A probiotic composition for preventing and treating intestinal inflammation and its application

Abstract

The present invention relates to a probiotic composition for preventing and treating intestinal inflammation and its application, wherein the probiotic composition for preventing and treating intestinal inflammation comprises Lactobacillus acidophilus LA85 strain and Bifidobacterium longum subspecies BL36 strain. The present invention creatively develops a new probiotic compounding method and a new strategy for preventing and treating intestinal inflammation, and finds that the LA85 strain and the BL36 strain can cooperate with each other, promote each other, and synergize in the effect of preventing and treating intestinal inflammation, which is specifically embodied in: the concentrations of proinflammatory cytokines TNF-α, IL-6 and IL-1β in mouse serum can be reduced, and the concentration of anti-inflammatory cytokine IL-10 can be increased; the concentration of urolithin A in mouse serum can be increased, and the concentration of serum reactive oxygen can be significantly reduced; the levels of intestinal barrier indicators tight junction protein-1, tight junction protein and occludin 1 in mouse serum can be significantly increased.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to a probiotic composition for preventing and treating intestinal inflammation and application thereof. Background Art

[0002] The intestine is the body's first line of defense. It is not only responsible for the selective absorption of nutrients, but also for resisting the invasion of inflammatory factors and harmful microorganisms. When the body is in a bad state or suddenly exposed to a large number of pathogens and virulence factors, it will cause intestinal inflammation and intestinal mucosal damage, leading to systemic inflammatory response syndrome and multiple organ failure. Lipopolysaccharide (LPS) is one of the stressors that induce intestinal mucosal damage. It can not only damage the intestine, but also cause bacterial translocation. The damaged gastrointestinal barrier will cause abnormal expression of intestinal tight junction proteins, allowing endotoxins, pathogens, etc. to pass through the intestinal wall, inducing activation of cell surface Toll-like receptor 4 (TLR4) to stimulate the downstream nuclear factor-κB (NF-κB) signaling pathway, and synthesizing and releasing cellular inflammatory factors, thereby aggravating intestinal damage.

[0003] Probiotics, especially Bifidobacterium and Lactobacillus, are well known for their ability to improve intestinal health and regulate immunity. On the one hand, probiotics can improve intestinal inflammation by alleviating existing intestinal damage, restoring damaged intestinal barriers and enhancing the immune system. On the other hand, they can increase the level of tight junction (TJ) proteins between intestinal epithelial cells, inhibit the nuclear factor-κB (NF-κB) signaling pathway, downregulate pro-inflammatory cytokines, and upregulate anti-inflammatory cytokines, thereby maintaining immune balance and protecting the immune barrier.

[0004] It can be seen that probiotics have the function of regulating intestinal health. Probiotics can relieve intestinal inflammation by promoting intestinal barrier function, regulating inflammatory markers, and regulating the balance of immune cells. In addition, probiotics do not promote the spread of antibiotic resistance, nor do they carry transferable antibiotic resistance. Therefore, probiotics are safe, green, and harmless antibiotic alternatives. It is very meaningful to find and develop more probiotic intervention strategies for the prevention and treatment of intestinal inflammation, especially probiotic combination strategies. Summary of the invention

[0005] In view of the deficiencies of the prior art, the object of the present invention is to provide a probiotic composition for preventing and treating intestinal inflammation and its application.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a probiotic composition for preventing and treating intestinal inflammation, wherein the probiotic composition for preventing and treating intestinal inflammation comprises Lactobacillus acidophilus with a preservation number of CGMCC No. 1.12735. Lactobacillus acidophilusLA85 strain and Bifidobacterium longum subsp. longum with a deposit number of CGMCC No. 24413 Bifidobacterium longum subsp. longum BL36 strain.

[0008] The present invention creatively develops a new probiotic compounding method and a new strategy for preventing and treating intestinal inflammation, namely, Lactobacillus acidophilus Lactobacillus acidophilus Bifidobacterium longum subsp. longum Bifidobacterium longum subsp. longum The BL36 strain was used in combination, and it was found that the two strains can cooperate with each other, promote each other, and synergize in the prevention and treatment of intestinal inflammation. Specifically, they can: (1) reduce the concentrations of proinflammatory cytokines TNF-α, IL-6, and IL-1β in mouse serum, and increase the concentration of anti-inflammatory cytokine IL-10; (2) increase the concentration of urolithin A in mouse serum and significantly reduce the concentration of serum reactive oxygen species; (3) significantly increase the levels of intestinal barrier indicators such as tight junction protein-1, tight junction protein, and occludin 1 in mouse serum.

[0009] When the amount of bacteria used is the same, the combination of the two bacteria significantly improves the above effects compared with the intervention of a single LA85 strain or a single BL36 strain. Therefore, the probiotic composition has a good prospect for preparing products for preventing, improving or treating intestinal inflammation. At the same time, both bacteria are probiotics, the product is highly safe, and it is not easy to develop resistance.

[0010] Preferably, the ratio of the viable count of the LA85 strain to that of the BL36 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, etc. Other specific point values ​​within this numerical range can be selected, and they will not be described one by one here.

[0011] In a second aspect, the present invention provides a probiotic for preventing and treating intestinal inflammation, wherein the strains in the probiotic include the probiotic composition described in the first aspect.

[0012] Preferably, in the probiotic agent, the total number of viable bacteria is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g, for example 1×10 9 CFU / g (CFU / mL), 2×10 9 CFU / g (CFU / mL), 5×10 9 CFU / g (CFU / mL), 8×10 9 CFU / g (CFU / mL), 1×10 10CFU / g (CFU / mL), 5×10 10 CFU / g (CFU / mL), 1×10 11 CFU / g (CFU / mL), 1×10 12 CFU / g (CFU / mL), 1×10 13 CFU / g (CFU / mL), etc. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0013] Preferably, the dosage form of the probiotic includes solution, lyophilized powder, capsule, tablet or granule.

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

[0015] The LA85 strain and the BL36 strain are inoculated into a culture medium respectively for activation and fermentation culture in sequence to obtain a fermentation broth; the fermentation broth is centrifuged respectively and resuspended with a solvent to obtain a LA85 bacterial suspension and a BL36 bacterial suspension; the LA85 bacterial suspension and the BL36 bacterial suspension are mixed according to the ratio of the number of live bacteria to obtain the probiotic agent.

[0016] Preferably, the probiotic is in the form of a freeze-dried powder, which is prepared by the following method:

[0017] The LA85 strain and the BL36 strain are inoculated into a culture medium respectively for activation and fermentation culture in sequence to obtain a fermentation broth; the fermentation broth is centrifuged respectively, mixed with a protective agent and freeze-dried to obtain LA85 bacterial powder and BL36 bacterial powder; the LA85 bacterial powder and the BL36 bacterial powder are mixed according to the ratio of the number of live bacteria to obtain the probiotic agent.

[0018] In a third aspect, the present invention provides use of the probiotic composition described in the first aspect or the probiotic agent described in the second aspect in the preparation of a medicament for preventing, improving or treating intestinal inflammation.

[0019] Preferably, the medicine further comprises excipients.

[0020] Preferably, the auxiliary material is selected from any one or a combination of at least two of fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, colorants, pH regulators, antioxidants, antibacterial agents or buffers.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention creatively develops a new probiotic compounding method and a new strategy for preventing and treating intestinal inflammation, namely, Lactobacillus acidophilus Lactobacillus acidophilus Bifidobacterium longum subsp. longum Bifidobacterium longum subsp. longum The BL36 strain was used in combination, and it was found that the two strains can cooperate with each other, promote each other, and synergize in the prevention and treatment of intestinal inflammation. Specifically, they can: (1) reduce the concentrations of proinflammatory cytokines TNF-α, IL-6, and IL-1β in mouse serum, and increase the concentration of anti-inflammatory cytokine IL-10; (2) increase the concentration of urolithin A in mouse serum and significantly reduce the concentration of serum reactive oxygen species; (3) significantly increase the levels of intestinal barrier indicators such as tight junction protein-1, tight junction protein, and occludin 1 in mouse serum.

[0023] When the amount of bacteria used is the same, the combination of the two bacteria significantly improves the above effects compared with the intervention of a single LA85 strain or a single BL36 strain. Therefore, the probiotic composition has a good prospect for preparing products for preventing, improving or treating intestinal inflammation. At the same time, both bacteria are probiotics, the product is highly safe, and it is not easy to develop resistance.

[0024] The LA85 strain involved in the present invention is classified and named as Lactobacillus acidophilus Lactobacillus acidophilus The depository is the General Microbiology Center of China Microbiological Culture Collection Administration. The deposit date is July 20, 2020. The deposit number is CGMCC No. 1.12735. The address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0025] The BL36 strain involved in the present invention is classified and named as Bifidobacterium longum subsp. longum, and the depository unit is the General Microbiology Center of China Microorganism Culture Collection Administration, the deposit time is February 21, 2022, the deposit number is CGMCC No. 24413, and the address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a statistical result diagram of interleukin 1β (IL-1β) level in the serum of each group of mice;

[0027] Figure 2 It is a statistical result diagram of interleukin 6 (IL-6) level in the serum of each group of mice;

[0028] Figure 3 It is a statistical result diagram of interleukin 10 (IL-10) level in the serum of each group of mice;

[0029] Figure 4 is a statistical result graph of the level of tumor necrosis factor α (TNF-α) in the serum of each group of mice;

[0030] Figure 5 It is a statistical result diagram of the level of reactive oxygen species (ROS) in the serum of each group of mice;

[0031] Figure 6 This is a statistical result chart of the level of Urolithin A in the serum of each group of mice;

[0032] Figure 7 It is a statistical result diagram of the level of tight junction protein-1 (ZO-1) in the serum of each group of mice;

[0033] Figure 8 This is the statistical result of the tight junction protein (Occludin) level in the serum of each group of mice;

[0034] Fig. 9 This is a statistical graph of the Claudin-1 levels in the serum of each group of mice. DETAILED DESCRIPTION

[0035] In order to further explain the technical means and effects adopted by the present invention, the technical solution of the present invention is further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0036] The culture medium involved in the following examples is as follows:

[0037] MRS medium (g / L): peptone 10g / L, beef extract 10g / L, glucose 15g / L, lactose 15g / L, yeast powder 5g / L, diammonium hydrogen citrate 2g / L, K2PO4·3H2O 2.6g / L, MgSO4·7H2O 0.1g / L, MnSO4 0.05g / L, Tween 80 1mL / L, cysteine ​​salt 0.5g / L.

[0038] The LA85 strain involved in the following examples is classified and named as Lactobacillus acidophilus Lactobacillus acidophilus The deposit date is July 20, 2020, and the deposit number is CGMCC No. 1.12735.

[0039] The BL36 strain involved in the present invention is classified as Bifidobacterium longum subsp. Bifidobacterium longum subsp. longum The deposit date is February 21, 2022, and the deposit number is CGMCC No. 24413.

[0040] The bacterial suspension involved in the following embodiments is as follows: the desired strain is inoculated into MRS liquid culture medium, and cultured at 37°C for 24 hours for activation to obtain an activation solution; the activation solution is inoculated into MRS liquid culture medium at an inoculum amount of 3% (v / v), and cultured at 37°C for 24 hours to obtain a bacterial solution; the bacterial solution is centrifuged at 6000g for 6 minutes, and the bacteria are resuspended in PBS to obtain. Example

[0041] This example explores the ability of the probiotic composition to improve the symptoms of intestinal inflammation model mice:

[0042] (1) Experimental animals: Male C57BL / 6 mice (6 weeks old) (70 mice, purchased from Shanghai Laboratory Animal Center) were housed in a controlled environment with room temperature maintained at 22±2°C and humidity at 55%±5%, following a 12-h light / dark cycle, and free access to food and water. All experimental procedures involving mice were in accordance with the ethical guidelines for the care and use of animals stipulated by the Shanghai Laboratory Animal Care and Animal Experimentation Center.

[0043] (2) Animal grouping: After one week of adaptive feeding, 70 mice were randomly divided into seven groups (10 mice in each group): blank control group (CON), model group (LPS), LA85 group (LA85), BL36 group (BL36), composite bacteria group 1 (LA85+BL36, the ratio of live bacteria was 1:1), composite bacteria group 2 (LA85+ATCC15707 group, the ratio of live bacteria was 1:1), and composite bacteria group 3 (ATCC4356+BL36 group, the ratio of live bacteria was 1:1).

[0044] (3) Animal modeling and intervention methods:

[0045] On days 1 to 21 of the formal experiment, the blank control group and the model group were fed normally + normal purified water, and the probiotic intervention groups were fed normally + probiotic drinking water (the amount of probiotics was 10 9 CFU / mouse / day); On the 22nd day of the formal experiment, mice in the model group and each probiotic intervention group were intraperitoneally injected with 1.5 mg / kg LPS, and samples were taken 24 hours after the injection.

[0046] (4) Sampling:

[0047] After the intervention, the mice were killed, blood was collected from the eyeballs, and serum was extracted after centrifugation at 1200×g for 10 min and stored at −80°C.

[0048] (5) Indicator analysis:

[0049] (5.1) Detection of inflammatory factor indicators:

[0050] Mouse inflammatory factor ELISA research kit was used to detect the levels of interleukin 1β (IL-1β), interleukin 6 (IL-6), interleukin 10 (IL-10) and tumor necrosis factor α (TNF-α) in the serum of each group of mice.

[0051] The results are as follows Figure 1-Figure 4 As shown, compared with the blank control group, the levels of TNF-α, IL-1β and IL-6 in the model group mice were significantly increased, and the level of IL-10 was significantly reduced. After probiotic intervention, the probiotic intervention group was able to have different degrees of reversal effects, and the reversal effect of the composite bacteria group 1 was better, which could significantly reduce the levels of pro-inflammatory factors TNF-α, IL-1β and IL-6 in mice, and significantly increase the level of anti-inflammatory factor IL-10, making the level of serum inflammatory factors in mice closer to normal levels. It shows that the probiotic composition involved in the present invention has a good effect in preventing and treating intestinal inflammation.

[0052] (5.2) Antioxidant factor detection:

[0053] Mouse antioxidant factor ELISA research kit was used to detect the levels of reactive oxygen species (ROS) and urolithin A in mouse serum.

[0054] The results are as follows Figure 5-Figure 6 As shown in the figure, compared with the blank control group, the ROS concentration in the serum of the model group mice increased while the urolithin A concentration decreased significantly. After probiotic intervention, the ROS concentration decreased significantly and the urolithin A concentration increased significantly, among which the composite bacteria group 1 had the most significant effect. This shows that the probiotic composition involved in the present invention has a good effect in preventing and treating intestinal inflammation.

[0055] (5.3) Intestinal barrier index detection:

[0056] The mouse intestinal barrier index ELISA research kit was used to detect the levels of tight junction protein-1 (ZO-1), tight junction protein (Occludin) and closed protein 1 (Claudin-1) in mouse serum.

[0057] The results are as follows Figure 7-Figure 9As shown, compared with the blank control group, the levels of tight junction protein-1 (ZO-1), tight junction protein (Occludin) and closed protein 1 (Claudin-1) in the serum of the model group mice were significantly reduced, indicating that the integrity of the epithelial barrier of the mice was damaged and the intestinal permeability was increased. After the intervention of probiotics, different degrees of reversal effects can occur, and the reversal effect of the composite bacteria group 1 is better, which can significantly improve the levels of tight junction protein-1 (ZO-1), tight junction protein (Occludin) and closed protein 1 (Claudin-1) in the intestinal barrier indicators of mice, indicating that the probiotic composition involved in the present invention has a good effect in preventing and treating intestinal inflammation.

[0058] The applicant declares that the present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0059] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0060] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A probiotic composition for preventing intestinal inflammation, characterized in that: The probiotic composition for preventing intestinal inflammation is composed of Lactobacillus acidophilus with a preservation number of CGMCC No. 1.12735. Lactobacillus acidophilus LA85 strain and Bifidobacterium longum subsp. longum with a deposit number of CGMCC No. 24413 Bifidobacterium longum subsp. longum BL36 strain composition.

2. The probiotic composition for preventing intestinal inflammation according to claim 1, characterized in that: The ratio of the viable count of the LA85 strain to that of the BL36 strain is 1:10-10:

1.

3. A probiotic for preventing intestinal inflammation, characterized in that: The strain in the probiotic comprises the probiotic composition according to claim 1 or 2.

4. The probiotic according to claim 3, characterized in that In the probiotics, the total number of viable bacteria is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g.

5. The probiotic according to claim 3, characterized in that The dosage form of the probiotics includes solution, lyophilized powder, capsule, tablet or granule.

6. The probiotic according to claim 5, characterized in that The dosage form of the probiotic is a solution, which is prepared by the following method: The LA85 strain and the BL36 strain are inoculated into a culture medium respectively for activation and fermentation culture in sequence to obtain a fermentation broth; the fermentation broth is centrifuged respectively and resuspended with a solvent to obtain a LA85 bacterial suspension and a BL36 bacterial suspension; the LA85 bacterial suspension and the BL36 bacterial suspension are mixed according to the ratio of the number of live bacteria to obtain the probiotic agent.

7. The probiotic according to claim 5, characterized in that The dosage form of the probiotic is a freeze-dried powder, which is prepared by the following method: The LA85 strain and the BL36 strain are inoculated into a culture medium respectively for activation and fermentation culture in sequence to obtain a fermentation broth; the fermentation broth is centrifuged respectively, mixed with a protective agent and freeze-dried to obtain LA85 bacterial powder and BL36 bacterial powder; the LA85 bacterial powder and the BL36 bacterial powder are mixed according to the ratio of the number of live bacteria to obtain the probiotic agent.

8. Use of the probiotic composition according to claim 1 or 2 or the probiotic agent according to any one of claims 3 to 7 in the preparation of a medicament for preventing or improving intestinal inflammation.

9. The use according to claim 8, characterized in that: The medicine also includes auxiliary materials.

10. The use according to claim 9, characterized in that: The auxiliary material is selected from any one or a combination of at least two of fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, colorants, pH regulators, antioxidants, antibacterial agents or buffers.

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