A strain of Bifidobacterium longum subspecies longum CN973 capable of stabilizing blood sugar and blood pressure and enhancing liver function and its application

Abstract

The present invention provides a strain of Bifidobacterium longum subsp. longum CN973 that stabilizes blood sugar and blood pressure and enhances liver function, and its application. The Bifidobacterium longum subsp. longum CN973 (Bifidobacterium longum subsp. longum CN973) is deposited in the China Center for Type Culture Collection, with a deposit date of May 28, 2024, and a deposit number of CCTCC NO: M 20241083. The strain has the effect of stabilizing blood sugar and blood pressure and enhancing liver function. Experimental results show that the Bifidobacterium longum subsp. longum CN973 has excellent acid and bile salt resistance and is safe for the human body; it has an outstanding antibacterial effect and can effectively inhibit Escherichia Ferguson, Vibrio parahaemolyticus and / or Streptococcus anginosus; in addition, it can effectively stabilize blood pressure and blood sugar, has an immunomodulatory effect, can enhance the body's immunity, and can improve the liver's antioxidant capacity and enhance liver function; especially when used in combination with Lactobacillus plantarum CN211, the two have a synergistic effect, and regulating immune function and improving liver antioxidant capacity are significantly better.

Description

Technical Field

[0001] The present invention belongs to the technical field of Bifidobacterium longum subspecies longum, and in particular relates to a strain of Bifidobacterium longum subspecies longum CN973 that stabilizes blood sugar and blood pressure and enhances liver function, and applications thereof. Background Art

[0002] Hypertension and hyperglycemia are two common chronic diseases that pose multiple risks to the human body, affecting multiple systems and organs, including the cardiovascular, renal, ocular, and nervous systems. Therefore, it is crucial for patients with hypertension and hyperglycemia to actively control their condition, maintain a good lifestyle and diet, and regularly monitor relevant indicators. At the same time, patients should also receive standardized treatment and management under the guidance of a doctor to reduce the risk of complications and improve their quality of life. In the daily management of hypertension and hyperglycemia, controlling blood pressure and blood sugar is crucial. Stable blood sugar and blood pressure can reduce the occurrence of most complications and significantly improve quality of life.

[0003] Immunity refers to the overall activity of the human immune system, which plays an important role in protecting the body from external pathogens, preventing the occurrence of diseases, and maintaining a healthy state. The functional characteristics of immunity mainly include: 1. Identification and defense against pathogens: Identifying and attacking invading pathogens, including bacteria, viruses, fungi, and parasites, by producing antibodies, cytokines, and other immune molecules. 2. Repairing damaged tissues: The immune system can identify and repair damaged tissues and cells to help the body recover. This is usually achieved by producing stem cells, regulating cell apoptosis, and promoting tissue regeneration. 3. Regulating autoimmunity: The immune system also needs to maintain tolerance to its own tissues while identifying and attacking foreign pathogens to avoid the occurrence of autoimmune diseases. Nutrition, genetics, environment, emotions, lifestyle habits, age, and other factors all affect immunity. Low immunity will not be able to effectively resist infection and disease, which will lead to the occurrence of many diseases. Summary of the Invention

[0004] Purpose of the invention: To address the problems existing in the prior art, the present invention provides a strain of Bifidobacterium longum subspecies longum CN973 and its application for stabilizing blood sugar and blood pressure and enhancing liver function.

[0005] Technical solution: To achieve the above-mentioned purpose, the present invention adopts the following technical solution:

[0006] In the first aspect, the present invention provides a strain of Bifidobacterium longum subsp. longum that stabilizes blood sugar and blood pressure and enhances liver function. The Bifidobacterium longum subsp. longum is Bifidobacterium longum subsp. longum CN973 (Bifidobacterium longumsubsp.longum CN973), which is deposited in the China Center for Type Culture Collection on May 28, 2024, and the deposit number is CCTCC NO: M 20241083.

[0007] In a second aspect, the present invention provides a composition comprising the Bifidobacterium longum subspecies longum CN973.

[0008] In a third aspect, the present invention provides the use of the Bifidobacterium longum subspecies longum CN973 and the composition in the preparation of a blood sugar stabilizing product; preferably, the blood sugar stabilizing product can alleviate elevated blood sugar caused by diabetic nephropathy.

[0009] In a fourth aspect, the present invention provides the use of the Bifidobacterium longum subspecies longum CN973 and the composition in the preparation of a blood pressure stabilizing product; preferably, the blood pressure stabilization can alleviate the increase in blood pressure caused by diabetic nephropathy.

[0010] In a fifth aspect, the present invention provides the use of the Bifidobacterium longum subspecies longum CN973 and the composition in the preparation of immunomodulatory products.

[0011] In a sixth aspect, the present invention provides the use of the Bifidobacterium longum subspecies longum CN973 and the composition in the preparation of a product for improving the antioxidant capacity of the liver.

[0012] In a seventh aspect, the present invention provides the use of the Bifidobacterium longum subspecies longum CN973 and the composition in the preparation of an antibacterial agent; preferably, the antibacterial agent can inhibit Escherichia fergusonii, Vibrio parahaemolyticus and / or Streptococcus anginosus.

[0013] In an eighth aspect, the present invention provides a composition comprising Bifidobacterium longum subspecies longum CN973 and Lactobacillus plantarum CN211, wherein the Lactobacillus plantarum CN211

[0014] (Lactiplantibacillus plantarum CN211), deposited in China Center for Type Culture Collection, the deposit date is April 7, 2024, and the deposit number is CCTCC NO: M 2024642.

[0015] In a ninth aspect, the present invention provides use of the composition comprising Bifidobacterium longum subsp. longum CN973 and Lactobacillus plantarum CN211 in the preparation of an immunomodulatory product.

[0016] In a tenth aspect, the present invention provides the use of the composition comprising Bifidobacterium longum subspecies longum CN973 and Lactobacillus plantarum CN211 in the preparation of a product for improving liver antioxidant capacity.

[0017] The products described in the present invention include food, antibacterial agents or medicines.

[0018] Beneficial Effects: Compared with the existing technology, the present invention provides a new strain of Bifidobacterium longum subspecies longum CN973, which has the effects of stabilizing blood sugar and blood pressure and enhancing liver function. Experimental results show that Bifidobacterium longum subspecies longum CN973 has excellent acid and bile salt resistance and is safe for the human body. It has outstanding antibacterial effects and can effectively inhibit Escherichia fergusonii, Vibrio parahaemolyticus, and / or Streptococcus anginosus. In addition, it can effectively stabilize blood pressure and blood sugar, has immunomodulatory effects, can enhance the body's immunity, and can improve the liver's antioxidant capacity and enhance liver function. In particular, when used in combination with Lactobacillus plantarum CN211, the two have a synergistic effect, significantly improving immune function and enhancing liver antioxidant capacity. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Gram stain of Bifidobacterium longum subsp. longum CN973.

[0020] Figure 2 This is the biological evolutionary tree of Bifidobacterium longum subspecies longum CN973.

[0021] Figure 3 Figure 5 is the fasting blood glucose values ​​of the tail vein of mice in each group at different time points.

[0022] Figure 4 is the diastolic blood pressure at the tail of mice in each group.

[0023] Figure 5 is the tail systolic blood pressure of mice in each group.

[0024] Figure 6 This is the statistical result of serum immunoglobulin IgG content in each group of mice.

[0025] Figure 7 This is the statistical result of serum immunoglobulin IgA content in each group of mice.

[0026] Figure 8 This is the statistical result of the total antioxidant capacity of the liver of each group of mice.

[0027] Figure 9 This is the statistical result of glutathione peroxidase content in the liver of mice in each group.

[0028] Figure 10 This is the statistical result of superoxide dismutase content in the liver of mice in each group. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is further described in detail below with reference to the accompanying drawings and embodiments.

[0030] Example 1 Screening of acid-resistant and bile-resistant strains

[0031] In order to further obtain healthy intestinal flora for regulating the stability of blood sugar and blood pressure, the dominant strains of healthy intestines were screened. Frozen healthy intestinal samples were taken, gradiently diluted and then spread on MRS agar medium for culture. The same sample was spread twice in parallel and cultured in an anaerobic environment. The culture temperature was 35-37 ° C. Single colony strains were selected from MRS agar medium and placed in 15 ml MRS liquid culture medium. The selected single colony was placed in a 35-37 ° C shaker culture for 8-10 hours to obtain fermentation liquid. By measuring the fermentation OD 600 Compare the fermentation liquid with the microscope to observe the bacterial count, discard the liquid fermentation effect is poor or no obvious bacterial amplification samples, take 0.5ml of the remaining fermentation liquid for strain sequencing identification, take 2ml and add 2ml 30%-40% glycerol solution, pre-cool and freeze to -80℃ for alternative strain storage, and continue to ferment the remaining fermentation liquid at 35-37℃ in a shaker, measure the pH value every 8h, and measure it for 3 times in a row. 600 Samples with higher final fermentation pH values ​​of 3.5-4.5 were selected as candidate strains, totaling 22 samples, numbered CN970-CN991.

[0032] Example 2 Screening of high membrane adhesion strains

[0033] The strains selected in Example 1 were subjected to an intestinal mucosal adhesion test. After each strain was cultured at 37°C for 3 hours, scanning electron microscopy analysis showed that a strain CN973 with high intestinal mucosal adhesion was selected, with an adhesion rate of 88.75% ± 7.4% to the intestinal mucosa, indicating a high mucosal adhesion rate.

[0034] Example 3 Preservation, identification and collection of bacterial strains

[0035] Combined with the evaluation results of the strain screening process in Example 1-2, the CN973 strain with both high acid resistance and high intestinal mucosal adhesion rate was preserved as the target strain. The Gram staining result was Gram-positive, such as Figure 1As shown. 16SrRNA molecular biological identification was carried out, and the 16SrDNA gene sequence of strain CN973 was amplified and sequenced using the published 16S universal primers (primer sequences: 8F: 5'-AGAGTTTGATCCTGGCTC A-3'; 1510R: 5'-GGTTACCTTGTTACGACTT-3'). The PCR amplification product was sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing and identification. The nucleotide sequence of 16SrDNA of strain CN973 is sequence 1 in the sequence table; after 16SrDNA gene alignment, the similarity rate with Bifidobacterium longum subsp.longum in Genebank was 98%; combined with microbial system identification, strain CN973 was determined to be a long subspecies of Bifidobacterium longum, named Bifidobacterium longum subsp.longum CN973 (Bifidobacterium longum subsp.longum CN973), and its 16S rDNA is shown in SEQ ID NO.1, and Gram staining is as shown in Figure 1 As shown, the biological evolution tree is as follows Figure 2 shown.

[0036] The biologically identified Bifidobacterium longum subsp. longum CN973 (Bifidobacterium longumsubsp.longum CN973) was deposited in the China Center for Type Culture Collection (CCTCC) on May 28, 2024, with the deposit number CCTCC NO:M 20241083. The deposit address is Wuhan University, Wuhan City, Hubei Province, People's Republic of China.

[0037] Example 4: Ability of Bifidobacterium longum subspecies longum CN973 to inhibit pathogenic bacteria

[0038] Bifidobacterium longum subspecies longum BB536 was selected as the control test strain of Bifidobacterium longum subspecies longum CN973 and inoculated into 50 ml of MRS liquid medium. After culturing at 37 ° C for 16 hours, the fermentation liquid was centrifuged at 10000 r / min for 10 minutes, and the supernatant and bacteria were collected respectively. The bacteria were washed twice with 0.9% sterile saline and then prepared into OD 600 It is a bacterial suspension of 0.5-0.8, stored at 4 ° C for future use.

[0039] Escherichia fergusonii ATCC 35469, Vibrio parahaemolyticus ATCC 17802, and Streptococcus anginosus ATCC 9895 were selected as test strains for in vitro antibacterial experiments. The fermentation supernatant and bacterial suspension of each strain were subjected to inhibition zone tests using the agar punch method. Erythromycin solution at a concentration of 0.05 mg / mL served as a positive control for inhibition against Escherichia fergusonii ATCC 35469, Vibrio parahaemolyticus ATCC 17802, and Streptococcus anginosus ATCC 9895. MRS culture medium adjusted to the same pH as the supernatant served as a blank control. The inhibition results for each strain are shown in Table 1.

[0040] The results showed that the bacterial suspension and fermentation supernatant of the CN973 strain had a certain inhibitory effect on Escherichia Ferguson, Vibrio parahaemolyticus and Streptococcus anginosus, and were significantly better than the BB536 strain, and can be used in inhibitory products for related pathogens.

[0041] Table 1 Evaluation of antibacterial ability

[0042]

[0043] Note: a is significantly different from BB536.

[0044] Example 5 Industrial fermentation of Bifidobacterium longum subspecies longum CN973 and preparation of lyophilized powder

[0045] (1) Culture medium configuration.

[0046] The configuration scheme for industrial high-density Bifidobacterium longum subspecies longum CN973 culture medium is as follows: soy peptone 16.5g / L, yeast extract powder 21.8g / L, maltose 16.7g / L, anhydrous glucose 10g / L, magnesium sulfate 0.15g / L, dipotassium hydrogen phosphate 2g / L, diammonium hydrogen citrate 2g / L, manganese sulfate 0.06g / L, L-cysteine ​​hydrochloride 1g / L, and Tween 80 1ml / L. All raw materials are food-grade.

[0047] (2) Liquid culture

[0048] After the culture medium is prepared, the pH value is adjusted to 6.8, sterilized at 121°C for 15 minutes, and cooled to 36°C-38°C. The fermenter is inoculated at a 3%-8% inoculum, preferably 5%, and cultured at a temperature of 35°C-37°C and a pH of 6.2-6.5. During the fermentation process, the fermenter is sealed and pressure is maintained with nitrogen. The fermenter is stirred slowly. The culture is continued for 8-16 hours. At 8 hours, the viable bacterial count of the fermentation broth is measured to be 2.6×10 8 CFU / mL, and the viable bacteria in the fermentation broth were measured at 16 h to be 3.5×10 9CFU / mL, the level of fermentation live bacteria is already relatively high, meeting the requirements of industrial high-density fermentation production.

[0049] (3) Centrifugation

[0050] In order to avoid the aging of bacteria caused by too long fermentation time, which will affect the activity of freeze-dried powder bacteria, the active bacteria should be kept at 10 8 CFU / mL-10 9 CFU / mL, based on OD 600 After the growth curve entered the plateau phase, the fermentation broth was quickly cooled to below 15°C and centrifuged at 5000 rpm using a tubular centrifuge to harvest the bacterial sludge.

[0051] (4) Protective agent emulsification and freeze-drying

[0052] The harvested sludge is diluted to a moisture content of 60%-80%. An equal volume of sludge preservative is added for emulsification. The sludge preservative consists of: 70% water, 10% skim milk powder, 5% sucrose, 5% mannitol, 1% Tween-80, 3% betaine, 0.5% sodium glutamate, and 5% soluble starch. After mixing thoroughly, the sludge is placed in a vacuum freeze dryer for freeze-drying. The freeze-dried material is ≤1 cm thick. The dried material is characterized by a moisture content of less than 5% and a water activity of 0.08-0.20aw.

[0053] The activity of freeze-dried bacterial powder can reach 5.5×10 11 CFU / g, Bifidobacterium longum subspecies longum CN973 is a high-yield strain and can be used for industrial fermentation production, for the preparation of food-added lactic acid bacteria powder or for the preparation of microecological intervention preparations.

[0054] Example 6 Effect of Bifidobacterium longum subspecies longum CN973 on blood glucose stability

[0055] Lepr gene mutant mice were constructed using C57BLKS / JGpt mice as the background and randomly divided into two groups, each with 6 mice, divided into a model group (G1) and an intervention group (G2). In addition, 6 normal mice were selected as a control group (G0). The mice were adaptively fed according to normal feeding conditions for one week, and tail vein blood was collected and fasting blood glucose levels were measured after 6 hours of fasting. The control group mice were then given 0.5 mL / day of purified water by gavage, and the experimental group was given 0.5 mL / day of Bifidobacterium longissimum subsp. CN973 bacterial solution (bacterial content of 1×10 8 CFU / mL) were gavaged, and after 30 days of intervention, the tail vein fasting blood glucose was tested every 10 days until 60 days of intervention. The changes in blood glucose in each group were as follows: Figure 3As shown in the figure (G0-0 represents the test results of the control group on day 0, G0-30 represents the test results of the control group on day 30, and so on, the same applies to G1 and G2). The results show that compared with the control group, the fasting blood glucose values ​​of the mice in the model group increased significantly, that is, the mice in the model group showed hyperglycemia. After intervention with Bifidobacterium longum subspecies longum CN973, the blood glucose values ​​of the model mice were alleviated, and the fluctuation range of blood glucose was small throughout the process, which can control blood glucose stability.

[0056] Example 7 Effect of Bifidobacterium longum subspecies longum CN973 on blood pressure stability

[0057] In Example 6, the tails of the mice were fixed before and 60 days after the intervention, and the blood pressure was measured using a non-invasive continuous blood pressure monitor. The diastolic and systolic blood pressure levels of the mice in each group were as follows: Figure 4 and Figure 5 As shown (G0-0 and other codes have the same meaning Figure 3 ), as shown in the figure, compared with the model group, the blood pressure level and diastolic pressure were reduced to varying degrees after the intervention of Bifidobacterium longum subsp. longum CN973. This shows that the Bifidobacterium longum subsp. longum CN973 involved in the present invention can significantly alleviate the elevated blood pressure in diabetic nephropathy-induced mice.

[0058] Example 8: Immunomodulatory Effects of Bifidobacterium longum Subspecies longum CN973

[0059] Forty-eight healthy female Kunming mice were randomly divided into six groups after adaptive feeding for two weeks. One group served as a blank control group, and the remaining five groups were orally gavaged with 100 mg / kg cyclophosphamide daily for three consecutive days to establish an immunocompromised mouse model. The mice in each group were then intervened according to the experimental method in Table 2, with the corresponding bacterial solution concentration of 1.0 × 10 9 CFU / day / mouse, intervention continued for 1 week, mice were sacrificed, and samples were collected. Blood was collected from the mouse orbits and allowed to stand at room temperature for 2 hours. The samples were centrifuged at 2000×g for 10 minutes, and the supernatant was used for enzyme-linked immunosorbent assay (ELISA) and biochemical analysis. Figure 6 、 Figure 7 As shown in the data, compared with the control groups BL1 and BL2, the levels of serum immunoglobulin IgG and serum immunoglobulin IgA in mice increased to varying degrees after probiotic intervention in each group, and the effect of the TE3 group was better than that of other single strain intervention effects or combined intervention effects, that is, the combination of Lactobacillus plantarum CN211 and Bifidobacterium longum subspecies longum CN973 can more significantly increase the expression level of immunoglobulins in serum and improve the body's immunity.

[0060] Table 2 Experimental groups and intervention methods

[0061]

[0062] Example 9 Analysis of the Antioxidant Capacity of Bifidobacterium longum Subspecies longum CN973 on Liver

[0063] Based on the experimental mouse intervention protocol of Example 8, the mouse liver organs were removed and rinsed with enzyme-free nucleic acid water. A 10% (w / v) homogenate of the liver was prepared by adding sterile 0.9% saline solution. The liver antioxidant indicators of total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-PX) and superoxide dismutase (SOD) were tested. The results were as follows: Figure 8 、 Figure 9 、 Figure 10 As shown in the figure, compared with the control group, after the intervention of each probiotic group, the total antioxidant capacity, glutathione peroxidase and superoxide dismutase content of mice were improved to varying degrees, and the effect of the plant lactobacillus CN211 and Bifidobacterium longum subspecies longum CN973 group was better than that of other single strains or combinations. It can be seen that the combination of Bifidobacterium longum subspecies longum CN973 and Lactobacillus plantarum CN211 involved in the present invention can significantly improve the liver's antioxidant capacity.

[0064] The above describes the implementation of the present invention in detail with reference to specific embodiments. However, the present invention is not limited to the above implementation. Various changes can be made within the scope of knowledge possessed by ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. A strain of Bifidobacterium longum subspecies longum that stabilizes blood sugar and blood pressure and enhances liver function, characterized by: The Bifidobacterium longum subsp. longum is Bifidobacterium longum subsp. longum CN973, which was deposited in the China Center for Type Culture Collection on May 28, 2024, with a deposit number of CCTCC NO: M20241083.

2. A composition, characterized in that The composition comprises the Bifidobacterium longum subspecies longum CN973 according to claim 1.

3. Use of the Bifidobacterium longum subspecies longum CN973 according to claim 1 and the composition according to claim 2 in the preparation of a blood sugar stabilizing product.

4. Use of the Bifidobacterium longum subspecies longum CN973 according to claim 1 and the composition according to claim 2 in the preparation of a product for stabilizing blood pressure.

5. Use of the Bifidobacterium longum subspecies longum CN973 according to claim 1 and the composition according to claim 2 in the preparation of an immunomodulatory product.

6. Use of the Bifidobacterium longum subspecies longum CN973 according to claim 1 and the composition according to claim 2 in the preparation of a product for improving the antioxidant capacity of the liver.

7. Use of the Bifidobacterium longum subspecies longum CN973 according to claim 1 and the composition according to claim 2 in the preparation of an antibacterial agent, characterized in that: The bacteriostatic agent can inhibit Escherichia Fergusonii, Vibrio parahaemolyticus and / or Streptococcus anginosus.

8. A composition, characterized in that The composition comprises the Bifidobacterium longum subspecies longum CN973 described in claim 1 and Lactobacillus plantarum CN211, wherein the Lactobacillus plantarum CN211 is deposited in the China Center for Type Culture Collection on April 7, 2024, with a deposit number of CCTCCNO: M 2024642.

9. Use of the composition according to claim 8 in the preparation of immunomodulatory products.

10. Use of the composition according to claim 8 in preparing a product for improving the antioxidant capacity of the liver.

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