Bifidobacterium animalis subsp. Lactis BA-C9 capable of improving diabetes and application thereof

Abstract

The invention discloses animal bifidobacterium subsp. Lactis BA-C9 capable of improving diabetes and application of the animal bifidobacterium subsp. Lactis BA-C9, the animal bifidobacterium subsp. Lactis BA-C9 is preserved in China Center for Type Culture Collection on March 11, 2024, and the preservation number is CCTCC NO: M2024457. The product prepared from the animal bifidobacterium subsp. Lactis BA-C9 has the beneficial effect of improving diabetes mellitus, can reduce fasting blood-glucose and liver injury, and has the capabilities of improving insulin resistance, glucose tolerance and dyslipidemia.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to animal Bifidobacterium lactis subspecies BA-C9 capable of improving diabetes and application thereof. Background Art

[0002] Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease characterized by persistent hyperglycemia and insulin resistance. Currently, the treatment of type 2 diabetes mainly includes lifestyle intervention (such as diet control and exercise), oral hypoglycemic drugs (such as metformin, glibenclamide and thiazolidinediones) and insulin therapy. However, traditional drug treatments, such as metformin and insulin, although they can effectively lower blood sugar, long-term use may be accompanied by side effects such as hypoglycemia, weight gain and gastrointestinal discomfort. In addition, many patients find it difficult to adhere to long-term drug treatment, so there is an increasing demand for more natural and side-effect-free treatment methods. Summary of the invention

[0003] The object of the present invention is to provide an animal Bifidobacterium lactis subspecies BA-C9 having the effect of improving diabetes and its application. The product prepared by the animal Bifidobacterium lactis subspecies BA-C9 of the present invention has the beneficial effect of improving diabetes, can reduce fasting blood sugar and liver damage, and has the ability to improve insulin resistance, glucose tolerance and dyslipidemia.

[0004] The technical solution of the present invention is: an animal Bifidobacterium lactis subspecies BA-C9 with the function of improving diabetes. The animal Bifidobacterium lactis subspecies BA-C9 has been deposited in the China Center for Type Culture Collection on March 11, 2024, with the deposit number: CCTCC NO: M 2024457.

[0005] The application of the above-mentioned animal Bifidobacterium lactis subspecies BA-C9 in the preparation of products for improving diabetic symptoms.

[0006] In the aforementioned application, the product is a bacterial agent, food and / or medicine.

[0007] In the aforementioned application, the food is one or more of fermented milk, cheese, milk-containing beverage, solid beverage, and milk powder; the food contains live bacterial powder and / or inactivated bacterial liquid of Bifidobacterium animalis subspecies lactis BA-C9.

[0008] In the aforementioned application, the medicine comprises live bacteria powder and / or inactivated bacterial liquid of animal Bifidobacterium lactis subspecies BA-C9, and / or other medicines compatible with the live bacteria powder and / or inactivated bacterial liquid of animal Bifidobacterium lactis subspecies BA-C9, as well as pharmaceutically acceptable carriers and / or excipients.

[0009] In the aforementioned application, the animal Bifidobacterium lactis subspecies BA-C9 is used to prepare a bacterial agent, food and / or medicine for lowering fasting blood sugar.

[0010] In the aforementioned application, the animal Bifidobacterium lactis subspecies BA-C9 is used to prepare a bacterial agent, food and / or medicine for improving insulin resistance and glucose tolerance.

[0011] In the aforementioned application, the animal Bifidobacterium lactis subspecies BA-C9 is used to prepare a bacterial agent, food and / or medicine for improving dyslipidemia.

[0012] In the aforementioned application, the animal Bifidobacterium lactis subspecies BA-C9 is used to prepare bacterial agents, foods and / or medicines for protecting liver damage.

[0013] In the aforementioned application, the inactivated bacterial solution of Bifidobacterium animalis subspecies lactis BA-C9 is prepared as follows:

[0014] 1 g of BA-C9 powder was dissolved in 500 mL of LB liquid culture medium, and after culturing at 37°C for 24 h, the bacterial suspension was inactivated at 121°C for 30 min under high temperature and high pressure, and centrifuged at 3000 rpm for 15 min at room temperature. After the centrifugation, the supernatant was removed, and an appropriate amount of PBS was added to wash away the residual culture medium, and the mixture was centrifuged again to remove the supernatant to obtain the inactivated animal Bifidobacterium lactis subsp. BA-C9 cells. 1 mL of physiological saline was added to every 50 mg of the cells to dissolve the inactivated animal Bifidobacterium lactis subsp. BA-C9 bacterial liquid.

[0015] Compared with the prior art, the animal bifidobacterium lactis subspecies BA-C9 of the present invention can effectively reduce the blood sugar level of diabetic mice by regulating fasting blood sugar, reducing liver injury reaction and improving dyslipidemia state. Experiments have shown that the live bacteria powder and inactivated bacteria liquid of the animal bifidobacterium lactis subspecies BA-C9 of the present invention all have the ability of good improvement of diabetic symptoms, can reduce the blood sugar level of diabetic mice, and have the advantages of safety, effectiveness and no obvious side effects. In addition, the bifidobacterium lactis subspecies BA-C9 of the present invention has good gastrointestinal tolerance, and is therefore easier to survive by stomach and small intestine after the product is taken orally or eaten. Therefore, the research and development of the live bacteria powder and inactivated bacteria liquid of the animal bifidobacterium lactis subspecies BA-C9 of the present invention has opened up a new direction for the improvement of diabetic symptoms. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a Gram-stained bacterial morphology of Bifidobacterium animalis subspecies lactis BA-C9 of the present invention;

[0017] Figure 2 The effect of the animal Bifidobacterium lactis subspecies BA-C9 of the present invention on the body weight, liver weight and liver-to-body ratio of mice;

[0018] Figure 3The effect of the animal Bifidobacterium lactis subspecies BA-C9 of the present invention on the fat weight of mice;

[0019] Figure 4 The effect of the animal Bifidobacterium lactis subspecies BA-C9 of the present invention on glucose tolerance and insulin resistance levels in mice;

[0020] Figure 5 The effect of the animal Bifidobacterium lactis subspecies BA-C9 of the present invention on fasting blood glucose and fasting insulin in mice;

[0021] Figure 6 The present invention is the effect of animal Bifidobacterium lactis subspecies BA-C9 on alanine aminotransferase and aspartate aminotransferase (ALT and AST) in mouse plasma;

[0022] Figure 7 The present invention is the effect of animal Bifidobacterium lactis subspecies BA-C9 on triglyceride (TG), total cholesterol (TC), high-density lipoprotein (HDL-C), and low-density lipoprotein (LDL-C) in mouse plasma;

[0023] Figure 8 The effect of the inactivated animal Bifidobacterium lactis subspecies BA-C9 of the present invention on the body weight, liver weight and liver-to-body ratio of mice;

[0024] Fig. 9 The effect of the inactivated animal Bifidobacterium lactis subspecies BA-C9 of the present invention on the fat weight of mice;

[0025] Fig.10 The effect of the inactivated animal Bifidobacterium lactis subspecies BA-C9 of the present invention on glucose tolerance and insulin resistance levels in mice;

[0026] Fig.11 The effect of the inactivated animal Bifidobacterium lactis subspecies BA-C9 of the present invention on fasting blood glucose and fasting insulin in mice;

[0027] Fig.12 The invention relates to the effect of the inactivated animal Bifidobacterium lactis subspecies BA-C9 on alanine aminotransferase and aspartate aminotransferase (ALT and AST) in mouse plasma;

[0028] Fig.13 The invention discloses the influence of the inactivated animal Bifidobacterium lactis subspecies BA-C9 on triglyceride (TG), total cholesterol (TC), high-density lipoprotein (HDL-C) and low-density lipoprotein (LDL-C) in mouse plasma. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with the accompanying drawings and embodiments, but they are not intended to limit the present invention.

[0030] Example 1: Identification of Bifidobacterium animalis subspecies lactis BA-C9;

[0031] A strain of Bifidobacterium animalis subsp. lactis was isolated and screened from the feces of healthy children. The strain was identified through bacterial morphology, physiology and culture characteristics combined with 16S rDNA sequencing, and named Bifidobacterium animalis subsp. lactis BA-C9. It was deposited in the China Center for Type Culture Collection on March 11, 2024, with the deposit number: CCTCC NO: M 2024457.

[0032] The sequenced DNA sequence is as follows:

[0033] CGGGCGCTTGCTGTCGGGGTGAGAGTGGCGAACGGGTGAGTAATGCGTGACCAA

[0034] CCTGCCCTGTGCACCGGAATAGCTCCTGGAAACGGGTGGTAATACCGGATGCTCCGCT

[0035] CCATCGCATGGTGGGGTGGGAAATGCTTTTTGCGGCATGGGATGGGGTCGCGTCCTATC

[0036] AGCTTGTTGGCGGGGTGATGGCCCACCAAGGCGTTGACGGGTAGCCGGCCTGAGAGGG

[0037] TGACCGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGG

[0038] GAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGCGGGATGGAGGCC

[0039] TTCGGGTTGTAAACCGCTTTTGTTCAAGGGCAAGGCACGGTTTCGGCCGTGTTGAGTG

[0040] GATTGTTCGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGT

[0041] GCGAGCGTTATCCGGATTTATTGGGCGTAAAGGGCTCGTAGGCGGTTCGTCGCGTCCG

[0042] GTGTGAAAGTCCATCGCCTAACGGTGGATCTGCGCCGGGTACGGGCGGGCTGGAGTGC

[0043] GGTAGGGGAGACTGGAATTCCCGGTGTAACGGTGGAATGTGTAGATATCGGGAAGAAC

[0044] ACCAATGGCGAAGGCAGGTCTCTGGGCCGTCACTGACGCTGAGGAGCGAAAGCGTGGG

[0045] GAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGATGCTGGATGT

[0046] GGGGCCCTTTCCACGGGTCCCGTGTCGGAGCCAACGCGTTAAGCATCCCGCCTGGGGA

[0047] GTACGGCCGCAAGGCTAAAACTCAAAGAAATTGACGGGGGCCCGCACAAGCGGCGGAG

[0048] CATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGTGCCGGA

[0049] TCGCCGTGGAGACACGGTTTCCCTTCGGGGCCGGTTCACAGGTGGTGCATGGTCGTCG

[0050] TCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCGCA

[0051] TGTTGCCAGCGGGTGATGCCGGGAACTCATGTGGGACCGCCGGGGTCAACTCGGAGGA

[0052] AGGTGGGGATGACGTCAGATCATCATGCCCCTTACGTCCAGGGCTTCACGCATGCTAC

[0053] AATGGCCGTACAACGCGGTGCGACACGGTGACGTGGGGCCGGATCGCTGAAAACCGGT

[0054] CTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGGCGGAGTCGCTAGTAATCG

[0055] CGGATCAGCAACGCCGCGGTGAATGCGTTCCCGGGCCTTGTACACACCGCCCGTCAAG

[0056] TCATGAAAGTGGGTAGCACCCGAAGCCGGTGGCCCGACCCTTGTGGGGGGAGCCGTCT

[0057] AAGGTGAGACTCGTGATGAAAAAT

[0058] 1.1. Colony characteristics;

[0059] After the isolated and purified strain was cultured in LB solid medium for 48 hours, the colonies were milky white, smooth, convex, shiny and soft, with smooth and neat edges without wrinkles. Figure 1 .

[0060] 1.2, State under microscope;

[0061] The colony of the isolated and purified strain was smeared: Gram staining was positive, no spores were formed, polymorphic rods were in Y-shaped, V-shaped, etc., Figure 1 .

[0062] Example 2: Animal Bifidobacterium lactis subspecies BA-C9 reduces blood sugar in diabetic mice;

[0063] 2.1 Experimental materials;

[0064] Twenty-four C57BL / 6 male mice were purchased from Shanghai Slake Experimental Animal Center (company license number: SCXK(Shanghai)2013-0016) and were kept in the SPF-level animal breeding room of the Experimental Animal Center of Zhejiang Chinese Medical University.

[0065] Reagents: ALT kit (Cat. No.: C009-2 Nanjing Jiancheng Bioengineering Institute), AST kit (Cat. No.: C010-2 Nanjing Jiancheng Bioengineering Institute), TG kit (Cat. No.: A110-1-1 Nanjing Jiancheng Bioengineering Institute), TC kit (Cat. No.: A111-1-1 Nanjing Jiancheng Bioengineering Institute), HDL-C kit (Cat. No.: A112-1-1 Nanjing Jiancheng Bioengineering Institute), LDL-C kit (Cat. No.: A113-1-1 Nanjing Jiancheng Bioengineering Institute)

[0066] Rearing environment: room temperature 20-25℃, light and dark alternation every 12 hours, relative humidity about 50%, feed, drinking water, etc. are uniformly matched, keep the indoor environment well ventilated, clean every day, and maintain a good hygienic environment in the cage. All experiments comply with the Animal Ethics Code and the relevant regulations of the Experimental Animal Ethics Committee, and are specifically carried out in accordance with IAC and other relevant standard operating procedures (SOP). After the experiment, blood was collected from the abdominal aorta under anesthesia, and the bodies were uniformly handled harmlessly.

[0067] 2.2 Methods:

[0068] Laboratory animal husbandry;

[0069] Rearing environment: room temperature 20-25℃, light and dark alternation every 12 hours, relative humidity about 50%, feed, drinking water, etc. are uniformly matched, keep the indoor environment well ventilated, clean every day, and maintain a good hygienic environment in the cage. All experiments comply with the Animal Ethics Code and the relevant regulations of the Experimental Animal Ethics Committee, and are specifically carried out in accordance with IAC and other relevant standard operating procedures (SOP). After the experiment, blood was collected from the abdominal aorta under anesthesia, and the bodies were uniformly handled harmlessly.

[0070] After one week of adaptation feeding in an SPF-level animal laboratory, C57BL / 6 mice were randomly divided into a normal control group (NFD group) (n=6) and a diabetic mouse model group (n=18). The mice were fasted for 12 hours before the experiment and intraperitoneally injected with STZ solution (prepared with pH=4.5 citric acid buffer) at a dose of 100 mg / kg for 5 consecutive days. Fasting blood glucose was tested 1 week and 2 weeks after the injection. The model was successful when the blood glucose value exceeded 11.0 mmol / L.

[0071] The mice with successful modeling were randomly divided into three groups, namely, high-fat model group (HFD group), animal Bifidobacterium group (HFD+BA-C9 group) and metformin group (HFD+Metformin group), with 6 mice in each group. The normal control group and model control group were given an equal amount of citric acid solution by gavage, and the mice in the animal Bifidobacterium group were given animal Bifidobacterium lactis subsp. BA-C9 by gavage (4×10 9CFU / mL, 0.25mL / day / mouse), and the metformin group was given metformin by gavage (100mg / kg, 0.25mL / day / mouse). The intervention lasted for 4 weeks. During the intervention period, the three groups were provided with the same ordinary feed, all mice were free to eat and drink, and their body weight was measured every week.

[0072] The feed consumed by the mice was replaced with new feed every two days, and the weight of the mice was recorded weekly to detect changes. After feeding, the mice were anesthetized with 1% pentobarbital intraperitoneal injection, and blood was drawn from the inferior vena cava to measure biochemical indicators such as ALT and AST.

[0073] 2.3、Indicator determination;

[0074] 2.3.1. Glucose tolerance test;

[0075] After fasting for 12 h, mice were intraperitoneally injected with 2.5 g / kg of glucose solution according to their body weight. Blood samples were collected at 0, 30, 60, and 120 min after the intraperitoneal injection, and the glucose content in the blood was measured using a blood glucose meter.

[0076] 2.3.2. Insulin resistance test;

[0077] After 4 hours of fasting, the mice were intraperitoneally injected with 0.75 U / kg of glucose solution according to their body weight. Blood samples were collected at 0, 30, 60, and 120 minutes after the intraperitoneal injection, and the glucose content in the blood was measured using a blood glucose meter.

[0078] 2.3.3, fasting blood glucose test;

[0079] After the mice were fasted for 12 hours but not water, they were placed in a fixed tube or appropriate fixture with their tails exposed. The tails of the mice were disinfected with 75% alcohol cotton balls to ensure the hygiene of blood collection. A blood collection needle was used to gently puncture the end of the tail, squeeze out a drop of blood, and place the blood drop on the test area of ​​the blood glucose test strip. The blood glucose meter was used for testing, and the results were read and recorded.

[0080] 2.3.4, fasting insulin test;

[0081] Take mouse plasma samples directly for sampling and determination. Add 10μL Assay Buffer and 10μL Matrix Solution to the blank wells, add 10μL of different concentrations of standard or QC and 10μL Matrix Solution to the wells of standard and QC, add 80μL Detection Antibody to each reaction well, seal with sealing film after adding the sample, shake gently on the decolorization shaker, and incubate at room temperature for 2h. Remove the sealing film, pour out the reaction liquid, and tap gently on toilet paper to remove the residual liquid. Add 300μL of wash buffer to each reaction well for washing 3 times. After removing the residual liquid in the well, add 100μL Enzyme Solution to each reaction well, seal with sealing film, shake gently on the decolorization shaker, and incubate at room temperature for 30min. Each reaction well was washed 6 times with 100μL of wash buffer. After removing the residual liquid in the well, 100μL of Substrate Solution was added to each reaction well. The ELISA strips were sealed with tin foil, and gently shaken on a decolorizing shaker. Incubated at room temperature for 30 minutes away from light. 100μL of Stop Solution was added to each reaction well, and the plate rack was gently tapped by hand to mix the liquid thoroughly and remove bubbles. The light absorption value of each well at a wavelength of 450nm was read with an ELISA reader, and the concentration value of the test sample was converted according to the standard curve.

[0082] 2.3.5. Plasma ALT test;

[0083] Mouse plasma samples were directly sampled for determination. The matrix solution was preheated at 37°C in advance. 20 μL matrix solution and 5 μL sample to be tested were added to the sample wells and mixed. 20 μL matrix solution was added to the control wells and incubated at 37°C for 30 min. 20 μL of 2,4-dinitrophenylhydrazine solution was added to the determination wells and control wells, and 5 μL sample to be tested was added to the control wells, mixed, and incubated at 37°C for 20 min. 200 μL of 0.4 mol / L sodium hydroxide solution was added to each well, mixed, and placed at room temperature for 15 min. The OD value of each well was measured by an enzyme marker at a wavelength of 510 nm, and the standard curve was checked to obtain the corresponding ALT / GPT activity unit.

[0084] 2.3.6. Plasma AST detection;

[0085] Mouse plasma samples were directly sampled for determination. The matrix solution was preheated at 37°C in advance. 20 μL matrix solution and 5 μL sample to be tested were added to the sample wells and mixed. 20 μL matrix solution was added to the control wells and incubated at 37°C for 30 min. 20 μL of 2,4-dinitrophenylhydrazine solution was added to the determination wells and control wells, and 5 μL sample to be tested was added to the control wells, mixed, and incubated at 37°C for 20 min. 200 μL of 0.4 mol / L sodium hydroxide solution was added to each well, mixed, and placed at room temperature for 15 min. The OD value of each well was measured by an enzyme marker at a wavelength of 510 nm, and the standard curve was checked to obtain the corresponding AST / GPT activity unit.

[0086] 2.3.7. Plasma TG detection;

[0087] Mouse plasma samples were directly sampled for determination. 2.5 μL of the sample to be tested and 250 μL of working solution were added to the sample well, 2.5 μL of distilled water and 250 μL of working solution were added to the blank well, 2.5 μL of calibrator and 250 μL of working solution were added to the standard well, the well plate was shaken to mix, incubated at 37°C for 10 minutes, and the OD value of each well was measured by an enzyme-labeled instrument at a wavelength of 500 nm. The standard curve was checked to obtain the corresponding triglyceride content.

[0088] 2.3.8. Plasma TC detection;

[0089] Take mouse plasma samples directly for measurement, add 2.5 μL of the sample to be tested and 250 μL of working solution to the sample well, add 2.5 μL of distilled water and 250 μL of working solution to the blank well, add 2.5 μL of calibrator and 250 μL of working solution to the standard well, shake the well plate to mix, incubate at 37°C for 10 minutes, and use a microplate reader to measure the OD value of each well at a wavelength of 500 nm. Check the standard curve to obtain the corresponding cholesterol content.

[0090] 2.3.9. Plasma HDL-C test;

[0091] Take mouse plasma samples directly for sampling and determination. Add 2.5μL of the sample to be tested and 180μL of reagent 1 to the sample well, add 2.5μL of distilled water and 180μL of reagent 1 to the blank well, add 2.5μL of calibration substance and 180μL of reagent 1 to the standard well, shake the plate to mix, incubate at 37℃ for 5 minutes, and measure the absorbance value A1 of each well with a microplate reader at a wavelength of 600nm. After the determination of A1, add 60μL of reagent 2 to the sample well, blank well, and calibration well, shake the plate to mix, incubate at 37℃ for 5-10 minutes, and measure the absorbance value A2 of each well with a microplate reader at a wavelength of 600nm. Calculate △A=A2-A1 to obtain the corresponding high-density lipoprotein cholesterol content.

[0092] 2.3.10. Plasma LDL-C test;

[0093] Take mouse plasma samples directly for sampling and determination. Add 2.5μL of the sample to be tested and 180μL of reagent 1 to the sample well, add 2.5μL of distilled water and 180μL of reagent 1 to the blank well, add 2.5μL of calibration substance and 180μL of reagent 1 to the standard well, shake the plate to mix, incubate at 37℃ for 5 minutes, and measure the absorbance value A1 of each well with a microplate reader at a wavelength of 600nm. After the determination of A1, add 60μL of reagent 2 to the sample well, blank well, and calibration well, shake the plate to mix, incubate at 37℃ for 5-10 minutes, and measure the absorbance value A2 of each well with a microplate reader at a wavelength of 600nm. Calculate △A=A2-A1 to obtain the corresponding low-density lipoprotein cholesterol content.

[0094] Experimental results and analysis:

[0095] Figure 2 The effect of the animal Bifidobacterium lactis subspecies BA-C9 of the present invention on the body weight, liver weight and liver-to-body ratio of mice; Figure 2 The results showed that animal Bifidobacterium BA-C9 can significantly increase the weight loss caused by diabetes. Compared with the NFD group, the body weight of diabetic mice (HFD group) was significantly reduced, and BA-C9 intervention can significantly increase the weight loss caused by diabetes. The liver-to-body ratio is an important indicator reflecting liver damage. The liver weight and liver index of mice in the HFD group were significantly higher than those in the NFD group. Compared with the model group (HFD group), the liver weight and liver index of mice in the HFD+BA-C9 group were significantly reduced.

[0096] Figure 3 The effect of the animal Bifidobacterium lactis subspecies BA-C9 of the present invention on the fat weight of mice; Figure 3 The results showed that animal Bifidobacterium BA-C9 can significantly reduce the fat gain caused by diabetes. Compared with the NFD group, the weight of white fat and beige fat in diabetic mice (HFD group) increased significantly, and the weight of brown fat decreased significantly. Compared with the HFD group, BA-C9 intervention can significantly reduce the increase in white fat and beige fat weight caused by diabetes, and reduce the weight of brown fat.

[0097] Figure 4 The effect of the animal Bifidobacterium lactis subspecies BA-C9 of the present invention on glucose tolerance and insulin resistance levels in mice; Figure 4 The results showed that animal Bifidobacterium BA-C9 could significantly improve insulin resistance and glucose tolerance caused by a high-fat diet.

[0098] Figure 5 The effect of the animal Bifidobacterium lactis subspecies BA-C9 of the present invention on fasting blood glucose and fasting insulin in mice; Figure 5The results showed that diabetic mice induced by high-fat diet combined with STZ had insulin resistance, which prompted compensatory insulin secretion to maintain blood sugar stability. Compared with the normal control group, high-fat diet combined with STZ could significantly increase the fasting blood glucose and fasting insulin levels of mice. Compared with the model group, animal Bifidobacterium BA-C9 intervention significantly reduced the increase in fasting blood glucose and fasting insulin levels caused by HFD+STZ.

[0099] Figure 6 The present invention is the effect of animal Bifidobacterium lactis subspecies BA-C9 on alanine aminotransferase and aspartate aminotransferase (ALT and AST) in mouse plasma; Figure 6 The results showed that animal Bifidobacterium BA-C9 can significantly reduce the increase of ALT and AST caused by HFD+STZ. Alanine aminotransferase and aspartate aminotransferase are recommended by the World Health Organization as the most sensitive detection indicators of liver function damage. Compared with the control group (NFD), the plasma ALT and AST levels of mice in the HFD group were significantly increased, while animal Bifidobacterium BA-C9 intervention significantly reduced the increase of ALT and AST caused by HFD+STZ.

[0100] Figure 7 The present invention is the effect of animal Bifidobacterium lactis subspecies BA-C9 on triglyceride (TG), total cholesterol (TC), high-density lipoprotein (HDL-C), and low-density lipoprotein (LDL-C) in mouse plasma; Figure 7 The results showed that animal Bifidobacterium BA-C9 can significantly reduce the dyslipidemia state caused by HFD+STZ. Dyslipidemia (such as high cholesterol, high triglycerides and low HDL cholesterol) is often associated with insulin resistance, which is the main feature of type 2 diabetes. Insulin resistance can lead to a decrease in the body's response to insulin, thereby affecting sugar and fat metabolism. This not only leads to increased blood sugar, but also disrupts lipid metabolism, leading to dyslipidemia. An 8-week high-fat diet led to a significant increase in the plasma TG, TC and LDL-C levels and a significant decrease in the HDL-C level in the HFD group mice. After intervention, animal Bifidobacterium BA-C9 significantly reduced the increase in TG, TC and LDL-C levels caused by the high-fat diet, and increased the plasma HDL-C level.

[0101] Example 3: Preparation of inactivated animal Bifidobacterium lactis subspecies BA-C9 bacterial solution:

[0102] 1 g of BA-C9 powder was dissolved in 500 mL of LB liquid culture medium, and after culturing at 37°C for 24 h, the bacterial suspension was inactivated at 121°C for 30 min under high temperature and high pressure, and centrifuged at 3000 rpm for 15 min at room temperature. After the centrifugation, the supernatant was removed, and an appropriate amount of PBS was added to wash away the residual culture medium, and the mixture was centrifuged again to remove the supernatant to obtain inactivated animal Bifidobacterium lactis subsp. BA-C9 cells. 1 mL of physiological saline was added to every 50 mg of cells to dissolve the cells to obtain an inactivated animal Bifidobacterium lactis subsp. BA-C9 bacterial solution.

[0103] Example 4: Inactivation of animal Bifidobacterium lactis subspecies BA-C9 bacterial liquid reduces blood sugar in diabetic mice:

[0104] 4.1 Experimental materials;

[0105] Twenty-four C57BL / 6 male mice were purchased from Shanghai Slake Experimental Animal Center (company license number: SCXK(Shanghai)2013-0016) and were kept in the SPF-level animal breeding room of the Experimental Animal Center of Zhejiang Chinese Medical University.

[0106] Reagents: ALT kit (Cat. No.: C009-2 Nanjing Jiancheng Bioengineering Institute), AST kit (Cat. No.: C010-2 Nanjing Jiancheng Bioengineering Institute), TG kit (Cat. No.: A110-1-1 Nanjing Jiancheng Bioengineering Institute), TC kit (Cat. No.: A111-1-1 Nanjing Jiancheng Bioengineering Institute), HDL-C kit (Cat. No.: A112-1-1 Nanjing Jiancheng Bioengineering Institute), LDL-C kit (Cat. No.: A113-1-1 Nanjing Jiancheng Bioengineering Institute)

[0107] 4.2 Methods:

[0108] Laboratory animal husbandry;

[0109] Rearing environment: room temperature 20-25℃, light and dark alternation every 12 hours, relative humidity about 50%, feed, drinking water, etc. are uniformly matched, keep the indoor environment well ventilated, clean every day, and maintain a good hygienic environment in the cage. All experiments comply with the Animal Ethics Code and the relevant regulations of the Experimental Animal Ethics Committee, and are specifically carried out in accordance with IAC and other relevant standard operating procedures (SOP). After the experiment, blood was collected from the abdominal aorta under anesthesia, and the bodies were uniformly handled harmlessly.

[0110] After one week of adaptation feeding in an SPF-level animal laboratory, C57BL / 6 mice were randomly divided into a normal control group (NFD group) (n=6) and a diabetic mouse model group (n=18). The mice were fasted for 12 hours before the experiment and intraperitoneally injected with STZ solution (prepared with pH=4.5 citric acid buffer) at a dose of 100 mg / kg for 5 consecutive days. Fasting blood glucose was tested 1 week and 2 weeks after the injection. The model was successful when the blood glucose value exceeded 11.0 mmol / L.

[0111] The mice with successful modeling were randomly divided into three groups, namely, high-fat model group (HFD group), animal Bifidobacterium group (HFD+BA-C9 group) and animal Bifidobacterium inactivated group (HFD+BA-C9 (heat-killed) group), with 6 mice in each group. The normal control group and model control group were given an equal amount of citric acid solution by gavage, and the mice in the animal Bifidobacterium group were given animal Bifidobacterium lactis subsp. BA-C9 by gavage (4×10 9 CFU / mL, 0.25mL / day / mouse), and the inactivated animal Bifidobacterium group was intragastrically administered with the inactivated animal Bifidobacterium lactis subspecies BA-C9 bacterial solution in Example 3. The intervention lasted for 4 weeks, during which the three groups were provided with the same common feed, all mice were allowed to eat and drink freely, and their body weight was measured every week.

[0112] The feed consumed by the mice was replaced with new feed every two days, and the weight of the mice was recorded weekly to detect changes. After feeding, the mice were anesthetized with 1% pentobarbital intraperitoneal injection, and blood was drawn from the inferior vena cava to measure biochemical indicators such as ALT and AST.

[0113] 4.3、Indicator determination;

[0114] 4.3.1. Glucose tolerance test;

[0115] After fasting for 12 h, mice were intraperitoneally injected with 2.5 g / kg of glucose solution according to their body weight. Blood samples were collected at 0, 30, 60, and 120 min after the intraperitoneal injection, and the glucose content in the blood was measured using a blood glucose meter.

[0116] 4.3.2. Insulin resistance test;

[0117] After 4 hours of fasting, the mice were intraperitoneally injected with 0.75 U / kg of glucose solution according to their body weight. Blood samples were collected at 0, 30, 60, and 120 minutes after the intraperitoneal injection, and the glucose content in the blood was measured using a blood glucose meter.

[0118] 4.3.3 Fasting blood glucose test;

[0119] After the mice were fasted for 12 hours but not water, they were placed in a fixed tube or appropriate fixture with their tails exposed. The tails of the mice were disinfected with 75% alcohol cotton balls to ensure the hygiene of blood collection. A blood collection needle was used to gently puncture the end of the tail, squeeze out a drop of blood, and place the blood drop on the test area of ​​the blood glucose test strip. The blood glucose meter was used for testing, and the results were read and recorded.

[0120] 4.3.4, Fasting insulin test;

[0121] Take mouse plasma samples directly for sampling and determination. Add 10μL Assay Buffer and 10μL Matrix Solution to the blank wells, add 10μL of different concentrations of standard or QC and 10μL Matrix Solution to the wells of standard and QC, add 80μL Detection Antibody to each reaction well, seal with sealing film after adding the sample, shake gently on the decolorization shaker, and incubate at room temperature for 2h. Remove the sealing film, pour out the reaction liquid, and tap gently on toilet paper to remove the residual liquid. Add 300μL of wash buffer to each reaction well for washing 3 times. After removing the residual liquid in the well, add 100μL Enzyme Solution to each reaction well, seal with sealing film, shake gently on the decolorization shaker, and incubate at room temperature for 30min. Each reaction well was washed 6 times with 100μL of wash buffer. After removing the residual liquid in the well, 100μL of Substrate Solution was added to each reaction well. The ELISA strips were sealed with tin foil, and gently shaken on a decolorizing shaker. Incubated at room temperature for 30 minutes away from light. 100μL of Stop Solution was added to each reaction well, and the plate rack was gently tapped by hand to mix the liquid thoroughly and remove bubbles. The light absorption value of each well at a wavelength of 450nm was read with an ELISA reader, and the concentration value of the test sample was converted according to the standard curve.

[0122] 4.3.5. Plasma ALT test;

[0123] Mouse plasma samples were directly sampled for determination. The matrix solution was preheated at 37°C in advance. 20 μL matrix solution and 5 μL sample to be tested were added to the sample wells and mixed. 20 μL matrix solution was added to the control wells and incubated at 37°C for 30 min. 20 μL of 2,4-dinitrophenylhydrazine solution was added to the determination wells and control wells, and 5 μL sample to be tested was added to the control wells, mixed, and incubated at 37°C for 20 min. 200 μL of 0.4 mol / L sodium hydroxide solution was added to each well, mixed, and placed at room temperature for 15 min. The OD value of each well was measured by an enzyme marker at a wavelength of 510 nm, and the standard curve was checked to obtain the corresponding ALT / GPT activity unit.

[0124] 4.3.6. Plasma AST detection;

[0125] Mouse plasma samples were directly sampled for determination. The matrix solution was preheated at 37°C in advance. 20 μL matrix solution and 5 μL sample to be tested were added to the sample wells and mixed. 20 μL matrix solution was added to the control wells and incubated at 37°C for 30 min. 20 μL of 2,4-dinitrophenylhydrazine solution was added to the determination wells and control wells, and 5 μL sample to be tested was added to the control wells, mixed, and incubated at 37°C for 20 min. 200 μL of 0.4 mol / L sodium hydroxide solution was added to each well, mixed, and placed at room temperature for 15 min. The OD value of each well was measured by an enzyme marker at a wavelength of 510 nm, and the standard curve was checked to obtain the corresponding AST / GPT activity unit.

[0126] 4.3.7. Plasma TG detection;

[0127] Mouse plasma samples were directly sampled for determination. 2.5 μL of the sample to be tested and 250 μL of working solution were added to the sample well, 2.5 μL of distilled water and 250 μL of working solution were added to the blank well, 2.5 μL of calibrator and 250 μL of working solution were added to the standard well, the well plate was shaken to mix, incubated at 37°C for 10 minutes, and the OD value of each well was measured by an enzyme-labeled instrument at a wavelength of 500 nm. The standard curve was checked to obtain the corresponding triglyceride content.

[0128] 4.3.8. Plasma TC detection;

[0129] Take mouse plasma samples directly for measurement, add 2.5 μL of the sample to be tested and 250 μL of working solution to the sample well, add 2.5 μL of distilled water and 250 μL of working solution to the blank well, add 2.5 μL of calibrator and 250 μL of working solution to the standard well, shake the well plate to mix, incubate at 37°C for 10 minutes, and use a microplate reader to measure the OD value of each well at a wavelength of 500 nm. Check the standard curve to obtain the corresponding cholesterol content.

[0130] 4.3.9. Plasma HDL-C test;

[0131] Take mouse plasma samples directly for sampling and determination. Add 2.5μL of the sample to be tested and 180μL of reagent 1 to the sample well, add 2.5μL of distilled water and 180μL of reagent 1 to the blank well, add 2.5μL of calibration substance and 180μL of reagent 1 to the standard well, shake the plate to mix, incubate at 37℃ for 5 minutes, and measure the absorbance value A1 of each well with a microplate reader at a wavelength of 600nm. After the determination of A1, add 60μL of reagent 2 to the sample well, blank well, and calibration well, shake the plate to mix, incubate at 37℃ for 5-10 minutes, and measure the absorbance value A2 of each well with a microplate reader at a wavelength of 600nm. Calculate △A=A2-A1 to obtain the corresponding high-density lipoprotein cholesterol content.

[0132] 4.3.10. Plasma LDL-C test;

[0133] Take mouse plasma samples directly for sampling and determination. Add 2.5μL of the sample to be tested and 180μL of reagent 1 to the sample well, add 2.5μL of distilled water and 180μL of reagent 1 to the blank well, add 2.5μL of calibration substance and 180μL of reagent 1 to the standard well, shake the plate to mix, incubate at 37℃ for 5 minutes, and measure the absorbance value A1 of each well with a microplate reader at a wavelength of 600nm. After the determination of A1, add 60μL of reagent 2 to the sample well, blank well, and calibration well, shake the plate to mix, incubate at 37℃ for 5-10 minutes, and measure the absorbance value A2 of each well with a microplate reader at a wavelength of 600nm. Calculate △A=A2-A1 to obtain the corresponding low-density lipoprotein cholesterol content.

[0134] Experimental results analysis:

[0135] Figure 8 The effect of the inactivated animal Bifidobacterium lactis subspecies BA-C9 of the present invention on the body weight, liver weight and liver-to-body ratio of mice; Figure 8 As shown, the results showed that the inactivated animal Bifidobacterium BA-C9 can significantly increase the weight loss caused by diabetes. Compared with the NFD group, the body weight of diabetic mice (HFD group) was significantly reduced, and the inactivated BA-C9 intervention can significantly increase the weight loss caused by diabetes. The liver-to-body ratio is an important indicator reflecting liver damage. The liver weight and liver index of mice in the HFD group were significantly higher than those in the NFD group. Compared with the model group (HFD group), the liver weight and liver index of mice in the HFD+BA-C9 (heat-killed) group were significantly reduced.

[0136] Fig. 9 The effect of the inactivated animal Bifidobacterium lactis subspecies BA-C9 of the present invention on the fat weight of mice; Fig. 9 The results showed that inactivated animal Bifidobacterium BA-C9 can significantly reduce the fat gain caused by diabetes. The weight of white fat and beige fat in the HFD group mice was significantly higher than that in the NFD group mice, and the weight of brown fat was significantly reduced. Compared with the HFD group, the inactivated BA-C9 intervention can significantly reduce the increase of white fat and beige fat weight caused by diabetes, and reduce the weight of brown fat.

[0137] Fig.10 The effect of the inactivated animal Bifidobacterium lactis subspecies BA-C9 of the present invention on glucose tolerance and insulin resistance levels in mice; Fig.10 The results showed that inactivated animal Bifidobacterium BA-C9 can significantly improve insulin resistance and glucose tolerance caused by a high-fat diet.

[0138] Fig.11The effect of the inactivated animal Bifidobacterium lactis subspecies BA-C9 of the present invention on fasting blood glucose and fasting insulin in mice; Fig.11 The results showed that the fasting blood glucose and fasting insulin levels of mice in the HFD group were significantly higher than those in the normal group. Intervention with animal Bifidobacterium BA-C9 and inactivated animal Bifidobacterium BA-C9 could significantly reduce the increased fasting blood glucose and fasting insulin levels caused by HFD+STZ.

[0139] Fig.11 The effect of the inactivated animal Bifidobacterium lactis subspecies BA-C9 of the present invention on fasting blood glucose and fasting insulin in mice; Fig.12 As shown, the results showed that animal Bifidobacterium BA-C9 itself has the ability to improve liver damage caused by the combined effects of high-fat diet and STZ. Alanine aminotransferase and aspartate aminotransferase are recommended by the World Health Organization as the most sensitive detection indicators of liver function damage. Compared with the control group (NFD), the plasma ALT and AST levels of mice in the HFD group were significantly increased, while the ALT and AST levels of mice in the HFD+BA-C9 (heat-killed) group were significantly lower than those in the HFD group.

[0140] Fig.13 The invention relates to the effect of the inactivated animal Bifidobacterium lactis subspecies BA-C9 on triglycerides (TG), total cholesterol (TC), high-density lipoprotein (HDL-C), and low-density lipoprotein (LDL-C) in mouse plasma; Fig.13 The results showed that inactivated animal Bifidobacterium BA-C9 can significantly reduce the dyslipidemia caused by HFD+STZ. The combined effect of 8 weeks of high-fat diet and STZ can significantly increase the plasma TG, TC and LDL-C levels of mice in the HFD group and reduce the HDL-C level. After intervention with animal Bifidobacterium BA-C9, the increase in TG, TC and LDL-C levels caused by high-fat diet was significantly reduced, and the plasma HDL-C level was increased.

[0141] Example 5: Animal Bifidobacterium lactis subspecies BA-C9 was used as the research object to simulate the gastrointestinal tolerance experiment, and Bifidobacterium lactis BL-99 and Bifidobacterium lactis HN019 were used as comparative examples.

[0142] Tolerance experiment in gastrointestinal fluid: simulated human gastrointestinal fluid was prepared, and animal Bifidobacterium lactis subspecies BA-C9, Bifidobacterium lactis BL-99 and Bifidobacterium lactis HN019 were treated in the simulated gastrointestinal fluid for different time periods; the plate counting method was used to determine the number of live bacteria of animal Bifidobacterium lactis subspecies BA-C9, Bifidobacterium lactis BL-99 and Bifidobacterium lactis HN019 after treatment in the simulated gastrointestinal fluid.

[0143] Preparation of simulated gastric juice: Use PBS at pH 2.5 to prepare a pepsin solution with a concentration of 3 mg / mL, i.e. artificial gastric juice, which is filtered and sterilized through a 0.22 μm microporous filter membrane for later use.

[0144] Preparation of simulated intestinal fluid: Prepare a trypsin solution with a concentration of 1 mg / mL using PBS at pH 8.0, add ox bile salt to make the final concentration of 1.8%, the obtained solution is the artificial intestinal fluid, and filter it through a 0.22 μm microporous filter membrane for sterilization before use.

[0145] Add sterile PBS (pH 7.3) to the powder of animal Bifidobacterium lactis subspecies BA-C9, Bifidobacterium lactis BL-99 and Bifidobacterium lactis HN019, shake to resuspend, take 500 μL of the resuspended solution and add it to 4.5 mL of artificial gastric juice, incubate in a 37°C incubator for 3 hours, and count by pouring plate method. Then take 500 μL of the bacterial suspension incubated in artificial gastric juice for 3 hours and add it to 4.5 mL of artificial intestinal juice, incubate in a 37°C incubator for 8 hours, and count by pouring plate method. Calculate the survival rate according to the following formula.

[0146] Survival rate = number of colonies after treatment (CFU / mL) / initial number of colonies (CFU / mL) × 100%.

[0147] The gastrointestinal tolerance results of Bifidobacterium animalis subspecies lactis BA-C9, Bifidobacterium lactis BL-99 and Bifidobacterium lactis HN019 are shown in Table 1:

[0148]

[0149] Table 1

[0150] From the table, it can be seen that the survival rate of the viable bacteria of animal Bifidobacterium lactis subspecies BA-C9 can still be maintained at 32.62% after 3 hours in gastric juice, while the survival rate of the viable bacteria after 8 hours of incubation in intestinal juice is reduced to 78.29% of the viable bacteria after gastric juice treatment. This shows that animal Bifidobacterium lactis subspecies BA-C9 has higher gastrointestinal tolerance and has higher tolerance to simulated gastrointestinal juice than Bifidobacterium lactis BL-99 and Bifidobacterium lactis HN019.

[0151] In summary, animal bifidobacterium lactis subspecies BA-C9 of the present invention can effectively reduce the blood sugar level of diabetic mice by regulating fasting blood sugar, reducing liver injury reaction and improving dyslipidemia state.Experiments have shown that the live bacteria type bacterium powder and inactivated bacteria liquid of animal bifidobacterium lactis subspecies BA-C9 of the present invention all have the ability of good improvement of diabetic symptoms, can reduce diabetic mice blood sugar level, have the advantages of safety, effective, no obvious side effects.Therefore the research and development of animal bifidobacterium lactis subspecies BA-C9 live bacteria type bacterium powder and inactivated bacteria liquid of the present invention, for the improvement of diabetic symptoms has opened up a new direction.

[0152] The above descriptions are only some preferred embodiments of the present disclosure and an explanation of the technical principles used. Those skilled in the art should understand that the scope of the invention involved in the embodiments of the present disclosure is not limited to the technical solutions formed by a specific combination of the above-mentioned technical features, but should also cover other technical solutions formed by any combination of the above-mentioned technical features or their equivalent features without departing from the above-mentioned inventive concept. For example, the above-mentioned features are replaced with the technical features with similar functions disclosed in the embodiments of the present disclosure (but not limited to) and the technical solutions formed.

Claims

1. An animal Bifidobacterium lactis subspecies BA-C9 having the function of improving diabetes, characterized in that: The animal Bifidobacterium lactis subspecies BA-C9 was deposited in the China Center for Type Culture Collection on March 11, 2024, with the deposit number: CCTCC NO: M2024457.

2. Use of the animal Bifidobacterium lactis subspecies BA-C9 according to claim 1 in the preparation of a product for improving diabetic symptoms.

3. The use according to claim 2, characterized in that: The product is a bacterial agent, food and / or medicine.

4. The use according to claim 3, characterized in that: The food is one or more of fermented milk, cheese, milk-containing beverage, solid beverage and milk powder; the food contains live bacterial powder and / or inactivated bacterial liquid of Bifidobacterium animalis subspecies lactis BA-C9.

5. The use according to claim 3, characterized in that: The medicine comprises live bacteria powder and / or inactivated bacterial liquid of animal Bifidobacterium lactis subspecies BA-C9, and / or other medicines compatible with the live bacteria powder and / or inactivated bacterial liquid of animal Bifidobacterium lactis subspecies BA-C9, as well as pharmaceutically acceptable carriers and / or excipients.

6. The use according to claim 2, characterized in that: The animal Bifidobacterium lactis subspecies BA-C9 is used for preparing bacterial agents, foods and / or medicines for reducing fasting blood sugar.

7. The use according to claim 2, characterized in that: The animal Bifidobacterium lactis subspecies BA-C9 is used for preparing bacterial agents, foods and / or medicines for improving insulin resistance and glucose tolerance.

8. The use according to claim 2, characterized in that: The animal Bifidobacterium lactis subspecies BA-C9 is used for preparing bacterial agents, foods and / or medicines for improving dyslipidemia.

9. The use according to claim 2, characterized in that: The animal Bifidobacterium lactis subspecies BA-C9 is used for preparing bacterial agents, foods and / or medicines for protecting liver damage.

10. The use according to claim 4, characterized in that: The preparation of the inactivated bacterial solution of the animal Bifidobacterium lactis subspecies BA-C9 is as follows: 1 g of BA-C9 powder was dissolved in 500 mL of LB liquid culture medium, and after culturing at 37°C for 24 h, the bacterial suspension was inactivated at 121°C for 30 min under high temperature and high pressure, and centrifuged at 3000 rpm for 15 min at room temperature. After the centrifugation, the supernatant was removed, and an appropriate amount of PBS was added to wash away the residual culture medium, and the mixture was centrifuged again to remove the supernatant to obtain the inactivated animal Bifidobacterium lactis subsp. BA-C9 cells. 1 mL of physiological saline was added to every 50 mg of the cells to dissolve the inactivated animal Bifidobacterium lactis subsp. BA-C9 bacterial liquid.

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