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WO2022142070A1 - Probiotique intestinal pour lutter contre des maladies métaboliques et son utilisation - Google Patents

Probiotique intestinal pour lutter contre des maladies métaboliques et son utilisation Download PDF

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WO2022142070A1
WO2022142070A1 PCT/CN2021/093854 CN2021093854W WO2022142070A1 WO 2022142070 A1 WO2022142070 A1 WO 2022142070A1 CN 2021093854 W CN2021093854 W CN 2021093854W WO 2022142070 A1 WO2022142070 A1 WO 2022142070A1
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composition
acetic acid
culture
indole
improving
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PCT/CN2021/093854
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Chinese (zh)
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郭非凡
胡小明
牛雨果
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中国科学院上海营养与健康研究所
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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
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
    • 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
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • 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 present invention belongs to the field of microbiology and metabolic endocrinology, and more particularly, the present invention relates to intestinal probiotics for improving metabolic diseases and applications thereof.
  • Nutritional status is closely related to health, and the incidence of various chronic metabolic diseases, such as type 2 diabetes mellitus, caused by imbalanced nutritional intake is rapidly increasing.
  • the ninth edition of the Diabetes Map of the Global Diabetes Alliance in 2019 shows that in 2019, there were 463 million people in the world aged 20-79 with diabetes, and it is expected that by 2030, there will be 578 million people with diabetes. By 2045, this number will reach 700 million, with an additional 374 million people at high risk [1].
  • Type 2 diabetes is one of the most common chronic metabolic diseases, accounting for more than 90% of diabetic patients, and is closely related to metabolic diseases such as obesity and hypertension [2].
  • One of the main causes of type 2 diabetes is insulin resistance, therefore, how to increase the body's insulin sensitivity is crucial for the treatment of type 2 diabetes.
  • metabolic diseases such as type 2 diabetes will become one of the main factors restricting the development of health care in China. Therefore, the research on the new mechanism and regulation of the imbalance of glucose metabolism homeostasis has important theoretical significance for the treatment of metabolic diseases such as diabetes, and meets the current major needs of the country.
  • the human gut contains about 500 to 1000 species of bacteria, with about 2 million genes, about 100 times the number of genes in humans, and their abundance is affected by many intrinsic factors (such as pH, intestinal peristalsis, mucus, antimicrobial peptides, etc.) and external factors (such as diet, antibiotics, laxatives, etc.) regulation [3,4].
  • the interaction between gut microbiota and the occurrence and development of metabolic diseases such as diabetes is a frontier field of metabolic research in the world. With the rapid development of next-generation metagenomic sequencing technology, more and more studies have shown that intestinal flora, as a very important "metabolic organ" in the body, is closely related to the occurrence of insulin resistance [5-7].
  • Nutritional and genetic factors are key determinants of gut microbiota composition.
  • the macronutrients required by the human body mainly refer to protein, sugar and fat.
  • people have a relatively full understanding of the relationship between the three types of macronutrients and health, especially excessive intake of sugar or fat is the main cause of diabetes and related metabolic diseases.
  • the purpose of the present invention is to provide intestinal probiotics for improving insulin resistance and lowering blood sugar and application thereof.
  • a composition comprising a composition selected from the group consisting of The composition of : a composition for inhibiting disorders of glucose metabolism; a composition for improving intestinal flora disorder.
  • the disorders of glucose metabolism include: insulin resistance, impaired glucose tolerance, diabetes, and hyperglycemia.
  • the composition for improving intestinal flora disorder includes: a composition for remodeling the normal ecology of the flora and increasing the amount of intestinal probiotics.
  • the intestinal probiotics include (but are not limited to): Alloprevotella and Lachnoclostridium.
  • the composition for improving intestinal flora disorder comprises: a composition for improving the alpha diversity of intestinal flora, the abundance of Bacteroidetes and the ratio of Bacteroidetes/Post-wall bacteria;
  • the increasing the alpha diversity of the intestinal flora comprises increasing the number of bacterial species or increasing the Shannon index.
  • novel metabolite of Blautella is: indole-3-acetic acid.
  • the Braute coccus uses tryptophan as a substrate to generate indole-3-acetic acid.
  • the broutella, its culture, its metabolites, or a mixture containing them activates insulin signaling in primary liver cells, thereby inhibiting glucose metabolism disorders or improving intestinal flora disorders; more
  • the brouterella, a culture thereof, a metabolite thereof, or a mixture containing them increases the phosphorylation level of protein kinase B (AKT) or glycogen synthase kinase 3 ⁇ (GSK3 ⁇ ) protein, activates hepatogen Insulin signaling in generation cells.
  • AKT protein kinase B
  • GSK3 ⁇ glycogen synthase kinase 3 ⁇
  • the concentration of the brouterella is 1 ⁇ 10 3 to 1 ⁇ 10 10 CFU/mL; preferably 1 ⁇ 10 6 to 1 ⁇ 10 9 CFU/mL (for example, 1 ⁇ 10 7 , 1 ⁇ 10 8 , 1 ⁇ 10 9 CFU/mL).
  • the composition includes: a pharmaceutical composition, a health product composition, a food composition, and a feed composition.
  • a method for improving intestinal flora disturbance comprising: administering Sonella, its culture, its metabolites, or a mixture containing them.
  • the method is a non-therapeutic method.
  • Brautia spp. for the production of indole-3-acetic acid; preferably, for the production of indole-3-acetic acid with tryptophan as a substrate.
  • a method for producing indole-3-acetic acid comprising: culturing Brauterella to produce indole-3-acetic acid; preferably, adding color to the culture system amino acid.
  • the final concentration of the tryptophan in the culture system is 1-100 ⁇ mol/L.
  • H H&E staining of ileum and colon tissue, the length of the scale is 50 ⁇ m;
  • A Flow chart of experimental operation: 6-week-old male C57BL/6J mice were fed food with 60% fat content for 12 weeks to create a mouse model of insulin resistance; LBC) or high temperature-killed (KBC) blautella, after 4 weeks, blood glucose and GTT, ITT tests were performed.
  • LBC low fat content
  • KBC high temperature-killed
  • F the ratio of Bacteroidetes and Firmicutes
  • FIG. 4 Insulin signaling of primary mouse liver cells activated by culture of Brauterella; inoculation of Blauterella in logarithmic growth phase at a ratio of 1:100, cultured under anaerobic conditions for 48 hours, and the culture The cells were mixed with complete cell culture medium at the proportions of 2%, 5% and 10%, respectively, to stimulate mouse liver primary cells for 48 hours; before the end of treatment, cells were stimulated with 100 nmol/L insulin for 20 minutes, and then the cells were collected.
  • Western blot technique was used to detect the phosphorylation levels of insulin receptor (IR), protein kinase B (AKT) and glycogen synthase kinase 3 ⁇ (GSK3 ⁇ ) proteins, and to analyze the activity of insulin signaling.
  • the experimental results are expressed as mean ⁇ standard error; the statistical method adopts one-way analysis of variance; *: P ⁇ 0.05 indicates significant difference.
  • Indole-3-acetic acid activates insulin signaling in mouse liver primary cells; 0, 10, 100 and 500 ⁇ mol/L of indole-3-acetic acid sodium salt (I3AA) were added to the complete cell culture medium, respectively ), stimulated mouse liver primary cells for 48h; before the end of treatment, cells were stimulated with 100nmol/L insulin for 20min, and then the cells were collected.
  • Western blot technique was used to detect the phosphorylation levels of insulin receptor (IR), protein kinase B (AKT) and glycogen synthase kinase 3 ⁇ (GSK3 ⁇ ) proteins, and to analyze the activity of insulin signaling.
  • the experimental results are expressed as mean ⁇ standard error; the statistical method adopts one-way analysis of variance; *: P ⁇ 0.05 means significant difference; P ⁇ 0.01 means extremely significant difference.
  • I3AA Indole-3-acetic acid relieves insulin resistance and lowers blood sugar
  • A Flow chart of experimental operation: 6-week-old male C57BL/6J mice were fed food with 60% fat content for 12 weeks to create a mouse model of insulin resistance; and then oral administration of 10 mg/kg body weight indole-3 - Sodium acetate (I3AA), after 4 weeks, blood glucose and GTT, ITT tests.
  • I3AA body weight indole-3 - Sodium acetate
  • the experimental results are expressed as the mean ⁇ standard error; the statistical method adopts unpaired Student's t-test or one-way ANOVA; *: P ⁇ 0.05 means significant difference and **: P ⁇ 0.01 means extremely significant difference.
  • the inventors After in-depth screening and testing, the inventors screened out a new type of bacteria that is conducive to improving intestinal flora disturbance, improving the body's glucose metabolism homeostasis, and improving insulin sensitivity, which is Blautia coccoides It has important application value for the intervention and treatment of metabolic diseases such as type 2 diabetes.
  • the inventors also found that the in vitro culture of the strain and its metabolite indole-3-acetic acid also have the effects of improving the body's glucose metabolism homeostasis and improving insulin sensitivity.
  • strains are isolated strains.
  • the term “comprising” means that the various ingredients can be used together in the mixture or composition of the present invention.
  • the terms “consisting essentially of” and “consisting of” are encompassed by the term “comprising”.
  • pharmaceutically acceptable carrier or “food pharmaceutically acceptable carrier” or “nutraceutically acceptable carrier” is the one used to transfer the Blautia coccoides or cell culture, cell metabolites of the present invention , cell culture supernatant or cell lysate is delivered to the object needing treatment, in terms of toxicity, side effects controllable, environmentally friendly or harmless to humans and animals solvent, suspending agent or excipient.
  • the carrier may be a liquid or a solid, preferably a carrier capable of maintaining the activity of the Blautia coccoides or cell culture, cell metabolites, cell culture supernatants or cell lysates to a higher degree.
  • the Blautia coccoides bacterial strain of the present invention is isolated from an anaerobic fermenter that processes manure. After comparing a large number of various strains, the inventors determined that it has a relatively ideal effect.
  • the Blautia coccoides belongs to a strictly anaerobic bacterium, belongs to Firmicutes, and belongs to the genus Blautia, and the fermentable substrates include glucose, arabinose, cellobiose, lactose, maltose, mannose, raffinose
  • the main end products of metabolism are acetic acid and succinic acid [8].
  • the present invention discloses for the first time that it is associated with metabolic diseases, especially type 2 diabetes.
  • the present inventors focused on Blautia coccoides, which has been reported to mainly metabolize carbohydrates to produce the end products short-chain fatty acids (acetic acid) and succinic acid.
  • the research results of the present inventors show that the insulin resistance model mice were continuously gavaged with the Blautia coccoides for four weeks, and the food and starvation blood glucose levels, HOMA-IR index, glucose tolerance (GTT) and insulin sensitivity of the animals in the experimental group were significantly reduced. Sexuality (ITT) was significantly improved.
  • the Blautia coccoides can also reshape the intestinal flora, improve the ⁇ diversity of the intestinal flora, the abundance of Bacteroidetes and the ratio of Bacteroidetes/Firmicutes in insulin resistance model mice, etc. .
  • the research results of the present inventors show that the in vitro culture of Blautia coccoides can increase the activity of insulin signaling in mouse liver primary cells stimulated by insulin; moreover, the in vitro incubation of Blautia coccoides with Tryptophan can metabolize it to produce indium Indole-3-acetic acid.
  • the described Blautia coccoides metabolite indole-3-acetic acid was given to insulin resistance model mice by gavage for 4 weeks, and the food and starvation blood glucose levels and HOMA-IR index of the experimental group were significantly reduced, and the glucose tolerance was significantly reduced. (GTT) and insulin sensitivity (ITT) were significantly improved.
  • the Blautia coccoides strain of the present invention is a living cell, and once the strain is obtained, the strain can be obtained in large quantities by means of inoculation, passage, regeneration and the like. This is usually by inoculating it into a solid plate medium or a liquid medium to expand the strain to obtain the living cells of the present invention.
  • the obtained living cells can be further subjected to laboratory domestication, genetic breeding and molecular genetic manipulation to obtain mutants and transformants.
  • the Blautia coccoides can also be utilized as bioengineered host cells for heterologous expression.
  • the isolated natural Blautia coccoides strain in addition to including the isolated natural Blautia coccoides strain, it also includes its variant, domesticated strain or genetically engineered strain.
  • the variant is a variant with the same function/improved function; preferably, the variant with the same function/improved function includes improving the disturbance of intestinal flora and inhibiting the disturbance of glucose metabolism.
  • the variant may be a variant formed by mutagenizing the strain.
  • Mutation breeding of strains refers to the use of physical and chemical factors to induce changes in the genetic characteristics of animals and plants, and then select individual plants/individuals that meet certain requirements of people from the mutant population, and then cultivate new varieties or germplasm breeding methods. .
  • the mutants of Blautia coccoides that have obtained the effect or stronger function can be screened by mutagenesis; alternatively, the function or effect that remains unchanged but the growth performance is more can also be screened by mutagenesis.
  • mutagenesis methods known to those skilled in the art can be used for mutagenesis, for example, including: starting strain culture, mutagenesis treatment, observation of a large number of colonies, comparison of properties of interest, and finally screening out the properties of interest to improve and modified strains.
  • Such mutagenesis can generally be performed in a high-throughput manner, preferably in repetitions over multiple or multiple generations.
  • strains The domestication of strains is different from the variation of strains, which generally refers to the method of directional selection of microorganisms through artificial measures to gradually adapt to a certain condition. It generally includes steps: starting strain culture with changes in various conditions/experimental parameters, observing a large number of colonies, comparing the properties of interest, and finally sorting out strains/communities with improved and modified properties of interest. Strains with higher tolerance and activity can be obtained through domestication.
  • the domestication of a strain generally requires two or more passages in order to adapt the strain to a certain condition or to produce an improvement in the performance of interest.
  • Bioengineered/genetically engineered strains based on the natural strains of the present invention are also included in the present invention.
  • the introduction of exogenous genes and the deletion/knockout of endogenous genes can be carried out in the Blautia coccoides, and the basic condition of the modification is that the strain of the present invention retains its function or activity.
  • the purpose of its transformation may include: improving the colonization ability of the strain of the present invention on the surface of the object to be inhibited, or making the strain of the present invention, on the basis of retaining its function or activity, further produce at least one other excellent performance: for example
  • the growth performance is more excellent, the environmental compatibility is more ideal (for example, when some toxic and side effects occur, it can be eliminated by genetic modification), and the anti-adversity ability is more remarkable.
  • one or some metabolic flows/signal pathways can be enhanced/weakened by analyzing the metabolic flow/signal pathways in the strain, so that the performance of the strain can be further optimized.
  • exogenous genes and deletion/knockout of endogenous genes are known in the art, such as using some viral constructs/vectors or non-viral constructs/vectors to carry the exogenous genes to be introduced (including Exogenous gene for overexpression, exogenous gene for gene knockout, exogenous gene for gene editing) expression cassette, the construct/vector is introduced into the strain, and the strain is targeted for modification .
  • the exogenous genes may include, but are not limited to, functional genes, enzyme genes, structural genes, miRNA, siRNA, shRNA, and the like.
  • strain transformation can also be performed by directly introducing exogenous proteins into the strain.
  • exogenous proteins can be directly introduced into the strain.
  • the bacterium of the present invention is cultivated in large quantities, and the method for obtaining its culture and metabolites is as follows: familiar to those skilled in the art. That is, cultures or metabolites of Brauterella obtained by these means, and compositions containing them or their uses are also included in the present invention.
  • the medium suitable for culturing the Brauterella of the present invention can be varied, and it should be understood that as long as it can provide the Brauterella with the necessary nutrients for growth and reproduction (such as including a carbon source) , nitrogen sources, etc.) are all encompassed by the present invention.
  • the medium can be solid or liquid; the medium can be commercial or laboratory-prepared, such as tryptic soy broth.
  • the culture media used in the prior art for culturing various other bacteria may be suitable for culturing the brouterella described in the present invention.
  • the present invention also provides the cell culture, cell metabolite, cell culture supernatant or cell lysate of the Blauterella, which has a product selected from the group consisting of The function of the next group: improve intestinal flora disorder and inhibit glucose metabolism disorder.
  • a cell culture is obtained by appropriately changing the culture medium or culturing process which can also be obtained.
  • the cell culture contains active strains or substances secreted by them, so that they also have functions or activities similar to those of active strains.
  • the cell metabolites are a class of substances produced or secreted by the Blautia coccoides during the culture process, which can be directly secreted into the culture medium by the cells, or separated from the cells after a certain treatment.
  • the cell product can be isolated, purified or concentrated.
  • the metabolite is indole-3-acetic acid.
  • the cell culture supernatant is the remaining culture solution after the cells and solid impurities are removed during the process of culturing the Blautia coccoides or after the culturing, which can be unconcentrated or concentrated.
  • cells and solid impurities can be removed by means such as centrifugation, filtration, and the like.
  • the cell lysate is a mixture formed by lysing cells by using a reagent for lysing cells during the process of culturing the Blautia coccoides or after the culturing is completed.
  • the cell lysate may be a product obtained after lysis with solid impurities removed. It can be a purified or concentrated product as required.
  • the present invention also provides the application of the Blautella bacteria of the present invention for improving insulin resistance, lowering blood sugar and improving intestinal flora disturbance.
  • the Blautella of the present invention its culture or production process can also be optimized, and the scope of consideration can include optimization of various culture conditions, optimization of various components of the medium, and the like. By identifying some factors that have a greater impact on the cultivation and inhibition or degradation of the strain. It should be understood that these modifications are also encompassed by the present invention.
  • the Blautia coccoides can achieve long-term growth and stable passage.
  • the culture medium and culture method used for culturing the Blautia coccoides are not limited to those disclosed in the present invention, and other conventional culture medium and culture methods for culturing bacteria can also be applied in the present invention. It can be understood that, on the basis of the examples provided in the present invention, various medium components may be replaced by other components with similar functions, and other components may be appropriately added according to the characteristics of specific strains under different circumstances. Or remove some of the components or change the content of some of the components.
  • the present invention also provides a method for improving intestinal flora disturbance, comprising administering the lauterella, a culture thereof or a mixture containing the same.
  • the Blautia coccoides strain of the invention has remarkable functions in improving glucose metabolism disorder and restoring intestinal flora ecology, and has wide-scale application potential.
  • the present invention also provides the use of Brauterella for the production of indole-3-acetic acid; preferably, for the production of indole-3-acetic acid with tryptophan as a substrate Acetic acid.
  • the present invention also provides a method for producing indole-3-acetic acid, comprising: culturing Brauterella to produce indole-3-acetic acid; preferably, in a culture system Add tryptophan.
  • the final concentration of the tryptophan in the culture system is 1-100 ⁇ mol/L.
  • indole-3-acetic acid Using the method, small, medium or large-scale production of indole-3-acetic acid can be carried out, thereby being applied to improve glucose metabolism disorder or improve intestinal flora disorder.
  • indole-3-acetic acid produced by the method of the present invention can also be applied to other applications known in the art, such as plant growth hormones and the like.
  • the culture system or fermentation system of the present invention can be scaled up for industrial production. Depending on the size of the system, those skilled in the art can make appropriate adjustments based on the general knowledge they have mastered to facilitate the growth or production of strains.
  • indole-3-acetic acid on a smaller scale is provided in the embodiments of the present invention, those skilled in the art can further optimize to improve the indole-3-acetic acid after reading the content disclosed in the present invention.
  • the output of indole-3-acetic acid realizes large-scale production, and these optimized processes according to the idea of the present invention should also be included in the present invention.
  • the present invention also provides a composition, which comprises the Blautella sp. or the cell culture, cell metabolite, cell culture supernatant or cell lysate of the present invention.
  • the composition comprises the cell culture, cell metabolite, cell culture supernatant or cell lysate.
  • the compositions include, but are not limited to, pharmaceutical compositions, health product compositions, food compositions, feed compositions, and the like.
  • the composition may further include: an industrially acceptable carrier, or a microorganism Academically acceptable carrier.
  • the effective dose of the brouterella of the present invention or said cell culture, cell metabolite, cell culture supernatant or cell lysate may vary depending on the mode of administration or the condition of the subject to be treated.
  • an "effective amount" refers to an amount that can produce a function or activity on the object to be treated and can be accepted by the object to be treated, which is generally controllable in terms of toxicity and side effects, and environmentally friendly or to humans and animals. harmless amount.
  • the conversion of the administered dose may vary according to the assessment of an experienced pharmacist.
  • compositions can be in a variety of forms including, but not limited to: lyophilisates, wettable powders, emulsifiable concentrates, aqueous solutions, emulsions, sprayable solutions, oily or aqueous dispersions, suspensions, powders, Granules, or microcapsules. It should be understood that any dosage form is desirable as long as the composition of the present invention can be delivered to the subject to be treated while maintaining all or part of its activity. Dosage forms for easy administration, such as suspensions, solutions, capsules, powders, lyophilisates, and the like are preferred.
  • the active ingredient content in the concentrated composition is relatively high, such as 20-90 wt %, while the active ingredient content in the diluted composition and the actually used composition is relatively low, usually 0.00005-0.5 wt %.
  • other suitable chemical agents synergists, trace elements, stabilizers, binders, wetting agents, dispersing agents, emulsifiers, penetrants, solvents, fillers and other common components may also be included.
  • the compositions of the present invention may also contain other active ingredients, such as digestion enhancers, and the like.
  • the interaction between the intestinal flora and the host is closely related to the metabolism of the body, and more and more attention has been paid by those skilled in the art.
  • the regulation of the intestinal flora is expected to become a new target for the prevention and treatment of diabetes and related metabolic diseases. Therefore, the present invention
  • the technical solution has good application value.
  • TLB Tryptic Soy Broth
  • ATCC medium 2722 formula: tryptic soy broth 30g, yeast extract 5g, L-cysteine hydrochloride 0.5g, heme 5mg (final concentration 5mg/L), vitamin K1 (final concentration 1mg/L) , an additional 15 g of agar powder was added to the solid medium; ddH 2 O was added to the volume to 1 L, sterilized by high temperature and high pressure, and deoxygenated in an anaerobic box for 2 days.
  • B. coccoides bacteria were inoculated at a ratio of 1:100, grown to log phase in 24-48 h, collected by centrifugation, and frozen in PBS containing 20% glycerol.
  • the bacteria were thawed in a 37°C water bath, and the bacterial solution was diluted with deoxygenated PBS at a 10-fold gradient to 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , respectively. , 10 -8 , and then the two concentrations of bacterial liquids of 10 -7 and 10 -8 were applied to solid petri dishes, and cultured strictly anaerobic; after 48h, the number of colonies was calculated, and the number of viable bacteria in the bacterial stock solution was calculated according to the dilution ratio. .
  • CFU Colony-Forming Units
  • Wild-type male C57BL/6 mice were purchased from Shanghai Slack Laboratory Animal Center.
  • IR mouse model established by continuous high-fat (60% fat) diet feeding for 4 months.
  • the experimental procedure was as follows: 6-week-old male C57BL/6J mice were first fed with high-fat food and corresponding control food for 12 weeks, and then gavaged with PBS containing 2% glycerol and B.coccoides (1 ⁇ 10 9 CFU/mouse) and active B. coccoides (1 ⁇ 10 9 CFU/mouse), each mouse was given 200 ⁇ L of bacterial solution by gavage, once a day for 4 weeks, while small Mice were maintained on a high-fat diet. Mice were housed in SPF animal room, and the feeding temperature was 25°C for 12 hours day and night. Before the experiment, they were fed with food and drinking water that met international standards. Finally, the mice were sacrificed with carbon dioxide, and various tissues were collected, frozen in liquid nitrogen, and stored in a -80°C refrigerator for later use.
  • blood sugar was collected from the tail of mice, and blood sugar was detected with Glucometer Elite monitor. Serum insulin levels were detected using the Mercodia Ultrasensitive Rat Insulin ELISA kit from ALPCO Diagnostics.
  • Glucose tolerance test C57BL/6 mice were starved overnight and injected with 2 g/kg glucose solution intraperitoneally, and blood glucose was detected by risostane rapid blood glucose tester and test strips at 0, 15, 30, 60, and 120 minutes, respectively. .
  • ITT Insulin tolerance test
  • the insulin resistance index (HOMA-IR) was calculated as [fasting blood glucose level (mmol/L)] ⁇ [fasting serum insulin ( ⁇ U/ml)]/22.5.
  • Collect intestinal contents such as ileum or cecum contents
  • add 1 mL of pre-cooled PBS then add glass beads, shake to break the stool, centrifuge at 800g, and take the supernatant. Then, the bacterial particles were pelleted by centrifugation at 8,000 ⁇ g for 5 min, and washed once with pre-cooled PBS.
  • Add 500 ⁇ L of CTAB lysate containing 2% concentration and 20 ⁇ L of 20 mg/ml proteinase K shake at 37 °C for 1 h, then add 56 ⁇ L of 20% SDS and incubate in a 65 °C water bath for 2 h (mix up and down every 30 min).
  • centrifuge at 12,000 ⁇ g for 15 min to remove the protein fraction transfer the supernatant to a new EP tube, add an equal volume of phenolic alcohol (25:24:1), mix well and let stand for 5 min, centrifuge at 12,000 ⁇ g for 5 min (repeatedly) three times); finally suck the supernatant, add 60% volume of isopropanol to precipitate DNA; wash once with 75% alcohol, add TE buffer to dissolve, and determine the concentration.
  • Eosin and hematoxylin (H&E) staining was performed using standard procedures for histological analysis of intestinal morphology.
  • a suitable concentration of discontinuous denaturing polyacrylamide gel SDS-PAGE for vertical electrophoresis, generally 10%. Start electrophoresis at 80 volts. After the front of the dye enters the separating gel, increase the voltage to 120 volts and continue electrophoresis until bromophenol blue reaches the bottom of the separating gel (10 ⁇ electrophoresis solution: Tris 30g, Glycine 144g, SDS 10g, add ddH 2 O to 1L).
  • the PVDF membrane was soaked in methanol for 15sec, and put into transfer buffer along with the gel to equilibrate for 15min (10 ⁇ transfer buffer: Tris 30g, Glycine 144g, methanol 200ml, add water to 1L); then follow the steps of anode-sponge-filter paper-PVDF
  • the sequence of membrane-glue-filter paper-sponge-cathode is placed in a transfer container, 4°C, 380mA.
  • the transfer time varies according to the molecular weight, generally 1.5-2h. After transferring the membrane, take out the PVDF membrane, mark the direction and marker, and cut it as needed.
  • Anti-phosphorylated(p)-IR(Tyr1150/1151), anti-IR, anti-p-Akt(Ser473), anti-Akt, anti-p-GSK3 ⁇ (Ser9) and anti-GSK3 ⁇ were purchased from Cell Signaling Technology Inc.
  • the inventors detected and found that compared with the control group, there were no significant differences in the basic phenotypes such as food intake, body weight, body temperature, body fat and lean meat weight, and blood sugar of the mice in the experimental group (Fig. 1A-F).
  • the tissue weight and H&E staining morphology of the colon and ileum also did not change significantly compared with the control group ( Figure 1G and H).
  • 6-week-old male C57BL/6J mice were fed with 60% fat content (HFD) and corresponding control food (normal fat content, NCD) for 16 weeks to obtain a mouse model of insulin resistance; oral administration was started 4 weeks before the end of the experiment After gavage of 2% glycerol in PBS, 1 x 109 CFU content of live or heat-killed Brauterella for 4 weeks, blood glucose and GTT, ITT tests were performed (Fig. 2A).
  • HFD 60% fat content
  • NCD normal fat content
  • the inventors detected and found that compared with the high-fat control group, the feeding and starvation blood glucose, HOMA-IR index, GTT and ITT of the mice in the live bacteria group were significantly improved (Figure 2B-F), but the dead bacteria treatment group did not There is an improvement in the corresponding indicators.
  • Example 3 Blautella improves intestinal flora disorder caused by high-fat diet
  • 6-week-old male C57BL/6J mice were fed with 60% fat content (HFD) and corresponding control food (normal fat content, NCD) for 16 weeks to obtain an insulin resistance mouse model.
  • HFD 60% fat content
  • NCD normal fat content
  • 4 weeks before the end of the experiment the live or high temperature inactivated Braute coccus in PBS containing 2% glycerol and 1 ⁇ 10 9 CFU was orally administered by oral gavage.
  • 16S rDNA technology was used to detect the structure and composition.
  • the complete medium was mixed to stimulate the primary mouse liver cells for 48h; before the end of the treatment, the cells were stimulated with 100nmol/L insulin for 20min, and then the cells were collected.
  • Western blot technique was used to detect the phosphorylation levels of insulin receptor (IR), protein kinase B (AKT) and glycogen synthase kinase 3 ⁇ (GSK3 ⁇ ) proteins, and to analyze the activity of insulin signaling.
  • IR insulin receptor
  • AKT protein kinase B
  • GSK3 ⁇ glycogen synthase kinase 3 ⁇
  • the inventors detected and found that compared with the control group, the phosphorylation levels of IR, AKT and GSK3 ⁇ proteins were significantly increased under the stimulation of the medium containing 10% of the culture, as shown in Figure 4, indicating that the culture of Brauterella Significantly activates insulin signaling in mouse liver primary cells.
  • the brouterella in the logarithmic growth phase was centrifuged, the supernatant was removed, and washed twice with isotonic potassium phosphate buffer (containing 40 mmol/L potassium phosphate, 10 mmol/L magnesium sulfate, pH adjusted to 7.0), Then, tryptophan with a final concentration of 10 ⁇ mol/L was added and incubated under anaerobic conditions at 37°C for 0, 0.5, 1, 2, 4 and 8 h respectively; ) technique to detect the concentration of indole-3-acetic acid and tryptophan.
  • isotonic potassium phosphate buffer containing 40 mmol/L potassium phosphate, 10 mmol/L magnesium sulfate, pH adjusted to 7.0
  • the inventors detected that: with the prolongation of incubation time, the metabolite indole-3-acetic acid (I3AA) appeared in the buffer, and the concentration gradually increased with the prolongation of time ( FIG. 5A ). At the same time, the tryptophan concentration in the buffer gradually decreased, and was basically consumed after 1 h (Fig. 5B), indicating that Brauterella had the ability to metabolize tryptophan to produce indole-3-acetic acid.
  • I3AA metabolite indole-3-acetic acid
  • I3AA indole-3-acetic acid sodium salt
  • the inventors detected and found that compared with the control group, under the stimulation of 500 ⁇ mol/L I3AA, the phosphorylation levels of AKT and GSK3 ⁇ proteins were significantly increased (Fig. 6), indicating that the metabolite indole-3- Acetic acid significantly activates insulin signaling in hepatic primary cells.
  • 6-week-old male C57BL/6J mice were fed with 60% fat content (HFD) and corresponding control food (normal fat content, NCD) for 16 weeks to create a mouse model of insulin resistance; oral administration was started 4 weeks before the end of the experiment 10 mg/kg body weight of indole-3-acetic acid sodium salt (I3AA) was administered by gavage for 4 weeks, then blood glucose and GTT and ITT tests were performed (Fig. 7A).
  • HFD 60% fat content
  • NCD normal fat content
  • the inventors detected and found that compared with the control group, the fed and starved blood glucose, starved serum insulin content, HOMA-IR index, GTT and ITT of the mice in the I3AA group were significantly improved (Fig. 7B-F). It shows that indole-3-acetic acid can relieve the body's insulin resistance and lower blood sugar.

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Abstract

L'invention concerne l'utilisation de Blautia coccoides dans la préparation d'une composition destinée à inhiber les troubles du glycométabolisme ou à lutter contre les troubles de la flore intestinale. L'invention concerne en outre l'utilisation de Blautia coccoides pour produire de l'acide indole-3-acétique.
PCT/CN2021/093854 2020-12-28 2021-05-14 Probiotique intestinal pour lutter contre des maladies métaboliques et son utilisation WO2022142070A1 (fr)

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