Strain with hypoglycemic effect as well as culture method and application thereof

Abstract

The invention belongs to the technical field of probiotics, and particularly relates to a bacterial strain with a hypoglycemic effect and a culture method and application thereof.The bacterial strain with the hypoglycemic effect is obtained by conducting specific hypoglycemic induction culture on bifidobacterium animalis subsp. Lactis HN1000, the hypoglycemic activity is further improved, and the hypoglycemic effect is good. When the bacterial strain is applied to preparation of probiotic tablets as an auxiliary hypoglycemic product, the bacterial strain and bacillus coagulans synergistically play a biological hypoglycemic role under an enteric coating layer formed by whey protein and sodium alginate, the in-vivo activity of probiotics is greatly improved, and meanwhile, the in-vivo hypoglycemic effect of the probiotics is improved. And with other nutritional active components including mulberry leaf extract, galactooligosaccharide, resistant starch and microcrystalline cellulose, sugar absorption inhibition and insulin sensitivity improvement are realized.

Description

Technical Field

[0001] This invention belongs to the field of probiotic technology, specifically relating to a strain with hypoglycemic effect, its culture method, and its application. Background Technology

[0002] With the rapid increase in the prevalence of diabetes, probiotic-based glycemic regulation therapy has become a research hotspot due to its advantages of mild effects and high safety. However, the current bottleneck restricting the development of hypoglycemic probiotics is mainly the insufficient hypoglycemic activity of probiotic strains: conventional probiotics have limited glucose metabolism capacity, making it difficult to effectively activate the host insulin signaling pathway in the complex intestinal environment, and the production of key metabolites such as butyrate is low, resulting in weak effects on improving hepatic gluconeogenesis and peripheral insulin resistance; secondly, gastric acid erosion and bile salt dissolution lead to extremely low survival rates of live bacteria reaching the intestine, resulting in low oral delivery efficiency, while traditional microsphere encapsulation technology, although it can improve gastric acid tolerance, often causes premature release of probiotics due to the swelling characteristics of the material, failing to achieve precise intestinal delivery of live bacteria and active ingredients; in addition, most solutions rely on single-pathway intervention of probiotics or plant extracts, with a single hypoglycemic target, and the high homogeneity of prebiotic selection limits the proliferation efficiency of hypoglycemic specific bacterial groups.

[0003] Therefore, there is an urgent need to develop an innovative solution that combines targeted strain enhancement, efficient gastrointestinal delivery, and multi-component synergy to overcome the technical bottlenecks in the application of probiotics for lowering blood sugar while ensuring safety. Summary of the Invention

[0004] To address the above issues, this invention provides a bacterial strain with hypoglycemic effects, its cultivation method, and its application. By designing a culture medium containing nutrient-active components such as wolfberry polysaccharides and chromium-rich yeast, the probiotic strain with hypoglycemic effects is subjected to targeted activity induction culture, deeply activating the glucose metabolism regulatory pathway of the probiotic strain and enhancing its endogenous hypoglycemic activity. Simultaneously, by utilizing the synergistic effect of the bacterial community and enteric coating technology, probiotic tablets that combine strain activity assurance and intestinal targeting are prepared, providing an efficient and stable microbial solution for adjuvant hypoglycemic and nutritional intervention.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides a bacterial strain with hypoglycemic effect. The strain is obtained by culturing Bifidobacterium animalis subsp. lactis HN1000 in a hypoglycemic induction medium. Bifidobacterium animalis subsp. lactis HN1000 is isolated from dairy products and deposited on June 5, 2024, at the China General Microbiological Culture Collection Center (CGMCC) located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. It is classified and named Bifidobacterium animalis subsp. lactis. The viability test result shows that it is viable, and the accession number is CGMCC NO.30862.

[0006] The present invention also provides a method for culturing a strain with hypoglycemic effect. The strain culturing method uses a hypoglycemic induction medium, which includes the following raw materials: MRS broth medium, wolfberry polysaccharide, bitter melon peptide powder, chromium-enriched yeast, xylooligosaccharide and distilled water, in a mass ratio of 55:8:10:0.5:17:910.

[0007] The specific steps for culturing the strain with hypoglycemic effect are as follows: S1: Dissolve MRS broth culture medium in 70℃ distilled water and autoclave to obtain basic broth. Dissolve MRS broth culture medium, wolfberry polysaccharide, bitter melon peptide powder and xylooligosaccharide in 70℃ distilled water, autoclave and then add chromium-enriched yeast to obtain hypoglycemic induction culture medium. S2: Bifidobacterium animalis subsp. lactis HN1000 was inoculated into basal broth for activation and anaerobic incubation at 37°C for 24 h to obtain seed culture. The seed culture was then inoculated into a mixed culture medium prepared by basal broth and hypoglycemic induction medium (mass ratio 1:1) at a 5% inoculation rate and incubated at 35°C and 5% dissolved oxygen for 12 h to allow Bifidobacterium animalis subsp. lactis HN1000 to slowly adapt to the functional substrate in the hypoglycemic induction medium and obtain pre-adapted bacterial culture. S3: The pre-adapted bacterial solution was inoculated into the hypoglycemic induction medium at a 10% inoculum and incubated at 30℃, 10% dissolved oxygen, and 150 rpm shaking for 12 h to induce culture, thus obtaining the first induction solution. The first induction solution was inoculated into the hypoglycemic induction medium at a 5% inoculum and the induction culture was repeated twice to obtain the third induction solution, which promoted the absorption and utilization of functional components by Bifidobacterium animalis subsp. lactis HN1000 and stimulated the hypoglycemic metabolic pathway. S4: Inoculate the three induction solutions into the fermenter at a 5% inoculation rate, and ferment for 8 hours at 37℃, anaerobic environment, 60 rpm rotation speed, and 0.3 L / min ventilation rate to obtain fermentation broth. Centrifuge the fermentation broth at 4℃ and 8000 rpm for 15 min, and collect the bacterial sludge precipitate, which is the strain with hypoglycemic effect.

[0008] The present invention also provides an application of a strain with hypoglycemic effect in the preparation of an auxiliary hypoglycemic product, wherein the hypoglycemic product is a probiotic tablet, and the strain with hypoglycemic effect in the application of preparing probiotic tablets is in the form of lyophilized probiotic powder, wherein the lyophilized probiotic powder is prepared from a strain with hypoglycemic effect, skim milk and monosodium glutamate in a mass ratio of 91.5:8:0.5.

[0009] The probiotic tablets comprise the following ingredients in parts by weight: 30-45 parts of probiotic freeze-dried powder, 10-14 parts of Bacillus coagulans freeze-dried powder, 20-30 parts of galactooligosaccharides, 12 parts of whey protein, 10 parts of sodium alginate, 6 parts of microcrystalline cellulose, 4 parts of mulberry leaf extract, 3 parts of resistant starch, and 0.5 parts of magnesium stearate.

[0010] The preparation method of the probiotic tablets is as follows: Step 1: Weigh 30-45 parts of probiotic freeze-dried powder, 10-14 parts of Bacillus coagulans freeze-dried powder, 4 parts of mulberry leaf extract and 3 parts of resistant starch, mix them well, and then use 10% (mass fraction) maltitol solution as a binder for wet granulation and drying to obtain composite particles. Step 2: Weigh 12 parts whey protein and 10 parts sodium alginate and dissolve them in 100 parts distilled water to obtain a coating solution. Place the composite particles in a fluidized bed and spray the coating solution onto the surface of the composite particles to perform enteric coating to obtain enteric particles. Step 3: Premix enteric granules, 20-30 parts of galactooligosaccharide and 6 parts of microcrystalline cellulose in a three-dimensional mixer for 10 minutes, then add 0.5 parts of magnesium stearate and mix for 3 minutes to improve the flowability of the material and obtain the total mixture. Compress the total mixture into tablets to obtain probiotic tablets.

[0011] The beneficial effects achieved by this invention are as follows: The blood glucose-lowering strain provided by this invention has a good blood glucose-lowering function, and its blood glucose-lowering effect is further enhanced during the cultivation process using the blood glucose-lowering induction medium. The wolfberry polysaccharide in the blood glucose-lowering induction medium acts as a metabolic activator, activating the strain's signaling pathway, enhancing the expression of glucose transporter proteins, and increasing glucose uptake capacity. The bitter melon peptide powder contains bitter melon extract analogues, which can induce the strain to secrete insulin-like active peptides and improve insulin receptor sensitivity. The bioactive chromium in chromium-enriched yeast promotes insulin signal transduction.

[0012] In the preparation of probiotic tablets, strains with hypoglycemic effects achieve the purpose of assisting in blood sugar reduction through the synergistic effect of multi-level formulation components. Bacillus coagulans and induced-cultured Bifidobacterium animalis subsp. lactis HN1000 form an acid-resistant symbiotic system, ensuring efficient delivery of live bacteria to the intestines and providing an anaerobic environment for the induced-cultured Bifidobacterium animalis subsp. lactis HN1000. Mulberry leaf extract contains DNJ (deoxynojirimycin), which can act as a potent α-glucosidase inhibitor, regulating blood sugar by inhibiting sugar conversion. The enteric coating layer formed by sodium alginate and whey protein controls the release of live bacteria and DNJ in the small intestine. Whey protein not only strengthens the gastric acid barrier as a coating layer but also provides nutrients, and its free form can promote incretin secretion. Galacto-oligosaccharides and mulberry leaf extract construct a microecological environment for sustained blood sugar reduction. Through the synergistic effect of these components, the system achieves the functional effects of inhibiting sugar absorption and improving insulin sensitivity. Attached Figure Description

[0013] Figure 1 Results of constructing the insulin-resistant cell model IR-HepG2; Figure 2 The results of the insulin sensitivity index study of the probiotic tablets prepared in Example 3 and Comparative Examples 1-2; Figure 3 The results show the inhibition rates of α-amylase and α-glucosidase activities of the strains before and after glucose-lowering induction culture. Figure 4 The results show the cytotoxicity of the fermentation supernatant of the strain before and after glucose-lowering induction culture on IR-HepG2 cells. Detailed Implementation

[0014] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0016] Unless otherwise specified, all methods described in the following embodiments are conventional. Unless otherwise specified, all raw materials used in the following embodiments are new materials purchased from the market, and all quantities are by weight. The live bacteria count of the lyophilized probiotic powder used is approximately 7 × 10⁻⁶. 10CFU / g, moisture ≤3%; the viable count of the Bacillus coagulans lyophilized powder used is approximately 1.0 × 10⁻⁶. 11 CFU / g; the wolfberry polysaccharide used is food-grade wolfberry extract with a polysaccharide content of 90%; the bitter melon peptide powder used is food-grade bitter melon enzymatic hydrolysate, mainly oligopeptides, with an effective substance content of 99% and a molecular weight ≤1000 Da; the chromium-enriched yeast used is enriched from brewer's yeast, with a chromium content ≥2000 μg / g and a moisture content ≤6%; the xylooligosaccharide used is food-grade XOS-95 type; the whey protein used is food-grade WPC80, with a protein content ≥80%, lactose content ≤6%, and fat ≤6%; the sodium alginate used is high-G type, with guluronic acid ≥65%; the mulberry leaf extract used is a food-grade nutritional fortifier; the resistant starch used is RS3 type, with an amylose content ≥35%.

[0017] In the following examples and comparative examples, the preparation method of the probiotic freeze-dried powder is as follows: S1: Dissolve 55 parts of MRS broth culture medium in 945 parts of 70℃ distilled water, and autoclave at 121℃ for 20 min to obtain the basic broth. Dissolve 55 parts of MRS broth culture medium, 8 parts of wolfberry polysaccharide, 10 parts of bitter melon peptide powder and 17 parts of xylooligosaccharide in 70℃ distilled water, autoclave at 121℃ for 20 min and then add 0.5 parts of chromium-enriched yeast to obtain the glucose-lowering induction culture medium. S2: Using an inoculation loop, *Bifidobacterium animalis* subsp. *lactum* HN1000 was inoculated into basal broth at a 3% inoculation rate for activation. The mixture was then anaerobically incubated at 37°C for 24 h to obtain a seed culture with a viable count of 6 × 10⁻⁶. 9 CFU / g, the seed culture was inoculated at a rate of 5% into a mixed medium made of basal broth and hypoglycemic induction medium (mass ratio 1:1) for subculture. The culture was incubated at 35℃ and 5% dissolved oxygen for 12 h to allow Bifidobacterium animalis subsp. lactis HN1000 to slowly adapt to the functional substrate in the hypoglycemic induction medium and obtain a pre-adapted bacterial culture. S3: The pre-adapted bacterial culture was inoculated into the hypoglycemic induction medium at a 10% inoculum and incubated at 30℃, 10% dissolved oxygen, and 150 rpm shaking for 12 h to induce culture, thus obtaining the first induction solution. The first induction solution was inoculated into the hypoglycemic induction medium at a 5% inoculum and the induction culture was repeated twice to ensure the stability of the metabolic phenotype, thus obtaining the third induction solution. This promoted the absorption and utilization of functional components by Bifidobacterium animalis subsp. lactis HN1000 and stimulated the hypoglycemic metabolic pathway. S4: The three induction solutions were inoculated into the fermenter at a 5% inoculation rate. Fermentation was carried out at 37°C in an anaerobic environment with a rotation speed of 60 rpm and a ventilation rate of 0.3 L / min for 8 h to obtain the fermentation broth. The fermentation broth was centrifuged at 4°C and 8000 rpm for 15 min to collect the bacterial sludge precipitate. Skim milk and sodium glutamate were added to the bacterial sludge precipitate in a mass ratio of 91.5:8:0.5 and then freeze-dried to obtain probiotic freeze-dried powder.

[0018] Example 1: This example provides a strain with hypoglycemic effect. The strain is Bifidobacterium animalis subsp. lactis HN1000, isolated from dairy products.

[0019] This embodiment also provides the application of a strain with hypoglycemic effect in the preparation of probiotic tablets. The probiotic tablets comprise the following raw materials in parts by weight: 30 parts of lyophilized probiotic powder, 14 parts of lyophilized Bacillus coagulans powder, 30 parts of galactooligosaccharides, 12 parts of whey protein, 10 parts of sodium alginate, 6 parts of microcrystalline cellulose, 4 parts of mulberry leaf extract, 3 parts of resistant starch, and 0.5 parts of magnesium stearate. The specific preparation method is as follows: Step 1: Weigh 30 parts of probiotic freeze-dried powder, 14 parts of Bacillus coagulans freeze-dried powder, 4 parts of mulberry leaf extract and 3 parts of resistant starch and mix them in a three-dimensional motion mixer at 20 rpm for 15 min. Then, use 11 parts of 10% maltitol solution as a binder for wet granulation. The extrusion granulation screen is 16 mesh and the extrusion pressure is 0.8 MPa. The mixture is then dried in a fluidized bed at 40℃ to obtain composite particles. Step 2: Weigh 12 parts of whey protein and 10 parts of sodium alginate and dissolve them in 100 parts of preheated distilled water to obtain a coating solution. Place the composite particles in a fluidized bed and spray the coating solution onto the surface of the composite particles for enteric coating. The inlet air temperature is 40℃, the atomization pressure is 1.0 bar, and the coating rate is 6 mL / min to obtain enteric particles. Step 3: Premix enteric granules, 30 parts of galactooligosaccharide and 6 parts of microcrystalline cellulose in a three-dimensional motion mixer for 10 min, then add 0.5 parts of magnesium stearate and mix for 3 min to improve the flowability of the material to obtain a total mixture. Compress the total mixture into tablets at a pressure of 8 kN to obtain probiotic tablets with a weight of 1000±30 mg and a hardness of 85±5 N.

[0020] Example 2: This example provides a strain with hypoglycemic effect, the strain being Bifidobacterium animalis subsp. lactis HN1000.

[0021] This embodiment also provides the application of a strain with hypoglycemic effect in the preparation of probiotic tablets. The probiotic tablets comprise the following raw materials in parts by weight: 38 parts of lyophilized probiotic powder, 12 parts of lyophilized Bacillus coagulans powder, 24 parts of galactooligosaccharides, 12 parts of whey protein, 10 parts of sodium alginate, 6 parts of microcrystalline cellulose, 4 parts of mulberry leaf extract, 3 parts of resistant starch, and 0.5 parts of magnesium stearate. The specific preparation method is as follows: Step 1: Weigh 38 parts of probiotic freeze-dried powder, 12 parts of Bacillus coagulans freeze-dried powder, 4 parts of mulberry leaf extract and 3 parts of resistant starch and mix them in a three-dimensional motion mixer at 20 rpm for 15 min. Then, use 14 parts of 10% maltitol solution as a binder for wet granulation. The extrusion granulation screen is 16 mesh and the extrusion pressure is 0.8 MPa. The mixture is then dried in a fluidized bed at 40℃ to obtain composite particles. Step 2: Weigh 12 parts of whey protein and 10 parts of sodium alginate and dissolve them in 100 parts of preheated distilled water to obtain a coating solution. Place the composite particles in a fluidized bed and spray the coating solution onto the surface of the composite particles for enteric coating. The inlet air temperature is 40℃, the atomization pressure is 1.0 bar, and the coating rate is 6 mL / min to obtain enteric particles. Step 3: Premix enteric granules, 24 parts of galactooligosaccharide and 6 parts of microcrystalline cellulose in a three-dimensional motion mixer for 10 min, then add 0.5 parts of magnesium stearate and mix for 3 min to improve the flowability of the material to obtain a total mixture. Compress the total mixture into tablets at a pressure of 8 kN to obtain probiotic tablets with a weight of 1000±30 mg and a hardness of 85±5 N.

[0022] Example 3: This example provides a strain with hypoglycemic effect, the strain being Bifidobacterium animalis subsp. lactis HN1000.

[0023] This embodiment also provides the application of a strain with hypoglycemic effect in the preparation of probiotic tablets. The probiotic tablets comprise the following raw materials in parts by weight: 44 parts of lyophilized probiotic powder, 10 parts of lyophilized Bacillus coagulans powder, 20 parts of galactooligosaccharides, 12 parts of whey protein, 10 parts of sodium alginate, 6 parts of microcrystalline cellulose, 4 parts of mulberry leaf extract, 3 parts of resistant starch, and 0.5 parts of magnesium stearate. The specific preparation method is as follows: Step 1: Weigh 44 parts of probiotic freeze-dried powder, 10 parts of Bacillus coagulans freeze-dried powder, 4 parts of mulberry leaf extract and 3 parts of resistant starch and mix them in a three-dimensional motion mixer at 20 rpm for 15 min. Then, use 16 parts of 10% maltitol solution as a binder for wet granulation. The extrusion granulation screen is 16 mesh and the extrusion pressure is 0.8 MPa. The mixture is then dried in a fluidized bed at 40℃ to obtain composite particles. Step 2: Weigh 12 parts of whey protein and 10 parts of sodium alginate and dissolve them in 100 parts of preheated distilled water to obtain a coating solution. Place the composite particles in a fluidized bed and spray the coating solution onto the surface of the composite particles for enteric coating. The inlet air temperature is 40℃, the atomization pressure is 1.0 bar, and the coating rate is 6 mL / min to obtain enteric particles. Step 3: Premix enteric granules, 20 parts of galactooligosaccharide and 6 parts of microcrystalline cellulose in a three-dimensional motion mixer for 10 min, then add 0.5 parts of magnesium stearate and mix for 3 min to improve the flowability of the material to obtain a total mixture. Compress the total mixture into tablets at a pressure of 8 kN to obtain probiotic tablets with a weight of 1000±30 mg and a hardness of 85±5 N.

[0024] Example 4: This example provides a strain with hypoglycemic effect, the strain being Bifidobacterium animalis subsp. lactis HN1000.

[0025] This embodiment also provides the application of a strain with hypoglycemic effect in the preparation of probiotic tablets. The probiotic tablets comprise the following raw materials in parts by weight: 45 parts of lyophilized probiotic powder, 14 parts of lyophilized Bacillus coagulans powder, 30 parts of galactooligosaccharides, 12 parts of whey protein, 10 parts of sodium alginate, 6 parts of microcrystalline cellulose, 4 parts of mulberry leaf extract, 3 parts of resistant starch, and 0.5 parts of magnesium stearate. The specific preparation method is as follows: Step 1: Weigh 45 parts of probiotic freeze-dried powder, 14 parts of Bacillus coagulans freeze-dried powder, 4 parts of mulberry leaf extract and 3 parts of resistant starch and mix them in a three-dimensional motion mixer at 20 rpm for 15 min. Then, use 17 parts of 10% maltitol solution as a binder for wet granulation. The extrusion granulation screen is 16 mesh and the extrusion pressure is 0.8 MPa. The mixture is then dried in a fluidized bed at 40℃ to obtain composite particles. Step 2: Weigh 12 parts of whey protein and 10 parts of sodium alginate and dissolve them in 100 parts of preheated distilled water to obtain a coating solution. Place the composite particles in a fluidized bed and spray the coating solution onto the surface of the composite particles for enteric coating. The inlet air temperature is 40℃, the atomization pressure is 1.0 bar, and the coating rate is 6 mL / min to obtain enteric particles. Step 3: Premix enteric granules, 30 parts of galactooligosaccharide and 6 parts of microcrystalline cellulose in a three-dimensional motion mixer for 10 min, then add 0.5 parts of magnesium stearate and mix for 3 min to improve the flowability of the material to obtain a total mixture. Compress the total mixture into tablets at a pressure of 8 kN to obtain probiotic tablets with a weight of 1000±30 mg and a hardness of 85±5 N.

[0026] Example 5: This example provides a strain with hypoglycemic effect, the strain being Bifidobacterium animalis subsp. lactis HN1000.

[0027] This embodiment also provides the application of a strain with hypoglycemic effect in the preparation of probiotic tablets. The probiotic tablets comprise the following raw materials in parts by weight: 30 parts of lyophilized probiotic powder, 10 parts of lyophilized Bacillus coagulans powder, 20 parts of galactooligosaccharides, 12 parts of whey protein, 10 parts of sodium alginate, 6 parts of microcrystalline cellulose, 4 parts of mulberry leaf extract, 3 parts of resistant starch, and 0.5 parts of magnesium stearate. The specific preparation method is as follows: Step 1: Weigh 30 parts of probiotic freeze-dried powder, 10 parts of Bacillus coagulans freeze-dried powder, 4 parts of mulberry leaf extract and 3 parts of resistant starch and mix them in a three-dimensional motion mixer at 20 rpm for 15 min. Then, use 9 parts of 10% maltitol solution as a binder for wet granulation. The extrusion granulation screen is 16 mesh and the extrusion pressure is 0.8 MPa. The mixture is then dried in a fluidized bed at 40℃ to obtain composite particles. Step 2: Weigh 12 parts of whey protein and 10 parts of sodium alginate and dissolve them in 100 parts of preheated distilled water to obtain a coating solution. Place the composite particles in a fluidized bed and spray the coating solution onto the surface of the composite particles for enteric coating. The inlet air temperature is 40℃, the atomization pressure is 1.0 bar, and the coating rate is 6 mL / min to obtain enteric particles. Step 3: Premix enteric granules, 20 parts of galactooligosaccharide and 6 parts of microcrystalline cellulose in a three-dimensional motion mixer for 10 min, then add 0.5 parts of magnesium stearate and mix for 3 min to improve the flowability of the material to obtain a total mixture. Compress the total mixture into tablets at a pressure of 8 kN to obtain probiotic tablets with a weight of 1000±30 mg and a hardness of 85±5 N.

[0028] The difference between Comparative Example 1 and Example 3 is that the Bifidobacterium animalis subsp. lactis HN1000 strain was not cultured in a hypoglycemic induction medium, that is, basal broth was used instead of hypoglycemic induction medium during the strain culture process, and the rest was the same as in Example 3. The difference between Comparative Example 2 and Example 3 is that an equal weight of edible starch was used instead of probiotic freeze-dried powder, while the rest of the parts were the same as in Example 3.

[0029] Construction of Insulin Resistance Cell Model Establishment of glucose standard curve: A 0.4 mg / mL glucose standard solution was prepared and serially diluted with glucose assay buffer to obtain standard solutions of different concentrations: 0, 0.01, 0.025, 0.05, 0.1, 0.2, 0.3, and 0.4 mg / mL. These solutions were then added to 96-well plates, and absorbance was measured at A510 nm. Using wells containing only glucose as the blank background, a standard curve was fitted and calculated.

[0030] Cell culture: HepG2 cells were seeded in culture flasks and cultured until 70%–90% confluence was achieved; then the culture medium was replaced with high-glucose DMEM, and 1×10⁻⁶ cells were added. -7 An IR-HepG2 insulin resistance model was established by inducing insulin resistance for 48 h with mol / L insulin.

[0031] HepG2 cells and IR-HepG2 cells were transferred to cell culture flasks and cultured in DMEM high-glucose medium. After two passages, cell counts were performed at a density of 5 × 10⁶ cells / year. 3 Cells were seeded at a concentration of [number] cells / well in 96-well plates and cultured overnight to obtain a blank control group (HepG2) and a model group (IR-HepG2). Each group had six replicates. After culturing in a cell incubator for 48 hours, glucose levels were measured using a microplate reader. Glucose consumption in different groups was calculated based on a glucose standard curve. Results are shown below. Figure 1 .

[0032] Model validation: The glucose consumption in the cell culture medium was detected using a glucose assay kit. If the glucose consumption in the model group was significantly reduced (P<0.05) compared with the normal HepG2 control group, it indicated that the model was successfully constructed.

[0033] Insulin sensitivity testing The probiotic tablets prepared in Example 3 and Comparative Examples 1-2 were pulverized, weighed, and prepared into a 1 mg / mL bacterial solution. IR-HepG2 cells were then taken and cultured at a concentration of 5 × 10⁻⁶. 3 Cells were seeded at a concentration of 10 μL / well in 96-well plates and cultured overnight. 10 μL of bacterial suspension prepared according to Example 3 and Comparative Examples 1-2 were added to each well, and the volume was brought up to 100 μL with pH 7.4 PBS buffer. This yielded the control group (IR-HepG2), Comparative Example 1 group (IR-HepG2 + Comparative Example 1 bacterial suspension), Comparative Example 2 group (IR-HepG2 + Comparative Example 2 bacterial suspension), and Example 3 group (IR-HepG2 + Example 3 bacterial suspension). After 48 h of incubation in the dark, glucose levels were measured. The insulin sensitivity index was calculated as: (Glucose consumption in experimental groups / Glucose consumption in control groups) × 100%. Results are shown in the table below. Figure 2 .

[0034] Study on the hypoglycemic ability of the strain Bacterial culture: Using a sterile inoculation loop, dip into Bifidobacterium animalis subsp. lactis HN1000 and the three induction solutions after incubation on glucose-lowering induction medium, respectively, and plot in sections on MRS agar plates (four-zone method). Immediately place in an anaerobic tank and incubate at 37°C for 48 h until single colonies form. Pick a single colony and inoculate it into the basic broth, and incubate anaerobically at 37°C for 24 h to obtain the fermentation broth. Centrifuge the fermentation broth using a sterile pipette, separate the supernatant, and filter it through a filter membrane to obtain the fermentation supernatant, which is divided into the original strain group and the induced strain group, and store at 4°C for later use.

[0035] Group the samples according to the following criteria, where the samples are either the original strain group or the fermentation supernatant of the induced strain group: A: Contains sample and α-amylase solution; B: Contains sample but does not contain α-amylase solution; C: Does not contain sample but contains α-amylase solution; D: Does not contain sample and α-amylase solution; E: Contains sample and α-glucosidase solution; F: Contains sample but does not contain α-glucosidase solution; G: Does not contain sample but contains α-glucosidase solution; H: Does not contain sample and α-glucosidase solution.

[0036] α-Amylase activity inhibition rate determination: 0.25 mL of fermentation supernatant from the original strain group and the induced strain group were mixed with an equal volume of 1 mg / mL α-amylase solution and incubated at 37℃ for 10 min. After the reaction, 0.5 mL of 1.5% soluble starch solution was added, and the reaction was allowed to proceed for 5 min. Then, 1 mL of DNS was added for colorimetric analysis. After mixing, the mixture was boiled in boiling water for 5 min, cooled with ice water, diluted 5-fold with pH 6.8 PBS buffer, and allowed to stand for 30 min. The absorbance was measured at 540 nm. The α-amylase activity inhibition rate was calculated according to the formula: α-amylase activity inhibition rate (%) = (1 - (AB) / (CD)) × 100%. The results are shown in the figure. Figure 3 .

[0037] Assay for α-glucosidase activity inhibition rate: 50 μL of pH 6.8 PBS buffer was mixed with 50 μL of 1.5 mM PNPG solution, followed by 25 μL of fermentation supernatant from the original and induced strain groups. After mixing, the mixture was reacted at 37℃ for 10 min. Then, 30 μL of 0.2 U / mL α-glucosidase solution was added, and the reaction was continued for 30 min. Finally, 50 μL of 0.2 M Na₂CO₃ solution was added to terminate the reaction. The absorbance at 405 nm was measured using a microplate reader. The α-glucosidase activity inhibition rate was calculated using the formula: α-glucosidase activity inhibition rate (%) = (1 - (EF) / (GH)) × 100%. The results are shown in the figure. Figure 3 .

[0038] Cell compatibility study IR-HepG2 cells were harvested at a rate of 5 × 10⁻⁶.3 Cells were seeded at a concentration of [number] cells / well into 96-well plates and cultured overnight until cell adhesion. The liquid in the wells was carefully aspirated, and then 5, 10, 15, 20, 30, and 50 μL of fermentation supernatant from the original and induced bacterial strains were added, respectively. The volume was then brought to 100 μL with pH 7.4 PBS buffer. The plates were incubated for 48 h, and cell proliferation was assessed using the MTT assay. Results are shown below. Figure 4 .

[0039] In the experiment to construct an insulin resistance cell model, the glucose standard curve equation was A = 4.378c + 0.005, R0 2 =0.9991, where A is the absorbance value and c is the glucose concentration. Figure 1 The results showed that, compared with the blank control group, the glucose consumption level in the model group was significantly reduced, and the model was successfully established.

[0040] Figure 2 The results of glucose consumption and insulin sensitivity index showed that Comparative Example 1 and Example 3 had higher insulin sensitivity, and Example 3 was more effective, indicating that the strain induced by hypoglycemic specificity had a stronger glucose consumption capacity and more sensitive insulin activity.

[0041] Figure 3 The results of the enzyme activity inhibition test showed that the inhibition rate of α-amylase and α-glucosidase activities in the fermentation supernatant of the induced strain group was 27.127% and 26.878% higher than that in the original strain group, respectively. The enzyme inhibition effect was significant, which is beneficial to reduce glucose production and play a role in lowering blood sugar.

[0042] Figure 4 MTT cell assay results showed that when the sample volume fraction was ≤20%, that is, when the volume of added fermentation supernatant was ≤20 μL, the cytotoxicity to IR-HepG2 cells was low, and the cells had good biocompatibility.

[0043] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0044] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A bacterial strain with hypoglycemic effect, characterized in that, The strain with hypoglycemic effect was obtained by culturing Bifidobacterium animalis subsp. lactis HN1000 in a hypoglycemic induction medium. The preservation number of Bifidobacterium animalis subsp. lactis HN1000 is CGMCC NO.30862. The hypoglycemic induction culture medium includes the following raw materials: MRS broth medium, wolfberry polysaccharide, bitter melon peptide powder, chromium-enriched yeast, xylooligosaccharide and distilled water.

2. A method for culturing a bacterial strain with hypoglycemic effect according to claim 1, characterized in that, The specific cultivation method and steps are as follows: S1: Weigh MRS broth medium, dissolve and sterilize to obtain basic broth, weigh MRS broth medium, wolfberry polysaccharide, bitter melon peptide powder and xylooligosaccharide, dissolve in distilled water and sterilize, then add chromium-enriched yeast to obtain hypoglycemic induction medium. S2: Take Bifidobacterium animalis subsp. lactis HN1000 and inoculate it into basal broth to activate it, and then inoculate the seed culture into a mixed medium made of basal broth and glucose-lowering induction medium for microaerobic incubation to obtain pre-adapted bacterial culture. S3: Inoculate the pre-adapted bacterial culture into the glucose-lowering induction medium and culture with aerobic shaking to obtain a first induction solution. Repeat the inoculation and aerobic shaking culture process twice to obtain a third induction solution. S4: Anaerobic fermentation of the three induction solutions, centrifugation, and collection of the precipitate yielded a strain with hypoglycemic effects.

3. The method for culturing the strain with hypoglycemic effect according to claim 2, characterized in that, The mass ratio of MRS broth medium, wolfberry polysaccharide, bitter melon peptide powder, chromium-enriched yeast, xylooligosaccharide and distilled water in the hypoglycemic induction medium is 55:8:10:0.5:17:

910. In step S2, the mass ratio of basic broth to hypoglycemic induction medium in the mixed culture medium is 1:1, and the temperature of the microaerobic incubation process is 35°C, the dissolved oxygen is 5%, and the incubation time is 12 h. In step S3, the temperature of the aerobic oscillation culture process is 30°C, the dissolved oxygen is 10%, the incubation time is 12 h, and the oscillation rate is 150 rpm.

4. The application of a bacterial strain with hypoglycemic effect according to claim 1, characterized in that, The strain with hypoglycemic effect is used to prepare probiotic tablets; The strain with hypoglycemic effect is in the form of lyophilized probiotic powder when used to prepare probiotic tablets. The probiotic freeze-dried powder is prepared from strains with hypoglycemic effects, skim milk, and monosodium glutamate; The probiotic tablets comprise the following ingredients in parts by weight: 30-45 parts of probiotic freeze-dried powder, 10-14 parts of Bacillus coagulans freeze-dried powder, 20-30 parts of galactooligosaccharides, 12 parts of whey protein, 10 parts of sodium alginate, 6 parts of microcrystalline cellulose, 4 parts of mulberry leaf extract, 3 parts of resistant starch, and 0.5 parts of magnesium stearate.

5. The application of the strain with hypoglycemic effect according to claim 4, characterized in that, The specific preparation method of the probiotic tablets is as follows: Step 1: Mix probiotic freeze-dried powder, Bacillus coagulans freeze-dried powder, mulberry leaf extract and resistant starch, then wet granulate and dry to obtain composite granules; Step 2: Weigh whey protein and sodium alginate and dissolve them to obtain a coating solution. Use the coating solution to coat the composite particles to obtain enteric-coated particles. Step 3: Premix the enteric-coated granules, galactooligosaccharides and microcrystalline cellulose, then add magnesium stearate and mix to obtain a total mixture. Compress the total mixture into tablets to obtain probiotic tablets.

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