Bifidobacterium adolescentis strain B24728 with the function of inhibiting α-glucosidase activity and secreting GLP-1 and its application

Abstract

The present invention provides a strain of Bifidobacterium adolescentis with the function of inhibiting α-glucosidase activity and secreting GLP-1 and its application, belonging to the field of functional microbial technology. The present invention isolates a strain of Bifidobacterium adolescentis B24728 from the feces of centenarians in Guangxi. The strain B24728 not only has the function of inhibiting α-glucosidase activity in vitro, but also has the function of high production of GLP-1, and animal experiments show that the strain B24728 has the efficacy of treating diabetes. At the same time, the strain B24728 is a human strain, has no species rejection reaction, and meets the safety requirements. Therefore, the strain B24728 and its culture can be used as food, health products or medicines for lowering blood sugar, and can be used to prepare medicines for the treatment of diabetes.

Description

Technical Field

[0001] The present invention belongs to the technical field of functional microorganisms, and specifically relates to a Bifidobacterium adolescentis strain B24728 having the function of inhibiting α-glucosidase activity and secreting GLP-1 and an application thereof. Background Art

[0002] Diabetes is a metabolic disease caused by insufficient secretion and / or impaired utilization of insulin. α-Glucosidase is an enzyme located in the brush border of the small intestinal mucosa. It participates in the digestion of carbohydrates and hydrolyzes oligosaccharides and disaccharides into glucose and other monosaccharides, leading to increased blood sugar. Therefore, inhibiting the activity of α-glucosidase can delay the absorption of glucose, thereby reducing postprandial blood sugar levels. Glucagon-like peptide-1 (GLP-1) is a 30-amino acid incretin secreted by intestinal L cells that inhibits glucose synthesis, increases insulin secretion in response to glucose, and inhibits the production of glucagon to achieve the effect of lowering blood sugar. Currently, GLP-1 analogs and dipeptidyl peptidase 4 (DPP-IV) inhibitors have been used as antidiabetic drugs, but their high cost, high price, huge economic burden and short half-life of GLP-1 limit their clinical application.

[0003] In addition, there are currently very 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. Moreover, the construction process of engineered bacteria involves multiple links such as genetic engineering and cell culture, which is technically difficult. There are differences in expression efficiency and stability among different engineered bacteria, which may lead to unstable production and quality of GLP-1 derivatives or analogues, and safety issues such as gene escape. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a Bifidobacterium adolescentis strain B24728, which inhibits the activity of α-glucosidase and the function of secreting GLP-1 and has a good hypoglycemic effect.

[0005] The present invention provides a Bifidobacterium adolescentis strain B24728, with a deposit number of GDMCC No: 65489.

[0006] The present invention provides a metabolite or culture of the Bifidobacterium adolescentis strain B24728.

[0007] The present invention provides a method for preparing the metabolites of the Bifidobacterium adolescentis strain B24728, comprising the following steps:

[0008] The Bifidobacterium adolescentis strain B24728 was inoculated into MPYG liquid medium for anaerobically culturing to obtain a culture;

[0009] The liquid phase of the culture is separated to obtain metabolites of Bifidobacterium adolescentis strain B24728.

[0010] Preferably, the separation of the liquid phase further includes concentration; the concentration multiple is 5 to 10 times.

[0011] The present invention provides a probiotic, comprising the Bifidobacterium adolescentis strain B24728, the metabolite or culture or the metabolite prepared by the preparation method.

[0012] The present invention provides an α-glucosidase inhibitor, wherein the active ingredients include the Bifidobacterium adolescentis strain B24728, the metabolite or culture, and the metabolite prepared by the preparation method.

[0013] The present invention provides use of the Bifidobacterium adolescentis strain B24728, the metabolite or culture, the metabolite prepared by the preparation method, the probiotic or the α-glucosidase inhibitor in preparing a product for lowering blood sugar.

[0014] Preferably, the product includes at least one of the following: medicine, health care product and food.

[0015] The present invention provides use of the Bifidobacterium adolescentis strain B24728, the metabolite or culture, the metabolite prepared by the preparation method, the probiotic or the α-glucosidase inhibitor in preparing a drug for preventing and / or treating diabetes.

[0016] Preferably, the drug comprises at least one of the following dosage forms: tablets, powders, granules, capsules and oral solutions.

[0017] The present invention provides a Bifidobacterium adolescentis strain B24728, with a deposit number of GDMCC No: 65489. The strain B24728 has the function of efficiently inhibiting α-glucosidase activity in vitro, and also has the function of efficiently secreting GLP-1. At the same time, animal experiments show that the strain B24728 can improve the weight loss of mice caused by diabetes, reduce the fasting blood sugar level, glycosylated hemoglobin, insulin and improve islet resistance and glucose tolerance of mice, promote intestinal GLP-1 secretion, and reduce pancreatic damage and maintain pancreatic function, thereby achieving the purpose of treating diabetes. In addition, the strain B24728 is isolated from the intestinal flora of centenarians in Guangxi, a human-derived strain, has no species rejection reaction, has the potential to colonize the human body, and its hemolytic test results show that the bacteria meet the safety requirements, so it can be used as food or medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a colony morphology diagram of Bifidobacterium adolescentis strain B24728;

[0019] Figure 2 This is a Gram staining microscopic examination of Bifidobacterium adolescentis strain B24728;

[0020] Figure 3 This is the gel electrophoresis diagram of the 16S rRNA gene of Bifidobacterium adolescentis strain B24728;

[0021] Figure 4 This is the phylogenetic tree of the 16S rRNA gene of Bifidobacterium adolescentis strain B24728;

[0022] Figure 5 The results of the inhibition rate of α-glucosidase activity by Bifidobacterium adolescentis strain B24728, ns in the figure means no significant difference, * means P < 0.05, **** means P < 0.0001;

[0023] Figure 6 The results of the GLP-1 secretion ability test of Bifidobacterium adolescentis strain B24728, * represents P < 0.05, ** represents P < 0.01;

[0024] Figure 7 The results of the weight changes of mice in each experimental group, * represents P < 0.05, ** represents P < 0.01;

[0025] Figure 8 The results of fasting blood glucose (FBG) and glycosylated hemoglobin (GHb) in mice of each experimental group. In the figure, ns means no significant difference, * means P < 0.05, *** means P < 0.001, **** means P < 0.0001;

[0026] Fig. 9 The results of insulin (INS) and insulin resistance index (HOMA-IR) of mice in each experimental group, ns in the figure means no significant difference, **** means P < 0.0001;

[0027] Fig.10 Oral glucose tolerance test (OGTT) results of mice in each experimental group, * represents P < 0.05, **** represents P < 0.0001;

[0028] Fig.11 The results of the determination of C-peptide (CP) and glucagon-like peptide-1 (GLP-1) levels in mice of each experimental group. In the figure, ns means no significant difference, * means P < 0.05, and *** means P < 0.001;

[0029] Fig.12 The H&E staining results of the pancreas of mice in each experimental group, among which the left picture is the normal group, the middle picture is the model group, and the right picture is the strain B24728 group.

[0030] Biomaterial Deposit Information

[0031] Bifidobacterium adolescentis strain B24728 was deposited on November 15, 2024 in the Guangdong Provincial Microbiological Culture Collection Center, abbreviated as GDMCC, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology, with the deposit number GDMCC No: 65489. DETAILED DESCRIPTION

[0032] The invention provides a Bifidobacterium adolescentis strain B24728, with a preservation number of GDMCC No: 65489.

[0033] In the present invention, the strain B24728 is isolated from the intestinal flora of centenarians in Guangxi. The colony morphology of strain B24728 is a circular colony with a diameter of 1 to 2 mm, which is slightly raised, the colony color is milky white, and its edges are smooth and neat. It is suitable for growth under facultative anaerobic conditions. Its optimal growth and fermentation temperature range is 35 to 40°C, and the optimal initial pH value is 6.5 to 7.0. The Gram staining of strain B24728 is blue-purple, and the strain shape is rod-shaped, which is judged to be a Gram-positive bacterium. After 16S rRNA detection, the sequencing result of 16SrRNA of strain B24728 is SEQ ID NO: 1, and the sequence comparison results show that it is similar to Bifidobacterium adolescentis ATCC 15703. T The sequence homology of the strain B24728 was 98.70%. Based on morphological and molecular identification, the strain B24728 belonged to Bifidobacterium adolescentis.

[0034] The present invention provides a metabolite or culture of the Bifidobacterium adolescentis strain B24728.

[0035] The present invention provides a method for preparing the metabolite or culture, comprising the following steps:

[0036] The activated Bifidobacterium adolescentis strain B24728 is inoculated into MPYG liquid medium for anaerobically culturing to obtain a culture;

[0037] The fermentation broth is separated into a liquid phase to obtain metabolites of Bifidobacterium adolescentis strain B24728.

[0038] In the present invention, the temperature of the anaerobic culture can be 36-38°C, or 37°C. The time of the anaerobic culture is 20-26h, or 22-24h. The inoculation amount of the inoculation is preferably 4%-6%, or 5%. The culture contains the bacterial cells of Bifidobacterium adolescentis strain B24728 and the metabolites of Bifidobacterium adolescentis strain B24728. The method for separating the liquid phase is preferably centrifugation and filtration. The speed of the centrifugation is preferably 8000-12000rpm, or 10000rpm. The time of the centrifugation is preferably 4-6min, or 5min. The filtration method is preferably membrane filtration. The pore size of the filter membrane is preferably 0.22μm. After collecting the liquid phase, it is preferably further concentrated. The concentration method preferably includes freeze drying. The multiple of the concentration is preferably 1:5-10, or 1:7-8.

[0039] The present invention provides a probiotic preparation, comprising the Bifidobacterium adolescentis strain B24728 or the metabolite or culture.

[0040] In the present invention, the probiotic preferably includes at least one of the following dosage forms: aqueous solution, powder and granule. The effective live bacteria concentration of the Bifidobacterium adolescentis strain B24728 is not less than 1×10 8 CFU / mL, can be 5×10 8 CFU / mL, 10×10 8 CFU / mL, 50×10 8 CFU / mL, 100×10 8 CFU / mL, 500×10 8 CFU / mL. The effective concentration of the metabolite or culture is not less than 10% to 99%, can be 15% to 80%, can also be 20% to 70%, can also be 30% to 50%. The present invention has no special restrictions on the preparation method of the probiotics, and the probiotic preparation method known in the art can be used.

[0041] The present invention provides an α-glucosidase inhibitor, wherein the active ingredients include the Bifidobacterium adolescentis strain B24728, the metabolite or culture, and the metabolite prepared by the preparation method.

[0042] In one embodiment of the present invention, the metabolites of the Bifidobacterium adolescentis strain B24728 were used as active ingredients to test the effect on α-glucosidase activity in vitro. The results showed that the strain B24728 inhibited α-glucosidase activity by 97.24%, which is close to acarbose currently used to treat diabetes, while other Bifidobacterium adolescentis strains had lower α-glucosidase activity, indicating that the Bifidobacterium adolescentis strain B24728 has a unique advantage in inhibiting α-glucosidase activity compared with other Bifidobacterium adolescentis strains.

[0043] In another embodiment of the present invention, qualitative and quantitative detection of secretions of Bifidobacterium adolescentis strain B24728 was also carried out, and the results showed that strain B24728 has the characteristic of high production of GLP-1, with a production of 1542pg / ml, and is significantly higher than that of Bifidobacterium adolescentis strains of the same species.

[0044] Since inhibiting α-glucosidase activity and GLP-1 are both important ways to lower blood sugar, and the Bifidobacterium adolescentis strain B24728 has the function of inhibiting α-glucosidase activity and secreting GLP-1 itself, the present invention provides the use of the Bifidobacterium adolescentis strain B24728, the metabolite or culture, the metabolite prepared by the preparation method, the probiotic agent or the α-glucosidase inhibitor in the preparation of a product for lowering blood sugar.

[0045] In the present invention, the product preferably includes at least one of the following: medicine, health care product and food. In the product, the effective live bacteria concentration of the Bifidobacterium adolescentis strain B24728 is not less than 1×10 8 CFU / mL, can be 5×10 8 CFU / mL, 10×10 8 CFU / mL, 50×10 8 CFU / mL, 100×10 8 CFU / mL, 500×10 8 CFU / mL. The effective mass concentration of the metabolite or culture is not less than 10% to 99%, can be 15% to 80%, can also be 20% to 70%, can also be 30% to 50%. The present invention has no special restrictions on the preparation method of the product, and methods well known in the art can be used. The blood sugar reduction includes fasting blood sugar content and postprandial blood sugar content.

[0046] In one embodiment of the present invention, the food comprises a solid beverage. The solid beverage comprises a fruit-flavored solid beverage. The fruit-flavored solid beverage comprises the following components by mass: 1 part of freeze-dried powder of Bifidobacterium adolescentis strain B24728, 0.5-1.0 g of galacto-oligosaccharide, 0.5-1.0 part of resistant dextrin, 0.5-1.0 part of Tremella extract, 0.5-1.0 part of Ganoderma lucidum extract, and 0.5-1.0 part of fruit powder. The fruit powder comprises blueberry fruit powder or cherry fruit powder.

[0047] The present invention provides use of the Bifidobacterium adolescentis strain B24728, the metabolite or culture, the metabolite prepared by the preparation method, the probiotic or the α-glucosidase inhibitor in preparing a drug for preventing and / or treating diabetes.

[0048] In the present invention, the drug preferably includes at least one of the following dosage forms: tablets, powders, granules, capsules and oral liquids. The present invention has no particular restrictions on the preparation method of the drug, and any method known in the art may be used. The effective live bacterial concentration of Bifidobacterium adolescentis strain B24728 in the drug is not less than 2×10 9 CFU / mL, can be 5×10 9 CFU / mL, 1×10 10 CFU / mL and 5×10 10 CFU / mL.

[0049] In one embodiment of the present invention, a type 2 diabetes mouse model induced by a high-fat (60%) diet is used as the implementation object, and the type 2 diabetes mouse model exhibits at least one of the following conditions: fasting blood glucose, glycosylated hemoglobin, and insulin are all increased, insulin resistance occurs, glucose tolerance decreases, intestinal secretion of GLP-1 levels decreases, and pancreatic damage occurs. After administering the Bifidobacterium adolescentis strain B24728, the fasting blood glucose level, glycosylated hemoglobin, and insulin of the mice can be reduced, islet resistance and glucose tolerance can be improved, intestinal GLP-1 secretion can be promoted, and pancreatic damage can be reduced, pancreatic function can be maintained, and the problem of weight loss in mice caused by diabetes can be reversed. It can be seen that the Bifidobacterium adolescentis strain B24728 has a good efficacy in treating diabetes.

[0050] The following is a detailed description of the Bifidobacterium adolescentis strain B24728 having the function of inhibiting α-glucosidase activity and secreting GLP-1 and its application provided by the present invention in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0051] The culture medium involved in the embodiment:

[0052] MPYG medium: 5 g tryptone, 3 g peptone, 2 g soy peptone, 1 g polypeptone, 10 g yeast extract, 5 g beef extract, 5 g glucose, 0.5 g maltose, 0.5 g cellobiose, 0.5 g soluble starch, 2 g dipotassium hydrogen phosphate, 0.5 g cysteine, 0.25 g sodium sulfide, 1 mg resazurin, 0.5 mL Tween 80, 0.5 mL glycerol, 1000 mL distilled water, pH 6.6-6.8. When preparing solid culture medium, add 15 g agar and sterilize at 121°C under high pressure for 15 min.

[0053] LB medium: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, 1000 mL of distilled water, pH 7.0. When preparing solid culture medium, add 15 g of agar and sterilize at 121°C for 15 min.

[0054] Example 1

[0055] Isolation, purification and identification of a strain of Bifidobacterium adolescentis B24728

[0056] (1) The intestinal feces of centenarians in Guangxi were used as samples. After dilution with physiological saline, streaking culture was performed, and a single colony was picked and inoculated into MPYG solid culture medium. The plate was placed in an anaerobic workstation and cultured at 37°C for 2-3 days. After the colonies grew, a single colony was picked and streaked for two generations to obtain strain B24728. A single colony on the final purification plate was picked for Gram staining microscopy.

[0057] Figure 1 The colony morphology of Bifidobacterium adolescentis B24728 is a round colony with a diameter of 1 to 2 mm, slightly raised, milky white in color, and smooth and neat edges. Figure 2 The bacteria are rod-shaped and Gram-stained blue-purple, so they are Gram-positive bacteria.

[0058] (2) Identification of the 16S rRNA gene of strain B24728: Pick a small amount of purified single colonies with a sterile bamboo stick, shake and mix in 50 μL sterile PBS (10 mmol / L, pH 7.4) buffer to prepare a bacterial suspension. Now prepare a 16S rRNA gene amplification system. The system is configured as follows: 1 μL each of primer 27F (5'-AGAGTTTG ATCCTGGCTCAG-3', SEQ ID NO: 2) and primer 1492R (5'-CTACGGCTACCTTGT TACGA-3', SEQ ID NO: 3), 12.5 μL of 2×Es Taq Mix, and ddH 209.5μL was mixed in a PCR tube, and finally 1μL of bacterial suspension was added to a PCR tube containing a 16S rRNA gene amplification system, and mixed, and placed in a PCR amplifier for amplification. The amplification program was 94℃ pre-denaturation for 5min, 94℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 1min, 30 cycles, and 72℃ final extension for 2min. After the amplification, the amplified liquid was taken for agarose gel electrophoresis (1% agarose gel), with a spot volume of 6μL, a voltage of 115V, and a time of 35min. Finally, a gel imager was used to observe whether there was a band at about 1500bp (see Figure 3T ) has a sequence homology of 98.70%. By constructing a phylogenetic tree such as Figure 4 It can be seen that this strain is clustered together with Bifidobacterium adolescentis, and combined with its physiological and biochemical characteristics, it is preliminarily judged that this strain is Bifidobacterium adolescentis.

[0059] Example 2

[0060] In vitro inhibition test of α-glucosidase by Bifidobacterium adolescentis strain B24728

[0061] 1. Reagent configuration:

[0062] pNPG solution: weigh 0.006 g pNPG into a 10 mL centrifuge tube, add an appropriate amount of 0.1 M pH 6.8 PBS solution to dissolve it, and then make up to 10 mL to prepare a 2 mmol / L solution.

[0063] 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.

[0064] Na 2 CO 3 Solution: weigh 1.05g Na 2 CO 3 In a 50mL centrifuge tube, add appropriate amount of deionized water to dissolve and make up to 50mL to prepare a 0.2mol / L solution.

[0065] Vitamin K1 solution: Dissolve 0.1 g of vitamin K1 in 20 mL of 95% ethanol and filter sterilize, then store at 4°C until use.

[0066] Hematin solution: Dissolve 0.05 g hematin in 1 mL of 1 mol / L NaOH and make up to 100 mL with distilled water. Filter and sterilize and refrigerate for later use.

[0067] 2. Experimental Procedure

[0068] The strain B24728 stored in a -80℃ preservation tube was inoculated into MPYG medium at 1% inoculation and cultured at 37℃ for 48h for three generations. After the strain was activated for three generations, the density of the bacterial suspension was adjusted to 1×10 9CFU / mL, centrifuged at 6000r / min for 10min at 4℃, and the fermentation supernatant was collected. The fermentation supernatant was filtered with a 0.22μm water filter membrane to obtain a fermentation supernatant sample. Using MPYG medium as a control, the ability of B24728 and the same batch of screened Bifidobacterium adolescentis strains H27 and L167, Bacteroides kingii 11-9 and Enterococcus N193 (control) to inhibit α-glucosidase activity was determined. 50μL 0.6U α-glucosidase solution and 25μL strain supernatant were added to a 96-well plate, incubated at 37℃ for 10min, 10μL 2mmol / L pNPG solution was added, and 85μL Na was added after incubation at 37℃ for 3min. 2 CO 3 The solution was used to terminate the reaction, and the absorbance at 405 nm was measured. Acarbose was used as a positive control, and the experiment was repeated three times independently to obtain the average value. The inhibition rate of α-glucosidase activity was calculated according to formula I.

[0069] Glucosidase inhibition rate (%) = 1-(A-B / (C-D) × 100% Formula I.

[0070] Where A: sample OD value.

[0071] B: Replace the α-glucosidase solution with 50 μL of 0.1 M pH 6.8 PBS buffer.

[0072] C: There is no sample that inhibits α-glucosidase in the system, and 25 μL 0.1M pH 6.8 PBS buffer is used to replace the strain supernatant.

[0073] D: For samples without α-glucosidase in the system, 25 μL 0.1M pH 6.8 PBS buffer was used to replace the strain supernatant, and 50 μL 0.1M pH 6.8 PBS buffer was used to replace the α-glucosidase solution.

[0074] Results Figure 5 The results showed that different strains of Bifidobacterium adolescentis had different inhibition rates on α-glucosidase activity, and there were significant differences in the inhibition rates between different strains of the same species. Strain B24728 inhibited α-glucosidase activity by 97.24%, which is close to acarbose currently used to treat diabetes.

[0075] Example 3

[0076] In vitro GLP-1 secretion test of Bifidobacterium adolescentis B24728

[0077] 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 determine the GLP-1 of the strain fermentation supernatant using an enzyme-linked immunosorbent assay (ELISA) kit, with blank culture medium MPYG as a negative control and the fermentation supernatant of Bifidobacterium adolescentis strain H27 screened in the same batch as a control.

[0078] Results Figure 6 The results showed that the amount of GLP-1 secreted and produced by strain B24728 reached 1542pg / ml, and was significantly improved compared with the GLP-1 secretion capacity of different strains of Bifidobacterium adolescentis of the same species.

[0079] Example 4

[0080] Preparation of freeze-dried powder of Bifidobacterium adolescentis B24728

[0081] Pick a single colony of strain B24728 after activation twice to 15ml of MPYG liquid medium, and culture it under anaerobic conditions at 37℃ for 12h to obtain seed solution 1. Inoculate seed solution 1 into 150ml of MPYG 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 MPYG 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 at 4℃, 5000rpm, and centrifuge for 5min. Weigh the wet weight of the bacteria. Mix the lyophilized protective agent and the bacteria in an equal volume ratio, divide into EP tubes, place at -20℃ for 30min, and then treat 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. Use the plate count method to perform the technique and store the bacterial powder at 4°C for later use. Resuspend it with sterile saline when using it.

[0082] The freeze-drying protective agent is prepared as follows: 15% trehalose, 15% maltodextrin, 3% gelatin, 1% glycerol, and sterilized at 115°C for 15 minutes. Vitamin C is first prepared to a concentration of 10%, dissolved, and then filtered on a clean bench. When used, it is diluted 200 times to a final concentration of 0.05%.

[0083] Example 5

[0084] Animal experiment function verification

[0085] 1. Animal Experiment Grouping and Dosing

[0086] 50 SPF-grade 6-week-old female C57BL / 6J mice (purchased from Beijing Sibeifu Biotechnology Co., Ltd.) were fed adaptively for 1 week. During the adaptation period, the light cycle was maintained at 12 h, the temperature was 20-26 ° C, and the humidity was 40%-60%. The mice were allowed to eat and drink freely. After the adaptation period, they were randomly divided into 4 groups, with 10-14 mice in each group:

[0087] Normal group: basic feed and 0.2 ml skim milk;

[0088] Model group: high-fat (60%) feed and 0.2 ml skim milk;

[0089] B24728 group: high-fat (60%) diet and freeze-dried bacterial powder (dissolved in 0.2 ml of saline to 2 x 10 9 cfu / ml);

[0090] The same batch of Bifidobacterium adolescentis H27 group: high-fat (60%) feed and freeze-dried bacterial powder (dissolved in 0.2 ml of saline to 2 × 10 9 CFU / ml.

[0091] After the adaptation period, fasting blood sugar was tested first, and mice with abnormal blood sugar were discarded. Mice with normal blood sugar were fed with 60% high-fat diet for 14 days. Then, STZ (dissolved in citric acid buffer to 80 mg / kg and 60 mg / kg) was injected intraperitoneally in the model group and the bacteria group on days 14 and 17, respectively, and citric acid buffer was injected intraperitoneally in the control group. The mice were fasted one day before the injection. The fasting blood sugar of mice was measured twice in a row on days 20 and 21. The model was considered to be successful if the fasting blood sugar concentration was greater than 7.0 mmol / L and the blood sugar 2 hours after the meal was greater than 11.0 mmol / L. The entire experimental period lasted for 9 weeks.

[0092] 2. Determination of relevant indicators

[0093] The type 2 diabetes model was established by high-fat and STZ to evaluate the hypoglycemic effect of Bifidobacterium adolescentis B24728, which has the function of highly inhibiting α-glucosidase activity and high production of GLP-1. The main indicators were body weight, blood sugar, glycosylated hemoglobin, GLP-1 and other indicators of mice.

[0094] Results Figure 7 to Figure 12The experimental results showed that strain B24728 can improve weight loss caused by diabetes. At the same time, strain B24728 can reduce the fasting blood sugar level of mice to normal blood sugar level (5.99mmol / L), which is no different from the normal group. Compared with the model group, glycosylated hemoglobin decreased by 64.59%, insulin decreased by 21.23%, and islet resistance index decreased by 50.96%, which were significantly different; the oral glucose tolerance of mice was improved, and the intestinal GLP-1 secretion was promoted to achieve the effect of lowering blood sugar; in addition, the C-peptide (CP) in the serum of mice increased by 281.72%. Combined with the pancreatic H&E pathological sections (the islet cells in the normal group were arranged in an orderly manner, the islet cells were in elliptical clusters, with clear boundaries and regular morphology; the islet structure in the model group was obviously atrophied and deformed, with blurred boundaries, reduced area, and inflammatory cell infiltration; compared with the model group, the islet structure of B24728 was relatively complete, and the islet cell morphology atrophy was reduced), it shows that B24728 can reduce pancreatic damage, promote the proliferation of islet β cells, and maintain the function of islet β cells (red arrow).

[0095] Example 6

[0096] Preparation method of solid beverage containing Bifidobacterium adolescentis strain B24728

[0097] Probiotic solid beverage (3g) composition:

[0098] Blueberry flavored solid beverage: 1 g of freeze-dried powder of Bifidobacterium adolescentis strain B24728 prepared in Example 4, 1.0 g of galacto-oligosaccharide, 1.0 g of resistant dextrin, 1.0 g of Tremella extract, 1.0 g of Ganoderma lucidum extract, and 1.0 g of blueberry fruit powder.

[0099] Cherry-flavored solid beverage: 1 g of freeze-dried powder of Bifidobacterium adolescentis strain B24728 prepared in Example 4, 0.5 g of galacto-oligosaccharide, 0.5 g of resistant dextrin, 0.5 g of Tremella extract, 0.5 g of Ganoderma lucidum extract, and 0.5 g of cherry fruit powder.

[0100] The solid beverages are accurately weighed according to their composition, and the weighed components are fully mixed to obtain two fruit-flavored solid beverages.

[0101] 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 Bifidobacterium adolescentis strain B24728, characterized in that The deposit number is GDMCC No:65489.

2. A metabolite or culture of the Bifidobacterium adolescentis strain B24728 according to claim 1.

3. The method for preparing the metabolites of Bifidobacterium adolescentis strain B24728 according to claim 2, characterized in that: The following steps are involved: The Bifidobacterium adolescentis strain B24728 was inoculated into MPYG liquid medium for anaerobically culturing to obtain a culture; The liquid phase of the culture is separated to obtain metabolites of Bifidobacterium adolescentis strain B24728.

4. The preparation method according to claim 3, characterized in that: The separation of the liquid phase also includes concentration; the multiple of concentration is 5 to 10 times.

5. A probiotic, characterized in that: It comprises the Bifidobacterium adolescentis strain B24728 according to claim 1, the metabolite or culture according to claim 2, or the metabolite prepared by the preparation method according to claim 3 or 4.

6. An α-glucosidase inhibitor, characterized in that The active ingredients include the Bifidobacterium adolescentis strain B24728 according to claim 1, the metabolite or culture according to claim 2, and the metabolite prepared by the preparation method according to claim 3 or 4.

7. Use of the Bifidobacterium adolescentis strain B24728 according to claim 1, the metabolite or culture according to claim 2, the metabolite prepared by the preparation method according to claim 3 or 4, the probiotic according to claim 5 or the α-glucosidase inhibitor according to claim 6 in the preparation of a product for lowering blood sugar.

8. The use according to claim 7, characterized in that: The product includes at least one of the following: medicine, health care product and food.

9. Use of the Bifidobacterium adolescentis strain B24728 according to claim 1, the metabolite or culture according to claim 2, the metabolite prepared by the preparation method according to claim 3 or 4, the probiotic according to claim 5 or the α-glucosidase inhibitor according to claim 6 in the preparation of a medicament for preventing and / or treating diabetes.

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

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