Bifidobacterium breve JYBF-220 and its postbiotic preparations and applications for maintaining healthy blood glucose levels

Abstract

This invention relates to the field of probiotics technology, specifically to *Bifidobacterium breve* JYBF-220, which maintains healthy blood sugar levels, and its subsequent biogenic preparations and applications. A strain of *Bifidobacterium breve* JYBF-220 that maintains healthy blood sugar levels, *Bifidobacterium breve* (… Bifidobacterium breve JYBF-220 was deposited on July 24, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO. 27991. Metabiotic preparations made from Bifidobacterium breve JYBF-220 can prevent and improve hyperglycemia, helping to maintain blood sugar levels at healthy levels.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of probiotics, in particular to a Bifidobacterium breve JYBF-220 for maintaining a healthy level of blood glucose, a postbiotic preparation thereof and an application thereof. BACKGROUND

[0002] Hyperglycemia refers to a pathological state in which the fasting blood glucose concentration is higher than the normal value or the 2-hour postprandial blood glucose is higher than the normal value. Long-term hyperglycemia can cause oxidative stress, chronic inflammation and metabolic disorders, thereby damaging blood vessels, nerves and the functions of multiple organs, and is an important risk factor for metabolic syndrome, type 2 diabetes and cardiovascular diseases. In recent years, with changes in dietary structure and lifestyle, the population of hyperglycemia continues to grow, and safe and effective control methods are urgently needed.

[0003] Current interventions for hyperglycemia mainly include lifestyle management, oral hypoglycemic drugs and new injection therapies, but all have certain deficiencies. Lifestyle interventions emphasize low-sugar, low-fat, high-fiber diets and regular exercise, but patient compliance is poor, it is difficult to adhere to for a long time, and the effect on moderate to severe hyperglycemia is limited. Oral hypoglycemic drugs (such as metformin, sulfonylureas, DPP-4 inhibitors, etc.) can effectively control blood glucose, but may cause gastrointestinal discomfort, hypoglycemia or urinary tract infections, and long-term use of some drugs may affect liver and kidney function. New GLP-1 receptor agonists (such as semaglutide) can balance hypoglycemic and cardiovascular protection, but are expensive and require injection, and some patients may experience nausea, diarrhea or urogenital tract infections. In addition, traditional Chinese medicine (such as berberine, pueraria extract) has certain hypoglycemic effect, but the onset is slow and the mechanism is not clear, and the clinical evidence still needs to be improved. SUMMARY

[0004] In view of the technical problems of poor compliance, obvious side effects and high treatment cost of existing hyperglycemia intervention methods, the present application provides a Bifidobacterium breve JYBF-220 for maintaining a healthy level of blood glucose, a postbiotic preparation thereof and an application thereof.

[0005] In a first aspect, the present application provides a Bifidobacterium breve JYBF-220 for maintaining a healthy level of blood glucose, wherein the Bifidobacterium breve JYBF-220 has the following characteristics: Bifidobacterium breve The Bifidobacterium breve JYBF-220 has been deposited with the China General Microbiological Culture Collection Center on July 24, 2023, at the address of No. 1, Beichen West Road, Haidian District, Beijing, with the accession number of CGMCC NO. 27991.

[0006] In a second aspect, the present application provides a probiotic preparation of the above-mentioned Bifidobacterium breve JYBF-220, and the preparation method is as follows: the Bifidobacterium breve JYBF-220 is activated on a MRS plate medium, and a single colony after activation is inoculated into a MRS liquid medium, and then the seed liquid is obtained by incubation at 37℃ under anaerobic conditions for 12 hours; the seed liquid is inoculated into the MRS liquid medium at an inoculation amount of 1%, and then the bacterial liquid obtained after incubation at 37℃ under anaerobic conditions for 24 hours is subjected to heat inactivation treatment at 115℃ for 30 minutes, and then the Bifidobacterium breve JYBF-220 probiotic preparation is obtained by concentration and freeze-drying.

[0007] Further, the number of bacterial bodies in the probiotic preparation is 1.0×10 10 CFU / g.

[0008] Further, the probiotic preparation further comprises a blood sugar raising index 0 auxiliary material.

[0009] The blood sugar raising index 0 auxiliary material comprises erythritol.

[0010] In a third aspect, the present application further provides an application of the above-mentioned probiotic preparation in the preparation of health care products for helping to maintain a healthy level of blood sugar.

[0011] The present application has the following beneficial effects:

[0012] The present application provides a Bifidobacterium breve JYBF-220, and the Bifidobacterium breve JYBF-220 probiotic preparation prepared from the bacterial strain can maintain blood sugar at a healthy level. Experimental results show that, in the screening process of α-amylase activity inhibition, the OD630 value of the heat-inactivated bacterial liquid of the Bifidobacterium breve JYBF-220 is higher than that of the negative control and other samples to be tested. The probiotic preparation of the Bifidobacterium breve JYBF-220 can maintain the fasting blood sugar of mice at a healthy level before the high blood sugar model mice are modeled. BRIEF DESCRIPTION OF DRAWINGS

[0013] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description, and obviously, other drawings can also be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0014] Figure 1 is the Western blotting map of the blank group, the JYBF-220 group and the model group in experimental example 5. DETAILED DESCRIPTION

[0015] In order for those skilled in the art to better understand the technical solutions in the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor should fall within the scope of protection of the present application.

[0016] Example 1 Isolation, screening and identification of strains

[0017] 1. Strain screening and purification

[0018] (1) Sampling: pickled jar beans were collected in Nantong, Jiangsu Province in November 2021.

[0019] (2) Strain isolation: 1 g of pickled jar beans was added to a sterile triangular flask containing sterilized physiological saline (0.85%), shaken to obtain a sample solution; the sample solution was diluted with sterilized physiological saline to prepare dilutions of different concentration gradients, 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 ; then a spreader was used to spread the seven dilutions of different concentration gradients on MRS plate culture medium, which was incubated at 37°C under anaerobic conditions for 48h;

[0020] The formula and preparation method of the MRS plate culture medium are as follows:

[0021] Formula: Proteose peptone 10 g, beef powder 5 g, sodium acetate trihydrate 5 g, potassium phosphate dibasic heptahydrate 2 g, Tween 80 1 mL, manganese sulfate tetrahydrate 0.05 g, triammonium citrate 2 g, glucose 20 g, magnesium sulfate heptahydrate 0.2 g, agar 15 g, distilled water 1000 mL; after mixing the raw materials in the above formula, stirring uniformly at natural pH, sterilization at 121°C, 0.1 MPa for 20 min; under sterile conditions, pour the sterilized medium into a sterile plate, and cool it down before use.

[0022] (3) Selection of colonies: select colonies according to the following colony characteristics:

[0023] Colony characteristics: round, neat edge, milky white.

[0024] (4) Isolation and purification: pick 5 single colonies with the above colony characteristics, inoculate on MRS plate culture medium by streaking method, incubate at 37°C under anaerobic conditions for 48h, then pick single colonies and store them in glycerol tubes at -80°C.

[0025] 2. Screening of strains with inhibitory effect on α-amylase activity

[0026] (1) Preparation of heat-inactivated bacterial solution

[0027] Five strains preserved at -80℃ were activated on MRS plate medium, and one single colony after activation was inoculated into 100 mL of MRS liquid medium at 37℃ for 12 h to prepare a seed solution. The seed solution was inoculated into MRS liquid medium at a 1% (v / v) inoculation amount, and then incubated at 37℃ for 24 h under anaerobic conditions to obtain a bacterial solution with a viable cell count of 1.0 x 10 10 CFU / mL (containing bacterial cells and metabolites). After heat inactivation (115℃, 30 min), five heat-inactivated bacterial solutions were obtained, designated as JYBF-220, JYBF-0215, JYBF-3284, JYBF-4215 and JYBF-5213.

[0028] The formula and preparation method of the MRS liquid medium are as follows:

[0029] Formula: Peptone 10 g, beef powder 5 g, sodium acetate trihydrate 5 g, potassium phosphate dibasic heptahydrate 2 g, Tween 80 1 mL, manganese sulfate tetrahydrate 0.05 g, triammonium citrate 2 g, glucose 20 g, magnesium sulfate heptahydrate 0.2 g, distilled water 1000 mL. After mixing the above ingredients, stirring was performed at natural pH, and sterilization was performed at 121℃, 0.1 MPa for 20 min. After cooling, sterile packaging was performed under sterile conditions for standby use.

[0030] (2) α-amylase activity inhibition test

[0031] Preparation of reagents:

[0032] 0.04% soluble starch: 0.04 g of soluble starch was weighed and added to 100 mL of distilled water. The solution was stirred and boiled with a glass rod until it became transparent. It was stored at room temperature.

[0033] 0.1 mg / mL α-amylase solution: 0.05 g of α-amylase (porcine pancreas) was accurately weighed into a 50 mL volumetric flask, which was then made up to volume with phosphate buffer at pH 6.9. The solution was filtered with a 0.45 μm microporous filter and used immediately after preparation.

[0034] Iodine staining solution: accurately weigh 11 g of crystalline iodine and 22 g of potassium iodide, add a small amount of distilled water to completely dissolve the iodine, then dilute to 500 mL in a volumetric flask, pour into a brown bottle as iodine storage solution, store at 4°C, then take 2 mL of iodine storage solution, add 20 g of potassium iodide, dissolve with distilled water, then dilute to 500 mL, pour into a brown bottle to obtain iodine staining solution, ready for use.

[0035] Test process:

[0036] Iodine and starch color development method was used for strain screening. 100 μL of 0.04% soluble starch was added as substrate in a 96-well plate, then 30 μL of heat-inactivated bacteria solution and 5 μL of 0.1 mg / mL α-amylase solution were added in turn to start the reaction. The 96-well plate was incubated in a constant temperature incubator at 37°C for 30 min, then 25 μL of iodine staining solution was added, and the absorbance value at 630 nm was measured in the enzyme marker.

[0037] The negative control was prepared by replacing the heat-inactivated bacteria solution with distilled water, and the other steps were the same as above. The positive control was prepared by replacing the heat-inactivated bacteria solution with acarbose (0.0166 g / mL), and the other steps were the same as above.

[0038] Test results:

[0039] α-amylase is a key enzyme involved in the process of carbohydrate metabolism in living organisms, which can catalyze the cleavage of carbohydrate glycosidic bond to release glucose. Starch will be hydrolyzed into monosaccharide substances under the action of amylase, which cannot react with iodine to develop color. Components that inhibit the activity of α-amylase can hinder the hydrolysis of amylase, so the OD630 value of the system that inhibits the activity of α-amylase will be higher than that of the negative control. The sample with OD630 value higher than that of the negative control contains components that inhibit the activity of α-amylase. The test results are shown in Table 1.

[0040] Table 1 α-amylase activity inhibition results

[0041]

[0042] As shown in Table 1, the OD630 value of JYBF-220 is the highest, indicating that among the five heat-inactivated bacteria solutions, JYBF-220 has the best α-amylase activity inhibition.

[0043] 3. Identification and preservation

[0044] The strain used to prepare JYBF-220 was sent for identification to Geneseeq Biotech (Shanghai) Co., Ltd. The primers used during the identification process are as follows:

[0045] 27F (Sequence 1): AGAGTTTGATCMTGGCTCAG;

[0046] 1492R (SEQ ID NO: 2): GGTTACCTTGTTACGACTT.

[0047] The strain was identified as Bifidobacterium breve (Bifidobacterium breve) Bifidobacterium breve ), and named Bifidobacterium breve JYBF-220.

[0048] The 16S rDNA gene sequence (SEQ ID NO: 3) of the Bifidobacterium breve (Bifidobacterium breve) Bifidobacterium breve ) JYBF-220 is:

[0049]

[0050] Bifidobacterium breve (ATCC 15700) was sent to China General Microbiological Culture Collection Center for preservation, and the preservation date was July 24, 2023, and the preservation number was CGMCC NO. 27992. Bifidobacterium breve

[0051] Example 2 Preparation of the postbiotic preparation of Bifidobacterium breve JYBF-220

[0052] Bifidobacterium breve JYBF-220 preserved at -80℃ was activated on MRS plate medium (formulation and preparation process see Example 1), and 1 single colony after activation was transferred to 100 mL of MRS liquid medium (formulation and preparation process see Example 1), and was cultured at 37℃ under anaerobic conditions for 12h to prepare a seed liquid. The seed liquid was inoculated in MRS liquid medium at an inoculation amount of 1% (v / v), and then was cultured at 37℃ under anaerobic conditions for 24h to obtain a bacterial liquid with a viable cell count of 1.0×10 10 CFU / mL (containing bacterial cells and metabolites). The bacterial liquid was heat-inactivated (115℃, 30min), concentrated, and freeze-dried to obtain the postbiotic preparation of Bifidobacterium breve JYBF-220.

[0053] The number of bacterial cells in the postbiotic preparation of Bifidobacterium breve JYBF-220 was 1.0×10 10 CFU / g.

[0054] Example 3 Preparation of the postbiotic preparation of Bifidobacterium breve JYBF-220

[0055] The postbiotic preparation of Bifidobacterium breve JYBF-220 also includes excipients. The postbiotic preparation of Bifidobacterium breve JYBF-220 prepared in Example 2 was mixed with erythritol, and a postbiotic preparation of Bifidobacterium breve JYBF-220 with a bacterial cell count of 1.0×10 10 CFU / g was also prepared.

[0056] Comparative Example 1

[0057] Bifidobacterium breve ATCC 15700 was activated on MRS plate medium (formulation and preparation method see Example 1), and 1 single colony after activation was transferred to 100 mL of MRS liquid medium (formulation and preparation process see Example 1), and was cultured at 37℃ under anaerobic conditions for 12h to prepare a seed liquid. The seed liquid was inoculated in MRS liquid medium at an inoculation amount of 1% (v / v), and then was cultured at 37℃ under anaerobic conditions for 24h to obtain a bacterial liquid with a viable cell count of 1.0×10 10 ​Bacterial solution (containing bacterial cells and their metabolites) at 1.0 x 10

[0058] The number of bacterial cells in the probiotic preparation of Bifidobacterium breve ATCC 15700 was 1.0 x 10 10 CFU / g.

[0059] Experimental Example 1: Preventive effect of probiotic preparation of Bifidobacterium breve JYBF-220 on blood glucose elevation in hyperglycemic mice

[0060] 1. Construction of hyperglycemic model mice

[0061] SPF Kunming mice from Beijing Vital River Laboratory Animal Technology Co., Ltd. were purchased, and 100 mice with a body weight of 20 ± 1 g were adaptively fed for one week. The feeding conditions were: sufficient food and water source, constant temperature of 25℃, 12h light, and 12h darkness.

[0062] After the mice were adapted to the environment for one week, they were fasted for 12h without water, and then the blood glucose of the mice was determined. The mice with abnormal blood glucose were removed. Among the remaining mice, 70 mice were randomly selected as experimental mice, and randomly divided into 7 groups, namely, model group, positive control group, blank group, JYBF-220 prevention group, JYBF-220 group, ATCC 15700 prevention group, and ATCC 15700 group, each group having 10 mice.

[0063] The mice in the JYBF-220 prevention group were administered with the probiotic preparation of Bifidobacterium breve JYBF-220 prepared in Example 2 (dissolved in normal saline, the addition amount was 5 million CFU per mouse per day) before modeling, and the concentration was: 5 x 10 7 CFU / mL; the mice in the ATCC 15700 prevention group were administered with the probiotic preparation of Bifidobacterium breve ATCC 15700 prepared in Comparative Example 1 (dissolved in normal saline, the addition amount was 5 million CFU per mouse per day) before modeling, and the concentration was: 5 x 10 7 CFU / mL.

[0064] The mice in the blank group, JYBF-220 group, ATCC 15700 group, positive control group, and model group were administered with the same amount of normal saline, and the other feeding conditions in each group were the same, and the mice were continuously fed for 14 days.

[0065] After the mice in each group were placed in the animal room 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 ratio of the two feeds is shown in Table 2. In the 8th week of high-fat feed, 40 mg / kg·bw of streptozotocin (STZ) was dissolved in sodium citrate buffer, and the model group, positive control group, JYBF-220 group, JYBF-220 prevention group, ATCC 15700 prevention group and ATCC 15700 group of mice were injected intraperitoneally for 5 consecutive days. At the same time, the mice in the blank group were injected with sodium citrate buffer as a control. During the experiment, the routine situation and external appearance were observed.

[0066] Table 2 Energy supply table of feed

[0067]

[0068] 2. Assess whether the mice are successfully modeled

[0069] (1) Observe external appearance

[0070] After modeling, the JYBF-220 group, positive control group, model group, ATCC 15700 prevention group and ATCC 15700 group of mice gradually darkened in color and lost luster, and the symptoms of thirst and hunger were abnormally obvious, and the mental state was unstable.

[0071] (2) Detection of successful high blood glucose mouse model

[0072] The normal range of fasting blood glucose (fasting blood glucose refers to blood glucose measured after at least 8 hours without food) in healthy adults is 3.9-6.1 mmol / L. Mice have a high basal metabolic rate, and their fasting blood glucose baseline is higher than that of humans (normal mouse fasting blood glucose is about 5-8 mmol / L), so the threshold is usually raised to ensure model stability during the study. In this experiment, the criteria for a high blood glucose mouse model were fasting blood glucose ≥11.1 mmol / L after 72 hours.

[0073] After 72 hours, the fasting blood glucose (FBG) of the mice in each group was determined by taking blood from the tail vein. The results are shown in Table 3.

[0074] The experimental data obtained are expressed as mean ± standard deviation, and the data were analyzed using SPSS 26.0 statistical software. When multiple group comparisons meet the normal distribution and homogeneity of variance, one-way ANOVA analysis is used, and P<0.05 is considered to have a statistically significant difference.

[0075] Table 3 Fasting blood glucose results

[0076]

[0077] Note: # indicates P<0.05 compared with the blank group.

[0078] As shown in Table 3, the fasting blood glucose of the blank group and the JYBF-220 prevention group was <11.1 mmol / L, and the fasting blood glucose of the JYBF-220 group, the model group, the positive control group, the ATCC 15700 prevention group and the ATCC 15700 group was >11.1 mmol / L. Compared with the blank group, the fasting blood glucose of the JYBF-220 group, the model group, the positive control group, the ATCC 15700 prevention group and the ATCC 15700 group was significantly increased (P<0.05), indicating that the high blood glucose mouse modeling was successful. There was no significant difference in the fasting blood glucose between the blank group and the JYBF-220 prevention group, indicating that the probiotic preparation of Bifidobacterium breve JYBF-220 can prevent blood glucose from rising and maintain the fluctuation of blood glucose at a healthy level (no significant difference compared with the blank group), while the probiotic preparation of Bifidobacterium breve ATCC 15700 does not have the effect of preventing blood glucose from rising.

[0079] Example 2 Effect of probiotic preparation of Bifidobacterium breve JYBF-220 on body weight of high blood glucose mice

[0080] The JYBF-220 group mice successfully modeled in Example 1 were gavaged with the probiotic preparation of Bifidobacterium breve JYBF-220 prepared in Example 2 dissolved in normal saline (the addition amount was 5 million CFU per mouse per day), the ATCC 15700 group mice were gavaged with the probiotic preparation of Bifidobacterium breve ATCC 15700 prepared in Comparative Example 1 dissolved in normal saline (the addition amount was 5 million CFU per mouse per day), the positive control group mice were gavaged with metformin hydrochloride (400 mg / kg / d) dissolved in normal saline, and the blank group and the model group mice were gavaged with the same amount of normal saline. Continuous gavage for 35 days, during which each group was fed with standard feed. The body weight of each group of mice was detected at a fixed time every week, and the results are shown in Table 4.

[0081] The obtained experimental data are expressed as mean ± standard deviation, and the data are analyzed by using SPSS 26.0 statistical software. When the comparison among multiple groups meets the normal distribution and homogeneity of variance, the single factor variance method (One-way ANOVA) is used for analysis, and P<0.05 is considered to have a statistically significant difference.

[0082] Table 4 Body weight results of mice in each group

[0083]

[0084] Note: # indicates P<0.05 compared with the blank group; * indicates P<0.05 compared with the model group.

[0085] As shown in Table 4, there was a significant difference in body weight between the blank group and the model group within 5 weeks after successful modeling (P<0.05).

[0086] In the last two weeks (2w) of modeling success, compared with the model group, the body weight of JYBF-220 group, positive control group mice and ATCC15700 group mice had no significant difference (P≥0.05). With the increase of days of JYBF-220 group gavage of postbiotic preparation of Bifidobacterium breve JYBF-220 and positive control group gavage of metformin hydrochloride, the body weight of JYBF-220 group and positive control group mice was significantly greater than that of the model group (P<0.05), indicating that after the success of modeling, the postbiotic preparation of Bifidobacterium breve JYBF-220 had a certain effect on improving the body weight of hyperglycemic mice, and the effect was comparable to that of metformin hydrochloride. However, the postbiotic preparation of Bifidobacterium breve ATCC 15700 had no significant effect on improving the body weight of hyperglycemic mice.

[0087] Example 3 Effect of postbiotic preparation of Bifidobacterium breve JYBF-220 on serum HbAlc, GLP-1 and LPS of hyperglycemic mice

[0088] Glycated hemoglobin (HbA1c) is the product of non-enzymatic reaction of glucose with hemoglobin in red blood cells, and the content of HbA1c is positively correlated with the average level of blood glucose in the body, which can reflect the blood glucose condition of the body.

[0089] Glucagon-like peptide 1 (GLP-1) is an intestinal insulinotropic hormone secreted by intestinal L cells, which stimulates pancreatic beta cells to secrete insulin in a glucose-dependent manner, so that the glucose absorbed through the intestinal epithelium is 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 beta cells, promoting satiety, delaying gastric emptying, protecting the heart and nerves, etc.

[0090] Lipopolysaccharide (LPS), also known as endotoxin, is an important component of the cell wall of gram-negative bacteria. Under normal circumstances, LPS usually exists only in the intestinal lumen, and the tight junctions between intestinal epithelial cells form an intestinal barrier to resist the transfer of intestinal bacteria and harmful components into the blood. However, for people with high blood sugar or those with insulin resistance, their intestinal barrier is more likely to be damaged due to metabolic disorders, inflammatory conditions or high-sugar high-fat diet, leading to increased intestinal permeability. At this time, LPS will break through the barrier and enter the blood circulation. Therefore, the increase of LPS concentration in the blood not only reflects the increase of intestinal permeability, but also reflects the condition of elevated blood glucose.

[0091] 1. Preparation of serum samples

[0092] After 35 days (at the end of the experiment of Experimental Example 2), the mice in each group were fasted for 12 h, and then anesthetized by intraperitoneal injection of 0.4% pentobarbital at a dose of 1 mL / g. The mice were placed on an ice tray in a supine position, and the head and limbs were fixed with transparent medical tape. After the eyeballs of the mice were removed 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 clot at room temperature for 20-30 min, and then centrifuged at 4°C and 3000 rpm for 10 min. The supernatant was aspirated with a pipette, and divided into two parts. One part was stored in a -20°C freezer, and the other part was stored in a -80°C freezer for later use. At the same time, the abdominal cavity of each mouse was quickly opened, and the intact liver was removed and rinsed in pre-cooled physiological saline to remove blood and connective tissue. The tissue was cut into small pieces of 5-10 mg, and then quickly placed in a cryogenic tube and frozen in liquid nitrogen. After 15 min, the tissue was transferred to a -80°C freezer for later protein detection.

[0093] 2. Detection and method of serum HbAlc, GLP-1, and LPS

[0094] The concentrations of HbAlc, GLP-1, and LPS in the serum of the mice in each group were detected using an ELISA kit purchased from Shanghai Keai Biological Technology Co., Ltd. according to the operating method described in the kit. The results are shown in Table 5.

[0095] The experimental data obtained are expressed as mean ± standard deviation. The data were analyzed using SPSS 26.0 statistical software. When the comparison between multiple groups meets the normal distribution and homogeneity of variance, the One-way ANOVA method was used for analysis. P<0.05 was considered to have a statistically significant difference.

[0096] Table 5. Detection results of serum HbAlc, GLP-1, and LPS

[0097]

[0098] Note: # indicates P<0.05 compared with the blank group; * indicates P<0.05 compared with the model group.

[0099] As shown in Table 5, the serum HbAlc, GLP-1 and LPS of the blank group mice and the model group mice were significantly different (P<0.05). Compared with the model group, the HbAlc concentration of the JYBF-220 group mice was significantly decreased, the GLP-1 concentration was significantly increased, and the LPS concentration was significantly decreased (P<0.05). The HbAlc concentration, GLP-1 concentration and LPS concentration had no significant difference compared with the blank group (P≥0.05), and the effect was equivalent to that of the positive control group, indicating that the postbiotic preparation of B. breve JYBF-220 had the effect of regulating the concentrations of HbAlc, GLP-1 and LPS in the serum of hyperglycemic mice, and could stabilize blood glucose at a healthy level. Compared with the model group, the HbAlc concentration of the ATCC15700 group mice was not significantly decreased, and the HbAlc concentration, GLP-1 concentration and LPS concentration had significant difference compared with the blank group (P<0.05).

[0100] Example 4 Effect of postbiotic preparation of B. breve JYBF-220 on glycated serum protein content of hyperglycemic mice

[0101] Serum glucose can undergo non-enzymatic glycation reaction with the amino group at the N-terminus of serum albumin and other protein molecules to produce high molecular ketamine. Such ketamine compounds are collectively referred to as glycated serum protein, of which serum albumin accounts for the largest proportion. Therefore, glycated serum protein is often used as an indicator to reflect the degree of serum albumin glycation. In an alkaline environment, the ketamine structure of glycated serum protein can undergo a reduction reaction with nitro tetrazolium blue NBT to generate 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 long-term blood glucose level for 2-3 months. It has important significance in blood glucose monitoring, especially for the evaluation of therapeutic effect and adjustment of treatment plan for patients with greater blood glucose fluctuations.

[0102] 1. Detection step

[0103] The serum of each group of mice stored in the -20°C refrigerator in experimental example 3 was taken out and completely thawed in the 4°C ultra-low temperature refrigerator, and the stock solution was directly determined. Take a 96-well operation plate, add 10 μL of double distilled water to the blank wells (3), add 10 μL of 2 mmol / L DMF standard solution to the standard wells (3), add bovine serum albumin to the standard blank wells (3), and add 10 μL of each group of mouse serum to the sample wells in turn, and label them in order. NBT color developing agent was preheated at 37°C, and then 200 μL of NBT color developing agent was added to each well (including blank wells, standard wells, sample wells and standard blank wells) in turn. Shake gently, water bath at 37°C for 15 min, take back, and add 10 μL of stabilizer to each well in turn (if the stabilizer is coagulated, it can be heated to transparent in a water bath before use). Shake gently, and use the enzyme marker to determine the absorbance value of each well at 546 nm wavelength. The results are shown in Table 6.

[0104] Notes: During the determination of the above biochemical indicators, the mouse serum should be placed in ice bath at 4°C to avoid hemolysis caused by repeated freezing and thawing.

[0105] Calculation formula:

[0106]

[0107] The calibration concentration is 2 mmol / L.

[0108] The obtained experimental data are expressed as mean ± standard deviation, and the data are analyzed using SPSS 26.0 statistical software. When the comparison between multiple groups meets the normal distribution and homogeneity of variance, the single factor ANOVA method is used for analysis, and P<0.05 is considered to have statistical difference.

[0109] Table 6 Concentration results of glycated serum protein

[0110]

[0111] Note: # indicates P<0.05 compared with the blank group; * indicates P<0.05 compared with the model group.

[0112] As shown in Table 6, the glycated serum protein concentration of the model group mice was significantly different from that of the blank group (P<0.05). The glycated serum protein concentration of the JYBF-220 group and the positive control group mice was significantly different from that of the model group (P<0.05), and was not significantly different from that of the blank group (P≥0.05), indicating that the postbiotic preparation of B. breve JYBF-220 had an effect on reducing the glycated serum protein concentration of hyperglycemic mice, could maintain blood glucose at a healthy level, and the postbiotic preparation of B. breve JYBF-220 and the positive control group had a comparable effect on the glycated serum protein concentration of hyperglycemic mice. Compared with the model group, the glycated serum protein concentration of the ATCC 15700 group mice was not significantly different (P≥0.05), indicating that the postbiotic preparation of B. breve ATCC 15700 had no obvious effect on reducing the glycated serum protein concentration of hyperglycemic mice.

[0113] Example 5 Effect of the postbiotic preparation of B. breve JYBF-220 on the expression of InsR, IRS-2 and PPARγ proteins in the liver of hyperglycemic mice

[0114] 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 gamma (PPARγ) belongs to nuclear receptor, and may play a role in adipocyte differentiation and glucose-lipid metabolism.

[0115] The liver tissue stored in the -80°C refrigerator in Example 4 was used to detect the content of InsR, IRS-2 and PPARγ proteins in the tissue by Western Blot radioimmunoassay, and the specific steps were as follows:

[0116] (1) Sample preparation: complete lysis. After lysis, centrifuge to take the supernatant, and after protein quantification, freeze and store; (2) protein quantification; (3) preparation of lower separation gel and preparation of upper concentrated gel; (4) sample loading and electrophoresis; (5) membrane transfer; (6) detection of proteins on the membrane: place the membrane in TBST and wash once, then place it in the ponceau staining working solution, shake at room temperature for 5 minutes, wash the membrane with water until the water is clear and the protein band is clear, and the PVDF membrane needs to be reactivated with methanol (before membrane transfer) and then washed with TBST and blocked; (7) membrane blocking and antibody incubation, blocking: 5% skim milk powder at room temperature for 1 hour or 4°C overnight; primary antibody: dilute the antibody, dilute the antibody to the required concentration in blocking solution, and incubate the membrane at room temperature for 2 hours or 4°C overnight; secondary antibody: wash the membrane incubated with the primary antibody with TBST, then dilute the HRP-labeled secondary antibody according to the amount, at a ratio of 1:1000, and incubate the membrane at 37°C for 1 hour; wash with TBST; (8) color development: ECL chemiluminescence detection. Place it in the imaging system for scanning; (9) use Image J software to compare gray values, and use the ratio of the gray value of the band to the gray value of the internal reference a-tubulin as the basis for relative quantitative analysis, and the results are shown in Figure 1 Table 7.

[0117] Microsoft excel 2021 was used for database processing, and SPSS 26.0 was used for statistical processing. The experimental results were expressed as mean ± standard deviation, one-way ANOVA was used for comparison between groups, LSD was used for multiple comparisons between groups, and P<0.05 was considered statistically significant.

[0118] Table 7 Protein expression results of liver InsR, IRS-2, and PPARy

[0119]

[0120] Note: # indicates P<0.05 compared with the blank group; * indicates P<0.05 compared with the model group.

[0121] As shown in Table 7, there were significant differences in the expression of InsR, IRS-2, and PPARγ proteins in the livers of mice in the control group and the model group (P < 0.05). The relative expression levels of InsR, IRS-2, and PPARγ proteins in the livers of mice in the JYBF-220 group were significantly higher than those in the model group (P < 0.05); however, there were no significant differences compared to the control group (P ≥ 0.05). This indicates that the post-biotic preparation of Bifidobacterium breve JYBF-220 is effective in increasing the expression of InsR, IRS-2, and PPARγ proteins in the livers of hyperglycemic mice, maintaining blood glucose levels at healthy levels. Furthermore, the post-biotic preparation of Bifidobacterium breve JYBF-220 is comparable in effect to metformin hydrochloride. Compared with the model group, the relative expression levels of InsR, IRS-2, and PPARγ proteins in the liver of mice in the ATCC 15700 group were not significantly increased (P≥0.05), indicating that the prebiotic preparation of Bifidobacterium breve ATCC 15700 had no significant effect on increasing the expression of InsR, IRS-2, and PPARγ proteins in the liver of hyperglycemic mice.

[0122] from Figure 1 The results show that the relative expression levels of InsR, IRS-2, and PPARγ proteins in the liver of mice in the JYBF-220 group were not significantly different from those in the blank group. This also indicates that the post-biotic preparation of Bifidobacterium breve JYBF-220 can increase the expression of InsR, IRS-2, and PPARγ proteins in the liver of hyperglycemic mice and maintain stable blood glucose levels at healthy levels.

[0123] Although the present invention has been described in detail with reference to the accompanying drawings and preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.

Claims

1. A Bifidobacterium breve JYBF-220 strain that maintains a healthy level of blood glucose, characterized in that, Bifidobacterium breve (ATCC 15698) Bifidobacterium breve Bifidobacterium breve (ATCC 15698) JYBF-220 was deposited with the China General Microbiological Culture Collection Center on July 24, 2023, at an address of No. 1, Beichen West Road, Yuanmingyuan District, Beijing, with a preservation number of CGMCC NO. 27991.

2. The postbiotic preparation of Bifidobacterium breve JYBF-220 according to claim 1, characterized by, The preparation method is as follows: the Bifidobacterium breve JYBF-220 is activated on MRS flat medium, and the activated single colony is inoculated into MRS liquid medium to obtain seed liquid after being cultured at 37°C under anaerobic conditions for 12 hours; The seed liquid is inoculated into MRS liquid medium at an inoculation amount of 1%, and the obtained bacterial liquid is cultured at 37°C under anaerobic conditions for 24 hours, and then subjected to heat inactivation treatment at 115°C for 30 minutes, and concentrated and freeze-dried to obtain the metaplasma preparation of the Bifidobacterium breve JYBF-220.

3. The postbiotic formulation of claim 2, wherein, The number of bacterial bodies in the postbiotic preparation is 1.0 x 10 10 CFU / g.

4. The postbiotic formulation of claim 2, wherein, The metaplasma preparation further comprises a blood glucose index 0 auxiliary material.

5. Use of the metaplasma preparation according to any one of claims 2-4 in the preparation of health care products for helping to maintain a healthy level of blood glucose.

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