Phytobacterium plantarum A21246 with function of secreting GLP-1 and application of phytobacterium plantarum A21246

Abstract

The invention belongs to the technical field of functional microorganisms, and discloses a lactobacillus plantarum A21246 with a GLP-1 secretion function and application of the lactobacillus plantarum A21246, the lactobacillus plantarum A21246 is disclosed for the first time, and it is verified through experiments that the lactobacillus plantarum A21246 can reduce the level of blood sugar and glycosylated hemoglobin, promote secretion of GLP-1 in intestinal tracts, improve the blood sugar level and the glycosylated hemoglobin level and improve the blood sugar level and the glycosylated hemoglobin level, so that the lactobacillus plantarum A21246 can be used for improving the blood sugar level and the glycosylated hemoglobin level. The weight loss caused by diabetes mellitus is effectively improved, the formation of liver fatty degeneration is remarkably inhibited, the liver injury is relieved, the abnormal glycolipid metabolism is improved (the content of cholesterol and triglyceride in serum can be remarkably reduced), and the pathological forms of the liver and pancreas are improved. The plant lactobacillus A21246 plays an important role in preventing or treating diabetes mellitus and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of functional microorganisms, and particularly relates to a lactobacillus plantarum A21246 with a GLP-1 secretion function and an application thereof. Background Art

[0002] Diabetes mellitus (DM) is a common disease that endangers human health and can lead to insulin deficiency, insulin resistance and impaired biological functions. Due to its high complications and mortality, diabetes is a major public health threat around the world (including China). The disease can lead to a variety of health problems and complications, such as heart disease, eye disease, renal impairment, nerve damage, etc. There are two types of diabetes; type 2 diabetes mellitus (T2DM) accounts for about 85% to 90% of all cases. So far, there are many different treatment options that can effectively treat type 2 diabetes, such as exercise therapy, reasonable nutritional therapy and drug therapy. Drugs used to treat type 2 diabetes include sulfonylureas, biguanides, nateglinide, glucosidase inhibitors and thiazolidinediones. Although these traditional oral hypoglycemic drugs play a hypoglycemic effect by improving insulin action and increasing the efficiency of insulin secretion, they also have certain side effects, such as gastrointestinal intolerance, leading to liver damage, weight gain or worse. Therefore, the development of new hypoglycemic drugs is of great significance.

[0003] Glucagon-like peptide-1 (GLP-1) is a peptide that can lower blood sugar in multiple ways and has become a hot topic of research, among which GLP-1 analogs and GLP-1 receptor agonists have received much attention. GLP-1 is secreted by intestinal L cells and can lower postprandial blood sugar. Its hypoglycemic mechanism mainly includes enhancing the biosynthesis and secretion of insulin, and also interacting with glucagon to produce a hypoglycemic effect, which in a sense exceeds the effect of insulin. In recent years, intestinal flora has been considered to be a major component of the human body's internal environment and one of the main environmental factors determining the severity of diabetes. Animal experiments and population-based studies have shown that there is a close relationship between intestinal flora and diabetes. Germ-free mice develop type 1 diabetes (T1D), indicating that intestinal microorganisms help change T1D susceptibility. In another experiment, patients with type 2 diabetes were compared with subjects with normal glucose tolerance, and it was found that four lactobacilli that were positively correlated with fasting blood sugar and glycated hemoglobin (HbA1c) increased, while five Clostridia decreased and were negatively correlated with fasting blood sugar, glycated hemoglobin and insulin. Currently, GLP-1 analogs and dipeptidyl peptidase 4 (DPP-IV) inhibitors have been used as anti-diabetic drugs by promoting insulin secretion. The short half-life of glucagon-like peptide-1 (GLP-1) limits its clinical application. In addition, there are currently few patents on probiotic strains that secrete GLP-1. Most of them achieve the effect of sustained expression of GLP-1 through the construction of engineered bacteria, but the cost is high and the technology is difficult. There are differences in the expression efficiency and stability of different engineered bacteria, which may lead to unstable production and quality of GLP-1 derivatives or analogs, and safety issues such as gene escape. Therefore, it is necessary to develop natural therapeutic strategies that can regulate the level of sustained intestinal expression of GLP-1. Summary of the invention

[0004] The present invention aims to provide a Lactobacillus plantarum A21246 that can naturally secrete GLP-1 and promote the continuous expression of GLP-1 in the intestine. A21246 is a beneficial probiotic in the human body, which can effectively colonize in the intestine and is conducive to the continuous expression of GLP-1 levels to achieve the therapeutic effect of lowering blood sugar. It can be used as an effective and safe food supplement or drug adjuvant to reduce the side effects caused by long-term use of drugs.

[0005] The first aspect of the present invention aims to provide a strain of Lactobacillus plantarum A21246.

[0006] The second aspect of the present invention aims to provide a method for preparing a culture of Lactobacillus plantarum A21246.

[0007] The third aspect of the present invention is to provide a culture of Lactobacillus plantarum A21246.

[0008] The fourth aspect of the present invention aims to provide a microbial agent.

[0009] The fifth aspect of the present invention aims to provide the use of the Lactobacillus plantarum A21246 of the first aspect of the present invention, the culture of Lactobacillus plantarum A21246 of the third aspect of the present invention, or the microbial agent of the fourth aspect of the present invention.

[0010] The sixth aspect of the present invention aims to provide a pharmaceutical composition.

[0011] The seventh aspect of the present invention aims to provide a method for preparing GLP-1.

[0012] In order to achieve the above object, the technical solution adopted by the present invention is:

[0013] The first aspect of the present invention provides a strain of Lactobacillus plantarum A21246, which is deposited in Guangdong Microbiological Culture Collection Center with a deposit number of GDMCC NO: 65488, a deposit date of November 15, 2024, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0014] In the present application, Lactobacillus plantarum A21246 was isolated from the feces of a centenarian. The results of morphological and 16SrDNA sequence analysis showed that the bacterium was Lactobacillus plantarum. The bacterium has the function of resistance to artificial gastrointestinal fluid, can effectively colonize in the intestine, and can secrete GLP-1. In vivo experiments verified that Lactobacillus plantarum A21246 can significantly reduce the blood sugar level of mice and promote the continuous secretion of GLP-1 level in the intestine to achieve the therapeutic effect of lowering blood sugar.

[0015] The second aspect of the present invention provides a method for preparing a culture of Lactobacillus plantarum A21246, comprising:

[0016] Provide liquid culture system;

[0017] The Lactobacillus plantarum A21246 according to the first aspect of the present invention is inoculated into the liquid culture system for culture.

[0018] In some embodiments of the present invention, the liquid culture system contains nutrients, and the nutrients include components such as protein (peptone), sugar, phosphorus, magnesium, etc. For example, the nutrients can be provided in the form of a liquid culture medium, and the liquid culture medium is selected from any one of MRS medium, BBL and MPYG.

[0019] In some embodiments of the present invention, the pH value of the liquid culture system is 6-7, preferably 6.2±0.2.

[0020] In some embodiments of the present invention, the culture condition is culturing under facultative anaerobic conditions at 35-40° C. for 10-40 h, preferably culturing under anaerobic conditions at 37° C. for 30 h.

[0021] The third aspect of the present invention provides a culture of Lactobacillus plantarum A21246, which is prepared by the preparation method of the second aspect of the present invention.

[0022] A fourth aspect of the present invention provides a microbial agent, wherein the microbial agent comprises the culture of Lactobacillus plantarum A21246 of the first aspect of the present invention or the culture of Lactobacillus plantarum A21246 of the third aspect of the present invention.

[0023] The active ingredients of the above-mentioned bacterial agent may be the above-mentioned Lactobacillus plantarum A21246, the culture of the above-mentioned Lactobacillus plantarum A21246 (such as fermentation metabolites), and the active ingredients of the above-mentioned bacterial agent may also contain other biological components or non-biological components. Those skilled in the art may determine the other active ingredients of the above-mentioned bacterial agent according to the effects of the bacterial agent.

[0024] The term "culture" refers to a liquid or solid product (all substances in the culture container, fermentation products) that has a microbial community after artificial inoculation and culture. That is, the product obtained by growing and / or amplifying microorganisms, which can be a biologically pure culture of microorganisms, or it can contain a certain amount of culture medium, metabolites or other components produced during the culture process.

[0025] In the above-mentioned microbial agent, in addition to the active ingredient, the microbial agent also contains a carrier. The carrier can be a commonly used and biologically inert carrier in the field (such as microorganisms, agriculture). The carrier can be a solid carrier or a liquid carrier; the solid carrier can be a mineral material, a plant material or a polymer compound; the mineral material can be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica and diatomaceous earth; the plant material can be at least one of corn flour, soybean flour and starch; the polymer compound can be polyvinyl alcohol and / or polyglycol; the liquid carrier can be an organic solvent or water; the organic solvent can be decane and / or dodecane.

[0026] The above-mentioned bacterial agents may be in various dosage forms, such as liquid, emulsion, suspension, powder, granule, capsule, tablet, wettable powder or water dispersible granule.

[0027] According to needs, a binder, a stabilizer (such as an antioxidant), a pH adjuster, etc. may be added to the bacterial agent.

[0028] In some embodiments of the present invention, the bacterial agent further comprises: nutrients. The nutrients include: at least one of proteins, carbohydrates, lipids, minerals, vitamins, plant extracts, amino acids, immunomodulators and milk substitutes.

[0029] The fifth aspect of the present invention provides the use of the Lactobacillus plantarum A21246 of the first aspect of the present invention, the culture of Lactobacillus plantarum A21246 of the third aspect of the present invention, or the microbial agent of the fourth aspect of the present invention in at least one of (1) to (4):

[0030] (1) preparing GLP-1;

[0031] (2) Prepare products that help maintain healthy blood sugar levels;

[0032] (3) preparing products that help regulate the function of intestinal flora;

[0033] (4) Preparation of products for the prevention and / or treatment of metabolic disorders.

[0034] In some embodiments of the present invention, the metabolic abnormality in (4) is abnormal liver lipid metabolism caused by type 2 diabetes.

[0035] In some embodiments of the present invention, the abnormal liver lipid metabolism includes at least one of abnormally increased cholesterol and triglyceride levels in the liver, abnormally increased glycosylated hemoglobin levels, and the formation of fatty degeneration of the liver.

[0036] In some embodiments of the present invention, the products include but are not limited to pharmaceutical compositions, foods and health products.

[0037] A sixth aspect of the present invention provides a pharmaceutical composition comprising the Lactobacillus plantarum A21246 of the first aspect of the present invention, the culture of Lactobacillus plantarum A21246 of the third aspect of the present invention, or the microbial agent of the fourth aspect of the present invention.

[0038] In some embodiments of the present invention, the number of viable bacteria of Lactobacillus plantarum A21246 contained in the pharmaceutical composition is (1-10)×10 9 CFU / mL (CFU / g).

[0039] In some embodiments of the present invention, the pharmaceutical composition may further contain other combined drugs, and the drugs are used to treat diabetes / abnormal liver lipid metabolism.

[0040] In some embodiments of the present invention, the combination drug is selected from one or more of insulin secretagogues (such as glibenclamide, gliclazide, glipizide, gliclazide, glimepiride, etc.), non-sulfonylurea insulin secretagogues (such as repaglinide, nateglinide, etc.), glycosidase inhibitors (voglibose, acarbose, miglitol, etc.), thiazolidinediones (such as rosiglitazone maleate, pioglitazone hydrochloride, etc.), DPP-4 enzyme inhibitors (such as alogliptin, sitagliptin phosphate, linagliptin, saxagliptin, vildagliptin, etc.), SCLT2 inhibitors (canagliflozin, dapagliflozin, empagliflozin, etc.), and insulin.

[0041] In this case, at least two active ingredients may be administered together or separately, and as part of the same pharmaceutical preparation or as different preparations. Thus, the composition used in the context of the present invention may be used in combination with an antidiabetic agent, including but not limited to metformin, sulfonylureas, glinides, DPP-IV inhibitors, glitazones or insulin. In certain embodiments, the compound or its salt or solvate is used in combination with insulin, DPP-IV inhibitors, sulfonylureas or metformin (especially sulfonylureas or metformin) to obtain adequate glycemic control. In certain preferred embodiments, the composition is used in combination with insulin or insulin analogs to obtain adequate glycemic control.

[0042] In some embodiments of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0043] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of fillers, disintegrants, diluents, dispersants, excipients, stabilizers, lubricants, binders, wetting agents, flavoring agents, suspending agents, solvents, sustained-release agents, emulsifiers, absorption enhancers, surfactants, preservatives, pigments, flavors and solvents.

[0044] In some embodiments of the present invention, the filler is selected from starch, sucrose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose or glucose; the binder is selected from cellulose derivatives, alginate, starch, water, dextrin, gelatin or polyvinyl pyrrolidone; the disintegrant is selected from microcrystalline cellulose, sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone, low-substituted hydroxypropyl cellulose or cross-linked sodium carboxymethyl cellulose; the lubricant is selected from stearic acid, polyethylene glycol, calcium carbonate, sodium bicarbonate, micro-powder silica gel, talc or magnesium stearate; the suspending agent is selected from micro-powder silica gel, beeswax, cellulose, solid polyethylene glycol : The wetting agent is selected from glycerol, Tween-80, hydrogenated castor oil or lecithin; the solvent is selected from ethanol, liquid polyethylene glycol, isopropanol, Tween-80, glycerol, propylene glycol or vegetable oil, and the vegetable oil is selected from soybean oil, castor oil, peanut oil, blending oil, etc.; the surfactant is selected from sodium dodecylbenzene sulfonate, stearic acid, polyoxyethylene-polyoxypropylene copolymer, fatty acid sorbitan or polysorbate (Tween), etc.; the flavoring agent is selected from aspartame, sucralose, flavor, stevioside, acesulfame potassium, citric acid or saccharin sodium; the preservative is selected from at least one of methyl paraben or propyl paraben.

[0045] In some embodiments of the present invention, the dosage form of the pharmaceutical composition is selected from tablets (such as ordinary tablets, double-layer tablets, multi-layer tablets, sustained-release tablets, single-chamber controlled-release tablets, double-chamber controlled-release tablets, microporous controlled-release tablets, sublingual tablets, orally rapidly disintegrating tablets, dispersible tablets, enteric-coated tablets), pills, powders, suspensions, gels, emulsions, creams, granules, nanoparticles, capsules (such as ordinary capsules, sustained-release capsules, controlled-release capsules, capsules containing micropellets or small tablets, pH-dependent capsules containing micropellets or small tablets, gastrointestinal composite capsules), suppositories, injections, sprays and injections.

[0046] The compositions of the present invention may be in unit dosage form. In this form, the composition is divided into unit doses containing appropriate quantities of the active ingredient. The unit dosage form may be a packaged preparation, the package containing discrete quantities of the preparation, such as packaged tablets, capsules, and powders in vials or ampoules. The unit dosage form may also be a capsule, cachet, or tablet itself, or it may be the appropriate number of any of these packaged forms.

[0047] The seventh aspect of the present invention provides a method for preparing GLP-1, comprising the following steps: culturing the plant lactobacillus A21246 of the first aspect of the present invention in a liquid culture system, collecting the supernatant and filtering and sterilizing it to obtain a sterile culture containing GLP-1.

[0048] In some embodiments of the present invention, the culture condition is culturing under facultative anaerobic conditions at 35-40° C. for 10-40 h, preferably culturing under anaerobic conditions at 37° C. for 30 h.

[0049] In some embodiments of the present invention, the liquid culture system contains nutrients, and the nutrients include components such as protein (peptone), sugar, phosphorus, magnesium, etc. For example, the nutrients can be provided in the form of a liquid culture medium, and the liquid culture medium is selected from any one of MRS medium, BBL and MPYG.

[0050] The beneficial effects of the present invention are:

[0051] The invention discloses Lactobacillus plantarum A21246 for the first time, and verifies through experiments that Lactobacillus plantarum A21246 can reduce blood sugar level and the content of glycosylated hemoglobin, promote the secretion of intestinal GLP-1, effectively improve weight loss caused by diabetes, significantly inhibit the formation of liver fatty degeneration, alleviate liver damage, improve abnormal glucose and lipid metabolism (can significantly reduce the content of cholesterol and triglyceride in serum), and improve the pathological morphology of liver and pancreas. The Lactobacillus plantarum A21246 plays an important role in preventing or treating diabetes and has wide application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 Phylogenetic tree analysis of Lactobacillus plantarum A21246.

[0053] Figure 2 Tolerance of Lactobacillus plantarum A21246 to simulated artificial gastrointestinal fluid.

[0054] Figure 3 This is the Elisa assay result of GLP-1 secretion by Lactobacillus plantarum A21246; wherein, the left figure is the GLP-1 standard curve, and the right figure is the assay result of GLP-1 secretion by Lactobacillus plantarum A21246. In the figure, ** represents P < 0.01.

[0055] Figure 4 The body weight changes of mice in each treatment group.

[0056] Figure 5 The changes in blood glucose levels of mice in each treatment group. In the figure, ns represents no significant difference, * represents P < 0.05, ** represents P < 0.01, and **** represents P < 0.001.

[0057] Figure 6 The changes of glycosylated hemoglobin in mice in each treatment group. In the figure, ** represents P < 0.01.

[0058] Figure 7 The intestinal GLP-1 levels of mice in each treatment group. In the figure, ns means no significant difference, and * means P < 0.05.

[0059] Figure 8 H&E staining analysis results of liver tissues of mice in each treatment group.

[0060] Fig. 9 The TC and TG levels in the liver of mice in each treatment group. In the figure, * represents P < 0.05, **** represents P < 0.001.

[0061] Fig.10 The results of H&E staining analysis of pancreatic tissues of mice in each treatment group. DETAILED DESCRIPTION

[0062] The present invention is further described in detail below through specific examples.

[0063] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0064] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0065] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0066] Example 1 Isolation and identification of Lactobacillus plantarum A21246 with the function of secreting GLP-1

[0067] Lactobacillus plantarum strain A21246 was isolated from the feces of a centenarian and cultured and purified using conventional MRS medium (10.0 g of peptone, 5.0 g of sodium acetate, 5.0 g of beef extract powder, 1.0 mL of Tween-80, 20.0 g of glucose, 2.0 g of K2HPO4, 4.0 g of yeast extract powder, 0.05 g of MnSO4·4H2O, 2.0 g of triammonium citrate, 0.2 g of MgSO4, 12.0 g of agar, 800 mL of distilled water was added, mixed until the solute was completely dissolved, the pH was adjusted to 6.2±0.2, the volume was made up to 1 L, and autoclaved at 121°C for 20 min).

[0068] The specific steps are: take a fresh stool sample from a centenarian and dilute it in a 10-fold gradient to 10 -5 , respectively, 10 -3 , 10 -4 , 10 -5Three tubes of dilution, 0.1 mL each, were spread on MRS solid medium for separation using the plate spreading method. After the plate was cultured at 37°C, 90% nitrogen + 10% carbon dioxide for 48 hours, a single colony of the suspected target strain was picked and transferred to a new MRS solid plate by streaking. After streaking purification for 2 generations, it was confirmed to be pure by microscopic examination, inoculated into a slant medium and cultured for 24 hours, and then stored in a 4°C refrigerator for later use.

[0069] The purified strain was molecularly identified by 16S rDNA method. The 16S rDNA sequence (such as SEQ ID NO: 1) was compared in the NCBI (National Center for Biotechnology Information) database, and it was found that the gene sequence of the strain had a homology of 99.66% with that of Lactobacillus plantarum among all similar sequences. Therefore, combined with the physiological and biochemical characteristics, the strain was identified as Lactobacillus plantarum and named A21246. A phylogenetic tree analysis was also performed ( Figure 1 ), the results showed that different plant lactobacillus differed in the evolution process, which was also verified in the subsequent hypoglycemic function experiment. The plant lactobacillus A21246 strain isolated from the intestines of centenarians was deposited in the Guangdong Microbial Culture Collection Center on November 15, 2024. The storage address is 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, and its storage number is GDMCCNo:65488.

[0070] 16S rDNA sequence of Lactobacillus plantarum A21246:

[0071]

[0072] Example 2 Experiment on resistance to artificial gastrointestinal fluid of Lactobacillus plantarum A21246 with GLP-1 secretion function

[0073] The activated bacterial solution was shaken and mixed, 1 mL of the bacterial suspension was added to 9 mL of artificial gastric juice (purchased from Regen Biotech, item number: CZ0213) (pH = 3.0), and cultured at 37°C for 3 hours. The artificial gastric juice at 0 hours, 1 hour, and 3 hours of culture was taken for plate count of live bacteria. The survival rate was calculated with 0 hours as the control. Survival rate of simulated artificial gastric juice (%) = logCFUN t / logCFUN0*100%, where N t Indicates the number of viable bacteria after culture th, and N0 indicates the number of viable bacteria at 0h. Take 1mL of artificial gastric juice after 3h of culture and add it to 9mL of artificial small intestinal fluid (purchased from Regen Biotechnology, product number: CZ0201) (pH=8.0), and culture it at 37℃ for 2h, 4h and 6h. Take artificial intestinal fluid cultured at 0h, 2h, 4h and 6h for plate viable bacteria count. Take 0h as the control and calculate the survival rate. Survival rate of simulated artificial intestinal fluid (%) = logCFUN t / logCFUN0*100%, where N t N represents the number of viable bacteria after culture for th, and N0 represents the number of viable bacteria at 0h.

[0074] The results showed that the survival rate of Lactobacillus plantarum A21246 was as high as 98.75% after 3 hours of treatment with artificial gastric juice at pH = 3, and 99.77% after 6 hours of treatment with artificial intestinal juice at pH = 8 (Table 1 and Figure 2 ). It can be seen that Lactobacillus plantarum A21246 can tolerate the low pH environment of gastric juice and enter the intestinal juice, and can proliferate in the intestinal juice, indicating that Lactobacillus plantarum A21246 has excellent tolerance to gastrointestinal juice, which is the premise of potential probiotics.

[0075] Table 1 Tolerance of Lactobacillus plantarum A21246 in simulated artificial gastrointestinal fluid

[0076]

[0077] Example 3 GLP-1 determination of Lactobacillus plantarum A21246 with GLP-1 secretion function

[0078] The activated bacterial solution was shaken and evenly mixed, 1 mL of bacterial suspension was centrifuged (6000 rpm, 1 min), the fermentation supernatant was collected, and the fermentation supernatant was filtered and sterilized using a 0.22 μm filter membrane. The treated fermentation supernatant was used to measure GLP-1 using an enzyme-linked immunosorbent assay (ELISA) kit, with blank culture medium MPYG as a control.

[0079] MPYG preparation: Tryptone 5.00g, Peptone 3.00g, Soybean Peptone 2.00g, Polypeptone 1.00g, Yeast Extract 10.00g, Beef Extract 5.00g, Glucose 5.00g, Tween 80 0.50mL, Maltose 0.50g, Cellobiose 0.50g, Soluble Starch 0.50g, Glycerol 0.50mL, K2HPO4 2.00g, Cysteine ​​0.50g, Na2S 0.25g, Resazurin 1.00mg, Salt Solution 40.00mL, Trace Element 10.00mL, Vitamin Solution 10.00mL, Hemin Chloride 10.00mL, Vitamin K1 Solution 0.20mL, Agar 15g.

[0080] Salt solution: CaCl2.2 H2O 0.25g, MgSO4.7 H2O 0.50g, K2HPO4 1.00g, KH2PO41.00g, NaHCO3 10.00g, NaCl 2.00g, constant volume 1000.00mL.

[0081] Trace element solution: nitrilotriacetic acid 1.50g, MgSO4.7H2O 3.00g, MnSO4.H2O0.50g, NaCl 1.00g, FeSO4.7H2O 0.10g, CoSO4.7H2O 0.18g, CaCl2.2H2O 0.10g, ZnSO4.7H2O 0.18g, CuSO4.5H2O 0.01g, KAl(SO4)2.12H2O 0.02g, H3BO3 0.01g, Na2MoO4.2H2O0.01g, NiCl2.6 H2O 0.03g, Na2SeO3.5 H2O 0.30mg, fixed volume 1000.00mL.

[0082] Vitamin solution: Biotin 2.00mg, Folic acid 2.00mg, Pyridoxine hydrochloride 10.00mg, Thiamine hydrochloride 5.00mg, Riboflavin 5.00mg, Niacin 5.00mg, D-calcium pantothenate 5.00mg, V B 12 0.10mg, 4-aminobenzoic acid 5.00mg, lipoic acid 5.00mg, fixed volume 1000.00mL.

[0083] Vitamin K1 solution (Hopebio HB8462-a, China): Dissolve 0.1 g vitamin K1 in 20 mL 95% ethanol and filter sterilize, then store at 4°C until use.

[0084] Hematin solution: Dissolve 0.05 g hematin in 1 mL of 1 mol / L NaOH, add distilled water to make up to 100 mL, filter and sterilize, and refrigerate for later use.

[0085] Test results such as Figure 3 As shown, Lactobacillus plantarum A21246 has a better function of secreting GLP-1.

[0086] Example 4 In vivo effect of Lactobacillus plantarum A21246 with GLP-1 secretion function

[0087] (1) Preparation of strain freeze-dried powder

[0088] Pick a single colony of the experimental strain after activation twice to 15mL of MRS liquid medium, and culture it under anaerobic conditions at 37℃ for 12h to obtain seed solution 1. Inoculate seed solution 1 into 150mL of MRS liquid medium at a 3% inoculation amount, and culture it under anaerobic conditions at 37℃ for 20-22h to obtain seed solution 2. Inoculate the seed solution into 300mL of MRS liquid medium at a 3% inoculation amount, and culture it under anaerobic conditions at 37℃ for 20-24h to obtain the bacterial solution after amplification culture. Collect the bacterial solution in a refrigerated centrifuge at 4℃, 5000rpm, centrifuge for 5min, and discard the supernatant. Wash the bacteria twice with physiological saline, centrifuge for 5min at 4℃, 5000rpm. Weigh the wet weight of the bacteria. Resuspend in a volume ratio of lyophilized protective agent: wet weight of bacteria = 1:1, divide into EP tubes, place at -20℃ for 30min, and then place at -80℃ for 1h. Place the packaged EP tubes into freeze-dried bottles, place the freeze-dried bottles into freeze dryers for freeze drying, and continue freeze drying for 30 hours to finally obtain freeze-dried bacterial powder. Count the bacterial powder using the plate count method and store the bacterial powder at 4°C for later use. Resuspend it with sterile saline when using it.

[0089] Preparation of lyophilized protective agent: weigh 12 g skim milk powder and add it to 100 mL distilled water, and sterilize it with high pressure steam at 115°C for 15 min.

[0090] (2) Animal experiments

[0091] 32 SPF-grade 6-week-old female C57BL / 6J were purchased from Beijing Sibeifu Biotechnology Co., Ltd. and adapted for 1 week. During the adaptation period, the light cycle was maintained at 12h, the temperature was 20-26℃, the humidity was 40%-60%, and the mice were allowed to eat and drink freely. After the adaptation period, they were randomly divided into 4 groups, namely the normal group: basic feed (purchased from Sibeifu rat maintenance feed (item number: SPF-F02-003)) and 0.2mL saline; the model group: high-fat (60%) feed and 0.2mL saline; the A21246 group: high-fat (60%) feed and 0.2mL bacterial powder resuspension (the bacterial powder was dissolved into 2x10 with 0.2mL saline). 9 CFU / mL); Lactobacillus plantarum 179-2 group: high-fat (60%) feed (purchased with the synergistic biological diet type XTHF60-1) and 0.2 mL of bacterial powder resuspension (the bacterial powder was dissolved in 0.2 ml of saline to 2x10 9 CFU / mL). 7-8 C57BL / 6J in each group. After the adaptation period, fasting blood glucose was tested first, and then the mice were fed with 60% high-fat feed for 14 days. Then, STZ (dissolved to 110 mg / kg in citric acid buffer) was intraperitoneally injected into the model group and product group on days 14 and 17, and citric acid buffer was intraperitoneally injected into the control group. The fasting blood glucose of mice was measured twice in a row on days 20 and 21. If the blood glucose concentration was greater than 11.0 mmol / L, the model was judged to be successful. After the model was successfully established, the plant lactobacillus strain was gavaged for 28 days.

[0092] (3) Determination of relevant indicators

[0093] The evaluation of whether Lactobacillus plantarum A21246, which has the function of secreting GLP-1, has a hypoglycemic effect mainly focuses on the detection of indicators such as mouse body weight, blood sugar, glycosylated hemoglobin, and GLP-1.

[0094] The results of the experiment showed that A21246 can improve the weight loss caused by diabetes ( Figure 4 ), which can reduce the blood sugar level in mice ( Figure 5 ) and glycosylated hemoglobin ( Figure 6 ), and can also promote intestinal GLP-1 secretion ( Figure 7 ). In terms of lipid accumulation caused by high fat, A21246 can significantly inhibit the formation of fatty degeneration in the liver ( Figure 8 ), significantly reducing cholesterol and triglyceride levels in the liver ( Fig. 9 ), reducing the abnormal glucose and lipid metabolism caused by diabetes. Through pancreatic H&E section analysis, A21246 has a certain effect on maintaining the shape of pancreatic islet cells ( Fig.10 ). The other control strain 179-2 did not have a significant blood sugar lowering function during the entire experimental period ( Figure 4), indicating that different strains of the same species of Lactobacillus plantarum have different functional effects.

[0095] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A strain of Lactiplantibacillus plantarum A21246, characterized in that: The plant lactobacillus A21246 is deposited in the Guangdong Provincial Microbiological Culture Collection Center with a deposit number of GDMCC NO: 65488 and a deposit date of November 15, 2024.

2. A method for preparing a culture of Lactobacillus plantarum A21246, comprising: Provide liquid culture system; The Lactobacillus plantarum A21246 of claim 1 is inoculated into the liquid culture system for cultivation.

3. The preparation method according to claim 2, characterized in that: The pH value of the liquid culture system is 2-9.

4. A culture of Lactobacillus plantarum A21246, prepared by the preparation method according to claim 2 or 3.

5. A microbial agent, characterized in that: The microbial agent comprises the Lactobacillus plantarum A21246 according to claim 1 or the Lactobacillus plantarum A21246 culture according to claim 4.

6. Use of the Lactobacillus plantarum A21246 of claim 1, the culture of Lactobacillus plantarum A21246 of claim 4, or the microbial agent of claim 5 in at least one of (1) to (4): (1) preparing GLP-1; (2) Prepare products that help maintain healthy blood sugar levels; (3) preparing products that help regulate the function of intestinal flora; (4) Preparation of products for the prevention and / or treatment of metabolic disorders.

7. The use according to claim 6, characterized in that: The metabolic abnormality described in (4) is abnormal liver lipid metabolism caused by type 2 diabetes.

8. A pharmaceutical composition comprising the Lactobacillus plantarum A21246 according to claim 1, the culture of Lactobacillus plantarum A21246 according to claim 4, or the microbial agent according to claim 5.

9. A method for preparing GLP-1, comprising the following steps: The Lactobacillus plantarum A21246 described in claim 1 is cultured in a liquid culture system, and the supernatant is collected to obtain GLP-1.

10. The method according to claim 9, characterized in that The culture condition is 35-40° C. under anaerobic conditions for 10-40 hours.

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