Bifidobacterium animalis subsp. Lactis YYSJ001 with hypoglycemic effect and application thereof

Abstract

The invention relates to a bifidobacterium animalis subsp. Lactis YYSJ001 with a blood sugar reducing effect and application of the bifidobacterium animalis subsp. Lactis YYSJ001. The classification name of the strain is bifidobacterium animalis subsp. Lactis, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.33471. The invention further relates to a preparation method of the bifidobacterium animalis subsp. Lactis YYSJ001. The bacterial strain has good artificial gastric juice resistance, artificial intestinal juice resistance and intestinal tract adhesion ability, has an inhibition effect on numerous pathogenic bacteria such as escherichia coli, staphylococcus aureus, salmonella, klebsiella pneumoniae and quail chicken enterococcus, and can stimulate intestinal cells to generate glucagon-like peptide-1, so that the bacterial strain can be applied to preparation of various pathogenic bacteria. Diseases of diabetic mice can be effectively improved, and the compound can be used for preparing products for preventing and treating diabetes or assisting in reducing blood sugar.

Description

Technical Field

[0001] The present invention belongs to the technical field of probiotics, and relates to a Bifidobacterium animalis subsp. lactis YYSJ001 with blood glucose lowering efficacy and its application. Background Art

[0002] Type II diabetes is a complex chronic metabolic disease, characterized by pancreatic islet β-cell dysfunction and reduced insulin sensitivity, resulting in elevated blood glucose levels. Its inducing factors are mostly related to lifestyle factors such as genetics, age, and obesity. Patients with type II diabetes with metabolic abnormalities often have complications such as diabetic retinopathy and kidney diseases.

[0003] Improving type II diabetes by effectively regulating the intestinal flora is an effective method. Probiotics are active microorganisms ingested by the body, which can effectively improve the balance of the intestinal flora and produce good health effects on the host. Bifidobacterium animalis subsp. lactis is a type of Gram-positive bacillus, mainly present in breast milk and the feces of infants and young children, and its abundance gradually decreases or even disappears with age. Bifidobacterium animalis subsp. lactis in the human intestine plays an important role in human health. However, there are relatively few types of Bifidobacterium animalis subsp. lactis that can safely and effectively lower blood glucose and improve diabetes. Therefore, more Bifidobacterium animalis subsp. lactis with blood glucose lowering potential need to be developed in this field. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a Bifidobacterium animalis subsp. lactis YYSJ001 with blood glucose lowering efficacy and its application.

[0005] To achieve the purpose of this invention, the following technical solutions are adopted:

[0006] In the first aspect, the present invention provides a Bifidobacterium animalis subsp. lactis YYSJ001 with blood glucose lowering efficacy. The taxonomic name of the Bifidobacterium animalis subsp. lactis YYSJ001 is Bifidobacterium animalis subsp. lactis, and the deposit number is CGMCC No. 33471, and the deposit date is January 20, 2025.

[0007] A new strain YYSJ001 with blood glucose lowering effect was isolated from infant feces and preserved. After sequencing analysis, the 16S rDNA sequence of this strain is shown as SEQ ID No:1. The sequenced sequence was compared with nucleic acid sequences in GeneBank, and the result shows that the strain is Bifidobacterium animalis subsp. lactis. This strain has good abilities of resisting artificial gastric juice, resisting artificial intestinal juice, and intestinal adhesion, and has inhibitory effects on many pathogenic bacteria, such as Escherichia coli, Staphylococcus aureus, Salmonella, Klebsiella pneumoniae, and Enterococcus gallinarum. This strain can stimulate intestinal cells to produce glucagon-like peptide-1 (GLP-1). This strain can effectively control the food intake, water intake, urine volume, and body weight of diabetic mice, and can reduce the fasting blood glucose value and insulin resistance index of diabetic mice, improve the glucose absorption and metabolism ability of diabetic mice, and slow down the impairment of islet β-cell function. It can improve the disordered state of blood lipid levels in diabetic mice, reduce the inflammation level of the diabetic mouse body, and increase the anti-inflammatory factor level in the mouse body. It can improve and restore the abundance and diversity of the intestinal flora in diabetic mice, reduce the abundance of intestinal pathogenic bacteria, and increase the abundance of intestinal beneficial bacteria, and can be used to prepare products for preventing and treating diabetes or assisting in lowering blood glucose.

[0008] In a second aspect, the present invention provides a probiotic agent with blood glucose lowering effect, and the active component in the probiotic agent includes Bifidobacterium animalis subsp. lactis YYSJ001 described in the first aspect.

[0009] Preferably, the viable count of Bifidobacterium animalis subsp. lactis YYSJ001 is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g, such as 1×10 9 CFU / g (CFU / mL), 5×10 9 CFU / g (CFU / mL), 1×10 10 CFU / g (CFU / mL), 5×10 10 CFU / g (CFU / mL), 1×10 11 CFU / g (CFU / mL), 5×10 11 CFU / g (CFU / mL), 1×10 12 CFU / g (CFU / mL), 5×10 12 CFU / g (CFU / mL), 1×10 13 CFU / g (CFU / mL), etc. Other specific point values within this numerical range can be selected and will not be elaborated one by one here.

[0010] Preferably, the dosage form of the probiotic agent includes solution, powder, tablet, capsule, and granule. The dosage form of the probiotic agent involved in the present invention is not limited, including the most common solution and lyophilized powder, or further prepared capsule, tablet, or granule.

[0011] Preferably, the probiotic agent also contains excipients, and the excipients include any one or a combination of at least two of carrier, diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, solubilizer, cosolvent, osmotic pressure regulator, surfactant, coating material, colorant, pH regulator, antioxidant, bacteriostatic agent, or buffer.

[0012] More preferably, the active component in the probiotic agent further includes metformin or its salt.

[0013] The above-mentioned YYSJ001 strain can not only be used alone to assist in improving diabetes or reducing blood sugar, but also can be used in combination with the drug metformin or its salt. Metformin is a first-line drug for the treatment of type II diabetes.

[0014] The probiotic agent involved in the present invention combines the Bifidobacterium animalis subsp. lactis YYSJ001 strain and the drug metformin or its salt. It is found that the two can cooperate and promote each other in reducing blood sugar and improving diabetes. Compared with the single strain intervention method or the single drug intervention method, the combination of the two active components can improve the effect of reducing blood sugar and improving diabetes with a lower intervention amount.

[0015] Preferably, the usage ratio of the Bifidobacterium animalis subsp. lactis YYSJ001 to metformin or its salt is (10 7 -10 9 ) CFU: (1 - 5) μg.

[0016] Among them, the specific point value in (10 7 -10 9 ) can be selected as 1×10 7 , 3×10 7 , 5×10 7 , 8×10 7 , 1×10 8 , 2×10 8 , 4×10 8 , 6×10 8 , 8×10 8 , 1×10 9 etc.

[0017] Among them, the specific point value in (1 - 5) can be selected as 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, etc.

[0018] In a third aspect, the present invention provides the use of Bifidobacterium animalis subsp. lactis YYSJ001 described in the first aspect or the probiotic agent described in the second aspect in the preparation of a preparation for promoting intestinal cells to secrete glucagon-like peptide-1.

[0019] The present invention also develops another use of the strain Bifidobacterium animalis subsp. lactis YYSJ001, that is, using it as a preparation for promoting intestinal cells to secrete glucagon-like peptide-1. According to the research results of the present invention, the strain Bifidobacterium animalis subsp. lactis YYSJ001 has an excellent effect of stimulating intestinal cells to produce glucagon-like peptide-1. Therefore, the results indicate that the strain Bifidobacterium animalis subsp. lactis YYSJ001 can also be used as a simple reagent for in vitro experiments in the scientific research field.

[0020] In a fourth aspect, the present invention provides the use of Bifidobacterium animalis subsp. lactis YYSJ001 described in the first aspect or the probiotic agent described in the second aspect in the preparation of a product for preventing and treating diabetes or assisting in reducing blood sugar.

[0021] In a fifth aspect, the present invention provides the use of Bifidobacterium animalis subsp. lactis YYSJ001 described in the first aspect or the probiotic agent described in the second aspect in the preparation of a product having any one or at least two of the following effects:

[0022] 1) Improving the ability of glucose absorption and metabolism; 2) Slowing down the impairment of pancreatic islet β cell function in diabetes; 3) Improving the blood lipid disorder in diabetes; 4) Reducing the inflammation level and enhancing the immunity; 5) Improving the richness and diversity of the intestinal flora in the diabetic body, reducing the abundance of intestinal pathogenic bacteria and increasing the abundance of intestinal beneficial bacteria; 6) Inhibiting pathogenic bacteria, and the antibacterial spectrum of the antibacterial includes Escherichia coli, Salmonella, Staphylococcus aureus, Klebsiella pneumoniae, Enterococcus gallinarum.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention isolated and preserved a new strain YYSJ001 with blood sugar lowering efficacy from infant feces. After sequencing analysis, its 16S rDNA sequence is as shown in SEQ ID No: 1. The sequenced sequence was compared with the nucleic acid sequence in GeneBank, and the results showed that the strain is Bifidobacterium animalis subsp. lactis. The strain has good abilities of resistance to artificial gastric juice, resistance to artificial intestinal juice, and intestinal adhesion ability, and has an inhibitory effect on many pathogenic bacteria, such as Escherichia coli, Staphylococcus aureus, Salmonella, Klebsiella pneumoniae, Enterococcus gallinarum; the strain can stimulate intestinal cells to produce glucagon-like peptide-1 (GLP-1); the strain can effectively improve the symptoms of diabetic mice and can be used to prepare a product for preventing and treating diabetes or assisting in reducing blood sugar. Description of the Drawings

[0025] Figure 1 It is a statistical result graph of the average food intake of each group of mice;

[0026] Figure 2 It is a statistical result graph of the average water intake of each group of mice;

[0027] Figure 3 It is a statistical result graph of the average body weight of each group of mice;

[0028] Figure 4 It is a statistical result graph of the measurement results of fasting blood glucose (FBG) of each group of mice;

[0029] Figure 5 It is a statistical result graph of the measurement results of glycated serum protein (GSP) of each group of mice;

[0030] Figure 6 It is a statistical result graph of the area under the oral glucose tolerance curve of each group of mice;

[0031] Figure 7 It is a statistical result graph of the insulin resistance index of each group of mice;

[0032] Figure 8 It is a statistical result graph of the measurement results of glucagon-like peptide-1 (GLP-1) of each group of mice;

[0033] Figure 9 It is a statistical result graph of the levels of blood lipid indexes of each group of mice;

[0034] Figure 10 It is a statistical result graph of the levels of serum inflammatory factors of each group of mice;

[0035] Figure 11 It is a statistical result graph of the Chao1 index and Simpson index of the intestinal flora of each group of mice;

[0036] Figure 12 It is a statistical result graph of the NMDS analysis results of the intestinal flora of each group of mice;

[0037] Figure 13 It is a statistical result graph of the relative abundances of the intestinal flora of each group of mice;

[0038] Different letters in the graph represent statistical differences between groups (p < 0.05);

[0039] The taxonomic naming of the YYSJ001 strain involved in the present invention is Bifidobacterium animalis subsp. lactis, the preservation time is January 20, 2025, the preservation number is CGMCC No. 33471, the preservation unit is the China General Microbiological Culture Collection Center, and the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Specific implementation manners

[0040] The technical solutions of the present invention will be further described below through specific implementation manners. Those skilled in the art should understand that the embodiments are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0041] The classification and naming of the YYSJ001 strain involved below is Bifidobacterium animalis subsp. lactis. The preservation time is January 20, 2025, and the preservation number is CGMCC No. 33471. Metformin hydrochloride (MET) is a product purchased from Macklin with the reagent model number M813341.

[0042] The preparation method of the YYSJ001 bacterial suspension involved in the following experiments: Inoculate the YYSJ001 strain into L-MRS liquid medium, culture at 37°C for 18 h for activation, and activate continuously for 2 times to obtain an activation solution; inoculate the activation solution into L-MRS liquid medium at an inoculation amount of 2% (v / v), and culture at 37°C for 15 h to obtain a bacterial solution; centrifuge the bacterial solution at 8000 rpm for 1 min to obtain bacterial cells, resuspend the bacterial cells with physiological saline and dilute as needed to obtain the YYSJ001 bacterial suspension.

[0043] L-MRS liquid medium: Peptone 10 g / L, Beef extract 10 g / L, Glucose 20 g / L, Sodium acetate 2 g / L, Yeast extract 5 g / L, Diammonium hydrogen citrate 2 g / L, Dipotassium hydrogen phosphate 2 g / L, Magnesium sulfate heptahydrate 0.58 g / L, Manganese sulfate 0.25 g / L, Tween 80 1 mL / L, Cysteine hydrochloride 0.1 g / L.

[0044] Example 1

[0045] Screening and identification of Bifidobacterium animalis subsp. lactis YYSJ001:

[0046] (1) Place the collected infant feces in a sterile sampling tube and transport it in an ice box. Dilute it step by step with 0.85% physiological saline under sterile conditions. Select an appropriate dilution gradient and spread it on an LMRS + lithium salt agar plate, and culture at 37°C for 48 hours. Visually observe the colony morphology and pick suspected single colonies, and conduct microscopic examination, preliminary screening and purification culture. After purification, culture in an LMRS liquid anaerobic tube at 37°C for 18 hours, centrifuge to remove the supernatant, resuspend in a sterile 25% glycerol aqueous solution, and store in an ultra-low temperature refrigerator.

[0047] (2) Liquidly expand the screened target strain, collect the bacterial cells, extract genomic DNA, amplify using universal primers, and amplify its 16S rDNA fragment. Detect the PCR amplification product by agarose gel electrophoresis and sequence the PCR product. Send the PCR product for sequencing to Wuhan Kingcare Biotechnology Co., Ltd. after detecting by gel electrophoresis. Compare the identified gene sequence with the NCBI database using the BLAST tool. According to the results of molecular biological identification, the Latin name of the strain category is Bifidobacterium animalis subsp. lactis, and it is determined that this strain is Bifidobacterium animalis subsp. lactis. Name this strain Bifidobacterium animalis subsp. lactis YYSJ001 and send it for preservation.

[0048] Example 2

[0049] Evaluation of the tolerance of Bifidobacterium animalis subsp. lactis YYSJ001 to artificial gastric juice and intestinal juice:

[0050] (1) Preparation of artificial gastric juice: Prepare 0.5% sodium chloride solution, adjust the pH to 3 with 1 mol / L HCl, add 0.3% pepsin, and then filter and sterilize with a 0.22 μm microporous filter membrane after fully dissolving for standby.

[0051] Preparation of artificial intestinal juice: Prepare 0.5% sodium chloride solution, adjust the pH value to 8 with 0.1 mol / L NaOH, add 0.1% trypsin, and then filter and sterilize with a 0.22 μm microporous filter membrane after fully dissolving for standby.

[0052] (2) After activating the strain to be tested for 3 generations, adjust the bacterial liquid concentration to 10 8 CFU / mL. Take 1 mL of the bacterial suspension, centrifuge to collect the bacterial cells, and separately inoculate them into 1 mL of the prepared artificial gastric juice or intestinal juice and mix well. Digest at 37 °C, and at the same time, take the digestive juices at 0 h and 3 h to detect the viable bacteria count and calculate the survival rate, with parallel determination 3 times. Among them, the strain survival rate (%) = N t / N 0 ×100%, where N 0 represents the viable bacteria count of the strain at 0 h, CFU / mL; N t represents the viable bacteria count of the strain at 3 h, CFU / mL.

[0053] The results show that the survival rate of strain YYSJ001 in artificial gastric juice is 99.67 ± 0.34%, and the survival rate in artificial intestinal juice is 94.20 ± 0.36%. It shows that Bifidobacterium animalis subsp. lactis YYSJ001 has good tolerance to gastric acid and intestinal juice.

[0054] Example 3

[0055] Evaluation of the intestinal colonization ability of Bifidobacterium animalis subsp. lactis YYSJ001:

[0056] (1) The human colon cancer cell line Caco-2 was obtained from the China Center for Type Culture Collection (Wuhan, China). Caco-2 cells were inoculated into DMEM medium supplemented with 20% (v / v) fetal bovine serum, transferred to a 12-well cell culture plate, and the cell seeding density was 2.4×10 5 cells / mL. The cells were incubated at 37°C with 5% CO 2 , and the medium was changed every other day until a confluent monolayer was obtained for further use;

[0057] (2) For the confluent monolayer of prepared Caco-2 cells, the medium was aspirated, and the cells were rinsed twice with PBS buffer. After aspirating the buffer, 1 mL / well of the prepared bacterial suspension (1×10 8 CFU / mL) was added. After mixing, the cells were co-incubated with the bacteria at 37°C with 5% CO 2 for 2 h;

[0058] (3) The culture supernatant was carefully removed, and the cells were rinsed 5 times with sterile PBS to remove non-adherent bacteria. 0.2 mL / well of trypsin cell digestive solution was added and the cells were digested for 5 min to detach the cells from the wells of the culture plate. The collected samples were serially diluted and viable cell counts were performed in triplicate. The adhesion ability (CFU / cell) = the number of bacteria adhered to the cells (CFU) / the number of cells in the well (cells).

[0059] The results showed that the adhesion ability of strain YYSJ001 to cells was 10.63±1.31 CFU / cell, indicating that Bifidobacterium animalis subsp. lactis YYSJ001 has good colonization ability on intestinal epithelial cells.

[0060] Example 4

[0061] Evaluation of the anti-pathogenic ability of Bifidobacterium animalis subsp. lactis YYSJ001:

[0062] The information of pathogenic bacteria used in this example: Escherichia coli (ATCC25922), Salmonella (ATCC14028), Staphylococcus aureus (ATCC25923), Klebsiella pneumoniae (GenBank: OR755477.1), Enterococcus gallinarum (GenBank: PP961529.1). The BHI medium was purchased from Qingdao Haibo Biotechnology Co., Ltd., and sterile defibrinated sheep blood was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0063] Cool the BHI + 5% sheep blood medium containing 1.5% agar to about 55°C, mix it with the bacterial suspensions of Escherichia coli, Salmonella, Staphylococcus aureus, Klebsiella pneumoniae, and Enterococcus gallinarum, so that the viable count of the indicator bacteria is 10 6 CFU / mL, then quickly pour it into a plate pre-placed with Oxford cups. After the medium cools and solidifies, take out the Oxford cups, and inject 200 μL of the bacterial solution of Bifidobacterium animalis subsp. lactis YYSJ001 (10 8 CFU / mL) into each well. After culturing at 37°C for 24 h, measure the diameter of the inhibition zone, and perform parallel determination 3 times. The results are shown in Table 1.

[0064] Table 1

[0065] Indicator bacteria Diameter of inhibition zone (mm) Escherichia coli 19.79±0.09 Salmonella 19.68±0.37 Staphylococcus aureus 17.15±0.19 Klebsiella pneumoniae 13.67±0.24 Enterococcus gallinarum 24.50±0.41

[0066] It can be seen from the data results in Table 1 that the YYSJ001 strain also has excellent effects in inhibiting pathogenic bacteria, which is beneficial to the intestinal microecological environment.

[0067] Example 5

[0068] Evaluation of the stimulation of glucagon-like peptide-1 production by Bifidobacterium animalis subsp. lactis YYSJ001:

[0069] Inoculate the mouse intestinal endocrine cell line (STC-1 cell line) into a 24-well plate and culture it until the density reaches 2×10 5 / well, and the cell culture reaches 80% confluence. Wash it 2 times with PBS buffer without Ca 2+ and Mg 2+ , and culture it in DMEM (0.5 mL) without glucose and glutamine for 30 min.

[0070] Take 0.5 mL of the YYSJ001 bacterial suspension (1×10 8 CFU / mL) and add it to the cell plate, and incubate it at 37°C for 4 h. Collect the supernatant into a 1.5 mL centrifuge tube, centrifuge it at 5900×g at 4°C for 10 min to remove the cell precipitate. Use a mouse GLP-1 ELISA kit to measure the content of glucagon-like peptide-1 (GLP-1), with the supernatant of STC-1 cells without inoculated bacterial suspension as the blank control, and at the same time use the commercially available Bifidobacterium animalis subsp. lactis ATCC700541 as a comparison. The results show that the GLP-1 concentration in the blank control group is 55.98 ± 0.38 b pg / mL, the GLP-1 concentration in the YYSJ001 group is 59.01 ± 0.38 a pg / mL, and the GLP-1 concentration in the ATCC700541 group is 56.21 ± 0.29 bpg / mL, indicating that compared with other Bifidobacterium animalis subsp. lactis strains, strain YYSJ001 can significantly promote the release of GLP-1 by STC-1 cells (p < 0.05).

[0071] Example 6

[0072] Evaluation of the improvement effect of Bifidobacterium animalis subsp. lactis YYSJ001 on diabetic mice:

[0073] (1) Experimental animals: Healthy male C57BL / 6j mice (5 - 6 weeks old; 19 ± 1 g) were purchased from Beijing Speyford Biotechnology Co., Ltd. The animal experiment ethics number is: Anping Center Animal (Fu) No. 202410241, and the experimental animal use license number is: SCXK (Jing) 2024 - 0001. These mice were raised in a controlled environment with the room temperature maintained at 20 - 26 °C, the humidity at 40 - 60%, following a 12 h light / dark cycle. They had free access to food and water. The bedding was changed 1 - 2 times a week.

[0074] (2) Animal grouping: After 1 week of adaptive feeding, the mice were randomly assigned to 8 groups of 8 mice each: normal group (NC group), model group (MC group), Bifidobacterium animalis subsp. lactis YYSJ001 group (YYSJ001 group, intervened with Bifidobacterium animalis subsp. lactis YYSJ001 bacterial solution), metformin hydrochloride group (MET group, intervened with metformin hydrochloride), probiotic ATCC700541 group (ATCC700541 group, intervened with commercially available Bifidobacterium animalis subsp. lactis bacterial solution), YYSJ001 + MET groups 1 - 2 (co - intervened with Bifidobacterium animalis subsp. lactis YYSJ001 bacterial solution and metformin hydrochloride in different ratios), and ATCC700541 + MET group (co - intervened with commercially available Bifidobacterium animalis subsp. lactis bacterial solution and metformin hydrochloride).

[0075] (3) Animal modeling and intervention methods:

[0076] The mice were modeled for diabetes according to the method described in the reference (F. Yan, N. Li, J. Shi, H. Li, Y. Yue, W. Jiao, N. Wang, Y. Song, G. Huo and B. Li, Lactobacillus acidophilus alleviates type 2 diabetes by regulating 2 hepatic glucose and lipid metabolism and gut microbiota in mice, Food Funct, 2019,).

[0077] After successful modeling, the mice in each group were intervened for 42 days. The mice in the normal group (NC group) and the model group (MC group) were gavaged with sterile normal saline once a day for 42 consecutive days;

[0078] The YYSJ001 group was gavaged with the suspension of Bifidobacterium animalis subsp. lactis YYSJ001 (the intervention dose was 6×10 9 CFU / d) once a day for 42 consecutive days;

[0079] The ATCC700541 group was gavaged with the suspension of Bifidobacterium animalis subsp. lactis ATCC700541 (the intervention dose was 6×10 9 CFU / d) once a day for 42 consecutive days;

[0080] The MET group was gavaged with metformin hydrochloride (MET) (the intervention dose was 0.2 g / kg / d) once a day for 42 consecutive days;

[0081] The YYSJ001+MET group 1 was gavaged with the suspension of Bifidobacterium animalis subsp. lactis YYSJ001 (the intervention dose was 6×10 7 CFU / d) and metformin hydrochloride (MET) (the intervention dose was 0.15 g / kg / d) once a day for 42 consecutive days;

[0082] The YYSJ001+MET group 2 was gavaged with the suspension of Bifidobacterium animalis subsp. lactis YYSJ001 (the intervention dose was 6×10 8 CFU / d) and metformin hydrochloride (MET) (the intervention dose was 0.15 g / kg / d) once a day for 42 consecutive days;

[0083] The ATCC700541+MET group was gavaged with the suspension of ATCC700541 (the intervention dose was 6×10 8 CFU / d) and metformin hydrochloride (MET) (the intervention dose was 0.15 g / kg / d) once a day for 42 consecutive days.

[0084] (4) Index analysis:

[0085] (4.1) Monitoring of food intake, water intake and body weight:

[0086] During the intervention period, the average food intake, average water intake and average body weight of the mice in each group were detected and counted weekly. The statistical results are as Figure 1 、 Figure 2 and Figure 3 shown.

[0087] It can be seen that compared with the normal group, the mice in the diabetes model group showed an increase in daily food intake, water intake, urine output, and a decrease in body weight. After 6 weeks of intervention in each group, the typical symptoms of diabetic mice were alleviated and improved to varying degrees. The food intake and water intake of the mice were controlled, and the body weight of the mice recovered. Among them, the effect of the YYSJ001 strain was better than that of the commercially available strain, indicating that the YYSJ001 strain involved in the present invention has better blood glucose-lowering ability. Moreover, the effect of the YYSJ001+MET group 1-2 was the best, indicating that when the YYSJ001 strain and metformin hydrochloride were used in combination, not only could the intervention doses of each be reduced, but also they had a more significant effect on improving diabetes than a single bacterial solution or a single drug, playing a synergistic promoting role.

[0088] (4.2) Effect on glucose metabolism:

[0089] (4.2.1) Fasting blood glucose value: During the period before treatment and 6 weeks of treatment, blood was collected from the tail vein of mice once a week to measure the fasting blood glucose value of the mice. The measurement results of the fasting blood glucose value (FBG) of the mice in each group are as Figure 4 shown. After the intervention ended, the glycated serum protein index of the mouse serum was measured. The glycated serum protein index reflects the average blood glucose level in the past 1-3 weeks, as Figure 5 shown.

[0090] It can be seen that after 6 weeks of different interventions, the fasting blood glucose values of diabetic mice decreased to varying degrees and approached the healthy group. Among them, the effect of the YYSJ001 strain was better than that of the commercially available strain, indicating that the YYSJ001 strain involved in the present invention has better blood glucose-lowering ability. Moreover, the effect of the YYSJ001+MET group 1-2 was the best, indicating that when the YYSJ001 strain and metformin hydrochloride were used in combination, not only could the intervention doses of each be reduced, but also they had a more significant effect on improving diabetes than a single bacterial solution or a single drug, playing a synergistic promoting role.

[0091] (4.2.2) Oral glucose tolerance: After 6 weeks of intervention, the oral glucose tolerance of mice was measured. The glucose dosage for the glucose tolerance test of mice was 2 g per kilogram of body weight, and a 20% glucose solution was prepared with physiological saline; the mice were fasted for 16 hours, and the mice maintained normal water intake during this period; before starting the glucose tolerance test, the weight of each mouse was weighed, blood was taken from the tip of the mouse's tail, and the fasting blood glucose was measured with a blood glucose meter, and the measured value was regarded as the blood glucose value at 0 min; glucose was administered by gavage and timing started from the moment it was completed. The blood glucose values of each mouse were measured at 15, 30, 60, 90, and 120 min, and the area under the oral glucose tolerance curve was calculated. The statistical results are as Figure 6 shown.

[0092] It can be seen that the area under the oral glucose tolerance curve of each intervention group increased to varying degrees compared with the model group and approached the healthy value, indicating that the YYSJ001 strain can effectively improve the glucose absorption and metabolism ability of diabetic mice.

[0093] (4.2.3) Insulin resistance index: The insulin resistance index (HOMA-IR) is a value calculated by a formula based on serum insulin levels and fasting blood glucose values. It is an indicator used to judge the degree of insulin resistance and can effectively reflect the function status of pancreatic islet β cells in the body. The statistical results are as Figure 7 shown.

[0094] It can be seen that the insulin resistance index of each intervention group decreased to varying degrees compared with the model group and approached the healthy value, indicating that the intervention of the YYSJ001 strain can effectively slow down the continuous damage of pancreatic islet β cell function, thereby effectively reducing the blood glucose value of mice, and its effect is better than that of the commercially available Bifidobacterium animalis subsp. lactis.

[0095] (4.2.4) Glucagon-like peptide-1 level: Glucagon-like peptide-1 (GLP-1) is an incretin hormone secreted by intestinal L cells, which has the effects of promoting insulin secretion and reducing blood glucose levels. After the experiment, the serum GLP-1 concentration of mice was measured. The results are as Figure 8 shown. YYSJ001 can significantly promote the production of GLP-1 by intestinal cells in T2D mice, with a better effect than the commercially available Bifidobacterium animalis subsp. lactis, and is consistent with the in vitro evaluation results.

[0096] (4.3) Effects on blood lipid levels:

[0097] After the intervention, the levels of the following indicators in the serum of mice in each group were measured, including: total triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C). The results are as Figure 9 shown.

[0098] It can be seen that compared with the mice in the model group, the blood lipid levels of the mice in each intervention group were regulated and improved to varying degrees, and the YYSJ001 strain was more obvious in reducing total cholesterol and high-density lipoprotein in the serum in the treatment of diabetic dyslipidemia than other strains of the same type.

[0099] (4.4) Effects on inflammatory factors:

[0100] After the intervention ended, whole blood was collected from the mice in each group and centrifuged to obtain serum. Mouse serum was assayed for inflammatory factors using an ELISA kit, including the concentration levels of lipopolysaccharide (LPS), tumor necrosis factor (TNF-α), interleukin-6 (IL-6), and interleukin-10 (IL-10), as Figure 10 shown.

[0101] As can be seen from the figure, compared with the mice in the model group, the levels of inflammatory factors in the mice in each intervention group were controlled to varying degrees, and the levels of anti-inflammatory factors were effectively increased. Moreover, the YYSJ001 strain was more effective than other strains of the same type in alleviating inflammation in diabetic mice, and its combination with MET was more effective than using the bacterial solution alone or MET alone.

[0102] (4.5) Effects on the intestinal flora:

[0103] After the intervention ended, the mice were sacrificed. After disinfecting the abdomen, the mice were dissected, and the cecal contents of the mice in the normal group (NC group), model group (MC group), MET group, and YYSJ001 group were taken for microbial diversity analysis. The V3-V4 hypervariable region of the 16S rDNA of all bacteria in the samples was sequenced on a sequencing platform to determine the characteristics of the intestinal microbiota.

[0104] (4.5.1) α-diversity reflects the species richness and species diversity of a sample. The Chao1 index and Simpson index are commonly used judgment indicators for the abundance and diversity of intestinal microbial colonies. The results are as Figure 11 shown. The results showed that the abundance and diversity of the intestinal flora in diabetic mice decreased compared with those in healthy mice. According to the Chao1 index analysis, the 6-week treatment with the YYSJ001 strain could increase and improve the abundance of the intestinal flora, reaching a significant difference level compared with the model group and showing no significant difference compared with the drug treatment group. According to the Shannon index analysis, after 6 weeks of intragastric administration of the YYSJ001 strain, the intestinal diversity of diabetic mice increased significantly and approached that of the healthy group. In summary, the YYSJ001 strain can increase the abundance and diversity of the intestinal flora in diabetic mice.

[0105] (4.5.2) β-diversity is used to compare the similarity in species diversity between different samples. Non-metric multidimensional scaling (NMDS) is a ranking method suitable for ecological research. It mainly simplifies the research objects (samples or variables) in multidimensional space to a low-dimensional space for positioning, analysis, and classification, while retaining the original relationships between the objects. When Stress is less than 0.2, it indicates that the NMDS analysis has a certain reliability. The closer the samples are to each other on the coordinate graph, the higher their similarity. Based on the unweighted NMDS analysis, as Figure 12As shown, the intervention of probiotic YYSJ001 and drug MET can improve the intestinal flora of diabetic mice and show differences from the model group, while approaching that of healthy mice. This indicates that strain YYSJ001 can effectively improve the intestinal flora of diabetic mice and tend towards normalcy.

[0106] (4.5.3) The impact on the intestinal flora at the genus level is as Figure 13 shown. The YYSJ001 group significantly increased the abundance of the genus Bifidobacterium in the intestinal flora of mice. It was found that the abundances of the genera Blautia, Olsenella, Dubosiella, and Lachnoclostridium in the intestines of type II diabetic mice were significantly lower compared to the healthy group, while in the YYSJ001 group and the MET group, their relative abundances were significantly increased and reached a significant difference level compared to the model group. There are research reports that an increase in the abundance of Blautia in metabolic diseases is beneficial for improving body glucose homeostasis, enhancing insulin sensitivity, and improving intestinal flora disorders. Additionally, the harmful intestinal flora genera Enterococcus, Aerococcus, and Corynebacterium were significantly increased in the model group compared to the healthy group, and showed a significant decrease in the YYSJ001 group and the MET group.

[0107] The present invention uses the above embodiments to illustrate the technical solutions of the present invention, but the present invention is not limited to the above embodiments, that is, it does not mean that the present invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvement to the present invention, the equivalent replacement of each raw material of the products of the present invention, the addition of auxiliary components, and the selection of specific methods, etc., all fall within the protection scope and the disclosure scope of the present invention.

[0108] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0109] In addition, it should be noted that in the above specific embodiments, the various specific technical features described can be combined in any appropriate manner without contradiction. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

Claims

1. An animal Bifidobacterium lactis subspecies YYSJ001 having blood sugar lowering effect, characterized in that: The classification name of the Bifidobacterium animalis subsp. lactis YYSJ001 is Bifidobacterium animalis subsp. lactis, the preservation number is CGMCC No.33471, and the preservation date is January 20, 2025.

2. A probiotic agent with blood sugar lowering effect, characterized in that: The active component in the probiotic comprises the animal Bifidobacterium lactis subspecies YYSJ001 described in claim 1.

3. The probiotic agent with hypoglycemic effect according to claim 2, characterized in that: The viable count of Bifidobacterium animalis subspecies YYSJ001 is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g.

4. The probiotic agent with hypoglycemic effect according to claim 2, characterized in that: The dosage form of the probiotics includes solution, powder, tablet or capsule.

5. The probiotic agent with hypoglycemic effect according to claim 2, characterized in that: The probiotics also contain excipients, which include any one or a combination of at least two of carriers, diluents, excipients, fillers, adhesives, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, antibacterial agents or buffers.

6. The probiotic agent with hypoglycemic effect according to claim 2, characterized in that: The active ingredient in the probiotic also includes metformin or its salt.

7. The probiotic agent with hypoglycemic effect according to claim 6, characterized in that: The ratio of animal Bifidobacterium lactis subspecies YYSJ001 to metformin or its salt is (10 7 -10 9 )CFU:(1-5)μg.

8. Use of the animal Bifidobacterium lactis subsp. YYSJ001 according to claim 1 or the probiotic according to any one of claims 2 to 7 in the preparation of a preparation for promoting the secretion of glucagon-like peptide-1 by intestinal cells.

9. Use of the animal Bifidobacterium lactis subsp. YYSJ001 according to claim 1 or the probiotic according to any one of claims 2 to 7 in the preparation of a product for preventing and treating diabetes or assisting in lowering blood sugar.

10. Use of the animal Bifidobacterium lactis subspecies YYSJ001 according to claim 1 or the probiotic agent according to any one of claims 2 to 7 in the preparation of a product having any one or at least two of the following effects: 1) Improve glucose absorption and metabolism; 2) Slow down the damage of diabetic pancreatic β-cell function; 3) Improve diabetic dyslipidemia; 4) Reduce inflammation level and improve immunity; 5) Improve the richness and diversity of intestinal flora in diabetic patients, reduce the abundance of intestinal pathogens and increase the abundance of beneficial intestinal bacteria; 6) Inhibit pathogens, and the antibacterial spectrum includes Escherichia coli, Salmonella, Staphylococcus aureus, Klebsiella pneumoniae, and Enterococcus gallinarum.

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