Compound probiotic capable of alleviating gouty arthritis and use thereof

Abstract

The present application provides a compound probiotic capable of alleviating gouty arthritis and the use thereof. The compound probiotic capable of alleviating gouty arthritis consists of a Bifidobacterium longum BL21 strain and a Lactobacillus salivarius LS97 strain. The present application has discovered that the two strains can work together, mutually promote each other, and exhibit a synergistic effect in alleviating gouty arthritis. The synergistic effect is specifically demonstrated by the following: the ability to reduce pain-sensitive reactivity; the ability to decrease the number of white blood cells and the aggregation of neutrophils in joints; the ability to lower the activity of myeloperoxidase in periarticular tissues; the ability to reduce the production of the inflammatory factors CXCL1 and IL-1β; and the ability to increase the expression level of the anti-inflammatory cytokine IL-10 in joint tissues.

Description

Composite probiotics for relieving gouty arthritis and application thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of probiotics, and relates to composite probiotics for relieving gouty arthritis and application thereof. BACKGROUND

[0002] Gout is a disease that seriously affects joint health, and its pathogenesis is closely related to uric acid metabolism disorder. When the uric acid level in the body of a patient increases, uric acid crystals will deposit in the joints and surrounding tissues, causing severe pain and inflammatory reactions. These uric acid crystals accumulate in the joints, especially in the joints of the toes, ankles, knees and fingers, causing acute arthritis attacks. Patients may experience sudden pain and swelling, and even severe pain upon slight touch. In addition to acute gouty arthritis, gout can also cause chronic arthritis, and long-term deposition of uric acid can form tophi around the joints, affecting joint function.

[0003] Therefore, gout is not only a joint disease, but also a disease that seriously affects the overall health and quality of life of patients, and timely diagnosis and treatment are crucial for relieving symptoms and preventing recurrence. Probiotics can promote the balance of beneficial flora in the intestinal tract, and may help improve uric acid metabolism, reduce its generation in the body or increase its excretion rate. Intestinal flora imbalance is related to the pathogenesis of gout, and probiotics can maintain the balance of intestinal flora, reduce the growth of harmful bacteria, and indirectly affect uric acid metabolism, thereby possibly having a positive impact on the prevention and relief of gout. Therefore, using probiotics to control intestinal microbiota may be an effective treatment or prevention measure that can improve hyperuricemia and relieve gout and other problems. SUMMARY

[0004] The present application provides composite probiotics for relieving gouty arthritis and application thereof, and specifically provides composite probiotics for relieving gouty arthritis, probiotic agents for relieving gouty arthritis, and application thereof in the preparation of products for preventing, improving or treating gouty arthritis.

[0005] In a first aspect, the present application provides composite probiotics for relieving gouty arthritis, which are composed of Bifidobacterium longum BL21 strain with a preservation number of CGMCC No.10452 and Lactobacillus salivarius LS97 strain with a preservation number of CGMCC No.16922.

[0006] The Bifidobacterium longum BL21 strain is preserved in the China General Microbiological Culture Collection Center, with a preservation time of January 27, 2015, a preservation number of CGMCC No.10452, and an address of No.3, Beichen West Road, Chaoyang District, Beijing; and the Lactobacillus salivarius LS97 strain is preserved in the China General Microbiological Culture Collection Center, with a preservation time of December 10, 2018, a preservation number of CGMCC No.16922, and an address of No.3, Beichen West Road, Chaoyang District, Beijing.

[0007] The present application creatively develops a brand-new probiotic compound mode and a brand-new strategy for preventing and treating gout, i.e., the Bifidobacterium longum BL21 strain and the Lactobacillus salivarius LS97 strain are compounded and used in combination, and it is found that the two can cooperate with and promote each other, and have a synergistic effect on relieving gouty arthritis, which is specifically shown as follows: (1) the pain sensitivity response force can be reduced; (2) the number of white blood cells and the aggregation of neutrophils in the joint can be reduced; (3) the activity of myeloperoxidase in the joint surrounding tissue can be reduced; (4) the production of inflammatory factors CXCL1 and IL-1β can be reduced; and (5) the expression level of anti-inflammatory cytokine IL-10 in the joint tissue can be increased.

[0008] In the case of using the same amount of bacteria, compared with the intervention mode of a single BL21 strain or a single LS97 strain, the compound of the two strains significantly improves the effect on relieving and improving gouty arthritis. Therefore, the compound probiotic bacteria has a good prospect for use in preparing a product for preventing, relieving or treating gouty arthritis. Meanwhile, both of the two strains are probiotic bacteria, the product has high safety, and is not easy to produce resistance.

[0009] Preferably, the ratio of the viable bacterial count of the BL21 strain to the viable bacterial count of the LS97 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, 10:1, and other specific point values in the numerical range can be selected, which will not be described here.

[0010] In a second aspect, the present application provides a probiotic agent for relieving gouty arthritis, and the strain in the probiotic agent comprises the compound probiotic bacteria of the first aspect.

[0011] Preferably, in the probiotic agent, the total number of viable bacteria is not less than 1 x 10 9 CFU / mL or 1 x 10 9 CFU / g, such as 1 x 10 9 CFU / g (CFU / mL), 2 x 10 9 CFU / g (CFU / mL), 5 x 10 9 CFU / g (CFU / mL), 8 x 10 9 CFU / g (CFU / mL), 1 x 10 10 CFU / g (CFU / mL), 5 x 10 10 CFU / g (CFU / mL), 1 x 10 11 CFU / g (CFU / mL), 1 x 10 12 CFU / g (CFU / mL), 1 x 10 13 CFU / g (CFU / mL), etc., and other specific point values within the numerical range can be selected, which will not be repeated here.

[0012] Preferably, the dosage form of the probiotic agent includes a freeze-dried powder, a capsule, a tablet or a granule.

[0013] The dosage form of the probiotic agent involved in the present application is not limited, including the most commonly used freeze-dried powder, or further prepared capsule, tablet or granule.

[0014] Preferably, the dosage form of the probiotic agent is a freeze-dried powder, which is prepared by the following preparation method:

[0015] The BL21 strain and the LS97 strain are inoculated into the culture medium respectively to carry out activation and fermentation culture in turn to obtain fermentation liquor; the fermentation liquor is centrifuged respectively, mixed with a freeze-drying protective agent and then freeze-dried to obtain BL21 bacterial powder and LS97 bacterial powder; the BL21 bacterial powder and the LS97 bacterial powder are mixed according to the viable bacterial number ratio to obtain the probiotic agent for relieving gouty arthritis.

[0016] Preferably, the freeze-drying protective agent is selected from any one or a combination of at least two of skimmed milk, gelatin, dextrin, acacia, dextran, sodium alginate, polyvinylpyrrolidone, sucrose, lactose, trehalose, sorbitol or xylitol.

[0017] Further preferably, after mixing the BL21 bacterial powder and the LS97 bacterial powder according to the viable bacterial number ratio, a functional aid is further mixed;

[0018] The functional adjuvant is selected from any one or a combination of at least two of oligofructose, oligogalactose, oligoxylan, oligoisomaltose, soybean oligosaccharide, inulin, spirulina, arthrospira, grifola frondosa polysaccharide, stachyose, polydextrose, alpha-lactalbumin or lactoferrin.

[0019] In a third aspect, the application provides use of the composite probiotic bacteria of the first aspect or the probiotic agent of the second aspect in the preparation of a product for preventing, improving or treating gouty arthritis.

[0020] Preferably, the product further contains an excipient.

[0021] Preferably, the excipient is selected from any one or a combination of at least two of a filler, a binder, a wetting agent, a disintegrant, an emulsifier, a cosolvent, a solubilizer, an osmotic pressure regulator, a colorant, a pH regulator, an antioxidant, a bacteriostatic agent or a buffer.

[0022] Compared with the prior art, the application has the following beneficial effects:

[0023] The application creatively develops a brand-new probiotic bacteria compounding method and a brand-new strategy for preventing and treating gout, i.e., the Bifidobacterium longum BL21 strain and the Lactobacillus salivarius LS97 strain are compounded and used in combination, and it is found that the two can cooperate with and promote each other and have a synergistic effect in relieving gouty arthritis, which is specifically manifested in the following aspects: (1) the pain sensitivity response force can be reduced; (2) the number of leukocytes and the aggregation of neutrophils in the joint can be reduced; (3) the activity of myeloperoxidase in the tissue around the joint can be reduced; (4) the production of inflammatory factors CXCL1 and IL-1β can be reduced; and (5) the expression level of the anti-inflammatory cytokine IL-10 in the joint tissue can be increased. In the case of using the same amount of bacteria, compared with the intervention mode of a single BL21 strain or a single LS97 strain, the compounding of the two bacteria significantly improves the effect of relieving and improving gouty arthritis. BRIEF DESCRIPTION OF DRAWINGS

[0024] FIG. 1 is a result statistical graph of the pain sensitivity response force of mice in each group.

[0025] FIG. 2 is a result statistical graph of the leukocyte count in the knee joint cavity fluid of mice in each group.

[0026] FIG. 3 is a result statistical graph of the neutrophil count in the knee joint cavity fluid of mice in each group.

[0027] FIG. 4 is a result statistical graph of the myeloperoxidase (MPO) level in the knee joint tissue of mice in each group.

[0028] Figure 5 is a graph showing the results of the levels of chemokine ligand 1 (CXCL1) in the knee joint tissues of mice in each group.

[0029] Figure 6 is a graph showing the results of the levels of interleukin-1β (IL-1β) in the knee joint tissues of mice in each group.

[0030] Figure 7 is a graph showing the results of the levels of interleukin-10 (IL-10) in the knee joint tissues of mice in each group. DETAILED DESCRIPTION

[0031] The technical solutions of the present application are further illustrated below by means of specific embodiments. Those skilled in the art should understand that the embodiments are only used to help understand the present application and should not be regarded as specific limitations on the present application.

[0032] The taxonomic name of the BL21 strain involved in the following examples is Bifidobacterium longum, and the accession number is CGMCC No. 10452.

[0033] The taxonomic name of the LS97 strain involved in the following examples is Lactobacillus salivarius, and the accession number is CGMCC No. 16922.

[0034] The culture medium involved in the following examples is as follows:

[0035] MRS culture medium (g / L): 10 g / L proteose peptone, 10 g / L beef extract, 15 g / L glucose, 15 g / L lactose, 5 g / L yeast powder, 2 g / L diammonium hydrogen citrate, 2.6 g / L K2PO4·3H2O, 0.1 g / L MgSO4·7H2O, 0.05 g / L MnSO4, 1 mL / L polysorbate 80 (Tween 80), 0.5 g / L cysteine amino acid salt.

[0036] The bacterial suspension involved in the following examples is as follows: inoculate the desired strain in MRS liquid medium, activate at 37°C for 24 h, continuously activate for 2 times to obtain an activation liquid; inoculate the activation liquid in MRS liquid medium at an inoculation amount of 2% (v / v), cultivate at 37°C for 24 h to obtain a bacterial liquid; centrifuge the bacterial liquid at 6000 g for 10 min, resuspend the bacterial body with PBS to obtain the bacterial suspension.

[0037] The experimental result data is statistically analyzed using graphpad, and compared with the model group, *** represents p<0.001, ** represents p<0.01, * represents p<0.05, and NS. represents no significant difference.

[0038] EMBODIMENT

[0039] This example explores the ability of the composite probiotic to improve the symptoms of gouty arthritis in a mouse model:

[0040] (1) Test animals: Healthy female 8-week-old SPF C57BL / 6J mice (48, purchased from Shanghai Experimental Animal Center). These mice were raised in a controlled environment, with room temperature maintained at 22±2°C and humidity at 55%±5%, following a 12h light / dark cycle. They had free access to food and water. All experimental procedures involving mice were in accordance with the ethical guidelines for animal care and use as specified by the Shanghai Experimental Animal Care and Animal Experiment Center.

[0041] (2) Animal grouping: After 1 week of adaptive feeding as described above, 48 mice were randomly divided into 6 groups (8 in each group): control group (Ctl group), model group (Treat group), BL21 group (referred to as S1 group), LS97 group (referred to as S2 group), composite bacteria group 1 (BL21+LS97 group, live bacteria ratio 1:1, referred to as S3 group), composite bacteria group 2 (ATCC15707+LS97 group, live bacteria ratio 1:1, referred to as S4 group).

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

[0043] The mice were anesthetized, and the right hind ankle joint of the mouse was disinfected with iodophor. The model group and each probiotic intervention group were injected with 200μL of monosodium urate suspension (concentration of 0.5mg / mL) into the joint cavity, and the control group was injected with 200μL of PBS solution into the joint cavity. At the same time, 15h after modeling, the intervention was performed: the control group and the model group were routinely fed, and each probiotic intervention group was gavaged with each group of bacteria suspension (the amount was 1×10 10 CFU / each / day) while being routinely fed, for a total of 7 days of intervention.

[0044] (4) Index detection:

[0045] (4.1) After 7 days of probiotic intervention, an electronic pressure gauge was used to assess the nociception of the mice. The withdrawal threshold caused by flexion was used to infer the behavioral response related to pain, and pain response was induced by applying progressive pressure on the tail or hind limbs of the animal. Once the animal showed a clear pain response, the corresponding applied pressure was recorded. Mice with gout pain tolerated lower pressure. The results were expressed as the average measurement value in grams.

[0046] The results are shown in Figure 1. Compared with the control group, the model group mice showed increased reactivity to mechanical stimulation of the inflamed limb. After intervention in each probiotic group, compared with the model group, the mice treated with probiotics showed varying degrees of increased tolerance to high nociceptive response, and the S3 group was more significant, indicating that the probiotics involved in this application can alleviate the symptoms of gout in mice.

[0047] (4.2) Knee joint leukocyte and neutrophil counts:

[0048] After 7 days of probiotic intervention, all mice were sacrificed by cervical dislocation, the knee joint cavities were removed and washed twice with PBS containing 3% bovine serum albumin (5 μL) before the synovial fluid was subjected to cell counting. The number of leukocytes from the joint cavity was determined in a cell counting chamber by May-Grunwald staining. The neutrophil content was calculated based on a standard curve based on myeloperoxidase activity.

[0049] The results are shown in Figures 2 and 3. Compared with the control group, the number of leukocytes and neutrophils in the model group increased significantly, and the number of neutrophils was considered an important pathological marker of gout inflammation, indicating that the model mice had specific and obvious gout symptoms. After intervention by each probiotic group, the number of leukocytes and neutrophils decreased to varying degrees, and the change in the S3 group was more significant, indicating that the probiotics involved in the present application can alleviate the gout symptoms of mice.

[0050] (4.3) Cytokine and chemokine detection:

[0051] The knee joint tissue around the mouse joints was collected and homogenized in PBS containing antiprotease, the sample was homogenized with 0.05% Tween 20, and the levels of myeloperoxidase (MPO), chemokine ligand 1 (CXCL1), interleukin-1β (IL-1β) and interleukin-10 (IL-10) in the supernatant after centrifugation of the homogenate were measured by ELISA.

[0052] The results are shown in Figures 4-7, respectively. Compared with the control group, the levels of myeloperoxidase (MPO), chemokine ligand 1 (CXCL1) and interleukin-1β (IL-1β) in the model group mice increased significantly, the high expression of MPO, CXCL1 and IL-1β was related to the aggregation of neutrophils in the knee joint, and the level of interleukin-10 (IL-10) in the model group mice decreased. After intervention by each probiotic group, the levels of myeloperoxidase (MPO), chemokine ligand 1 (CXCL1) and interleukin-1β (IL-1β) decreased significantly, and the level of interleukin-10 (IL-10) increased significantly, the increase in IL-10 level is usually associated with spontaneous resolution of gout, and the change in the S3 group was more significant, indicating that the probiotics involved in the present application can alleviate the gout symptoms of mice.

[0053] The applicant declares that the technical solutions of the present application are illustrated by the above-mentioned embodiments, but the present application is not limited to the above-mentioned embodiments, that is, the present application does not mean that it must rely on the above-mentioned embodiments to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific modes, etc. fall within the protection scope and disclosure scope of the present application.

[0054] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details in the above-mentioned embodiments, and various simple modifications can be made to the technical solutions of the present application within the technical concept of the present application, and these simple modifications all belong to the protection scope of the present application.

[0055] In addition, it should be noted that each specific technical feature described in the above-mentioned specific embodiments can be combined in any appropriate manner without contradiction, and in order to avoid unnecessary repetition, the present application will not further describe various possible combination manners.

Claims

1. A probiotic compound for relieving gouty arthritis, which is composed of a Bifidobacterium longum BL21 strain with a preservation number of CGMCC No. 10452 and a Lactobacillus salivarius LS97 strain with a preservation number of CGMCC No. 16922.

2. The probiotic complex for alleviating gouty arthritis according to claim 1, wherein, The ratio of viable cell count of the BL21 strain to that of the LS97 strain is 1: 10-10:

1. 3.A probiotic agent for relieving gouty arthritis, wherein the strain comprises the probiotic compound of claim 1 or 2.

4. The probiotic agent for alleviating gouty arthritis according to claim 3, wherein, In the probiotic agent, the total number of viable bacteria is not less than 1 x 10 9 CFU / mL or 1 x 10 9 CFU / g.

5. The probiotic agent for alleviating gouty arthritis according to claim 3, wherein, The dosage form of the probiotic agent comprises a lyophilized powder, a capsule, a tablet or a granule.

6. The probiotic agent for alleviating gouty arthritis according to claim 5, wherein The dosage form of the probiotic agent is a lyophilized powder, which is prepared by the following method: The BL21 strain and the LS97 strain are inoculated into culture media respectively and activated and fermented in turn to obtain fermentation broth; the fermentation broth is centrifuged and mixed with a lyophilization protective agent to be lyophilized to obtain BL21 bacterial powder and LS97 bacterial powder; the BL21 bacterial powder and the LS97 bacterial powder are mixed according to the ratio of viable cell count to obtain the probiotic agent for relieving gouty arthritis.

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

8. The probiotic agent for alleviating gouty arthritis according to claim 6, wherein The BL21 bacterial powder and the LS97 bacterial powder are mixed according to the ratio of viable cell count and then mixed with a functional aid; The functional aid is selected from any one or a combination of at least two of fructooligosaccharide, galactooligosaccharide, xylooligosaccharide, isomaltooligosaccharide, soybean oligosaccharide, inulin, spirulina, arthrospira, grifola frondosa polysaccharide, stachyose, polydextrose, α-lactalbumin or lactoferrin. 9.Use of the probiotic compound of claim 1 or 2 or the probiotic agent of any one of claims 3-8 in the preparation of a product for preventing, improving or treating gouty arthritis.

10. Use according to claim 9, wherein, The product further contains an auxiliary material.

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