Application of bifidobacterium breve BB990 in metabolism field

Abstract

The invention belongs to the technical field of biological medicines, discloses application of bifidobacterium breve BB990 in the field of metabolism, and particularly discloses bifidobacterium breve BB990 which is preserved in Guangdong Microorganism Culture Collection Center, the preservation number is GDMCC No.63259, and the preservation date is March 10, 2023. The invention further discloses a preparation method of the bifidobacterium breve BB990. The bifidobacterium breve BB990 provided by the invention has the effects of regulating gastrointestinal disorder, enhancing intestinal immune function, inhibiting harmful bacteria, improving intestinal flora structure, maintaining intestinal steady state, regulating immunity, improving immune curative effect, regulating body glycometabolism and liver metabolism and the like. Experiments prove that the bifidobacterium breve BB990 can effectively prevent and treat metabolic diseases, such as non-alcoholic fatty liver, type II diabetes and the like, the treatment effect of the bifidobacterium breve BB990 is remarkably superior to that of other bifidobacterium breve (such as bifidobacterium breve 206), and the bifidobacterium breve BB990 has a good prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to the application of Bifidobacterium breve BB990 in the field of metabolism. Background Art

[0002] The incidence of metabolic diseases, including obesity, non-alcoholic fatty liver disease, insulin resistance, type 2 diabetes, atherosclerosis and polycystic ovary syndrome, has increased dramatically worldwide. Metabolic diseases (such as obesity, polycystic ovary syndrome, non-alcoholic fatty liver disease, hyperglycemia, hyperlipidemia) are closely related to intestinal microorganisms. The host's genes, diet, and immunity shape the microbiota at all times. The microbiota also interacts with the host through its metabolites, thereby affecting the host's health.

[0003] Diabetes is a metabolic disease characterized by hyperglycemia, caused by partial or complete insulin deficiency. Diabetes has become the third major chronic disease threatening human health after cancer and cardiovascular disease, and more than 90% of patients have type 2 diabetes mellitus (T2DM). The onset of type 2 diabetes is usually slow, and insulin resistance is one of the important characteristics of type 2 diabetes. It is most common in the elderly, but due to rising obesity levels, lack of exercise and improper diet, it is also increasing in children, adolescents and young people. Globally, the prevalence of type 2 diabetes is on the rise. The aging population, economic development and increasing urbanization have led to a more sedentary lifestyle and more unhealthy food consumption related to obesity, which will exacerbate this upward trend. At present, the main hypoglycemic drugs for the treatment of diabetes are: biguanides, sulfonylureas, thiazolidinediones, glinides, α-glucosidase inhibitors, and dipeptidyl peptidase-4 inhibitors. However, these drugs cannot fundamentally cure diabetes. At the same time, various therapeutic drugs have adverse reactions: such as hypoglycemia and weight gain (such as insulin and insulin secretagogues), gastrointestinal reactions (such as biguanides, glucosidase inhibitors and GLP-1 receptor agonists), weight gain, edema or adverse cardiac events (such as thiazolidinediones TZDs), etc. In addition, long-term use of metformin can cause irritation to the gastrointestinal tract of some patients and may affect the patient's absorption of vitamin B12. Rosiglitazone can cause liver damage and edema. It causes the patient to develop tolerance to the disease, so that only taking large doses of hypoglycemic drugs can effectively reduce blood sugar. However, long-term use of insulin will also make patients dependent on drugs, which is still a temporary solution, and the patient's blood sugar is difficult to be effectively controlled and prone to recurrence. Therefore, it is necessary to develop more therapeutic strategies that can effectively prevent or treat type 2 diabetes. The use of drugs to treat diabetes does not exclude the importance of other measures used to combat this disease. Recent studies have shown that chronic inflammation caused by endotoxins entering the blood due to intestinal flora imbalance is one of the important factors in the development of metabolic diseases such as obesity and diabetes. Imbalanced flora can cause systemic chronic inflammation, leading to pancreatic β-cell damage and decreased insulin resistance, while affecting the absorption of sugar by other cells in the body for energy conversion, making it difficult for the human body to function normally, and finally causing a series of complications.

[0004] The prevalence of non-alcoholic fatty liver disease (NAFLD) has been increasing year by year. NAFLD refers to a clinical pathological syndrome characterized by diffuse hepatocyte macrovesicular steatosis and lipid metabolism disorders in addition to alcohol and other clear liver-damaging factors. Its disease spectrum includes simple fatty liver, nonalcoholic steatohepatitis (NASH), liver fibrosis and cirrhosis. The global prevalence of NAFLD is 25% to 30%; NAFLD seriously threatens human health. It is not only closely related to type 2 diabetes mellitus (T2DM), but also can predict the occurrence of T2DM and cardiovascular disease (CVD). At present, the main treatment goals of NAFLD are to reduce the activity of the disease, delay the progression of liver fibrosis, cirrhosis and even liver cancer, and reduce the occurrence of complications such as cardiovascular disease. Its main treatment methods include lifestyle improvement, drug therapy and surgical treatment. At present, no specific drug for the treatment of NAFLD has been approved for marketing. Improving metabolic syndrome and its related complications through lifestyle intervention is the most important and basic means of treating NAFLD. For patients who cannot be improved by lifestyle intervention, the use of drugs targeting metabolic syndrome can improve insulin resistance and dyslipidemia, but the therapeutic effect on NASH and liver fibrosis is still uncertain; the treatment of liver-protective drugs is mainly anti-inflammatory and anti-oxidative stress, but the efficacy is uncertain. In my country, the incidence of NAFLD has also shown a trend of gradual increase and younger age. In recent years, the relationship between NAFLD and intestinal flora has received increasing attention. There is a two-way communication pathway between the intestine and the liver called the gut-liver axis (GLA), one of which is the portal vein. The portal vein provides most of the blood supply to the liver, so the intestinal flora and the liver are closely connected and influence each other. There are 500 to 1500 species of bacteria in the human gastrointestinal tract, and the bacteria in the intestinal system weigh 1 to 2 kg. Healthy intestinal flora plays a vital role in the body's immune regulation. A large number of studies have shown that intestinal flora imbalance plays an important role in the pathogenesis of human liver disease, especially NAFLD. Based on this, this study selected NAFLD patients as research subjects to observe the effect of probiotics-assisted treatment of NAFLD, aiming to provide new ideas for the treatment of NAFLD.

[0005] An unhealthy lifestyle is a major risk factor for metabolic diseases. In recent years, increasing evidence has shown that intestinal flora and its metabolites also play a vital role in the occurrence and development of many metabolic diseases, including obesity, type 2 diabetes, non-alcoholic fatty liver disease and cardiovascular disease. There are many microorganisms in the intestine, which are key mediators of host energy acquisition and can metabolize dietary nutrients into many bioactive substances, thus serving as a bridge for the interaction between intestinal flora and the host. The intestinal flora is regulated by host genes, immune responses and dietary factors, and the metabolic and immune potential of the intestinal flora determines its importance in host health and disease. Therefore, targeting the intestinal flora and its related metabolic pathways is expected to become an effective strategy for the treatment of many metabolic diseases. Intestinal microorganisms and their metabolites play an important role in the occurrence and development of many metabolic diseases, and will also produce a series of potential mechanisms and new technologies for targeted therapeutic drugs, providing guidance for future research in the emerging field of intestinal microorganisms related to the development of human metabolic diseases.

[0006] The intestinal flora of healthy individuals maintains ecological balance. Once the internal and external environment of the body changes, resulting in a disruption of balance, it is easy to cause dysbacteriosis. More and more studies have shown that intestinal flora plays a key role in the health of the body, can affect the body's metabolism, and is directly related to obesity, cardiovascular disease and type 2 diabetes. Probiotics are a type of microorganism that has a beneficial effect on the host, can colonize the host's intestines and maintain the balance of flora.

[0007] In view of this, the present invention is proposed. Summary of the invention

[0008] The first aspect of the present invention aims to provide Bifidobacterium breve BB990.

[0009] The second aspect of the present invention is to provide a microbial agent.

[0010] The third aspect of the present invention is to provide a composition.

[0011] The fourth aspect of the present invention aims to provide use of the Bifidobacterium breve BB990 of the first aspect of the present invention, the microbial agent of the second aspect of the present invention or the composition of the third aspect of the present invention in the preparation of a treatment or / prevention of metabolic diseases.

[0012] The fifth aspect of the present invention aims to provide a product.

[0013] In order to achieve the above object, the technical solution adopted by the present invention is:

[0014] The first aspect of the present invention provides a Bifidobacterium breve BB990, characterized in that the Bifidobacterium breve BB990 is deposited in Guangdong Provincial Microbiological Culture Collection Center, with a deposit number of GDMCC No. 63259 and a deposit date of March 10, 2023.

[0015] The second aspect of the present invention provides a microbial agent, which includes the Bifidobacterium breve BB990 bacterial body, metabolites, fermentation culture, fermentation supernatant or any combination thereof according to the first aspect of the present invention.

[0016] The active ingredient of the above-mentioned bacterial agent may be the above-mentioned Bifidobacterium breve BB990, the culture (such as a fermentation product) of the above-mentioned Bifidobacterium breve BB990 and / or the fermentation supernatant of the above-mentioned Bifidobacterium breve BB990. The active ingredient of the above-mentioned bacterial agent may also contain other biological components or non-biological components. The other active ingredients of the above-mentioned bacterial agent can be determined by those skilled in the art according to the effect of the bacterial agent.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] According to needs, a binder, a stabilizer (such as an antioxidant), a pH adjuster, etc. may be added to the bacterial agent.

[0021] 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.

[0022] The third aspect of the present invention provides a composition, wherein the pharmaceutical composition comprises Bifidobacterium breve BB990 according to the first aspect of the present invention and / or the microbial agent according to the second aspect of the present invention.

[0023] In some embodiments of the present invention, the composition further comprises a pharmaceutically acceptable excipient.

[0024] 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.

[0025] 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.

[0026] In some embodiments of the present invention, the dosage form of the 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.

[0027] 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.

[0028] In some embodiments of the present invention, the bacterial count (live bacteria count or inactivated bacteria count) of Bifidobacterium breve BB990 in the composition is greater than 10 6 CFU / mL.

[0029] In some embodiments of the present invention, the bacterial count (live bacteria count or inactivated bacteria count) of Bifidobacterium breve BB990 in the composition is greater than 10 7 CFU / mL.

[0030] In some embodiments of the present invention, the composition may further include a combined drug, which includes a drug for treating or preventing metabolic diseases.

[0031] 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.

[0032] The fourth aspect of the present invention provides use of the Bifidobacterium breve BB990 of the first aspect of the present invention, the microbial agent of the second aspect of the present invention, or the composition of the third aspect of the present invention in the preparation of a product for treating or / preventing metabolic diseases.

[0033] In some embodiments of the present invention, the metabolic disease includes at least one of non-alcoholic fatty liver disease, insulin resistance, type II diabetes, atherosclerosis and polycystic ovary syndrome.

[0034] In some embodiments of the present invention, the metabolic disease includes non-alcoholic fatty liver disease, insulin resistance and / or type 2 diabetes.

[0035] In some embodiments of the present invention, the product can be used to relieve elevated blood sugar in diabetic patients, relieve impaired glucose tolerance, and relieve insulin secretion disorders in diabetic patients, and has the potential to improve glucose tolerance, relieve impaired glucose tolerance, and improve insulin secretion.

[0036] In some embodiments of the present invention, the product includes a reagent, a medicine, a food or a health food.

[0037] A fifth aspect of the present invention provides a product, comprising the Bifidobacterium breve BB990 of the first aspect of the present invention, the microbial agent of the second aspect of the present invention, or the composition of the third aspect of the present invention.

[0038] In some embodiments of the present invention, the product has at least one of the following functions: helps maintain healthy blood sugar levels, helps maintain healthy blood lipid levels, and helps control body fat.

[0039] In some embodiments of the present invention, the product includes a reagent, a medicine, a food or a health food.

[0040] The beneficial effects of the present invention are:

[0041] The Bifidobacterium breve BB990 provided by the present invention has the functions of regulating gastrointestinal disorders, enhancing intestinal immune function, inhibiting harmful bacteria, improving intestinal flora structure, maintaining intestinal homeostasis, immunomodulation, improving immune efficacy, regulating body sugar metabolism and liver metabolism, etc. Experiments have shown that Bifidobacterium breve BB990 can effectively prevent and treat metabolic diseases, such as non-alcoholic fatty liver disease, type II diabetes, etc., and its therapeutic effect is significantly better than other Bifidobacterium breve (such as Bifidobacterium breve 206), and has good prospects.

[0042] Specifically, in the treatment of type 2 diabetes, experimental results show that the Bifidobacterium breve BB990 provided by the present invention can significantly reduce the fasting blood sugar of diabetic mice, significantly improve the glucose tolerance of diabetic mice, relieve the symptoms of insulin secretion disorders and impaired glucose tolerance caused by high-fat diet feeding, relieve the decline of Lee's index of diabetic mice, promote the growth of mice and relieve their emaciation, and have a good diabetes treatment effect. In the treatment of non-alcoholic fatty liver, experimental results show that the Bifidobacterium breve BB990 provided by the present invention can significantly improve the liver index of fatty liver mice, relieve the accumulation of fat (white fat, brown fat) in the liver of fatty liver mice, significantly relieve the formation of fatty liver in mice on a high-fat diet, inhibit weight gain, reduce fat content, and reduce body fat rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 is the fasting blood glucose of mice.

[0044] Figure 2 This is the OGTT change curve of mice.

[0045] Figure 3 is the area under the curve of OGTT in mice.

[0046] Figure 4 This is the weight change curve of mice.

[0047] Figure 5 The curve is the daily percentage change of mouse body weight.

[0048] Figure 6 The percentage of body weight change of mice at the end of the experiment.

[0049] Figure 7 The fasting blood glucose of mice was the experimental endpoint.

[0050] Figure 8 This is the mouse OGTT curve at the end point of the experiment.

[0051] Fig. 9 The area under the curve of OGTT in mice is the endpoint of the experiment.

[0052] Fig.10 Lee's index of mice was the end point of the experiment.

[0053] Fig.11 ALT statistics of model mice and healthy mice at the end of modeling.

[0054] Fig.12 AST statistics of model mice and healthy mice at the end of modeling.

[0055] Fig.13 ALT / AST statistics of model mice and healthy mice at the end of modeling.

[0056] Fig.14 TG statistics of model mice and healthy mice at the end of modeling.

[0057] Fig.15 T-CHO statistics of model mice and healthy mice at the end of modeling.

[0058] Fig.16 This is the weight change curve of mice.

[0059] Fig.17 is the percentage change of mouse body weight per day.

[0060] Fig.18 The liver index of mice with fatty liver was the experimental endpoint.

[0061] Fig.19 Lee's index of fatty liver mice is the experimental endpoint.

[0062] Fig. 20 The fat content of mice with fatty liver was the experimental endpoint.

[0063] Fig.21 The body fat percentage of mice with fatty liver was the experimental endpoint.

[0064] Fig. 22 The white fat content of fatty liver was the experimental endpoint.

[0065] Fig.23 The endpoint of the experiment is the percentage of white fat to mouse body weight in mice with fatty liver.

[0066] Fig.24 The brown fat content of mice with fatty liver was the experimental endpoint.

[0067] Fig.25 The percentage of brown fat to mouse body weight in mice with fatty liver was the experimental endpoint. DETAILED DESCRIPTION

[0068] The present invention is further described in detail below through specific examples.

[0069] 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.

[0070] 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.

[0071] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0072] Example 1 Isolation and purification of Bifidobacterium breve BB990

[0073] Bifidobacterium breve BB990 is a bacterium isolated from the intestinal microorganisms of healthy people. Strain BB990 is deposited in Guangdong Microbiological Culture Collection Center with the deposit number GDMCC No.63259 and the deposit date March 10, 2023. The deposit address is Guangdong Institute of Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The 16S sequence of strain BB990 is shown in SEQ ID NO:1.

[0074] ACCTTAGACGGCTCCCTCCCGCAAGGGGTTAGGCCACCGGCTTCGGGTGCTGCCCACTTTCATGACTTGACGGGCGGTGTGTACAAGGCCCGGGAACGCATTCACCGCGACGTTGCTGATTCGCGATTACTAGCGACTCCGCCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGACCGGTTTTCAGGGATCCGCTCCAGCTCGCACTGTCGCATCCCGTTGTACCGGCCATTGTAGCATGCGTGAAGCCCTGGACGTAAGGGGCATGATGATCTGACGTCATCCCCACCTTCCTCCGAGTTAACCCCGGCGGTCCCCCGTGAGTTCCCGGCACAATCCGCTGGCAACACGGGGCGAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACGACCATGCACCACCTGTGAACCCGCCCCGAAGGGAAACCCCATCTCTGGGATCGTCGGGAACATGTCAAGCCCAGGTAAGGTTCTTCGCGTTGCATCGAATTAATCCGCATGCTCCGCCGCTTGTGCGGGCCCCCGTCAATTTCTTTGAGTTTTAGCCTTGCGGCCGTACTCCCCAGGCGGGATGCTTAACGCGTTAGCTCCGACACGGAACCCGTGGAACGGGCCCCACATCCAGCATCCACCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTAACGGCCCAGAGACCTGCCTTCGCCATTGGTGTTCTTCCCGATATCTACACATTCCACCGTTACACCGGGAATTCCAGTCTCCCCTACCGCACTCAAGCCCGCCCGTACCCGGCGCGGATCCACCGTTAAGCGATGGACTTTCACACCGGACGCGACGAACCGCCTACGAGCCCTTTACGCCCAATAATTCCGGATAACGCTTGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGTGCTTATTCGAAAGGTACACTCAACACAAAGTGCCTTGCTCCCTAACAAAAGAGGTTTACAACCCGAAGGCCTCCATCCCTCACGCGGCGTCGCTGCATCAGGCTTGCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTATCTCAGTCCCAATGTGGCCGGTCGCCCTCTCAGG CCGGCTACCCGTCGAAGCCATGGTGGGCCGTTACCCGCCATCAAGCTGATAGGACGCGACCCCATCCCATGCCGCAAAGGCTTTCCCAACACACCATGCGGTGATGGAGCATCCGGCATTACCACCCGTTTCCAGGAGCTATTCCGGTGCATGGGGCAGGTCGGTCACGCATTACTCACCCGTTCGCCACTCTCACCACCAAGCAAAGCCCGATGGATCCC (SEQ ID NO:1).

[0075] The general process of separation and purification of Bifidobacterium breve BB990 is as follows:

[0076] After the healthy donor signed the informed consent for stool collection with the company, 1 gram of fresh stool sample was taken from the donor and placed in an anaerobic operating table. The sample was shaken for 1 minute using a vortex shaker to mix well. Then, 1 mL of the mixed sample was taken and added to 9 mL of normal saline and mixed to obtain 10 -1 Then, perform gradient dilutions until 10 -6 Dilution for spreading on plates.

[0077] From 10 -6 Take 100 μl of the diluent and spread it evenly on the anaerobic blood plate. The plate is cultured anaerobically at 37°C on the anaerobic workbench for 3 to 5 days. Observe the growth of the isolated strains and use a sterilized toothpick to pick a single colony for plate streak purification and culture.

[0078] Example 2 Preparation of Bifidobacterium breve bacterial solution

[0079] After the glycerol tube of Bifidobacterium breve BB990 or Bifidobacterium breve 206 (purchased from Guangdong Microbiological Collection Center, item number: GIM1.206) was completely thawed at 37°C, it was inoculated on the modified YCFA solid culture medium in the anaerobic workstation for resuscitation and activation. The activated strain was picked and inoculated into the modified YCFA liquid culture medium for 48 hours. The bacterial solution was transferred to the modified YCFA liquid culture medium at a ratio of 1.0% (v / v) and cultured on the anaerobic operating table. After 48 hours of culture, all the bacterial solution was divided into 50mL centrifuge tubes and centrifuged at 8000g and 4°C for 10 minutes. Half of the precipitate was resuspended with DMEM culture medium and the concentration of the bacterial suspension was adjusted to 1.0×10 9 CFU / mL, and set aside (for Example 3). The other half of the precipitate was resuspended in modified YCFA liquid medium, and the concentration of the bacterial suspension was adjusted to 1.0×10 10 CFU / mL, for future use (for Example 4). The prepared bacterial solution was dispensed into sterile cryopreservation tubes, with 1.6 mL of bacterial solution in each tube. Refrigerated at 4°C.

[0080] Example 3 Application of strain Bifidobacterium breve BB990 in the treatment of type 2 diabetes

[0081] The characteristics of the type 2 diabetes model (T2DM): "three more and one less", i.e., more drinking, more eating, more urination, and weight loss. Model construction method: high-sugar and high-fat feed feeding combined with streptozotocin (STZ) injection. Continuously feed high-sugar and high-fat feed, and drink normal drinking water; after feeding for 12 weeks, a single intraperitoneal injection or intravenous injection of STZ solution at a dose of 30 mg / kg body weight. Successful model establishment criteria: fasting blood glucose ≥ 6.78 mmol / L.

[0082] In order to verify the therapeutic effect of strain Bifidobacterium breve BB990 on type 2 diabetes, this example uses C57B / 6 mice to construct a model. This experiment has passed the ethical review of the Guangdong Daoke Medical Biological Animal Ethics Committee. 50 C57BL / 6 mice were selected. The mice were randomly divided into 5 groups, namely, a healthy control group, a model group, a metformin group, a Bifidobacterium breve BB990 group, and a Bifidobacterium breve 206 (purchased from Guangdong Provincial Microbiological Collection Center, item number: GIM1.206) group, with 10 mice in each group. C57BL / 6 mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., and metformin was purchased from Merck Pharmaceuticals (Jiangsu) Co., Ltd.

[0083] At the beginning of modeling, mice were fed with a high-fat diet (purchased from Synergy Biotech) for 12 weeks. After treatment with 30 mg / kg STZ, the fasting blood glucose and glucose tolerance of mice were tested. The results showed that the fasting blood glucose of mice in the diabetic model group was significantly lower than that in the healthy control group. Figure 1 As shown, the OGTT change curve of mice is as follows Figure 2 As shown, the area under the OGTT curve of mice is Figure 3 As shown in the figure, the results showed that the fasting blood glucose of the diabetic model mice was significantly increased compared with the healthy control group. The glucose tolerance of the diabetic model mice was significantly reduced. Therefore, it is speculated that the diabetic model mice have symptoms such as impaired glucose tolerance.

[0084] After modeling, the mice were divided into three groups: healthy control group, model group, Bifidobacterium breve BB990 group, Bifidobacterium breve 206 group, and metformin group. The mice were given normal saline, normal saline, and Bifidobacterium breve BB990 suspension (1.0×10 9 CFU / mL), Bifidobacterium breve 206 bacterial suspension (1.0×10 9 CFU / mL), metformin (160 mg / kg / day), gavage at regular intervals every day, 0.2 mL each time, once a day, for 6 weeks. Figure 4 The daily weight changes of mice during the administration period are shown in Figure 5 As shown in the figure, there was no significant weight loss during the experiment, no death, and the activity and mental state of the mice were normal. The diet and drinking water were relatively stable, the excretion was normal, the skin and breathing were normal, and no tremor, spasm, movement disorder, abnormal posture, proptosis, hair, eyes, skin and mucous membrane changes, convulsions, salivation, diarrhea, local damage and other toxic phenomena were found. The percentage change of mouse weight at the end of the experiment is as follows Figure 6 As shown in the figure, the fasting blood glucose of mice at the end of the experiment was Figure 7 As shown, the OGTT curve of mice at the end of the experiment is as follows Figure 8 As shown, the area under the OGTT curve of mice at the end of the experiment is as follows Fig. 9 As shown in the figure, the Lee's index of mice at the end of the experiment is as follows: Fig.10 shown.

[0085] The percentage change of mouse body weight at the end of the experiment was as follows Figure 6 The results showed that compared with the diabetic model group, metformin, Bifidobacterium breve BB990, and Bifidobacterium breve 206 significantly alleviated the weight loss of the experimental mice, and Bifidobacterium breve BB990 was significantly better than Bifidobacterium breve 206 in alleviating the weight loss of diabetic mice. Figure 7 The results showed that compared with the diabetic model group, metformin, Bifidobacterium breve BB990, and Bifidobacterium breve 206 significantly reduced the fasting blood sugar of diabetic mice, and there was no statistical difference between Bifidobacterium breve BB990 and the healthy group of mice, but there was significant statistical significance compared with the Bifidobacterium breve 206 group of mice (P>0.05), that is, Bifidobacterium breve BB990 has a better effect on reducing the fasting blood sugar of diabetic mice than Bifidobacterium breve 206. The experimental endpoint detected the glucose tolerance OGTT curve of mice and the area of ​​the OGTT curve Figure 8 , Fig. 9 The results showed that metformin, Bifidobacterium breve BB990, and Bifidobacterium breve 206 significantly improved the glucose tolerance of diabetic mice and alleviated the symptoms of insulin secretion disorders and impaired glucose tolerance caused by high-fat diet feeding. Bifidobacterium breve BB990 was statistically significant compared with Bifidobacterium breve 206 (P>0.05), that is, Bifidobacterium breve BB990 has better effects than Bifidobacterium breve 206 in improving the glucose tolerance of diabetic mice, alleviating the insulin secretion disorders and impaired glucose tolerance caused by high-fat diet feeding. Lee's index of mice at the end of the experiment Fig.10 The results showed that metformin and Bifidobacterium breve BB990 could alleviate the decline of Lee's index in diabetic mice, promote the growth of mice and alleviate their emaciation, and had a good effect on the treatment of diabetes. However, there was no statistical significance between Bifidobacterium breve 206 and the model group.

[0086] In summary, the weight of Bifidobacterium breve BB990 did not change much during the administration period, the food intake decreased, and glucose tolerance was significantly improved. It is believed that long-term administration of Bifidobacterium breve BB990 significantly alleviated the increase in blood sugar in diabetic mice, alleviated impaired glucose tolerance, and alleviated the insulin secretion disorder of diabetic mice. It has the potential to improve glucose tolerance, alleviate impaired glucose tolerance, and improve insulin secretion. It provides a useful reference for further research and development of diabetes treatment.

[0087] Example 4 Application of strain Bifidobacterium breve BB990 in the treatment of non-alcoholic fatty liver disease

[0088] Non-alcoholic fatty liver disease is a common chronic liver disease, the main feature of which is the accumulation of fat in liver cells, leading to abnormal liver function. Usually, patients have no history of drinking or drink less, and the degree of liver inflammation and fibrosis is mild. The disease is often associated with metabolic abnormalities such as obesity, type 2 diabetes and hyperlipidemia. Carbon tetrachloride can affect the fat metabolism and oxidative stress response of the liver, causing fat to accumulate in the liver and form fat masses. At the same time, carbon tetrachloride can also promote the proliferation and differentiation of adipocytes, leading to an increase in the number and size of adipocytes, aggravating the degree of fatty liver.

[0089] To verify the therapeutic effect of strain Bifidobacterium breve BB990 on non-alcoholic fatty liver disease, this example uses C57B / 6 mice to construct a model. This experiment passed the ethical review of the Guangdong Dao Ke Medical Biological Animal Ethics Committee. C57BL / 6 mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. 50 C57BL / 6 mice were selected. The mice were randomly divided into 5 groups, namely, a healthy control group, a model group, an obeticholic acid group, a Bifidobacterium breve BB990 group, and a Bifidobacterium breve 206 group, with 10 mice in each group. C57BL / 6 mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0090] During the modeling period, the mice were continuously fed with a high-sugar and high-fat diet (purchased from Synergy Biotech). After 12 weeks of feeding, 0.5 mL / kg of 4% carbon tetrachloride edible oil was injected weekly for a total of 5 weeks. During this period, the drinking water was normal drinking water.

[0091] Successful model establishment criteria: Determine whether the NASH model is successfully established through biochemical index detection, comparison between the experimental group and the control group, and other aspects of evaluation. Specific criteria: Increased liver function indicators such as AST (aspartate aminotransferase), ALT (alanine aminotransferase), TG (triglycerides), etc.

[0092] After the model was established, the blood biochemical indicators (ALT, AST, ALT / AS, TG, T-CHO) of the mice were tested. The results are as follows Figure 11 to Figure 15 As shown, the results showed that compared with the healthy control group, the ALT, AST, ALT / AS, TG, and T-CHO (total cholesterol) in the model group were higher than those in the healthy control group, and the comparison between the two was statistically significant.

[0093] After modeling, the mice were divided into three groups: healthy control group, model group, Bifidobacterium breve BB990 group, Bifidobacterium breve 206 group, and obeticholic acid group. They were given normal saline, normal saline, and Bifidobacterium breve BB990 suspension (1.0×10 10 CFU / mL), Bifidobacterium breve 206 bacterial suspension (1.0×10 10 CFU / mL), obeticholic acid (170 mg / kg / day), daily regular gavage, 0.2 mL each time, once a day, for 5 weeks.

[0094] The body weight of mice during the administration period was Figure 16-17As shown. During the administration period, there was no significant weight loss in the experimental animals in each group, no death occurred, and the activity and mental state of the mice were normal. The diet and drinking water were relatively stable, the excretion was normal, the skin and breathing were normal, and no tremor, spasm, movement disorder, abnormal posture, no protrusion of the eyeball, no changes in hair, eyes, skin and mucous membranes, no convulsions, salivation, diarrhea and local damage and other toxic phenomena were found. No toxicity-related symptoms occurred in each group during the experiment, and the long-term administration was safe.

[0095] The liver index of mice at the end of the experiment was Fig.18 As shown in the figure, the Lee's index of mice at the end of the experiment is as follows: Fig.19 As shown in the figure, the fat content of mice at the end of the experiment was Fig. 20 As shown in the figure, the body fat percentage of mice at the end of the experiment was Fig.21 As shown in Figure 2, the white fat content of mice at the end of the experiment was Fig. 22 As shown in the figure, the percentage of white fat and body weight of mice at the end of the experiment is as follows Fig.23 As shown in Figure 2, the brown fat content of mice at the end of the experiment was Fig.24 As shown in Figure 2, the percentage of brown fat and body weight of mice at the end of the experiment was Fig.25 shown.

[0096] The liver index of mice at the end of the experiment was Fig.18 The results showed that Bifidobacterium breve BB990 and obeticholic acid could significantly improve the liver index of fatty liver mice, alleviate the accumulation of fat in the liver of fatty liver mice, and significantly alleviate the formation of fatty liver in mice fed a high-fat diet; while Bifidobacterium breve 206 had no statistically significant difference compared with the model group. Fig.19 The results showed that Bifidobacterium breve BB990 and obeticholic acid could alleviate the increase of Lee's index in mice with fatty liver, inhibit the growth of body size and alleviate the obesity of mice with fatty liver, and had a good therapeutic effect on fatty liver. Bifidobacterium breve 206 had no statistical significance compared with the model group. Fig. 20 The body fat percentage of mice at the end of the experiment was Fig.21 The results showed that Bifidobacterium breve BB990, obeticholic acid, and Bifidobacterium breve 206 can inhibit fat accumulation in mice with fatty liver, improve the obesity level of mice with fatty liver, and affect body shape. Compared with Bifidobacterium breve 206, Bifidobacterium breve BB990 is more effective in treating fatty liver than Bifidobacterium breve 206, and the comparison between the two is statistically significant. The white fat content of mice at the end of the experiment is as follows Fig. 22 At the end of the experiment, the percentage of white fat and body weight of mice was as follows Fig.23The results showed that Bifidobacterium breve BB990, obeticholic acid, and Bifidobacterium breve 206 could inhibit the accumulation of white fat in mice with fatty liver. Compared with Bifidobacterium breve 206, Bifidobacterium breve BB990 was more effective in alleviating the accumulation of white fat in fatty liver than Bifidobacterium breve 206, and the difference between the two was statistically significant. Fig.24 At the end of the experiment, the percentage of brown fat in mice to their body weight was Fig.25 The results showed that Bifidobacterium breve BB990, obeticholic acid, and Bifidobacterium breve 206 can all inhibit the accumulation of brown fat in mice with fatty liver. Compared with Bifidobacterium breve 206, Bifidobacterium breve BB990 was better than Bifidobacterium breve 206 in alleviating the accumulation of brown fat in fatty liver, and the comparison between the two was statistically significant.

[0097] In summary, obeticholic acid and Bifidobacterium breve BB990 showed significant efficacy in inhibiting weight gain, reducing fat content, reducing body fat percentage, inhibiting white fat formation and brown fat content. Taking Bifidobacterium breve BB990 can significantly inhibit weight gain caused by a high-fat diet, reduce fat content, reduce body fat percentage, inhibit white fat formation and brown fat content, and alleviate the formation of fatty liver. These results provide a useful reference for further research and development of non-alcoholic fatty liver treatment.

[0098] 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 Bifidobacterium breve BB990, characterized in that The Bifidobacterium breve BB990 is deposited in the Guangdong Provincial Microbiological Culture Collection Center with the deposit number GDMCC No.63259 and the deposit date of March 10, 2023.

2. A microbial agent, characterized in that: The microbial agent comprises the bacterial body, metabolites, fermentation culture, fermentation supernatant or any combination thereof of Bifidobacterium breve BB990 according to claim 1.

3. A composition, characterized in that The composition comprises the Bifidobacterium breve BB990 according to claim 1 and / or the microbial agent according to claim 2.

4. The composition according to claim 3, characterized in that The composition further comprises pharmaceutically acceptable excipients and / or probiotics.

5. The composition according to claim 4, characterized in that 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.

6. The composition according to any one of claims 3 to 5, characterized in that The number of Bifidobacterium breve BB990 in the composition is greater than 10 6 CFU / mL; Preferably, the combination group further comprises at least one combined drug; Preferably, the combined drug includes a drug for treating or preventing metabolic diseases.

7. Use of the Bifidobacterium breve BB990 according to claim 1, the microbial agent according to claim 2, or the composition according to any one of claims 3 to 6 in the preparation of a product for treating or / preventing metabolic diseases.

8. The use according to claim 7, characterized in that: The metabolic disease includes at least one of non-alcoholic fatty liver disease, insulin resistance, type II diabetes, atherosclerosis and polycystic ovary syndrome.

9. A product comprising the Bifidobacterium breve BB990 according to claim 1, the microbial agent according to claim 2, or the composition according to any one of claims 3 to 6.

10. The product according to claim 9, characterized in that The product has at least one of the following functions: helps maintain healthy blood sugar levels, helps maintain healthy blood lipid levels, and helps control body fat.

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