Bacteroides parapseudovaccae for alleviating insulin resistance and its application

Abstract

The present invention discloses Parabacteroides distasonis NSP007 for alleviating insulin resistance and its application, belonging to the field of microbial technology. Parabacteroides distasonis NSP007 of the present invention can inhibit the weight gain and body fat accumulation of IR mice without affecting food intake; improve the glucose tolerance and insulin sensitivity of IR mice, while reducing the serum insulin and insulin resistance index of mice; regulate the lipid metabolism disorder of IR mice and relieve liver tissue damage; improve the intestinal barrier function of IR mice. The Parabacteroides distasonis NSP007 of the present invention is used for preparing a pharmaceutical composition for alleviating IR, and has very broad application prospects.

Description

Technical Field

[0001] The invention relates to Parabacteroides gibbsii for alleviating insulin resistance and application thereof, belonging to the technical field of microorganisms. Background Art

[0002] Insulin resistance (IR) is a symptom of increased compensatory insulin secretion. IR is an important pathological basis of type 2 diabetes mellitus (T2DM). The occurrence of T2DM and various metabolic diseases is often related to IR. The number of patients with T2DM will reach 700 million in 2045, which will bring a huge burden to people's lives. At present, insulin-sensitizing drugs are mainly used to treat IR, but long-term medication has many side effects. Chronic inflammation is one of the important factors leading to the occurrence of IR. Mechanistically, long-term high-fat diet can lead to intestinal flora imbalance, destroy intestinal barrier function, further cause bacterial endotoxin (LPS) in the intestine to leak into the blood and activate toll-like receptor 4 (TLR-4), induce the production of tumor necrosis factor α (TNF-α), and cause inflammatory response. TNF-α directly affects insulin signal transduction, thereby hindering the cell's uptake of glucose and inducing IR. Therefore, by improving the intestinal barrier function, the leakage of LPS can be reduced from the root, thereby slowing down the process of IR. Studies have shown that a variety of intestinal flora (Akkermansia, Lactobacillus, Bifidobacterium) can have beneficial effects on the host's intestinal barrier function and sugar metabolism. Parabacteroides gibbsii is an important and common bacteria in the adult intestine. It has the probiotic function of inhibiting intestinal pathogens and is a highly promising next-generation "probiotic".

[0003] The patent with the publication number CN108289917A records a system and method for treating microecological imbalance with a fecal bacterial community containing Parabacteroides goldsteinii; the patent with the publication number CN116555096A records that a strain of Parabacteroides goldsteinii has the ability to rapidly convert ginsenoside Re into ginsenoside Rg2 and protopanaxatriol PPT; the patent with the publication number CN116751699A records a strain of Porcine Parabacteroides distasonis with antibacterial effects and its application; the patent with the publication number CN113876815A records the application of a strain of Parabacteroides distasonis in the treatment and prevention of male reproductive toxicity drugs; the patent with the publication number CN111728986A records that a compound bacterium agent containing Parabacteroides goldsteinii can effectively reduce intestinal permeability and reduce the level of intestinal inflammation; the patent with the publication number CN113651896A records an extracellular polysaccharide of Parabacteroides distasonis, its extraction method and application; the patent with the publication number CN110839693A records the application of a strain of Parabacteroides goldsteinii in the prevention or treatment of obesity or related diseases; the patent with the publication number CN107550942A records the application of a strain of Parabacteroides goldsteinii in the treatment and prevention of metabolic diseases; the patent with the publication number CN112322545A records a strain of Parabacteroides distasonis LCG-06, its compound bacterium agent compounded with bile acids and its application; the patent with the publication number CN113750121A records the application of a strain of Parabacteroides distasonis in the preparation of pharmaceutical preparations for Alzheimer's disease; the patent with the publication number CN112410242A records a strain of Parabacteroides distasonis isolated from rectal cancer tumor tissue and its application in inhibiting the proliferation of colorectal cancer cell lines; the patent with the publication number CN113143971A records the application of a bile acid compound bacterium agent containing a strain of Parabacteroides distasonis in the preparation of a preparation for preventing and treating pet tear stains; Parabacteroides goldsteinii has a variety of probiotic effects. Although some studies have shown that Parabacteroides goldsteinii can alleviate the symptoms of obesity or metabolic diseases, IR is an important pathological basis for various metabolic diseases such as obesity and T2DM, and impaired intestinal barrier is an important inducement for the occurrence of IR. Currently, there is no report indicating the improvement effect of Parabacteroides goldsteinii on IR and impaired intestinal barrier function.

[0004] Therefore, there is an urgent need for a beneficial bacterium that can relieve impaired intestinal barrier function and IR caused by a high-fat diet. Summary of the Invention

[0005] In order to relieve the impaired intestinal barrier and IR caused by a high-fat diet, the present invention screened a strain of Parabacteroides goldsteinii from the feces of T2DM patients and proved that it has the effect of comprehensively improving the impaired intestinal barrier function and IR. The present invention provides important theoretical support and guiding significance for the intervention of pre-diabetes.

[0006] The present invention provides a strain of Parabacteroides distasonis NSP007, which was deposited in the Institute of Microbiology, Guangdong Academy of Sciences on August 25, 2021. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 61888.

[0007] The Parabacteroides distasonis NSP007 is isolated from the fermentation broth of fecal samples of T2DM patients from Jiangxi Province. The strain is sequenced and analyzed, and its 16S rDNA sequence is shown in SEQID NO.1. The sequence obtained by sequencing is compared with the nucleic acid sequence of Parabacteroides distasonis in NCBI, and the result shows that the similarity with the nucleic acid sequence of Parabacteroides distasonis is as high as 99%. It is named Parabacteroides distasonis NSP007. At the same time, it should be pointed out that Parabacteroides distasonis PD26 and Parabacteroides distasonis NSP007 on the biological material preservation certificate are different naming methods for the same strain of bacteria. The text of the present invention adopts the naming of Parabacteroides distasonis NSP007.

[0008] The Parabacteroides gibbsii NSP007 has the following properties: bacterial characteristics: it is a Gram-negative rod-shaped bacterium, without spores, with a diameter of 0.8-1.6×1.2-12μm, a colony diameter of 1-2mm on an anaerobic blood agar plate, a round front shape, a convex middle, neat edges, slightly white, opaque, and a moist and smooth surface. Growth characteristics: the strain is an obligate anaerobe, with an optimal growth temperature of 36°C-38°C, an optimal growth pH of 6.6-7.0, and good growth in a medium containing glucose, and can enter the late logarithmic phase or the early stable phase in 12-24h.

[0009] The present invention also provides a microbial agent, which contains the Parabacteroides distasonis NSP007.

[0010] In one embodiment of the present invention, the viable count of Parabacteroides distasonis NSP007 in the microbial agent is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g.

[0011] The present invention also provides a product, which contains the above-mentioned Parabacteroides distasonis NSP007.

[0012] In one embodiment of the present invention, in the product, the viable count of Parabacteroides distasonis NSP007 is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g.

[0013] In one embodiment of the present invention, the product is a microbial inoculant, a medicine, a health product or a feed additive.

[0014] In one embodiment of the present invention, the medicine contains Parabacteroides distasonis NSP007, a drug carrier and / or a pharmaceutical excipient.

[0015] In one embodiment of the present invention, the dosage form of the medicine or the health product includes dosage forms such as granules, capsules, tablets, pills or oral liquids.

[0016] In one embodiment of the present invention, the pharmaceutical excipient is a pharmaceutically acceptable excipient.

[0017] In one embodiment of the present invention, the acceptable excipients include one or more commonly used thickeners, antioxidants, acid-base regulators, emulsifiers, preservatives, fillers, binders, wetting agents, disintegrants, lubricants and flavoring agents, etc.

[0018] In one embodiment of the present invention, the filler is starch, sucrose, lactose, calcium sulfate and / or microcrystalline cellulose.

[0019] In one embodiment of the present invention, the binder is a cellulose derivative, alginate, gelatin and / or polyvinylpyrrolidone.

[0020] In one embodiment of the present invention, the wetting agent is water, ethanol, starch and / or syrup.

[0021] In one embodiment of the present invention, the disintegrant is sodium carboxymethyl starch, carboxypropyl cellulose, cross-linked carboxymethyl cellulose, agar, calcium carbonate and / or sodium bicarbonate.

[0022] In one embodiment of the present invention, the lubricant is talc, calcium stearate, magnesium stearate, colloidal silica and / or polyethylene glycol.

[0023] In one embodiment of the present invention, the flavoring agent is simple syrup, sucrose, lecithin, orange peel syrup, cherry syrup, lemon, fennel, peppermint oil, sodium alginate, gum arabic, gelatin, methylcellulose, sodium carboxymethylcellulose, citric acid, tartaric acid, and / or sodium bicarbonate.

[0024] The present invention also provides the application of the above-mentioned Parabacteroides distasonis NSP007, or the above-mentioned microbial agent in the preparation of a medicament for preventing and / or treating IR.

[0025] In one embodiment of the present invention, in the medicament, the viable count of Parabacteroides distasonis NSP007 is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g.

[0026] In one embodiment of the present invention, the medicament comprises Parabacteroides distasonis NSP007, a pharmaceutical carrier, and / or a pharmaceutical excipient.

[0027] In one embodiment of the present invention, the dosage form of the medicament includes dosage forms such as granules, capsules, tablets, pills, or oral liquids.

[0028] In one embodiment of the present invention, the pharmaceutical excipient is a pharmaceutically acceptable excipient.

[0029] In one embodiment of the present invention, the acceptable excipients include one or more commonly used thickeners, antioxidants, acid-base regulators, emulsifiers, preservatives, fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents, etc.

[0030] In one embodiment of the present invention, the filler is starch, sucrose, lactose, calcium sulfate, and / or microcrystalline cellulose.

[0031] In one embodiment of the present invention, the binder is a cellulose derivative, alginate, gelatin, and / or polyvinylpyrrolidone.

[0032] In one embodiment of the present invention, the wetting agent is water, ethanol, starch, and / or syrup.

[0033] In one embodiment of the present invention, the disintegrant is sodium carboxymethyl starch, carboxypropyl cellulose, cross-linked carboxymethyl cellulose, agar, calcium carbonate, and / or sodium bicarbonate.

[0034] In one embodiment of the present invention, the lubricant is talc, calcium stearate, magnesium stearate, colloidal silica, and / or polyethylene glycol.

[0035] In one embodiment of the present invention, the flavoring agent is simple syrup, sucrose, lecithin, orange peel syrup, cherry syrup, lemon, fennel, peppermint oil, sodium alginate, gum arabic, gelatin, methylcellulose, sodium carboxymethylcellulose, citric acid, tartaric acid, and / or sodium bicarbonate.

[0036] The present invention also provides the use of the above-mentioned Parabacteroides distasonis NSP007, or the above-mentioned microbial agent in the preparation of a health product for maintaining a healthy blood glucose level.

[0037] Beneficial effects

[0038] 1. The present invention has screened out a strain of Parabacteroides distasonis NSP007, which has the effect of alleviating IR, specifically manifested as follows:

[0039] 1) It can reduce the body fat of IR mice and inhibit the increase in body weight of mice without affecting the food intake of mice.

[0040] 2) It can improve the glucose tolerance, insulin tolerance, hyperinsulinemia, and insulin resistance of high-fat-induced IR mice.

[0041] 3) It can reduce liver fibrosis and lipid deposition in high-fat diet-induced IR mice and lower the levels of free fatty acids and blood lipids in serum.

[0042] 4) It can reduce the contents of pro-inflammatory factors IL-10 and IL-1β and the level of LPS in serum of high-fat diet-induced IR mice.

[0043] 5) It can increase the expression of genes and proteins related to intestinal barrier function in high-fat diet-induced IR mice.

[0044] 2. Parabacteroides distasonis is a potential probiotic with great research value, and its various probiotic effects have been widely reported. The inventors of the present invention have found through a large number of creative experimental studies that the Parabacteroides distasonis NSP007 of the present invention can effectively alleviate the impairment of intestinal barrier function and IR caused by a high-fat diet, and can be applied to drugs for preventing T2DM and treating IR and related diseases.

[0045] Biological material preservation

[0046] A strain of Parabacteroides distasonis NSP007 was deposited at the Institute of Microbiology, Guangdong Academy of Sciences on August 25, 2021. Its taxonomic name is Parabacteroides distasonis, and the deposit number is GDMCC No: 61888. The deposit address is the 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences. Description of the Drawings

[0047] Figure 1 : Photographs of the liver (left), H&E staining of liver tissue (middle), and Oil Red O staining of liver tissue (right) of IR mice after intervention with Parabacteroides distasonis NSP007. The scale bar is 50 μm.

[0048] Figure 2 : Immunohistochemical staining of F4 / 80 in the liver of IR mice after intervention with Parabacteroides distasonis NSP007. The scale bar is 50 μm.

[0049] Figure 3 : Immunohistochemical staining of Claudin-1, Muc-2, Occludin, and ZO-1 in intestinal tissue of IR mice after intervention with Parabacteroides distasonis NSP007. The scale bar is 50 μm. Detailed Implementation Modes

[0050] The mice involved in the following examples were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd. and were raised at 25 ± 2°C, with a constant humidity of 50 ± 5%, a light cycle of 12 hours (8:00 - 20:00), in a soundproof environment, with free access to food and water. After one week of adaptive feeding, the experiments were started. The insulin kit (purchased from crystal chem, catalog number: 90080), blood glucose test strips, and blood glucose meter involved in the following examples were purchased from Roche; the TNF-α inflammatory factor kit (catalog number: FMS-ELM028) and IL-1β inflammatory factor kit (catalog number: FMS-ELM002) were purchased from Nanjing Fumas Biotechnology Co., Ltd.; the endotoxin (LPS) detection kit (catalog number: F10621-B) was purchased from Wuhan Huamei Bio-Engineering Co., Ltd.; the free fatty acid (FFA) kit (catalog number: A042-2-1), total cholesterol (TC) kit (catalog number: A111-1), high-density lipoprotein (HDL-c) kit (catalog number: A112-1), low-density lipoprotein (LDL-c) kit (catalog number: A113-1-1), and triglyceride TG kit (A110-1-1) were purchased from Nanjing Jiancheng Bioengineering Institute; the Trizol reagent (catalog number: 15596026) involved in the following examples was purchased from Thermo Fisher Scientific, USA; the reverse transcription kit (catalog number: RR047A) and real-time quantitative kit (catalog number: RR820A) involved in the following examples were purchased from Takara Biotechnology (Beijing) Co., Ltd.; the culture medium components involved in the following examples were all purchased from Shanghai Yuanye Co., Ltd.; the high-fat diet involved in the following examples was purchased from Research Diets, USA (catalog number: D12492). The normal diet involved in the following examples was purchased from Wuhan Slack Co., Ltd. (breeding diet for mice and rats). The primers involved in the following examples were all ordered from Shanghai Sangon Biotech Co., Ltd.

[0051] Parabacteroides distasonis NSP007 involved in the following examples is a different naming of the same strain as Parabacteroides distasonis PD26 on the biological material preservation certificate. In the main text of the present invention, the naming of Parabacteroides distasonis NSP007 is adopted.

[0052] The culture media involved in the following examples:

[0053] Preparation of the activation medium (g / L): The components include carbon sources: pectin 0.047, xylan 0.047, arabinogalactan, amylopectin 0.04, soluble starch 0.392; nitrogen sources: bacteriological peptone 24, tryptone 24; inorganic salts: magnesium sulfate heptahydrate 0.5, potassium dihydrogen phosphate 2.5, sodium chloride 4.5, calcium chloride dihydrate 0.45, ferric sulfate heptahydrate 0.005; bile salts 0.4, anaerobic agent cysteine hydrochloride 0.2, and acid-base buffer (MES) 19.52. First, the above components are formulated and the pH is adjusted to 6, followed by deoxygenation and sterilization (121 °C, 15 min). After sterilization, the medium is transferred to an anaerobic glove box, and 1 μg of heat-labile hemin, 1 μg of vitamin K3 (VK3), and 0.1 mL of vitamin mixture (Wolfe's Vitamin Solution) are added to 1 L of the medium through a 0.22-μm filter membrane, and deoxygenation is carried out overnight in the anaerobic glove box to obtain the activation liquid medium.

[0054] Preparation of the enrichment medium: Each liter of the enrichment medium is composed of 350 mL of solution A, 150 mL of solution B, 500 mL of solution C, 1 mL of solution D, and 0.08 mL of vitamin mixture (Wolfe's Vitamin Solution). The formula (g / L) includes: Solution A: bacteriological peptone 68.57, tryptone 68.57, bile salts 1.14, anaerobic agent cysteine hydrochloride 1.43, magnesium sulfate 1.14, potassium hydrogen phosphate 5.48, sodium chloride 12.86, calcium chloride 0.97, ferric sulfate heptahydrate 0.014; Solution B: acid-base buffer (MES) 130, Solution C: polysaccharide from Dendrobium officinale 10, Solution D: hemin 10 mg, vitamin K3 (VK3) 8 mg. First, the components that can be autoclaved (solutions A - C) are formulated, the pH is adjusted to 6, followed by deoxygenation, and then sterilization (121 °C, 15 min). After sterilization, the medium is transferred to an anaerobic glove box overnight. Finally, solution D and Wolfe's Vitamin Solution are filtered through a 0.22-μm filter membrane and added to the medium in proportion to obtain the enrichment medium.

[0055] BHI liquid medium (g / L): Peptone 10.0, bovine heart infusion powder 17.5, sodium chloride 5.0, glucose 2.0, disodium hydrogen phosphate 2.5, dissolved in 1 L of distilled water, and 0.5 g of cysteine hydrochloride is added, mixed evenly, and then the pH is adjusted to 7.2 - 7.6. After sterilization at 115 - 121 °C for 15 - 20 min, the BHI liquid medium is obtained.

[0056] Preparation of BHI solid medium: Add 1.5 - 2% agar to the BHI liquid medium. Mix evenly, then adjust its pH to 7.2 - 7.6, and sterilize at 115 - 121 °C for 15 - 20 min to obtain the BHI solid medium.

[0057] BHI selective medium (g / L): Add 5 mg / L of hemin, 10 mg / L of vitamin K1, 7.5 mg / L of vancomycin, and 100 mg / L of kanamycin, which are sterilized by membrane filtration, to the BHI solid medium. Then the BHI selective medium is obtained.

[0058] The detection methods involved in the following examples are as follows:

[0059] Determination of the weights of mouse epididymal fat and liver tissues: After the experiment at the 5th week ended, the mice were anesthetized and sacrificed, and the intact epididymal fat and liver of the mice were isolated, weighed, and recorded.

[0060] Epididymal index (%) = (weight of epididymal fat (g) / body weight of mouse (g)) × 100

[0061] Observation of tissue morphology and immunohistochemistry of mouse liver and colon:

[0062] (1) H&E staining: Embed the tissue in paraffin. Before staining, dewax the paraffin sections, stain them with hematoxylin and eosin respectively, and observe microscopically after mounting the dehydrated sections.

[0063] (2) Oil Red O staining: Embed the tissue in paraffin, cut it into sections after freezing, immerse the sections in Oil Red staining solution, counterstain with hematoxylin, and observe microscopically after mounting with glycerin gelatin.

[0064] (3) Immunohistochemistry: Embed the tissue in paraffin, block with hydrogen peroxide, perform antigen repair, add the primary antibody and secondary antibody in sequence, stain with DAB, counterstain with hematoxylin, and observe microscopically after mounting the dehydrated sections.

[0065] (4) Percentage of immunohistochemical positive area of intestinal barrier-related proteins (%): Calculate the percentage of the positive area through ImageJ 1.53k software.

[0066] Real-time fluorescence quantitative PCR:

[0067] First, extract the RNA in the tissue using the Trizol method. The specific operation refers to the instruction manual of the kit. Measure the RNA concentration and purity using NanoDrop and agarose gel electrophoresis. Further, perform reverse transcription. Add 1 μg of total RNA in the tissue to 2 μL of 5×gDNA Buffer, 1 μL of gDNA Eraser, and make up to 10 μL with RNase-Free ddH2O. Mix briefly and centrifuge, then incubate at 42 °C for 2 min to remove genomic DNA in the sample. Then add 1 μL of PrimeScript RT Enzyme MixⅠ, 1 μL of RT Primer Mix, 4 μL of 5×PrimeScript Buffer 2, and make up to 20 μL with RNase-Free ddH2O. React at 37 °C for 15 min, and then react at 85 °C for 5 s to complete the reverse transcription.

[0068] Take the product after reverse transcription, dilute it 10 times as the template for real-time fluorescence quantitative PCR. qPCR reaction system (20 μL): 2 μg of cDNA template, 10 μL of SYBR dye solution, 0.8 μL of primer, and make up the remaining volume with ddH2O. Reaction conditions: 25 - 50 °C, 1.6 °C / s, hold at 50 °C for 2 min; 50 - 95 °C, 1.6 °C / s, hold at 95 °C for 30 s; amplification (95 °C for 20 s, 60 °C for 1 min, a total of 40 cycles). Set 2 sub-wells for each gene, and use 2−ΔΔCt for data processing. -ΔΔCT Perform relative quantitative analysis.

[0069] Table 1 Primers used in real-time fluorescence quantitative PCR

[0070]

[0071] Oral glucose tolerance test (OGTT): After the animals are fasted for 6 h, measure the basal blood glucose at 0 min, then intragastrically administer 1.5 g / kg of glucose. Collect blood from the tail vein of the mice and measure the blood glucose at 15, 30, 60, and 90 min after glucose gavage using a Roche blood glucose meter. Plot the blood glucose-time curve, and calculate the area under the curve (AUC) for each group based on the blood glucose values at each time point. Plot the blood glucose-time curve, and calculate the area under the curve AUC for each group based on the blood glucose values at each time point.

[0072] AUC calculation method (the same below): AUC (mg*min / dL) = (BG0 + BG 15 ) × 15 / 2 + (BG 15 + BG 30 ) × 15 / 2 + (BG 30 + BG 60 ) × 30 / 2 + (BG 60 + BG 90 ) × 30 / 2

[0073] BG0, BG 15 , BG 30 , BG 60 , BG 90 represent the blood glucose at 0 min, 15 min, 30 min, 60 min, and 90 min after treatment.

[0074] Insulin tolerance test (ITT): After the animals were fasted for 6 h, the basal blood glucose at 0 min was measured, and then insulin (0.8 U / kg) was intraperitoneally injected. Blood was collected from the tail vein of the mice at 15, 30, 60, and 90 min after insulin injection, and the blood glucose was measured using a Roche blood glucose meter. The blood glucose-time curve was plotted, and the area under the curve (AUC) of each group was calculated based on the blood glucose values at each time point (the same as above).

[0075] Determination of serum insulin: After the experiment, the mice were anesthetized and sacrificed, and the serum was obtained. The insulin content in the serum was measured according to the instructions of the kit.

[0076] Determination of fasting blood glucose: After the animals were fasted for 6 h, blood was collected from the tail vein of the mice, and the blood glucose was measured using a Roche blood glucose meter.

[0077] Calculation method of insulin resistance index (HOMA-IR):

[0078] HOMA-IR = fasting serum insulin (μU / mL) × fasting blood glucose (mmol / L) / 22.5

[0079] Determination of FFA, TG, TC, HDL-c, LDL-c, LPS, TNF-α, IL-1β, and fasting insulin: Refer to the instructions of the corresponding kits.

[0080] Example 1: Isolation and screening of Parabacteroides gilvus NSP007

[0081] 1. Sample collection

[0082] Fecal samples of T2DM patients in Jiangxi region were collected. The samples were placed in preservation tubes, and 5 times the weight of the protective solution was added (preparation of the protective agent: weigh 1 g / L of cysteine hydrochloride and 200 - 300 g / L of glycerol, dissolve them uniformly in PBS (1×), sterilize at 115 - 121 °C for 15 - 20 min), and stored in an insulated box with dry ice. After being brought back to the laboratory, they were quickly placed in a -80 °C refrigerator for isolation and screening.

[0083] 2. Enrichment of fecal bacteria

[0084] Take out the above fecal bacteria solution from the -80°C refrigerator. After thawing, centrifuge it at low speed and low temperature (500g, 5min, 4°C) to obtain the supernatant. Then filter the supernatant through a 100μm filter membrane to remove impurities. Inoculate the supernatant fecal bacteria solution into the activated medium (fecal bacteria solution: activated medium = 1:9, (v / v)). Incubate it at 37°C with 140rpm for 16h, and then inoculate it into the enrichment medium with an inoculation ratio of 10% (v / v). Incubate it at 37°C with 140rpm for 24h to obtain the fecal bacteria solution enriched with Dendrobium officinale polysaccharide. The above operations are carried out in a sterile anaerobic environment.

[0085] 3. Isolation and purification of Bacteroides

[0086] (1) Gradient dilution of fecal bacteria solution: In a sterile anaerobic environment, take the above-enriched fecal bacteria solution and add it to 9mL of normal saline to obtain the first gradient dilution solution. Pipette 1mL of the first gradient dilution solution into 9mL of normal saline to obtain the second gradient dilution solution, and so on. A total of 5 gradient dilution solutions are prepared.

[0087] (2) Spread plate culture: Pipette 100μL of all the above gradient dilution solutions respectively onto the BHI solid medium, spread them, and then incubate them under anaerobic conditions at 37°C for 48h to obtain the dilution spread plates.

[0088] (3) Purification culture: Pick the pure single colonies with neat edges, slightly white, opaque, moist and smooth surfaces, and consistent morphology on the solid medium and inoculate them into 5mL of liquid BHI selective medium. Incubate them under anaerobic conditions at 37°C for 24h to obtain the purified culture solution.

[0089] 4. Strain preservation and identification

[0090] Centrifuge the purified culture solution with the best growth obtained in step 3 at 8000rpm for 10min, discard the supernatant to obtain the bacterial cells. Perform PCR using the bacterial 16S rDNA PCR specific primers (see Table 2). After the PCR product is analyzed by nucleic acid electrophoresis and confirmed, send the amplified product to the company for sequencing. Its 16S rDNA sequence is shown in SEQ ID NO.1, and the sequencing result is compared and analyzed with the sequences in the NCBI database; the result shows that the nucleic acid sequence similarity with Parabacteroides distasonis is as high as 99%, and it is named Parabacteroides distasonis NSP007.

[0091] Table 2 Primer names

[0092]

[0093] Example 2: Effects of Parabacteroides distasonis NSP007 on body weight, body fat and diet of IR mice fed a high-fat diet

[0094] The specific steps are as follows:

[0095] 1. Preparation of the cryopreservative for Parabacteroides goldsteinii NSP007:

[0096] (1) Cultivation method: In a sterile anaerobic environment, streak the Parabacteroides goldsteinii NSP007 strain on BHI solid medium, and culture it anaerobically for 48 h. After single colonies are formed, pick a single colony and inoculate it into BHI liquid medium, and culture it anaerobically at 37 °C for 16 - 24 h until the stationary phase is reached. At this time, the OD value is 0.8 - 1.0, and the seed liquid is prepared.

[0097] (2) Preparation of the cryoprotectant: Weigh 1 g / L of cysteine hydrochloride and 200 - 300 g / L of glycerol, dissolve them evenly in distilled water, and sterilize at 115 - 121 °C for 15 - 20 min.

[0098] (3) Preparation of the cryogen: After centrifuging the Parabacteroides goldsteinii NSP007 seed liquid cultured to the stationary phase in step (1) (8000 rpm, 10 min, 4 °C), wash it 1 - 2 times with sterile phosphate buffer (pH 7.2), and resuspend the bacterial liquid with the cryoprotectant prepared in step (2) to obtain the cryopreservative for Parabacteroides goldsteinii NSP007, and store it at -80 °C for standby.

[0099] 2. Preparation of the Parabacteroides goldsteinii NSP007 bacterial agent:

[0100] (1) Activation of the strain: Streak the cryopreservative for Parabacteroides goldsteinii NSP007 prepared in step 1 on BHI solid medium, and culture it anaerobically for 48 h. After single colonies are formed, inoculate the single colony into BHI liquid medium, and culture it anaerobically at 37 °C for 16 - 24 h until the stationary phase is reached (OD value is 0.8 - 1.0).

[0101] (2) Preparation of the bacterial agent: Take 100 μL of the culture solution obtained in step (1) with different dilution multiples and spread it on BHI solid medium, count the number of colonies on the BHI solid plate, and calculate the number of viable bacteria in the liquid medium in step (1). After washing 1 - 2 times with sterile phosphate buffer (pH 7.2), prepare the bacterial liquid into a preparation with a concentration of 1×10 9 CFU / mL, and the gavage volume is 0.1 mL.

[0102] 3. Experimental method:

[0103] Process of the intervention treatment experiment:

[0104] In this invention, a method of feeding with a high-fat diet was used to induce insulin resistance (IR) in mice. Thirty-two healthy male C57BL / 6J mice at 6 weeks of age were randomly divided into four groups (8 mice in each group): a normal group (denoted as N for convenience of description), a high-fat diet insulin resistance group (model group, denoted as M), a live Bacteroides paramagnus NSP007 group (Bacteroides paramagnus group, denoted as LPD), and a heat-inactivated Bacteroides paramagnus NSP007 group (Bacteroides paramagnus group, denoted as KPD). After 8 weeks of high-fat diet, the fasting blood glucose, fasting serum insulin levels of the mice in each high-fat diet group were measured, and the area under the oral glucose tolerance curve and insulin resistance index were calculated. The results showed that the above indexes of the mice in the high-fat diet group were significantly higher than those of the normal group mice, indicating that the establishment of the high-fat-induced insulin resistance mouse model was successful (8 mice in each group); the specific indexes are shown in Table 3:

[0105] Table 3 Indexes for successful establishment of the high-fat-induced insulin resistance mouse model

[0106]

[0107] The experimental procedure is shown in Table 4. After a one-week adaptation period; Normal group (N): fed with normal feed and given free access to water during the modeling period, continued to be fed with normal feed during the treatment period, gavaged once a day with 0.1 mL of sterile phosphate buffer, and given free access to water;

[0108] During the treatment period, while high-fat feed was used to intervene in IR, Bacteroides paramagnus NSP007 was used for treatment. The process of the intervention treatment experiment:

[0109] Model group (M): gavaged once a day with 0.1 mL of sterile phosphate buffer during the intervention period;

[0110] Bacteroides paramagnus NSP007 group (LPD): gavaged once a day with 0.1 mL of Bacteroides paramagnus NSP007 bacterial solution (bacterial concentration: 1×10 9 CFU / mL) during the intervention period;

[0111] Heat-inactivated Bacteroides paramagnus NSP007 group (KPD): gavaged once a day with 0.1 mL of heat-inactivated Bacteroides paramagnus NSP007 bacterial solution (bacterial concentration: 1×10 9 CFU / mL) during the intervention period.

[0112] After 5 weeks of intervention, the mice were anesthetized and sacrificed. Blood was taken from the mouse orbits, the blood was collected, centrifuged at 3000 rpm for 15 min, and mouse serum was obtained. The serum, epididymal fat, and liver were stored at -80 °C for subsequent analysis.

[0113] Table 4 Experimental procedure

[0114]

[0115] 4. Effects of Parabacteroides gilvus NSP007 on body weight, body fat, and diet of IR mice on a high-fat diet

[0116] The specific experimental procedure was the same as in Steps 1-3, except that during the intervention period, each group of mice was weighed every 4 days. After the experiment ended, the mice were sacrificed, and their livers and epididymal fat were collected and weighed. The epididymal index was calculated based on the body weight and the weight of the epididymal fat. The experimental results are shown in Tables 5, 6, and 7. The intervention with LPD had no significant effect on the energy intake of the mice, but the intervention with LPD significantly inhibited the weight gain, liver weight gain, and epididymal fat accumulation of the IR mice.

[0117] Table 5 Effects of Parabacteroides gilvus during the intervention period on the energy intake (kcal / day / mouse) of IR mice

[0118]

[0119]

[0120] Table 6 Effects of Parabacteroides gilvus during the intervention period on the body weight (g) of IR mice

[0121]

[0122] Table 7 Effects of Parabacteroides gilvus on the organ weights and indices of IR mice

[0123]

[0124] Example 3: Effects of Parabacteroides gilvus NSP007 on insulin resistance in high-fat-induced IR mice

[0125] The specific steps are as follows:

[0126] The specific experimental method was the same as in Example 2, except that 3 days before the mice were sacrificed, an oral glucose tolerance test (OGTT) and an insulin tolerance test (ITT) were performed on each group of mice. After the experiment ended, the mice were anesthetized and sacrificed, and their sera were taken to measure insulin and calculate their insulin resistance index (HOMA-IR). The results are shown in Tables 8-10.

[0127] The results showed that the areas under the curves of OGTT and ITT in the LPD group were significantly lower than those in the M group and the KPD group. In addition, the HOMA-IR in the LPD group was also significantly lower than that in the M group and the KPD group. Compared with the M group, the areas under the curves of OGTT, ITT, and HOMA-IR decreased by 26.6%, 24.4%, and 58.5% respectively. The fasting insulin and insulin resistance index in the LPD group both recovered to levels comparable to those of the control group. This indicates that the Parabacteroides gilvus NSP007 of the present invention can improve the glucose tolerance and insulin sensitivity of IR mice, and alleviate hyperinsulinemia and insulin resistance.

[0128] Table 8 Effects of Parabacteroides gilvus on Glucose Tolerance (mg / dL) of IR Mice

[0129]

[0130] Table 9 Effects of Parabacteroides gilvus on Insulin Tolerance (mg / dL) of IR Mice

[0131]

[0132] Table 10 Effects of Parabacteroides gilvus on Insulin Resistance of IR Mice

[0133]

[0134] Example 4: Effects of Parabacteroides gilvus NSP007 on Hepatic Injury and Lipid Metabolism in High-Fat-Induced IR Mice

[0135] The specific steps are as follows:

[0136] The specific experimental method was the same as that in Example 2, except that after the experiment, mouse serum was collected to measure the levels of free fatty acids (FFA), triglycerides (TG), total cholesterol (TC), high-density lipoprotein (HDL-c), and low-density lipoprotein (LDL-c) in the mouse serum; the mouse livers were photographed and stained to observe the morphological changes of the livers of each group of mice. The results are shown in Table 11 and Figure 1 as follows.

[0137] Table 11 The results showed that the levels of FFA, TG, TC, and LDL-c in the LPD group were significantly lower than those in the M group and the KPD group. Compared with the M group, the levels of FFA, TG, TC, and LDL-c decreased by 24.8%, 32.6%, 23.6%, and 17.4% respectively; and the FFA and TG in the LPD group recovered to levels comparable to those of the control group. This indicates that LPD significantly improved the lipid metabolism disorder in IR mice.

[0138] Figure 1The results showed that there were obvious diffuse fatty degeneration (vacuolization) and fibrosis in the livers of the M group. After treatment with Parabacteroides gilvus NSP007, the liver morphology, size, fibrosis and fat accumulation of the mice were significantly improved compared with those of the M group and the KPD group, and were similar to those of the control group in morphology. This indicates that LPD can alleviate liver fibrosis and lipid deposition in IR mice induced by a high-fat diet, and reduce the levels of free fatty acids and blood lipids in the serum.

[0139] Table 11 Effects of Parabacteroides gilvus on lipid metabolism in IR mice

[0140]

[0141]

[0142] Example 5: Effects of Parabacteroides gilvus NSP007 on systemic inflammation and liver inflammation in high-fat-induced IR mice

[0143] The specific experimental method was the same as that in Example 2, except that after the experiment, the serum of the mice was collected to measure the levels of free TNF-α, IL-1β and LPS in the serum of the mice, and the livers of the mice were subjected to immunohistochemical staining to observe the aggregation behavior of macrophages in the livers of each group of mice. The results are shown in Table 12 and Figure 2 as follows.

[0144] Table 12 The results showed that the levels of TNF-α, IL-1β and LPS in the LPD group were significantly lower than those in the M group and the KPD group. Compared with the M group, the levels of TNF-α, IL-1β and LPS decreased by 26.8%, 42.5% and 45.8% respectively; and the levels of IL-1β and LPS in the LPD group recovered to the level equivalent to that of the control group. This indicates that LPD significantly improved the systemic inflammation in IR mice.

[0145] Figure 2 The results showed that there was obvious macrophage aggregation behavior in the livers of the M group. After LPD intervention, the macrophage aggregation behavior was significantly reduced. This indicates that LPD significantly improved the liver inflammation in IR mice.

[0146] Table 12 Effects of Parabacteroides gilvus on systemic inflammation in IR mice

[0147]

[0148] Example 6: Effects of Parabacteroides gilvus NSP007 on intestinal barrier function in high-fat-induced IR mice

[0149] The specific experimental method was the same as that in Example 2, except that after the experiment, the mouse colon tissues were collected, the expression levels of intestinal barrier-related genes (Claudin1, Muc2, Occludin, Zo1) in the mouse colon tissues were measured, and immunohistochemical staining was performed on the mouse colon to observe the expression levels of intestinal barrier-related proteins (Claudin-1, Muc2, Occludin, ZO-1) in the colon of each group of mice. The results are shown in Tables 13, 14 and Figure 3 as follows.

[0150] Table 13 results showed that the mRNA expression levels of Claudin1, Muc2, Occludin and Zo1 in the LPD group were significantly higher than those in the M group and the KPD group. Compared with the M group, the mRNA levels of Claudin1, Muc2, Occludin and Zo1 increased by 70.6%, 135.0%, 69.0% and 122.8% respectively, indicating that LPD can effectively improve intestinal barrier function.

[0151] Table 14 and Figure 3 the results showed that the protein expression levels of Claudin-1, Muc2, Occludin and ZO-1 in the LPD group were significantly higher than those in the M group and the KPD group. Compared with the M group, the protein levels of Claudin-1, Muc2, Occludin and ZO-1 increased by 24.3%, 73.1%, 44.8% and 267.9% respectively; and the expression levels of each gene and protein in the LPD group returned to the level equivalent to that of the control group. This indicates that LPD can effectively improve intestinal barrier function.

[0152] Table 13 Effects of Parabacteroides gilvus on the expression levels of intestinal barrier-related genes in IR mice

[0153]

[0154]

[0155] Table 14 Effects of Parabacteroides gilvus on the expression levels of intestinal barrier-related proteins (positive area, %) in IR mice (related to Figure 3 ...)

[0156]

[0157] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A strain of Parabacteroides distasonis NSP007 was deposited at the Institute of Microbiology, Guangdong Academy of Sciences on August 25, 2021. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 61888.

2. A microbial inoculant, characterized in that, Contains the Parabacteroides gibbsii according to claim 1.

3. The microbial inoculum according to claim 2, wherein In the microbial inoculum, the viable count of Parabacteroides gilvus NSP007 is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g.

4. A product containing the Parabacteroides goldsteinii as described in claim 1, characterized in that, The product is a microbial agent, medicine, health product or feed additive.

5. The product according to claim 4, characterized in that, In the said product, the viable count of Parabacteroides gilvus is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g.

6. The product according to claim 4, wherein The medicine comprises the Parabacteroides gibbsii described in claim 1, and further comprises a drug carrier and / or a pharmaceutical excipient.

7. The product according to claim 4, characterized in that, The dosage form of the medicine includes granules, capsules, tablets, pills or oral liquids.

8. Use of the Parabacteroides gibbsii described in claim 1, or the microbial agent described in claim 2 or 3 in the preparation of a medicine for treating insulin resistance.

9. Use of the Parabacteroides gibbsii described in claim 1, or the microbial agent described in claim 2 or 3 in the preparation of a health product for maintaining a healthy blood sugar level.

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