Probiotic composition for preventing and treating intestinal inflammation and use thereof

Abstract

Provided are a probiotic composition for preventing and treating intestinal inflammation and a use thereof. The probiotic composition for preventing and treating intestinal inflammation comprises a Lactobacillus acidophilus LA85 strain and a Bifidobacterium longum subsp. BL36 strain. Provided are a new probiotic formulation method and a new strategy for preventing and treating intestinal inflammation. It has been found that the LA85 strain and the BL36 strain can cooperate with each other, promote each other, have a synergistic effect in preventing and treating intestinal inflammation, can reduce the concentrations of the pro-inflammatory cytokines TNF-α, IL-6, and IL-1β in the serum of mice, and increase the concentration of the anti-inflammatory cytokine IL-10; can increase the concentration of urolithin A in the serum of mice and significantly reduce the concentration of reactive oxygen species in the serum; and can significantly increase the levels of the intestinal barrier indicators tight junction protein-1, tight junction protein, and Claudin-1 in the serum of mice.

Description

Probiotic composition for preventing and treating intestinal inflammation and application thereof TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and relates to a probiotic composition for preventing and treating intestinal inflammation and application thereof. BACKGROUND

[0002] The intestinal tract is the first line of defense of the body, not only responsible for the selective absorption of nutrients, but also responsible for resisting the invasion of inflammatory factors and harmful microorganisms. When the body is in an unhealthy state or suddenly contacts a large number of pathogenic bacteria and virulence factors, intestinal inflammation and intestinal mucosal damage will be caused, and then systemic inflammatory response syndrome and multiple organ failure will be caused. Lipopolysaccharide (LPS) is one of the stressors that induce intestinal mucosal damage, which can not only damage the intestinal tract, but also cause bacterial translocation. The damaged gastrointestinal barrier will cause abnormal expression of intestinal tight junction proteins, so that endotoxins, pathogens and the like can pass through the intestinal wall, induce activation of Toll-like receptor 4 (TLR4) on the surface of cells, excite the downstream nuclear factor-kappa B (NF-kappa B) signaling pathway, and synthesize and release cell inflammatory factors, and then aggravate the damage to the intestinal tract.

[0003] Probiotics, especially Bifidobacterium and Lactobacillus, are 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 the damaged intestinal barrier and enhancing the immune system, and on the other hand, can increase the level of tight junction (TJ) proteins between intestinal epithelial cells, and also can inhibit the nuclear factor-kappa B (NF-kappa B) signaling pathway, down-regulate pro-inflammatory cytokines and up-regulate anti-inflammatory cytokines, so as to maintain immune balance and protect the immune barrier.

[0004] It can be seen that probiotics have the effect of regulating intestinal health. Probiotics can alleviate intestinal inflammation by promoting intestinal barrier function, regulating inflammatory markers and regulating immune cell balance. In addition, probiotics will not promote the spread of antibiotic resistance, nor will they carry transferable antibiotic resistance. Therefore, probiotics are safe, green and pollution-free alternatives to antibiotics, and it is particularly meaningful to find and develop more probiotic intervention strategies for preventing and treating intestinal inflammation, especially probiotic combination strategies. SUMMARY

[0005] The present application provides a probiotic composition for preventing and treating intestinal inflammation and application thereof.

[0006] In a first aspect, the present application provides a probiotic composition for preventing and treating intestinal inflammation, which comprises a Lactobacillus acidophilus LA85 strain with a preservation number of CGMCC No. 1.12735 and a Bifidobacterium longum subsp. longum BL36 strain with a preservation number of CGMCC No. 24413.

[0007] The preservation unit of the Lactobacillus acidophilus LA85 strain is the General Microbiological Center of China Microorganism Culture Collection Committee, the preservation time is July 20, 2020, the preservation number is CGMCC No. 1.12735, and the address is No. 3, Beichen West Road, Chaoyang District, Beijing;

[0008] The preservation unit of the Bifidobacterium longum subsp. longum BL36 strain is the General Microbiological Center of China Microorganism Culture Collection Committee, the preservation time is February 21, 2022, the preservation number is CGMCC No. 24413, and the address is No. 3, Beichen West Road, Chaoyang District, Beijing.

[0009] The present application creatively develops a brand-new probiotic compound mode and a brand-new strategy for preventing and treating intestinal inflammation, i.e. the Lactobacillus acidophilus LA85 strain and the Bifidobacterium longum subsp. longum BL36 strain are compounded and used together, and it is found that the two can cooperate with each other, promote each other, and synergistically enhance the effect in preventing and treating intestinal inflammation, which is specifically embodied in: (1) the concentration of pro-inflammatory cytokines TNF-α, IL-6 and IL-1β in the serum of mice can be reduced, and the concentration of anti-inflammatory cytokine IL-10 can be increased; (2) the concentration of urolithin A in the serum of mice can be increased, and the concentration of serum reactive oxygen species can be significantly reduced; (3) the levels of intestinal barrier indicators, tight junction protein-1, tight junction protein and occludin 1 in the serum of mice can be significantly increased.

[0010] Under the condition of using the same amount of bacteria, compared with the intervention mode of single LA85 strain or single BL36 strain, the compounding of the two strains significantly improves the above effects. Therefore, the probiotic composition has a good prospect for use in preparing products for preventing, improving or treating intestinal inflammation. At the same time, both of the two strains are probiotics, the product has high safety, and is not easy to produce resistance.

[0011] Preferably, the ratio of viable cell number of the LA85 strain to 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, and other specific point values within the numerical range can be selected, which will not be repeated here.

[0012] In a second aspect, the present application provides a probiotic agent for preventing and treating intestinal inflammation, wherein the strains in the probiotic agent comprise the probiotic composition of the first aspect.

[0013] Preferably, in the probiotic agent, the total number of viable cells 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 10 CFU / 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), and other specific point values within the numerical range can be selected, which will not be repeated here.

[0014] Preferably, the dosage form of the probiotic agent comprises a solution, a lyophilized powder, a capsule, a tablet, or a granule.

[0015] Preferably, the dosage form of the probiotic agent is a solution, which is prepared by the following method:

[0016] The LA85 strain and the BL36 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 LA85 bacterial suspension and BL36 bacterial suspension; the LA85 bacterial suspension and the BL36 bacterial suspension are mixed according to the ratio of viable cell number to obtain the probiotic agent.

[0017] Preferably, the dosage form of the probiotic agent is a lyophilized powder, which is prepared by the following method:

[0018] The LA85 strain and the BL36 strain are inoculated into culture media to perform activation and fermentation culture in sequence to obtain fermentation liquor; the fermentation liquor is centrifuged and mixed with a protective agent to perform freeze-drying to obtain LA85 bacterial powder and BL36 bacterial powder; the LA85 bacterial powder and the BL36 bacterial powder are mixed according to the number of viable bacteria to obtain the probiotic agent.

[0019] In a third aspect, the application provides use of the probiotic composition of the first aspect or the probiotic agent of the second aspect in the preparation of a drug for preventing, improving or treating intestinal inflammation.

[0020] Preferably, the drug further comprises 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 compound mode and a brand-new strategy for preventing and treating intestinal inflammation, i.e., the Lactobacillus acidophilus LA85 strain and the Bifidobacterium longum subsp.longum BL36 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 preventing and treating intestinal inflammation, which is embodied in the following aspects: (1) the concentration of pro-inflammatory cytokines TNF-α, IL-6 and IL-1β in the serum of mice can be reduced, and the concentration of anti-inflammatory cytokine IL-10 can be increased; (2) the concentration of urolithin A in the serum of mice can be increased, and the concentration of serum reactive oxygen species can be significantly reduced; (3) the levels of intestinal barrier indicators, i.e., tight junction protein-1, tight junction protein and occludin 1, in the serum of mice can be significantly increased.

[0024] In the case of using the same amount of bacteria, compared with the intervention mode of a single LA85 strain or a single BL36 strain, the compounding of the two bacteria significantly improves the above effects. Therefore, the probiotic composition has a good prospect when used in the preparation of a product for preventing, improving or treating intestinal inflammation. Meanwhile, both of the bacteria are probiotics, the product has high safety, and is not prone to resistance. BRIEF DESCRIPTION OF DRAWINGS

[0025] FIG. 1 is a statistical result graph of the interleukin 1β (IL-1β) level in the serum of mice in each group.

[0026] Figure 2 is a statistical result graph of interleukin 6 (IL-6) levels in the serum of mice in each group.

[0027] Figure 3 is a statistical result graph of interleukin 10 (IL-10) levels in the serum of mice in each group.

[0028] Figure 4 is a statistical result graph of tumor necrosis factor alpha (TNF-a) levels in the serum of mice in each group.

[0029] Figure 5 is a statistical result graph of reactive oxygen species (ROS) levels in the serum of mice in each group.

[0030] Figure 6 is a statistical result graph of urolithin A (Urolithin A) levels in the serum of mice in each group.

[0031] Figure 7 is a statistical result graph of tight junction protein-1 (ZO-1) levels in the serum of mice in each group.

[0032] Figure 8 is a statistical result graph of tight junction protein (Occludin) levels in the serum of mice in each group.

[0033] Figure 9 is a statistical result graph of claudin-1 (Claudin-1) levels in the serum of mice in each group. DETAILED DESCRIPTION

[0034] In order to further illustrate the technical means adopted by the present application and its effects, the technical solutions of the present application will be further described below in combination with the preferred embodiments of the present application, but the present application is not limited in the scope of the embodiments.

[0035] The culture medium involved in the following examples is as follows:

[0036] 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 Tween 80, 0.5 g / L cysteine amino acid salt.

[0037] The LA85 strain involved in the following examples is named Lactobacillus acidophilus, and the preservation time is July 20, 2020, and the preservation number is CGMCC No. 1.12735.

[0038] The taxonomic designation of the BL36 strain involved in the present application is Bifidobacterium longum subsp. longum, which was deposited on February 21, 2022, and has the accession number CGMCC No. 24413.

[0039] The bacterial suspension involved in the following examples: inoculate the desired strain into MRS liquid medium, cultivate at 37°C for 24h for activation, and obtain an activation liquid; inoculate the activation liquid into MRS liquid medium at an inoculation amount of 3% (v / v), cultivate at 37°C for 24h, and obtain a bacterial liquid; centrifuge the bacterial liquid at 6000g for 6min, resuspend the bacterial body with PBS, and obtain the bacterial suspension.

[0040] Example

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

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

[0043] (2) Animal grouping: after 1 week of adaptive feeding of the mice as described above, 70 mice were randomly divided into 7 groups (10 in each group): blank control group (CON), model group (LPS), LA85 group (LA85), BL36 group (BL36), complex bacteria group 1 (LA85+BL36, live bacteria ratio 1:1), complex bacteria group 2 (LA85+ATCC15707 group, live bacteria ratio 1:1), and complex bacteria group 3 (ATCC4356+BL36 group, live bacteria ratio 1:1).

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

[0045] During the first 21 days of the formal test, the blank control group and the model group were normally fed + normal purified water, and each probiotic intervention group was normally fed + probiotic water (the amount of probiotic bacteria was 10 9 CFU / each / day); on the 22nd day of the formal test, the model group and each probiotic intervention group of mice were injected intraperitoneally with 1.5mg / kg LPS, and sampled 24h after injection.

[0046] (4) Sampling:

[0047] After the intervention, the mice were sacrificed, the eyeballs were bled, centrifuged at 1200xg for 10 min, and the serum was extracted and stored at -80°C.

[0048] (5) Index analysis:

[0049] (5.1) Inflammatory factor index detection:

[0050] 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 mice in each group were detected using a mouse inflammatory factor ELISA research kit.

[0051] The results are shown in Figures 1-4, respectively. 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 decreased. After probiotic intervention, the probiotic intervention group could reverse the effect to varying degrees, and the reversal effect of the compound 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, so that the serum inflammatory factor level of mice was closer to the normal level. It shows that the probiotic composition involved in the present application has a good effect on preventing and treating intestinal inflammation.

[0052] (5.2) Antioxidant factor detection:

[0053] The levels of reactive oxygen species (ROS) and urolithin A (Urolithin A) in the serum of mice were detected using a mouse antioxidant factor ELISA research kit.

[0054] The results are shown in Figures 5-6, respectively. Compared with the blank control group, the concentration of ROS in the serum of mice in the model group was increased, and the concentration of urolithin A was significantly decreased. After probiotic intervention, the concentration of ROS was significantly decreased, and the concentration of urolithin A was significantly increased, among which the effect of the compound bacteria group 1 was the most significant. It shows that the probiotic composition involved in the present application has a good effect on preventing and treating intestinal inflammation.

[0055] (5.3) Intestinal barrier index detection:

[0056] The levels of tight junction protein-1 (ZO-1), tight junction protein (Occludin) and occluding protein 1 (Claudin-1) in the serum of mice were detected using a mouse intestinal barrier index ELISA research kit.

[0057] The results are shown in Figures 7-9. Compared with the blank control group, the serum levels of tight junction protein-1 (ZO-1), tight junction protein (Occludin), and blocking protein 1 (Claudin-1) in the model group mice were significantly reduced, indicating impaired epithelial barrier integrity and increased intestinal permeability. Probiotic intervention resulted in varying degrees of reversal effects, with the compound probiotic group 1 showing a better reversal effect. It significantly increased the levels of the intestinal barrier markers tight junction protein-1 (ZO-1), tight junction protein (Occludin), and blocking protein 1 (Claudin-1) in mice, demonstrating that the probiotic composition involved in this application has a good effect in preventing and treating intestinal inflammation.

[0058] 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.

[0059] 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.

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

Claims

1. A probiotic composition for preventing and treating intestinal inflammation, comprising a Lactobacillus acidophilus LA85 strain with a preservation number of CGMCC No. 1.12735 and a Bifidobacterium longum subsp. longum BL36 strain with a preservation number of CGMCC No. 24413.

2. The probiotic composition for the prevention and treatment of intestinal inflammation according to claim 1, wherein, The ratio of viable cell number of the LA85 strain to the BL36 strain is 1:10-10:

1. 3.A probiotic agent for preventing and treating intestinal inflammation, wherein the strain comprises the probiotic composition of claim 1 or 2.

4. The probiotic agent of 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 of claim 3, wherein, The dosage form of the probiotic agent comprises a solution, a lyophilized powder, a capsule, a tablet or a granule.

6. The probiotic agent of claim 5, wherein, The dosage form of the probiotic agent is a solution, which is prepared by the following method: The LA85 strain and the BL36 strain are inoculated into culture media respectively and activated and fermented in turn to obtain fermentation broth; the fermentation broth is centrifuged respectively, resuspended with a solvent to obtain LA85 bacterial suspension and BL36 bacterial suspension; the LA85 bacterial suspension and the BL36 bacterial suspension are mixed according to the ratio of viable cell number to obtain the probiotic agent.

7. The probiotic agent of claim 5, wherein, The dosage form of the probiotic agent is a lyophilized powder, which is prepared by the following method: The LA85 strain and the BL36 strain are inoculated into culture media respectively and activated and fermented in turn to obtain fermentation broth; the fermentation broth is centrifuged respectively, mixed with a protective agent and then 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 viable cell number to obtain the probiotic agent. 8.Use of the probiotic composition of claim 1 or 2 or the probiotic agent of any one of claims 3-7 in the preparation of a medicament for preventing, improving or treating intestinal inflammation.

9. Use according to claim 8, wherein, The medicament further comprises an excipient.

10. Use according to claim 9, wherein, 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.

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