Probiotic agent comprising akkermansia muciniphila strain akk11 for ameliorating hypertension and use thereof

Abstract

Provided is a probiotic agent comprising Akkermansia muciniphila strain Akk11 for ameliorating hypertension. Strains in the probiotic agent include Akkermansia muciniphila strain Akk11 and Lactobacillus rhamnosus strain LRa05. The two strains have potential interactions, can cooperate with each other, and work synergistically to ameliorate hypertension. Specifically, the strains significantly enhance the blood pressure regulation ability and optimize the blood pressure regulation mechanism; significantly regulate the levels of protein in urine and serum creatinine, which are renal function markers, effectively improve renal function, and alleviate kidney injuries caused by hypertension; and significantly optimize oxidative stress responses and enhance the body's defense against oxidation.

Description

A probiotic containing Akk11 strain of Akkermansia myxophilus for improving hypertension and its application Technical Field

[0001] This application belongs to the field of probiotic preparation technology, and relates to a probiotic preparation containing Akk11 strain of Acermetophilus for improving hypertension and its application. Background Technology

[0002] Traditional treatments for hypertension primarily rely on medication, such as ACE inhibitors and calcium channel blockers. However, long-term drug therapy can not only cause side effects but also lead to increased patient dependence. Therefore, researchers and medical experts have been seeking safer alternative treatments.

[0003] Probiotics, as biological agents that regulate the balance of the human gut microbiota, are increasingly recognized for their potential in disease prevention and treatment. Probiotic strains have shown the potential to lower blood pressure through various mechanisms, such as improving gut microbiota balance, promoting the growth of beneficial microorganisms, reducing toxins produced by harmful bacteria, and enhancing intestinal barrier function. Furthermore, probiotics can indirectly regulate blood pressure by influencing the gut-brain axis to regulate autonomic nervous system function.

[0004] While second-generation probiotics like *Ackermania myxotropicis* are widely used, reports and strategies for their application in treating hypertension are still scarce. Therefore, developing more probiotic agents that can effectively improve hypertension would not only provide new strategies for its prevention and treatment but also help expand the applications of *Ackermania myxotropicis*.

[0005] Summary of the Invention

[0006] This application provides a probiotic agent containing Akk11 strain of Akermania myxophilus for improving hypertension and its application, specifically providing a probiotic agent containing Akk11 strain of Akermania myxophilus for improving hypertension and its application in the preparation of drugs for preventing, improving or treating hypertension.

[0007] In one aspect, this application provides a probiotic agent for improving hypertension containing the Akkermansia muciniphila strain Akk11, wherein the strains in the probiotic agent include Akkermansia muciniphila strain Akk11 and Lactobacillus rhamnosus strain LRa05; wherein the Akkermansia muciniphila strain Akk11 has the accession number CCTCC NO. M2024119, the accession date is January 15, 2024, and the depositary institution is the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China; the Lactobacillus rhamnosus strain LRa05 has the accession number CGMCC. No. 24377, deposited on January 24, 2022, by the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. This application develops a novel probiotic compounding method, which combines Akkermansia muciniphila Akk11 strain and Lactobacillus rhamnosus LRa05 strain. It was found that the two strains have potential interactions and can cooperate to synergistically enhance the efficacy in improving hypertension. Specifically, they significantly enhance the ability to regulate blood pressure and optimize the blood pressure regulation mechanism; significantly regulate the levels of renal function markers such as urinary protein and serum creatinine, effectively improve renal function, and reduce kidney damage caused by hypertension; and significantly optimize the oxidative stress response and enhance the body's antioxidant defense capabilities.

[0008] When using the same bacterial count, the combination of the two strains significantly enhanced the aforementioned efficacy compared to using either a single Akk11 strain or a single LRa05 strain. Therefore, this probiotic preparation provides a new strategy for the prevention, relief, or treatment of hypertension. Since both Akkermansia myxophilus and Lactobacillus rhamnosus are probiotics, their use in the preparation of related products is highly safe and unlikely to induce dependence.

[0009] Preferably, the ratio of viable counts of the *Akkermansia muciniphila* Akk11 strain to *Lactobacillus rhamnosus* LRa05 strain is 1:10-10:1, for example, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0010] Preferably, the total number of live bacteria in the probiotic agent is not less than 1×10⁻⁶. 8 CFU / mL or 1×10 8 CFU / g, for example 1×10 8 CFU / mL (CFU / g), 1×10 9 CFU / mL (CFU / g), 5×10 9 CFU / mL (CFU / g), 1×10 10 CFU / mL (CFU / g), 5×10 10 CFU / mL (CFU / g), 1×10 11 CFU / mL (CFU / g), 1×10 12 CFU / mL (CFU / g) or 1×10 13 CFU / mL (CFU / g), etc.; other specific point values ​​within this range can be selected, which will not be elaborated here.

[0011] Preferably, the dosage form of the probiotic agent includes solutions, lyophilized powders, capsules, tablets, or granules. The dosage form of the probiotic agent involved in this application is not limited, including the most commonly used solutions, lyophilized powders, or further prepared capsules, tablets, or granules.

[0012] Preferably, the probiotic agent is in the form of a solution, which is prepared by the following method:

[0013] The Akk11 strain and the LRa05 strain were inoculated into the culture medium and activated and fermented sequentially to obtain fermentation broth. The fermentation broth was centrifuged and resuspended in solvent to obtain Akk11 bacterial suspension and LRa05 bacterial suspension. The Akk11 bacterial suspension and LRa05 bacterial suspension were mixed according to the live bacteria ratio to obtain the probiotic agent.

[0014] Preferably, the probiotic agent is in the form of a lyophilized powder, which is prepared by the following method:

[0015] Akk11 and LRa05 strains were inoculated into culture medium and activated and fermented sequentially to obtain fermentation broth. The fermentation broth was centrifuged, mixed with a protectant, and then freeze-dried to obtain Akk11 powder and LRa05 powder. The Akk11 powder and LRa05 powder were mixed according to the live cell count ratio to obtain the probiotic agent.

[0016] Preferably, the protective agent is selected from any one or a combination of at least two of the following: skim milk, gelatin, dextrin, gum arabic, dextran, sodium alginate, polyvinylpyrrolidone, sucrose, lactose, trehalose, sorbitol, or xylitol.

[0017] Secondly, this application provides the use of the probiotic agent described in the first aspect in the preparation of a medicament for the prevention, improvement or treatment of hypertension.

[0018] Preferably, the drug further includes excipients selected from any one or a combination of at least two of the following: fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, colorants, pH regulators, antioxidants, antibacterial agents, or buffers.

[0019] Preferably, the drug further includes a functional adjuvant selected from any one or a combination of at least two of the following: fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, inulin, trachomaconazole, polydextrose, α-lactalbumin, or lactoferrin.

[0020] Compared with the prior art, this application has the following advantages:

[0021] This application develops a novel probiotic compounding method, which combines Akkermansia muciniphila Akk11 strain and Lactobacillus rhamnosus LRa05 strain. It was found that the two strains have potential interactions and can cooperate to synergistically enhance the efficacy of improving hypertension. Specifically, it can: (1) significantly enhance blood pressure regulation capacity and optimize blood pressure regulation mechanism; (2) significantly regulate the levels of renal function markers such as urinary protein and serum creatinine, effectively improve renal function, and reduce kidney damage caused by hypertension; and (3) significantly optimize oxidative stress response and enhance the body's antioxidant defense capacity.

[0022] When using the same bacterial count, the combination of the two strains significantly enhanced the aforementioned efficacy compared to using either a single Akk11 strain or a single LRa05 strain. Therefore, this probiotic preparation provides a new strategy for the prevention, relief, or treatment of hypertension. Since both Akkermansia myxophilus and Lactobacillus rhamnosus are probiotics, their use in the preparation of related products is highly safe and unlikely to induce dependence. Attached Figure Description

[0023] Figure 1 shows the statistical results of tail artery systolic blood pressure in each group of rats.

[0024] Figure 2 shows the statistical results of plasma norepinephrine levels in rats of each group.

[0025] Figure 3 shows the statistical results of plasma adrenaline levels in rats of each group.

[0026] Figure 4 shows the statistical results of creatinine levels in the serum of rats in each group.

[0027] Figure 5 shows the statistical results of urinary protein content in the urine of rats in each group.

[0028] Figure 6 shows the statistical results of malondialdehyde levels in the kidney tissue of rats in each group.

[0029] Figure 7 shows the statistical results of superoxide dismutase levels in the kidney tissue of rats in each group.

[0030] Figure 8 shows the statistical results of glutathione levels in the kidney tissue of rats in each group. Detailed Implementation

[0031] The technical solution of this application will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely to help understand this application and should not be regarded as specific limitations on this application.

[0032] The culture medium formulations involved in the following examples are as follows:

[0033] MRS medium: peptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, sodium acetate 2 g / L, yeast extract 5 g / L, diammonium hydrogen citrate 2 g / L, K2PO4·3H2O 2.6 g / L, MgSO4·7H2O 0.1 g / L, MnSO4 0.05 g / L, polysorbate 80 1 mL / L, cysteine ​​hydrochloride 0.5 g / L.

[0034] The Akk11 strain involved in the following examples is classified as Akkermansia muciniphila Akk11, deposited at the China Center for Type Culture Collection (CCTCC) on January 15, 2024, with accession number CCTCC NO: M2024119, and located at Wuhan University, Wuhan, China.

[0035] The LRa05 strain involved in the following examples is classified as Lactobacillus rhamnosus. It is deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 24, 2022, with accession number CGMCC No. 24377. The address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0036] The following method involves the preparation of bacterial suspension: the desired bacterial strain is inoculated into liquid culture medium and activated by culturing at 37°C for 24 hours. This activation is repeated twice to obtain an activated solution. The activated solution is inoculated into liquid culture medium at an inoculation rate of 5% (v / v) and cultured at 37°C for 24 hours to obtain a bacterial suspension. The bacterial suspension is centrifuged at 5000 rpm for 10 minutes at 4°C, filtered, and the bacterial cells are obtained. The bacterial cells are resuspended in PBS solution to obtain the final product.

[0037] The experimental results were statistically analyzed using ggplot2 in R. Compared with the control group, ### represents p<0.001; compared with the model group, *** represents p<0.001, ** represents p<0.01, * represents p<0.05, and NS. represents no significant difference.

[0038] Example

[0039] This example investigates the ability of the strain to improve symptoms in a rat model of hypertension:

[0040] (1) Experimental animals: Healthy SPF-grade male Dahl salt-sensitive rats, 8 weeks old (56 rats, purchased from Shanghai Chengxi Biotechnology Co., Ltd.). These rats were raised in a controlled environment with room temperature maintained at 20-24℃ and humidity at 50-60%, following a 12-hour light / dark cycle. They were allowed to eat and drink freely.

[0041] (2) Animal grouping: After acclimatization feeding for 1 week, rats were randomly divided into 7 groups of 8 rats each: control group (CTL), high salt model group (MC group), Akk11 strain group (Akk11 group, designated as S1 group), LRa05 strain group (LRa05 group, designated as S2 group), commercially available Akkermansia myxophilus group (BNCC341917 group, designated as S3 group), compound bacteria group 1 (Akk11+LRa05 group, viable bacteria ratio of 1:1, designated as S4 group), and compound bacteria group 2 (BNCC341917+LRa05 group, viable bacteria ratio of 1:1, designated as S5 group).

[0042] (3) Animal modeling and intervention methods:

[0043] Rats in the CTL group were fed a diet containing 0.3% sodium chloride, while rats in the MC group and the probiotic intervention group were fed a diet containing 8% sodium chloride to establish a salt-sensitive hypertension model. In addition, rats in the intervention group were also given oral probiotic solution (total probiotic dose 10...). 8 The intervention lasted for 4 weeks in all groups of rats (CFU / day / rat). During the experiment, elevated systolic blood pressure in the tail artery and renal fibrosis were observed in the rats, indicating that the model was successfully established.

[0044] (4) Indicator Analysis:

[0045] (4.1) Effect on systolic blood pressure of rat tail artery:

[0046] After the intervention, the tail artery systolic pressure of rats in each group was measured at the same time between 8 and 9 a.m. in a resting state using a multi-parameter monitor.

[0047] The results are shown in Figure 1. As can be seen, compared with the CTL group, the systolic blood pressure of the tail artery in the MC group was significantly increased, indicating that the model successfully induced a hypertensive state. After receiving probiotic intervention in each group, the systolic blood pressure of the tail artery in the rats was significantly reduced, especially in the S4 group where the effect was most pronounced. This suggests that the probiotic intervention involved in this application can alleviate the increase in blood pressure caused by the hypertension model, and that strains Akk11 and LRa05 have a potential synergistic effect on the above-mentioned efficacy.

[0048] (4.2) Effects on plasma norepinephrine and epinephrine levels in rats:

[0049] After the intervention, 200 μL of blood was collected from each rat between 10:00 and 11:00 AM, and plasma was also collected from each group of rats. The levels of plasma norepinephrine (NE) and epinephrine (EPI) were calculated according to the ELISA kit instructions.

[0050] The results are shown in Figures 2 and 3. As can be seen, compared with the CTL group, the plasma norepinephrine (NE) and epinephrine (EPI) levels in the MC group mice were significantly increased, which is consistent with the typical physiological response of enhanced sympathetic nerve activity under hypertension. After probiotic intervention, the levels of these two important stress hormones significantly decreased, approaching the levels of the healthy control group, indicating that the probiotics involved in this application have the potential to regulate endocrine responses, thereby alleviating hypertension symptoms. This reduction effect was particularly significant in the S4 group.

[0051] (4.3) Effect on serum creatinine levels in rats:

[0052] After the intervention, rats were anesthetized by intraperitoneal injection of 10% chloral hydrate. 5 mL of blood from the abdominal aorta was collected, centrifuged at 4000 r / min for 10 minutes, and the serum was separated. The serum creatinine level was detected using a serum creatinine assay kit.

[0053] The results are shown in Figure 4. As can be seen, the MC group rats exhibited significant renal function impairment under hypertension, specifically a significant increase in serum creatinine levels, which was significantly different from the CTL group. After probiotic intervention, the rats' serum creatinine levels decreased significantly, indicating that probiotics have the potential to protect renal function and improve kidney health. This reduction effect was particularly pronounced in the S4 group, more effectively alleviating kidney damage caused by hypertension.

[0054] (4.4) Effect on the content of urinary protein in rat urine:

[0055] After the intervention, 24-hour urine samples were collected from each group of rats, and urine protein was detected using a urine protein quantification test kit.

[0056] The results are shown in Figure 5. As can be seen, the MC group rats exhibited significant renal function impairment under hypertension, specifically a significant increase in urinary protein levels, which was significantly different from the CTL group. After probiotic intervention, the urinary protein levels in the rats decreased significantly, indicating that probiotics have the potential to protect renal function and improve kidney health. This reduction effect was particularly pronounced in the S4 group, more effectively alleviating kidney damage caused by hypertension.

[0057] (4.5) Effects on oxidative stress indices:

[0058] After the intervention, the rats were sacrificed, and the right kidney tissue was collected and the tissue homogenate supernatant was prepared. The levels of malondialdehyde, superoxide dismutase and glutathione in the rat kidney tissue were detected according to the instructions of the malondialdehyde, superoxide dismutase and glutathione detection kit.

[0059] The results, as shown in Figures 6, 7, and 8, indicate that under hypertension-induced oxidative stress, the malondialdehyde (MDA) level in the MC group rats was significantly increased, while the levels of superoxide dismutase (SOD) and glutathione (GSH) were significantly decreased, reflecting the exacerbation of oxidative stress and the weakening of antioxidant defense mechanisms. After probiotic intervention, the MDA level in hypertensive rats was significantly reduced, while the levels of superoxide dismutase (SOD) and glutathione (GSH) were significantly increased, indicating that probiotics effectively alleviated oxidative damage caused by hypertension. These effects were particularly pronounced in the S4 group, demonstrating that the probiotic agent involved in this application plays an important promoting role in the antioxidant defense system and helps restore cellular antioxidant balance.

[0060] The applicant declares that this application illustrates the technical solution of this application through the above embodiments, but this application is not limited to the above embodiments, that is, it does not mean that this application must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this application, equivalent substitutions of the raw materials of this application's products, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this application.

[0061] The preferred embodiments of this application have been described in detail above. However, this application is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this application, various simple modifications can be made to the technical solution of this application, and these simple modifications all fall within the protection scope of this application.

[0062] It should also be noted that the various specific technical features described in the above embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this application will not describe the various possible combinations separately.

Claims

1. A probiotic agent for improving hypertension comprising Akkermansia muciniphila Akk 11 strain, characterized by, The strains in the probiotic agent include Akkermansia muciniphila Akk11 strain with a preservation number of CCTCC NO: M2024119 and Lactobacillus rhamnosus LRa05 strain with a preservation number of CGMCC No. 24377.

2. The probiotic agent of claim 1, wherein, The ratio of the viable cell counts of the Akkermansia muciniphila Akk11 strain and the Lactobacillus rhamnosus LRa05 strain is 1:10-10:

1.

3. The probiotic agent of claim 1, wherein, In the probiotic agent, the total number of viable bacteria is not less than 1 x 10 8 CFU / mL or 1 x 10 8 CFU / g.

4. The probiotic agent of claim 1, wherein, The dosage form of the probiotic agent includes a solution, a lyophilized powder, a capsule, a tablet or a granule.

5. The probiotic agent of claim 4, wherein, The dosage form of the probiotic agent is a solution, which is prepared by the following method: The Akk11 strain and the LRa05 strain are inoculated into culture media respectively for activation and fermentation culture in sequence to obtain fermentation broth; the fermentation broth is centrifuged respectively, resuspended with a solvent to obtain Akk11 bacterial suspension and LRa05 bacterial suspension; the Akk11 bacterial suspension and the LRa05 bacterial suspension are mixed according to the viable cell count ratio to obtain the probiotic agent.

6. The probiotic agent of claim 4, wherein, The dosage form of the probiotic agent is a lyophilized powder, which is prepared by the following method: The Akk11 strain and the LRa05 strain are inoculated into culture media respectively for activation and fermentation culture in sequence to obtain fermentation broth; the fermentation broth is centrifuged respectively, mixed with a protective agent and then freeze-dried to obtain Akk11 bacterial powder and LRa05 bacterial powder; the Akk11 bacterial powder and the LRa05 bacterial powder are mixed according to the viable cell count ratio to obtain the probiotic agent.

7. The probiotic agent of claim 6, wherein, The protective agent is selected from any one or a combination of at least two of skimmed milk, gelatin, dextrin, gum arabic, dextran, sodium alginate, polyvinylpyrrolidone, sucrose, lactose, trehalose, sorbitol or xylitol.

8. Use of the probiotic agent in any one of claims 1-7 in the preparation of a drug for preventing, improving or treating hypertension.

9. Use according to claim 8, wherein, The drug further includes an auxiliary material selected from any one or a combination of at least two of a filler, a binder, a wetting agent, a disintegrant, an emulsifying agent, a co-solvent, a solubilizing agent, an osmotic pressure regulator, a coloring agent, a pH regulator, an antioxidant, a bacteriostatic agent or a buffer.

10. Use according to claim 8, wherein, The drug further includes a functional aid selected from any one or a combination of at least two of oligofructose, oligogalactose, oligoxylan, inulin, grifolan, polydextrose, alpha-lactalbumin or lactoferrin.

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