Bifidobacterium animalis lactis and application thereof

Abstract

This invention provides a hypoglycemic subspecies of Bifidobacterium animalis (Liver) and its applications. The Bifidobacterium animalis (Liver) subspecies is named Tianshan-1 and is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35821 on September 5, 2025. Cell experiments show that Tianshan-1 significantly promotes the secretion of GLP-1 from human colorectal adenocarcinoma cells NCI-H716 and upregulates GCG gene expression levels. Animal experiments confirm that this strain significantly increases serum GLP-1 concentration in diabetic mice and reduces fasting blood glucose and glycated hemoglobin levels, with no significant difference in hypoglycemic effect compared to the positive control. This strain is a food-grade safe probiotic with a clear mechanism of action and stable effects, providing a safe, long-lasting, and industrially viable new probiotic resource for diabetes prevention and treatment, with broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and specifically relates to a strain of Bifidobacterium lactis with hypoglycemic effect and its application. Background Technology

[0002] Type 2 diabetes is a metabolic disease caused by insulin resistance or insufficient insulin secretion. It is characterized by elevated blood glucose levels and clinically manifests as the typical "three highs and one low" symptoms: polyphagia, polydipsia, polyuria, and weight loss. Type 2 diabetes significantly increases the risk of cardiovascular diseases such as coronary heart disease, myocardial infarction, and stroke, seriously threatening patients' lives and health.

[0003] The main interventions for diabetes currently include medication, diet, and exercise, with oral hypoglycemic agents being the core treatment. Clinically, insulin secretagogues such as sulfonylureas and meglitinides are commonly used. While they can control blood sugar to some extent, they have significant limitations: these drugs carry a high risk of hypoglycemia, and also suffer from low bioavailability and rapid drug release, requiring more frequent administration to maintain efficacy. Furthermore, long-term use can easily lead to adverse consequences such as drug dependence and worsening of cardiovascular and cerebrovascular complications.

[0004] Glucagon-like peptide-1 (GLP-1) is secreted by intestinal L cells and can activate downstream signaling pathways by binding to receptors on the surface of pancreatic β cells, promoting calcium ion influx and triggering insulin exocytosis, playing a key role in blood glucose homeostasis regulation and diabetes prevention and treatment.

[0005] Existing studies have confirmed that some probiotics can promote the secretion of GLP-1 in the body. For example, the P9 protein secreted by Akkermansia muciniphila (AKK) can induce GLP-1 secretion in a calcium-dependent manner by binding to the intercellular cell adhesion molecule-2 (ICAM-2) receptor, and its outer membrane proteins also have GLP-1 secretion-promoting activity. However, Akkermansia is a next-generation probiotic and cannot be directly applied in the food industry, thus its application scenarios and scope are strictly limited.

[0006] Therefore, screening lactic acid bacteria strains that are safe for use in food and have a strong ability to promote GLP-1 secretion is of great practical significance for developing new diabetes intervention programs. Summary of the Invention

[0007] In view of the problems existing in the prior art, the purpose of this invention is to provide a blood sugar lowering effect of Bifidobacterium lactis subspecies and its application.

[0008] Although existing research has confirmed that some probiotics can improve diabetes-related symptoms to a certain extent, the effects vary significantly between different studies, and the hypoglycemic efficacy is highly uneven among different species and strains. The inventors found in previous research that the main reasons for the unstable and varied hypoglycemic effects of probiotics are twofold: First, probiotic functions are highly specific. While some health regulatory mechanisms are common in many types of probiotics, core functions such as hypoglycemia and promoting GLP-1 secretion have strict species and strain specificity, and the function of a single strain is difficult to replace by similar species. Second, diabetes itself is a highly heterogeneous metabolic disease, and the pathogenesis and pathological processes differ significantly among individuals, further amplifying the volatility of probiotic application effects.

[0009] Based on the above problems, the inventors conducted targeted screening and functional verification of food-grade safe probiotics, focusing on core targets such as promoting GLP-1 secretion, regulating GCG gene expression, and lowering blood glucose and glycated hemoglobin. Through extensive cell experiments and animal model screening, a stable and functionally defined *Bifidobacterium animalis* subspecies *Tianshan-1* was finally obtained. This strain can efficiently promote the secretion of GLP-1 by intestinal cells, activating the physiological hypoglycemic pathway at the molecular level. It achieves a stable and safe hypoglycemic effect in a diabetic mouse model, effectively overcoming the shortcomings of existing probiotics such as unstable hypoglycemic effects and limited applicability. This provides a stable and reliable new strain resource for developing food-grade, strain-specific hypoglycemic probiotic products. In view of this, the inventors provide the following technical solution of this invention: The first aspect of this invention provides a strain of Bifidobacterium animalis subsp. lactis Tianshan-1 with hypoglycemic effect, which is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 35821 and deposit date of September 5, 2025.

[0010] The second aspect of the present invention provides a probiotic preparation, wherein the probiotic preparation uses the Bifidobacterium animalis subsp. Tianshan-1, its culture, lysate or extract as active substances as described in claim 1.

[0011] Furthermore, the probiotic agent is a live bacteria preparation or a dead bacteria preparation; preferably, the active substance of the probiotic agent is a live bacterial lysate of Bifidobacterium animalis subsp. lactis Tianshan-1.

[0012] The third aspect of this invention provides the application of the Bifidobacterium lactis subsp. Tianshan-1 described in the first aspect, or the probiotic agent described in the second aspect, in the preparation of products with hypoglycemic function.

[0013] Furthermore, the dosage form of the product includes capsules, tablets, granules, powders, or sustained-release formulations.

[0014] Furthermore, the product is a pharmaceutical product.

[0015] Furthermore, the product is one or more of the following: (1) The product can promote the secretion of GLP-1 by cells; (2) The product can significantly upregulate the expression level of the GCG gene; (3) The product can significantly increase the concentration of GLP-1 in serum; (4) The product can significantly reduce fasting blood glucose levels; (5) The product can significantly reduce glycated hemoglobin levels.

[0016] Information on strain preservation: Tianshan-1, a subsp. *Bifidobacterium animalis*, is classified as *Bifidobacterium animalis* subsp. *lactis*. The depository institution is the China General Microbiological Culture Collection Center (CGMCC); address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China; accession number: CGMCC No. 35821; deposit date: September 5, 2025.

[0017] The advantages of this invention compared to the prior art are as follows: 1. This invention screened and obtained a strain of Bifidobacterium animalis subsp. lactis with hypoglycemic effect, preservation number CGMCC No. 35821; classification name: Bifidobacterium animalis subsp. lactis; 2. This invention has demonstrated through cell experiments that Tianshan-1 live bacteria lysate can significantly increase the secretion of GLP-1 in NCI-H716 colorectal adenocarcinoma cells, with better effects than other components such as live bacteria, dead bacteria, and supernatant. Moreover, the effect is not significantly different from that of the positive control AKK, demonstrating a stable and efficient ability to promote GLP-1 secretion. 3. Tianshan-1 can significantly increase the expression level of GCG gene and also upregulate the expression of PC1 gene. It promotes the synthesis and secretion of GLP-1 at the gene transcription level. Its mechanism of action is clear and its target is well-defined, which is different from the vague blood sugar lowering effect of ordinary probiotics. 4. Animal experiments have shown that Tianshan-1 live bacterial lysate can significantly reduce fasting blood glucose in diabetic mice and significantly reduce glycated hemoglobin levels, achieving both immediate blood glucose reduction and long-term blood glucose homeostasis regulation, and improving core pathological indicators of diabetes. At the same time, Tianshan-1 can significantly increase serum GLP-1 concentration in diabetic mice, thereby lowering blood glucose by activating the body's own insulin secretion pathway, avoiding the risk of hypoglycemia that is easily caused by traditional hypoglycemic drugs, and thus having higher safety. 5. The Bifidobacterium animalis subspecies Tianshan-1 described in this invention is not a next-generation probiotic and can be directly applied to the development of food, health products and pharmaceuticals. It solves the application limitations of strains such as AKK bacteria that cannot be used in food and has broad prospects for industrialization. Attached Figure Description

[0018] The present invention will be further described below with reference to the accompanying drawings and embodiments: Figure 1 The effects of different probiotic components on the viability of NCI-H716 cells are shown; where, live lysate: live bacteria lysate, live supernatant: live bacteria supernatant, dead lysate: dead bacteria lysate, and dead supernatant: dead bacteria supernatant. Figure 2 To demonstrate the effects of different probiotic components on GLP-1 secretion by cells; wherein, live lysate: live bacteria lysate, live supernatant: live bacteria supernatant, dead lysate: dead bacteria lysate, and dead supernatant: dead bacteria supernatant. P < 0.01, P < 0.001; Figure 3 This diagram illustrates the effects of different probiotic components on GLP-1 secretion-related genes; where: GCG: glucagon; PC1: proprotein convertase subtilisin / kexin1; live lysate: live bacterial lysate; dead lysate: dead bacterial lysate. P < 0.01; Figure 4 The effect of Tianshan-1 live bacterial lysate on fasting blood glucose in mice is shown; where live lysate: live bacterial lysate, dead lysate: dead bacterial lysate. Figure 5 The effect of Tianshan-1 live bacterial lysate on glycated hemoglobin in mice is shown; where live lysate: live bacterial lysate, dead lysate: dead bacterial lysate. Figure 6 The effect of Tianshan-1 live bacterial lysate on GLP-1 secretion in mouse serum is shown; where live lysate: live bacterial lysate, dead lysate: dead bacterial lysate. Detailed Implementation

[0019] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0020] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0021] The present invention will be described in detail below through embodiments. It should be understood that the following embodiments are only used to exemplify and further explain and illustrate the content of the present invention, and are not intended to limit the present invention.

[0022] In the following examples, *Bifidobacterium animalis* subspecies *Tianshan-1* was provided by Maiquer Group Co., Ltd. This strain was derived from the intestines of healthy humans and is deposited at the China General Microbiological Culture Collection Center (CGMCC). All other chemical reagents used were purchased from reputable chemical reagent suppliers and were of analytical purity.

[0023] Example 1 This embodiment provides a probiotic preparation with a hypoglycemic effect. The active ingredient of the preparation is *Bifidobacterium animalis* subsp. *lactamase* Tianshan-1. The preparation method of the preparation includes: The Tianshan-1 strain was activated, cultured on a large scale, and centrifuged to collect bacterial cells, which were then freeze-dried to prepare bacterial powder. The viable cell content per 2g of bacterial powder was [missing information]. CFU should be stored at low temperature for future use.

[0024] Test Example 1: Effects of Probiotics on the Viability of NCI-H716 Cells Bifidobacterium lactis subspecies Tianshan-1 was prepared into six components: live bacteria, live bacterial lysate, live bacterial supernatant, dead bacteria, dead bacterial lysate, and dead bacterial supernatant. AKK bacteria components were used as positive controls. The CCK-8 assay was used to detect the effect of each component on the viability of human colorectal adenocarcinoma NCI-H716 cells. Specifically: (1) Human colorectal adenocarcinoma cells NCI-H716 in the logarithmic growth phase were taken, and the cell concentration was adjusted with complete culture medium containing 10% fetal bovine serum. The cells were then seeded at an appropriate density in 96-well cell culture plates and incubated at 37°C with 5% saline solution. Culture in an incubator until the cells adhere to the wall.

[0025] (2) Discard the original culture medium and add treatment culture medium containing the above different test components respectively. At the same time, set up a blank control group. Set up 3 replicates for each group and continue to culture.

[0026] (3) After the culture is completed, add CCK-8 detection reagent to each well and incubate in the dark for an appropriate time; use an ELISA reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm.

[0027] With the cell viability of the blank control group as 100%, the relative cell viability of each group was calculated according to the formula: Relative cell viability (%) = (OD value of test group - OD value of blank well) / (OD value of control group - OD value of blank well) × 100% like Figure 1 The results showed that Tianshan-1 live bacteria sludge and live bacteria lysate could enhance cell viability, while live bacteria supernatant and dead bacteria supernatant both had a certain inhibitory effect on cell viability, and there was no obvious cytotoxicity overall.

[0028] Test Example 2: Effect of Probiotics on GLP-1 Secretion in NCI-H716 Cells Bifidobacterium animalis subsp. *Tianshan-1* was prepared into six components: live bacteria, live bacteria lysate, live bacteria supernatant, dead bacteria, dead bacteria lysate, and dead bacteria supernatant. AKK bacteria were used as a positive control for the corresponding component. The GLP-1 content in the cell supernatant of each group was detected by ELISA. Specifically: (1) Logarithmic growth phase human colorectal adenocarcinoma cells NCI-H716 were seeded into culture plates and adhered to the plates. Tianshan-1 live bacteria were then added to each plate. CFU / mL), viable bacterial lysis buffer, viable bacterial supernatant ( The study included CFU / mL of dead bacteria, dead bacterial lysate, and dead bacterial supernatant. A blank control group and positive control groups for each component of AKK bacteria were also set up. Each group had 3 replicates and was cultured for the specified time.

[0029] (2) After the culture is completed, the cell culture supernatant is collected, the precipitate is removed by low-temperature centrifugation, and the supernatant is used for GLP-1 content detection.

[0030] (3) Using the GLP-1 enzyme-linked immunosorbent assay kit, the sample was added, incubated, washed and colored according to the instructions; the OD value of each well was measured at 450 nm wavelength using an ELISA reader, and the GLP-1 concentration in each sample was calculated according to the standard curve.

[0031] like Figure 2The results showed that the live bacterial lysate of Tianshan-1 was the most effective in promoting GLP-1 secretion in NCI-H716 cells, significantly better than other components such as live bacteria, dead bacteria, and supernatant. Moreover, there was no significant difference in the effect of dead bacterial lysate compared with the positive control AKK bacteria, indicating that this strain can efficiently induce GLP-1 secretion.

[0032] Test Example 3: Effects of Probiotics on GLP-1 Related Gene Expression This test case uses qPCR to detect the relative expression levels of GCG and PC1, key genes in GLP-1 synthesis. Specifically: (1) Take NCI-H716 cells in the logarithmic growth phase, seed them into culture plates, and after adhesion, add Tianshan-1 live bacterial lysis buffer ( The bacteria were treated with CFU / mL, and a blank control group and an AKK lysate positive control group were set up. Each group had 3 replicates and were cultured for the specified time.

[0033] (2) Discard the culture medium, wash the cells with PBS, add lysis buffer, and extract total RNA from the cells using a kit; detect the RNA concentration and purity to ensure that it meets the requirements of quantitative real-time PCR detection.

[0034] (3) Using total RNA as a template, cDNA was synthesized according to the instructions of the reverse transcription kit; the relative expression levels of GCG and PC1 genes in NCI-H716 cells were detected by real-time quantitative PCR (qPCR) with β-actin as an internal reference gene.

[0035] (4) The relative expression level of the target gene was calculated using the 2^(-ΔΔCt) method, and normalization was performed using the blank group as a reference.

[0036] like Figure 3 The results showed that the live bacterial lysate of Tianshan-1 significantly upregulated GCG gene expression, with no significant difference compared to the positive control; it also showed an upregulation trend for PC1 gene expression, but the difference did not reach statistical significance.

[0037] Test Example 4: The Effect of Probiotics on Blood Glucose in Diabetic Mice This test case used SPF-grade diabetic mice to investigate the hypoglycemic effect of Bifidobacterium lactis subsp. Tianshan-1, specifically including: 1. Laboratory animals and grouping SPF-grade diabetic model mice were randomly divided into 5 groups after adaptive feeding: blank group, diabetic model group, and high-dose Tianshan-1 live bacteria lysate group. CFU / kg bw), low-dose group of Tianshan-1 live bacteria lysate ( CFU / kg bw), AKK dead bacteria lysis buffer positive control group, several in each group.

[0038] 2. Intervention methods Mice in each group were continuously administered the corresponding dose by gavage for a certain period of time. The blank group and the model group were given the same amount of physiological saline. During this period, the mice were given normal drinking water and feeding, and the breeding environment was kept consistent.

[0039] 3. Indicator Testing (1) Fasting blood glucose measurement Before the end of the intervention, the mice were fasted for 12 hours but allowed free access to water. Blood was collected from the tail vein and fasting blood glucose was measured using a blood glucose meter.

[0040] (2) Glycated hemoglobin determination Blood was collected from the orbital region of mice, placed in anticoagulant tubes, and glycated hemoglobin levels were detected using a kit.

[0041] (3) Serum GLP-1 concentration measurement After the blood was allowed to stand at room temperature, it was centrifuged to separate the serum, and the serum GLP-1 concentration was detected using an ELISA kit.

[0042] After intervention, it was found that fasting blood glucose levels in the model group were significantly higher than those in the control group. Figure 4 As shown, compared with the model group, high-dose Tianshan-1 live bacterial lysate significantly reduced fasting blood glucose in mice, and there was no significant difference compared with the positive control AKK dead bacterial lysate group.

[0043] like Figure 5 As shown, glycated hemoglobin in the model group was significantly higher than that in the control group. Compared with the model group, high-dose Tianshan-1 live bacterial lysate significantly reduced glycated hemoglobin in mice, and there was no significant difference compared with the positive control AKK dead bacterial lysate group.

[0044] like Figure 6 As shown, serum GLP-1 levels in the model group mice were significantly lower than those in the control group. Compared with the model group, high-dose Tianshan-1 live bacterial lysate significantly increased serum GLP-1 levels in mice, and there was no significant difference compared with the positive control AKK dead bacterial lysate group.

[0045] Finally, it should be noted that the above description is only used to illustrate the technical solutions of the present invention and is not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention.

Claims

1. A strain of *Bifidobacterium animalis* subsp. *lactis* Tianshan-1 with hypoglycemic effect, characterized in that... The *Bifidobacterium animalis* subsp. *lactum* is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35821 and deposit date of September 5, 2025.

2. A probiotic preparation, characterized in that, The probiotic agent uses the Bifidobacterium lactis subspecies Tianshan-1, its culture, lysate, or extract as active substances as described in claim 1.

3. The probiotic agent according to claim 2, characterized in that, The probiotic agent is a live bacteria preparation or a dead bacteria preparation.

4. The application of Bifidobacterium animalis subsp. Tianshan-1 as described in claim 1, or the probiotic agent as described in claim 2, in the preparation of products with hypoglycemic function.

5. The application according to claim 4, characterized in that, The dosage forms of the product include capsules, tablets, granules, powders, or sustained-release formulations.

6. The application according to claim 4, characterized in that, The product in question is a pharmaceutical product.

7. The application according to claim 4, characterized in that, The product is one or more of the following: (1) The product can promote the secretion of GLP-1 by cells; (2) The product can significantly upregulate the expression level of the GCG gene; (3) The product can significantly increase the concentration of GLP-1 in serum; (4) The product can significantly reduce fasting blood glucose levels; (5) The product can significantly reduce glycated hemoglobin levels.

Images