Application of bifidobacterium breve i1088 metagen in preparation of products for regulating blood sugar

Abstract

The invention relates to the technical field of microbial engineering, and particularly discloses application of bifidobacterium breve i1088 metagen in preparation of a blood sugar regulating product. The bifidobacterium breve i1088 metabolite can play a biological function through a key metabolite 5-HIAA, stimulate human colorectal adenocarcinoma cells to secrete GLP-1 and up-regulate GCG gene expression so as to regulate the blood sugar level, has multiple biological activities (such as oxidation resistance and glycosidase activity inhibition), and can be used for preparing a medicine for treating cancer. The method has obvious advantages and effects in the fields of food science, biotechnology, nutrition and health care and the like. Animal experiments prove that the bifidobacterium breve i1088 metagen has the effect of remarkably reducing the fasting blood-glucose of a diabetic mouse; the bifidobacterium breve i1088 metagen shows good alpha-glucosidase and alpha-amylase inhibitory activity in an in-vitro experiment, and can be used for development and application of hypoglycemic products.

Description

Technical Field

[0001] This invention relates to the field of microbial engineering technology, and in particular to the application of a postbiotic, Bifidobacterium breve i1088, in the preparation of blood glucose regulating products. Background Technology

[0002] Glucagon-like peptide-1 (GLP-1) is an incretin secreted by intestinal L cells. It promotes insulin secretion from pancreatic β cells, inhibits glucagon secretion, delays gastric emptying, and lowers blood glucose levels, playing an important role in the prevention and treatment of type 2 diabetes mellitus (T2DM) and related complications. While GLP-1 receptor agonists can effectively maintain healthy blood glucose levels in current clinical treatments, their high cost, the need for injection, and the potential for adverse reactions with long-term use limit their widespread adoption in routine health management.

[0003] In recent years, the relationship between gut microbiota and glucose metabolism has received widespread attention. Studies have shown that some probiotics can indirectly affect incretin levels by regulating the gut environment and host metabolic pathways, thereby participating in the regulation of glycemic homeostasis. However, most commercially available probiotics focus on improving digestion and absorption and alleviating intestinal inflammation. Reports on probiotics that can simultaneously promote GLP-1 secretion, upregulate related gene expression, and possess multiple biological activities (such as hypoglycemic, antioxidant, and glycosidase inhibition) are rare. Furthermore, the key target functional components of probiotics remain unclear. Therefore, developing novel strains with multiple functions suitable for developing hypoglycemic products, while simultaneously identifying key functional components, has become an important research direction in food science and nutrition. Summary of the Invention

[0004] In view of this, the present invention provides an application of Bifidobacterium breve i1088 postbiotic in the preparation of blood glucose regulating products.

[0005] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows: In a first aspect, the present invention provides the application of Bifidobacterium breve i1088 postbiotic in the preparation of blood glucose regulating products, wherein the Bifidobacterium breve i1088 postbiotic includes a live lysate of Bifidobacterium breve i1088.

[0006] The present invention relates to *Bifidobacterium breve* i1088 as a postbiotic. *Bifidobacterium breve* i1088 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on May 8, 2023, with accession number CGMCC NO. 27281 and Latin name [missing information]. Bifidobacterium breve i1088.

[0007] Preferably, the active substance in the live lysate of Bifidobacterium breve i1088 includes 5-hydroxyindoleacetic acid.

[0008] Preferably, the lysate of the live Bifidobacterium breve i1088 can promote the secretion of glucagon-like peptide-1 by cells and enhance the gene transcription level of glucagon.

[0009] More preferably, the cells that secrete glucagon-like peptide-1 include human colorectal adenocarcinoma cells.

[0010] In this invention, Bifidobacterium breve i1088 can metabolize high levels of 5-hydroxyindoleacetic acid (5-HIAA), which can further promote the secretion of GLP-1 by human colorectal adenocarcinoma cells (NCI-H716 cells) and increase glucagon (GCG) gene expression, providing a new intervention method for people with abnormal glucose metabolism.

[0011] Preferably, the live lysate of Bifidobacterium breve i1088 has inhibitory effects on the activity of α-glucosidase and α-amylase.

[0012] In this invention, the live lysate of Bifidobacterium breve i1088 can simultaneously promote GLP-1 secretion and inhibit the activity of α-glucosidase and α-amylase, thus synergistically regulating blood glucose levels through multiple pathways.

[0013] Preferably, the method for preparing the live lysate of Bifidobacterium breve i1088 includes the following steps: Bifidobacterium breve i1088 was cultured in a glucose-containing medium until the logarithmic growth phase, then isolated. The obtained bacterial cells were resuspended, broken, and separated, and the resulting supernatant was the live bacterial lysate of Bifidobacterium breve i1088.

[0014] In this invention, Bifidobacterium breve i1088 can efficiently produce 5-HIAA through glucose metabolism. Under the action of glucose, the production efficiency of 5-HIAA can be further improved, and the activity stability of the live bacterial lysate of Bifidobacterium breve i1088 can be further increased.

[0015] More preferably, the culture is an anaerobic culture, in which 0.03% to 0.08% cysteine ​​salt is added to the culture medium.

[0016] In this invention, adding a specific amount of cysteine ​​salt to the culture medium can provide an anaerobic environment.

[0017] More preferably, the culture medium includes MRS culture medium.

[0018] More preferably, the crushing is performed using intermittent ultrasonic crushing, with an ultrasonic power of 180W~220W, a single ultrasonic time of 1.5s~2.5s, and an interval of 2.5s~3.5s.

[0019] For example, after culturing to the logarithmic growth phase and separating the cells, the resulting bacterial cells are washed with PBS buffer and then resuspended in PBS buffer.

[0020] More preferably, the number of bacterial cells in the *Bifidobacterium breve* i1088 live bacterial lysate before lysis is not less than 10. 7 CFU / mL (more preferably 10) 9 (CFU / mL or higher).

[0021] For example, the supernatant (live lysate of Bifidobacterium breve i1088) can be treated by spray drying, freeze drying or microencapsulation to maintain its activity during food preparation and storage.

[0022] Preferably, the blood sugar regulating products include hypoglycemic functional low-temperature yogurt, functional dairy products, formula milk powder, or health foods.

[0023] Using Bifidobacterium breve i1088 as a postbiotic in the aforementioned blood sugar regulating products can balance safety and functionality, expanding the application scope of probiotics in the field of food health.

[0024] Secondly, the present invention provides a blood sugar regulating product, comprising the aforementioned Bifidobacterium breve i1088 postbiotic.

[0025] Preferably, the mass percentage of the postbiotic Bifidobacterium breve i1088 is 0.1% to 20%.

[0026] More preferably, the mass percentage of the postbiotic Bifidobacterium breve i1088 is 0.4% to 11%.

[0027] The present invention has the following beneficial effects: Compared with existing technologies, the application of Bifidobacterium breve i1088 postbiotic in the preparation of blood glucose regulating products provided by this invention allows the live lysate of Bifidobacterium breve i1088 to exert its biological functions through the key metabolite 5-HIAA, stimulating human colorectal adenocarcinoma cells to secrete GLP-1 and upregulate GCG gene expression, thereby regulating blood glucose levels. It also has multiple biological activities (such as antioxidant and glycosidase inhibition), and has significant advantages in the fields of food science, biotechnology and nutrition and health care.

[0028] Bifidobacterium breve i1088 can be safely used under food-grade conditions, and the strain has high stability, making it convenient for the development of functional foods and health products. The live lysate of Bifidobacterium breve i1088 can significantly promote the secretion of GLP-1 by intestinal epithelial cells, thereby giving it multiple physiological functions. Specifically, these include: (1) enhancing glucose-dependent insulin secretion and improving pancreatic β-cell function; (2) inhibiting glucagon secretion, reducing hepatic glucose output, and maintaining blood glucose homeostasis; (3) delaying gastric emptying, enhancing satiety, and helping to control postprandial blood glucose; (4) centrally inhibiting appetite by acting on hypothalamic neurons to reduce food intake; (5) cardiovascular protection, improving cardiac function and reducing the risk of cardiovascular events; and (6) neuroprotection, showing potential application value in neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.

[0029] This invention verifies through animal experiments that the live lysate of Bifidobacterium breve i1088 has a significant effect on reducing fasting blood glucose in diabetic mice; the live lysate of Bifidobacterium breve i1088 exhibits good α-glucosidase and α-amylase inhibitory activity in in vitro experiments, and can be used for the development and application of hypoglycemic products. Attached Figure Description

[0030] Figure 1 This is a comparison chart of GLP-1 secretion levels in NCI-H716 cells in different groups in Example 2 of the present invention; Figure 2 This is a comparison chart of GCG gene expression levels in NCI-H716 cells in different groups in Example 2 of the present invention; Figure 3 These are the top 50 differential metabolites screened in Example 3 of the present invention. In red, the differential metabolites that are significantly enriched in the live lysate of Bifidobacterium breve i1088 compared with other groups are indicated. Figure 4 This is a comparison chart of the amount of GLP-1 secreted by cells after intervention with thiazolic acid standard in Example 3 of the present invention; Figure 5 This is a comparison chart of the amount of GLP-1 secreted by cells after intervention with sphingosine standard in Example 3 of the present invention; Figure 6 This is a comparison chart of the amount of GLP-1 secreted by cells after intervention with indole-3-acetic acid standard in Example 3 of the present invention; Figure 7 This is a comparison chart of the amount of GLP-1 secreted by cells after intervention with choline standard in Example 3 of the present invention; Figure 8 This is a comparison chart of the amount of GLP-1 secreted by cells after intervention with 5-HIAA standard in Example 3 of the present invention; Figure 9This is a comparison chart of the amount of GLP-1 secreted by cells after intervention with 3-hydroxy-2-methyl-4(1H)-pyridone standard in Example 3 of the present invention; Figure 10 This is a comparison chart of fasting blood glucose levels in mice before and after the intervention period in Example 4 of the present invention; Figure 11 This is a comparison chart of serum GLP-1 levels in mice in different groups after intervention in Example 4 of the present invention. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0032] Example 1 This embodiment provides a method for preparing a live lysate of Bifidobacterium breve i1088, comprising the following steps: S100. Add 0.1g of Bifidobacterium breve i1088 bacterial powder to MRS medium modified with 0.05% cysteine ​​salt, and culture anaerobically at 37℃ for 24h to obtain the first generation bacterial solution.

[0033] S200. Add 0.5 mL of the first-generation bacterial culture to MRS medium modified with 0.05% cysteine ​​salt, and culture anaerobically at 37°C for 24 h to obtain the second-generation bacterial culture.

[0034] S300. Add 0.5 mL of second-generation bacterial culture to MRS medium modified with 0.05% cysteine ​​salt, and culture anaerobically at 37°C for 16 h to obtain third-generation bacterial culture (logarithmic growth phase).

[0035] S400. Centrifuge the third-generation bacterial culture at 10000 r / min for 15 min, wash the bacterial cells three times with PBS buffer, and then resuspend the washed bacterial cells with PBS buffer to obtain a concentration of 10. 10 CFU / mL live bacterial culture.

[0036] S500. Place the live bacterial culture in an ice bath and sonicate to disrupt the cells (ultrasonic power of 200W, single sonication time of 2s, 3s interval followed by 2s sonication, repeating this process, for a total sonication time of approximately 15min). After observing under a microscope that no intact bacterial cells are found, centrifuge the disrupted culture at 4℃ and 10000r / min for 20min, collect the supernatant, and filter it through a 0.22μm microporous membrane (such as a disposable cellulose acetate membrane) to obtain a live bacterial lysate of Bifidobacterium breve i1088.

[0037] Comparative Example 1 This comparative example provides a method for preparing a lysate of dead Bifidobacterium breve i1088, comprising the following steps: S100: Take the third-generation bacterial culture prepared by S300 in Example 1, inactivate it in a water bath at 65°C for 30 min, centrifuge at 10000 r / min for 15 min, wash the inactivated bacterial cells three times with PBS buffer, and then resuspend the washed inactivated bacterial cells with PBS buffer to obtain a concentration of 10. 10 Dead bacterial solution at CFU / mL.

[0038] Method for determining inactivation: Spread 100 μL of dead bacterial solution onto mMRS solid medium and incubate at 37°C for 48 h. No sterile growth indicates successful inactivation.

[0039] S200. Place the dead bacterial solution in an ice bath and sonicate to disrupt the cells (ultrasonic power of 200W, single sonication time of 2s, 3s interval, and sonication for another 2s, repeating this process, with a total sonication time of approximately 15min). After observing under a microscope that no intact bacterial cells are found, centrifuge the lysate at 4℃ and 10000r / min for 20min, collect the supernatant, and filter it through a 0.22μm microporous membrane to obtain the dead bacterial lysate of Bifidobacterium breve i1088.

[0040] Example 2: Verification of GLP-1 secretion-promoting efficacy 1. Cell Culture NCI-H716 cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and placed in a constant temperature cell culture incubator at 37°C with 5% CO2 and saturated humidity. The medium was changed or passaged every 2 to 3 days.

[0041] 2. Experimental Grouping The experimental group information is shown in Table 1.

[0042] Table 1. Experimental grouping information for Example 2

[0043] Berberine (BBR) has been shown to enhance GLP-1 secretion by activating the GLP-1 / GLP-1R / PKA signaling pathway in intestinal L cells and pancreatic β cells; it was used as a positive control in this embodiment. Akkermansia muciniphila (AKK) has also been reported to stimulate GLP-1 secretion from intestinal L cells and improve insulin sensitivity and glucose tolerance; it was used as a positive control strain in this embodiment. The preparation methods for live bacterial suspensions, live bacterial lysates, and dead bacterial lysates of AKK, Bifidobacterium shortis JMCC1103, and commercial Bifidobacterium shortis V are as described in Example 1 and Comparative Example 1.

[0044] 3. Intervention Experiment NCI-H716 cells were seeded in 12-well substrate-coated plates at a cell density of 1.5 × 10⁻⁶ cells / well. 6 CFU / well; resuspend the intervention material (i.e., groups 1-14 in Table 1) in KRB buffer, incubate for 2 h, centrifuge, and collect the supernatant in a 1.5 mL centrifuge tube containing 50 μg / mL benzyl sulfonyl fluoride (PMSF) and 10 μg / mL dipeptidyl peptidase-4 (DPP-4) inhibitor.

[0045] The level of GLP-1 secreted by cells was detected according to the instructions of the ELISA kit. Each group had three replicates, and the results were calculated using a 2x2 method. -△△Ct The test results are shown in Table 2 and Figure 1 As shown.

[0046] Table 2. Results of GLP-1 fold positivity in NCI-H716 cells in each group.

[0047] As can be seen from the table above, group 7 (short-lived Bifidobacterium i1088 live bacterial lysate) had the best effect on promoting GLP-1 secretion in NCI-H716 cells; the effects of live bacterial suspension and dead bacterial lysate were not as good as those of live bacterial lysate, indicating that the key component in short-lived Bifidobacterium i1088 live bacterial lysate may be a heat-sensitive intracellular metabolite.

[0048] GCG gene expression was detected using RT-qPCR. Primer sequences are shown in Table 3, and test results are shown in Table 4. Figure 2 As shown. Primer reference: Cui C, Wu SL, Chen JJ, Gongpan P, Guan M, Geng CA. Sesquiterpenoids from Alpinia oxyphylla with GLP-1 Stimulative Effects through Ca 2+ / CaMKII and PKA Pathways and Multiple-Enzyme Inhibition. J AgricFood Chem. 2023 Nov 1;71(43):16148-16159.

[0049] Table 3 Primer sequences

[0050] Table 4. Results of GCG gene expression levels in NCI-H716 cells of each group

[0051] As can be seen from the table above, group 7 (short bifidobacteria i1088 live bacterial lysate) showed the best effect in promoting GCG gene expression in NCI-H716 cells.

[0052] Example 3: Analysis of Key Active Factors For the same concentration (1.5×10), 9 Non-targeted metabolomics component analysis was performed using the following samples (CFU / mL): Bifidobacterium breve i1088 live bacterial lysate (Group A), Bifidobacterium breve i1088 live bacterial suspension (Group B), Bifidobacterium breve JMCC 1103 live bacterial lysate (Group C), and commercial Bifidobacterium breve V live bacterial lysate (Group D). Data were preprocessed using log10, and Z-score normalization was performed for each metabolite. Each group had three replicates (groups were designated A1, A2, A3, B1, B2, B3, C1, C2, C3, D1, D2, and D3). The specific procedures were as follows: Filtered samples (Groups A through D) were transferred to 2 mL high-performance liquid chromatography (HPLC) vials for analysis. Metabolomics analysis was performed using an ultra-high performance liquid chromatography-electrospray ionization-quadrupole-mass spectrometry (UHPLC system: Shimadzu Nexera X2 LC-30AD) and Q-Exactive Plus.

[0053] For liquid chromatography: an ACQUITY UPLC® HSS T3 column (2.1 × 100 mm, 1.8 μm) was used; the flow rate was 0.3 mL / min; the mobile phase consisted of: phase A being an aqueous solution containing 0.1% formic acid, and phase B being 100% acetonitrile. The gradient elution program was as follows: 0 min–2 min, phase A 100%; 2 min–6 min, phase A 100% → 52%, phase B 0% → 48%; 6 min–10 min, phase A 52% → 0%, phase B 48% → 100%; 10 min–12 min, phase B 100%; 12.01 min–15 min, phase A 100%.

[0054] For mass spectrometry: Electrospray ionization (ESI) in positive and negative ion modes was used to acquire mass spectrometry data separately. The heated electrospray ionization source (HESI) was set as follows: spray voltage: 3.8 kV for positive ion mode, 3.2 kV for negative ion mode; capillary temperature: 320 °C; sheath gas (nitrogen) flow rate: 30 (any unit); auxiliary gas flow rate: 5 (any unit); probe heating temperature: 350 °C; S-lens RF level: 50. The instrument was set to acquire full-scan mass spectra within a mass-to-charge ratio (m / z) range of 70 Da to 1050 Da. The resolution for full-scan mass spectra was set to 70,000 at a mass-to-charge ratio of 200, and the resolution for secondary mass spectrometry (MS / MS) was set to 17,500 at a mass-to-charge ratio of 200. The maximum injection time for mass spectrometry was set to 100 ms, and the maximum injection time for secondary mass spectrometry was set to 50 ms. The isolation window for the secondary mass spectrometer (MS2) was set to a mass-to-charge ratio of 2, and the normalized collision energies (segmented) were set to 20, 30, and 40 for fragmentation.

[0055] 1. Non-targeted omics detection of differentially expressed metabolites The principles for identifying differentially expressed metabolites are: (1) Variable Important for the Projection (VIP) ≥ 1; (2) Fold Change (FC) ≥ 1.2 or ≤ 0.83; (3) q-value < 0.05. The top 50 differentially expressed metabolites selected are as follows: Figure 3 As shown in the figure. Further analysis revealed that, compared to groups B, C, and D, group A was enriched with higher levels of thiazolinone, sphingosine, choline, 5-hydroxyindoleacetic acid (5-HIAA), indole-3-acetic acid, and 3-hydroxy-2-methyl-4(1H)-pyridinone. These significantly enriched metabolites may play a key role in its biological effects.

[0056] 2. Verification of key active ingredients The key differentially expressed metabolites identified above were validated: cells were treated with differentially expressed metabolite standards at concentrations of 10000 μg / mL, 1000 μg / mL, 100 μg / mL, and 1 μg / mL, respectively, and the amount of GLP-1 secreted by the cells was measured. Live lysate of *Bifidobacterium breve* i1088 was used as a positive control, and the untreated cell group served as a blank calibration. The experimental results are as follows: Figures 4-9As shown in the analysis results, 5-HIAA is the key active substance in the lysate of live Bifidobacterium breve i1088 that promotes GLP-1 secretion.

[0057] Existing research indicates that 5-HIAA derived from gut microbiota can improve insulin resistance and glucose intolerance induced by a high-fat diet by activating the hepatic AhR / TSC2 / mTORC1 pathway, thus possessing potential anti-type 2 diabetes activity. This study found that *Bifidobacterium breve* i1088 produces higher levels of 5-HIAA through metabolism, and 5-HIAA, as a key active ingredient, can directly act on human colorectal cancer cell lines, thereby promoting the secretion of more GLP-1 by intestinal cells.

[0058] Example 4: Validation of the efficacy of promoting GLP-1 secretion in reducing postprandial blood glucose levels in diabetic rats 1. Experimental Grouping Mice strain: C57BL / 6J, 6 weeks old, male, 10 mice per group. Experimental group information is shown in Table 5. Berberine dosage was 100 mg / kg / day, and the intervention dose for all other groups was 10 mg / kg / day. 10 CFU / kg body weight, conversion method: mouse Km value / human Km value = 0.081, the corresponding human dose for a 60kg body weight is 5 billion / day.

[0059] Table 5. Experimental grouping information for Example 4

[0060] 2. Experimental Design Experimental period: 1 week of acclimatization feeding, 8 weeks of high-fat diet modeling, and 8 weeks of experimental intervention. Husbandry environment: 12 hours of light per day, temperature controlled at 23℃±1℃, and relative humidity controlled at 50%±5%. Mice in all groups had normal access to water. Diet: Normal diet (Research diets, D12450J, Beijing Bai'ao Biotechnology Co., Ltd.), and high-fat diet (Research diets, D12450, Beijing Bai'ao Biotechnology Co., Ltd.). The high-fat diet provided 60% of the energy from fat (i.e., 60% of the total energy in the high-fat diet came from fat).

[0061] One week of adaptation feeding: The blank control group mice were fed a normal diet for one week, while the model group was fed a high-fat diet in a gradient for one week (0d~3d: 25% high-fat diet + 75% normal diet, 3d~5d: 50% high-fat diet + 50% normal diet, 5d~7d: 75% high-fat diet + 25% normal diet, after 7d: 100% high-fat diet).

[0062] High-fat diet feeding stage (modeling): Each mouse was fed 4g of feed per day. The blank control group was given normal feed, while the model group was given a high-fat diet for 8 weeks. When the weight of the model group mice was significantly different from that of the blank control group, they were induced with STZ (Sigma, 1%, pH=4.5, dissolved in 0.1M citrate buffer, injection dose of 45mg / kg~50mg / kg). The blank control group was injected with citrate buffer for 5 days. Fasting blood glucose was measured by taking blood from the tail tip of the mice. A fasting blood glucose concentration ≥11.1mmol / L indicated successful modeling.

[0063] Intervention period: The blank control group and the model group were given normal saline by gavage daily, while the other groups were given the corresponding sample by gavage daily for a total of 8 weeks.

[0064] Fasting blood glucose levels were measured in mice before and after the intervention period by collecting blood from the tail vein. The results are shown in Table 6. Figure 10 As shown.

[0065] Table 6 Fasting blood glucose levels in mice before and after intervention.

[0066] The GLP-1 level in the serum of mice after the intervention period was detected according to the instructions of the ELISA kit. The relative fold increase of serum GLP-1 levels compared with the blank control group is shown in Table 7. Figure 11 As shown.

[0067] Table 7. Serum GLP-1 levels in mice after intervention (equivalent to blank control group)

[0068] From Tables 6 to 7 and Figures 10-11 As can be seen, the live lysate of Bifidobacterium breve i1088 effectively reduced fasting blood glucose levels in diabetic model mice by increasing serum GLP-1 levels.

[0069] Example 5: Verification of the inhibitory effect on α-glucosidase and α-amylase activities The inhibitory effects of groups 6-14 (samples) in Table 1 on the activities of α-glucosidase and α-amylase were tested respectively, with 3 replicates for each group.

[0070] α-Glucosidase: Add 20 μL of the same concentration (1.5 × 10⁻⁶) to each well of a 96-well plate. 10The bacterial culture sample (CFU / mL) and 40 μL of p-nitrophenyl-α-D-glucopyranoside (PNPG) were mixed thoroughly and incubated at 37°C for 10 min. Then, 10 μL of α-glucosidase was added, mixed thoroughly, and incubated at 37°C for another 10 min. Finally, 140 μL of sodium carbonate solution was added to terminate the reaction, and the absorbance at 400 nm was measured.

[0071] α-Amylase: 100 μL of the same concentration (1.5 × 10⁻⁶) was added to each container. 10 The bacterial culture sample (CFU / mL) was mixed with 100 μL of α-amylase and incubated at 37°C for 30 min. Then, 200 μL of soluble starch was added and incubated at 37°C for 10 min. 100 μL of the mixed solution was taken and 400 μL of 3,5-dinitrosalicylic acid (DNS) reagent was added. After boiling in a water bath for 5 min, the absorbance was measured at a wavelength of 540 nm.

[0072] Blank control group: The reaction system contains no sample, but contains enzyme and substrate, reflecting the maximum reactivity of the enzyme.

[0073] Blank control background group: The reaction system does not contain enzymes or samples (it is replaced with phosphate buffer) to eliminate the influence of background light absorption.

[0074] Sample group: The reaction system contains the sample, enzyme, and substrate, and is used to determine the inhibitory effect of the sample on enzyme activity.

[0075] Sample background group: The reaction system does not contain enzymes, but contains the sample and substrate, and is used to eliminate the influence of the sample itself on the colorimetric reaction or light absorption.

[0076] The inhibition rate was calculated using the following formula: Inhibition rate = [1 - (A3 - A4) / (A1 - A2)] × 100%. The test results are shown in Table 8. In the formula, A1 is the absorbance of the blank control group, A2 is the absorbance of the blank control background group, A3 is the absorbance of the sample group, and A4 is the absorbance of the sample background group.

[0077] Table 8. Inhibitory effects of each sample on the activities of α-glucosidase and α-amylase.

[0078] In the table, different letters indicate significant differences between the corresponding groups, P < 0.05.

[0079] As shown in the table above, the live lysate of *Bifidobacterium breve* i1088 exhibited the best performance in inhibiting the activities of α-glucosidase and α-amylase, demonstrating potential glycemic regulation capabilities. α-glucosidase is mainly distributed in the brush border membrane of small intestinal epithelial cells and is responsible for breaking down oligosaccharides or disaccharides into absorbable monosaccharides, promoting carbohydrate absorption. Inhibiting its activity can slow down the digestion rate, thereby reducing postprandial and fasting blood glucose. Therefore, the development of natural, efficient, and safe α-glucosidase inhibitors has always been a focus of diabetes research. Inhibiting α-amylase activity can effectively prevent the digestion and hydrolysis of carbohydrates in food in the body, thereby reducing sugar intake and controlling blood glucose levels.

[0080] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of Bifidobacterium breve i1088 probiotic in the preparation of a product for regulating glycemia, characterized in that, The Bifidobacterium breve i1088 probiotic includes a Bifidobacterium breve i1088 live bacteria lysate.

2. Use of Bifidobacterium breve i1088 probiotic for the preparation of a product for regulating glycemia according to claim 1, characterized in that, The active substance of the Bifidobacterium breve i1088 live bacteria lysate includes 5-hydroxyindoleacetic acid.

3. Use of Bifidobacterium breve i1088 probiotic for the preparation of a product for regulating glycemia according to claim 1 or 2, characterized in that, The Bifidobacterium breve i1088 live bacteria lysate can promote cells to secrete glucagon-like peptide-1 and enhance the gene transcription level of glucagon. The Bifidobacterium breve i1088 live bacteria lysate has an inhibitory effect on the activities of alpha-glucosidase and alpha-amylase.

4. Use of Bifidobacterium breve i1088 probiotic for the preparation of a product for regulating glycemia according to claim 3, characterized in that, The cell secreting glucagon-like peptide-1 includes human colorectal adenocarcinoma cells.

5. Use of Bifidobacterium breve i1088 probiotic for the preparation of a product for regulating glycemia according to claim 1 or 2, characterized in that, The preparation method of the Bifidobacterium breve i1088 live bacteria lysate includes the following steps: The Bifidobacterium breve i1088 is cultured in a glucose-containing culture medium to the logarithmic growth phase, isolated, and the obtained bacterial cell is resuspended, broken, and separated, and the obtained supernatant is the Bifidobacterium breve i1088 live bacteria lysate.

6. The use of Bifidobacterium breve i1088 probiotic for the preparation of a product for regulating glycemia according to claim 5, characterized in that, The culture medium includes an MRS culture medium. The breaking adopts intermittent ultrasonic breaking, the ultrasonic power is 180W-220W, the single ultrasonic time is 1.5s-2.5s, and the interval time is 2.5s-3.5s.

7. Use of Bifidobacterium breve i1088 probiotic for the preparation of a product for regulating glycemia according to claim 5, characterized in that, The number of Bifidobacterium breve i1088 live bacteria before cell disruption is not less than 10 7 CFU / mL.

8. Use of Bifidobacterium breve i1088 probiotic for the preparation of a product for regulating glycemia according to claim 1, characterized in that, The blood sugar regulating product includes a hypoglycemic functional low-temperature yogurt, a functional dairy product, a formula milk powder, or a health food.

9. A blood glucose regulating product, characterized in that, The Bifidobacterium breve i1088 probiotic in any one of claims 1-8 is included.

10. The blood glucose regulation product of claim 9, wherein, The mass ratio of the Bifidobacterium breve i1088 probiotic is 0.1%-20%.

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