mammals
  • mammals include humans, laboratory animals (e.g., primates, rats, mice), domestic animals (e.g., cattle, sheep, goats, pigs), and household pets (e.g., dogs, cats, rodents) .
  • the object can be a person.
  • the subject may be a non-human mammal, including, but not limited to, dog, cat, cow, horse, pig, donkey, goat, camel, mouse, rat, guinea pig, sheep, camel, monkey, gorilla, or chimpanzee.
  • a subject or patient may be healthy or may have a metabolic disease at any stage of development.
  • treating a subject for a disease or “treating” a subject having or suspected of having a disease refers to administering a pharmaceutical treatment to the subject, such as administering one or more agents, thereby reducing or preventing At least one symptom of the disease worsens.
  • “treating” means inter alia delaying progression, accelerating remission, inducing remission, increasing remission, accelerating recovery, increasing the efficacy of alternative therapies or reducing resistance to alternative therapies, or combinations thereof.
  • prevention is art-recognized and is used in connection with conditions such as local recurrence are well known in the art and include administration that reduces the onset or delays the onset of symptoms of a medical condition in a subject relative to a subject not receiving the composition.
  • the present invention develops bacterial strains (MNH05026, MNH22004, MNH27256) containing species of the genus Megasphaera and new compositions. It can be used to treat and prevent cancer, autoimmune and inflammatory diseases, metabolic diseases, neurodegenerative diseases and psychiatric diseases, especially directly by short-chain fatty acids (SCFA) and/or medium-chain fatty acids (MCFA), or through SCFA and /or MCFA-mediated inhibition of histone deacetylase (HDAC) activity to achieve the purpose of treating the above diseases.
  • SCFA short-chain fatty acids
  • MCFA medium-chain fatty acids
  • HDAC histone deacetylase
  • Bacterial strains from the genus Megacoccus have been identified that efficiently produce a number of SCFA and MCFA, including acetic acid, butyric acid, valeric acid, and caproic acid.
  • Megasphaera Known species under the genus Megasphaera include Megasphaera elsdenii, Megasphaera stantonii, Megasphaera massiliensis, Megasphaera indica, Megasphaera paucivorans, Megasphaerasueciensis, Megasphaera micronuciformis, Megasphaera hexanoica, and Megasphaera cerevisiae.
  • Megasphaera elsdenii model strain DSM 20460 Megasphaera indica model strain NMBHI-10, Megasphaera massiliensis model strain NP3 and strain NCIMB42787 (see CN112601534A), Megasphaera cerevisiae model strain DSM 20462, Megasphaera paucivorans model strain DSM 16981, Megasphaera sueciensis type strain DSM 17042 , Megasphaera micronuciformis model strain DSM 17226, Megasphaera hexanoica model strain MH, all have the ability to produce short-chain fatty acids, especially butyric acid (see "Megasphaera hexanoica sp.nov., a medium-chain carboxylic acid-producing bacterium isolated from a cow rumen ; DOI: 10.1099/ijseMegasphaera 0.001888” Table 1).
  • the present invention When conducting drug screening, the present invention first predicts and analyzes the butyric acid-producing ability of strains belonging to the genus Megasphaera through genomic data analysis at the genetic level. Based on the determination of the butyric acid-producing path and the integrity of the butyric acid-producing path, the method is used to Determine the key enzymes that can express butyrate production to further lock the genetic components; select bacterial strains containing genetic components/key enzymes (proteins), and verify the acid production ability at the experimental data level. Subsequently, the selected candidate strains will be subjected to in vitro experiments and mouse experiments to verify their effectiveness in tumor suppression and metabolic disease prevention and treatment.
  • bacterial strains MNH05026, MNH22004, and MNH27256 belonging to the genus Megasphaera were selected for anti-tumor experiments and metabolic disease prevention and treatment experiments.
  • the bacterial strain of the genus Megasphaera of the present invention (MNH05026) has a deposit number of GDMCC No: 62001, and its 16S rRNA is SEQ ID NO: 1;
  • MNH22004 Another bacterial strain of the genus Megasphaera of the present invention (MNH22004), the deposit number is GDMCC No: 62000, and its 16S rRNA is SEQ ID NO: 2;
  • Another bacterial strain of the genus Megasphaera of the present invention (MNH27256), the deposit number is GDMCC No: 61999, and its 16S rRNA is SEQ ID NO: 3.
  • Figure 1 shows the colony morphology of strains MNH 05026, MNH22004 and MNH27256 cultured on anaerobic blood plates for 48 hours;
  • Figure 1A shows the colony morphology of strain MNH 05026 cultured on anaerobic blood plates for 48 hours;
  • Figure 1B shows the colony morphology of strain MNH22004 cultured on anaerobic blood plates for 48 hours.
  • Figure 1C shows the colony morphology of strain MNH27256 cultured on anaerobic blood plates for 48 hours;
  • Figure 2 shows the microscopic morphology of strains MNH 05026 and MNH22004.
  • Figures 2C and 2D show the microscopic morphology of MNH27256 under different magnifications.
  • Figure 2A shows the microscopic morphology of strain MNH05026
  • Figure 2B shows the strain Microscopic morphology of MNH22004
  • Figures 2C and 2D show the microscopic morphology of MNH27256 under different magnifications;
  • Figure 3 shows the Gram staining microscopic morphology of strains MNH 05026, MNH22004 and MNH27256; wherein: Figure 3A shows the Gram staining microscopic morphology of strain MNH 05026; Figure 3B shows the Gram staining microscopic morphology of strain MNH22004 respectively; Figure 3C They are the Gram stain microscopic morphology of strain MNH27256;
  • Figure 4 shows the stained microscopic morphology of spores of strains MNH 05026, MNH22004 and MNH27256; wherein: Figure 4A shows the stained microscopic morphology of spores of strain MNH 050266; Figure 4B shows the stained microscopic morphology of spores of strain MNH22004; Figure 4C shows the stained microscopic morphology of spores of strain MNH27256 form;
  • Figure 5 shows the results of different concentrations of NaCl tolerance of strains MNH 05026, MNH22004 and MNH27256; wherein: Figures 5A, 5B and 5C are the results of different concentrations of NaCl tolerance of strains MNH 05026, MNH22004 and MNH27256 respectively;
  • Figure 6 shows the results of different pH tolerances of strains MNH 05026, MNH22004 and MNH27256; wherein: Figures 6A, 6B and 6C are the results of different pH tolerances of strains MNH 05026, MNH22004 and MNH27256 respectively;
  • Figure 7 shows the results of different concentrations of bile salt tolerance of strains MNH 05026, MNH22004 and MNH27256; wherein: Figures 7A, 7B and 7C are the results of different concentrations of bile salt tolerance of strains MNH 05026, MNH22004 and MNH27256 respectively;
  • Figure 8 shows the results of culturing strain MNH 05026 in medium API 20
  • the bacterial strains MNH05026, MNH22004, and MNH27256 of the genus Megasphaera of the present invention can all express butyrate-acetyl CoA-transferase A subunit (EC2.8.3.9 enzyme, see Uniprot.org for specific explanations).
  • the GENE IDs of the bacterial strains MNH05026, MNH22004, and MNH27256 expressing EC2.8.3.9 of the present invention involve: 650027236, 641897133, 650594268, 642201644, 650018449, 650536170, 251 1555023, 646248671 (For the sequence corresponding to GENE ID, see Integrated Microbial Genome (IMG); http://img.jgi.doe.gov).
  • the inventor counted the strains with anti-tumor effects that have been documented and confirmed in literature or patents under the genus Megasphaera, extracted the whole-gene data of these strains that have been published in the database, and conducted whole-genome data analysis.
  • the 4-aminobutyrate_pathway is higher than 70%; MNH22004 and MNH27256 both have complete butyrate-producing pathways (that is, the butyrate-producing pathway is 100% complete); MNH05026 has at least 70% completeness in the Pyruvate_pathway pathway and 4aminobutyrate_pathway The pathways have over 80% integrity.
  • Microbial SCFA has been fully studied by the existing technology for the treatment or prevention of obesity, diabetes and other metabolic diseases; the existing technology "Gut microbial metabolites in obesity, NAFLD and T2DM" shows that: produced by the fermentation of carbohydrates related to weight control Metabolites include acetic acid, propionic acid, butyric acid, and succinic acid; acetate and butyrate have also been shown to induce satiety through central mechanisms and increase thermogenesis in adipose tissue and liver, as well as induce adipose tissue browning and leanness.
  • GLP-1 glucagon-like peptide 1
  • PYY peptide YY
  • butyric acid is also a metabolite that has been fully proven in the existing technology to inhibit HDAC enzyme activity (Human gut bacteria as potent class I hisstone deacetylase inhibitors in vitro through production of butyric acid and valeric acid, https://doi.org/10.1371/journal.pone.0201073), and existing studies have shown that inhibiting HDAC activity can effectively treat metabolic diseases, such as diabetes. Diabetes is a group of diseases in which low levels of insulin and/or peripheral insulin resistance lead to hyperglycemia.
  • HDAC inhibition is also a promising treatment for advanced diabetic complications such as diabetic nephropathy and retinal ischemia (Christensen et al., 2011, Mol Med, 17(5-6), 370-390).
  • the present invention also discloses preventing and/or treating metabolic diseases by inhibiting histone acetylase activity (HDAC) using SCFA and/or MCFA produced by strains MNH05026, MNH22004, and MNH27256; preferably, the histone acetylase It is a class I, II, III and IV histone acetylase.
  • HDAC histone acetylase activity
  • MNH05026, MNH22004, and MNH27256 of the genus Megasphaera of the present invention are producers of butyric acid. According to the known effects of butyric acid, MNH05026 also has the ability to reduce the impermeability of the blood-brain barrier with neuroprotective effects (Michel and Prat (2016 )Ann Transl Med.4(1):15).
  • the bacterial strains MNH05026, MNH22004, MNH27256 of the genus Megasphaera of the present invention and their compositions can cause an increase in the expression of pro-inflammatory molecules, such as pro-inflammatory cytokines, in PBMC (see Figure 13); administration of the pharmaceutical composition of the present invention can Resulting in an increase in IL-1 ⁇ expression in PBMCs.
  • IL-1 ⁇ is a pro-inflammatory cytokine.
  • the production and secretion of IL-1 ⁇ is regulated by the inflammasome, a protein complex associated with the activation of inflammatory responses. Since administration of the composition of the present invention has been shown to increase the expression of IL-1 ⁇ , the composition of the present invention may be used to treat diseases characterized by decreased expression of IL-1 ⁇ .
  • the inventors also determined that Megasphaera sp. MNH05026, MNH22004, and MNH27256 can reduce LPS-induced inflammation.
  • the Megasphaera strain of the present invention and its compositions are particularly beneficial in inducing the production of various SCFAs such as butyric acid and inhibiting HDAC activity, thereby achieving the effect of preventing and treating metabolic diseases.
  • the Megasphaera strains and their compositions of the present invention can prevent and treat metabolic diseases alone or synergistically in combination with active substances such as Semaglutide in preclinical murine syngeneic metabolic disease models.
  • the Megasphaera strain and its composition of the present invention are used to treat or prevent metabolic diseases, such as liver diseases, obesity, cardiovascular diseases, cardiovascular and cerebrovascular diseases, hyperlipidemia, diabetes, impaired glucose tolerance, etc.
  • type II diabetes impaired glucose tolerance, insulin resistance, weight control, overweight, glycemic control, prediabetes, obesity, hyperglycemia, hyperinsulinemia, fatty liver, alcoholic steatohepatitis, Hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia
  • Acetic acid, butyric acid, valeric acid, and caproic acid have been shown to mediate autoimmune and inflammation-related diseases. These effects can increase the production of immunomodulatory regulatory T cells (Tregs) and anti-inflammatory cytokines by reducing the synthesis of inflammatory cytokines. to fulfill.
  • Tregs immunomodulatory regulatory T cells
  • MNH05026, MNH22004 and MNH27256 can produce acetic acid, butyric acid, valeric acid and caproic acid.
  • MNH05026, MNH22004, and MNH27256 can increase anti-inflammatory cytokines and attenuate LPS-induced inflammation.
  • Acetic acid and butyric acid have been shown to treat and prevent obesity, type 2 diabetes, and non-alcoholic fatty liver disease.
  • administration of a composition comprising Megacoccus sp.: MNH05026, MNH22004, and MNH27256 can reduce HDAC activity.
  • the present invention provides compositions comprising Megacoccus spp. MNH05026, MNH22004 and MNH27256 Methods for treating or preventing inflammatory bowel disease mediated by HDAC activity. Inhibiting HDAC activity has been shown to inhibit the production of pro-inflammatory cytokines in the gastrointestinal tract. Therefore, the Megasphaera strains and compositions thereof of the present invention can be used to treat inflammatory diseases. In particular, the Megasphaera strains of the present invention and compositions thereof may be used to treat or prevent conditions associated with the pathogenesis of increased colonic pro-inflammatory cytokines. In some embodiments, the Megasphaera strains of the invention and compositions thereof are used to treat or prevent inflammatory bowel disease.
  • the Megasphaera strains of the invention and compositions thereof are used to treat or prevent ulcerative colitis. In some embodiments, the Megasphaera strains of the invention and compositions thereof are used to treat or prevent Crohn's disease. In certain embodiments, the invention provides strains comprising Megacoccus and compositions thereof. MNH05026, MNH22004 and MNH27256 are used to treat or prevent inflammatory diseases. In a preferred embodiment, the present invention provides strains comprising Macrococcus and compositions thereof, MNH05026, MNH22004 and MNH27256 for use in the treatment or prevention of colitis.
  • the bacterial strain in the composition is Macrococcus strain MNH05026.
  • the bacterial strain in the composition is a new species under Megasphaera, which Closely related strains may also be used, such as strains having a 16S rRNA gene sequence that is at least 95%, 96%, 97%, 98%, 98.63%, 99%, 99.7% or 99.9% identical to SEQ ID NO: 1 .It may also be a strain having a 16S rRNA gene sequence that is at least 95%, 96%, 97%, 98%, 98.63%, 99%, 99.5% or 99.9% identical to SEQ ID NO:2. It may also be a strain having a 16S rRNA gene sequence identical to SEQ ID NO:2.
  • the strain used in the present invention has the 16S rRNA gene sequence represented by SEQ ID NO:1.
  • the present invention provides a food product comprising the above-described Megasphaera strain and compositions thereof.
  • the present invention provides methods of treating or preventing diseases or conditions mediated by HDAC activity, comprising administering bacterial strains of the genus Megalococcus and compositions thereof.
  • the present invention also provides strains and compositions comprising such cells or biologically pure cultures of such cells.
  • the present invention also provides a cell of the genus Megacoccus, such as MNH05026, MNH22004, MNH27256 strains or derivatives thereof, for use in the treatment of, in particular, the diseases described herein.
  • the Megacoccus bacterial strain and its pharmaceutical composition of the present invention can be used to prevent and/or treat metabolic diseases, including but not limited to: type II diabetes, impaired glucose tolerance, insulin resistance, weight control, overweight, and blood sugar control. , prediabetes, 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, nephropathy, diabetic neuropathy, Diabetic retinopathy, skin disease, indigestion, or edema.
  • metabolic diseases including but not limited to: type II diabetes, impaired glucose tolerance, insulin resistance, weight control, overweight, and blood sugar control.
  • prediabetes obesity, hyperglycemia, hyper
  • the Megacoccus bacterial strain of the present invention includes species having at least 95% identity with SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.
  • Bacterial strains of the present invention include Megasphaera elsdenii, Megasphaera stantonii, Megasphaera indica, Megasphaera paucivorans, Megasphaera sueciensis, Megasphaera micronuciformis, Megasphaera hexanoica, Megasphaera cerevisiae or have at least SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 95% identical species strain that expresses EC2.8.3.9;
  • the bacterial strain regulates the expression of EC2.8.3.9 through GENE ID: 650027236, 641897133, 650594268, 642201644, 650018449, 650536170, 2511555023 and 646248671.
  • the present invention relates to bacterial strains and compositions thereof capable of modulating at least one short chain fatty acid or short chain fatty acid salt including acetic acid, butyric acid, valeric acid, valeric acid or caproic acid.
  • Short-chain fatty acids usually exist in the form of short-chain fatty acid salts when they have therapeutic effects in the body. Through verification, it was found that acetate, propionate and butyrate are the main short-chain fatty acid salts.
  • the present invention relates to bacterial strains having the characteristics of SEQ ID NO: 1 and having at least 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 98.65%, 99%, 99.5% or 99.9 % identity of 16S rRNA sequence;
  • the bacterial strains and their compositions involved in the present invention are used for the treatment or prevention of autoimmune diseases or inflammatory diseases, such as: asthma, arthritis, psoriasis, graft-versus-host disease, inflammatory bowel disease, Crohn's disease , ulcerative colitis, allograft rejection, chronic inflammatory bowel disease, systemic lupus erythematosus, psoriasis, rheumatoid arthritis, multiple sclerosis or Hashimoto's disease; allergic diseases such as food Allergies, hay fever or asthma; Clostridium difficile infection; the inflammatory diseases include meningitis, myelitis or TNF-mediated inflammatory diseases, such as inflammatory diseases of the gastrointestinal tract, such as pouchitis, cardiovascular inflammation disease, such as atherosclerosis, or inflammatory lung disease, such as chronic obstructive pulmonary disease.
  • autoimmune diseases or inflammatory diseases such as: asthma, arthritis, psoriasis, graft-versus-host disease,
  • the bacterial strains and their compositions involved in the present invention are used for the treatment or prevention of neurological diseases, such as depression, anxiety, post-traumatic stress disorder, obsessive-compulsive disorder; Parkinson's disease, brain atrophy, vascular or arteriosclerotic Parkinson's disease, mild cognitive impairment disorders, HIV-related cognitive impairment, Alzheimer's disease (AD).
  • neurological diseases such as depression, anxiety, post-traumatic stress disorder, obsessive-compulsive disorder
  • Parkinson's disease brain atrophy, vascular or arteriosclerotic Parkinson's disease, mild cognitive impairment disorders, HIV-related cognitive impairment, Alzheimer's disease (AD).
  • compositions involved in the present invention include pharmaceutically acceptable carriers;
  • the carrier is selected from one or more of diluents, dispersants, excipients, stabilizers, lubricants, and disintegrants.
  • the dosage forms of the medicines involved in the present invention include any one of liquid preparations, solid preparations, capsule preparations, sustained-release preparations and nano-preparations.
  • the dosage of bacterial liquid used in the animal experiment of the present invention can be based on the mouse's weight, and the dosage of bacterial liquid can be 0.04-0.3ml/10g body weight; it can also be 0.2ml/mouse, and the above-mentioned gavage
  • the strains MNH05026, MNH22004, and MNH27256 of the present application can be used alone or in combination with other drugs to achieve the improvement of liver and kidney function in the present invention, as well as the therapeutic effects of diabetes, obesity, fatty liver and other diseases. And the effect is remarkable.
  • the solid MM01 culture medium involved in this article consists of: peptone 5g/L, tryptic casein 5g/L, yeast powder 10g/L, beef extract 5g/L, glucose 5g/L, K 2 HPO 4 2g/L , sodium acetate 2g/L, Tween 80 1mL/L, heme 5mg/L, L-cysteine hydrochloride 0.5g/L, vitamin K1 1uL/L, inorganic salt solution 8ml/L, inorganic salt solution
  • Each 1L includes (calcium chloride 0.25g, dipotassium hydrogen phosphate 1g, potassium dihydrogen phosphate 1g, magnesium sulfate 0.5g, sodium bicarbonate 10g, sodium chloride 2g), agar 15g/L.
  • the liquid MM01 culture medium involved in this article consists of: peptone 5g/L, tryptic casein 5g/L, yeast powder 10g/L, beef extract 5g/L, glucose 5g/L, K 2 HPO 4 2g/L , sodium acetate 2g/L, Tween 80 1mL/L, heme 5mg/L, L-cysteine hydrochloride 0.5g/L, vitamin K1 1uL/L, inorganic salt solution 8ml/L, inorganic salt solution
  • Each 1L includes (calcium chloride 0.25g, dipotassium hydrogen phosphate 1g, potassium dihydrogen phosphate 1g, magnesium sulfate 0.5g, sodium bicarbonate 10g, sodium chloride 2g).
  • AC liquid medium contains per liter: peptone, 20g; glucose, 5g; yeast extract, 3g; beef extract powder, 3g; vitamin C, 0.2g; pH7.0.
  • the anaerobic blood plate formula (purchased from Huankai Microorganisms) contained in this article (per liter): 10.0g casein trypsin digestion; 3.0g heart trypsin digestion; 1.0g corn starch; 5.0g meat stomach enzymatic digestion; Yeast extract powder 5.0g; sodium chloride 5.0g; agar 15.0g; sterile defibrinated sheep blood 50-100mL; distilled water 1000mL; final pH 7.3 ⁇ 0.2.
  • the tryptone soy peptone liquid culture medium TSB liquid culture medium (purchased from Huankai Microorganisms) in this article contains components of the formula (per liter): tryptone 17.0g; soybean papain hydrolyzate 3.0g; dipotassium hydrogen phosphate 2.5 g; sodium chloride 5.0g; glucose 2.5g; final pH 7.3 ⁇ 0.2.
  • the intestinal strain MNH05026 involved in the present invention was isolated from the fecal sample of a healthy female volunteer in Guangzhou City, Guangdong province.
  • the donor takes 2 to 5 grams of fresh feces from himself and puts it into a sample collection and storage tube. After shaking and homogenizing, the processed feces sample is placed in an ice box and sent to the laboratory within 24 hours for bacterial strain isolation.
  • the dripping amount is 100 ⁇ L/dish. Spread evenly and wait until After the plate surface is dry, the plate is inverted and cultured at 37°C for 3 to 5 days. Observe the growth status of the strains in the isolation medium and pick single colonies with sterilized toothpicks to purify the strains.
  • the purified strains are placed at 37°C and cultured anaerobically. Purely cultured strains are prepared into bacterial liquid with 20% glycerol or water and stored at -86°C.
  • the strain identification method was based on the 16S rRNA gene sequence identification method of the strain.
  • the intestinal strain MNH22004 involved in the present invention was isolated from the fecal sample of a healthy female volunteer in Guangzhou City, Guangdong province. Strain MNH22004 was isolated using the above method.
  • the intestinal strain MNH27256 involved in the present invention was isolated from the fecal sample of a healthy female volunteer in Guangzhou City, Guangdong province. Strain MNH27256 was isolated using the above method.
  • Strain MNH05026 (referred to as A026) has been deposited in the Guangdong Provincial Microbial Culture Collection Center.
  • the preservation name is Megasphaera sp.MNH05026
  • the preservation number is GDMCC No: 62001
  • the preservation time is October 18, 2021
  • the preservation address Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City.
  • the proposed classification name is Megasphaera sp.
  • the strain MNH22004 of the present invention has been deposited in the Guangdong Provincial Microbial Culture Collection Center.
  • the preservation name is Megasphaera sp.MNH22004
  • the preservation number is GDMCC NO: 62000
  • the preservation time is October 18, 2021
  • the preservation address Xianlie Middle Road, Guangzhou City 5th Floor, Building 59, Courtyard No. 100, Institute of Microbiology, Guangdong Academy of Sciences, the proposed classification name is Megasphaera sp.
  • the strain MNH27256 of the present invention has been deposited in the Guangdong Provincial Microbial Culture Collection Center.
  • the preservation name is Megasphaera sp.MNH27256
  • the preservation number is GDMCC NO: 61999
  • the preservation time is October 18, 2021
  • the preservation address Xianlie Middle Road, Guangzhou City 5th Floor, Building 59, Courtyard No. 100, Institute of Microbiology, Guangdong Academy of Sciences, the proposed classification name is Megasphaera sp.
  • Strain MNH05026 (referred to as A026) was inoculated into anaerobic blood plate culture medium. After anaerobic culture at 37°C for 48 hours, visible colonies were formed on the anaerobic blood plate culture medium. The colonies were round, with regular and smooth edges, and a diameter of approximately 1mm, light yellow, opaque, no secretion is formed around the colony; the strain is Gram-negative; microscopic morphological observation shows that the strain has no spores, no flagella, no movement, a spherical shape, and a diameter of about 5 to 10 ⁇ m. (See Figures 1A to 4A).
  • Strain MNH22004 was inoculated into anaerobic blood plate culture medium. After anaerobic culture at 37°C for 24 hours, visible colonies were formed on the anaerobic blood plate culture medium. The colonies were round, with regular and smooth edges, about 3mm in diameter, yellow, opaque, and There is no secretion formation around (see Figure 1B for the colony morphology photo); the strain is Gram-negative (the Gram-stained microscopic morphological photo of the strain is shown in Figure 3B, and the spore-stained microscopic morphological photo of the strain is shown in Figure 4B); microscopic morphological observation revealed that the strain No spores, no flagella, non-motile, short rod-shaped, about 5 ⁇ 10 ⁇ 15 ⁇ m in size (see Figure 2B for microscopic morphological photos of colonies).
  • the strain MNH27256 was inoculated into the anaerobic blood plate culture medium. After anaerobic cultivation at 37°C for 24 hours, visible colonies were formed on the anaerobic blood plate culture medium. The colonies were round, with regular and smooth edges, about 2 mm in diameter, yellow, opaque, and There is no secretion formation around (the colony morphology photo is shown in Figure 1C); the strain is Gram-negative (the Gram-stained microscopic morphological photo of the strain is shown in Figure 3C, and the spore-stained microscopic morphological photo is shown in Figure 4C); microscopic morphological observation revealed that the strain No spores, no flagella, non-motile, spherical, about 5 ⁇ m in diameter (see Figures 2C and 2D for microscopic morphological photos of colonies).
  • Strain MNH05026 has a temperature growth range of 30 to 42°C, and the optimal growth is 37°C; it can grow in the range of pH 6.0 to 8.0, and the optimal growth pH is 6.0 to 7.0 (see Figure 6A); the highest Tolerant to 1% NaCl (see Figure 5A); strain MNH05026 can survive and grow in the bile salt concentration range of 0 to 0.15%, but cannot grow when the bile salt concentration is greater than or equal to 0.2% (see Figure 7A). Strain MNH05026 cannot grow under aerobic conditions but grows well under anaerobic conditions and is an obligate anaerobic bacterium.
  • Strain MNH22004 has a temperature growth range of 30 to 42°C, and the optimal growth is 37°C; it can grow in the range of pH 5.0 to 10.0, and the optimal growth pH is 7.0 to 8.0; it can tolerate up to 2% NaCl; strain MNH22004 can exist The strain could survive and grow within the range of 0-0.2% bile salt concentration, but the growth trend of the strain weakened as the bile salt concentration increased. Strain MNH22004 cannot grow under aerobic conditions but grows well under anaerobic conditions and is an obligate anaerobic bacterium. The tolerance of strains to NaCl, pH and bile salts are shown in Figures 5B to 7B.
  • Strain MNH27256 has a growth temperature range of 30 to 42°C, and the optimal growth is 37°C; it can grow in the range of pH 5.0 to 10.0, and the optimal growth pH is 7.0 to 8.0; it can tolerate up to 2% NaCl; strain MNH27256 can grow in The strain could survive and grow within the range of 0-0.4% bile salt concentration, but the growth trend of the strain weakened as the bile salt concentration increased. Strain MNH27256 cannot grow under aerobic conditions but grows well under anaerobic conditions and is an obligate anaerobic bacterium. The tolerance of strains to NaCl, pH and bile salts are shown in Figures 5C to 7C.
  • API 20A test strain According to the instructions, use API 20A reagent strip (BioMérieux) for testing. The culture conditions of the strain were 37°C and anaerobic.
  • Antibiotic susceptibility testing of strains MNH05026, MNH22004, and MNH27256 The antibiotic susceptibility testing of strains MNH05026, MNH22004, and MNH27256 was performed using the disk diffusion method. The test results are shown in Table 3. Strain MNH05026 is resistant to gentamicin, erythromycin, chloramphenicol, tetracycline, penicillin, ciprofloxacin, and Compound Xinnuo It was sensitive to antibiotics such as ampicillin, ampicillin, lincomycin and ceftriaxone; strain MNH05026 was not resistant to any of the antibiotics used.
  • Strain MNH22004 is resistant to penicillin and ampicillin; it is sensitive to antibiotics such as gentamicin, erythromycin, chloramphenicol, tetracycline, ciprofloxacin, cotrimoxazole, ceftriaxone, and lincomycin.
  • Strain MNH27256 is resistant to penicillin, ampicillin, and ceftriaxone; it is sensitive to antibiotics such as gentamicin, erythromycin, chloramphenicol, tetracycline, ciprofloxacin, cotrimoxazole, and lincomycin.
  • Fresh cultures of strains MNH05026, MNH22004, and MNH27256 were taken to extract the genomic DNA of the strains.
  • the extracted genomic DNA of the strain was used as a template to amplify the 16S rRNA gene.
  • the primer pairs used in the 16S rRNA gene PCR of the present invention are as follows:
  • PCR reaction program is as follows: PCR reaction cycle: pre-denaturation: 94°C, 4mim; denaturation: 94°C, 50sec; annealing: 52°C, 40sec; extension: 72°C, 70sec; final extension: 72°C, 10min; cycle 36 times.
  • the PCR product is purified and submitted to Genewise for 16S rRNA gene sequencing. Submit the 16S rRNA gene sequence of the strain returned by sequencing to the NCBI Basic Local Alignment Search Tool, perform strain 16S rRNA gene analysis, and confirm the strain classification information.
  • the 16S rRNA gene amplification product of strain MNH05026 was sent for sequencing to obtain the 16S rRNA gene sequence (SEQ ID NO: 1).
  • the measured sequence and the data in GenBank were analyzed by BLAST.
  • the comparison results showed that strain MNH05026 belongs to the genus Megasphaera. , the strains with the highest similarity are Megasphaera hexanoica (96.50%) and Megasphaera elsdenii (94.43%).
  • the 16S rRNA gene sequence similarity between strain MNH05026 and other strains of the genus Megasphaera is less than 94.30%; according to Kim et al.
  • MNH05026 can be significantly distinguished from other known strains of the genus Megasphaera, so MNH05026 of the present invention is a new species strain of the genus Megasphaera.
  • MNH22004 SEQ ID NO:2
  • MNH27256 SEQ ID NO:3
  • the strains with higher similarity to MNH22004 are Megasphaera micronuciformis (90.92%), Megasphaera massiliensis (90.63%), Megasphaera hexanoica (90.63%), Megasphaera elsdenii (90.61%), Megasphaera paucivorans (90.59%), Megasphaera indica (90.29 %), Megasphaera sueciensis (90.18%); the 16S rRNA gene sequence similarity between strain MNH22004 and Megasphaera genus and other known strains is less than 90%; lower ( ⁇ 95%) 16S rRNA gene sequence similarity This indicates that strain MNH22004 may be a new species of the Megasphaera family.
  • the strain with the highest similarity to MNH27256 is Megasphaera stantonii AJH120 (MG811574), and the 16S rRNA gene similarity between the two is 98.41%; the 16S rRNA gene sequence similarity between strain MNH27256 and other known strains of the genus Megasphaera is less than 94 %, indicating that MNH27256 may be a new species strain of the genus Megasphaera.
  • Strain MNH22004 formed a separate branch with Megasphaera stantonii AJH120T (Bootstrap support value 100). Strain MNH27256 and Megasphaera stantonii AJH120 form a clade together (Bootstrap support value is 100).
  • MNH05026, MNH22004, and MNH27256 do not belong to strains under the species Megasphaera_massiliensis and Megasphaera elsdenii, and there are obvious differences in genomics with Megasphaera_massiliensis and Megasphaera elsdenii.
  • MNH05026 SEQ ID NO: 1
  • MNH22004 SEQ ID NO: 2
  • MNH27256 SEQ ID NO: 3
  • the genome correlation analysis based on average nucleotide identity (ANI) in this application shows that MNH05026, MNH22004, and MNH27256 belong to new species in the genus Megacoccus, while MNH22004 and MNH27256 belong to the genus Megalococcus.
  • the genome of the original strain MNH05026 was sequence fragmented by ultrasonic method, and the fragment length range was ⁇ 350bp, and then use a standard DNA library construction kit (NEB UltraTM) to construct an Illumina sequencing library.
  • the constructed sequencing library was subjected to paired-end 150bp sequencing using NovaSeq (Illumina). Sequencing yielded 1.46Gbp data, of which Q20 accounted for 97.08%.
  • MNH22004 and MNH27256 used the same method to sequence and obtained 1.33Gbp and 1.39Gbp data respectively, of which Q20 accounted for 97.27% and 96.95% respectively.
  • the original genome sequencing data is filtered using fastp (version: 0.20.0), and the filtering parameters are: "--poly_g_min_len 10 --poly_x_min_len 10 -q 15 -u 40 -n 5 -l 50".
  • the filtered raw data were genome assembled using SPAdes (version: v3.14.0) with the assembly parameter "--isolate--cov-cutoff 10".
  • the genome assembly resulted in the total gene lengths of MNH05026, MNH22004 and MNH27256 being 2.79Mbp, 2.58Mbp and 2.59Mbp respectively, the N50 lengths being 59.8kbp, 157.9kbp and 92.9kbp respectively, and the GC contents being 50.46%, 52.86% and 52.85% respectively.
  • Genomic genes were predicted and analyzed using the prokaryotic analysis software genome annotation process prokka (version: 1.14.5), with the parameter "--gcode 11--evalue 1e-09".
  • a total of 2568 CDS sequences were predicted for MNH05026, with the average CDS sequence length being 957 bp.
  • the model strain with the highest genome similarity is Megasphaera elsdenii, with an average nucleotide similarity (ANI) of 78.58% and a gene coverage of 30.27%. Therefore, it can be identified as a new species under the genus Megasphaera elsdenii.
  • a total of 2353 CDS sequences were predicted for MNH22004, with the average CDS sequence length being 967 bp.
  • the model strain with the highest genome similarity to the experimental strain MNH22004 is Megasphaera elsdenii, with an average nucleotide similarity (ANI) of 79.49% and a gene coverage of 42.37%.
  • strain MNH22004 may be a new species in the genus Megasphaera.
  • a total of 2335 CDS sequences were predicted for MNH27256, with the average CDS sequence length being 975 bp.
  • the model strain with the highest genome similarity to the experimental strain MNH27256 is Megasphaera elsdenii, with an average nucleotide similarity (ANI) of 79.38% and a gene coverage of 41.04%.
  • antibiotic resistance genes in the genome were analyzed using the RGI pipeline (version: 4.2.2), in which the antibiotic resistance gene database is CARD (version: 3.0.0, https://card.mcmaster.ca/analyze/rgi). No antibiotic resistance genes were identified in MNH05026.
  • the drug resistance gene information compared between MNH22004 and MNH27256 is shown in Table 6.
  • VFDB virulence factor database
  • AntiSMASH5 (version: 5.1.1) was used to analyze potential secondary metabolism gene clusters in the genome. MNH05026 did not map secondary metabolism gene clusters. The comparison results between MNH22004 and MNH05026 are shown in Table 8.
  • the strain MNH05026 (referred to as A026) was inoculated on the TSA plate, and after anaerobic cultivation at 37°C for 48 hours, the bacterial cells were collected, and the bacterial fatty acids were extracted and methylated; Microbial ID, Inc., Newark, USA, was used. ,Del) (Kroppenstedt, 1985; Meier et al., 1993) fully automated bacterial identification system was used to analyze the fatty acid components of strain MNH05026 (see Table 11A). MNH22004 and MNH27256 were analyzed using the same method, and the results are shown in Tables 11B and 11C respectively.
  • the main fatty acids (>10%) of MNH05026 are twelve-carbon saturated fatty acids (C12:0 11.98%) and nineteen-carbon unsaturated fatty acids (C18:1CIS 9FAME 11.28%); the remaining types of fatty acids and their contents are detailed in Table 11A .
  • the main fatty acids (>10%) of MNH22004 are twelve-carbon saturated fatty acid (C12:0 14.43%), sixteen-carbon saturated fatty acid (C16:0 13.87%), 3-hydroxy fourteen-carbon saturated fatty acid (C14:0 3OH 11.84%) ) and 18-carbon monounsaturated fatty acid (C18:1CIS 9 10.14%); other types of fatty acids and their contents are detailed in Table 11B.
  • the main fatty acids (>10%) of MNH27256 are twelve-carbon saturated fatty acid (C12:0 15.54%), eighteen-carbon monounsaturated fatty acid (C18:1CIS 9 15.03%) and 3-hydroxy fourteen-carbon saturated fatty acid (C14:0 3OH 13.22%); other types of fatty acids and their contents are detailed in Table 11C.
  • strain MNH05026 belongs to a new species strain of the genus Megasphaera.
  • the present invention temporarily names it Megasphaera sp.MNH05026 (patented strain The deposit number is GDMCC No:62001).
  • Strains MNH22004 and MNH27256 both belong to different strains of the same new species in the genus Megasphaera.
  • the present invention temporarily names them as Megasphaera sp. MNH22004 (patent strain preservation number is GDMCC NO: 62000) and Megasphaera sp. MNH27256 (patent The strain preservation number is GDMCC NO:61999).
  • MNH05026 as an example for description, MNH22004 and MNH27256 are measured using the same method.
  • Preparation of bacterial cells Inoculate strain MNH05026 into TSB liquid culture medium, culture it anaerobically at 37°C for 48 hours, collect the bacterial cells by centrifugation, and store the collection at -86°C for later use.
  • Preparation of standard products Weigh acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid and caproic acid standard products, and use ethyl acetate to prepare 0.1 ⁇ g/mL, 0.5 ⁇ g/mL, 1 ⁇ g/mL, 5 ⁇ g /mL, 10 ⁇ g/mL, 20 ⁇ g/mL, 50 ⁇ g/mL, 100 ⁇ g/mL eight mixed standard concentration gradients. Take 600 ⁇ L of the standard, add 25 ⁇ L of 4-methylpentanoic acid with a final concentration of 500 ⁇ M as the internal standard, mix well, add it to the injection bottle, and enter the GC-MS detection. The injection volume is 1 ⁇ L, and the split ratio is 10:1. Split injection.
  • Metabolite extraction Thaw the sample on ice, put 80 mg of the sample into a 2 mL glass centrifuge tube, add 900 ⁇ L of 0.5% phosphoric acid to resuspend, shake and mix for 2 min, centrifuge at 14000g for 10 min, take 800 ⁇ L of the supernatant, and add an equal amount of ethyl acetate Extract the ester, shake and mix for 2 minutes, centrifuge at 14000g for 10 minutes, take 600 ⁇ L of the upper organic phase, add 4-methylpentanoic acid with a final concentration of 500 ⁇ M as the internal standard, mix and add to the injection bottle, enter the GC-MS detection, and the injection volume 1 ⁇ L, split ratio 10:1, split injection.
  • Sample detection and analysis Agilent DB-WAX capillary column (30m ⁇ 0.25mm ID ⁇ 0.25 ⁇ m) gas chromatography system was used to separate the samples.
  • Programmed temperature rise initial temperature is 90°C, raised to 120°C at 10°C/min, then to 150°C at 5°C/min, and finally to 250°C at 25°C/min, and maintained for 2 minutes.
  • the carrier gas was helium with a flow rate of 1.0 mL/min.
  • Mass spectrometry analysis was performed using Agilent 7890A/5975C GC/MS.
  • the inlet temperature is 250°C
  • the ion source temperature is 230°C
  • the transmission line temperature is 250°C
  • the quadrupole temperature is 150°C.
  • Electron impact ionization (EI) source full scan and SIM scanning mode, electron energy 70eV.
  • MSD ChemStation software was used to extract the chromatographic peak area and retention time. Draw a standard curve and calculate the content of short-chain fatty acids in the sample (see Table 12).
  • Strain MNH05026 can synthesize caproic acid, acetic acid, isovaleric acid, butyric acid and isobutyric acid in large amounts, and valeric acid and propionic acid in small amounts during the growth process.
  • Strain MNH22004 can synthesize acetic acid, propionic acid, isovaleric acid, butyric acid and isobutyric acid in large amounts during the growth process, especially it has a high ability to synthesize butyric acid; it can synthesize caproic acid and valeric acid in small or very small amounts.
  • Strain MNH27256 can synthesize acetic acid, propionic acid, isovaleric acid, butyric acid and isobutyric acid in large amounts during the growth process, especially it has a high ability to synthesize butyric acid; it can synthesize caproic acid and valeric acid in small or very small amounts.
  • the meaning of "high yield” in this application is that the output is not less than 200ug/g; therefore, it can be determined that MNH05026, MNH22004, and MNH27256 in this application have high yields of acetic acid and butyric acid.
  • the term "high yield of acetic acid and/or butyric acid” refers to the yield of acetic acid and/or butyric acid ⁇ 200 ⁇ g/g in the short-chain fatty acid (SCFA) assay.
  • the acetic acid output is ⁇ 250ug/g; preferably, the acetic acid output is ⁇ 300 ⁇ g/g; preferably, the acetic acid output is ⁇ 400 ⁇ g/g; and/or, preferably, the butyric acid output is ⁇ 300 ⁇ g/g, ⁇ 400 ⁇ g/g, ⁇ 500 ⁇ g/g, ⁇ 600 ⁇ g/g, ⁇ 700 ⁇ g/g, ⁇ 800 ⁇ g/g, ⁇ 900 ⁇ g/g, ⁇ 1000 ⁇ g/g, ⁇ 1100 ⁇ g/g, ⁇ 1200 ⁇ g/g, ⁇ 1300 ⁇ g/g, ⁇ 1400 ⁇ g/g, ⁇ 1500 ⁇ g/g or ⁇ 1600 ⁇ g/g.
  • MNH05026 as an example for description, MNH22004 and MNH27256 use the same method to prepare bacterial cells.
  • each tube is filled with 0.2ml of bacterial solution, and each strain is filled with 10 ml of bacterial solution. Tube. Store frozen at -80°C. After it is completely frozen (24 hours), take out a tube of frozen bacteria, thaw it to room temperature, and use the dilution coating method to measure the number of viable bacteria (CFU) for later use.
  • CFU viable bacteria
  • a high-fat diet-induced obesity mouse model was used to conduct experiments on the improvement of liver and kidney function and related diseases with MNH05026. This invention has passed the ethical review of the Experimental Animal Care and Use Committee of Munn Biotechnology.
  • Experimental mice were C57BL/6J mice, 5-6 weeks old, 20 in total, purchased from Guangdong Yaokang Biotechnology Co., Ltd.
  • Test strain After thawing the MNH05026 strain glycerol cryovial at 37°C, inoculate it on the M01 plate in an anaerobic workstation for activation, inoculate the activated strain into the M01 liquid medium, and culture it anaerobically. , obtain a sufficient amount of culture, centrifuge and concentrate the cultured bacterial liquid, and then resuspend the bacteria in a solvent to obtain a test substance whose purity and viable bacterial count (2.9 ⁇ 10 9 CFU/mL) meet the requirements of animal experiments.
  • Negative control substance Use PBS-Cys containing 25% glycerol as a negative control substance
  • mice 5-6 week old C57BL/6J male mice were fed a high-fat diet for 10 weeks after the quarantine period. 16 mice with a weight range of 35.50g-44.49g were selected and randomly selected according to weight. Stratified grouping, 8 animals/group, divided into 2 groups (experimental group and control group). After grouping (D1), intragastric administration was started. The amount of bacterial liquid administered to the experimental group was 0.2 mL/animal. The experimental group was given MNH05026, and the control group was given negative control substance, twice a day for a total of 21 days. , During the experiment, the mice had free access to water and food, and a 12h/12h day-night cycle was adopted.
  • MNH22004 uses the same methods as 1) to 4) to conduct relevant experiments. The experimental results are shown in Figure 10.
  • Figure 10 shows the effect of strains MNH05026 and MNH22004 on the liver weight of obese mice induced by high-fat diet
  • Figure 11 is a graph showing the effect of strain MNH05026 on the serum alanine aminotransferase (ALT) content of high-fat diet-induced obese mice;
  • MNH05026 can reduce the content of alanine aminotransferase (ALT) in serum.
  • alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are markers of liver cell damage.
  • Figure 11 Results It shows that MNH05026 can reduce the ALT content in serum, indicating that MNH05026 can improve liver function abnormalities caused by high-fat diet, and can significantly improve fatty liver and liver function abnormalities caused by high-fat diet.
  • MNH05026 and MNH22004 can significantly reduce liver weight, and MNH05026 can also reduce serum ALT content, indicating that MNH05026 and MNH22004 can significantly improve fatty liver and abnormal liver function caused by high-fat diet.
  • Example 6 Use of strains MNH05026 and MNH27256 to prevent or treat high-fat diet-induced obesity mouse model Use of in vivo tests in diabetes
  • Test strain After melting the MNH05026 strain glycerol cryovial at 37°C, inoculate it on the M01 plate in an anaerobic workstation for activation, and The activated strain was inoculated into M01 liquid medium and cultured anaerobically to obtain a sufficient number of cultures. The cultured bacterial liquid was centrifuged and concentrated and then resuspended with a solvent to obtain the purity and viable bacterial count (2.9 ⁇ 10 9 CFU/mL) test substances that meet the requirements for animal experiments.
  • the MNH27256 strain glycerol cryogenic tube After thawing the MNH27256 strain glycerol cryogenic tube at 37°C, inoculate it on the M01 plate in an anaerobic workstation for activation. Inoculate the activated strain into the M01 liquid medium and culture it anaerobically to obtain a sufficient number of cultures. The cultured bacterial liquid is centrifuged and concentrated, and then the bacteria are resuspended in a solvent to obtain a test substance whose purity and viable bacterial count (7.6 ⁇ 10 9 CFU/mL) meet the requirements of animal experiments.
  • mice 5-6 week old C57BL/6J male mice were fed a high-fat diet for 10 weeks after the quarantine period. 16 mice with a weight range of 35.50g-44.49g were selected and randomly selected according to weight. Stratified grouping, 8 animals/group, divided into 2 groups (experimental group and control group). After grouping (D1), intragastric administration was started. The experimental group was given MNH05026. The amount of intragastric bacterial liquid in the experimental group was 0.2 mL/animal. The control group was given negative control substance twice a day for a total of 21 days. , During the experiment, the mice had free access to water and food, and a 12h/12h day-night cycle was adopted.
  • MNH27256 used the same methods as 1) to 4) above to conduct relevant experiments.
  • Figure 12 shows the effects of strains MNH05026 and MNH27256 on fasting blood glucose (FGB) in high-fat diet-induced obese mice.
  • Figure 12C shows the results of MNH27256 on glucose tolerance (OGTT) in high-fat diet-induced obese mice.
  • Figure 12D shows the effect of MNH27256 on high-fat diet. Results of the area under the glucose tolerance curve of induced obese mice);
  • MNH05026 and MNH27256 strains can reduce FBG in obese mice induced by high-fat diet, and the hypoglycemic effect of MNH05026 is better than that of MNH27256. Based on this, MNH27256 was given priority for further experiments to explore its effect on OGTT in obese mice induced by high-fat diet.
  • Oral glucose tolerance test On the 17th day of administration (D17), measure the OGTT after fasting for 12 hours (such as fasting from 20:30:00 in the evening to 08:30:00 the next day). Weigh the fasting body weight of the mice, and administer glucose according to the fasting body weight of the mice. The dose of glucose administered is 2g/kg (glucose g/mouse fasting body weight kg). Measure fasting blood glucose, 15min, 30min, after giving sugar. Blood glucose levels at 60min, 90min, and 120min. Each mouse was timed strictly and the blood glucose value was measured accurately at 6 time points. The measurement results are shown in Figures 12C and 12D.
  • Figures 12C-12D are the results of the oral glucose tolerance test of MNH27256 on high-fat diet-induced obese mice.
  • the OGTT test is a glucose load test used to understand the function of pancreatic beta cells and the body's ability to regulate blood sugar; to observe patient tolerance
  • the ability to detect glucose is currently recognized as a diagnostic indicator for the diagnosis of diabetes.
  • glucose metabolism is disordered, blood sugar rises sharply after oral administration of a certain amount of glucose, or the rise is not obvious, but cannot be reduced to the fasting level or the original level within a short period of time.
  • This is abnormal glucose tolerance or impaired glucose tolerance; abnormal glucose tolerance indicates The body's ability to metabolize glucose is reduced, which is common in type 2 diabetes and obesity.
  • Figure 12C shows the oral glucose tolerance results
  • Figure 12D shows the area under the oral glucose tolerance curve results.
  • MNH27256 can effectively improve blood sugar elevation and improve glucose tolerance levels.
  • the main manifestation is that the AUC level of OGTT is reduced and has the use of preventing or treating diabetes, indicating that the strain has the use of preventing or treating diabetes, especially diabetes caused by obesity, type II diabetes and/or non-alcoholic fatty liver disease/non-alcoholic Diabetes in patients with steatohepatitis.
  • mice were BKS-Leprem2Cd479/Gpt(db/db) mice, 5-6 weeks old, 24 in total, purchased from Guangdong Yaokang Biotechnology Co., Ltd.
  • Test strain After thawing the MNH05026 strain glycerol cryovial at 37°C, inoculate it on the M01 plate in an anaerobic workstation for activation, inoculate the activated strain into the M01 liquid medium, and culture it anaerobically. , obtain a sufficient amount of culture, centrifuge and concentrate the cultured bacterial liquid, and then resuspend the bacteria in a solvent to obtain a test substance whose purity and viable bacterial count (6.3 ⁇ 10 9 CFU/mL) meet the requirements of animal experiments.
  • MNH27256 was activated and cultured using the same method as MNH05026 to obtain a test substance whose purity and viable bacterial count (6.1 ⁇ 10 9 CFU/mL) met the requirements for animal experiments.
  • Negative control substance Use PBS containing 0.05% L-Cys HCl as a negative control substance
  • MNH27256 uses the same methods as 1) to 4) above to perform relevant experimental settings.
  • mice were randomly divided into 3 groups according to body weight, 8 mice/group. They are: negative control group (HFD-Control), positive control group (Metformin), combined medication group (MNH05026+Metformin, MNH27256+Metformin). Administration was started after grouping (D1). The negative control group was given negative control substance, the positive control group was given Metformin (250mg/kg), and the combined drug group was given corresponding bacterial solution (0.2mL/mouse) and Metformin (250mg).
  • mice were fed the same size Rats were maintained on a diet, with free access to water and food, using a 12h/12h day-night cycle.
  • An OGTT measurement was performed on the 27th day of drug administration (D27), and a fasting blood glucose was measured on the 28th day of drug administration (D28).
  • D27 27th day of drug administration
  • D28 28th day of drug administration
  • D29 anatomical specimens were collected from all groups, and the data were summarized and analyzed for fasting blood glucose and OGTT. Waiting for data results.
  • Oral glucose tolerance test On the 27th day of administration (D27), measure the OGTT after fasting for 12 hours (such as fasting from 20:30:00 in the evening to 08:30:00 the next day). Weigh the fasting body weight of the mice, and administer glucose according to the fasting body weight of the mice. The dose of glucose administered is 2g/kg (glucose g/mouse fasting body weight kg). Measure fasting blood glucose and blood glucose values 15min, 30min, 60min, 90min, and 120min after sugar administration. Each mouse was timed strictly and the blood glucose value was measured accurately at 6 time points.
  • mice used in this experiment carry mutations in the Leptin gene, thus showing symptoms of type II diabetes.
  • DB/DB mice with BKS background are susceptible to diabetes, develop severe symptoms of diabetes, and suffer severe damage to pancreatic islet B cells.
  • point mutations in the leptin receptor lead to disorders of the leptin signaling pathway, which can also lead to obesity, insulin resistance, hyperglycemia, fatty liver and other symptoms in mice.
  • the oral glucose tolerance test is a glucose load test used to understand the function of pancreatic beta cells and the body's ability to regulate blood sugar. It observes the patient's ability to tolerate glucose and is currently a recognized diagnostic indicator for the diagnosis of diabetes.
  • glucose metabolism is disordered, blood sugar rises sharply after oral administration of a certain amount of glucose, or the rise is not obvious, but cannot be reduced to the fasting level or the original level within a short period of time.
  • Figure 13 shows the effect of MNH05026 combined with Metformin on oral glucose tolerance (OGTT) in type II diabetic mice.
  • Figure 13A shows the effect of MNH05026 combined with Metformin on the oral glucose tolerance (OGTT) of type II diabetic mice.
  • Figure 13B shows the area under the oral glucose tolerance curve results of experimental mice in each treatment group;
  • Figure 13A shows the test results of the improvement effect of MNH05026 in combination with Metformin on oral glucose tolerance in type II diabetic mice.
  • Figure 13B shows the area under the oral glucose tolerance curve results of mice in each experimental group (data adopts mean ⁇ standard deviation (Mean ⁇ SD) shows that the statistical analysis uses the One-Way ANOVA method and Dunnett's multiple comparisons test (*p ⁇ 0.05). The results show that the combination of strain MNH05026 and Metformin is more effective in improving blood sugar elevation than using Metformin alone, and more significantly improves glucose tolerance. The main manifestation is that the blood sugar values measured at 5 time points in the combined group after sugar administration were all low.
  • the blood glucose value of the combination group was significantly reduced, and finally the AUC level was significantly reduced.
  • the combination of MNH05026 and Metformin can better control blood sugar and prevent and control diabetes, especially type II diabetes.
  • this experiment also shows that the combination of MNH05026 and Metformin can effectively improve insulin resistance and improve glucose tolerance, which is useful for preventing or treating type II diabetes. Diabetes can achieve better results and is useful in preventing or treating diabetes, especially diabetes caused by obesity, type II diabetes and/or diabetes in patients with fatty liver/steatohepatitis.
  • Figure 14 shows the results of the effects of MNH05026 and MNH27256 combined with Metformin on fasting blood glucose in type II diabetic mice;
  • Figure 14 shows the effect of the combination of MNH05026 and Metformin on fasting blood glucose, and the effect of the combination of MNH27256 and Metformin on fasting blood glucose.
  • the data are displayed as mean ⁇ SD.
  • Statistical analysis was performed using the One-Way ANOVA method and Dunnett's multiple comparisons test. **p ⁇ 0.01.
  • Metformin is an AMPK agonist, which plays a role in lowering blood sugar and treating diabetes by promoting the secretion of GLP-1. This shows that the strain MNH05026 of the present application can promote the therapeutic effect of Metformin and enhance the promotion of GLP-1. Secreted to achieve the effect of lowering blood sugar and treating diabetes.
  • this experiment further used MNH27256 alone and in combination with Metform to treat hyperglycemia in type II diabetic mice to verify the effect of MNH27256 on preventing or treating diabetes.
  • This invention has passed the ethical review and supervision of the Munn Biotech Laboratory Animal Care and Use Committee (IACUC).
  • mice were C57BL/6J mice, 5-6 weeks old, 54 in total, purchased from Guangdong Yaokang Biotechnology Co., Ltd.
  • Test strain After thawing the MNH27256 strain glycerol cryovial at 37°C, inoculate it on the M01 plate in an anaerobic workstation for activation, inoculate the activated strain into the M01 liquid medium, and culture it anaerobically. , obtain a sufficient amount of culture, centrifuge and concentrate the cultured bacterial liquid, and then resuspend the bacteria in a solvent to obtain a test substance whose purity and viable bacterial count (1.25 ⁇ 10 10 CFU/mL) meet the requirements of animal experiments.
  • Negative control substance Use PBS-Cys containing 25% glycerol as a negative control substance
  • mice 54 C57BL/6J male mice aged 5-6 weeks were fed a high-fat diet for 6 weeks after the quarantine period, and then intraperitoneally injected (i.p.) STZ for 8 consecutive days starting on the 43rd day, 1 dose per day. Second-rate. On D43, all mice were intraperitoneally injected (i.p.) with STZ injection at a dose of 50 mg/kg, and from D44 to D50, all mice were intraperitoneally injected (i.p.) with STZ injection at a dose of 25 mg/kg. Body weight and random blood glucose were measured on D44, D46, D48, and D50, and fasting blood glucose of animals fasted for 12 hours was measured on D49 and D51.
  • mice with qualified blood glucose values were selected and transferred to subsequent intervention trials. Randomly stratified and grouped according to fasting blood glucose, 8 animals/group, divided into 4 groups: negative control group (Control), positive control group (Metformin), MNH27256 group, and combined medication group (MNH27256+Metformin). Administration was started after grouping (D1).
  • the negative control group was given negative control substance
  • the positive control group was given Metformin (250mg/kg)
  • the MNH27256 group was given MNH27256 bacterial liquid (0.2mL/mouse)
  • the combined drug group was given With MNH27256 bacterial liquid (0.2mL/mouse) and Metformin (250mg/kg), MNH27256 bacterial liquid is administered twice a day, and Metformin is administered once a day.
  • the administration method is intragastric administration.
  • the drug was administered for 28 days. During the experiment, the mice were fed high-fat feed throughout the experiment, with free access to water and food, and a 12h/12h day and night cycle. Fasting blood glucose data were measured once a week during the administration period.
  • Figure 15A is a graph showing changes in fasting blood glucose of type II diabetic mice using MNH27256 alone and in combination with Metformin;
  • Figure 15A is a fasting blood glucose change curve
  • Figure 15B is the fasting blood glucose value at the intervention endpoint.
  • MNH27256 has the effect of reducing fasting blood sugar when used alone
  • MNH27256 has the effect of reducing fasting blood sugar when used in combination with Metformin. It has a more obvious blood sugar lowering effect.
  • the combination of MNH27256 and Metformin can obviously promote the blood sugar-lowering effect of Metformin.
  • MNH27256 has a blood sugar-lowering effect comparable to that of Metformin, and the blood sugar value at the end of use is also comparable to that of the Metformin group.
  • MNH27256 alone is not as good as Metformin, it has value in long-term blood sugar control, and strain MNH27256 can promote the therapeutic effect of Metformin by further promoting the secretion of GLP-1, achieving blood sugar lowering and treatment Effects of diabetes. It shows that MNH27256 alone or in combination with Metformin has the effect of reducing fasting blood sugar, and can be used to prevent or treat diabetes.
  • Example 8 The performance of strains MNH05026, MNH22004 and MNH27256 in high-fat diet-induced obesity mouse model In vivo experiments to verify the application of corresponding strains in the treatment and prevention of obesity and related diseases
  • mice 5-6 week old C57BL/6J male mice were fed a high-fat diet for 10 weeks after the quarantine period. 16 mice with a weight range of 35.50g-44.49g were selected and randomly selected according to weight. Stratified grouping, 8 animals/group, divided into 2 groups (experimental group and control group). Administration began after grouping (D1). The experimental group was administered MNH05026 bacterial solution (0.2 mL/mouse), and the control group was administered negative control substance twice a day for a total of 21 days. During the experiment, the mice were free Drinking water and food intake adopt a 12h/12h day and night cycle. During the trial, a general clinical observation was conducted after each virtual administration or administration.
  • the weight of the animals was measured every Monday and Thursday, on the day of the start of dosing (D1), and on the 21st day of dosing (D21); the weight of the animals was weighed before anatomy at the end of the test.
  • the end point of this trial is the day after the end of drug administration (D22).
  • the end point of the trial is to conduct anatomy and sample collection according to the protocol.
  • the data are summarized and analyzed for body weight and weight change percentage, fasting blood glucose measurement results, various anatomy data results, and serum test data results. All data are expressed in the form of Mean ⁇ SD, and graphed and statistically analyzed using GraphPad Prism 8.0.2 software.
  • the t test (Student’s t test) analysis method was used. No significance is not indicated; significant differences are indicated by *, *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001.
  • Figure 16 shows the weight gain of strains MNH05026, MNH27256 and MNH22004 on high-fat diet-induced obese mice
  • Figure 16A shows the effect of MNH05026 and MNH22004 on weight gain (i.e. changes in weight gain/loss) of obese model mice.
  • Figure 16C shows the corresponding mouse weight gain value at the end of the experiment (21 days). The results showed that compared with the control group, both MNH05026 and MNH22004 could significantly reduce the weight gain of high-fat diet-induced obese mice, achieving the purpose of controlling weight and preventing excessive weight gain; and within the first 7 days after administration, MNH05026 and MNH22004 can slightly reduce the weight of obese mice to a certain extent.
  • Figure 16B is a graph showing the effect of MNH27256 on the weight gain of obese mice.
  • MNH05026 can stably control the weight of obese mice during the entire experimental period (a total of 21 days) after administration. At the end of the experiment, the weight of the mice did not change. There was a significant increase, and the overall body weight of the mice decreased to varying degrees during the entire experimental period compared with before administration, with the greatest degree of weight loss on the 9th day after administration, which was about 2.6%.
  • MNH05026, MNH22004 and MNH27256 can significantly inhibit the weight gain caused by high-fat diet, and MNH27256 can also reduce the weight of obese mice to a certain extent, improve the weight gain caused by high-fat diet, and has the potential to prevent and treat obesity. Effect.
  • Example 9 Strains MNH05026, MNH22004, and MNH27256 were respectively used in combination with Semaglutide in high-fat and high-fat diets. In vivo test of NAFLD/NASH mouse model induced by sugar-high cholesterol diet to verify its effectiveness in treatment and prevention Applications in NAFLD/NASH and related diseases
  • Experimental mice are C57BL/6J male mice, 5-6 weeks old, 24 in total, purchased from Guangdong Yaokang Biological Technology Co., Ltd.
  • Test strains After thawing the glycerol cryovials of MNH05026, MNH22004 and MNH27256 strains at 37°C, inoculate them on M01 plates in an anaerobic workstation for activation, and inoculate the activated strains into M01 liquid culture medium Medium, anaerobically culture, obtain a sufficient number of cultures, centrifuge and concentrate the cultured bacterial liquid, and re-suspend the bacteria with a solvent to obtain a subject with a purity and viable bacterial count (1.5 ⁇ 10 9 CFU/mL) that meets the requirements of animal experiments. test object.
  • Control substance Use PBS-Cys containing 25% glycerol as a negative control substance; use Semaglutide as a positive control substance.
  • mice 24 5-6 week old C57BL/6J male mice were fed a high-fat, high-sugar and high-cholesterol diet for 26 weeks after the quarantine period. They were randomly divided into stratified groups according to body weight, 8 mice/group. Divided into 3 groups, namely negative control group (Control), positive control group (Semaglutide) and combined treatment group (corresponding strain + Semaglutide). Administration was started after grouping (D1).
  • the negative control group was given negative control substance (PBS-Cys), which was administered by gavage, 0.2 mL/animal, twice a day; the positive control group was given Semaglutide, which was administered by subcutaneous injection, with a dose of 30 nmol.
  • mice had free access to water and food, and a 12h/12h day-night cycle was adopted.
  • a general clinical observation was conducted after each dose. The weight of the animals was measured twice a week during the dosing period, and before autopsy at the end of the test. The end point of this trial is the day after the end of drug administration (D61).
  • the end point of the trial is anatomy and collection of materials according to the protocol, and the results of data analysis are summarized.
  • Figure 17 shows the body weight changes of strains MNH05026, MNH22004, and MNH27256 respectively combined with Semaglutide in NAFLD/NASH mice induced by a high-fat, high-sugar, high-cholesterol diet;
  • Figures 17A-17C and Table 13 The results of the combined use of Macrococcus and Semaglutide on the body weight of NAFLD/NASH mice induced by a high-fat, high-sugar, high-cholesterol diet are shown in Figures 17A-17C and Table 13.
  • Figure 17A shows the changes in body weight of NAFLD/NASH mice induced by a high-fat, high-sugar, high-cholesterol diet when the Megacoccus strains MNH05026, MNH22004, and MNH27256 were combined with Semaglutide respectively.
  • Figure 17B shows the changes in the body weight of the Megacoccus strains MNH05026, MNH22004, The weight values of mice on day 60 when MNH27256 was combined with Semaglutide respectively.
  • Figure 17C shows the weight gain of mice when MNH22004 was combined with Semaglutide.
  • Table 13 shows some of the data extracted from Figure 17A.
  • Semaglutide is a GLP-1 receptor agonist and is a commonly used weight loss drug in clinical practice. Combining Figure 17A, Figure 17B and Table 13, it can be found that the Semaglutide treatment group can quickly reduce weight in the early stage of taking it, and the effect is more obvious.
  • the body begins to develop a certain degree of resistance to Semaglutide. The mice begin to regain weight between 30 and 60 days and the rate of rebound is higher than that of the combined group.
  • Semaglutide The weight gain value of the mice in the treatment group was -21.30%, and the corresponding gain values for MNH05026, MNH22004, and MNH27256 were -21.56%, -25.60%, and -24.02%, that is, the MNH05026, MNH27256, and MNH22004 combined with Semaglutide groups on day 33
  • the weight gain of obese mice can be further reduced, that is, the combined use group begins to show better weight control effects than Semaglutide alone. Megacoccus sp.
  • Strain MNH05026, MNH27256, and MNH22004 can reduce the body's resistance to Semaglutide to a certain extent when used for weight loss, and can improve the therapeutic effect of Semaglutide in the treatment of obesity.
  • the combination of Megacoccus strains MNH05026, MNH22004, and MNH27256 with Semaglutide has the effect of preventing and treating obesity in obese patients.
  • Figure 18A is a graph showing the effects of MNH05026, MNH22004 and MNH27256 combined with Semaglutide on liver weight in mice with NAFLD/NASH induced by a high-fat, high-sugar, high-cholesterol diet;
  • FIGS 18A-18D The results of the combined use of Macrococcus and Semaglutide on liver weight and liver function damage in NAFLD/NASH mice induced by a high-fat, high-sugar, high-cholesterol diet are shown in Figures 18A-18D.
  • the results in Figure 18A show that the combination of MNH05026, MNH22004, MNH27256 and Semaglutide can significantly reduce the liver weight of NAFLD/NASH model mice induced by a high-fat, high-sugar, high-cholesterol diet, which is more effective than the positive control substance Semaglutide. It shows that the combined use of Megacoccus and Semaglutide can improve the prevention and treatment effect of Semaglutide on liver function damage and better improve liver function.
  • the combination of MNH05026 and Semaglutide can relatively more significantly reduce the liver function of NAFLD/NASH model mice induced by a high-fat, high-sugar, high-cholesterol diet, and has better prevention and Effectiveness in treating liver damage.
  • ALT and AST are markers of liver cell damage
  • alkaline phosphatase (ALP) is an enzyme mainly distributed in the liver and is distributed On the microvilli on the sinusoidal side and capillary bile duct side of liver cells, bile is discharged into the small intestine. When bile is not discharged smoothly and the pressure in the capillary bile duct increases, a large amount of alkaline phosphatase will be produced. Alkaline phosphatase is high.
  • ALT, AST, and ALP reflect liver disease caused by liver cell damage, cholestasis, or both.
  • Figure 18C and Figure 18D show that the combination of MNH05026, MNH22004, MNH27256 and Semaglutide can significantly reduce aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) in serum.
  • AST aspartate aminotransferase
  • ALT alanine aminotransferase
  • ALP alkaline phosphatase
  • strains MNH05026, MNH22004, and MNH27256 of this application can be used in combination with Semaglutide to prevent or treat liver damage, prevent or treat chronic hepatitis, cirrhosis, or alcoholic hepatitis, prevent or treat Intrahepatic cholestasis, prevent or treat extrahepatic cholestasis; have better effect on improving liver function in patients with NAFLD/NASH; strains MNH05026, MNH22004, MNH27256 combined with Semaglutide have the effect of preventing or treating fatty liver in patients with NAFLD/NASH , improve liver function in NAFLD/NASH patients, prevent or treat liver damage in NAFLD/NASH patients, prevent or treat intrahepatic cholestasis in NAFLD/NASH patients, and prevent or treat extrahepatic cholestasis in NAFLD/NASH patients.
  • Oral glucose tolerance is used to measure pancreatic beta cell function and the body's ability to regulate blood sugar. It is currently a recognized diagnostic indicator for the diagnosis of diabetes. When glucose metabolism is disordered, blood sugar rises sharply after oral administration of a certain amount of glucose, or the rise is not obvious, but cannot be reduced to the fasting level or the original level within a short period of time. This is abnormal glucose tolerance or impaired glucose tolerance; abnormal glucose tolerance indicates The body's ability to metabolize glucose is reduced, which is common in type II diabetes and obesity.
  • Figure 19 shows the effect of MNH05026, MNH22004 and MNH27256 combined with Semaglutide on the oral glucose tolerance of the NAFLD/NASH mouse model induced by a high-fat, high-sugar, high-cholesterol diet;
  • Figure 19E shows that the use of Semaglutide alone or the combination of MNH27256 and Semaglutide has no significance in reducing fasting blood glucose in NAFLD/NASH mice, but the combination of MNH05026 and MNH22004 with Semaglutide has a significant effect in reducing the fasting blood glucose in NAFLD/NASH mice, and MNH05026 is more significant.
  • the combination of MNH05026, MNH22004, and MNH27256 with Semaglutide can further promote the control level of Semaglutide on the OGTT and FBG of NAFLD/NASH model mice induced by high-fat, high-sugar, and high-cholesterol diet, and more significantly reduce their OGTT and FBG. It can better prevent and treat diabetes in patients with NAFLD/NASH, improve blood sugar control function, and can also be used for type II diabetes and diabetes caused by obesity.
  • MNH05026 has the best control effect in combination with Semaglutide
  • MNH22004 has the second best control effect
  • MNH27256 has the weakest control effect. Products with different dosages can be developed for strains with different control effects. patients with different disease courses.
  • Figure 20 respectively shows the effects of MNH05026, MNH22004 and MNH27256 combined with Semaglutide on the subcutaneous fat weight (subcutaneous fat pad), inguinal fat pad (Inguinal fat pad) and brown fat of the NAFLD/NASH mouse model induced by a high-fat, high-sugar and high-cholesterol diet.
  • MNH05026, MNH22004, and MNH27256 with Semaglutide can significantly reduce local subcutaneous fat, inguinal fat, epididymal fat, and brown fat.
  • MNH05026 has the best overall control effect, followed by MNH27256, and MNH22004 is the weakest. That is to say, the application of megacoccus MNH05026, MNH22004, MNH27256 combined with Semaglutide can significantly reduce the fat accumulation in NAFLD/NASH model mice induced by high-fat, high-sugar, and high-cholesterol diet, which is better or equivalent to the effect of the positive control substance Semaglutide. It has an ideal effect in preventing and treating obesity and fat accumulation in NAFLD/NASH patients.
  • triglyceride triglyceride
  • HDL total cholesterol
  • LDL low-density lipoprotein
  • TG mainly participates in energy metabolism in the human body and produces heat energy. Excessive TG content in the blood can cause blood viscosity, causing lipids to deposit on the blood vessel walls, gradually forming small plaques, which is atherosclerosis.
  • Increased LDL-C is a major and independent risk factor for the occurrence and development of atherosclerosis; the increased level of LDL-C is also an indicator of coronary heart disease. Because HDL-C can transport cholesterol in the blood vessel wall to the liver for catabolism (ie, reverse cholesterol transport), it can reduce the deposition of cholesterol in the blood vessel wall and play an anti-atherosclerotic role. The results are shown in Figures 21A-21D.
  • Figure 21 shows the effect of MNH05026 combined with Semaglutide on triglyceride (TG), total cholesterol (CHO), high-density lipoprotein (HDL), low-density lipoprotein ( LDL);
  • strain MNH05026 and Semaglutide can significantly reduce the three blood lipids of TG, CHO and HDL in NAFLD/NASH model mice induced by high-fat, high-sugar and high-cholesterol diet. It shows that the combination of strain MNH05026 and Semaglutide can reduce the total cholesterol, triglyceride and low-density lipoprotein content in the serum of obese mice to varying degrees, and has the potential to prevent and treat cardiovascular disease, atherosclerosis, coronary heart disease, and reduce The effect of blood lipids also shows that the combination of strain MNH05026 and Semaglutide has the effect of treating/preventing cardiovascular and cerebrovascular diseases.
  • strain MNH05026 and Semaglutide can significantly reduce the low-density lipoprotein content in the serum of mice on a high-fat diet, and has the ability to prevent or prevent cardiovascular and cerebrovascular diseases. Used to treat hyperlipidemia and atherosclerosis. It can further reduce serum total cholesterol, suggesting that the combination of strain MNH05026 and Semaglutide can prevent and treat cardiovascular diseases, lower blood lipids, treat/prevent cardiovascular and cerebrovascular diseases, and prevent or treat atherosclerosis, coronary heart disease, Hyperlipidemia uses.
  • Figure 22 shows the effect of MNH05026, MNH22004 and MNH27256 in combination with Semaglutide on liver steatosis (Steatosis), liver lobular inflammation (Lobular inflammation) and liver ballooning (Ballooning) in NAFLD/NASH mice induced by a high-fat, high-sugar and high-cholesterol diet. And the impact map of liver NAS score and fibrosis;
  • the results of Figure 22B show that the combination of MNH05026, MNH22004, MNH27256 and Semaglutide can significantly reduce liver lobular inflammation (Lobular inflammation) in NAFLD/NASH model mice induced by a high-fat, high-sugar, high-cholesterol diet, and is more effective than the positive control substance Semaglutide.
  • the results in Figure 22C show that the combination of MNH05026, MNH22004, MNH27256 and Semaglutide can significantly reduce liver ballooning in NAFLD/NASH model mice induced by a high-fat, high-sugar, high-cholesterol diet, which is more effective than the positive control substance Semaglutide.
  • the results in Figure 22D show that the combination of MNH05026, MNH22004, MNH27256 and Semaglutide can significantly reduce the liver NAS score (NAS score) of NAFLD/NASH model mice induced by a high-fat, high-sugar, high-cholesterol diet, which is more effective than the positive control substance Semaglutide.
  • the results of Figure 22E show that the combination of MNH05026, MNH27256 and Semaglutide can reduce the liver fibrosis score (Fibrosis) of NAFLD/NASH model mice induced by a high-fat, high-sugar, high-cholesterol diet, which is more effective than the positive control substance Semaglutide.
  • the above data shows that the combination of Megacoccus and Semaglutide can improve the prevention and treatment of liver steatosis, liver lobular inflammation, liver ballooning, liver NAS score and fibrosis by Semaglutide. effect and has the potential to treat NAFLD/NASH.
  • Example 10 The effects of strains MNH22004 and MNH05026 on histone deacetylases (Histone deacetylases, HDAC) activity inhibition
  • MNH22004 and MNH27256 are different strains of the same species, so MNH22004 was selected as a representative and MNH05026 was selected to verify whether the Megacoccus strain has an inhibitory effect on histone deacetylase activity.
  • This study used the purchased HDAC Inhibitor Drug Screening Kit from Abcam Company ( Fluorometric) kit for in vitro detection of HDAC activity inhibition.
  • MNH22004 culture supernatant Inoculate strain MNH22004 into liquid culture medium, culture anaerobically at 37°C for 48 hours, centrifuge to remove bacterial cells, filter the culture supernatant with a 0.22 ⁇ m filter, aliquot, and freeze the collection at -80°C. ,stand-by.
  • MNH05026 culture supernatant inoculate strain MNH05026 into liquid culture medium, culture anaerobically at 37°C for 48 hours, centrifuge to remove bacterial cells, filter the culture supernatant with a 0.22 ⁇ m filter, aliquot, and freeze the collection at -80°C. ,stand-by.
  • Test sample preparation 1) Control group, MM01 culture medium was diluted 10 times with PBS to obtain MM01 containing 10%; 2) Positive control group: TSA, an inhibitor of HDAC, was diluted with PBS to a final concentration of 40 ⁇ M; 3) MNH22004 Group, MNH22004 culture supernatant was diluted 10 times with PBS to obtain experimental samples containing 10% bacterial supernatant. 4) MNH05026 group, the MNH05026 culture supernatant was diluted 10 times with PBS to obtain an experimental sample containing 10% bacterial supernatant.
  • Preparation of HDACs detection reaction reagents Prepare an appropriate amount of detection reaction system according to the kit instructions. Each reaction requires 50 ⁇ L reaction reagents.
  • Diabetes is a group of diseases in which low levels of insulin and/or peripheral insulin resistance lead to hyperglycemia.
  • Inhibiting HDAC activity has been proposed to treat diabetes, including inhibiting Pdxl by inhibiting HDAC activity (Park et al., 2008, J Clin Invest, 118, 2316-24) and enhancing the expression of the transcription factor Ngn3 to increase the endocrine pool.
  • Progenitor cells Haumaitre et al., 2008, Mol Cell Biol, 28, 6373-83
  • enhanced insulin expression Molsey et al., 2003, J Biol Chem, 278, 19660-6), etc.
  • HDAC inhibition is also a promising treatment for advanced diabetic complications such as diabetic nephropathy and retinal ischemia (Christensen et al., 2011, Mol Med, 17(5-6), 370-390). Therefore, the compositions of the present invention can be used to treat or prevent diabetes mediated by HDAC activity.
  • HDAC histone deacetylase
  • VPA valproic acid
  • Histone deacetylase inhibitors have received increasing attention for their role in improving pancreatic ⁇ -cell function, protecting them from inflammatory factors, improving peripheral tissue insulin resistance, regulating the immune system to improve metabolism, and slowing down diabetic complications. , is expected to become a new drug for the prevention and treatment of diabetes and even gestational diabetes.
  • composition of the present invention is used to treat or prevent diabetes.
  • the compositions of the invention are used to treat or prevent type I diabetes.
  • the compositions of the invention are used to treat or prevent type II diabetes.
  • compositions of the invention are used to treat or prevent diabetes, wherein said treatment or prevention is accomplished by reducing or preventing HDAC activation.
  • the composition of the present invention has HDAC inhibitory activity.
  • the HDACS inhibitory activity it can be used for neurological diseases and inflammatory bowel diseases such as IBD, cardiovascular diseases (HDAC inhibition also results in beneficial outcomes in various types of neurodegenerative diseases, inflammation disorders, and cardiovascular diseases.
  • HDAC and HDAC Inhibitor From Cancer to Cardiovascular Diseases, https://pubmed.ncbi.nlm.nih.gov/26865995/), it can be determined that the strain of the present invention can also be used to treat the nervous system mediated by HDAC activity diseases, as well as inflammatory bowel diseases such as IBD, cardiovascular disease.

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Abstract

提供了保藏于广东省微生物菌种保藏中心，保藏编号分别为GDMCC No:62001、GDMCC No：62000、GDMCC No：61999的巨球型菌属（Megasphaera sp.）菌株，所述菌株的16S rRNA分别如SEQ ID NOs：1-3所示，所述细菌菌株高产丁酸和/或乙酸，还提供了所述菌株在制备预防和/或治疗代谢疾病的药物中的用途。

Description

药物组合物、巨球型菌及其应用
相关申请
本申请要求于2022年4月28日提交的专利申请号为202210470811.2、2022年4月28日提交的专利申请号为202210471327.1、2022年4月28日提交的专利申请号为202210471322.9、2023年1月9日提交的专利申请号为202310029400.4、2023年1月9日提交的专利申请号为202310029410.8以及2023年1月9日提交的专利申请号为202310029842.9的优先权，以上专利申请的公开内容以引用方式全文并入本文。
技术领域
本发明涉及生物医药领域，尤其是涉及一种药物组合物、巨球型菌及其应用。
背景技术
肠道微生物组由来自数万亿细菌、古细菌、真菌、病毒和其他微真核定殖者的集体基因组和功能物质组成，已被广泛认为是“第二大脑”，在调节人类健康和疾病发展以及临床实践中发挥多种功能。肠道是人体内最大的微生态环境，参与人体的营养吸收、能量代谢、组织器官发育、免疫防御和内分泌调控等等多种重要的生理过程。人体肠道内有大量的共生微生物，其携带的基因信息总量为人自身基因组信息的50-100倍，即“肠道微生物组(gut micorbiome)”。肠道微生物组是人体最大、最直接的外环境，对维持人体的健康发挥较为重要的作用。越来越多的证据揭示了特定肠道微生物、通过微生物代谢活动释放产物以及宿主在操纵宿主免疫、代谢和癌症发展中的保护性相互作用。随着分子生物学、基因组学、生物信息分析技术、高通量测序技术及微生物培养技术等技术的高速发展，肠道菌群对肠道和肠道外疾病的影响及作用越来越明确。基于微生物组的疗法，许多不同的细菌物种可以影响人体消化系统、循环系统和神经系统，与人类各种疾病密切相关，包括癌症、感染、胃肠道炎症、自身免疫、代谢紊乱、中枢神经系统和精神疾病。研究肠道菌群与人体健康和疾病的相互关系，不仅是重要的科学研究，更具有临床诊断、治疗、乃至转化的重要意义和价值。
目前，已经有关于肠道微生物组在营养障碍、代谢异常及复杂疾病(比如肥胖、糖尿病、炎性肠病及代谢类等)方面的研究报道，但目前研究的微生物种类仅涉及到特定的极少数物种。如在治疗肥胖相关疾病时，肠道微生物中通常集中在双歧杆菌属、拟杆菌属和乳杆菌属等益生菌领域，而其他肠道微生物还有待开发形成活菌药物用于代谢类疾病的治疗或预防。
巨球型菌属主要存在于人类或动物肠道中，属于严格厌氧的微生物，并且对营养及培养环境要求极高，生长周期长。另外，在进行药效验证的时候，该菌属的细菌因为厌氧而难以通过体外细胞实验进行验证；在动物体内实验过程中，该菌属的细菌由于培养困难、厌氧程度高而很难保持稳定的活菌数，且存在重复性不足的问题。这些体内外相关的技术难题均限制了巨球型菌属新菌种的发现及应用。
另外，现有技术中如益生菌领域或者FMT菌群移植，通常是利用多种微生物混合作用于肠道，也有现有技术通过多种细菌混合作为治疗代谢疾病的药物。然而多细菌作用于适应症的机理更为复杂，细菌之间的彼此影响也未有成熟的研究；作为活菌药物而 言，多细菌的使用会打破肠道菌群的稳态，相比较而言，单菌对肠道菌群的稳态影响较小。此外，混合细菌的使用，还需要额外考虑细菌是否会影响彼此的活性，同时也无法明确混合细菌中的具体那种细菌能直接产生疗效。
微生物的SCFA已被现有技术充分研究用于治疗或预防肥胖、糖尿病等代谢疾病。现有技术中“Gut microbial metabolites in obesity,NAFLD and T2DM”表明：与体重控制有关的碳水化合物发酵产生的代谢物包括乙酸，丙酸，丁酸，琥珀酸；乙酸盐和丁酸盐也被证明通过中枢机制诱导饱腹感，并增加脂肪组织和肝脏的产热，以及诱导脂肪组织褐变和瘦素分泌。此外，乙酸、丙酸和丁酸以G蛋白偶联受体(GPR)依赖的方式刺激饱腹激素胰高血糖素样肽1(GLP1)和肽YY(PYY)的分泌。
2017年的一项小鼠研究表明，长期口服丁酸盐可预防饮食诱导的肥胖、NAFLD进展和胰岛素抵抗。这些效应主要与食物摄取量的减少有关，通过丁酸盐诱导的下丘脑表达神经肽Y的食欲神经元活性的抑制，以及脑干孤束核和迷走神经背侧复合体的神经活性的降低。此外，腹腔注射乙酸、丙酸和丁酸已被证明可以通过迷走神经传入刺激相关的机制抑制小鼠的能量摄入，丙酸酯和丁酸酯通过诱导肠道糖异生(IGN)来预防肥胖和胰岛素抵抗。
综上表明，菌株的SCFA中乙酸(乙酸盐)、丁酸(丁酸盐)、丙酸(丙酸盐)的产生能力能够表明其在肥胖、糖尿病、脂肪肝等中的治疗潜力。因此，进一步发现、挖掘和研究具有代谢类预防或治疗潜力的微生物菌株具有重要的应用价值和市场前景。
有鉴于此，特提出本发明。
发明内容
本发明的一个方面，涉及一种药物组合物，包括巨球型菌属(Megasphaera)的细菌；
所述巨球型菌属的细菌的16S rRNA的序列与SEQ的一致性≥95％；
所述SEQ包括SEQ ID No:1、SEQ ID No:2和SEQ ID No:3中的至少一个。
所述的组合物能够用于抑制食欲、防治和/或减轻代谢疾病；
优选地，所述抑制食欲包括：降低摄食量、降低食欲中的至少一种；
优选地，所述代谢疾病为肝脏类疾病、肥胖类、心血管疾病、心脑血管疾病、高血脂，糖尿病、葡萄糖耐量受损中的至少一类；
可选地，所述肝脏类疾病可选为脂肪肝、NAFLD、NASH、降低肝重量、肝功能异常中的至少一种；
可选地，所述肝脏类疾病包括高脂饮食引起的、高胆固醇饮食引起的、高糖饮食引起的、高脂高胆固醇引起的、高脂高糖引起的和/或高脂高胆固醇高糖引起的；
可选地，所述肥胖类疾病可选为高脂饮食引起的肥胖、高胆固醇引起的肥胖、高糖饮食引起的肥胖、高脂高胆固醇引起的肥胖、高脂高糖引起的肥胖、高脂高胆固醇高糖引起的肥胖、NAFLD/NASH患者的肥胖；
可选地，所述心血管疾病或心脑血管疾病可选为动脉粥样硬化和/或NAFLD/NASH患者的心血管疾病和/或NAFLD/NASH患者的心脑血管疾病和/或高胆固醇疾病；
所述肝脏类疾病包括高脂饮食、高胆固醇饮食、高糖饮食中的至少一种引起的肝脏类疾病或肝功能异常的疾病；进一步地，包括高脂饮食引起的、高胆固醇饮食引起的、高糖饮食引起的、高脂高胆固醇引起的、高脂高糖引起的和/或高脂高胆固醇高糖引起的 肝脏类或肝功能异常的疾病；
可选地，所述肥胖类疾病包括高脂饮食、高胆固醇饮食、高糖饮食中的至少一种引起的肥胖；进一步地，包括高脂饮食引起的肥胖、高胆固醇引起的肥胖、高糖饮食引起的肥胖、高脂高胆固醇引起的肥胖、高脂高糖引起的肥胖、高脂高胆固醇高糖引起的肥胖、NAFLD/NASH患者的肥胖；
可选地，所述心血管疾病或心脑血管疾病可选为动脉粥样硬化和/或NAFLD/NASH患者的心血管疾病和/或NAFLD/NASH患者的心脑血管疾病和/或高胆固醇疾病；
可选地，所述糖尿病包括肥胖引起的糖尿病、II型糖尿病、NAFLD/NASH患者的糖尿病；
可选地，所述糖尿病包括高脂饮食、高胆固醇饮食、高糖饮食中的至少一种引起的糖尿病；进一步地，包括高脂饮食引起的糖尿病、高胆固醇引起的糖尿病、高糖饮食引起的糖尿病、高脂高胆固醇引起的糖尿病、高脂高糖引起的糖尿病、高脂高胆固醇高糖引起的糖尿病、NAFLD/NASH患者的糖尿病。
优选地，所述代谢疾病为I型糖尿病、II型糖尿病、妊娠糖尿病、糖耐量受损、胰岛素抵抗、体重控制、超重、血糖控制、糖尿病前期、肥胖症、高血糖症、高胰岛素血症、脂肪肝、酒精性脂肪性肝炎、高胆固醇血症、高血压、高脂蛋白血症、高脂血症、高甘油三酯血症、尿毒症、酮症酸中毒、低血糖、血栓性疾病、血脂异常、非酒精性脂肪性肝病(NAFLD)、非酒精性脂肪性肝炎(NASH)、动脉粥样硬化、肾病、糖尿病性神经病、糖尿病性视网膜病、皮肤病、消化不良或水肿中的至少一种。
所述药物组合物还包括第二活性物质或联用药物，所述第二活性物质或联用药物包括GLP-1受体激动剂，GLP-1受体和GCG受体双重激动剂，GLP-1受体、GIP受体和GCG受体三重激动剂，AMPK激动剂或促进GLP-1分泌的活性药物、DPP-4受体抑制剂、PPAR受体激动剂、PPARα受体激动剂、PPARδ受体激动剂、PPARγ受体激动剂、PPARα/δ受体双重激动剂、PPARα/γ受体双重激动剂、PPARα/δ/γ受体三重激动剂、CRTC、PGC-1α、SREBP、FXR、FGF21、ASK1、THR-β、LXR、NF-κB、SNDRI、MC4R、PNLIP、MOA、DRI等机制/靶点中的至少一种活性药物；
优选地，所述GLP-1受体激动剂包括以下任意一种：Semaglutide、liraglutide、exenatide或beinaglutide；
优选地，所述AMPK激动剂或促进GLP-1分泌的活性药物包括Metformin、Semaglutide和/或liraglutide；
一些疾病或病症的发病机制的特征是微生物群稳定性降低。这种疾病和病症的例子是IBS、IBD、糖尿病(例如2型糖尿病)、过敏性疾病、自身免疫疾病和代谢疾病/病症。本发明的细菌菌株还能通过调节微生物群的稳定性来治疗或预防疾病。
本发明的另一个方面，还涉及所述的药物组合物或所述的细菌菌株或所述的巨球型菌属菌在制备治疗和/或预防疾病的药物中的应用；
优选地，所述疾病包括肿瘤、感染性疾病、代谢疾病、身免疫性疾病、炎性疾病或神经性疾病中的至少一种；
优选地，所述代谢疾病包括：肝脏类疾病、肥胖类、心血管疾病、心脑血管疾病、高血脂、糖尿病、葡萄糖耐量受损中的至少一种；
可选地，所述肝脏类疾病可选为脂肪肝、NAFLD、NASH、降低肝重量、肝功能异常中的至少一种；
可选地，所述肝脏类疾病包括高脂饮食引起的、高胆固醇饮食引起的、高糖饮食引起的、高脂高胆固醇引起的、高脂高糖引起的和/或高脂高胆固醇高糖引起的；
可选地，所述肥胖类疾病可选为高脂饮食引起的肥胖、高胆固醇引起的肥胖、高糖饮食引起的肥胖、高脂高胆固醇引起的肥胖、高脂高糖引起的肥胖、高脂高胆固醇高糖引起的肥胖、NAFLD/NASH患者的肥胖；
可选地，所述心血管疾病或心脑血管疾病可选为动脉粥样硬化和/或NAFLD/NASH患者的心血管疾病和/或NAFLD/NASH患者的心脑血管疾病和/或高胆固醇疾病；
可选地，所述糖尿病包括肥胖引起的糖尿病、II型糖尿病、NAFLD/NASH患者的糖尿病；
可选地，所述糖尿病可选为高脂饮食引起的糖尿病、高胆固醇引起的糖尿病、高糖饮食引起的糖尿病、高脂高胆固醇引起的糖尿病、高脂高糖引起的糖尿病、高脂高胆固醇高糖引起的糖尿病、NAFLD/NASH患者的糖尿病。
优选地，所述巨球型菌属的细菌的16S rRNA的序列与SEQ的一致性≥95％；
所述SEQ包括SEQ ID No:1、SEQ ID No:2和SEQ ID No:3中的至少一个。
与现有技术相比，本发明的有益效果为：
本发明从人体肠道中分离、筛选出多株属于巨球型菌属(Megasphaera)的厌氧菌株，属于天然菌株(非克隆和加工过的菌株)；通过鉴定，确定其属于Megasphaera属下的新物种；该多株菌株都能表达EC2.8.3.9酶，产丁酸通路完整性能至少达到70％以上，能够有效产丁酸及其他一些SCFA，有效防治代谢疾病。
本发明利用Megasphaera菌属在产丁酸上相似的共性，进一步通过基因和丁酸通路分析，进行菌株的候选药物筛选，提高筛选效率，同时筛选出的菌株经过验证都能能够有效用于代谢疾病的防治；筛选到的细菌菌株可通过调节短链脂肪酸/短链脂肪酸盐来抑制抑制组蛋白乙酰酶活性达到防治代谢疾病的目的；
本发明所提供的微生物制剂，含有所述巨球型菌或所述巨球型菌的代谢产物，具有防治代谢疾病的功效。本发明所提供的预防和/或治疗防治代谢疾病的药物，包含所述的从人体肠道中分离、筛选的巨球型菌或所述巨球型菌的代谢产物，能够用于疾病的治疗，同时具有较低的副作用。
本发明所提供的药物组合物，将所述的含有巨球型菌的药物与治疗代谢疾病的其他药物联用，能够达到较好的代谢疾病治疗效果。本发明提供的药物组合物，能够调节至少一种短链脂肪酸或调节乳酸盐，用于代谢疾病的治疗。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为菌株MNH 05026、MNH22004和MNH27256在厌氧血平板培养48h菌落形态；
图2为菌株MNH 05026、MNH22004和MNH27256的显微形态；
图3为菌株MNH 05026、MNH22004和MNH27256革兰氏染色显微形态；
图4为菌株MNH 05026、MNH22004和MNH27256芽孢染色显微形态；
图5为菌株MNH 05026、MNH22004和MNH27256的不同浓度NaCl耐受性的结果；
图6为菌株MNH 05026、MNH22004和MNH27256的不同pH耐受性的结果；
图7为菌株MNH 05026、MNH22004和MNH27256的不同浓度胆盐耐受性的结果；
图8为菌株MNH 05026在培养基API 20中培养的结果；
图9A为菌株MNH 05026的16S rRNA基因系统发育树；
图9B为菌株MNH 22004的16S rRNA基因系统发育树；
图9C为菌株MNH 27256的16S rRNA基因系统发育树；
图10为菌株MNH05026、MNH22004对高脂饮食诱导肥胖小鼠肝脏重量的影响图；
图11为菌株MNH05026对高脂饮食诱导肥胖小鼠血清中谷丙转氨酶(ALT)含量的影响图；
图12为菌株MNH05026和MNH27256对高脂饮食诱导肥胖小鼠空腹血糖(FGB)的影响，
图13为MNH05026与Metformin联用对II型糖尿病小鼠的口服葡萄糖耐量(OGTT)的影响结果；其中，图13中(A)为MNH05026与Metformin联用对II型糖尿病小鼠的口服葡萄糖耐量(OGTT)的影响结果，图13中(B)为各处理组实验小鼠的口服葡萄糖耐量曲线下面积结果；
图14为MNH05026和MNH27256分别与Metformin联用对II型糖尿病小鼠的空腹血糖影响结果；
图15为MNH27256单独使用及其与Metformin联用对II型糖尿病小鼠的空腹血糖变化曲线图；
图16为菌株MNH05026、MNH27256和MNH22004对高脂饮食诱导的肥胖小鼠的体重获得图；
图17为菌株MNH05026、MNH22004、MNH27256分别与Semaglutide联用对高脂高糖高胆固醇饮食诱导NAFLD/NASH小鼠的体重变化图；
图18为MNH05026、MNH22004和MNH27256分别与Semaglutide联用对高脂高糖高胆固醇饮食诱导NAFLD/NASH小鼠的肝重影响图；
图19为MNH05026、MNH22004和MNH27256与Semaglutide联用对高脂高糖高胆固醇饮食诱导NAFLD/NASH小鼠模型口服糖耐量的影响图；其中，图19中(A)为MNH05026、MNH22004和MNH27256与Semaglutide联用对高脂高糖高胆固醇饮食诱导NAFLD/NASH小鼠模型口服糖耐量Day30的影响图，图19中(B)为MNH05026与Semaglutide联用对高脂高糖高胆固醇饮食诱导NAFLD/NASH小鼠模型口服糖耐量Day55的影响，图19中(C)、(D)为图19中(A)、(B)对应的口服葡萄糖耐量曲线下面积结果图，图19中(E)为MNH05026、MNH22004和MNH27256与Semaglutide联用对高脂高糖高胆固醇饮食诱导NAFLD/NASH小鼠模型口服糖耐量Day30时的空腹血糖值图；
图20为MNH05026、MNH22004和MNH27256分别与Semaglutide联用对高脂高糖高胆固醇饮食诱导的NAFLD/NASH小鼠模型皮下脂肪重量(subcutaneous fat pad)、腹股沟脂肪重量(Inguinal fat pad)、棕色脂肪重量(brown adipose tissue)和附睾脂肪重量(epididymis fat pad)的影响；
图21为MNH05026与Semaglutide联用对高脂高糖高胆固醇饮食诱导的 NAFLD/NASH小鼠模型甘油三酯(TG)、总胆固醇(CHO)、高密度脂蛋白(HDL)、低密度脂蛋白(LDL)的影响；
图22为MNH05026、MNH22004和MNH27256分别与Semaglutide联用对高脂高糖高胆固醇饮食诱导NAFLD/NASH小鼠的肝脏脂肪变性(Steatosis)、肝小叶炎症(Lobular inflammation)、肝脏气球样变(Ballooning)及肝脏NAS score及纤维化(Fibrosis)的影响图；
图23为MNH22004、MNH05026对去乙酰化酶活性的抑制作用效果图。
具体实施方式
下面将结合附图和具体实施方式对本发明的技术方案进行清楚、完整地描述，但是本领域技术人员将会理解，下列所描述的实施例是本发明一部分实施例，而不是全部的实施例，仅用于说明本发明，而不应视为限制本发明的范围。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
本领域已知，可以通过传统分类方法和分子生物学方法对菌种进行分类鉴定。传统分类方法包括，例如细胞形态观察、革兰氏染色、鞭毛染色、各种代谢实验等。分子生物学方法包括核糖体RNA序列测定法、基于全基因组测序的测定方法等。
16S rRNA是原核生物的一种核糖体RNA，16S rRNA基因由可变区和保守区组成，保守区为所有细菌共有，而可变区在不同细菌之间存在不同程度的差异。通过比较细菌的16S rRNA的基因序列，根据序列差异基数按其进化距离，可以绘出生物进化树。
两个核酸分子的序列之间的“同一性”可以使用已知的计算机算法确定，例如“FASTA”程序，GCG程序包，BLASTN或FASTA。商业或公开可用的程序还可以是例如DNAStar“MegAlign”程序。
随着二代及三代测序技术的快速发展，基于全基因组测序的菌种鉴定成为可能，也使得菌种鉴定结果更为准确。细菌基因组平均核苷酸同一性(Average nucleotide identity，ANI)是指两细菌基因组之间同源基因的相似性。ANI值可以通过BLAST等方法计算。在细菌分类学领域，普遍认为ANI值需要达到95％以上才认定为属于同一个菌种(Jain C,Rodriguez-R L M,Phillippy A M,et al.High throughput ANI analysis of 90K prokaryotic genomes reveals clear speciesboundaries[J].Nature Communications,2018,9(1):5114.)。
目前已有各类成熟的ANI值的计算工具，比如本地运算软件Jspecies(/jspecies)和Gegenees(/documentation.html)，在线计算工具ANI caculator(http:/enveomics.gatech.edu/)、EzGenome(/ezgenome/ani)和ANItools。
利用以上方法，本领域技术人员可以判断一个分离菌株是否属于本发明人所发现的Megasphaera属中的新菌种。例如，当与保藏编号为GDMCC No:62001、GDMCC No：62000、GDMCC No：61999的菌株的平均核苷酸同一性ANI值为至少95％，例如95.5％、96％、96.5％、97％、97.5％、98％、98.5％、99％、99.5％、99.6％、99.7％、99.8％、99.9％或100％时，可以确定属于同一个菌种。
利用本文所述的Megasphaera菌菌种可以制备药物组合物，例如通过使用药学上可接受的辅料进行制备。所述药物组合物包含药学有效量的所述菌种的Megasphaera菌，例如保藏编号为GDMCC No:62001、GDMCC No：62000、GDMCC No：61999的菌株。 同样，保藏编号为GDMCC No:62001、GDMCC No：62000、GDMCC No：61999的菌株所属的Megasphaera菌种也可以制备药物组合物，例如通过使用药学上可接受的辅料进行制备，其包含药学有效量的该菌种的Megasphaera菌。
可以使用的合适的药学上可接受的辅料，例如载体、赋形剂、稀释剂、润滑剂、润湿剂、乳化剂、悬浮液稳定剂、防腐剂、甜味剂以及香料。例如，药学上可接受的辅料为乳糖、葡萄糖、蔗糖、山梨糖醇、甘露糖、淀粉、玉米淀粉、海藻糖、果糖、维生素C钠盐、L-半胱氨酸盐酸盐、脱脂奶粉、海藻酸钠、氯化钙、羧甲基纤维素钠、阿拉伯胶、磷酸钙、藻酸盐、明胶、硅酸钙、细结晶纤维素、聚乙烯吡咯烷酮、纤维素、水、糖浆、甲基纤维素、羟基苯甲酸甲酯、羟基苯甲酸丙酯、滑石、硬脂酸镁和矿物油中的一种或多种。
本发明含义内的“上清液”是指根据本发明的细菌菌株的培养上清液，任选地包含所述菌株的化合物和/或细胞碎片，和/或由所述菌株分泌的代谢物和/或分子。
本发明提供的药物组合物可包含药学上可接受的赋形剂、稀释剂或载体。用于治疗用途的可接受的载体或稀释剂在制药领域是众所周知。合适的载体的例子包括乳糖、淀粉、葡萄糖、甲基纤维素、硬脂酸镁、甘露糖醇或山梨糖醇等。合适的稀释剂的例子包括乙醇、甘油和水。药物载体、赋形剂或稀释剂的选择可以根据预期的给药途径和标准药物实践来选择。药物组合物可以包含作为载体、赋形剂或稀释剂的或除载体、赋形剂或稀释剂之外的任何合适的粘合剂、润滑剂、助悬剂、涂层剂或增溶剂等。合适的粘合剂的实例包括淀粉、明胶、天然糖以及天然或合成树胶。其中天然糖例如葡萄糖、无水乳糖、自由流动乳糖、β-乳糖或玉米甜味剂。天然或合成树胶例如阿拉伯树胶、黄芪胶、海藻酸钠、羧甲基纤维素或聚乙二醇。合适的润滑剂的例子包括油酸钠、硬脂酸钠、硬脂酸镁、苯甲酸钠、乙酸钠或氯化钠等。药物组合物中可以提供防腐剂、稳定剂、染料甚至调味剂。防腐剂的例子包括苯甲酸钠、山梨酸或对羟基苯甲酸酯。也可以使用抗氧化剂和悬浮剂。也可以使用抗氧化剂和悬浮剂。
菌种分类学相关内容
16S rRNA在分类学的应用内容：
判断标准：当两菌株16S rRNA基因序列之间相似性低于97％～98.65％时，可判断它们归属于不同的种
“一致性”或“一致性”计算方法：
两个核酸分子的核酸序列之间的“一致性”可以使用已知的计算机算法，例如“FASTA”程序，使用例如Pearson等人中的默认参数，确定为一致性百分比。(其他程序包括GCG程序包(Devereux,J.,et al.,Nucleic Acids Research 12(I):387(1984)),BLASTP,BLASTN,FASTA)。例如，NCBI数据库的BLAST功能、其他商业或公开可用的程序包括DNAStar“MegAlign”程序。
糖尿病相关
胰岛素抵抗是指各种原因使胰岛素促进葡萄糖摄取和利用的效率下降，机体代偿性的分泌过多胰岛素产生高胰岛素血症，以维持血糖的稳定，胰岛素抵抗易导致代谢综合征和2型糖尿病。
本专利应用4种代谢疾病相关的模型：高脂饮食(HFD)诱导的小鼠肥胖模型、高脂高果糖高胆固醇诱导的小鼠NASH模型、高脂饮食联合链脲佐菌素(Streptozotocin)(HFD-STZ)诱导的小鼠二型糖尿病模型、瘦素受体基因缺陷小鼠模型(db/db)，此4种 模型均为常用的代谢疾病类小鼠模型，模型小鼠通常伴随肥胖、胰岛素抵抗、高血糖、高血脂、高胆固醇、NAFLD/NASH等代谢疾病。
谷丙转氨酶(ALT)与谷草转氨酶(AST)为肝细胞损伤的敏感标志，在机体肝脏功能异常(损伤)或存在肝脏疾病(NAFLD/NASH)时，谷丙转氨酶(ALT)与谷草转氨酶(AST)的含量升高，另外NAFLD/NASH患者的肝脏存在大量的脂肪积累，肝脏重量会增加，因此当药物干预后ALT、AST水平降低及肝脏重量减轻能显示药物存在一定的治疗改善效果。
肝肾功能相关疾病：
肝肾疾病是指发生在严重肝病中的功能性急性肾衰竭，失代偿期肝硬化，由于有效循环血容量不足、前列腺素减少等原因，可产生肝肾综合征。本发明重点进行了肝脏相关疾病，主要是NAFLD/NASH的相关研究。
非酒精性脂肪性肝病(NAFLD)是指过多的脂肪以甘油三酯的形式堆积在肝脏中(脂肪变性)，尚有一些NAFLD的患者中除了有过多脂肪外尚有肝细胞损伤和炎症(脂肪性肝炎)，即NASH，NASH被广泛认为是代谢综合征的肝脏表现，如2型糖尿病，胰岛素抵抗，中央性肥胖，高脂血症(低高密度脂蛋白胆固醇，高甘油三酯血症)和高血压。
TG主要参与人体内能量代谢，产生热能。血中TG含量过高可导致血液黏稠，使脂质在血管壁上沉积，渐渐形成小斑块，即动脉粥样硬化。LDL-C增高是动脉粥样硬化发生、发展的主要、独立危险因素；LDL-C的增高水平也是衡量冠心病的指标。由于HDL-C能将血管壁内胆固醇转运至肝脏进行分解代谢(即胆固醇逆转运)，可减少胆固醇在血管壁的沉积，起到抗动脉粥样硬化作用。
糖尿病包括1型糖尿病(T1D)、2型糖尿病(T2D)和妊娠期糖尿病(GDM)。1型糖尿病是由于自身免疫损害或特发性原因引起的，以胰岛功能绝对破坏为特点的糖尿病，多发生在儿童和青少年，必须用胰岛素治疗才能获得满意疗效，否则将危及生命。2型糖尿病是一种以碳水化合物/脂肪代谢异常为特征的多因子综合征，通常包括高血糖、高血压和胆固醇异常。2型糖尿病是胰岛素不能有效发挥作用(与受体结合含量少)所致，因此不仅要检查空腹血糖，而且要观察餐后2小时血糖，特别应做胰岛功能检查。妊娠期间的糖尿病有两种情况，一种为妊娠前已确诊患糖尿病，称“糖尿病合并妊娠”；另一种为妊娠前糖代谢正常或有潜在糖耐量减退、妊娠期才出现或确诊的糖尿病，又称为“妊娠期糖尿病(GDM)”，糖尿病孕妇中80％以上为GDM。
口服葡萄糖耐量用于测定胰岛β细胞功能和机体对血糖的调节能力，是目前公认用于诊断糖尿病的一项诊断指标。当糖代谢紊乱时，口服一定量的葡萄糖后血糖急剧升高，或升高不明显，但短时间内不能降至空腹水平或原来水平，此为糖耐量异常或糖耐量降低；糖耐量异常表明机体对葡萄糖的代谢能力下降，常见于2型糖尿病和肥胖症。
HOMA-IR是用于评价个体的胰岛素抵抗水平的指标，目前已广泛用于临床评价糖尿病人胰岛素敏感性，其计算方法为：空腹血糖水平(FPG，mmol/L)×空腹胰岛素水平(FINS，μU/mL)/22.5。正常个体的HOMA-IR指数为1。随着胰岛素抵抗水平升高，HOMA-IR指数将高于1。
肠道中的L细胞能够分泌胰高血糖素样肽-1(GLP-1)，其可以促进胰岛β细胞产生胰岛素，抑制胰岛α细胞产生胰高血糖素，进而调节机体的血糖平衡，改善机体葡萄糖耐量。发明人发现，克里斯滕森菌具有提高胰高血糖素样肽-1(GLP-1)的分泌水平，从而 调节机体血糖平衡，改善机体葡萄糖耐量，进一步改善机体的胰岛素敏感性和瘦素敏感性，进而实现预防和/或治疗糖尿病和/或高血脂症的作用。
炎症：
脂多糖(lipopolysaccharide，LPS)又称为细胞内毒素，是一种磷脂，构成革兰氏阴性细菌的外细胞壁。除了保证细菌结构的完整性，脂多糖还能保护这些细菌免遭胆囊分泌的胆汁盐分解。通常情况下，脂多糖被肠壁细胞的紧密连接阻隔在血流之外。如果脂多糖进入到血液，就会在动物体内诱发强烈的炎症反应。所以血液中的脂多糖水平能够反应炎症水平。
抵抗素与炎症：
抵抗素为一种脂肪组织分泌的激素或脂肪因子，与肥胖和胰岛素抵抗有关。在人类中，抵抗素已被表征为免疫细胞特别是巨噬细胞表达和分泌的激素，并且与许多炎症反应有关，包括由于巨噬细胞浸润引起的脂肪组织炎症。抵抗素可以通过抵抗素诱导的炎症在肥胖和胰岛素抵抗的发生和发展中发挥重要作用。抵抗素还与其他慢性疾病有关，例如心血管疾病和癌症，在许多研究中，抵抗素已被提出作为代谢相关疾病的重要的生物标志物。
肥胖症相关：
肥胖是指一定程度的明显超重与脂肪层过厚，是体内脂肪，尤其是甘油三酯积聚过多而导致的一种状态，是对健康构成风险的脂肪异常或过度积累。由于食物摄入过多或机体代谢的改变而导致体内脂肪积聚过多造成体重过度增长并引起人体病理、生理改变或潜伏。体重指数超过25被视为超重，超过30被视为肥胖。肥胖会增加患许多身体和精神疾病的风险。它主要与代谢综合征、包括2型糖尿病、高血压、高胆固醇血症和高甘油三酯血症等疾病的组合有关。总的来说，肥胖对健康的影响分为两大类:可归因于体脂增加的疾病(如骨关节炎、阻塞性睡眠呼吸暂停等)和脂肪细胞数量增加的疾病(糖尿病、血脂异常、癌症、心血管疾病、非酒精性脂肪肝或非酒精性脂肪肝炎等)。
高血糖素肽：
胰高糖素样肽-1(glucagon-like peptide-1，GLP-1)是一种主要由肠道L细胞所产生的激素，属于肠促胰素(incretin)。胰高糖素样肽-1受体激动剂(GLP-1RA)是近年来的新型降糖药，通过激活GLP-1受体，以葡萄糖浓度依赖的方式增强胰岛素分泌，抑制胰高糖素分泌，并能够延缓胃排空，通过中枢性的食欲抑制减少进食量，从而达到降低血糖，减肥等作用。
瘦素：
瘦素(Leptin)是一种由脂肪组织分泌的激素，它在血清中的含量与动物脂肪组织大小成正比。瘦素作用于位于中枢神经系统的受体(Leptin Receptor)从而调控生物的行为以及新陈代谢。当动物体的体脂减少或处于低能量的状态下(例如饥饿)，血清中瘦素的含量会明显下降，从而激发动物的觅食行为，同时降低自身能量消耗。反之，当生物体的体脂增加时，血清中瘦素含量升高，进而抑制进食并且加速新陈代谢。瘦素就是通过这样的负反馈机制来调控生物体的能量平衡以及体重。
瘦素抵抗：
随着脂肪量的不断增长，瘦素不断分泌，长期大量瘦素的刺激让大脑对瘦素不再敏感，医学上称为“瘦素抵抗”。
肝脏功能损伤相关疾病包括如下疾病中的至少一种：脂肪肝、非酒精性脂肪性肝病、 非酒精性脂肪性肝炎、肝纤维化、肝硬化和肝癌。
肥胖相关疾病包括如下疾病中的至少一种：肥胖症、代谢综合症、心血管疾病、高脂血症、高胆固醇血症、高血压、胰岛素抗性综合征、肥胖相关的胃食管返流症和脂肪性肝炎。
糖尿病相关疾病包括如下疾病中的至少一种：II型糖尿病、胰岛素抗性综合征、葡萄糖不耐症、高血脂絮乱、糖尿病肾病变并发症、糖尿病神经病变、糖尿病眼睛病变、心血管疾病、糖尿病足。
上述“肥胖相关疾病”可选自如下疾病：过食症(overeating)、暴食症(binge eating)、易饥症、高血压、糖尿病、血浆胰岛素浓度升高、胰岛素抗性、高脂血症、代谢综合征、胰岛素抗性综合征、肥胖相关的胃食管返流症、动脉硬化症、高胆固醇血症、高尿酸血症、下背痛、心脏肥大和左心室肥大、脂肪代谢障碍、非酒精性脂肪性肝炎、心血管疾病，和多囊性卵巢综合征，以及具有这些肥胖相关疾病包括希望减轻体重的那些对象。
第二活性物质为其他代谢疾病治疗剂，如GLP-1受体激动剂，GLP-1受体和GCG受体双重激动剂，GLP-1受体、GIP受体和GCG受体三重激动剂，AMPK激动剂或促进GLP-1分泌的活性药物；也可以是包括二甲双胍、磺脲类、氯茴苯酸、噻唑烷二酮类、DPP-4抑制剂、GLP-1受体激动剂、SGLT2抑制剂、胰岛素、吡格列酮、罗格列酮、己酮可可碱、omega-3-脂肪酸、他汀类药物、依折麦布、或熊去氧胆酸、Semaglutide、liraglutide、exenatide或beinaglutide；。
在一些实施方案中，该方法进一步包括向受试者施用益生元。在一些实施方案中，益生元是低聚果糖、低聚半乳糖、反式低聚半乳糖、低聚木糖、壳低聚糖、低聚大豆、低聚龙胆糖、低聚异麦芽糖、低聚甘露糖、低聚麦芽糖、低聚甘露糖、乳果糖、乳糖蔗糖、帕拉金糖、糖基蔗糖、瓜尔豆胶、阿拉伯胶、塔加糖、直链淀粉、支链淀粉、果胶、木聚糖或环糊精。
“给药”或“施用”泛指将组合物(例如，药物组合物)给予受试者的途径。给药途径的实例包括口服给药、直肠给药、局部给药、吸入(鼻)或注射。注射给药包括静脉内(IV)、肌肉内(IM)、瘤内(IT)和皮下(SC)给药。本文所述的药物组合物可以通过任何有效途径以任何形式施用，包括但不限于瘤内、口服、肠胃外、肠内、静脉内、腹膜内、局部、透皮(例如，使用任何标准贴剂)、皮内、眼内、(鼻内、局部、非口腔，例如气雾剂、吸入、皮下、肌内、口腔、舌下、(经)直肠、阴道、动脉内和鞘内。在一个优选的实施方案中，本文所述的药物组合物经口、直肠、瘤内、局部、膀胱内、通过注射到引流淋巴结中或引流淋巴结附近、静脉内、通过吸入或气雾剂、或皮下施用。在另一个优选的实施方案中，本文所述的药物组合物经口、瘤内或静脉内施用。
术语“增加”或“提升”或“提高”是指改变，使得根据情况，与预处理状态相比，治疗后差异为至少10％、20％、30％、40％、50％、60％、70％、80％、90％、2倍、4倍、10倍、100倍、103倍、104倍、105倍、106倍和/或107倍。可能增加的特性包括免疫细胞、细菌细胞、基质细胞、髓源性抑制细胞、成纤维细胞、代谢物的数量；细胞因子的水平；或其他物理参数(例如肿瘤大小)。
术语“减少”或“降低”或“下降”是指改变，使得根据情况，与治疗前状态相比，治疗后差异为至少10％、20％、30％、40％、50％、60％、70％、80％、90％、1/100、1/1000、1/10,000、1/100,000、1/1,000,000或检测不到。可能减少的特性包括免疫细胞、细菌细胞、基质细胞、髓源性抑制细胞、成纤维细胞、代谢物的数量；细胞因子的水平；或其 他物理参数(例如耳朵厚度(例如，在DTH动物模型中)或肿瘤大小)。
术语“胰岛素抵抗”在本领域具有其共同含义。胰岛素抵抗是一种生理状况，其中天然激素胰岛素在降低血糖方面变得不那么有效。由此产生的血糖升高可能会使血糖水平超出正常范围，并导致不良健康影响，例如代谢综合征、血脂异常和随后的2型糖尿病。如本文所用，术语“胰岛素抵抗相关并发症”和“胰岛素抵抗相关病症”包括但不限于代谢综合征、血脂异常和2型糖尿病，以及内分泌疾病中的胰岛素抵抗(例如肥胖受试者患有2型糖尿病)、1型糖尿病、库欣病和脂肪代谢障碍综合征)。
如本文所用，“代谢性疾病”或“代谢疾病”是指一起发生的一组病症，增加了心脏病、中风和II型糖尿病的风险。这些情况包括血压升高、高血糖、脂肪过多、肥胖以及胆固醇或甘油三酯水平异常。在一些实施方案中，代谢疾病是II型糖尿病、葡萄糖耐量降低、胰岛素抵抗、体重控制、超重、血糖控制、糖尿病前期、肥胖症、高血糖症、高胰岛素血症、脂肪肝、非酒精性脂肪性肝炎、高胆固醇血症、高血压、高脂蛋白血症、高脂血症、高甘油三酯血症、酮症酸中毒、低血糖症、血栓性疾病、血脂异常、非酒精性脂肪肝(NAFLD)或相关疾病。在一些实施方案中，相关疾病是心血管疾病、动脉粥样硬化、肾病、肾病、
如本文所用，“代谢产物”是指在任何细胞或微生物代谢反应中用作底物或作为产物化合物、组合物、分子、来自任何细胞或微生物代谢反应的离子、辅因子、催化剂或营养物质。
“菌株”是指具有遗传特征的细菌物种的成员，使得它可以与相同细菌物种的密切相关的成员区分开来。遗传特征可以是至少一个基因的全部或部分不存在、至少一个调节区(例如，启动子、终止子、核糖开关、核糖体结合位点)的全部或部分不存在、不存在(至少一种天然质粒的“治愈”)、至少一种重组基因的存在、至少一种突变基因的存在、至少一种外源基因(来自另一物种的基因)的存在、至少一个突变的调节区(例如，启动子、终止子、核糖开关、核糖体结合位点)，至少一种非天然质粒的存在，至少一种抗生素抗性盒的存在，或其组合。不同菌株之间的遗传特征可以通过PCR扩增来鉴定，任选地随后对感兴趣的基因组区域或整个基因组进行DNA测序。在一种菌株(与同一物种的另一种菌株相比)获得或失去抗生素抗性或获得或失去生物合成能力(例如营养缺陷型菌株)的情况下，可以通过选择或使用抗生素的反选择来区分菌株或营养/代谢物。
本发明含义内的“上清液”是指根据本发明的细菌菌株的培养上清液，任选地包含所述菌株的化合物和/或细胞碎片，和/或由所述菌株分泌的代谢物和/或分子。
术语“受试者”或“患者”是指任何哺乳动物。描述为“有需要”的受试者或患者是指需要治疗(或预防)疾病的人。哺乳动物(即哺乳动物)包括人类、实验室动物(例如灵长类动物、大鼠、小鼠)、家畜(例如牛、绵羊、山羊、猪)和家庭宠物(例如狗、猫、啮齿动物)。对象可以是人。受试者可以是非人类哺乳动物，包括但不限于狗、猫、牛、马、猪、驴、山羊、骆驼、小鼠、大鼠、豚鼠、羊、骆驼、猴子、大猩猩或黑猩猩。受试者或患者可能是健康的，或可能在任何发育阶段患有代谢疾病。
如本文所用，术语“治疗”受试者的疾病或“治疗”患有或怀疑患有疾病的受试者是指向受试者施用药物治疗，例如施用一种或多种药剂，从而减少或防止疾病的至少一种症状恶化。因此，在一个实施方案中，“治疗”尤其是指延迟进展、加速缓解、诱导缓解、增加缓解、加速恢复、增加替代疗法的功效或降低对替代疗法的抗性，或它们的组合。
如本文所用，术语“预防”是本领域公认的，并且当与诸如局部复发等病症相关使用 时，在本领域中是众所周知的，并且包括施用降低相对于未接受该组合物的受试者，受试者的医学病症症状的发生或延迟该症状的发作。
本发明开发出包含Megasphaera属下物种的细菌菌株(MNH05026、MNH22004、MNH27256)及新组合物。可用于治疗和预防癌症、自身免疫和炎症性疾病、代谢疾病、神经退行性疾病和精神疾病，特别是由短链脂肪酸(SCFA)和/或中链脂肪酸(MCFA)直接作用，或通过SCFA和/或MCFA介导来抑制组蛋白去乙酰化酶(HDAC)活性达到治疗上述疾病的目的。已经确定来自巨球型菌属的细菌菌株能够确定菌株能有效地产生许多SCFA和MCFA，包括乙酸、丁酸、戊酸和己酸。这些短链脂肪酸已被证明可以改善多种疾病的症状，包括提高免疫疗法的抗肿瘤功效、胃肠道传染病、炎症性肠病(IBD)、代谢疾病、自身免疫疾病、神经退行性疾病和精神疾病。
巨球型菌属下已知的种包括Megasphaera elsdenii、Megasphaera stantonii、Megasphaera massiliensis、Megasphaera indica、Megasphaera paucivorans、Megasphaerasueciensis、Megasphaera micronuciformis、Megasphaera hexanoica、Megasphaera cerevisiae。其中，Megasphaera elsdenii模式菌株DSM 20460，Megasphaera indica模式菌株NMBHI-10，Megasphaera massiliensis模式菌株NP3以及菌株NCIMB42787(参见CN112601534A)，Megasphaera cerevisiae模式菌株DSM 20462，Megasphaera paucivorans模式菌株DSM 16981，Megasphaera sueciensis模式菌株DSM 17042，Megasphaera micronuciformis模式菌株DSM 17226，Megasphaera hexanoica模式菌株MH，都具有产短链脂肪酸能力，尤其是丁酸(参见“Megasphaera hexanoica sp.nov.,a medium-chain carboxylic acid-producing bacterium isolated from a cow rumen；DOI：10.1099/ijseMegasphaera 0.001888”表1)。
本发明在进行药物筛选时，首先在基因层面通过基因组数据分析，对Megasphaera菌属下的菌株产丁酸能力进行预测和分析，根据产丁酸路径的确定以及产丁酸路径的完整性，通过确定能够表达产丁酸的关键酶来进一步锁定基因元件；将包含基因元件/关键酶(蛋白)的细菌菌株挑选出来，并在实验数据层面进行了产酸能力验证。后续将筛选出来的候选菌株进行体外实验和小鼠实验进行验证其用于肿瘤抑制和代谢疾病防治的效果。按照该筛选逻辑，筛选出Megasphaera菌属下的细菌菌株MNH05026、MNH22004、MNH27256进行抗肿瘤实验和代谢疾病防治实验，实验证明本发明的三个Megasphaera菌种都有抗肿瘤和代谢疾病防治的效果。
本发明Megasphaera菌属的细菌菌株(MNH05026)，保藏编号为GDMCC No:62001，其16S rRNA为SEQ ID NO:1；
本发明Megasphaera菌属的另一细菌菌株(MNH22004)，保藏编号为GDMCC No:62000，其16S rRNA为SEQ ID NO:2；
本发明Megasphaera菌属的另一细菌菌株(MNH27256)，保藏编号为GDMCC No:61999，其16S rRNA为SEQ ID NO:3。
图1为菌株MNH 05026、MNH22004和MNH27256在厌氧血平板培养48h菌落形态；其中：图1A为菌株MNH 05026在厌氧血平板培养48h菌落形态；图1B为菌株MNH22004在厌氧血平板培养48h菌落形态；图1C为菌株MNH27256在厌氧血平板培养48h菌落形态；
图2为菌株MNH 05026、MNH22004的显微形态，图2C和2D为MNH27256在不同倍镜下的显微形态；其中：图2A为菌株MNH05026的显微形态，图2B为菌株 MNH22004的显微形态，图2C和2D为MNH27256在不同倍镜下的显微形态；
图3为菌株MNH 05026、MNH22004和MNH27256革兰氏染色显微形态；其中：图3A为菌株MNH 05026革兰氏染色显微形态；图3B分别为菌株MNH22004革兰氏染色显微形态；图3C分别为菌株MNH27256革兰氏染色显微形态；
图4为菌株MNH 05026、MNH22004和MNH27256芽孢染色显微形态；其中：图4A为菌株MNH 050266芽孢染色显微形态；图4B为菌株MNH22004芽孢染色显微形态；图4C为菌株MNH27256芽孢染色显微形态；
图5为菌株MNH 05026、MNH22004和MNH27256的不同浓度NaCl耐受性的结果；其中：图5A、5B和5C分别为菌株MNH 05026、MNH22004和MNH27256的不同浓度NaCl耐受性的结果；
图6为菌株MNH 05026、MNH22004和MNH27256的不同pH耐受性的结果；其中：图6A、6B和6C分别为菌株MNH 05026、MNH22004和MNH27256的不同pH耐受性的结果；
图7为菌株MNH 05026、MNH22004和MNH27256的不同浓度胆盐耐受性的结果；其中：图7A、7B和7C分别为菌株MNH 05026、MNH22004和MNH27256的不同浓度胆盐耐受性的结果；
图8为菌株MNH 05026在培养基API 20中培养的结果；
基因层面：一方面，本发明的Megasphaera菌属的细菌菌株MNH05026、MNH22004、MNH27256都能表达丁酸-乙酰CoA-转移酶A亚基(EC2.8.3.9酶，具体释义参见Uniprot.org)，可以预测其具有产短链脂肪酸的能力，如丁酸；本发明细菌菌株MNH05026、MNH22004、MNH27256表达EC2.8.3.9的GENE ID涉及:650027236、641897133、650594268、642201644、650018449、650536170、2511555023、646248671(GENE ID对应的序列参见Integrated Microbial Genome(IMG)；http://img.jgi.doe.gov)。
另一方面，发明人统计了Megasphaera属下文献或专利中已记载和证实的具有抗肿瘤作用的菌株,提取数据库中已经公开的这些菌株的全基因数据，进行全基因组数据分析，发明人发现这些菌株在丙酮酸途径(Pyruvate_pathway)和/或4-氨基丁酸途径(4aminobutyrate_pathway)的完整度，仅2株菌株在(丙酮酸路径)产丁酸路径完整度低于70％；但这两株菌在4-氨基丁酸途径(4aminobutyrate_pathway)高于70％；MNH22004、MNH27256都有完整的产丁酸路径(即产丁酸通路100％完整)；MNH05026在Pyruvate_pathway通路具有至少70％完整度、在4aminobutyrate_pathway通路具有80％以上的完整度。
实验数据层面：利用本发明实施例3介绍的测定短链脂肪酸(SCFA)的方法证实了MNH05026、MNH22004、MNH27256都能够有效产丁酸(表1)，实验结果与基因分析的预测吻合。且后续通过实验证实，能够有效产丁酸的Megashpaera菌属下的细菌菌株MNH05026、MNH22004、MNH27256都能有效抑制肿瘤，能用于预防、治疗癌症，同时也都能有效防治代谢疾病。
表1本发明的各菌株SCFA产量结果：
本发明公开的细菌菌株MNH05026、MNH22004、MNH27256除了具备产丁酸能力外，还具有良好的产乙酸等多种SCFA的能力。而微生物的SCFA已被现有技术充分研究用于治疗或预防肥胖，糖尿病等代谢疾病；现有技术中“Gut microbial metabolites in obesity,NAFLD and T2DM”表明：与体重控制有关的碳水化合物发酵产生的代谢物包括乙酸，丙酸，丁酸，琥珀酸；乙酸盐和丁酸盐也被证明通过中枢机制诱导饱腹感，并增加脂肪组织和肝脏的产热，以及诱导脂肪组织褐变和瘦素分泌。此外，乙酸、丙酸和丁酸以G蛋白偶联受体(GPR)依赖的方式刺激饱腹激素胰高血糖素样肽1(GLP-1)和肽YY(PYY)的分泌。
除了利用上述途径发挥治疗代谢疾病的作用外，丁酸(丁酸盐)还是现有技术中已经得到充分验证的具有抑制HDAC酶活性的代谢产物(Human gut bacteria as potent class I histone deacetylase inhibitors in vitro through production of butyric acid and valeric acid，https://doi.org/10.1371/journal.pone.0201073)，而已有研究表明，通过抑制HDAC活性能有效治疗代谢疾病，如糖尿病。糖尿病是其中低水平的胰岛素和/或外周胰岛素抵抗导致高血糖症的一组疾病。已提出抑制HDAC通过多种机制来治疗糖尿病，包括Pdxl的抑制(Park等，2008，J Clin Invest，118，2316-24)，增强转录因子Ngn3的表达以增加内分泌库。祖细胞(Haumaitre等，2008，Mol Cell Biol，28，6373-83)和增强胰岛素表达(Molsey等，2003，J Biol Chem，278，19660-6)等。HDAC抑制也是用于晚期糖尿病并发症如糖尿病性肾病和视网膜缺血的有希望的治疗方法(Christensen等，2011，Mol Med，17(5-6)，370-390)。
因此，本发明还公开了通过利用菌株MNH05026、MNH22004、MNH27256产生的SCFA和/或MCFA抑制组蛋白乙酰酶活性(HDAC)来实现预防和/或治疗代谢疾病；优选的，所述组蛋白乙酰酶为I类、II类、III类、IV类组蛋白乙酰酶。同时进行了MNH05026、MNH22004、MNH27256三株菌株的代谢产物(如丁酸)对HDAC抑制效果的验证实验。
本发明的Megasphaera菌属的细菌菌株MNH05026、MNH22004、MNH27256是丁酸的生产者，根据丁酸的已知作用，MNH05026还具有减少具有神经保护作用的血脑屏障的不可渗透(Michel and Prat(2016)Ann Transl Med.4(1)：15)。
本发明的Megasphaera菌属的细菌菌株MNH05026、MNH22004、MNH27256及其组合物能导致PBMC中促炎性分子，例如促炎性细胞因子的表达增加(参见图13)；施用本发明的药物组合物可导致PBMC中IL-1β表达的增加。IL-1β是一种促炎性细胞因子。IL-1β的产生和分泌受炎性小体调节，炎性小体是一种与炎症反应的激活相关的蛋白质复合物。由于显示出施用本发明的组合物可增加IL-1β的表达，因此本发明的组合物可用于治疗以IL-1β的表达降低为特征的疾病。
此外，发明人还确定了Megasphaera sp.MNH05026、MNH22004、MNH27256可以减轻LPS诱导的炎症。
在一些实施方案中，已发现本发明的Megasphaera菌属菌株及其组合物特别有益于诱导丁酸等多种SCFA的产生并抑制HDAC活性，从而达到防治代谢疾病的效果。在某些实施方案中，本发明的Megasphaera菌属菌株及其组合物可以在临床前鼠同基因代谢疾病模型中单独发挥防治代谢疾病的功效和与Semaglutide等活性物质联用协同发挥作用。本发明的Megasphaera菌属菌株及其组合物用于治疗或预防代谢疾病，如肝脏类疾病、肥胖类、心血管疾病、心脑血管疾病、高血脂，糖尿病、葡萄糖耐量受损等。具体包括但不限于：II型糖尿病、糖耐量受损、胰岛素抵抗、体重控制、超重、血糖控制、糖尿病前期、肥胖症、高血糖症、高胰岛素血症、脂肪肝、酒精性脂肪性肝炎、高胆固醇血症、高血压、高脂蛋白血症、高脂血症、高甘油三酯血症、尿毒症、酮症酸中毒、低血糖、血栓性疾病、血脂异常、非酒精性脂肪性肝病(NAFLD)、非酒精性脂肪性肝炎(NASH)、动脉粥样硬化、肾病、糖尿病性神经病、糖尿病性视网膜病、皮肤病、消化不良或水肿。
乙酸、丁酸、戊酸和己酸已显示能介导自身免疫、炎性相关疾病,这些作用可以通过减少炎性细胞因子的合成增加免疫调节调节性T细胞(Tregs)和抗炎细胞因子的产生来实现。MNH05026、MNH22004和MNH27256可生产乙酸、丁酸、戊酸和己酸。MNH05026、MNH22004、MNH27256可以增加抗炎细胞因子并减弱LPS诱导的炎症。乙酸和丁酸已被证明在治疗和预防肥胖症、2型糖尿病、非酒精性脂肪肝病。NAFLD、心脏代谢疾病和相关并发症方面具有保护作用。乙酸和丁酸的这些保护作用可以通过增加棕色脂肪组织的产热和白色脂肪组织的褐变、减少肝脏和脂肪组织中的脂质积累、增强肠道完整性和发挥免疫调节作用来实现。发明人已经发现Megasphaera sp.(MNH05026、MNH22004、MNH27256)不仅可以产生乙酸和丁酸，还可以诱导抗炎因子的产生并降低LPS诱导的疾病。
在一些实施方案中，施用包含巨球型菌属的组合物：MNH05026、MNH22004和MNH27256，能够降低HDAC活性。
在一些实施方案中，本发明提供了包含巨球型菌属的组合物。MNH05026、MNH22004和MNH27256用于治疗或预防由HDAC活性介导的炎症性肠病的方法。已显示抑制HDAC活性可抑制胃肠道中促炎细胞因子的产生。因此，本发明的Megasphaera菌属菌株及其组合物可用于治疗炎性疾病。特别地，本发明的Megasphaera菌属菌株及其组合物可用于治疗或预防与增加的结肠促炎细胞因子发病机制相关的病症。在一些实施方案中，本发明的Megasphaera菌属菌株及其组合物用于治疗或预防炎症性肠病。在一些实施方案中，本发明的Megasphaera菌属菌株及其组合物用于治疗或预防溃疡性结肠炎。在一些实施方案中，本发明的Megasphaera菌属菌株及其组合物用于治疗或预防克罗恩病。在某些实施方案中，本发明提供了包含巨球型菌属的菌株及其组合物。MNH05026、MNH22004和MNH27256用于治疗或预防炎症性疾病。在优选的实施方案中，本发明提供了包含巨球型菌属的菌株及其组合物，MNH05026、MNH22004和MNH27256用于治疗或预防结肠炎。
在本发明的某些实施方案中，组合物中的细菌菌株是巨球型菌属的菌株MNH05026。
在本发明的某些实施方案中，组合物中的细菌菌株是Megasphaera下的新物种，其 具有，也可以使用密切相关的菌株，例如具有与SEQ ID NO:1至少95％、96％、97％、98％、98.63％、99％、99.7％或99.9％相同的16S rRNA基因序列的菌株.也可以是具有与SEQ ID NO:2至少95％、96％、97％、98％、98.63％、99％、99.5％或99.9％相同的16S rRNA基因序列的菌株.也可以是具有与SEQ ID NO:3至少95％、96％、97％、98％、98.5％、98.63％、99％、99.5％或99.9％相同的16S rRNA基因序列的菌株。优选地，用于本发明的菌株具有由SEQ ID NO：1表示的16S rRNA基因序列。
在某些实施方案中，本发明提供了一种包含上述Megasphaera菌株及其组合物的食品。
此外，本发明提供治疗或预防由HDAC活性介导的疾病或病症的方法，包括施用包含巨球型菌属细菌菌株及其组合物。本发明还提供了包含此类细胞或此类细胞的生物学纯培养物的菌株及其组合物。本发明还提供了一种巨球型菌属的细胞，如MNH05026、MNH22004、MNH27256菌株或其衍生物，用于治疗，特别是用于本文所述的疾病。
本发明的巨球型菌属细菌菌株及其药物组合物能够用于预防和/或治疗代谢疾病，包括但不限于：II型糖尿病、糖耐量受损、胰岛素抵抗、体重控制、超重、血糖控制、糖尿病前期、肥胖症、高血糖症、高胰岛素血症、脂肪肝、酒精性脂肪性肝炎、高胆固醇血症、高血压、高脂蛋白血症、高脂血症、高甘油三酯血症、尿毒症、酮症酸中毒、低血糖、血栓性疾病、血脂异常、非酒精性脂肪性肝病(NAFLD)、非酒精性脂肪性肝炎(NASH)、动脉粥样硬化、肾病、糖尿病性神经病、糖尿病性视网膜病、皮肤病、消化不良或水肿。
优选的，本发明巨球型菌属细菌菌株包括与SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3具有至少95％一致性的物种。
本发明的细菌菌株包括Megasphaera elsdenii、Megasphaera stantonii、Megasphaera indica、Megasphaera paucivorans、Megasphaera sueciensis、Megasphaera micronuciformis、Megasphaera hexanoica、Megasphaera cerevisiae或与SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3具有至少95％一致性的物种菌株，所述物种菌株能表达EC2.8.3.9；
优选的，细菌菌株通过GENE ID:650027236、641897133、650594268、642201644、650018449、650536170、2511555023和646248671来调控表达EC2.8.3.9。
本发明涉及的细菌菌株及其组合物能够调节至少一种短链脂肪酸或短链脂肪酸盐，所述短链脂肪酸包括乙酸、丁酸、戊酸、丁戊酸或己酸。短链脂肪酸在体内起治疗作用时通常以短链脂肪酸盐的形式存在，通过验证发现乙酸盐、丙酸盐和丁酸盐为主要的短链脂肪酸盐。
本发明涉及的细菌菌株具有与SEQ ID NO:1，具有至少95％、95.5％、96％、96.5％、97％、97.5％、98％、98.5％、98.65％、99％、99.5％或99.9％一致性的16S rRNA序列；
本发明涉及的细菌菌株及其组合物用于治疗或预防自身免疫性疾病或炎性疾病，例如：哮喘，关节炎，银屑病，移植物抗宿主病，炎性肠病，克罗恩病，溃疡性结肠炎，同种异体移植物排斥，慢性炎症性肠病、系统性红斑狼疮、银屑病、类风湿性关节炎、多发性硬化症或桥本氏病；过敏性疾病，如食物过敏、花粉症或哮喘；艰难梭菌感染；所述炎性疾病包括脑膜炎、脊髓炎或TNF介导的炎性疾病，例如胃肠道的炎性疾病，例如储袋炎、心血管炎性疾病，例如动脉粥样硬化，或炎性肺病，例如慢性阻塞性肺病。
本发明涉及的细菌菌株及其组合物用于治疗或预防神经性疾病，例如抑郁症，焦虑，创伤后应激障碍，强迫症；帕金森，脑萎缩，血管或动脉硬化性帕金森病，轻度认知障 碍，HIV相关的认知损害，阿尔默兹海默症(AD)。
本发明涉及的组合物包含药学上可接受的载体；
优选地，所述载体选自稀释剂、分散剂、赋形剂、稳定剂、润滑剂、崩解剂中的一种或几种。
本发明涉及的药物的剂型包括液体制剂、固体制剂、胶囊制剂、缓释制剂和纳米制剂中的任一种。
本发明动物实验所使用的菌液剂量可以是按照小鼠体重进行菌液灌胃，灌胃剂量为0.04～0.3ml/10g体重；也可以是按照0.2ml/只小鼠进行灌胃，上述灌胃方式进行的实验，都表明本申请的菌株MNH05026、MNH22004、MNH27256都能单独或与其他药物联用取得本发明中关于肝肾功能的改善、及糖尿病、肥胖、脂肪肝等疾病的治疗效果，且效果显著。
本文涉及的固体MM01培养基，其组分为：蛋白胨5g/L，胰化酪蛋白5g/L，酵母粉10g/L，牛肉膏5g/L，葡萄糖5g/L，K2HPO4 2g/L，乙酸钠2g/L，吐温80 1mL/L，血红素5mg/L，L-半胱氨酸盐酸盐0.5g/L，维生素K1 1uL/L，无机盐溶液8ml/L，无机盐溶液每1L包括(氯化钙0.25g，磷酸氢二钾1g，磷酸二氢钾1g，硫酸镁0.5g，碳酸氢钠10g，氯化钠2g),琼脂15g/L。
本文涉及的液体MM01培养基，其组分为：蛋白胨5g/L，胰化酪蛋白5g/L，酵母粉10g/L，牛肉膏5g/L，葡萄糖5g/L，K2HPO4 2g/L，乙酸钠2g/L，吐温80 1mL/L，血红素5mg/L，L-半胱氨酸盐酸盐0.5g/L，维生素K1 1uL/L，无机盐溶液8ml/L，无机盐溶液每1L包括(氯化钙0.25g，磷酸氢二钾1g，磷酸二氢钾1g，硫酸镁0.5g，碳酸氢钠10g，氯化钠2g)。
AC液体培养基，每升含：蛋白胨，20g；葡萄糖，5g；酵母提取物，3g；牛肉浸粉，3g；维生素C，0.2g；pH7.0。
本文的厌氧血平板(购自环凯微生物)配方(每升)含量：酪蛋白胰酶消化物10.0g；心胰酶消化物3.0g；玉米淀粉1.0g；肉胃酶消化物5.0g；酵母浸出粉5.0g；氯化钠5.0g；琼脂15.0g；无菌脱纤维羊血50-100mL；蒸馏水1000mL；最终pH7.3±0.2。
本文的胰酪大豆胨液体培养基TSB液体培养基(购自环凯微生物)，配方(每升)含量的组分：胰酪胨17.0g；大豆木瓜蛋白酶水解物3.0g；磷酸氢二钾2.5g；氯化钠5.0g；葡萄糖2.5g；最终pH 7.3±0.2。
实施例1菌株MNH05026、MNH22004、MNH27256分离
本发明涉及到的肠道菌株MNH05026分离自广东省广州市的一位健康女性自愿者的粪便样本。捐赠者自取新鲜粪便2～5克，放入样本收集保存管中，震荡匀质后将处理好的粪便样本置于冰盒中，并于24小时内送达实验室进行菌株分离。生物安全柜中分装生理盐水，9ml/管；准备菌株分离培养基厌氧血琼脂平板(购自环凯微生物科技有限公司)，并提前24h将其转入厌氧工作站内，标记样品信息，培养基类型，分离日期等。
取新鲜粪便样本置于厌氧操作站中(Don Whitley Scientific H35)使用漩涡振荡器震荡1min，混匀，吸取1mL样本到9mL生理盐水中，混匀为10-1稀释液，然后梯度稀释至10-6稀释液，备用。
取10-6稀释液滴于厌氧血琼脂平板分离培养基，滴加量为100μL/皿，涂布均匀，待 平板表面干燥后，平板倒置，37℃培养3～5天。观察分离培养基菌株生长状况并用灭菌牙签挑取单菌落，进行菌株纯化，纯化菌株置于37℃，厌氧培养。将纯培养菌株制备成20％甘油或水的菌液，-86℃低温保存。采用基于菌株16S rRNA基因序列鉴定方法进行菌株鉴定。
本发明涉及到的肠道菌株MNH22004分离自广东省广州市的一位健康女性自愿者的粪便样本。采用上述方法分离菌株MNH22004。
本发明涉及到的肠道菌株MNH27256分离自广东省广州市的一位健康女性自愿者的粪便样本。采用上述方法分离菌株MNH27256。
保藏情况：菌株MNH05026(简称A026)，现已保藏于广东省微生物菌种保藏中心，保藏名称为Megasphaera sp.MNH05026，保藏编号为GDMCC No:62001，保藏时间2021年10月18日，保藏地址：广州市先烈中路100号大院59号楼5楼，广东省科学院微生物研究所，提议的分类名称为Megasphaera sp.。
本发明的菌株MNH22004，现已保藏于广东省微生物菌种保藏中心，保藏名称为Megasphaera sp.MNH22004，保藏编号为GDMCC NO：62000，保藏时间2021年10月18日，保藏地址：广州市先烈中路100号大院59号楼5楼，广东省科学院微生物研究所，提议的分类名称为Megasphaera sp.。
本发明的菌株MNH27256，现已保藏于广东省微生物菌种保藏中心，保藏名称为Megasphaera sp.MNH27256，保藏编号为GDMCC NO:61999，保藏时间2021年10月18日，保藏地址：广州市先烈中路100号大院59号楼5楼，广东省科学院微生物研究所，提议的分类名称为Megasphaera sp.。
形态特征：菌株MNH05026(简称A026)，接种于厌氧血平板培养基，37℃厌氧培养48h后，在厌氧血平板培养基上形成可见菌落，菌落圆形，边缘规则且光滑，直径约1mm，淡黄色，不透明，菌落周边无分泌物形成；菌株革兰氏阴性；显微形态观察发现菌株无芽孢，无鞭毛，不运动，球形，直径约5～10μm。(见图1A～4A)。
菌株MNH22004，接种于厌氧血平板培养基，37℃厌氧培养24h后，在厌氧血平板培养基上形成可见菌落，菌落圆形，边缘规则且光滑，直径约3mm，黄色，不透明，菌落周边无分泌物形成(菌落形态照片见图1B)；菌株革兰氏阴性(菌株革兰氏染色显微形态照片见图3B，菌株芽孢染色显微形态照片图4B)；显微形态观察发现菌株无芽孢，无鞭毛，不运动，短杆状，大小约5×10～15μm(菌落显微形态照片见图2B)。
将菌株MNH27256接种于厌氧血平板培养基，37℃厌氧培养24h后，在厌氧血平板培养基上形成可见菌落，菌落圆形，边缘规则且光滑，直径约2mm，黄色，不透明，菌落周边无分泌物形成(菌落形态照片见图1C)；菌株革兰氏阴性(菌株革兰氏染色显微形态照片见图3C，菌株芽孢染色显微形态照片图4C)；显微形态观察发现菌株无芽孢，无鞭毛，不运动，球状，直径约5μm(菌落显微形态照片见图2C和2D)。
菌株生理生化特征：菌株MNH05026，温度生长范围30～42℃，最适生长是37℃；在pH 6.0到8.0的范围内可以生长，最适生长pH为6.0～7.0(见图6A)；最高可耐受1％NaCl(见图5A)；菌株MNH05026能在胆盐浓度为0～0.15％范围内存活生长，在胆盐浓度大于等于0.2％时不能生长(见图7A)。菌株MNH05026在有氧的条件下不能生长，在厌氧条件下生长良好，属于专性厌氧细菌。
菌株MNH22004，温度生长范围30～42℃，最适生长是37℃；在pH5.0到10.0的范围内可以生长，最适生长pH为7.0～8.0；最高可耐受2％NaCl；菌株MNH22004能在 胆盐浓度为0～0.2％范围内存活生长，但菌株生长趋势随胆盐浓度增大而减弱。菌株MNH22004在有氧的条件下不能生长，在厌氧条件下生长良好，属于专性厌氧细菌。菌株对NaCl、pH以及胆盐的耐受性见图5B至图7B。
菌株MNH27256，生长温度范围30～42℃，最适生长是37℃；在pH 5.0到10.0的范围内可以生长，最适生长pH为7.0～8.0；最高可耐受2％NaCl；菌株MNH27256能在胆盐浓度为0～0.4％范围内存活生长，但是菌株生长趋势随胆盐浓度增大而减弱。菌株MNH27256在有氧的条件下不能生长，在厌氧条件下生长良好，属于专性厌氧细菌。菌株对NaCl、pH以及胆盐的耐受性见图5C至图7C。
API 20A测试菌株生理生化：根据说明书，使用API 20A试剂条(BioMérieux)进行测试。菌株的培养条件为37℃，厌氧。
实验结果：菌株MNH05026无法利用API 20A基础培养基生长(见图8)，MNH22004、MNH27256API 20A测试结果分别见表2-A和表2-B。
表2-A菌株MNH 22004API 20A测试结果：
表2-B菌株MNH27256API 20A测试结果：
菌株MNH05026、MNH22004、MNH27256抗生素敏感性测试：使用纸片扩散法进行菌株MNH05026、MNH22004和MNH27256抗生素敏感性测试。测试结果见表3。菌株MNH05026对庆大霉素、红霉素、氯霉素、四环素、青霉素、环丙沙星、复方新诺 明、氨苄西林、林可霉素和头孢曲松等抗生素敏感；菌株MNH05026对所用抗生素均未发现抗性。菌株MNH22004对青霉素和氨苄西林具有耐药性；对庆大霉素、红霉素、氯霉素、四环素、环丙沙星、复方新诺明、头孢曲松和林可霉素等抗生素敏感。菌株MNH27256对青霉素、氨苄西林和头孢曲松具有耐药性；对庆大霉素、红霉素、氯霉素、四环素、环丙沙星、复方新诺明和林可霉素等抗生素敏感。
表3菌株MNH05026、MNH22004、MNH27256抗生素敏感性测试结果
实施例2菌株MNH05026、MNH22004、MNH27256鉴定
取菌株MNH05026、MNH22004、MNH27256新鲜培养物，进行菌株基因组DNA的提取。采用提取的菌株基因组DNA作为模板进行16S rRNA基因扩增。
本发明16S rRNA基因PCR使用的引物对如下：
27F：AGAGTTTGATCMTGGCTCAG和1492R：TACGGYTACCTTGTTACGACTT。
PCR反应程序如下：PCR反应循环：预变性：94℃，4mim；变性：94℃，50sec；退火52℃，40sec；延伸：72℃，70sec；终延伸：72℃，10min；循环36次。
PCR扩增完成后，将PCR产物进行纯化，交由金唯智公司进行16S rRNA基因测序。将测序返回的菌株16S rRNA基因序列提交到NCBI Basic Local Alignment Search Tool，进行菌株16S rRNA基因分析，确认菌株分类信息。
菌株MNH05026的16S rRNA基因扩增产物送出测序，得到16S rRNA基因序列(SEQ ID NO:1)，将测得的序列与GenBank中的数据通过BLAST进行分析，比对结果表明菌株MNH05026属于Megasphaera菌属，与其相似性最高的菌株是Megasphaera hexanoica(96.50％)和Megasphaera elsdenii(94.43％)，菌株MNH05026与Megasphaera菌属其它菌株16S rRNA基因序列相似性均低于94.30％；依据Kim等人(2014)通过对数千个基因组和16S rRNA基因序列的统计分析发现，当两菌株16S rRNA基因序列之间相似性低于98.65％时，可判断它们归属于不同的种这个判断标准，本发明中的实验菌株MNH05026与Megasphaera菌属其它已知的菌株能显著区分开来，因此本发明的MNH05026是Megasphaera菌属的一个新物种菌株。
基于同样的分析，MNH22004(SEQ ID NO:2)，MNH27256(SEQ ID NO:3)测得的序列与GenBank中的数据通过BLAST进行分析，比对结果表明MNH22004(SEQ ID NO:2)和MNH27256(SEQ ID NO:3)同属于Megasphaera菌属。其中，与MNH22004相似性较高的菌株是Megasphaera micronuciformis(90.92％)，Megasphaera massiliensis(90.63％)，Megasphaera hexanoica(90.63％)，Megasphaera elsdenii(90.61％)，Megasphaera paucivorans(90.59％)，Megasphaera indica(90.29％)，Megasphaera sueciensis(90.18％)；菌株MNH22004与Megasphaera菌属与其它已知的菌株其它菌株16S rRNA基因序列相似性均低于90％；较低(<95％)的16S rRNA基因序列相似性表明菌株MNH22004可能是Megasphaera菌科的一个新物种。与MNH27256相似性最高的菌株是Megasphaera stantonii AJH120(MG811574)，二者之间的16S rRNA基因相似性为98.41％；菌株MNH27256与Megasphaera菌属其它已知的菌株16S rRNA基因序列相似性均低于94％，表明MNH27256可能是Megasphaera菌属的一个新物种菌株。
将MNH05026、MNH22004和MNH27256与从GenBank等数据库中调取的Megasphaera菌属及其近缘物种相关菌株的16S rRNA基因序列进行比较(表4)，以及从NCBI下载Megasphaera菌属下Megasphaera_massiliensis、Megasphaera elsdenii菌株的全基因数据与本发明的MNH05026、MNH22004、MNH27256进行比对(表5)；并构建系统进化树。从系统进化树可以看出菌株MNH05026与Megasphaera菌属菌株Megasphaera hexanoica共同形成一个单独的分枝(Bootstrap支持值为99)。菌株MNH22004与Megasphaera菌属菌株Megasphaera stantonii AJH120T共同形成一个单独的分枝(Bootstrap支持值为100)。菌株MNH27256与Megasphaera菌属菌株Megasphaera stantonii AJH120共同形成一个进化分枝(Bootstrap支持值为100)。将MNH05026、MNH 22004和MNH 27256从NCBI数据库中获得的16S rRNA基因序列相似性较高模式菌株的序列进行多序列比对，然后采用最大似然法，利用软件MEGA 5构建系统发育树，图9A为MNH05026的系统发育树，图9B为MNH 22004的系统发育树，图9C为MNH 27256的系统发育树，图中发育树节点只显示Bootstrap值大于50％数值，上标的“T”表示模式菌株。
表4 16S rRNA基因序列比较结果：
表5与Megasphaera elsdenii、Megasphaera_massiliensis两两比对结果：
从表5可知，MNH05026、MNH22004、MNH27256不属于Megasphaera_massiliensis、Megasphaera elsdenii物种下的菌株，与Megasphaera_massiliensis、Megasphaera elsdenii在基因组学上存在明显差异。
结合表4和表5进一步可知，虽然MNH05026(SEQ ID NO:1)、MNH22004(SEQ ID NO:2)、MNH27256(SEQ ID NO:3)同属于Megasphaera菌属。根据ANI>95％为同一物种，本申请基于平均核苷酸一致性(ANI)的基因组相关性分析表明，MNH05026与MNH22004、MNH27256属于巨球型菌属中的新物种，而MNH22004与MNH27256属于巨球型菌属中的物种，且属于同种不同株的两株菌。
MNH05026、MNH22004、MNH27256基因组分析：
MNH05026原始菌株的基因组通过超声波法进行序列片段化，片段化长度范围～ 350bp，然后利用标准DNA建库试剂盒(NEB UltraTM)构建Illumina测序文库。将构建好的测序文库利用NovaSeq(Illumina)进行双端150bp测序。测序得到1.46Gbp数据，其中Q20占比为：97.08％。MNH22004和MNH27256采用同样的方法，分别测序得到1.33Gbp和1.39Gbp数据，其中Q20占比分别为97.27％和96.95％。
基因组原始测序数据使用fastp(版本：0.20.0)进行数据过滤，过滤参数：“--poly_g_min_len 10 --poly_x_min_len 10 -q 15 -u 40 -n 5 -l 50”。过滤后的原始数据使用SPAdes(版本：v3.14.0)进行基因组组装，组装参数“--isolate--cov-cutoff 10”。基因组组装得到MNH05026、MNH22004和MNH27256基因总长度分别为2.79Mbp、2.58Mbp和2.59Mbp，N50长度分别为59.8kbp、157.9kbp和92.9kbp，GC含量分别为50.46％、52.86％和52.85％。
基因组基因使用原核分析软件基因组注释流程prokka(版本：1.14.5)进行基因组基因预测分析，参数“--gcode 11--evalue 1e-09”。MNH05026总共预测得到2568条CDS序列，其中平均CDS序列长度为957bp。基因组相似度最高的模式菌株为Megasphaera elsdenii，其平均核苷酸相似度(ANI)为78.58％，基因覆盖度为30.27％，因此，可以认定为巨球型菌属下的一个新种。MNH22004总共预测得到2353条CDS序列，其中平均CDS序列长度为967bp。与实验菌株MNH22004基因组相似度最高的模式菌株为Megasphaera elsdenii，二者之间平均核苷酸相似度(ANI)为79.49％，基因覆盖度为42.37％；同时结合菌株MNH22004的16S rRNA基因分析结果，可以确认菌株MNH22004可能是Megasphaera菌属中的一个新物种。MNH27256总共预测得到2335条CDS序列，其中平均CDS序列长度为975bp。与实验菌株MNH27256基因组相似度最高的模式菌株为菌Megasphaera elsdenii，二者之间平均核苷酸相似度(ANI)为79.38％，基因覆盖度为41.04％。
基因组中潜在的抗生素耐药基因使用RGI流程分析(版本：4.2.2)，其中抗生素耐药基因数据库为CARD(版本：3.0.0，https://card.mcmaster.ca/analyze/rgi)。MNH05026未比对出抗生素耐药基因。MNH22004和MNH27256比对出的耐药基因信息见表6。
表6耐药基因信息：
对基因组中潜在的毒力因子及相关基因的分析采用的是NCBI blastp(版本为：2.7.1+)比对毒力因子数据库VFDB(virulence factor database，http://www.mgc.ac.cn/cgi-bin/VFs/v5/main.cgi，更新日期为2019年9月19日)。详细比对结果见表7。
表7 MNH05026、MNH22004、MNH27256潜在毒性基因列表：

对基因组中潜在的次级代谢基因簇的分析采用的是antiSMASH5(版本为：5.1.1)。MNH05026未比对出次级代谢基因簇，MNH22004和MNH05026比对结果见表8。
表8.MNH22004和MNH05026潜在次级代谢基因簇：
对基因组中潜在的初级代谢基因簇的分析采用的是gutSMASH5(版本为：1.0.0)。详细比对结果见表9-A、9-B和9-C。
表9-A MNH05026潜在初级代谢基因簇列表：

表9-B.MNH22004潜在初级代谢基因簇：

表9-C.MNH27256潜在初级代谢基因簇：

对菌株产丁酸能力进行评估，采用现有技术(Vital M,Howe C,Tiedje MegasphaeraRevealing the Bacterial Butyrate Synthesis Pathways by Analyzing(Meta)genomic Data[J].Mbio,2014,5(2):1-11.)中的产丁酸通路有关基因作为参考数据库，用NCBI blastp(版本为：2.7.1+)将菌株的基因组序列和参考数据库进行比对，详细比对结果见表10，进而计算产丁酸通路的完整性，通过计算发现MNH05026菌株产丁酸通路的完整性为80％,MNH27256和MNH22004产丁酸通路的完整性为100％。
表10 MNH05026、MNH22004、MNH27256潜在产丁酸基因列表：
菌株MNH05026、MNH22004、MNH27256脂肪酸组分分析：
将菌株MNH05026(简称A026)接种在TSA平板上，37℃厌氧培养48小时后，收集菌体，进行菌体脂肪酸提取和甲基化处理；使用美国MIDI公司的(Microbial ID,Inc.,Newark,Del)(Kroppenstedt,1985；Meier et al.,1993)全自动细菌鉴定系统进行菌株MNH05026脂肪酸组分分析(见表11A)。MNH22004和MNH27256采用同样的方法进行分析，结果分别见表11B和11C。
从结果看，MNH05026主要脂肪酸(>10％)是十二碳饱和脂肪酸(C12:0 11.98％)和十九碳不饱和脂肪酸(C18:1CIS 9FAME 11.28％)；其余类型脂肪酸及含量详见表11A。MNH22004主要脂肪酸(>10％)是十二碳饱和脂肪酸(C12:0 14.43％)、十六碳饱和脂肪酸(C16:0 13.87％)、3-羟基十四碳饱和脂肪酸(C14:0 3OH 11.84％)和十八碳单不饱和脂肪酸(C18:1CIS 9 10.14％)；其余类型脂肪酸及含量详见表11B。MNH27256主要脂肪酸(>10％)是十二碳饱和脂肪酸(C12:0 15.54％)、十八碳单不饱和脂肪酸(C18:1CIS 9 15.03％)和3-羟基十四碳饱和脂肪酸(C14:0 3OH 13.22％)；其余类型脂肪酸及含量详见表11C。
表11A菌株MNH05026脂肪酸组分表：




表11B菌株MNH 22004脂肪酸组分表：



表11C菌株MNH27256脂肪酸组分表：



综合形态特征、显微特征、生理生化特征、16S rRNA基因序列和基因组信息等数据，显示菌株MNH05026属于Megasphaera菌属的一个新物种菌株，本发明暂时将其命名为Megasphaera sp.MNH05026(专利菌种保藏编号为GDMCC No:62001)。菌株MNH22004和MNH27256都属于Megasphaera菌属中的同一个新物种下不同菌株，本发明暂时将其分别命名为Megasphaera sp.MNH22004(专利菌种保藏编号为GDMCC NO:62000)和Megasphaera sp.MNH27256(专利菌种保藏编号为GDMCC NO:61999)。
实施例3短链脂肪酸(SCFA)测定：
以MNH05026为例进行描述，MNH22004和MNH27256采用相同的方法进行测定。
菌体制备:将菌株MNH05026接种在TSB液体培养基中，37℃厌氧培养48小时，离心收集菌体，收集物置于-86℃低温保存，待用。
标准品配制:称量乙酸、丙酸、丁酸、异丁酸、戊酸、异戊酸和己酸标准品，用乙酸乙酯配制成0.1μg/mL、0.5μg/mL、1μg/mL、5μg/mL、10μg/mL、20μg/mL、50μg/mL、100μg/mL八个混合标准浓度梯度。取600μL标准品，加入25μL终浓度为500μM的4-甲基戊酸作为内标，混匀加入进样瓶，进入GC-MS检测，进样量1μL，分流比10:1，分流进样。
代谢物提取:将样品冰上解冻，取80mg样品于2mL玻璃离心管中，加入900μL 0.5％的磷酸重悬，震荡混匀2min，14000g离心10min，取上清液800μL，加入等量的乙酸乙酯提取，震荡混匀2min，14000g离心10min，取600μL上层有机相，加入终浓度为500μM的4-甲基戊酸作为内标，混匀加入进样瓶，进入GC-MS检测，进样量1μL，分流比10:1，分流进样。
样本检测分析:采用Agilent DB-WAX毛细管柱(30m×0.25mm ID×0.25μm)气相色谱系统对样本进行分离。程序升温:初始温度90℃，以10℃/min升温至120℃，再以5℃/min升温至150℃，最后以25℃/min升温至250℃，并维持2min。载气为氦气载气流速1.0mL/min。
采用Agilent 7890A/5975C气质联用仪进行质谱分析。进样口温度250℃，离子源温度230℃，传输线温度250℃，四极杆温度150℃。电子轰击电离(EI)源，全扫及SIM扫描方式，电子能量70eV。
采用MSD ChemStation软件提取色谱峰面积及保留时间。绘制标准曲线，计算样品中短链脂肪酸的含量(见表12)。
表12短链脂肪酸(SCFA)产量结果：
结论：菌株MNH05026在生长过程中可以大量合成己酸、乙酸、异戊酸、丁酸和异丁酸，少量合成戊酸和丙酸。菌株MNH22004在生长过程中可以大量合成乙酸、丙酸、异戊酸、丁酸和异丁酸，特别是其具有较高的合成丁酸的能力；少量或极少合成己酸和戊酸。菌株MNH27256在生长过程中可以大量合成乙酸、丙酸、异戊酸、丁酸和异丁酸，特别是其具有较高的合成丁酸的能力；少量或极少合成己酸和戊酸。本申请中“高产”的含义是产量不低于200ug/g；因此，可以确定本申请的MNH05026、MNH22004、MNH27256高产乙酸，丁酸。
本发明中，术语“高产乙酸和/或丁酸”是指短链脂肪酸(SCFA)测定中乙酸和/或丁酸的产量≥200μg/g。优选地，乙酸产量≥250ug/g；优选地，乙酸产量≥300μg/g；优选地，乙酸产量≥400μg/g；和/或，优选地，丁酸产量≥300μg/g、≥400μg/g、≥500μg/g、≥600μg/g、≥700μg/g、≥800μg/g、≥900μg/g、≥1000μg/g、≥1100μg/g、≥1200μg/g、≥1300μg/g、≥1400μg/g、≥1500μg/g或≥1600μg/g。
实施例4用于细胞筛选平台的菌体制备方法
以MNH05026为例进行描述，MNH22004和MNH27256采用相同的方法进行菌体制备。
用无菌牙签挑取MNH05026单克隆到10ml的AC液体培养基(每升含：蛋白胨，20g；葡萄糖，5g；酵母提取物，3g；牛肉浸粉，3g；维生素C，0.2g；pH7.0)37℃厌氧培养1天，取1ml培养液进行质谱鉴定，鉴定结果正确后，吸取2ml菌悬液转接入80ml AC液体培养基中于厌氧操作台37℃培养。培养24小时后，吸取30ml菌悬液转接入400ml AC液体培养基中于厌氧操作台37℃培养。培养24小时后，取1ml菌液进行质谱鉴定，鉴定结果正确后，将全部菌液转移到1L离心瓶中，6000rpm，4℃离心30min。取沉淀用AC液体培养基：无菌甘油(4：1)重悬。
将MNH05026菌液甘油混合液吸取0.1ml菌株冻存液加入0.9ml生理盐水中进行梯度稀释，稀释至10-8梯度，取浓度为10-6、10-7、10-8梯度的稀释液100μl分别加入厌氧血平板培养基上，加入玻璃珠摇匀，采用平板计数法测定其活菌数CFU。
将制作好的菌株冻存液分装于无菌冻存管中，每管分装0.2ml菌液，每株菌分装10 管。冷冻于-80℃冷冻保存。待完全冷冻后(24h)，取出一管冻存细菌，融化至室温，并采用稀释涂布法测量其活菌数(CFU)，备用。
实施例5菌株MNH05026、MNH22004在高脂饮食诱导肥胖小鼠模型的体内试验的肝 肾功能及相关疾病的实验
利用高脂饮食诱导肥胖小鼠模型进行MNH05026对肝肾功能及相关疾病的改善实验。本发明已通过慕恩生物实验动物管理和使用委员会伦理审查。
1)实验动物：实验用鼠为C57BL/6J小鼠，鼠龄5-6周，总共20只，购自广东药康生物科技有限公司。
2)供试菌株：将MNH05026菌种甘油冻存管在37℃下融化后，于厌氧工作站中接种于M01平板上进行活化，将活化后的菌株接种于M01液体培养基中，厌氧培养，获得足够数量的培养物，将培养好的菌液离心浓缩后用溶媒将菌体重悬，得到纯度和活菌数(2.9×109CFU/mL)满足动物实验要求的受试物。
3)阴性对照品：用含25％甘油的PBS-Cys作为阴性对照品
4)实验过程：5-6周龄的C57BL/6J雄性小鼠，检疫期结束后，高脂饮食饲喂10周，选取体重范围在35.50g-44.49g的小鼠16只，根据体重进行随机分层分组，8只/组，分为2组(实验组及对照组)。分组后(D1)开始灌胃给药，实验组灌胃菌液的量为0.2mL/只，实验组给与MNH05026，对照组给与阴性对照品，每天2次给药，共给药21天，实验期间小鼠自由饮水及采食，采用12h/12h昼夜循环。试验期间，每次虚拟给药或给药结束后进行1次一般临床观察。给药期每周一和周四、开始给药当天(D1)、给药第21天(D21)称量动物体重；试验终点解剖前称量动物体重。本试验终点为给药结束次日(D22)，试验终点根据方案进行解剖取材，汇总数据分析体重及体重变化百分比、空腹血糖测定结果、各解剖数据结果及血清检测数据结果。所有数据采用Mean±SD形式表示，用GraphPad Prism 8.0.2软件绘图和统计分析。对于两两比较，采用t检验(Student’s t test)分析方法。无显著性不表示；差异显著性用*表示，*p＜0.05，**p＜0.01，***p＜0.001，****p＜0.0001。
MNH22004采用1)～4)同样的方法进行相关实验，实验结果见图10。
图10为菌株MNH05026、MNH22004对高脂饮食诱导肥胖小鼠肝脏重量的影响图；
图10中结果显示MNH05026和MNH22004都能显著降低肝脏重量，且MNH05026和MNH22004的作用效果相当，说明MNH05026和MNH22004都能显著改善高脂饮食引起的脂肪肝。
基于图10的研究结果，进一步以MNH05026为代表探究了巨球型菌属细菌在治疗或预防肝脏功能损伤的用途，结果如图11所示。
图11为菌株MNH05026对高脂饮食诱导肥胖小鼠血清中谷丙转氨酶(ALT)含量的影响图；
结果显示MNH05026能降低血清中谷丙转氨酶(ALT)的含量，图11中，丙氨酸氨基转移酶(ALT)和天冬氨酸氨基转移酶(AST)是肝细胞损伤的标志物，图11结果表明MNH05026能降低血清中ALT含量，说明MNH05026能改善高脂饮食引起的肝功能异常，能显著改善高脂饮食引起的脂肪肝及肝功能异常。综上，MNH05026和MNH22004都能显著降低肝脏重量，且MNH05026还能降低血清中ALT含量，说明MNH05026和MNH22004能显著改善高脂饮食引起的脂肪肝及肝功能异常。
实施例6.菌株MNH05026和MNH27256用于预防或治疗高脂饮食诱导肥胖小鼠模型的 体内试验的糖尿病的用途
1)实验动物、2)阴性对照品同实施例5；3)供试菌株：将MNH05026菌种甘油冻存管在37℃下融化后，于厌氧工作站中接种于M01平板上进行活化，将活化后的菌株接种于M01液体培养基中，厌氧培养，获得足够数量的培养物，将培养好的菌液离心浓缩后用溶媒将菌体重悬，得到纯度和活菌数(2.9×109CFU/mL)满足动物实验要求的受试物。
将MNH27256菌种甘油冻存管在37℃下融化后，于厌氧工作站中接种于M01平板上进行活化，将活化后的菌株接种于M01液体培养基中，厌氧培养，获得足够数量的培养物，将培养好的菌液离心浓缩后用溶媒将菌体重悬，得到纯度和活菌数(7.6×109CFU/mL)满足动物实验要求的受试物。
4)实验过程：5-6周龄的C57BL/6J雄性小鼠，检疫期结束后，高脂饮食饲喂10周，选取体重范围在35.50g-44.49g的小鼠16只，根据体重进行随机分层分组，8只/组，分为2组(实验组及对照组)。分组后(D1)开始灌胃给药，实验组给与MNH05026，实验组灌胃菌液的量为0.2mL/只，对照组给与阴性对照品，每天2次给药，共给药21天，实验期间小鼠自由饮水及采食，采用12h/12h昼夜循环。试验期间，每次虚拟给药或给药结束后进行1次一般临床观察。给药期每周一和周四、开始给药当天(D1)、给药第21天(D21)称量动物体重；试验终点解剖前称量动物体重。本试验终点为给药结束次日(D22)，试验终点根据方案进行解剖取材，汇总数据分析体重及体重变化百分比、空腹血糖测定结果、各解剖数据结果及血清检测数据结果。所有数据采用Mean±SD形式表示，用GraphPad Prism 8.0.2软件绘图和统计分析。对于两两比较，采用t检验(Student’s t test)分析方法。无显著性不表示；差异显著性用*表示，*p＜0.05，**p＜0.01，***p＜0.001，****p＜0.0001。
MNH27256采用上述1)～4)同样的方法进行相关实验。
5)实验结果
5.1)对高脂饮食诱导肥胖小鼠空腹血糖FBG的影响
MNH05026和MNH27256对高脂饮食诱导肥胖小鼠空腹血糖(FBG)的影响结果分别见图12A和图12B。(数据采用平均值±标准差(Mean±SD)显示。统计分析采用t检验(Student’s t test)分析方法；**，与HFD-Control组相比p＜0.01。)
图12为菌株MNH05026和MNH27256对高脂饮食诱导肥胖小鼠空腹血糖(FGB)的影响，图12C为MNH27256对高脂饮食诱导肥胖小鼠葡萄糖耐量(OGTT)结果，图12D为MNH27256对高脂饮食诱导的肥胖小鼠葡萄糖耐量曲线下面积结果)；
图12A和图12B的结果显示MNH05026和MNH27256都能降低高脂饮食诱导肥胖小鼠的空腹血糖，说明MNH05026和MNH22004都能显著改善高脂饮食引起的血糖升高，具有治疗糖尿病的作用。
5.2)菌株MNH27256对高脂饮食诱导肥胖小鼠口服葡萄糖耐量(OGTT)的影响
基于MNH05026和MNH27256菌株都能降低高脂饮食诱导的肥胖小鼠的FBG，且MNH05026的降血糖效果较MNH27256的降血糖效果更佳。基于此，优先选择以MNH27256为代表进行进一步实验，探究其对高脂饮食诱导的肥胖小鼠OGTT的影响。
口服葡萄糖耐量检查：给药第17天(D17)，测定禁食12h的OGTT(如晚上20:30:00禁食至次日08:30:00)。称量小鼠禁食体重，按照小鼠禁食体重值灌胃葡萄糖，灌胃葡萄糖剂量为2g/kg(葡萄糖g/小鼠禁食体重kg)。测定空腹血糖、给糖后15min、30min、 60min、90min、120min血糖值。每只小鼠严格计时，准确按照6个时间点测定血糖值。测定结果见图12C和12D。
图12C-12D为MNH27256对高脂饮食诱导的肥胖小鼠的口服葡萄糖耐量试验结果，OGTT实验是一种葡萄糖负荷实验，用以了解胰岛β细胞功能和机体对血糖的调节能力；观察病人耐受葡萄糖的能力，是目前公认用于诊断糖尿病的一项诊断指标。当糖代谢紊乱时，口服一定量的葡萄糖后血糖急剧升高，或升高不明显，但短时间内不能降至空腹水平或原来水平，此为糖耐量异常或糖耐量降低；糖耐量异常表明机体对葡萄糖的代谢能力下降，常见于2型糖尿病和肥胖症；图12C为口服葡萄糖耐量结果，图12D为口服葡萄糖耐量曲线下面积结果，MNH27256能有效改善血糖升高，提高了糖耐量水平，主要表现为使得OGTT的AUC水平降低具有预防或治疗糖尿病的用途，表明菌株具有预防或治疗糖尿病的用途，尤其是肥胖引起的糖尿病、II型糖尿病和/或具有非酒精性脂肪肝/非酒精性脂肪肝炎患者的糖尿病。
实施例7.菌株MNH05026和MNH27256分别与Metformin联合治疗II型糖尿病模型 小鼠的高血糖
1)实验动物：实验用鼠为BKS-Leprem2Cd479/Gpt(db/db)小鼠，鼠龄5-6周，总共24只，购自广东药康生物科技有限公司。
2)供试菌株：将MNH05026菌种甘油冻存管在37℃下融化后，于厌氧工作站中接种于M01平板上进行活化，将活化后的菌株接种于M01液体培养基中，厌氧培养，获得足够数量的培养物，将培养好的菌液离心浓缩后用溶媒将菌体重悬，得到纯度和活菌数(6.3×109CFU/mL)满足动物实验要求的受试物。
MNH27256采用与MNH05026同样的方法进行活化和培养、得到纯度和活菌数(6.1×109CFU/mL)满足动物实验要求的受试物。
3)阴性对照品：用含0.05％L-Cys HCl的PBS作为阴性对照品
4)Metformin(阳性对照)：250mg/kg,灌胃
MNH27256采用上述1)～4)同样的方法进行相关实验设置。
5)实验过程：5-6周龄的BKS-Leprem2Cd479/Gpt(db/db)雄性小鼠24只，检疫期结束后，根据体重进行随机分层分组，8只/组，分为3组,分别为：阴性对照组(HFD-Control)、阳性对照组(Metformin)、联合用药组(MNH05026+Metformin、MNH27256+Metformin)。分组后(D1)开始给药，阴性对照组给与阴性对照品，阳性对照组给与Metformin(250mg/kg),联合用药组给与对应菌液(0.2mL/只小鼠)和Metformin(250mg/kg)，菌液(MNH05026和MNH27256)为每天2次给药，Metformin为每天1次给药，给药方式均为灌胃给药，共给药28天，实验期间小鼠全程饲喂大小鼠维持饲料，自由饮水及采食，采用12h/12h昼夜循环。在给药第27天(D27)进行一次OGTT测定，给药第28天(D28)测量一次空腹血糖,给药结束次日(D29)对所有组别进行解剖取材，汇总数据分析空腹血糖、OGTT等数据结果。所有数据采用Mean±SD形式表示，用GraphPad Prism 8.0.2软件绘图和统计分析。对于三个以上的组相互比较，采用单因素方差分析(One-Way ANOVA)方法与搭配Dunnett多重比较分析(Dunnett’s multiple comparisons test)进行分析。无显著性不表示，差异显著性用*表示，*p＜0.05，**p＜0.01，***p＜0.001，****p＜0.0001。
6)实验结果：
6.1)MNH05026与Metformin联用对II型糖尿病小鼠口服葡萄糖耐量的改善效果：
口服葡萄糖耐量检查(OGTT)：给药第27天(D27)，测定禁食12h的OGTT(如晚上20:30:00禁食至次日08:30:00)。称量小鼠禁食体重，按照小鼠禁食体重值灌胃葡萄糖，灌胃葡萄糖剂量为2g/kg(葡萄糖g/小鼠禁食体重kg)。测定空腹血糖、给糖后15min、30min、60min、90min、120min血糖值。每只小鼠严格计时，准确按照6个时间点测定血糖值。
本实验采用的模型小鼠(BKS-Leprem2Cd479/Gpt(db/db)小鼠)携带有Leptin基因的突变,从而表现出Ⅱ型糖尿病的症状。BKS背景DB/DB小鼠为糖尿病易感性，出现重度糖尿病症状，胰岛B细胞严重受损。
此外，Leptin受体点突变导致leptin信号通路障碍，从而也会导致小鼠出现肥胖、胰岛素抵抗、高血糖、脂肪肝等症状。
口服葡萄糖耐量试验是一种葡萄糖负荷实验，用以了解胰岛β细胞功能和机体对血糖的调节能力；观察病人耐受葡萄糖的能力，是目前公认用于诊断糖尿病的一项诊断指标。当糖代谢紊乱时，口服一定量的葡萄糖后血糖急剧升高，或升高不明显，但短时间内不能降至空腹水平或原来水平，此为糖耐量异常或糖耐量降低；糖耐量异常表明机体对葡萄糖的代谢能力下降，常见于2型糖尿病和肥胖症。
图13为MNH05026与Metformin联用对II型糖尿病小鼠的口服葡萄糖耐量(OGTT)的影响结果。
图13A为MNH05026与Metformin联用对II型糖尿病小鼠的口服葡萄糖耐量(OGTT)的影响结果，图13B为各处理组实验小鼠的口服葡萄糖耐量曲线下面积结果；
图13A为MNH05026与Metformin联用对II型糖尿病小鼠口服葡萄糖耐量的改善效果试验结果，图13B为各实验组小鼠的口服葡萄糖耐量曲线下面积结果(数据采用平均值±标准差(Mean±SD)显示，统计分析采用单因素方差分析(One-Way ANOVA)方法与搭配Dunnett多重比较分析(Dunnett’s multiple comparisons test)进行分析，*p＜0.05)。结果表明菌株MNH05026与Metformin联合较单独使用Metformin能更加有效的改善血糖升高，更显著的提高了糖耐量水平，主要表现为给糖后联合用药组在5个时间点测得的血糖值皆低于阳性对照组，且在给糖后60分钟和给糖后90分钟这一时间段内，联合用药组的血糖值显著降低，最后变现为AUC水平显著降低。这说明MNH05026与Metformin联和更好的控制血糖并预防和控制糖尿病，尤其是II型糖尿病；此外该实验也表明MNH05026与Metformin联和能有效改善胰岛素抵抗、改善糖耐量，对于预防或治疗II型糖尿病能够达到更好的效果，具有预防或治疗糖尿病的用途，尤其是肥胖引起的糖尿病、II型糖尿病和/或具有脂肪肝/脂肪肝炎患者的糖尿病。
6.2)MNH05026和MNH27256分别与Metformin联用对II型糖尿病小鼠空腹血糖的影响实验结果：
图14为MNH05026和MNH27256分别与Metformin联用对II型糖尿病小鼠的空腹血糖影响结果；
图14为MNH05026与Metformin联用对于空腹血糖的影响结果，MNH27256与Metformin联用对于空腹血糖影响结果，数据采用平均值±标准差(Mean±SD)显示。统计分析采用单因素方差分析(One-Way ANOVA)方法与搭配Dunnett多重比较分析(Dunnett’s multiple comparisons test)进行分析。**p＜0.01。
Metformin属于AMPK激动剂，其通过促进GLP-1分泌而发挥降低血糖治疗糖尿病的作用，表明本申请的菌株MNH05026能够促进Metformin的治疗作用，提升促进GLP-1 分泌实现降血糖以及治疗糖尿病的效果。图14结果显示，MNH05026和MNH27256分别与Metformin联用能更显著的降低II型糖尿病小鼠的空腹血糖，且MNH05026与Metformin联用比MNH27256与Metformin联用能更明显降低空腹血糖，表明本申请的菌株MNH05026和MNH27256都能够通过进一步促进GLP-1的分泌起到促进Metformin的治疗效果的作用，实现降血糖以及治疗糖尿病的目的。且MNH05026与Metformin联用的降血糖效果更显著，可以针对血糖值更高的患者使用。
6.3)MNH27256单独及与Metformin联合治疗II型糖尿病模小鼠的高血糖：
基于MNH27256与Metformin联用具有更显著的降血糖效果，本实验进一步进行MNH27256单独使用及其与Metform联合使用治疗II型糖尿病小鼠的高血糖，以验证MNH27256对预防或治疗糖尿病的效果。本发明已通过慕恩生物实验动物管理和使用委员会(IACUC)伦理审查并监督实行。
1)实验动物：实验用鼠为C57BL/6J小鼠，鼠龄5-6周，总共54只，购自广东药康生物科技有限公司。
2)供试菌株：将MNH27256菌种甘油冻存管在37℃下融化后，于厌氧工作站中接种于M01平板上进行活化，将活化后的菌株接种于M01液体培养基中，厌氧培养，获得足够数量的培养物，将培养好的菌液离心浓缩后用溶媒将菌体重悬，得到纯度和活菌数(1.25×1010CFU/mL)满足动物实验要求的受试物。
3)阴性对照品：用含25％甘油的PBS-Cys作为阴性对照品
4)Metformin(阳性对照)：250mg/kg,灌胃
5)实验过程：5-6周龄的C57BL/6J雄性小鼠54只，检疫期结束后，高脂饮食饲喂6周，于第43天开始连续8天腹腔注射(i.p.)STZ，每天1次。D43所有小鼠腹腔注射(i.p.)剂量为50mg/kg的STZ注射液，D44～D50所有小鼠腹腔注射(i.p.)剂量为25mg/kg的STZ注射液。在D44、D46、D48、D50测定体重及随机血糖，在D49及D51测定动物禁食12h的空腹血糖。在D51，以D50的随机血糖≥13.9mM为主要参考标准，以两次空腹血糖≥11.1mM为次要参考标准，选择血糖值合格的32只小鼠转移至后续干预试验。根据空腹血糖进行随机分层分组，8只/组，分为4组,分别为：阴性对照组(Control)、阳性对照组(Metformin)、MNH27256组，联合用药组(MNH27256+Metformin)。分组后(D1)开始给药，阴性对照组给与阴性对照品，阳性对照组给与Metformin(250mg/kg),MNH27256组给与MNH27256菌液(0.2mL/只小鼠)，联合用药组给与MNH27256菌液(0.2mL/只小鼠)和Metformin(250mg/kg)，MNH27256菌液为每天2次给药，Metformin为每天1次给药，给药方式均为灌胃给药，共给药28天，实验期间小鼠全程饲喂高脂饲料，自由饮水及采食，采用12h/12h昼夜循环。给药期间每周测定一次空腹血糖数据结果。所有数据采用Mean±SD形式表示，用GraphPad Prism 8.0.2软件绘图和统计分析。对于三个以上的组相互比较，采用单因素方差分析(One-Way ANOVA)方法与搭配Dunnett多重比较分析(Dunnett’s multiple comparisons test)进行分析。无显著性不表示，差异显著性用*表示，*p＜0.05，**p＜0.01，***p＜0.001，****p＜0.0001。
图15A为MNH27256单独使用及其与Metformin联用对II型糖尿病小鼠的空腹血糖变化曲线图；
图15A为空腹血糖变化曲线图，图15B为干预终点空腹血糖值。从图15A可知，MNH27256单独使用时即具有降低空腹血糖的效果，MNH27256与Metformin联用后具 有更明显的降血糖效果。另外，MNH27256与Metformin联用后明显能促进Metformin的降血糖效果，且在使用初期(前8天)MNH27256具有与Metformin相当的降血糖效果，在使用终点时的血糖值也与Metformin组相当。说明MNH27256单独使用的整体效果虽不如Metformin，但其在长周期控血糖方面具有使用价值，且菌株MNH27256能够通过进一步促进GLP-1的分泌起到促进Metformin的治疗效果的作用，实现降血糖以及治疗糖尿病的效果。表明MNH27256单用及与Metformin联合均具有降低空腹血糖的效果，具有预防或治疗糖尿病的用途。
实施例8.菌株MNH05026、MNH22004和MNH27256在高脂饮食诱导肥胖小鼠模型的 体内试验以验证对应菌株在治疗和预防肥胖以及相关疾病中的应用
1)实验动物、2)供试菌株、3)阴性对照品同实施例5；
4)实验过程：5-6周龄的C57BL/6J雄性小鼠，检疫期结束后，高脂饮食饲喂10周，选取体重范围在35.50g-44.49g的小鼠16只，根据体重进行随机分层分组，8只/组，分为2组(实验组及对照组)。分组后(D1)开始给药，实验组给与MNH05026菌液(0.2mL/只小鼠)，对照组给与阴性对照品，每天2次给药，共给药21天，实验期间小鼠自由饮水及采食，采用12h/12h昼夜循环。试验期间，每次虚拟给药或给药结束后进行1次一般临床观察。给药期每周一和周四、开始给药当天(D1)、给药第21天(D21)称量动物体重；试验终点解剖前称量动物体重。本试验终点为给药结束次日(D22)，试验终点根据方案进行解剖取材，汇总数据分析体重及体重变化百分比、空腹血糖测定结果、各解剖数据结果及血清检测数据结果。所有数据采用Mean±SD形式表示，用GraphPad Prism 8.0.2软件绘图和统计分析。对于两两比较，采用t检验(Student’s t test)分析方法。无显著性不表示；差异显著性用*表示，*p＜0.05，**p＜0.01，***p＜0.001，****p＜0.0001。
菌株MNH05026、MNH22004和MNH27256分别都采用上述1)～4)的方法进行相关实验。
图16为菌株MNH05026、MNH27256和MNH22004对高脂饮食诱导的肥胖小鼠的体重获得图；
图16A为MNH05026、MNH22004对肥胖模型小鼠体重获得(即体重增长/降低变化情况)的影响结果，图16C为其实验终点(21天)时对应的小鼠体重获得值。结果显示与对照组相比，MNH05026、MNH22004都能显著降低高脂饮食诱导肥胖小鼠的体重获得，达到控制体重、防止体重过快增长的目的；且在给药后的前7天内，MNH05026和MNH22004都能在一定程度上略微降低肥胖小鼠体重。图16B为MNH27256对肥胖小鼠体重获得的影响变化图，显示MNH05026在给药后的整个实验周期内(共21天)一直都能稳定的控制肥胖小鼠的体重，在实验终点小鼠体重未出现显著增长，且整个实验期间小鼠的整体体重较给药前有不同程度的降低，以给药后第9天的体重降低程度最大，约为2.6％左右。综上可知，MNH05026、MNH22004和MNH27256都能显著抑制高脂饮食引起的体重增长，且MNH27256还能在一定程度上降低肥胖小鼠的体重，改善高脂饮食引起的体重增长，具有预防及治疗肥胖的效果。
实施例9.菌株MNH05026、MNH22004、MNH27256分别与Semaglutide联用在高脂高 糖高胆固醇饮食诱导NAFLD/NASH小鼠模型的体内试验以验证其在治疗和预防 NAFLD/NASH以及相关疾病中的应用
1)实验动物：实验用鼠为C57BL/6J雄性小鼠，5-6周龄，共24只，购自广东药康 生物科技有限公司。
2)供试菌株：分别将MNH05026、MNH22004和MNH27256菌种甘油冻存管在37℃下融化后，于厌氧工作站中接种于M01平板上进行活化，将活化后的菌株接种于M01液体培养基中，厌氧培养，获得足够数量的培养物，将培养好的菌液离心浓缩后用溶媒将菌体重悬，得到纯度和活菌数(1.5×109CFU/mL)满足动物实验要求的受试物。
3)对照品：用含25％甘油的PBS-Cys作为阴性对照品；用Semaglutide作为阳性对照品。
4)实验过程：24只5-6周龄的C57BL/6J雄性小鼠，检疫期结束后，高脂高糖高胆固醇饮食饲喂26周，根据体重进行随机分层分组，8只/组，分为3组，分别为阴性对照组(Control)、阳性对照组(Semaglutide)以及联合治疗组(对应菌株+Semaglutide)。分组后(D1)开始给药，阴性对照组给予阴性对照品(PBS-Cys)，灌胃给药，0.2mL/只，每天两次；阳性对照组给予Semaglutide，皮下注射给药，剂量为30nmol/kg，每3天一次；联合治疗组给予Semaglutide(剂量和使用频次与阳性对照组相同)和对应菌液(0.2mL/只，每天两次)，灌胃给药；试验周期为60天。实验期间小鼠自由饮水及采食，采用12h/12h昼夜循环。试验期间，每次给药结束后进行1次一般临床观察。给药期每周称量2次动物体重，试验终点解剖前称量动物体重。本试验终点为给药结束次日(D61)，试验终点根据方案进行解剖取材，汇总数据分析结果。所有数据采用Mean±SD形式表示，用GraphPad Prism 8.0.2软件绘图和统计分析。统计分析采用单因素方差分析(One-Way ANOVA)方法与搭配Dunnett多重比较分析(Dunnett’s multiple comparisons test)进行分析。无显著性不表示；差异显著性用*表示，*：p＜0.05，**：p＜0.01，***：p＜0.001，****：p＜0.0001。
菌株MNH05026、MNH22004和MNH27256分别都采用上述1)～4)的方法进行相关实验。
5)实验结果及分析：
5.1)本发明的巨球型菌与Semaglutide联用对NAFLD/NASH模型小鼠体重的影响：
图17为菌株MNH05026、MNH22004、MNH27256分别与Semaglutide联用对高脂高糖高胆固醇饮食诱导NAFLD/NASH小鼠的体重变化图；
巨球型菌与Semaglutide联用对高脂高糖高胆固醇饮食诱导NAFLD/NASH小鼠体重的影响结果见图17A-17C及表13。其中，图17A为巨球型菌菌株MNH05026、MNH22004、MNH27256分别和Semaglutide联用对高脂高糖高胆固醇饮食诱导NAFLD/NASH小鼠体重变化图，图17B为巨球型菌菌株MNH05026、MNH22004、MNH27256分别和Semaglutide联用在第60天时的小鼠体重值，图17C为MNH22004与Semaglutide联用对小鼠的体重获得图，表13为图17A中提取的部分数据。Semaglutide属于GLP-1受体激动剂，属于临床使用普遍的减肥药物，结合图17A、图17B和表13可以发现，Semaglutide治疗组在服用前期，能快速降低体重，且效果更为明显，然而随着时间的推移，当服用超过30天之后，机体开始对Semaglutide出现一定的耐药性，小鼠在30-60天之间体重开始反弹且反弹的速度高于联用组，干预33d时，Semaglutide处理组的小鼠体重获得值为-21.30％，MNH05026、MNH22004、MNH27256对应的获得值为-21.56％、-25.60％和-24.02％，即MNH05026、MNH27256和MNH22004与Semaglutide联用组在第33天之后，可以在Semaglutide减肥的基础上，进一步降低肥胖小鼠的体重获得，即连用组开始表现出比Semaglutide单用更好的体重控制效果。表明巨球型菌菌 株MNH05026、MNH27256、MNH22004在减肥的用途中具有一定的降低机体对Semaglutide的耐药程度，且能提高Semaglutide在肥胖治疗上的治疗效果。巨球型菌菌株MNH05026、MNH22004、MNH27256与Semaglutide联用具有预防及治疗肥胖患者的肥胖症的效果。在高脂高糖高胆固醇饮食诱导NAFLD/NASH模型小鼠中，巨球型菌菌株MNH05026、MNH22004、MNH27256与Semaglutide联用能够降低体重，减少体重获得，能够预防及治疗高脂高糖高胆固醇饮食引起的肥胖，具有预防和治疗NAFLD/NASH患者的肥胖症的效果。
表13 MNH05026、MNH22004、MNH27256与Semaglutide联用降低肥胖小鼠体重:
5.2)本发明的巨球型菌与Semaglutide联用对肝重、肝功能损伤的影响：
图18A为MNH05026、MNH22004和MNH27256分别与Semaglutide联用对高脂高糖高胆固醇饮食诱导NAFLD/NASH小鼠的肝重影响图；
巨球型菌与Semaglutide联用对高脂高糖高胆固醇饮食诱导NAFLD/NASH小鼠肝重及肝脏功能损伤的影响结果见图18A-18D。图18A结果显示MNH05026、MNH22004、MNH27256与Semaglutide联用能显著降低高脂高糖高胆固醇饮食诱导NAFLD/NASH模型小鼠的肝脏重量，比阳性对照品Semaglutide的效果更佳。说明巨球型菌与Semaglutide联用后，能提升Semaglutide对肝功能损伤的防治效果，更好的改善肝脏功能。其中，相较MNH22004和MNH27256与Semaglutide的联用效果，MNH05026与Semaglutide的联用能相对更为显著降低高脂高糖高胆固醇饮食诱导NAFLD/NASH模型小鼠的肝功能，具有更好的预防及治疗肝功能损伤的效果。
丙氨酸氨基转移酶(ALT)和天冬氨酸氨基转移酶(AST)是肝细胞损伤的标志物，而碱性磷酸酶(ALP)是一种主要在肝脏中分布的酶，而且是分布在肝细胞的血窦侧和毛细胆管侧的微绒毛上，经胆汁排入小肠，当胆汁排出不畅，毛细胆管内压力增加的时候，会产生大量的碱性磷酸酶，碱性磷酸酶高常常见于慢性肝炎、肝硬化或者酒精性肝炎；ALP活性升高较大也表明胆汁淤积，可能是肝内胆汁淤积或肝外胆汁淤积。因此，ALT，AST，ALP三个指标反馈肝细胞损伤、胆汁淤积或两者兼而有之的肝病。图18B、 图18C、图18D中显示，MNH05026、MNH22004、MNH27256与Semaglutide联用能显著降低血清中天门冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)和碱性磷酸酶(ALP)的含量，比阳性对照品Semaglutide的效果更佳，提示本申请的菌株MNH05026、MNH22004、MNH27256与Semaglutide联用可以预防或治疗肝脏损伤，预防或治疗慢性肝炎、肝硬化或酒精性肝炎，预防或治疗肝内胆汁淤积，预防或治疗肝外胆汁淤积；具有更好的改善NAFLD/NASH患者肝功能的效果；菌株MNH05026、MNH22004、MNH27256与Semaglutide联用具有预防或治疗NAFLD/NASH患者的脂肪肝的效果，改善NAFLD/NASH患者肝脏功能、可以预防或治疗NAFLD/NASH患者的肝脏损伤，预防或治疗NAFLD/NASH患者的肝内胆汁淤积，预防或治疗NAFLD/NASH患者的肝外胆汁淤积。
5.3)本发明的巨球型菌与Semaglutide联用对高脂高糖高胆固醇饮食诱导 NAFLD/NASH模型小鼠血糖的影响：
口服葡萄糖耐量用于测定胰岛β细胞功能和机体对血糖的调节能力，是目前公认用于诊断糖尿病的一项诊断指标。当糖代谢紊乱时，口服一定量的葡萄糖后血糖急剧升高，或升高不明显，但短时间内不能降至空腹水平或原来水平，此为糖耐量异常或糖耐量降低；糖耐量异常表明机体对葡萄糖的代谢能力下降，常见于II型糖尿病和肥胖症。
图19为MNH05026、MNH22004和MNH27256与Semaglutide联用对高脂高糖高胆固醇饮食诱导NAFLD/NASH小鼠模型口服糖耐量影响图；
巨球型菌与Semaglutide联用对高脂高糖高胆固醇饮食诱导NAFLD/NASH小鼠口服葡萄糖糖耐量和空腹血糖的影响结果见图19A-19E。从图19A-19D结果显示，与单独使用Semaglutide相比，MNH05026、MNH22004、MNH27256分别与Semaglutide联用都具有更加显著的改善小鼠的口服糖耐量(OGTT)，且三株巨球型菌联用Semaglutide后改善OGTT的效果相当。图19E可知，单独使用Semaglutide或者MNH27256与Semaglutide联用对降低NAFLD/NASH小鼠的空腹血糖没有显著性，但是MNH05026和MNH22004联用Semaglutide对降低NAFLD/NASH小鼠的空腹血糖有显著性，且MNH05026的显著性更强。
综上可知，MNH05026、MNH22004、MNH27256与Semaglutide联用能进一步促进Semaglutide对高脂高糖高胆固醇饮食诱导NAFLD/NASH模型小鼠的OGTT和FBG的控制水平，更显著的降低其OGTT和FBG，具有更好的预防及治疗NAFLD/NASH患者的糖尿病的效果，改善血糖控制功能，同时也可以用于II型糖尿病和肥胖引起的糖尿病。且三株巨球型菌中，尤以MNH05026与Semaglutide联用的防效最佳、MNH22004的防效次之、MNH27256的防效最弱，针对不同防效的菌株，可以开发不同剂量的产品适用不同病程的患者。
5.4)本发明的巨球型菌与Semaglutide联用对高脂高糖高胆固醇饮食诱导 NAFLD/NASH模型小鼠局部体脂脂肪重量的影响：
本申请进一步探究MNH05026、MNH22004、MNH27256与Semaglutide联用对于小鼠皮下各脂肪重量的影响，医学上通过药物进行减重，并不能得出身体局部脂肪随之减少，为了评价本申请的巨球型菌在减肥过程中对于局部脂肪的影响而开展本实验，实验测定了MNH05026、MNH22004和MNH27256分别与Semaglutide联用对高脂高糖高胆固醇饮食诱导的NAFLD/NASH小鼠模型皮下脂肪重量(subcutaneous fat pad)、腹股沟脂肪重量(Inguinal fat pad)、棕色脂肪重量(brown adipose tissue)和附睾脂肪重量 (epididymis fat pad)的影响，实验结果见图20A-20D。
图20别为MNH05026、MNH22004和MNH27256分别与Semaglutide联用对高脂高糖高胆固醇饮食诱导的NAFLD/NASH小鼠模型皮下脂肪重量(subcutaneous fat pad)、腹股沟脂肪重量(Inguinal fat pad)、棕色脂肪重量(brown adipose tissue)和附睾脂肪重量(epididymis fat pad)的影响；
结果表明，MNH05026、MNH22004、MNH27256与Semaglutide联用能够对于局部的皮下脂肪、腹股沟脂肪、附睾脂肪、棕色脂肪都能显著降低。其中，尤以MNH05026的整体防效最佳，MNH27256次之，MNH22004最弱。即本申请的巨球型菌MNH05026、MNH22004、MNH27256与Semaglutide联用能显著降低高脂高糖高胆固醇饮食诱导NAFLD/NASH模型小鼠的脂肪积累，比阳性对照品Semaglutide的效果更佳或相当，具有较理想的预防及治疗NAFLD/NASH患者肥胖症及脂肪堆积的功效。
5.5)本发明的巨球型菌与Semaglutide联用对高脂高糖高胆固醇饮食诱导NAFLD/NASH模型小鼠血脂的影响：
以MNH05026为代表，将其与Semaglutide联用验证其对高脂高糖高胆固醇饮食诱导的NAFLD/NASH小鼠模型血脂四项：甘油三酯(TG)、总胆固醇(CHO)、高密度脂蛋白(HDL)、低密度脂蛋白(LDL)的影响；TG主要参与人体内能量代谢，产生热能。血中TG含量过高可导致血液黏稠，使脂质在血管壁上沉积，渐渐形成小斑块，即动脉粥样硬化。LDL-C增高是动脉粥样硬化发生、发展的主要、独立危险因素；LDL-C的增高水平也是衡量冠心病的指标。由于HDL-C能将血管壁内胆固醇转运至肝脏进行分解代谢(即胆固醇逆转运)，可减少胆固醇在血管壁的沉积，起到抗动脉粥样硬化作用。结果见图21A-21D。
图21为MNH05026与Semaglutide联用对高脂高糖高胆固醇饮食诱导的NAFLD/NASH小鼠模型甘油三酯(TG)、总胆固醇(CHO)、高密度脂蛋白(HDL)、低密度脂蛋白(LDL)的影响；
结果表明，菌株MNH05026与Semaglutide联用能显著降低高脂高糖高胆固醇饮食诱导NAFLD/NASH模型小鼠血脂的TG、CHO、HDL三项。表明菌株MNH05026与Semaglutide联用能在不同程度上降低肥胖小鼠血清中的总胆固醇、甘油三脂及低密度脂蛋白的含量，具有预防及治疗心血管疾病、动脉粥样硬化、冠心病、降血脂的效果、同时还表明菌株MNH05026与Semaglutide联用具有治疗/预防心脑血管疾病的效果，菌株MNH05026与Semaglutide联用能显著降低高脂饮食小鼠血清中的低密度脂蛋白含量，具有预防或治疗高血脂、动脉粥样硬化的用途。能更进一步降低血清总胆固醇，提示菌株MNH05026与Semaglutide联用能预防及治疗心血管疾病、降血脂的效果、治疗/预防心脑血管疾病的效果、具有预防或治疗动脉粥样硬化、冠心病、高血脂的用途。
5.6)本发明的巨球型菌与Semaglutide联用对肝脏脂肪变性(Steatosis)、肝小叶炎症(Lobular inflammation)、肝脏气球样变(Ballooning)及肝脏NAS score及纤维化(Fibrosis)的影响：
图22为MNH05026、MNH22004和MNH27256分别与Semaglutide联用对高脂高糖高胆固醇饮食诱导NAFLD/NASH小鼠的肝脏脂肪变性(Steatosis)、肝小叶炎症(Lobular inflammation)、肝脏气球样变(Ballooning)及肝脏NAS score及纤维化(Fibrosis)的影响图；
巨球型菌与Semaglutide联用对高脂高糖高胆固醇饮食诱导NAFLD/NASH小鼠的 肝脏脂肪变性(Steatosis)、肝小叶炎症(Lobular inflammation)_、肝脏气球样变(Ballooning)及肝脏NAS score及纤维化(Fibrosis)的影响见图22A-22E。图22A结果显示MNH05026、MNH22004、MNH27256与Semaglutide联用能显著降低高脂高糖高胆固醇饮食诱导NAFLD/NASH模型小鼠的肝脏肝脏脂肪变性(Steatosis)，比阳性对照品Semaglutide的效果更佳。图22B结果显示MNH05026、MNH22004、MNH27256与Semaglutide联用能显著降低高脂高糖高胆固醇饮食诱导NAFLD/NASH模型小鼠的肝脏肝小叶炎症(Lobular inflammation)，比阳性对照品Semaglutide的效果更佳。图22C结果显示MNH05026、MNH22004、MNH27256与Semaglutide联用能显著降低高脂高糖高胆固醇饮食诱导NAFLD/NASH模型小鼠的肝脏气球样变(Ballooning)，比阳性对照品Semaglutide的效果更佳。图22D结果显示MNH05026、MNH22004、MNH27256与Semaglutide联用能显著降低高脂高糖高胆固醇饮食诱导NAFLD/NASH模型小鼠的肝脏NAS评分(NAS score)，比阳性对照品Semaglutide的效果更佳。图22E结果显示MNH05026、MNH27256与Semaglutide联用能降低高脂高糖高胆固醇饮食诱导NAFLD/NASH模型小鼠的肝脏纤维化评分(Fibrosis)，比阳性对照品Semaglutide的效果更佳。以上数据说明巨球型菌与Semaglutide联用后，能提升Semaglutide对肝脏脂肪变性(Steatosis)、肝小叶炎症(Lobular inflammation)、肝脏气球样变(Ballooning)及肝脏NAS score及纤维化(Fibrosis)防治效果，具有治疗NAFLD/NASH潜力。
实施例10.菌株MNH22004、MNH05026对组蛋白去乙酰化酶(Histone deacetylases， HDAC)活性的抑制作用
MNH22004和MNH27256为同种不同株，因此选取MNH22004为代表、以及选择MNH05026来验证巨球菌菌株是否对组蛋白去乙酰化酶活性具抑制作用，本研究使用购买的Abcam公司的HDAC Inhibitor Drug Screening Kit(Fluorometric)试剂盒进行体外的HDAC活性抑制作用的检测。
MNH22004培养上清制备:将菌株MNH22004接种在液体培养基中，37℃厌氧培养48小时，离心去除菌体，培养上清用0.22μm滤器进行过滤，分装，收集物置于-80℃冷冻保存，待用。
MNH05026培养上清制备:将菌株MNH05026接种在液体培养基中，37℃厌氧培养48小时，离心去除菌体，培养上清用0.22μm滤器进行过滤，分装，收集物置于-80℃冷冻保存，待用。
检测样品准备：1)Control组，MM01培养基用PBS进行10倍稀释得到含10％的MM01；2)阳性对照组：TSA，HDAC的抑制剂，用PBS稀释成终浓度为40μM；3)MNH22004组，MNH22004培养上清用PBS进行10倍稀释得到含10％细菌上清的实验样品。4)MNH05026组，MNH05026培养上清用PBS进行10倍稀释得到含10％细菌上清的实验样品。
HDACs检测反应试剂配制：根据试剂盒说明书配制适量的检测反应体系，每个反应需50μL反应试剂。
HDACs活性检测：在96孔白板中加入50μL检测样品，然后分别加入50μL反应试剂，充分混匀，37℃孵育30min，加入10μL Lysine Developer到反应孔中并充分混匀，终止反应。板子置于37℃孵育30min。最后利用酶标仪检测样品的荧光强度。酶标仪设置，Ex.＝350-380nm，Em,＝440-460nm。样品的HDACs抑制活性分析，将Control 的荧光强度值设定为100％，阳性对照组和MNH22004的荧光强度分别除以Control再乘以100％，可得到相对的HDACs活性。
结果显示(见图23)，与Control组的HDACs活性相比较，MNH22004和MNH05026的上清都对HDAC活性具显著的抑制作用，与HDACs抑制剂对照组TSA的作用相似。这些结果提示，MNH22004和MNH05026都能够抑制HDAC活性，MNH22004和MNH05026都能通过抑制HDAC活性，用于预防或治疗HDAC活性介导的疾病。
糖尿病是其中低水平的胰岛素和/或外周胰岛素抵抗导致高血糖症的一组疾病。已提出抑制HDAC活性来治疗糖尿病，包括通过抑制HDAC活性来实现Pdxl的抑制(Park等，2008，J Clin Invest，118，2316-24)，增强转录因子Ngn3的表达以增加内分泌库。祖细胞(Haumaitre等，2008，Mol Cell Biol，28，6373-83)和增强胰岛素表达(Molsey等，2003，J Biol Chem，278，19660-6)等。HDAC抑制也是用于晚期糖尿病并发症如糖尿病性肾病和视网膜缺血的有希望的治疗方法(Christensen等，2011，Mol Med，17(5-6)，370-390)。因此，本发明的组合物可用于治疗或预防HDAC活性介导的糖尿病。
研究表明组蛋白脱乙酰酶(HDAC)抑制剂丙戊酸(VPA)部分恢复了糖尿病EPC-EV损伤的H3K9Ac水平、内皮细胞管状形成和活性，并增强了细胞存活和增殖基因Pdgfd和Sox12的表达(doi:10.7150/thno.70821.)
组蛋白去乙酰化酶抑制剂(HDACi)在改善胰岛β细胞功能，保护其免受炎症因子攻击，改善外周组织胰岛素抵抗，调节免疫系统而改善代谢，减缓糖尿病并发症等方面的作用日益受到重视，有望成为糖尿病甚至是妊娠期糖尿病预防和治疗的新型药物。
本发明的组合物用于治疗或预防糖尿病。在优选的实施方案中，本发明的组合物用于治疗或预防I型糖尿病。在优选的实施方案中，本发明的组合物用于治疗或预防II型糖尿病。在某些实施方案中，本发明的组合物用于治疗或预防糖尿病，其中所述治疗或预防通过减少或预防HDAC活化来实现。
本发明的组合物具有HDAC抑制活性，根据HDACS抑制活性可以用于神经系统型疾病以及炎性肠病如IBD，心血管疾病(HDAC inhibition also results in beneficial outcomes in various types of neurodegenerative diseases,inflammation disorders,and cardiovascular diseases.HDAC and HDAC Inhibitor:From Cancer to Cardiovascular Diseases，https://pubmed.ncbi.nlm.nih.gov/26865995/)，可以确定本发明的菌株还可用于治疗HDAC活性介导的神经系统型疾病以及炎性肠病如IBD，心血管疾病。
菌株MNH05026 16S rRNA序列SEQ ID NO:1——参见序列表中的序列1；
菌株MNH22004 16S rRNA序列SEQ ID NO:2——参见序列表中的序列2；
菌株MNH27256 16S rRNA序列SEQ ID NO:3——参见序列表中的序列3；
以上各实施例仅用以说明本发明的技术方案，而非对其限制；本领域的普通技术人员应当理解：在不背离本发明的精神和范围的情况下，可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。

Claims (32)

  1. 一种巨球型菌属细菌菌株，所述细菌菌株的16S rRNA序列具有与16S rRNA序列SEQ ID NO:1、SEQ ID NO:2和/或SEQ ID NO:3具有至少95％、96％、96.5％、97％、98％、98.65％、99％、99.5％、99.9％或100％一致性，所述细菌菌株用于预防和/或治疗代谢性疾病；
    优选地,用于预防和/或治疗代谢性疾病的巨球型菌属细菌菌株选自Megasphaera stantonii、Megasphaera indica、Megasphaera paucivorans、Megasphaera sueciensis、Megasphaera micronuciformis、Megasphaera hexanoica、Megasphaera cerevisiae、Megasphaera hominis、Megasphaera butyrica、Megasphaera hutchinsoni、Megasphaera lornae、Megasphaera vaginalis中的任意一种或多种。
  2. 根据权利要求1所述的细菌菌株，其特征在于，所述细菌菌株包含与SEQ ID NO:2和/或SEQ ID NO:3具有至少97％、97.5％、98％、98.5％、98.65％、99％、99.5％或99.9％一致性的16S rRNA序列。
  3. 如权利要求1所述的细菌菌株，其特征在于，所述细菌菌株具有至少70％完整度的产丁酸路径；优选的，所述产丁酸路径为丙酮酸途径和/或4氨基丁酸途径；
    优选地，所述细菌菌株高产丁酸和/或乙酸；
    优选地,所述细菌菌株选自Megasphaera stantonii、Megasphaera indica、Megasphaera paucivorans、Megasphaera sueciensis、Megasphaera micronuciformis、Megasphaera hexanoica、Megasphaera cerevisiae、Megasphaera hominis、Megasphaera butyrica、Megasphaera hutchinsoni、Megasphaera lornae、Megasphaera vaginalis中的任意一种或多种。
  4. 根据权利要求1-3任一项所述的细菌菌株，其特征在于，所述细菌菌株保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC No:62001、GDMCC No：62000和/或GDMCC No：61999；
    优选地，所述微生物菌株与保藏编号为GDMCC No:62001、GDMCC No：62000和/或GDMCC No：61999的巨球型菌属细菌菌株的平均核苷酸一致性ANI值至少为79％、80％、85％、88％、90％、95％、96％、97％、98％、99％、99.5％、99.8％、99.9％或100％；
    优选地，所述平均核苷酸一致性ANI值至少为79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、95.5％、96％、96.5％、97％、97.5％、98％、98.5％、99％、99.5％、99.6％、99.7％、99.8％、99.9％或100％。
  5. 一种微生物菌剂，其特征在于，所述微生物菌剂包含权利要求1-4任一项所述的巨球菌属细菌菌株、巨球菌属细菌菌株上清液、巨球菌属细菌菌株提取物、巨球菌属细菌菌株的纯培养物或巨球菌属细菌菌株的分离培养物或巨球菌属细菌菌株的代谢产物中的至少一种；
    优选地，所述细菌菌株的代谢产物含有(a)～(d)中至少一种：(a)乙酸或乙酸盐，(b)丙酸或丙酸盐，(c)丁酸或丁酸盐，(d)戊酸或戊酸盐；
    优选地，所述细菌菌株高产乙酸和/或丁酸。
  6. 一种培养物，其为权利要求1-4任一项权利要求所述的菌株的培养物；
    优选地，所述培养物为纯培养物或分离培养物；
    优选地，所述培养物为权利要求1-4中任一项权利要求所述的菌株的发酵培养液或发酵培养液的上清。
  7. 一种组合物，其特征在于，所述组合物包含权利要求5所述的微生物菌剂或权利要求6所述的培养物或权利要求1-4任一项所述的细菌菌株，和，赋形剂、稀释剂或载体中的至少一种；
    优选地，所述微生物菌剂的剂型为悬浮剂、颗粒剂、片剂、栓剂、散剂、胶囊、微胶囊、口服液或粉剂；
    优选地，所述组合物可以是食品或营养品或膳食补充剂或特医食品或益生菌饮料或益生菌粉；
    优选地，所述组合物可通过冻干、升华干燥或喷雾干燥得到的。
  8. 根据权利要求7所述的组合物，其特征在于，所述组合物还包括GLP-1受体激动剂，GLP-1受体和GCG受体双重激动剂，GLP-1受体、GIP受体和GCG受体三重激动剂，AMPK激动剂或促进GLP-1分泌的活性药物中的至少一种；
    优选地，所述GLP-1受体激动剂包括以下任意一种：Semaglutide、liraglutide、exenatide或beinaglutide；
    优选地，所述AMPK激动剂或促进GLP-1分泌的活性药物包括Metformin、Semaglutide和/或liraglutide；
    可选的，所述组合物的施用方式包括：先施用第一活性物质，再施用第二活性物质或联用药物；或先施用第二活性物质或联用药物，再施用第一活性物质；或第一第二活性物质或联用药物同时施用；或第一活性物质和第二活性物质或联用药物按照各自的使用频率施用，或还可以施用第一活性物质后降低第二活性药物的使用剂量或频率。
  9. 一种用于代谢性疾病的药物组合物，其特征在于，所述药物组合物包括第一活性物质，所述第一活性物质为权利要求1-4任一项所述的细菌菌株、权利要求5所述的微生物菌剂、权利要求6所述的培养物或权利要求7所述的组合物中的至少一种；
    优选地，所述药物组合物包括具有(a)～(c)至少一种功能的药物组合物：(a)抑制食欲，(b)预防代谢性疾病，(c)治疗代谢性疾病；
    更优选地，所述抑制食欲包括，降低摄食量和/或降低食欲；
    更优选地，所述代谢性疾病包括：肝脏类疾病、肥胖及肥胖类相关疾病、心血管疾病、糖尿病、血脂异常、心脑血管疾病、葡萄糖不耐受、动脉粥样硬化、冠心病或高血压、I型糖尿病、II型糖尿病、糖耐量受损、胰岛素抵抗、肥胖症、高血糖症、高胰岛素血症、脂肪肝、酒精性脂肪性肝炎、高胆固醇血症、高血压、高脂蛋白血症、高脂血症、高甘油三酯血症、尿毒症、酮症酸中毒、低血糖、血栓性疾病、血脂异常、非酒精性脂肪性肝病(NAFLD)、非酒精性脂肪性肝炎(NASH)、动脉粥样硬化、肾病、糖尿病性神经病、糖尿病性视网膜病中的至少一种；
    其中，所述肝脏类疾病为脂肪肝、NAFLD/NASH、肝功能异常、肝外胆汁淤积、肝炎、肝损伤、肝内胆汁淤积的至少一种；
    可选地，所述肝脏类疾病的诱因包括，高脂饮食、高胆固醇饮食、高糖饮食、高血脂、高血糖或高胆固醇中的至少一种；
    可选地，所述肝脏类疾病包括高脂饮食引起的、高胆固醇饮食引起的、高糖饮食引 起的、高脂高胆固醇引起的、高脂高糖引起的和/或高脂高胆固醇高糖引起的疾病；
    其中，所述肥胖及肥胖类相关疾病为超重、肥胖症、代谢综合症、心血管疾病、心脑血管疾病、高脂血症、高胆固醇血症、高血压、胰岛素抗性综合征、肥胖相关的胃食管返流症、脂肪性肝炎；
    可选地，所述肥胖类疾病的诱因包括，高脂饮食、高糖饮食、高胆固醇、高血脂、高血糖、NAFLD或NASH中的至少一种；
    可选地，所述肥胖类疾病包括高脂饮食引起的肥胖、高胆固醇引起的肥胖、高糖饮食引起的肥胖、高脂高胆固醇引起的肥胖、高脂高糖引起的肥胖、高脂高胆固醇高糖引起的肥胖，或者，NAFLD或NASH患者的肥胖；
    可选地，所述心血管疾病或心脑血管疾病的诱因包括，动脉粥样硬化、NAFLD、NASH、高血脂、高血糖或高胆固醇中的至少一种；
    可选地，所述心血管疾病或心脑血管疾病为动脉粥样硬化、冠心病、NAFLD或NASH患者的心血管疾病、NAFLD或NASH患者的心脑血管疾病，和/或，高胆固醇疾病；
    其中，所述糖尿病为I型糖尿病、II型糖尿病、妊娠糖尿病、HDAC活性介导的糖尿病、糖尿病性肾病、糖尿病性神经病、糖尿病性视网膜病、胰岛B细胞受损引起的糖尿病、胰岛素耐性引起的糖尿病、肥胖引起的的糖尿病；
    可选地，所述糖尿病的诱因包括，胰岛细胞功能障碍、胰岛素分泌下降、胰岛素抵抗增加、高脂饮食、高糖饮食、高胆固醇、高血脂、高血糖、NAFLD或NASH中的至少一种；
    可选地，所述糖尿病包括以下至少一种引起的：高脂饮食引起的、高糖饮食引起的、高胆固醇饮食引起的。
  10. 根据权利要求9所述的代谢性疾病的药物组合物，其特征在于，还包括第二活性物质，所述第二活性物质包括GLP-1受体激动剂，GLP-1受体和GCG受体双重激动剂，GLP-1受体、GIP受体和GCG受体三重激动剂，AMPK激动剂或促进GLP-1分泌的活性药物、DPP-4受体抑制剂、PPAR受体激动剂、PPARα受体激动剂、PPARδ受体激动剂、PPARγ受体激动剂、PPARα/δ受体双重激动剂、PPARα/γ受体双重激动剂、PPARα/δ/γ受体三重激动剂、CRTC、PGC-1α、SREBP、FXR、FGF21、ASK1、THR-β、LXR、NF-κB、SNDRI、MC4R、PNLIP、MOA、DRI等机制/靶点中的至少一种活性药物；
    优选地，所述GLP-1受体激动剂包括以下任意一种：Semaglutide、liraglutide、exenatide或beinaglutide；
    优选地，所述AMPK激动剂或促进GLP-1分泌的活性药物包括Metformin、Semaglutide和/或liraglutide；
    可选的，所述组合物的施用方式包括：先施用第一活性物质，再施用第二活性物质或联用药物；或先施用第二活性物质或联用药物，再施用第一活性物质；或第一第二活性物质或联用药物同时施用；或第一活性物质和第二活性物质或联用药物按照各自的使用频率施用；或还可以施用第一活性物质后降低第二活性药物的使用剂量，或还可以施用第一活性物质后降低第二活性药物的使用频率。
  11. 根据权利要求1-4任一项所述的巨球菌属细菌菌株、或权利要求5所述的微生物菌剂、权利要求6所述的培养物或权利要求7或8所述的组合物，或权利要求9或10 所述的代谢性疾病的药物组合物在制备预防和/或治疗代谢疾病的药物中的用途；
    所述细菌菌株和/或细菌菌株的代谢产物和/或细菌菌株的上清液抑制HDAC活性；
    优选地，所述细菌菌株和/或细菌菌株的代谢产物和/或细菌菌株的上清液用于治疗或预防HDAC活性介导的疾病；
    可选地，所述HDAC活性介导的疾病包括代谢性疾病；
    进一步地，所述代谢性疾病为：肝脏类疾病、肥胖及肥胖类相关疾病、心血管疾病、糖尿病、血脂异常、心脑血管疾病、葡萄糖不耐受、动脉粥样硬化、冠心病或高血压、I型糖尿病、II型糖尿病、糖耐量受损、胰岛素抵抗、肥胖症、体重控制、超重、血糖控制、糖尿病前期、高血糖症、高胰岛素血症、脂肪肝、酒精性脂肪性肝炎、高胆固醇血症、高血压、高脂蛋白血症、高脂血症、高甘油三酯血症、尿毒症、酮症酸中毒、低血糖、血栓性疾病、血脂异常、非酒精性脂肪性肝病(NAFLD)、非酒精性脂肪性肝炎(NASH)、动脉粥样硬化、肾病、糖尿病性神经病、糖尿病性视网膜病中的至少一种；
    优选地，所述抑制HDAC活性包括通过调节短链脂肪酸/短链脂肪酸盐来抑制HDAC活性；所述短链脂肪酸包括乙酸、丁酸、戊酸中的一种或多种；更优选的，所述短链脂肪酸为丁酸；所述短链脂肪酸盐包括乙酸盐、丙酸盐、戊酸盐中的一种或多种；更优选的，所述短链脂肪酸为丁酸盐；优选的，所述组蛋白乙酰酶为I类、II类、III类和/或IV类组蛋白乙酰酶；
  12. 根据权利要求11所述的用途，其特征在于，所述微生物菌株、上清液、培养物、提取物或所述微生物菌株的代谢产物使得GLP-1表达增加。
  13. 巨球型菌属细菌菌株和/或包含巨球型菌属细菌菌株的组合物在制备预防或治疗代谢性疾病的药物中的用途；
    所述细菌菌株具有与16S rRNA序列SEQ ID NO:1、SEQ ID NO:2和/或SEQ ID NO:3具有至少95％、96％、96.5％、97％、97.5％、98％、98.5％、98.65％、99％、99.5％或99.9％或100％一致性；所述包含巨球菌属细菌菌株的组合物包括所述细菌菌株、菌株上清液、菌株培养物、菌株提取物或所述细菌菌株的代谢产物中的至少一种；
    优选地，所述细菌菌株选自Megasphaera stantonii、Megasphaera indica、Megasphaera paucivorans、Megasphaera sueciensis、Megasphaera micronuciformis、Megasphaera hexanoica、Megasphaera cerevisiae、Megasphaera hominis、Megasphaera butyrica、Megasphaera hutchinsoni、Megasphaera lornae、Megasphaera vaginalis中的任意一种或多种。
    优选地，所述包含巨球菌属细菌菌株的组合物还包括赋形剂、稀释剂或载体中的至少一种。
  14. 根据权利要求13所述的用途，其特征在于，所述细菌菌株保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC No:62001、GDMCC No：62000和/或GDMCC No：61999；
    优选地，所述细菌菌株与保藏编号为GDMCC No:62001、GDMCC No：62000和/或GDMCC No：61999的巨球型菌属细菌菌株的平均核苷酸一致性ANI值至少为79％；
    优选地，所述平均核苷酸一致性ANI值为79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、95.5％、96％、96.5％、97％、97.5％、98％、98.5％、99％、99.5％、99.6％、99.7％、99.8％、99.9％或100％。
  15. 根据权利要求13或14所述的用途，其特征在于，所述代谢性疾病为HDAC活性介导的代谢性疾病；
    优选为HDAC活性介导的糖尿病，更优选地，所述代谢性疾病为HDAC活性介导的I型糖尿病或II型糖尿病或晚期糖尿病或糖尿病性肾病或糖尿病性视网膜缺血症。
  16. 根据权利要求13～15任一项所述的用途，其特征在于，所述代谢性疾病包括糖尿病，其表现为低水平的胰岛素和/或外周胰岛素抵抗导致高血糖症。
  17. 根据权利要求13或14或15所述的用途，其特征在于，所述代谢性疾病为代谢障碍引起的疾病，优选地，代谢障碍包括：(1)糖代谢紊乱引起的糖尿病，或(2)糖耐量异常或糖耐量降低引起的糖尿病，或(3)胰岛素B细胞受损引起的糖尿病，或(4)胰岛素抵抗引起的糖尿病。
  18. 根据权利要求13～17任一项所述的用途，其特征在于，所述糖尿病包括I型糖尿病、II型糖尿病或妊娠糖尿病。
  19. 根据权利要求13或14所述的用途；
    所述代谢性疾病包括：肥胖及肥胖相关疾病；例如肥胖症、冠心病、动脉粥样硬化、脂肪肝、酒精性脂肪性肝炎、高胆固醇血症、高血压、高脂蛋白血症、高脂血症、高甘油三酯血症、尿毒症、酮症酸中毒、血栓性疾病、血脂异常、非酒精性脂肪性肝病(NAFLD)、非酒精性脂肪性肝炎(NASH)、动脉粥样硬化、肾病中的至少一种。
  20. 根据权利要求13-19任一项所述的用途；
    所述药物剂型为儿童适用剂型或成人适用剂型。
  21. 根据权利要求13-20任一项所述的用途；所述细菌菌株包括活细菌、减毒细菌、被杀死的细菌、冻干细菌、经辐照的细菌。
  22. 根据权利要求13-21任一项所述的用途；所述药物的剂型选择经胃肠道给药剂型、或非经胃肠道给药剂型。
  23. 根据权利要求13-22任一项所述的用途；所述药物为口服药物或注射剂。
  24. 根据权利要求13-23任一项所述的用途；所述药物还包括一种或多种第二活性成分。
  25. 根据权利要求24所述的用途；所述第二活性成分包括以下物质中的至少一种：GLP-1受体激动剂，GLP-1受体和GCG受体双重激动剂，GLP-1受体、GIP受体和GCG受体三重激动剂，AMPK激动剂或促进GLP-1分泌的活性药物；
    优选地，所述第二活性成分包括以下物质中的一种或多种：二甲双胍、磺脲类、氯茴苯酸、噻唑烷二酮类、DPP-4抑制剂、GLP-1受体激动剂、SGLT2抑制剂、胰岛素、吡格列酮、罗格列酮、己酮可可碱、omega-3-脂肪酸、他汀类药物、依折麦布、或熊去氧胆酸、Semaglutide、liraglutide、exenatide、beinaglutide。
  26. 根据权利要求15或权利要求16所述的用途；所述细菌菌株促进或提升或增强第二活性成分在代谢疾病中的作用。
  27. 一种包含巨球型菌属细菌菌株的药物组合物，所述细菌菌株选自与16S rRNA序列SEQ ID NO:1、SEQ ID NO:2和/或SEQ ID NO:3具有至少95％、96％、96.5％、97％、97.5％、98％、98.5％、98.65％、99％、99.5％或99.9％或100％一致性，或选自巨球菌属中能表达EC2.8.3.9和/或具有70％以上完整产定酸通路的菌株；
    优选地，所述细菌菌株选自Megasphaera stantonii、Megasphaera indica、Megasphaera paucivorans、Megasphaera sueciensis、Megasphaera micronuciformis、Megasphaera  hexanoica、Megasphaera cerevisiae、Megasphaera hominis、Megasphaera butyrica、Megasphaera hutchinsoni、Megasphaera lornae、Megasphaera vaginalis中的至少一种；
    优选的，细菌菌株通过GENE ID:650027236、641897133、650594268、642201644、650018449、650536170、2511555023和/或646248671来调控表达EC2.8.3.9。
  28. 根据权利要求27所述的包含巨球型菌属细菌菌株的药物组合物，所述组合物能够调节至少一种短链脂肪酸或调节短链脂肪酸盐，所述短链脂肪酸包括乙酸、丁酸、戊酸、丁戊酸或己酸；所述短链脂肪酸盐包括乙酸盐、丙酸盐或丁酸盐；
    优选的所述组合物能够用于代谢性疾病的治疗。
  29. 根据权利要求27-28任一项所述的包含巨球型菌属细菌菌株的药物组合物，其特征在于如下的(1)至(9)项中的任意一项或者多项：
    (1)所述药物组合物为婴儿适用剂型、儿童适用剂型或成人适用剂型；
    (2)所述药物组合物为经胃肠道给药剂型或非经胃肠道给药剂型；
    (3)所述药物组合物为口服剂或注射剂；
    (4)所述菌株至少能够部分地在受试者的肠道内增殖；
    (5)所述菌株或所述菌株的代谢产物或者所述的培养物在药物组合物中的质量百分比含量为1％-80％、2％-70％、5％-60％、10％-50％、20％-40％、45％、50％、55％或60％；
    (6)所述菌株的形式选自：活细菌、减毒细菌、被杀死的细菌、冻干细菌或经辐照的细菌。
    (7)按重量每g药物组合物中包含1×103至1×1013个菌落形成单位(CFU)的菌株；
    (8)所述药物组合物为粉剂或液体剂型；
    (9)所述药物组合物被制备成冻干粉，片剂、胶囊剂、颗粒剂或针剂。
  30. 一种根据权利要求1-4任意一项所述细菌菌株在制备药物中的用途；
    其中，所述药物用于选自如下至少一项：
    减少肝脏重量；
    治疗初期脂肪肝炎病灶；
    减缓肝脏细胞脂肪堆积；
    降低血清AST、ALT；
    减少腹部白色脂肪炎症性病变；
    减轻哺乳动物的体重；
    减少哺乳动物的摄食量；
    降低哺乳动物的体脂；
    降低哺乳动物血清中如下至少一种指标的水平：总胆固醇水平、低密度脂蛋白和甘油三酯水平；
    提高哺乳动物血清高密度脂蛋白的水平；
    改善哺乳动物口服葡萄糖耐量受损；
    降低哺乳动物的空腹血糖；
    降低哺乳动物HOMA-IR指标；
    修复消化道黏膜损伤；
    治疗或预防冠心病；
    治疗或预防动脉粥样硬化；
    治疗或预防高血糖；
    治疗或预防高血脂；
    治疗或预防高胆固醇；
    治疗或预防肝功能损伤；
    治疗或预防脂肪肝；
    治疗或预防NAFLD或NASH；
    治疗或预防高血压；
    治疗或预防糖尿病，优选为妊娠糖尿病或II型糖尿病或HDAC活性介导的糖尿病；
    治疗或预防肥胖症；
    治疗或预防代谢综合症；
    治疗或预防局部皮脂过多、腹股沟脂肪过度、附睾脂肪过多和/或棕色脂肪过多。
  31. 一种治疗或预防代谢性疾病的方法，包括给予有需求的受试者以有效量的权利要求1-4任一项所述的细菌菌株、权利要求5所述的微生物菌剂、权利要求6所述的培养物或权利要求7所述的组合物的步骤；
    优选地，所述代谢性疾病为：肝脏类疾病、肥胖及肥胖类相关疾病、心血管疾病、糖尿病、血脂异常、心脑血管疾病、葡萄糖不耐受、动脉粥样硬化、冠心病或高血压、I型糖尿病、II型糖尿病、糖耐量受损、胰岛素抵抗、肥胖症、体重控制、超重、血糖控制、糖尿病前期、高血糖症、高胰岛素血症、脂肪肝、酒精性脂肪性肝炎、高胆固醇血症、高血压、高脂蛋白血症、高脂血症、高甘油三酯血症、尿毒症、酮症酸中毒、低血糖、血栓性疾病、血脂异常、非酒精性脂肪性肝病(NAFLD)、非酒精性脂肪性肝炎(NASH)、动脉粥样硬化、肾病、糖尿病性神经病、糖尿病性视网膜病中的至少一种；
    优选地，所述菌株或者所述菌株的代谢产物或者培养物通过选自如下的至少一种方式治疗或预防代谢性疾病：(a)减轻体重；(b)降低空腹血糖；(c)降低血脂；(d)抑制HDAC活性；(e)通过SCFA中的乙酸或乙酸盐、丙酸或丙酸盐、丁酸或丁酸盐、戊酸或戊酸盐中的至少一种抑制HDAC活性；(f)通过SCFA中的乙酸或乙酸盐、丙酸或丙酸盐、丁酸或丁酸盐、戊酸或戊酸盐中的至少一种短链脂肪酸或短链脂肪酸盐抑制肿瘤；(g)通过调节受试者肠道菌群调节作用发挥治疗作用。
  32. 根据权利要求31所述的方法，其中，所述菌株或者所述菌株的代谢产物或者权利要求6所述的培养物通过抑制HDAC活性实现治疗或预防代谢性疾病。
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