Bifidobacterium animalis subsp. Lactis strain B21247 with hypoglycemic function and application thereof

Abstract

The invention provides a bifidobacterium animalis subsp. Lactis strain B21247 with a hypoglycemic function and application thereof, and belongs to the technical field of functional microorganisms. The invention relates to a bifidobacterium animalis subsp. Lactis strain B21247 with a hypoglycemic function. The preservation number of the bifidobacterium animalis subsp. Lactis strain B21247 is GDMCC No: 65075. The strain B21247 has a good intestinal colonization function, and has the activity of inhibiting alpha-glucosidase in vitro. Meanwhile, a type 2 diabetes mouse model is used as an experimental model to verify that the strain B21247 achieves the effect of reducing blood sugar by adjusting glycosylated hemoglobin, reducing pancreatic beta cell damage and increasing secretion of intestinal GLP-1, and a new thought is provided for reducing blood sugar or treating diabetes.

Description

Technical Field

[0001] The invention belongs to the technical field of functional microorganisms, and in particular relates to a Bifidobacterium animalis subsp. lactis strain B21247 with blood sugar lowering function and an application thereof. Background Art

[0002] Diabetes is a metabolic disease characterized by chronic hyperglycemia caused by the long-term interaction of genetic and environmental factors. It is a disease that affects normal physiological activities due to disorders in the metabolism of sugar, fat, and protein caused by defects in insulin secretion and (or) utilization. Its prevalence is on the rise in both developed and developing countries. If diabetes is not treated, a series of complications will occur in the long-term disease state, such as cardiovascular and cerebrovascular diseases, retinopathy, diabetic nephropathy, etc.

[0003] At present, drug treatment is still a common choice for patients with diabetes. The main therapeutic drugs are insulin and oral hypoglycemic drugs, which are roughly divided into sulfonylureas, biguanides, traditional Chinese medicine preparations, other hypoglycemic drugs and auxiliary drugs. Oral hypoglycemic drugs can regulate blood sugar to a certain extent, but most drugs are accompanied by some adverse effects during treatment, such as weight gain, diarrhea, nausea, abdominal distension, acidosis, gastrointestinal dysfunction, hepatocellular and cholestatic liver damage, etc. Domestic and foreign studies have found that intestinal flora is related to diabetes and its complications. The structurally disordered intestinal flora and its metabolites can pass through the damaged intestinal mucosal barrier into the blood, causing systemic chronic low-grade inflammation, leading to the destruction and apoptosis of pancreatic β cells and insulin resistance, thereby inducing diabetes, and even leading to the occurrence and development of diabetes-related complications. Some articles have shown that probiotics can increase the number of beneficial bacteria in the intestine, promote the metabolism and absorption of glucose in food, and reduce the absorption and conversion of glucose in the intestine, thereby lowering blood sugar levels. However, the functional microbial strains currently disclosed do not have a significant effect on lowering blood sugar. Summary of the invention

[0004] In view of this, the object of the present invention is to provide an animal Bifidobacterium lactis subsp. lactis strain B21247, which has the function of inhibiting α-glucosidase activity in vitro and hypoglycemic activity in vivo, and can also improve the glucose and lipid metabolism caused by diabetes.

[0005] The invention provides a Bifidobacterium animalis subspecies lactis strain B21247, with a preservation number of GDMCC No: 65075.

[0006] The present invention provides a metabolite of the animal Bifidobacterium lactis subspecies strain B21247.

[0007] The present invention provides an α-glucosidase inhibitor, comprising the animal Bifidobacterium lactis subspecies strain B21247, the culture of the animal Bifidobacterium lactis subspecies strain B21247 or the metabolite.

[0008] The present invention provides a probiotic, comprising the animal Bifidobacterium lactis subspecies strain B21247 or a culture of the animal Bifidobacterium lactis subspecies strain B21247.

[0009] Preferably, it comprises lyophilized powder and / or aqueous solution.

[0010] The present invention provides the use of the animal Bifidobacterium lactis subspecies B21247, the culture of the animal Bifidobacterium lactis subspecies B21247, the metabolite, the α-glucosidase inhibitor or the probiotic in the preparation of a product for lowering blood sugar and / or blood lipids.

[0011] The present invention provides use of the animal Bifidobacterium lactis subspecies B21247, the culture of the animal Bifidobacterium lactis subspecies B21247, the metabolite, the α-glucosidase inhibitor or the probiotic in preparing a product for preventing and / or treating diabetes.

[0012] Preferably, the product comprises a pharmaceutical product.

[0013] The present invention provides a medicine for preventing and / or treating diabetes, comprising the animal Bifidobacterium lactis subspecies strain B21247, the culture of the animal Bifidobacterium lactis subspecies strain B21247, the metabolite, the α-glucosidase inhibitor or the probiotic and a pharmaceutically acceptable excipient.

[0014] Preferably, the drug comprises at least one of the following dosage forms: tablets, powders, chewable tablets, capsules, granules and oral liquids.

[0015] The present invention provides a strain B21247 of animal Bifidobacterium lactis subsp., with a deposit number of GDMCC No: 65075. The strain B21247 is isolated from the feces of a centenarian, belongs to Gram-positive bacteria, and is identified by 16S rDNA as belonging to animal Bifidobacterium lactis subsp. The survival rate of the strain B21247 after being treated in artificial gastric juice with a pH value of 3 for 3 hours is as high as 101.24%, and the survival rate after being treated in artificial intestinal juice with a pH value of 8 for 6 hours is 97.26%, indicating that the strain B21247 has good resistance to artificial gastrointestinal juice, and the strain B21247 also has good cell adhesion. It can be seen that the strain B21247 has good intestinal colonization ability. The strain B21247 has the function of inhibiting α-glucosidase activity in vitro, and has hypoglycemic and hypolipidemic functions in vivo, can improve the sugar and lipid metabolism function caused by a high-fat diet, improve the morphology and quantity of pancreatic β cells, reduce the impaired function of pancreatic β cells, and promote the production of GLP-1 in vivo, GLP-1 can inhibit the secretion of glucagon by pancreatic α cells, and by reducing the secretion of glucagon, GLP-1 helps to further reduce blood sugar levels. In the embodiment of the present invention, the type 2 diabetes mouse model established with a high-fat diet and streptozotocin (STZ) is used as an experimental subject, and the experimental verification results show that animal bifidobacterium lactis subspecies B21247 can significantly reduce the blood sugar level of mice, and further explore the possible mechanism: by regulating glycosylated hemoglobin, reducing pancreatic β cell damage and increasing the secretion of GLP-1, and then achieving the effect of hypoglycemia, so as to provide a new idea for the application of animal bifidobacterium lactis subspecies in hypoglycemia, and also provide a possibility for the development and promotion of related hypoglycemic microecological products. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the Gram staining observation result of Bifidobacterium animalis subsp. lactis strain B21247;

[0017] Figure 2 The survival rate (left) and cell adhesion growth (right) of Bifidobacterium animalis subsp. lactis strain B21247 in simulated artificial gastrointestinal fluid;

[0018] Figure 3 This is the observation result of bedding during mouse rearing;

[0019] Figure 4 The results of the measurement of blood glucose (left), glycosylated hemoglobin (middle), and GLP-1 content (right) in mice;

[0020] Figure 5 This is the result of OGTT test in mice;

[0021] Figure 6 is the area under the OGTT curve of mice;

[0022] Figure 7 This is the result of H&E staining analysis of mouse liver tissue;

[0023] Figure 8 The results of total cholesterol (TC) and triglyceride (TG) determination in mouse liver tissue;

[0024] Fig. 9 The results of H&E staining analysis of mouse pancreatic tissue.

[0025] Biological deposit information

[0026] Bifidobacterium animalis subsp. lactis strain B21247 was deposited in the Guangdong Microbial Culture Collection Center on August 29, 2024. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology, and the deposit number is GDMCC No: 65075. DETAILED DESCRIPTION

[0027] The invention provides an animal Bifidobacterium lactis subspecies strain B21247 with blood sugar lowering function, and the preservation number is GDMCC No: 65075.

[0028] In the present invention, the strain B21247 is isolated and purified from the feces of a centenarian, and Gram staining shows that the strain B21247 belongs to Gram-positive bacteria. The present invention uses 16S rDNA molecular identification to show that the sequence homology of the strain B21247 to the animal Bifidobacterium lactis subsp. is the highest, at 99.86%. Therefore, the strain B21247 is identified as animal Bifidobacterium lactis subsp.

[0029] In the present invention, the strain B21247 has a strong resistance to artificial gastrointestinal fluid. The survival rate is as high as 101.24% after being treated in artificial gastric fluid with a pH value of 3 for 3 hours, and the survival rate is 97.29% after being treated in artificial intestinal fluid with a pH value of 8 for 8 hours, indicating that the strain B21247 has good resistance to artificial gastrointestinal fluid. At the same time, the strain B21247 also has good cell adhesion. Taking human normal colon epithelial cells NCM460 as an example, the adhesion rate is greater than 60%, which indicates that the strain B21247 has good intestinal colonization ability and can be colonized in the intestine through oral administration.

[0030] The present invention provides a metabolite of the animal Bifidobacterium lactis subspecies strain B21247.

[0031] In the present invention, the metabolites of the animal Bifidobacterium lactis subsp. lactis strain B21247 preferably include fermentation supernatant. The method for preparing the metabolites is preferably to inoculate strain B21247 into MPYG medium for activation culture and collect the fermentation supernatant. The inoculation amount of the activated strain B21247 is preferably 0.8% to 1.2%, more preferably 1%. The culture temperature is preferably 36 to 38°C, more preferably 37°C. The culture time is 18 to 22h, more preferably 20h. The number of activation cultures is preferably 3 times. After the activation culture, the density of the bacterial suspension is preferably adjusted to 1×10 9 CFU / mL, and then solid-liquid separation. The solid-liquid separation method is preferably centrifugation and membrane filtration. The centrifugal speed is preferably 6000rpm. The centrifugal temperature is preferably 3-5°C, more preferably 4°C. The centrifugal time is preferably 9-11min, more preferably 10min. The pore size of the membrane filtration is preferably 0.22μm.

[0032] In the present invention, the metabolites of the strain B21247 have the function of inhibiting the activity of α-glucosidase in vitro, and the inhibition rate of α-glucosidase is 55.68%. It can be seen that the strain B21247 can be used to prepare α-glucosidase inhibitors.

[0033] The present invention provides an α-glucosidase inhibitor, comprising the animal Bifidobacterium lactis subspecies strain B21247, the culture of the animal Bifidobacterium lactis subspecies strain B21247 or the metabolite.

[0034] The present invention provides a probiotic, comprising the animal Bifidobacterium lactis subspecies strain B21247 or a culture of the animal Bifidobacterium lactis subspecies strain B21247.

[0035] In the present invention, the probiotic preferably includes a lyophilized powder and / or an aqueous solution. The preparation method of the lyophilized powder preferably includes the following steps: inoculating the activated strain B21247 in a BBL liquid culture medium for anaerobically culturing to obtain a seed solution 1; inoculating the seed solution 1 into a new BBL liquid culture medium for anaerobically culturing to obtain a seed solution 2; inoculating the seed solution 2 into a BBL liquid culture medium for anaerobically culturing to obtain a bacterial solution; separating the bacterial body from the bacterial solution, mixing the bacterial body with a lyophilized protective agent, and vacuum freeze-drying to obtain a lyophilized powder. The method of the anaerobic culture is preferably static culture. The temperature of the anaerobic culture is preferably 36 to 38°C, more preferably 37°C. The time of the anaerobic culture is preferably 12 to 24h, more preferably 20 to 22h. Before mixing the bacterial body with the lyophilized protective agent, the bacterial body is preferably washed 2 to 3 times with physiological saline to remove the culture medium. The method for separating the bacterial body is preferably centrifugation. The speed of the centrifugation is preferably 5000rpm. The time of the centrifugation is preferably 4 to 6min, more preferably 5min. The centrifugation temperature is preferably 3-5°C, more preferably 4°C. The freeze-drying protectant is preferably a skim milk aqueous solution with a volume percentage of 12%. The vacuum freeze-drying time is preferably 25-35h, more preferably 30h. The preparation method of the aqueous solution preferably includes the following steps: preparing a bacterial solution according to the preparation method of a freeze-dried powder, mixing the bacterial solution with glycerol to obtain an aqueous solution. The amount of glycerol added accounts for 40% to 90% of the total volume of the aqueous solution, and can be 50%, 60%, 70%, or 80%.

[0036] The present invention provides the use of the animal Bifidobacterium lactis subspecies B21247, the culture of the animal Bifidobacterium lactis subspecies B21247, the metabolite, the α-glucosidase inhibitor or the probiotic in the preparation of a product for lowering blood sugar and / or blood lipids.

[0037] In the present invention, a type 2 diabetes mouse model is established with a high-fat diet (fat content 60%) and streptozotocin (STZ), and the results of measuring the blood sugar level of mice show that the blood sugar degradation rate of the animal bifidobacterium lactis subspecies strain B21247 group is 57.72%, and the blood sugar degradation rate of the animal bifidobacterium lactis subspecies strain Bal23036-1 is only 36.67%, which shows that the animal bifidobacterium lactis subspecies strain B21247 has a better advantage in reducing blood sugar than the common animal bifidobacterium strain. At the same time, by detecting the GHb and GLP-1 levels of indicators of long-term blood sugar levels, the GHb level of the animal bifidobacterium lactis subspecies strain B21247 group is reduced by 73.24%, while the GHb of the Bal23036-1 group is reduced by 42.45%; the animal bifidobacterium lactis subspecies strain B21247 can improve the GLP-1 level, and the improvement rate is 143.64%, and Bal23036-1 cannot promote the intestinal secretion of GLP-1. The results of the oral glucose tolerance test (OGTT) showed that the animal Bifidobacterium lactis subspecies strain B21247 could significantly enhance the glucose tolerance of mice, alleviate the impaired function of pancreatic β cells, and improve the body's ability to regulate blood sugar. In addition, the animal Bifidobacterium lactis subspecies strain B21247 group reduced the amount of urine excreted by mice compared with the model group. This indicates that the animal Bifidobacterium lactis subspecies strain B21247 has a better effect in lowering blood sugar than other animal Bifidobacterium lactis subspecies strains.

[0038] In the present invention, a type 2 diabetic mouse model was established by high-fat diet (fat content 60%) and streptozotocin (STZ), and a pathological evaluation of liver tissue was performed, total cholesterol and triglycerides in mouse liver tissue were measured, and pathological changes in liver tissue were detected. The results showed that a large number of diffuse lipid vesicles were observed in the model group, occupying most of the hepatocyte cytoplasm, and a large number of inflammatory cells were infiltrated; compared with the model group, the Bal23036-1 and B21247 groups could reduce the size and number of lipid vesicles, and significantly inhibit the formation of fatty degeneration in the liver. Bal23036-1 and B21247 reduced cholesterol levels by 23.93% and 31.48%, respectively, and reduced triglyceride levels by 15.2% and 52.29%, respectively. Since the accumulation of triglycerides in the liver is the main cause of fatty degeneration, animal Bifidobacterium lactis subspecies strain B21247 has a better effect than Bal23036-1 in improving dyslipidemia caused by diabetes, and can significantly reverse fatty degeneration.

[0039] Based on the fact that the strain B21247 has the effects of inhibiting α-glucosidase activity in vitro and lowering blood sugar in a type 2 diabetes mouse model and improving dyslipidemia caused by diabetes, the present invention provides the use of the animal Bifidobacterium lactis subspecies strain B21247, the culture of the animal Bifidobacterium lactis subspecies strain B21247, the metabolite, the α-glucosidase inhibitor or the probiotic in the preparation of a product for preventing and / or treating diabetes.

[0040] In the present invention, the product preferably includes a medicine. The viable count of the animal Bifidobacterium lactis subsp. lactis strain B21247 in the medicine is 2×10 9 CFU / ml or more, more preferably (5 to 10)×10 9 CFU / ml. The drug is preferably taken orally. The drug dosage form preferably includes at least one of the following dosage forms: tablets, powders, chewable tablets, capsules, granules and oral liquids. The present invention has no special restrictions on the dosage form of the drug, and the preparation method of the drug dosage form known in the art can be used.

[0041] In the present invention, the type 2 diabetes mouse model was used as the experimental subject, and the pancreatic tissue pathology of the mouse was detected. The results showed that compared with the model group, animal Bifidobacterium lactis subspecies B21247 has the effect of maintaining the normal morphology and quantity of pancreatic islet β cells, reducing the impaired function of pancreatic islet cells, and compared with strain Bal23036-1, B21247 pancreatic islet structure is more complete, pancreatic islet cells are oval clusters, with clear boundaries, abundant cell numbers, reduced inflammatory cell infiltration, and closer to the normal group. At the same time, animal Bifidobacterium lactis subspecies B21247 can promote the production of GLP-1 in the body, and GLP-1 can inhibit the secretion of glucagon by pancreatic islet α cells. By reducing the secretion of glucagon, GLP-1 helps to further reduce blood sugar levels, while strain Bal23036-1 does not have this function.

[0042] The present invention provides a medicine for preventing and / or treating diabetes, comprising the animal Bifidobacterium lactis subspecies strain B21247, the culture of the animal Bifidobacterium lactis subspecies strain B21247, the metabolite, the α-glucosidase inhibitor or the probiotic and a pharmaceutically acceptable excipient.

[0043] In the present invention, the dosage form and administration method of the drug are the same as those of the drug in the above technical solution, and will not be described in detail here.

[0044] The following is a detailed description of the animal Bifidobacterium lactis subsp. lactis strain B21247 with blood sugar lowering function and its application provided by the present invention in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0045] Example 1

[0046] Isolation and identification of Bifidobacterium animalis subsp. lactis strain B21247

[0047] The feces of centenarians were used as the isolation sample and cultured and purified using conventional BBL medium (15.0 g peptone, 20.0 g glucose, 2 g yeast extract powder, 0.5 g soluble starch, 5 g sodium chloride, 0.5 g L-cysteine, 5 g tomato extract powder, 2 g liver extract powder, 1.0 g Tween 80, 20.0 g agar powder, 1000 mL distilled water, pH 7.0 ± 0.1, autoclaved at 121 ° C for 20 min). The specific steps are: take a fresh feces sample from a centenarian and dilute it to 10 by 10 times. -5 , take 10 respectively -3 , 10 -4 , 10 -5 Three tubes of dilution, 0.1 ml each, were spread on BBL solid medium for separation by 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 BBL solid plate by streaking. After streaking purification for 2 generations, it was confirmed to be pure by microscopic examination, inoculated into slant medium and cultured for 24 hours, and then stored in a 4°C refrigerator for later use.

[0048] The purified strains were subjected to Gram staining and microscopic examination. Figure 1 .Depend on Figure 1 It can be seen that the purified strain belongs to Gram-positive bacteria.

[0049] The isolated pure strains were molecularly identified using the 16S rDNA molecular identification method, and the sequence is as follows:

[0050]

[0051] The 16S rDNA sequence of the isolated strain was compared in the NCBI (National Center for Biotechnology Information) database. The results showed that the gene sequence of the strain had a homology of 99.86% with the animal Bifidobacterium lactis subspecies among all similar sequences. Therefore, combined with the physiological and biochemical characteristics, the strain was identified as the animal Bifidobacterium lactis subspecies and named B21247. The animal Bifidobacterium lactis subspecies B21247 strain isolated from the intestines of centenarians has been deposited in the Guangdong Microbial Culture Collection Center on August 26, 2024, and its deposit number is GDMCC No: 65075.

[0052] Example 2

[0053] Experiment on resistance to artificial gastrointestinal fluid and cell adhesion of Bifidobacterium lactis strain B21247

[0054] 1. Artificial gastrointestinal fluid resistance test

[0055] The activated bacterial suspension of animal Bifidobacterium lactis subsp. lactis strain B21247 was shaken and evenly mixed, 1 ml of the bacterial suspension was taken into 9 ml of artificial gastric juice (pH=3.0), and cultured at 37°C for 3 hours. The artificial gastric juices cultured at 0 hours, 1 hour, and 3 hours were taken for plate count of live bacteria. The cultured at 0 hours was used as a control, and the survival rate was calculated according to Formula I.

[0056] Simulated artificial gastric fluid survival rate (%) = logNt / logN0×100% Formula I.

[0057] Where N t N0 represents the number of viable bacteria (CFU) after culture for th hour, and N0 represents the number of viable bacteria (CFU) at 0h.

[0058] Take 1 ml of artificial gastric juice after 3 hours of culture and add it to 9 ml of artificial intestinal juice (pH=8.0), and culture it at 37°C for 2 hours, 4 hours, 6 hours and 8 hours. Take artificial intestinal juice after 0 hours, 2 hours, 4 hours, 6 hours and 8 hours of culture to count the viable bacteria on the plate. Take 0 hours as the control and calculate the survival rate according to formula II.

[0059] Survival rate in simulated intestinal fluid (%) = logN t / logN0×100% Formula II.

[0060] Where N t N0 represents the number of viable bacteria (CFU) after culture for th hour, and N0 represents the number of viable bacteria (CFU) at 0h.

[0061] The results are shown in Table 1 and Figure 2Middle left picture.

[0062] Table 1 Tolerance of B21247 simulated artificial gastrointestinal fluid

[0063]

[0064] The results showed that the survival rate of strain B21247 was as high as 101.24% after 3 hours of treatment with artificial gastric juice at pH 3, and 97.29% after 6 hours of treatment with artificial intestinal juice at pH 8. This shows that strain B21247 can tolerate the low pH environment of gastric juice and enter the intestine, indicating that strain B21247 has excellent tolerance to gastrointestinal juice, which is the premise of potential probiotics.

[0065] 2. Cell Adhesion Test

[0066] NCM460 normal intestinal epithelial cell culture and plate seeding: The revived NCM460 cell line was digested and seeded in a 6-well plate (including cell slides). 3×10 5 / well, and cultured overnight in a 37°C, 5% CO2 incubator.

[0067] Preparation of bacterial suspension and cell treatment: The activated bacterial suspension was shaken and collected, washed twice with PBS, and resuspended in high-glucose DMEM medium to adjust the bacterial concentration to 1×10 8 CFU / ml. Add the bacterial solution to a 6-well plate, 1 ml / well, and repeat three wells for each sample. Incubate the bacterial solution with the cells for 2 hours. Take out the 6-well plate and wash it 5 times with PBS to wash off the non-adherent cells as much as possible. Then fix the cell slide with an alcohol lamp and perform Gram staining. Observe the cell adhesion of the experimental strain in multiple fields of view, and calculate the cell adhesion rate according to formula III.

[0068] Bacterial adhesion rate = number of adhered cells / total number of cells in the field of view × 100% Formula III.

[0069] Figure 2 The middle right picture shows a field of view of bacterial adhesion experiment observation. The number of B21247 adhered to each NCM460 cell is >50, and the adhesion rate is 60%, indicating that strain B21247 has good adhesion to NCM460 cells.

[0070] Example 3

[0071] In vitro inhibition test of α-glucosidase by Bifidobacterium animalis subsp. lactis strain B21247

[0072] 1. Reagent Preparation

[0073] pNPG solution: weigh 0.006 g pNPG into a 10 mL centrifuge tube, add an appropriate amount of 0.1 M PBS solution with a pH value of 6.8 to dissolve it, and then make up to 10 mL to prepare a 2 mmol / L solution.

[0074] α-Glucosidase solution: Weigh 2 mg (0.002 g) of 50 U / mg α-glucosidase into a 1.5 mL centrifuge tube, add 1 mL of 0.01 M pH 6.8 phosphate buffer to dissolve, prepare a 100 U / mL stock solution, store at -80°C, and dilute to 0.6 U before use.

[0075] Na2CO3 solution: weigh 1.05g Na2CO3 into a 50mL centrifuge tube, add appropriate amount of deionized water to dissolve and dilute to 50mL to make a 0.2mol / L solution.

[0076] MPYG: 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 800.50ml, Maltose 0.50g, Cellobiose 0.50g, Soluble Starch 0.50g, Glycerol 0.50ml, K2HPO42.00g, Cysteine ​​0.50g, Na2S 0.25g, Resazurin 1.00mg, Salt Solution 40.00ml, Trace Elements 10.00ml, Multivitamin Solution 10.00ml, Hemin Chloride 10.00ml, Vitamin K1 Solution 0.20ml, Agar 15g.

[0077] Salt solution: CaCl2·2H2O 0.25g, MgSO4·7H2O 0.50g, K2HPO4 1.00 g, KH2PO4 1.00g, NaHCO3 10.00 g, NaCl 2.00g, make up to 1000.00ml with water.

[0078] Trace elements: nitrilotriacetic acid 1.50g, MgSO4·7H2O 3.00g, MnSO4·H2O 0.50g, NaCl1.00g, 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, H3BO30.01 g, Na2MoO4·2H2O 0.01g, NiCl2·6H2O 0.03g.Na2SeO3·5H2O 0.30mg, fixed volume to 1000.00ml.

[0079] Multivitamin solution: vitamin H 2.00 mg, fulvic acid 2.00 mg, pyridoxine hydrochloride 10.00 mg, thiamine·2H2O 5.00 mg, riboflavin 5.00 mg, niacin 5.00 mg, calcium pantothenate 5.00 mg, vitamin B12 0.10 mg, p-aminobenzoic acid 5.00 mg, lipoic acid 5.00 mg, made up to volume with water 1000.00 ml.

[0080] Vitamin K1 solution (Hopebio HB8462-a, China): 0.1 g vitamin K1 was dissolved in 20 mL 95% ethanol aqueous solution and filtered for sterilization, and stored at 4°C for later use.

[0081] Hematin solution: Dissolve 0.05 g hematin in 1 mL of 1 mol / L NaOH and add distilled water to make up to 100 mL. Filter and sterilize and refrigerate for later use.

[0082] 2. In vitro determination of the inhibitory activity of α-glucosidase by strains

[0083] The animal Bifidobacterium lactis subsp. lactis strain B21247 stored in a -80°C bacterium preservation tube was inoculated into MPYG medium at 1% inoculation and cultured at 37°C for 20 h for a total of three generations. After the strain was activated for three generations, the density of the bacterial suspension was adjusted to 1×10 9 CFU / mL, centrifuge at 6000r / min for 10min at 4℃, and collect the fermentation supernatant. The fermentation supernatant was filtered with a 0.22μm water filter to obtain a fermentation supernatant sample, with MPYG as a control. Add 50μL 0.6U α-glucosidase solution and 25μL fermentation supernatant to a 96-well plate, incubate at 37℃ for 10min, add 10μL 2mmol / L pNPG solution, incubate at 37℃ for 3min, then add 85μL Na2CO3 solution to terminate the reaction, and measure the absorbance at 405nm. Calculate the α-glucosidase inhibition rate according to formula IV.

[0084] Α-glucosidase inhibition rate (%) = 1-(AB) / (CD)×100% Formula IV.

[0085] A Positive control: sample OD;

[0086] B Positive blank control: 50 μL 0.1 M PBS buffer with pH 6.8 was used instead of the α-glucosidase solution;

[0087] C Negative control: (no sample inhibiting α-glucosidase in the system) replace the strain supernatant with 25 μL 0.1 M PBS buffer, pH 6.8;

[0088] D. Negative blank control: (no α-glucosidase activity in the system) replace the strain supernatant with 25 μL 0.1 M PBS buffer at pH 6.8, and replace the α-glucosidase solution with 50 μL 0.1 M PBS buffer at pH 6.8.

[0089] The results showed that the inhibition rate of strain B21247 on α-glucosidase was 55.68%.

[0090] Example 4

[0091] Animal Experiments on Bifidobacterium lactis subsp. lactis strain B21247

[0092] (1) Preparation of strain freeze-dried powder

[0093] Pick the animal Bifidobacterium lactis subspecies strain B21247 and the common animal Bifidobacterium strain Bal23036-1 single colony after activation twice and inoculate them into 15ml BBL liquid culture medium, and culture them statically at 37℃ anaerobic conditions for 12h to obtain seed liquid 1. Inoculate seed liquid 1 into 150ml BBL liquid culture medium at a 3% inoculation amount, and culture them statically at 37℃ anaerobic conditions for 20-22h to obtain seed liquid 2. Inoculate seed liquid 2 into 300ml BBL liquid culture medium at a 3% inoculation amount, and culture them statically at 37℃ anaerobic conditions for 20-24h to obtain the bacterial liquid after amplification culture. Collect the bacterial liquid 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. Preparation of lyophilized protective agent: weigh 12g skim milk powder and add it to 100ml distilled water, and sterilize it with high pressure steam at 115℃ for 15min. Resuspend the prepared lyophilized protective agent and wet weight of bacteria in a volume ratio of 1:1, divide into EP tubes, place at -20℃ for 30min, and then place at -80℃ for 1h. Put the divided EP tubes into freeze-dried bottles, put the freeze-dried bottles into the freeze dryer for freeze drying, and continue freeze drying for 30h to finally obtain freeze-dried bacterial powder. Use the plate count method to perform the technique and store the bacterial powder at 4℃ for standby use, and resuspend it with sterile saline when using.

[0094] (2) Animal experiments

[0095] Thirty-six 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 12 h, the temperature was 20-26 ° C, 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 (normal group: basic feed and 0.2 ml skim milk; model group: high-fat (60%) feed (purchased from Jiangsu Collaborative Biology) and 0.2 ml skim milk; B21247 group: high-fat (60%) feed and strain freeze-dried powder (dissolved in 0.2 ml saline to 2×10 9 CFU / ml); Bal23036-1 group (common animal Bifidobacterium): high-fat (60%) feed and strain freeze-dried powder (dissolved in 0.2 ml of saline to 2×10 9 CFU / ml), 8 to 10 mice per group. After the adaptation period, fasting blood sugar was tested first, and then the mice were fed with 60% high-fat feed for 14 days. Then, STZ (dissolved in citric acid buffer to 100 mg / kg) was injected intraperitoneally in the model group and product group on days 14 and 17, and citric acid buffer was injected intraperitoneally in the control group. The fasting blood sugar of mice was measured twice in a row on days 20 and 21. The model was considered successful if the blood sugar concentration was greater than 11.0 mmol / L. The freeze-dried bacterial powder was continuously gavaged for 35 days.

[0096] (3) Determination of biochemical indices for hypoglycemic effect in vivo by Bifidobacterium animalis subsp. lactis strain B21247

[0097] A. Blood glucose measurement in mice

[0098] Results Figure 3 During the intragastric administration, it was observed that the humidity of the litter in the B21247 group was significantly lower than that in the model group, indicating that the phenomenon of increased urine volume caused by diabetic urine was improved in this group.

[0099] At the same time, the glucose detection kit of Leigen Biotechnology was used to detect glucose in mouse serum according to the instructions of the kit. Figure 4. The results showed that after oral administration of animal Bifidobacterium lactis subspecies Bal23036-1, the blood glucose degradation rate was 36.67%, compared with 57.72% for animal Bifidobacterium lactis subspecies B21247, indicating that animal Bifidobacterium lactis subspecies B21247 of the same species but different strains had better blood glucose lowering effect. Glycated hemoglobin (GHb) is one of the indicators for detecting long-term blood glucose levels. After oral administration of Bal23036-1 and B21247, GHb levels decreased by 42.45% and 73.24%, respectively; at the same time, strain B21247 could significantly increase intestinal GLP-1 levels, up to 143.64%, while Bal 23036-1 could not promote intestinal secretion of GLP-1, and from the humidity of the litter, it was observed that Bal 23036-1 had a higher humidity than B21247. The above results showed that B21247 had a better effect in lowering blood glucose.

[0100] B. Oral glucose tolerance test (OGTT) in mice

[0101] OGTT is a glucose load test used to understand the function of pancreatic β cells, the degree of insulin resistance, and the body's ability to regulate blood sugar. It is usually performed one week before the end of the experiment. The subjects fasted overnight the day before the test, and then were given 2g / kg of glucose. The blood sugar levels at 0min, 30min, 60min, and 120min were measured using test strips and a blood glucose meter (Roche blood glucose meter).

[0102] Results Figure 5 and Figure 6 Compared with the normal group, the blood sugar level in the model group rose rapidly and to a greater extent, and the blood sugar level dropped relatively slowly within 120 minutes, and it was unable to return to normal blood sugar levels, indicating that glucose tolerance had decreased. The B21247 group was able to improve the blood sugar level to a certain extent, and there was a statistical difference (p<0.5), indicating that B21247 can improve the impaired function of pancreatic β cells and enhance the ability of pancreatic β cells to respond to glucose load.

[0103] C. Mouse liver pathological analysis

[0104] During the dissection, part of the mouse liver tissue was fixed with 4% paraformaldehyde fixative for 48 h, embedded in paraffin, and sliced ​​into 4-5 μm sections using a paraffin slicer (Kodi KD2260), followed by H&E staining for histopathological evaluation.

[0105] Results Figure 7 and Figure 8. The results showed that H&E staining of mouse liver tissue showed that compared with the normal group, a large number of diffuse lipid vesicles were visible in the model group, occupying most of the hepatocyte cytoplasm, and a large number of inflammatory cells infiltrated; compared with the model group, the Bal23036-1 and B21247 groups could reduce the size and number of lipid vesicles and significantly inhibit the formation of fatty degeneration in the liver, but the size of lipid droplets showed that B21247 had a better effect. Hepatic fatty degeneration is mainly caused by the accumulation of triglycerides in the liver. After gavage with the strains, the strain Bal23036-1 and strain B21247 reduced cholesterol levels by 23.93% and 31.48%, respectively, and reduced triglyceride levels by 15.2% and 52.29%, respectively, and B21247 had significant differences, further indicating that the animal Bifidobacterium lactis subsp. strain B21247 has a better effect in improving dyslipidemia caused by diabetes and can significantly reverse fatty degeneration.

[0106] D. Pathological analysis of mouse pancreatic tissue

[0107] During the dissection, part of the mouse pancreatic tissue was fixed with 4% paraformaldehyde fixative for 48 h, embedded in paraffin, and sliced ​​into 4-5 μm sections using a paraffin slicer (Kodi KD2260), followed by H&E staining for histopathological evaluation.

[0108] Results Fig. 9 . The results showed that the pancreatic histological structure of the normal group was normal, the islet cells were arranged in an orderly manner, the islet cells were in oval clusters, with clear boundaries, abundant cells, regular morphology, and uniform distribution. The islet structure of the model group was significantly atrophied and deformed, with blurred boundaries, reduced area, disordered cell arrangement, fragmented or dissolved nuclei, inflammatory cell infiltration, and reduced islet cell number. Compared with the model group, the pancreatic structure of the Bal23036-1 and B21247 groups was significantly restored, but the pancreatic islet structure of B21247 was more complete, the islet cells were in oval clusters, with clear boundaries, abundant cells, and reduced inflammatory cell infiltration, which was closer to the normal group, indicating that B21247 had a significant improvement effect on maintaining the shape of islet cells.

[0109] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A strain B21247 of Bifidobacterium animalis subsp. lactis, characterized in that: The deposit number is GDMCC No:65075.

2. A metabolite of the Bifidobacterium animalis subsp. lactis strain B21247 according to claim 1.

3. An α-glucosidase inhibitor, characterized in that The method comprises the animal Bifidobacterium lactis subspecies B21247 strain according to claim 1, the culture of the animal Bifidobacterium lactis subspecies B21247 strain according to claim 1 or the metabolite according to claim 2.

4. A probiotic, characterized in that: The method comprises the animal Bifidobacterium lactis subspecies B21247 strain according to claim 1 or a culture of the animal Bifidobacterium lactis subspecies B21247 strain according to claim 1.

5. The probiotic according to claim 4, characterized in that: Including lyophilized powder and / or aqueous solution.

6. Use of the animal Bifidobacterium lactis subspecies B21247 strain according to claim 1, the culture of the animal Bifidobacterium lactis subspecies B21247 strain according to claim 1, the metabolite according to claim 2, the α-glucosidase inhibitor according to claim 3 or the probiotic according to claim 4 in the preparation of a product for lowering blood sugar and / or blood lipids.

7. Use of the animal Bifidobacterium lactis subspecies B21247 strain according to claim 1, the culture of the animal Bifidobacterium lactis subspecies B21247 strain according to claim 1, the metabolite according to claim 2, the α-glucosidase inhibitor according to claim 3 or the probiotic according to claim 4 in the preparation of a product for preventing and / or treating diabetes.

8. The use according to claim 6 or 7, characterized in that: The products include pharmaceutical products.

9. A drug for preventing and / or treating diabetes, characterized in that: The invention comprises the animal Bifidobacterium lactis subspecies B21247 strain according to claim 1, the culture of the animal Bifidobacterium lactis subspecies B21247 strain according to claim 1, the metabolite according to claim 2, the α-glucosidase inhibitor according to claim 3 or the probiotic according to claim 4 and a pharmaceutically acceptable excipient.

10. The drug according to claim 9, characterized in that: The medicine comprises at least one of the following dosage forms: tablets, powders, chewable tablets, capsules, granules and oral liquids.

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