Bifidobacterium breve JYBF-220 capable of maintaining healthy level of blood sugar as well as postbiotic preparation and application of bifidobacterium breve JYBF-220

Abstract

The invention relates to the technical field of probiotics, in particular to bifidobacterium breve JYBF-220 capable of maintaining the healthy level of blood sugar and a postbiotic preparation and application of the bifidobacterium breve JYBF-220. The invention relates to a strain of bifidobacterium breve JYBF-220 capable of maintaining the healthy level of blood sugar, the bifidobacterium breve JYBF-220 is preserved in China General Microbiological Culture Collection Center on July 24, 2023, the preservation address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO.27991. The invention further discloses a preparation method of the bifidobacterium breve JYBF-220. The invention further discloses a preparation method of the bifidobacterium breve JYBF-220. The metagen preparation prepared from the bifidobacterium breve JYBF-220 can prevent and improve hyperglycemia and is beneficial to maintaining blood sugar at a healthy level.

Description

Technical Field

[0001] The present invention relates to the technical field of probiotics, and particularly to Bifidobacterium breve JYBF-220 for maintaining a healthy blood glucose level, its postbiotics preparation and application. Background Art

[0002] Hyperglycemia refers to a pathological state in which the fasting blood glucose concentration is higher than the normal value or the blood glucose 2 hours after a meal is higher than the normal value. Long-term hyperglycemia can trigger oxidative stress, chronic inflammation and metabolic disorders, and then damage the functions of blood vessels, nerves and multiple organs, and is an important risk factor for metabolic syndrome, type 2 diabetes and cardiovascular diseases. In recent years, with the changes in diet structure and lifestyle, the number of people with hyperglycemia has been continuously increasing, and there is an urgent need for safe and effective regulatory means.

[0003] At present, the main intervention methods for hyperglycemia mainly include lifestyle management, oral hypoglycemic drugs and new injection therapies, but they all have certain deficiencies. Lifestyle intervention emphasizes a low-sugar, low-fat, high-fiber diet and regular exercise, but the compliance of patients is poor, it is difficult to adhere to for a long time, and the effect on moderate and severe hyperglycemia is limited. Oral hypoglycemic drugs (such as metformin, sulfonylureas, DPP-4 inhibitors, etc.) can effectively regulate blood glucose, but may cause side effects such as gastrointestinal discomfort, hypoglycemia or urinary tract infections. Long-term use of some drugs may also affect liver and kidney functions. The new GLP-1 receptor agonists (such as semaglutide) can take into account both blood glucose lowering and cardiovascular protection, but they are expensive and need to be administered by injection, and some patients will have adverse reactions such as nausea, diarrhea or urinary and reproductive tract infections. In addition, traditional Chinese medicines (such as berberine, Pueraria lobata extract) have a certain blood glucose lowering effect, but the onset is slow and the mechanism is not clear, and the clinical evidence still needs to be improved. Summary of the Invention

[0004] Aiming at the technical problems of poor compliance, obvious side effects and high treatment costs existing in the existing hyperglycemia intervention methods, the present invention provides Bifidobacterium breve JYBF-220 for maintaining a healthy blood glucose level, its postbiotics preparation and application.

[0005] In the first aspect, the present invention provides a strain of Bifidobacterium breve JYBF-220 for maintaining a healthy blood glucose level, Bifidobacterium breve ( Bifidobacterium breve ) JYBF-220 was deposited at the China General Microbiological Culture Collection Center on July 24, 2023. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO. 27991.

[0006] In a second aspect, the present invention provides a postbiotic preparation of the above-mentioned Bifidobacterium breve JYBF-220. The preparation method is to activate Bifidobacterium breve JYBF-220 on an MRS plate medium, pick the activated single colony and inoculate it into an MRS liquid medium, and statically culture it at a constant temperature of 37°C for 12 h under anaerobic conditions to obtain a seed solution; inoculate the seed solution into the MRS liquid medium at an inoculation amount of 1%, and statically culture it at a constant temperature of 37°C for 24 h under anaerobic conditions. The obtained bacterial solution is subjected to heat inactivation treatment, concentrated, and freeze-dried to obtain the postbiotic preparation of Bifidobacterium breve JYBF-220.

[0007] Further, the number of bacteria in the postbiotic preparation is 1.0×10 10 CFU / g.

[0008] Further, the postbiotic preparation further includes an excipient with a glycemic index of 0.

[0009] The excipient with a glycemic index of 0 includes erythritol.

[0010] In a third aspect, the present invention also provides an application of the above-mentioned postbiotic preparation in the preparation of a health food or drug that helps maintain a healthy blood glucose level. Helping to maintain a healthy blood glucose level includes preventing and improving hyperglycemia.

[0011] Further, preventing hyperglycemia includes maintaining fasting blood glucose at a healthy level.

[0012] Further, improving hyperglycemia includes improving the symptom of weight loss.

[0013] Further, improving hyperglycemia includes reducing the serum HbAlc concentration, increasing the GLP-1 concentration, or / and reducing the LPS concentration.

[0014] Further, improving hyperglycemia includes reducing the concentration of glycated serum protein.

[0015] Further, reducing hyperglycemia includes increasing the protein expression of insulin receptor, insulin receptor substrate 2, and / or peroxisome proliferator-activated receptor γ.

[0016] The beneficial effects of the present invention are as follows: The present invention provides a strain of Bifidobacterium breve JYBF-220. The postbiotics preparation prepared from this strain can maintain blood glucose at a healthy level. Experiments have shown that during the screening process for α-amylase activity inhibition, the OD630 value of the heat-inactivated bacterial solution of Bifidobacterium breve JYBF-220 is higher than that of the negative control and other samples to be tested. Administering the postbiotics preparation of Bifidobacterium breve JYBF-220 by gavage to hyperglycemic model mice before modeling can maintain the fasting blood glucose of the mice at a healthy level. After successful modeling of hyperglycemic model mice, the postbiotics preparation of Bifidobacterium breve JYBF-220 can improve the symptom of weight loss caused by hyperglycemia, and improve hyperglycemic symptoms by reducing the serum HbAlc concentration, increasing the GLP-1 concentration, reducing the LPS concentration, reducing the glycated serum protein concentration, and increasing the protein expression of insulin receptor (InsR), insulin receptor substrate 2 (IRS-2), and peroxisome proliferator-activated receptor γ (PPARγ), which helps to maintain blood glucose stability at a healthy level. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0018] Figure 1 It is the Western blot of the blank group, JYBF-220 group, and model group in Experimental Example 5. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0019] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0020] Example 1 Isolation, Screening, and Identification of Bacterial Strains 1. Strain Screening and Purification (1) Sampling: Pickled cowpeas, collected in Nantong City, Jiangsu Province in November 2021.

[0021] (2) Strain isolation: Take 1 g of pickled cowpeas and add it to a sterile triangular flask containing sterile normal saline (0.85%), shake to obtain a sample solution; dilute the sample solution with sterile normal saline to prepare dilution solutions with different concentration gradients, which are 10 -1 、10-2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 ; Then, use a spreader to spread the diluents with 7 different concentration gradients onto MRS plate medium respectively, and culture it at 37 °C under anaerobic conditions for 48 h; Among them, the formula and preparation method of MRS plate medium are as follows: Formula: 10 g of peptone, 5 g of beef powder, 5 g of sodium acetate trihydrate, 2 g of dipotassium hydrogen phosphate heptahydrate, 1 mL of Tween 80, 0.05 g of manganese sulfate tetrahydrate, 2 g of ammonium citrate, 20 g of glucose, 0.2 g of magnesium sulfate heptahydrate, 15 g of agar, 1000 mL of distilled water; After mixing the raw materials in the above formula, stir evenly at natural pH, sterilize at 121 °C and 0.1 MPa for 20 min, then it is ready; Under sterile conditions, pour the sterilized medium into a sterilized petri dish, and let it cool for later use.

[0022] (3) Select colonies: Select colonies according to the following colony characteristics: Colony characteristics: The colonies are round, with neat edges and milky white.

[0023] (4) Isolation and purification: Pick 5 single colonies with the above colony characteristics, inoculate them onto MRS plate medium by the streaking method, culture at 37 °C under anaerobic conditions for 48 h, then pick single colonies and store them at -80 °C in glycerol tubes.

[0024] 2. Screening of strains with inhibitory effect on α - amylase activity (1) Preparation of heat - inactivated bacterial liquid Activate 5 strains stored at -80 °C on MRS plate medium respectively. Pick 1 activated single colony and transfer it to 100 mL of MRS liquid medium, and culture it statically at 37 °C for 12 h to obtain a seed solution. Inoculate the seed solution into MRS liquid medium according to an inoculation amount of 1% (v / v), and then culture it statically at 37 °C under anaerobic conditions for 24 h to obtain a bacterial liquid with a viable count of 1.0×10 10 CFU / mL (containing bacteria and their metabolites). After heat - inactivating the bacterial liquid (115 °C, 30 min), 5 kinds of heat - inactivated bacterial liquids are obtained, which are named JYBF - 220, JYBF - 0215, JYBF - 3284, JYBF - 4215 and JYBF - 5213 respectively.

[0025] Among them, the formula and preparation method of MRS liquid medium are as follows: Formula: 10 g of peptone, 5 g of beef powder, 5 g of sodium acetate trihydrate, 2 g of dipotassium hydrogen phosphate heptahydrate, 1 mL of Tween 80, 0.05 g of manganese sulfate tetrahydrate, 2 g of ammonium citrate, 20 g of glucose, 0.2 g of magnesium sulfate heptahydrate, 1000 mL of distilled water; After mixing the raw materials in the above formula, stir evenly under natural pH, sterilize at 121 °C and 0.1 MPa for 20 min, and after cooling, dispense under sterile conditions for standby.

[0026] (2)α-Amylase activity inhibition test Preparation of reagents: 0.04% soluble starch: Weigh 0.04 g of soluble starch, add 100 mL of distilled water, stir and boil with a glass rod until the solution is transparent, and store at room temperature; 0.1 mg / mL α-amylase solution: Accurately weigh 0.05 g of α-amylase (porcine pancreas) and add it to a 50 mL volumetric flask, then make up the volume with phosphate buffer solution with a pH of 6.9, and then filter it with a 0.45 μm microporous filter membrane for standby. This solution should be prepared and used immediately; Iodine staining solution: Accurately weigh 11 g of crystalline iodine and 22 g of potassium iodide and mix them, add a small amount of distilled water to completely dissolve the iodine, make up the volume in a 500 mL volumetric flask and pour it into a brown bottle for storage at 4 °C as iodine stock solution. Then take 2 mL of iodine stock solution, add 20 g of potassium iodide, dissolve it with distilled water, and then make up the volume to 500 mL, pour it into a brown bottle to obtain iodine staining solution for standby.

[0027] Test procedure: The iodine-starch colorimetric method was used for strain screening. Add 100 μL of 0.04% soluble starch as a substrate to a 96-well plate, and then sequentially add 30 μL of heat-inactivated bacterial solution and 5 μL of 0.1 mg / mL α-amylase solution to initiate the reaction. Incubate the 96-well plate in a constant temperature incubator at 37 °C for 30 min, then add 25 μL of iodine staining solution, and measure the absorbance at 630 nm in an enzyme-linked immunosorbent assay reader.

[0028] The negative control was to replace the heat-inactivated bacterial solution with distilled water, and the others were the same as above. The positive control was to replace the heat-inactivated bacterial solution with acarbose (0.0166 g / mL), and the others were the same as above.

[0029] Test results: α-Amylase is a key enzyme involved in the process of carbohydrate metabolism in organisms, which can catalyze the cleavage of glycosidic bonds of carbohydrates and release glucose. Starch will be hydrolyzed into monosaccharide substances under the action of amylase and will not react with iodine to show color. However, the components that inhibit the activity of α-amylase can hinder the hydrolysis of amylase. Therefore, the OD630 value of the system that inhibits the activity of α-amylase will be higher than that of the negative control, that is, the samples with an OD630 value higher than that of the negative control contain components that inhibit the activity of α-amylase. The test results are shown in Table 1.

[0030] Table 1 Inhibition results of α-amylase activity

[0031] As can be seen from Table 1, the OD630 value of JYBF-220 is the highest, indicating that among these 5 kinds of heat-inactivated bacterial solutions, JYBF-220 has the best inhibitory effect on α-amylase activity.

[0032] 3. Identification and preservation The strain for preparing JYBF-220 was sent for identification. The identification unit: Sangon Biotech (Shanghai) Co., Ltd. During the identification process, the primers used were as follows: 27F (Sequence 1): AGAGTTTGATCMTGGCTCAG; 1492R (Sequence 2): GGTTACCTTGTTACGACTT.

[0033] The strain was identified as Bifidobacterium breve ( Bifidobacterium breve ), named Bifidobacterium breve JYBF-220.

[0034] The 16S rDNA gene sequence (Sequence 3) of this Bifidobacterium breve ( Bifidobacterium breve ) JYBF-220 is:

[0035] Bifidobacterium breve ( Bifidobacterium breve ) JYBF-220 was sent to the General Microbiology Center of the China Committee for Culture Collection of Microorganisms for preservation. The preservation date was July 24, 2023, and the preservation number was CGMCC NO. 27991.

[0036] Example 2 Preparation of postbiotics of Bifidobacterium breve JYBF-220 Bifidobacterium breve JYBF-220 preserved at -80 °C was activated on an MRS plate medium (the formula and preparation process are shown in Example 1). One activated single colony was picked and transferred into 100 mL of MRS liquid medium (the formula and preparation process are shown in Example 1). It was statically cultured at 37 °C for 12 h under anaerobic conditions to obtain a seed solution. The seed solution was inoculated into the MRS liquid medium at an inoculation amount of 1% (v / v), and then statically cultured at 37 °C for 24 h under anaerobic conditions to obtain a bacterial solution with a viable count of 1.0×10 10 CFU / mL (containing bacterial cells and their metabolites). The bacterial solution was heat-inactivated (115 °C, 30 min), concentrated, and freeze-dried to obtain the postbiotics of Bifidobacterium breve JYBF-220.

[0037] The number of bacterial cells in the postbiotics of Bifidobacterium breve JYBF-220 was 1.0×10 10 CFU / g.

[0038] Example 3 Preparation of postbiotics of Bifidobacterium breve JYBF-220 The postbiotics of Bifidobacterium breve JYBF-220 also included excipients. The postbiotics of Bifidobacterium breve JYBF-220 prepared in Example 2 were mixed with erythritol, and postbiotics of Bifidobacterium breve JYBF-220 with a bacterial cell number of 1.0×10 10 CFU / g could also be prepared.

[0039] Comparative Example 1 Bifidobacterium breve ATCC 15700 was activated on an MRS plate medium (the formula and preparation method are shown in Example 1). One activated single colony was picked and transferred into 100 mL of MRS liquid medium (the formula and preparation process are shown in Example 1). It was statically cultured at 37 °C for 12 h under anaerobic conditions to obtain a seed solution. The seed solution was inoculated into the MRS liquid medium at an inoculation amount of 1% (v / v), and then statically cultured at 37 °C for 24 h under anaerobic conditions to obtain a bacterial solution with a viable count of 1.0×10 10Bacterial suspension (containing bacteria and their metabolites) at CFU / mL. The bacterial suspension was heat-inactivated (115 °C, 30 min), concentrated, and freeze-dried to obtain a postbiotic preparation of Bifidobacterium breve ATCC 15700.

[0040] The number of bacteria in the postbiotic preparation of Bifidobacterium breve ATCC 15700 was 1.0×10 10 CFU / g.

[0041] Experimental Example 1: Preventive effect of the postbiotic preparation of Bifidobacterium breve JYBF-220 on blood glucose elevation in hyperglycemic mice 1. Construction of hyperglycemic model mice 100 SPF-grade Kunming mice purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., with a body weight of 20±1 g, were fed adaptively for one week. The feeding conditions were: sufficient food and water, constant temperature of 25 °C, 12 h light, and 12 h darkness.

[0042] After the mice had adapted to the environment for one week, they were fasted for 12 h without water, and then their blood glucose was measured. Mice with abnormal blood glucose were excluded. Among the remaining mice, 70 were randomly selected as experimental mice and randomly divided into 7 groups, namely the model group, the drug group, the blank group, the JYBF-220 prevention group, the JYBF-220 group, the ATCC 15700 prevention group, and the ATCC 15700 group, with 10 mice in each group.

[0043] Mice in the JYBF-220 prevention group were intragastrically administered the postbiotic preparation of Bifidobacterium breve JYBF-220 prepared in Example 2 dissolved in physiological saline (dosage: 5 million CFU / mouse per day) before modeling, with a concentration of: 5×10 7 CFU / mL; mice in the ATCC 15700 prevention group were intragastrically administered the postbiotic preparation of Bifidobacterium breve ATCC 15700 prepared in Comparative Example 1 dissolved in physiological saline (dosage: 5 million CFU / mouse per day) before modeling, with a concentration of: 5×10 7 CFU / mL.

[0044] Mice in the blank group, the JYBF-220 group, the ATCC 15700 group, the drug group, and the model group were intragastrically administered the same amount of physiological saline, and the other feeding conditions of each group were the same. They were continuously fed for 14 days.

[0045] After the mice in each group were placed in the animal house for 3 days of adaptive feeding, the mice in the blank group were fed with standard feed, and the remaining 6 groups of mice were fed with high-fat feed. The energy supply ratios of the two feeds are shown in Table 2. In the 8th week of high-fat feed feeding, streptozotocin (STZ) at 40 mg / kg·bw was dissolved in sodium citrate buffer, and the mice in the model group, drug group, JYBF-220 group, JYBF-220 prevention group, ATCC 15700 prevention group and ATCC 15700 group were intraperitoneally injected continuously for 5 days. At the same time, the mice in the blank group were used as a control and only injected with sodium citrate buffer. During the experiment, their general conditions and external manifestations were observed.

[0046] Table 2 Feed energy supply table

[0047] 2. Evaluate whether the mouse model is successfully established (1)Observe external manifestations After modeling, the hair colors of the mice in the JYBF-220 group, drug group, model group, ATCC 15700 prevention group and ATCC 15700 group became darker and dull, the phenomena of polydipsia and hunger were extremely obvious, and the mental state was unstable.

[0048] (2)Detection of successful hyperglycemia mouse model The normal range of fasting blood glucose in healthy adults (fasting blood glucose refers to the blood glucose measured after at least 8 hours of fasting) is 3.9 - 6.1 mmol / L. The basal metabolic rate of mice is high, and the fasting blood glucose baseline is higher than that of humans (the fasting blood glucose of normal mice is about 5 - 8 mmol / L). Therefore, the threshold is usually increased during the study to ensure the stability of the model. In this experiment, the judgment standard for the hyperglycemia mouse model is fasting blood glucose ≥ 11.1 mmol / L after 72 h.

[0049] After 72 h, blood was collected from the tail vein to measure the fasting blood glucose (FBG) of the mice in each group, and the results are shown in Table 3.

[0050] All the obtained experimental data were expressed as mean ± standard deviation. The data were analyzed using SPSS 26.0 statistical software. When the comparison between multiple groups met the normal distribution and homogeneity of variance, one-way analysis of variance (One-way ANOVA) was used for analysis. P < 0.05 was considered that the result had statistical significance.

[0051] Table 3 Fasting blood glucose results

[0052] Note: # indicates comparison with the blank group, P < 0.05.

[0053] As shown in Table 3, the fasting blood glucose of mice in the blank group and the JYBF-220 prevention group was < 11.1 mmol / L, while that in the JYBF-220 group, model group, drug group was > 11.1 mmol / L. Compared with the blank group, the fasting blood glucose of mice in the JYBF-220 group, model group, drug group, ATCC 15700 prevention group and ATCC 15700 group was significantly increased (P < 0.05), indicating that the model of hyperglycemic mice was successfully established. There was no significant difference in the fasting blood glucose between the blank group and the JYBF-220 prevention group, indicating that the postbiotics preparation of Bifidobacterium breve JYBF-220 could prevent blood glucose from rising and maintain the blood glucose fluctuation at a healthy level (no significant difference compared with the blank group), while the postbiotics preparation of Bifidobacterium breve ATCC 15700 had no effect on preventing blood glucose from rising.

[0054] Experimental Example 2 Effect of the postbiotics preparation of Bifidobacterium breve JYBF-220 on the body weight of hyperglycemic mice The mice in the JYBF-220 group with successful modeling in Experimental Example 1 were intragastrically administered with the postbiotics preparation of Bifidobacterium breve JYBF-220 prepared in Example 2 dissolved in normal saline (the addition amount was 5 million CFU / mouse per day), the mice in the ATCC 15700 group were intragastrically administered with the postbiotics preparation of Bifidobacterium breve ATCC 15700 prepared in Comparative Example 1 dissolved in normal saline (the addition amount was 5 million CFU / mouse per day), the mice in the drug group were intragastrically administered with metformin hydrochloride (400 mg / kg / d) dissolved in normal saline, and the mice in the blank group and the model group were intragastrically administered with the same amount of normal saline. They were continuously intragastrically administered for 35 days, and during this period, each group was fed with standard feed. The body weight of mice in each group was detected at a fixed time every week, and the results are shown in Table 4.

[0055] All the obtained experimental data were expressed as mean ± standard deviation, and SPSS 26.0 statistical software was used to analyze the data. When the comparison among multiple groups met the normal distribution and homogeneity of variance, one-way ANOVA was used for analysis, and P < 0.05 was considered that the result had statistical difference.

[0056] Table 4 Body weight results of mice in each group

[0057] Note: # indicates P < 0.05 compared with the blank group; * indicates P < 0.05 compared with the model group.

[0058] As shown in Table 4, within 5 weeks after successful modeling, there was always a significant difference in the body weight between the blank group and the model group (P < 0.05).

[0059] Within the two weeks (2w) after successful model establishment, compared with the model group, there were no significant differences in the body weights of the mice in the JYBF-220 group, the drug group, and the ATCC15700 group (P≥0.05). As the number of days of intragastric administration of the postbiotics preparation of Bifidobacterium breve JYBF-220 in the JYBF-220 group and metformin hydrochloride in the drug group increased, the body weight gain of the mice in the JYBF-220 group and the drug group was significantly greater than that of the model group (P<0.05), indicating that after successful model establishment, the postbiotics preparation of Bifidobacterium breve JYBF-220 had a certain effect on increasing the body weight of hyperglycemic mice, and the effect was comparable to that of metformin hydrochloride. However, the postbiotics preparation of Bifidobacterium breve ATCC 15700 had no significant effect on increasing the body weight of hyperglycemic mice.

[0060] Example 3 Effects of the postbiotics preparation of Bifidobacterium breve JYBF-220 on serum HbAlc, GLP-1, and LPS in hyperglycemic mice Glycated hemoglobin (HbA1c) is the product of the non-enzymatic reaction between glucose and hemoglobin in red blood cells. The content of HbA1c is positively correlated with the average blood glucose level of the body and can reflect the blood glucose situation of the body; Glucagon-like peptide 1 (GLP-1) is an incretin hormone secreted by intestinal L cells. It stimulates pancreatic β cells to secrete insulin in a glucose-dependent manner, enabling glucose absorbed through the intestinal epithelium to be absorbed into peripheral tissues through the action of insulin. GLP-1 plays an important role in maintaining postprandial glucose homeostasis, promoting the regeneration and differentiation of pancreatic β cells, promoting satiety, delaying gastric emptying, and protecting the heart and nerves; Lipopolysaccharide (LPS), also known as endotoxin, is an important component of the cell wall of Gram-negative bacteria. Under normal conditions in the body, LPS usually only exists in the intestinal lumen. Tight junctions between intestinal epithelial cells form an intestinal barrier to prevent the transfer of intestinal bacteria and harmful components into the blood. However, for hyperglycemic individuals or those with a tendency to insulin resistance, their intestinal barrier is more likely to be damaged due to factors such as metabolic disorders, inflammatory states, or high-sugar and high-fat diets, resulting in increased intestinal permeability. At this time, LPS will break through the barrier and enter the blood circulation. Therefore, an increase in the LPS concentration in the blood not only reflects increased intestinal permeability but also reflects the situation of elevated blood glucose.

[0061] 1. Preparation of serum samples Thirty-five days later (after the experiment in Experimental Example 2 ended), the mice in each group were fasted for 12 h. After anesthetizing the mice by intraperitoneal injection of 0.4% pentobarbital at a dose of 1 mL / g, the mice were placed on an ice tray in the supine position, and the head and limbs were fixed with transparent medical tape. After removing the eyeballs of the mice with forceps, the mice were sacrificed by cervical dislocation, and about 1 mL of peripheral blood was collected with an EP tube. The blood was allowed to stand at room temperature for 20 - 30 min until coagulation, and then centrifuged at low temperature for 10 min (4 °C, 3000 rpm). The supernatant was aspirated with a pipette, and the supernatant was divided into two portions. One portion was stored frozen at -20 °C in a refrigerator, and the other portion was stored frozen at -80 °C in a refrigerator for later use. At the same time, the abdominal cavities of the mice in each group were quickly opened, the intact livers were taken out, rinsed in pre-cooled physiological saline to remove blood and connective tissue, the tissues were cut into small pieces of 5 - 10 mg, placed in a cryotube, and then quickly frozen in liquid nitrogen and stored at -80 °C in a refrigerator after 15 min for the preparation of subsequent protein detection.

[0062] 2. Detection and methods of serum HbAlc, GLP-1, and LPS The ELISA kits purchased from Shanghai Keaibo Biotechnology Co., Ltd. were used to detect the concentrations of HbAlc, GLP-1, and LPS in the sera of the mice in each group according to the operation methods described in the kit instructions. The results are shown in Table 5.

[0063] All the obtained experimental data were expressed as mean ± standard deviation, and the data were analyzed using SPSS 26.0 statistical software. When the comparison among multiple groups satisfied the normal distribution and homogeneity of variance, one-way analysis of variance (One-way ANOVA) was used for analysis. A P value < 0.05 was considered to indicate a statistically significant result.

[0064] Table 5 Detection results of serum HbAlc, GLP-1, and LPS

[0065] Note: # indicates P < 0.05 compared with the blank group; * indicates P < 0.05 compared with the model group.

[0066] As shown in Table 5, there were significant differences in serum HbAlc, GLP-1, and LPS between the blank group and the model group of mice (P < 0.05). Compared with the model group, the HbAlc concentration in the JYBF-220 group of mice decreased significantly, the GLP-1 concentration increased significantly, and the LPS concentration decreased significantly (P < 0.05); and there were no significant differences in the HbAlc concentration, GLP-1 concentration, and LPS concentration compared with the blank group (P ≥ 0.05), which was equivalent to the effect of the drug group, indicating that the postbiotic preparation of Bifidobacterium breve JYBF-220 has the effect of regulating the concentrations of HbAlc, GLP-1, and LPS in the serum of hyperglycemic mice and can stabilize blood glucose at a healthy level. Compared with the model group, the HbAlc concentration in the ATCC 15700 group of mice did not decrease significantly, and there were still significant differences in the HbAlc concentration, GLP-1 concentration, and LPS concentration compared with the blank group (P < 0.05).

[0067] Example 4 Effect of the postbiotic preparation of Bifidobacterium breve JYBF-220 on the content of glycated serum protein in hyperglycemic mice Serum glucose can undergo a non-enzymatic glycation reaction with the amino groups at the N-terminus of serum albumin and other protein molecules to produce high-molecular-weight ketoamines. These ketoamine compounds are collectively called glycated serum proteins. Among them, serum albumin accounts for the largest proportion, so glycated serum protein is often used as an index to reflect the degree of glycation of serum albumin. In an alkaline environment, the ketoamine structure of glycated serum protein can react with nitroblue tetrazolium NBT to produce formazan. After color development, fructosamine DMF is used as a standard color reference for colorimetry. By measuring the absorbance, the content of glycated serum protein can be calculated. Since the half-life of serum protein is about 2-3 weeks, the level of glycated serum protein can reflect the average blood glucose level of patients in the past 2-3 weeks, making up for the time interval that fasting blood glucose only reflects instantaneous blood glucose and glycated hemoglobin reflects the long-term blood glucose level of 2-3 months. It is of great significance in blood glucose monitoring, especially suitable for the efficacy evaluation and treatment plan adjustment of patients with large blood glucose fluctuations.

[0068] 1. Detection steps Take out the sera of each group of mice frozen in the -20°C refrigerator in Experimental Example 3 and thaw them completely in a -80°C ultra-low temperature refrigerator, and directly measure the stock solution. Take a 96-well operation plate, add 10 μL of double-distilled water to the blank wells (3 wells), add 10 μL of 2 mmol / L DMF standard solution to the standard wells (3 wells), add bovine serum albumin to the standard blank wells (3 wells), and sequentially add 10 μL of the sera of each group of mice to the sample wells, and label them in order. Preheat the NBT color reagent at 37°C, and then sequentially add 200 μL of the NBT color reagent to each well (including blank wells, standard wells, sample wells, and standard blank wells). Gently shake well, incubate in a water bath at 37°C for 15 min, take it back, and sequentially add 10 μL of the stabilizer (if the stabilizer solidifies, it can be heated in a water bath until transparent before use) to each well. Gently shake well, and measure the absorbance value of each well at a wavelength of 546 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The results are shown in Table 6.

[0069] Precautions: During the determination of the above biochemical indexes, the sera of mice should be placed in an ice bath at 4°C to avoid hemolysis caused by repeated freezing and thawing.

[0070] Calculation formula:

[0071] Among them, the concentration of the calibrator is 2 mmol / L.

[0072] All the obtained experimental data are expressed as mean ± standard deviation. The data are analyzed using SPSS 26.0 statistical software. When the comparison among multiple groups meets the normal distribution and homogeneity of variance, one-way analysis of variance (One-way ANOVA) is used for analysis. P < 0.05 is considered to have a statistically significant result.

[0073] Table 6 Results of glycated serum protein concentration

[0074] Note: # indicates comparison with the blank group, P < 0.05; * indicates comparison with the model group, P < 0.05.

[0075] As shown in Table 6, the concentration of glycated serum protein in the model group of mice was significantly different from that in the blank group (P < 0.05). The concentration of glycated serum protein in the JYBF-220 group and the drug group of mice was significantly different from that in the model group (P < 0.05), and not significantly different from that in the blank group (P ≥ 0.05). This indicates that the postbiotics preparation of Bifidobacterium breve JYBF-220 is effective in reducing the concentration of glycated serum protein in hyperglycemic mice, can maintain blood glucose at a healthy level, and the postbiotics preparation of Bifidobacterium breve JYBF-220 has an equivalent effect on the concentration of glycated serum protein in hyperglycemic mice as the drug group. Compared with the model group, the concentration of glycated serum protein in the ATCC 15700 group of mice was not significantly different (P ≥ 0.05), indicating that the postbiotics preparation of Bifidobacterium breve ATCC 15700 has no obvious effect on reducing the concentration of glycated serum protein in hyperglycemic mice.

[0076] Example 5 Effect of Postbiotics Preparation of Bifidobacterium breve JYBF-220 on the Protein Expression of InsR, IRS-2, and PPARγ in the Liver of Hyperglycemic Mice Insulin receptor (InsR) belongs to transmembrane protein, usually expressed on the cell membrane, and may be involved in glucose metabolism in the liver. Insulin receptor substrate 2 (IRS-2) is also a key protein in signal transduction, mainly in the cytoplasm, and is related to glucose metabolism. Peroxisome proliferator-activated receptor γ (PPARγ) belongs to nuclear receptor and may play a role in adipocyte differentiation and glucose and lipid metabolism.

[0077] The liver tissues stored in the -80 °C refrigerator in Example 4 were used to detect the protein contents of InsR, IRS-2, and PPARγ in the tissues by Western Blot radioimmunoassay. The specific steps are as follows: (1)Sample preparation: Complete lysis. After lysis, centrifuge to obtain the supernatant, perform protein quantification, and then store it frozen; (2)Protein quantification; (3)Preparation of the lower separating gel and preparation of the upper stacking gel; (4)Loading and electrophoresis; (5)Transferring the membrane; (6)Detection of proteins on the membrane: Wash the membrane once with TBST, then place it in the Ponceau S staining working solution, shake and stain at room temperature for 5 minutes, wash the membrane with water until the water becomes clear and colorless and the protein bands are clear. The PVDF membrane needs to be reactivated with methanol (activated before membrane transfer), then washed with TBST and blocked; (7)Blocking of the membrane and antibody incubation, Blocking: Block with 5% non-fat milk powder at room temperature for 1 hour or overnight at 4°C; Primary antibody: Dilute the antibody, add the antibody to the blocking solution and dilute it to the required concentration, and incubate with the membrane at room temperature for 2 hours or incubate overnight at 4°C; Secondary antibody: Wash the membrane incubated with the primary antibody with TBST, then dilute the HRP-labeled secondary antibody at a ratio of 1:1000 according to the amount used, and incubate with the membrane at 37°C for 1 h; Wash with TBST; (8)Color development: ECL chemiluminescence detection. Place it in an imaging system for scanning; (9)Compare the gray values with Image J software. The ratio of the gray value of the band to the gray value of the internal reference α-tubulin is used as the basis for relative quantitative analysis. The results are shown in Figure 1 , Table 7.

[0078] The database was processed using Microsoft excel 2021, and then statistical processing was performed using SPSS 26.0. The experimental result data were expressed as mean ± standard deviation. One-way analysis of variance (one-way ANOVA) was used for comparison between groups, and the least significant difference method (LSD) was used for pairwise multiple comparisons between groups. Statistical significance was considered when P < 0.05.

[0079] Table 7 Results of protein expression of InsR, IRS-2, and PPARγ in the liver

[0080] Note: # indicates comparison with the blank group, P < 0.05; * indicates comparison with the model group, P < 0.05.

[0081] As shown in Table 7, there were significant differences in the protein expressions of InsR, IRS-2, and PPARγ in the livers of mice in the blank group and the model group (P < 0.05). The relative protein expressions of InsR, IRS-2, and PPARγ in the livers of mice in the JYBF-220 group were significantly increased compared with those in the model group (P < 0.05); there were no significant differences compared with the blank group (P ≥ 0.05), indicating that the postbiotics preparation of Bifidobacterium breve JYBF-220 was effective in increasing the protein expressions of InsR, IRS-2, and PPARγ in the livers of hyperglycemic mice, could maintain blood glucose at a healthy level, and the effect of the postbiotics preparation of Bifidobacterium breve JYBF-220 was equivalent to that of metformin hydrochloride. Compared with the model group, the relative protein expressions of InsR, IRS-2, and PPARγ in the livers of mice in the ATCC 15700 group were not significantly increased (P ≥ 0.05), indicating that the postbiotics preparation of Bifidobacterium breve ATCC 15700 had no obvious effect on increasing the protein expressions of InsR, IRS-2, and PPARγ in the livers of hyperglycemic mice.

[0082] It can be seen from Figure 1 that the relative protein expressions of InsR, IRS-2, and PPARγ in the livers of mice in the JYBF-220 group were not much different from those in the blank group, which also indicated that the postbiotics preparation of Bifidobacterium breve JYBF-220 could increase the protein expressions of InsR, IRS-2, and PPARγ in the livers of hyperglycemic mice and maintain blood glucose stability at a healthy level.

[0083] Although the present invention has been described in detail by referring to the accompanying drawings and in combination with the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, those of ordinary skill in the art can make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions should all be within the scope of the present invention. / Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered by the protection scope of the present invention.

Claims

1. Bifidobacterium breve JYBF-220 for maintaining healthy blood glucose levels, characterized in that, Bifidobacterium breve ( Bifidobacterium breve ) JYBF-220 was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on July 24, 2023. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO. 27991.

2. A postbiotic preparation of Bifidobacterium breve JYBF-220 as described in claim 1, characterized in that, The preparation method is to activate Bifidobacterium breve JYBF-220 on MRS plate medium, pick a single colony after activation and inoculate it into MRS liquid medium, and statically culture it at a constant temperature of 37°C for 12 h under anaerobic conditions to obtain a seed solution; Inoculate the seed solution into MRS liquid medium according to an inoculation amount of 1%, and statically culture it at a constant temperature of 37°C for 24 h under anaerobic conditions. The obtained bacterial liquid is subjected to heat inactivation treatment, concentrated and freeze-dried to obtain a postbiotic preparation of Bifidobacterium breve JYBF-220.

3. The postbiotic preparation according to claim 2, characterized in that, The number of bacteria in the postbiotic preparation is 1.0×10 10 CFU / g.

4. The postbiotic preparation according to claim 2, characterized in that, The postbiotic preparation also includes an excipient with a glycemic index of 0.

5. Use of a postbiotic preparation according to any one of claims 2-4 in the preparation of a health food or drug that helps maintain a healthy blood glucose level, characterized in that, Helping to maintain a healthy blood glucose level includes preventing and improving hyperglycemia.

6. The application according to claim 5, wherein Preventing hyperglycemia includes maintaining fasting blood glucose at a healthy level.

7. The application according to claim 5, characterized in that, Improving hyperglycemia includes improving symptoms of weight loss.

8. The application according to claim 5, characterized in that, Improving hyperglycemia includes reducing the serum HbAlc concentration, increasing the GLP-1 concentration or / and reducing the LPS concentration.

9. The application according to claim 5, wherein Improving hyperglycemia includes reducing the concentration of glycated serum protein.

10. The application according to claim 5, characterized in that, Improving hyperglycemia includes increasing the protein expression of insulin receptor, insulin receptor substrate 2 and / or peroxisome proliferator-activated receptor γ.

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