Lactobacillus rhamnosus MBP01 for preventing and treating irritable bowel syndrome and composition thereof

Abstract

Provided are Lactobacillus rhamnosus MBP01 for preventing and treating irritable bowel syndrome and a composition thereof. Lactobacillus rhamnosus MBP01 is deposited under the accession number GDMCC No: 64725. In IBS model mice, the strain can ameliorate the weight loss trend, increase food intake and the water content of fecal matter, alleviate damage to colon tissue, down-regulate the levels of IL-1β, TNF-α, PAR-2, and serum corticosterone, and increase the level of IL-10. Moreover, a high concentration of MBP01 can increase the expression of tight junction proteins. MBP01 exhibits a positive regulatory effect on the intestinal function of IBS model mice and can be used for preventing and treating irritable bowel syndrome.

Description

Lactobacillus rhamnosus MBP01 and its composition for the prevention and treatment of irritable bowel syndrome Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to Lactobacillus rhamnosus MBP01 and its composition for the prevention and treatment of irritable bowel syndrome. Background Technology

[0002] Irritable bowel syndrome (IBS) is a functional bowel disorder characterized by chronic or recurrent abdominal pain accompanied by changes in bowel habits. It lacks morphological and biochemical abnormalities and primarily manifests as relief or reduction of abdominal pain and discomfort after defecation. This syndrome is characterized by persistent or intermittent symptoms and is characterized by a complex pathogenesis and numerous influencing factors. IBS can damage the intestinal mucosa, induce inflammation, disrupt the epithelial barrier, and lead to visceral hypersensitivity.

[0003] Damage to the intestinal mucosa can lead to the activation and infiltration of immune cells in the intestine, thereby releasing a large number of pro-inflammatory cytokines, which further aggravates the inflammation in the intestine, leading to intestinal mucosal erosion, which in turn exacerbates the damage to the intestine.

[0004] Inflammation is a response of the body's immune system to damage. The infiltration of immune cells into the gut leads to the secretion of large amounts of pro-inflammatory cytokines, further exacerbating immune system disorders and damaging the body's immune organs. IL-6 and TNF-α are recognized and widely studied pro-inflammatory cytokines associated with intestinal inflammation. IL-6 promotes the survival of intestinal TH1 and TH2 cells and prevents T cell apoptosis, thereby driving intestinal inflammation. Overexpression of TNF-α can directly induce intestinal epithelial cell apoptosis and significantly weaken intestinal barrier function. Conversely, as an anti-inflammatory cytokine, IL-10 plays a crucial role in controlling and preventing intestinal inflammation. IL-10 is believed to block macrophage metabolism and promote damaged mitophagy, thereby alleviating inflammation.

[0005] The intestinal barrier function plays a vital role in the human body. Exposure of intestinal epithelial cells to pro-inflammatory cytokines (such as TNF-α) leads to cell death, altered production of secretory mucins, and damage to the epithelial barrier. Occludin, a membrane protein and representative of tight junction structures, typically maintains the stability of intestinal tight junctions, ensuring normal intestinal barrier function. Studies related to IBS have shown that low-grade intestinal inflammation in IBS is characterized by impaired intestinal barrier function and decreased expression of tight junction proteins.

[0006] Visceral hypersensitivity is another typical clinical symptom of IBS besides low-grade inflammation. Studies have shown that visceral hypersensitivity is essentially a disorder of the peripheral and central nervous systems. In the gastrointestinal tract, mast cells are activated and release trypsin-like proteins, which in turn activate PAR-2 ​​on the surface of intestinal nerve cells, ultimately causing a sustained state of neural excitation that feeds back to the gastrointestinal tract, resulting in impaired gastrointestinal motility.

[0007] Studies have demonstrated that dietary intervention, fiber, and probiotics are effective treatment options for IBS. Probiotics can improve gut microbiota imbalance, regulate gastrointestinal motility, reduce visceral hypersensitivity, positively impact epithelial permeability, and reduce immune mucosal activation. Lactobacillus rhamnosus GG (LGG) is an L-lactic acid-producing, non-spore-forming, anaerobic, acid-resistant Gram-positive gut commensal bacterium. LGG possesses high acid resistance, high bile resistance, strong adhesion, and favorable growth characteristics, making it one of the most widely studied probiotic strains. Its functional properties mainly include regulating gut microbiota, preventing and treating diarrhea, promoting toxin elimination, and enhancing immunity, thus possessing high application value. Technical issues

[0008] To address the aforementioned issues, this invention provides a strain of *Lactaseibacillus rhamnosus*, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 5, 2024, with accession number GDMCC No: 64725. This strain has demonstrated good efficacy in the prevention and / or treatment of irritable bowel syndrome. Technical solutions

[0009] On one hand, the present invention provides a strain of Lacticaseibacillus rhamnosus, the preservation number of which is GDMCC No: 64725.

[0010] In another aspect, the present invention provides a culture obtained by culturing the aforementioned Lactaseibacillus rhamnosus.

[0011] In another aspect, the present invention provides the application of the aforementioned Lacticaseibacillus rhamnosus or its culture in the preparation of products for the prevention and treatment of irritable bowel syndrome.

[0012] Specifically, the irritable bowel syndrome is diarrhea-predominant, constipation-predominant, alternating, or mixed.

[0013] Preferably, the irritable bowel syndrome is constipation type.

[0014] Specifically, the products include, but are not limited to, pharmaceuticals, food, health food, and special purpose formula food.

[0015] Furthermore, the food products mentioned include, but are not limited to, compressed candy, yogurt, canned goods, biscuits, chocolate, pastries, cream, cheese, dairy products, milk powder, ice cream, popsicles, jam, fruit puree, candied fruit, preserved fruit, dried fruit, bread, egg rolls, protein drinks, lactic acid bacteria drinks, plant protein drinks, carbonated drinks, coffee, and puffed foods.

[0016] Furthermore, the aforementioned health food contains at least 1×10 7 CFU / g of Lactobacillus rhamnosus.

[0017] Furthermore, the health food also includes conventional excipients for health foods, including but not limited to fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional fortifiers.

[0018] Furthermore, the health food products mentioned include emulsion products, solution products, powder products, and solid products.

[0019] Furthermore, the product in question is a medicine.

[0020] Specifically, the viable count of *Lactobacillus rhamnosus* in the drug is not less than 1 × 10⁻⁶. 7 CFU / g.

[0021] Preferably, the viable count of *Lactaseibacillus rhamnosus* can be 1 × 10⁻⁶. 7 CFU / g-1×10 12 CFU / g.

[0022] Specifically, the drug also includes pharmaceutically acceptable excipients.

[0023] Preferably, the pharmaceutically acceptable excipients are selected from one or more combinations of wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, lubricants, antibacterial agents, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners, and flavorings.

[0024] More preferably, the pharmaceutically acceptable excipient is selected from at least one of lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, magnesium stearate, and mineral oil.

[0025] Specifically, the dosage forms of the drug include, but are not limited to: tablets, liquids, capsules, powders, suppositories, or granules.

[0026] Specifically, the administration method of the drug may be selected from oral, intravenous, local, intradermal, or subcutaneous injection.

[0027] In another aspect, the present invention provides a drug for the prevention and treatment of irritable bowel syndrome, the drug comprising the aforementioned Lactobacillus rhamnosus or its culture.

[0028] In another aspect, the present invention provides a method for preparing the aforementioned drug, the method comprising culturing Lactobacillus rhamnosus.

[0029] Specifically, the culture medium for culturing Lacticaseibacillus rhamnosus includes carbon, nitrogen, and / or phosphorus sources necessary for microbial proliferation, survival, or growth, and optionally may also contain growth additives such as trace elements that are beneficial to microbial proliferation, survival, or growth.

[0030] More specifically, those skilled in the art can prepare the “culture” or “microbial culture” described herein using any standard or known static drying or liquid fermentation technique known in the art for use in the compositions or products described herein. Optimal conditions for microbial culture may depend on the specific strain. Those skilled in the art will be able to determine suitable nutrients and conditions. Microorganisms can grow in aerobic, anaerobic, or facultative anaerobic liquid cultures on a culture medium.

[0031] Preferably, the culture medium for culturing Lacticaseibacillus rhamnosus includes, but is not limited to, MRS medium or modified MRS medium. Beneficial effects

[0032] The technical effects achieved by this invention are as follows:

[0033] (1) The MBP01 provided by the present invention can improve the weight loss trend of mice with irritable bowel syndrome, increase food intake and fecal water content.

[0034] (2) The MBP01 provided by the present invention can effectively alleviate colon damage in mice with irritable bowel syndrome, reduce inflammatory infiltration, protect the structural integrity of colon tissue, and thus restore intestinal function.

[0035] (3) The MBP01 provided by the present invention can effectively downregulate the levels of IL-1β, TNF-α, PAR-2 ​​and serum corticosterone and increase the level of IL-10.

[0036] (4) The MBP01 provided by the present invention can increase the expression of tight junction proteins and restore the intestinal barrier function damage caused by intestinal inflammation.

[0037] Preservation Instructions

[0038] Strain name: MBP01;

[0039] Accession number: GDMCC No: 64725;

[0040] Classification and naming: Lacticaseibacillus rhamnosus;

[0041] Date of deposit: June 5, 2024;

[0042] Preservation institution: Guangdong Provincial Center for Microbial Culture Collection;

[0043] Abbreviation of depositary institution: GDMCC;

[0044] Address of the storage unit: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0045] Figure 1 shows the changes in body weight and food intake of mice, where A represents the body weight of female mice; B represents the body weight of male mice; C represents the food intake of female mice; and D represents the food intake of male mice.

[0046] Figure 2 shows the changes in fecal water content and fecal particle number in mice. In the figure, A represents the fecal particle number of female mice; B represents the fecal particle number of male mice; C represents the fecal water content of female mice; and D represents the fecal water content of male mice.

[0047] Figure 3 shows stained sections of mouse colon tissue, where A represents colon tissue from female mice and B represents colon tissue from male mice.

[0048] Figure 4 shows the measurement of inflammatory factor levels in mice, where A represents IL-10 in female mice; B represents IL-10 in male mice; C represents IL-1β in female mice; D represents IL-1β in male mice; E represents TNF-α in female mice; F represents TNF-α in male mice; G represents IL-6 in female mice; and H represents IL-6 in male mice.

[0049] Figure 5 shows the determination of mouse cytokine levels, where A is the OCC of female mice; B is the OCC of male mice; C is the PAR-2 ​​of female mice; D is the PAR-2 ​​of male mice; E is the CORT of female mice; F is the CORT of male mice; G is the MCT of female mice; and H is the MCT of male mice.

[0050] Figure 6 shows the gene expression levels in the mouse colon, where A represents female PAR-2 ​​mice; B represents male PAR-2 ​​mice; C represents female OCC mice; and D represents male OCC mice. Embodiments of the present invention

[0051] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0052] Example 1

[0053] The main experimental materials and reagents used in this invention are shown in Table 1:

[0054] Table 1 Experimental Materials and Reagents

[0055]

[0056] The main instruments and equipment used in this invention are shown in Table 2:

[0057] Table 2 Instruments and Equipment

[0058]

[0059] 1.1 Experimental Methods

[0060] 1.1.1 Animal Model

[0061] Twenty-five male and twenty-five female C57BL / 6J mice were randomly divided into five groups: normal group, model group, positive control group, high-dose MBP01 group (MBP01-H), and low-dose MBP01 group (MBP01-L). Each group consisted of five male and five female mice, designated as: normal female, model female, positive female, MBP01-H female, MBP01-L female, normal male, model male, positive male, MBP01-H male, and MBP01-L male. From day 1 to 7 after the start of the experiment, except for the normal group, all other groups were fasted for 12 hours and then given 0.1 mL of TNBS solution via enema to establish the model. The MBP01-H group and the MBP01-L group were given 0.2 mL of live bacteria solution (1×10⁻⁶) by gavage daily. 9 CFU / kg, 1×10 7The positive control group received linaclotide (100 μg / g) by gavage daily after modeling. The experiment lasted for 29 days. Mouse weight and food intake were recorded every other day. The number of fecal pellets was recorded every two days, and fresh feces were collected, weighed in a dry EP tube, and the wet weight of the feces was recorded. The feces were then dried in a drying oven and weighed to determine the moisture content.

[0062] Fecal moisture content (%) = [(fecal wet weight - fecal dry weight) / fecal wet weight] × 100%.

[0063] 1.1.2 Measurement of cytokine levels in mouse serum and colon

[0064] After the experimental period, whole blood was collected via orbital sampling. The blood was allowed to stand at 4°C for half an hour, then centrifuged (4°C, 14000 r / min, 15 min). The supernatant serum was collected in centrifuge tubes and stored at -80°C. Mouse colon tissue was weighed and homogenized with sterile saline. The supernatant was collected after centrifugation and used later. Following the kit instructions, ELISA was used to determine serum corticosterone levels (CORT), and colonic levels of interleukins (IL-1β, IL-6, IL-10), tumor necrosis factor (TNF-α), occludin (OCC), PAR-2 ​​(PAR-2), and mast cell trypsin (MCT).

[0065] 1.1.3 Stained sections of colon tissue

[0066] Approximately 1 cm of the proximal colon of mice was removed by laparotomy. The colon was cleaned of any remaining contents with pre-cooled physiological saline at 4°C and cut along the longitudinal axis. The colon was then fixed in 4% polymethanol solution for 48 hours, followed by dehydration, embedding, sectioning, HE staining, and finally observation under a microscope. Images were collected and analyzed.

[0067] 1.1.4 Determination of mRNA in mouse intestinal tissue.

[0068] Mouse colon tissue was weighed, washed with sterile saline, and placed into homogenization tubes. Trizol was added to homogenize and extract RNA from the mouse colon tissue. The concentration and purity of the RNA extract were then measured using a spectrophotometer. The RNA extract was reverse transcribed into cDNA according to the reverse transcription kit instructions. After reverse transcription, a cDNA reaction mixture was prepared in an eight-tube strip containing 10 μL of the reaction mixture (Mastermix), 0.4 μL each of the upstream and downstream primers, 1 μL of template cDNA, and 8.2 μL of sterile, enzyme-free water. The reaction mixture was then placed on a real-time quantitative PCR instrument for amplification and detection.

[0069] Table 3 Primer sequences

[0070]

[0071] 1.1.5 Data Processing

[0072] Results in this experiment are expressed as mean ± standard deviation (x ± S), and one-way ANOVA was used for inter-group comparisons. The same lowercase letter 'a' or 'e' indicates no significant difference between the two groups, while different lowercase letters indicate a significant difference between the two groups (P < 0.05).

[0073] 1.2 Results and Analysis

[0074] 1.2.1 Results of mouse body weight changes

[0075] As shown in Figure 1, compared with the normal group, the body weight and food intake of mice modeled with TNBS were significantly reduced. During the drug administration period, the body weight of mice in the positive group gradually increased after drug treatment, with a faster growth rate. Compared with the model group, the MBP01-H and MBP01-L groups showed significantly increased body weight gain and food intake after intervention with Lactobacillus rhamnosus MBP01. This indicates that irritable bowel syndrome can lead to decreased food intake, reduced digestive capacity, and impaired digestion and absorption in animals; and that administration of Lactobacillus rhamnosus MBP01 can improve these symptoms.

[0076] 1.2.2 Changes in fecal water content and fecal particle number in mice

[0077] As shown in Figure 2, after the experimental model was established, the fecal water content and the number of fecal particles decreased in mice in all groups except the normal group. However, after intervention with drugs and MBP01, the fecal water content increased and the number of fecal particles increased.

[0078] 1.2.3 Histopathological observation of mouse colon tissue

[0079] As shown in Figure 3, HE pathological examination of colon tissue revealed that the colonic mucosa, submucosa, and muscularis propria of the normal group animals were structurally intact with clear texture. The mucosal epithelial cells showed no inflammatory response or damage, and the goblet cells were neatly arranged. In the colon of the mice after the model was established, inflammatory cell infiltration was observed, the submucosa was loose and somewhat edematous, and the goblet cells were loosely arranged. Compared with the model group, the colonic damage and inflammatory infiltration were reduced in the drug group and the MBP01 group, indicating that treatment with MBP01 can alleviate colonic damage caused by IBS to some extent.

[0080] 1.2.4 Measurement of inflammatory factor levels

[0081] As shown in Figure 4, compared with the normal group, the levels of IL-10 and TNF-α in female mice were significantly increased in the model group (P < 0.05), while the levels of IL-1β in female mice and TNF-α in male mice were decreased in the model group (P < 0.05), and the level of IL-1β in male mice remained unchanged. Compared with the model group, the levels of IL-1β, TNF-α, and IL-10 in female mice were significantly decreased in the MBP01 group (P < 0.05), while the level of IL-10 in the MBP01-L male group was significantly increased (P < 0.05). No obvious trend was observed in the IL-6 factor in this study, and it was not statistically significant (P > 0.05).

[0082] 1.2.5 Determination of trypsin-like proteins, PAR-2, tight junction proteins, and serum corticosterone levels in mouse colonic mast cells.

[0083] As shown in Figure 5, compared with the normal group, the levels of tight junction protein, PAR-2, and CORT in female mice were significantly increased in the model group (P < 0.05), while the levels of MCT and CORT in male mice were significantly decreased in the model group (P < 0.05). Compared with the model group, the levels of PAR-2 ​​and CORT in the MBP01 group were significantly decreased (P < 0.05), the level of MCT was significantly increased (P < 0.05), and no obvious trend was observed in the level of tight junction protein.

[0084] 1.2.6 Determination of PAR-2 ​​and tight junction protein expression levels in mouse colon

[0085] As shown in Figure 6, analysis of the expression levels of tight junction protein and PAR-2 ​​in the mouse colon revealed that, compared to the normal group, the expression levels of tight junction protein and PAR-2 ​​in male mice were significantly reduced in the model group (P < 0.05). Compared to the model group, MBP01-H significantly reduced the expression of tight junction protein and PAR-2 ​​in female mice (P < 0.05) and increased the expression of tight junction protein and PAR-2 ​​in male mice, while no obvious trend was observed in the gene expression of tight junction protein and PAR-2 ​​in the MBP01-L group.

[0086] 1.3 Analysis and Discussion

[0087] In this invention, it was observed that the condition of the model group mice differed significantly from that of the normal group mice. The model group mice exhibited a manic state in the early stages of modeling, transitioning to a sluggish state in the later stages, accompanied by weight loss, reduced food intake, decreased fecal particle count, and lower water content. After intervention with drugs and MBP01, the overall physical characteristics of the animals improved significantly; their activity level increased, food intake increased markedly, and their weight increased, along with an increase in fecal particle count and water content. This indicates that *Lactobacillus rhamnosus* MBP01 can improve the condition of mice with irritable bowel syndrome and has a certain therapeutic effect.

[0088] In the colon of IBS model mice, abundant inflammatory cell infiltration was observed, with loosely arranged goblet cells and weak intercellular junctions. After MBP01 intervention, inflammatory infiltration in the colon tissue of mice was reduced, intercellular junctions were strengthened, and goblet cells were more neatly arranged. This indicates that Lactobacillus rhamnosus MBP01 can effectively alleviate colonic damage in irritable bowel syndrome mice, reduce inflammatory infiltration, protect the structural integrity of colonic tissue, and thus restore intestinal function.

[0089] To further investigate the alleviating effect of MBP01 on low-grade inflammation in IBS, the expression of pro-inflammatory and anti-inflammatory cytokines in the mouse colon was examined. The results showed that TNBS enema modeling induced autoimmune dysregulation in mice, with a significant increase in TNF-α levels in the colon of female mice. This indicates that intestinal immune homeostasis was affected, and the mice exhibited intestinal inflammation symptoms. However, treatment with *Lactobacillus rhamnosus* MBP01 significantly reduced the expression levels of TNF-α and IL-1β in the mouse colon, while significantly increasing IL-10 levels in the MBP01-L male group. This suggests that *Lactobacillus rhamnosus* MBP01 has the efficacy of alleviating low-grade intestinal inflammation symptoms in IBS.

[0090] In this invention, the expression level of tight junction protein in the colon of mice was determined by PCR. The results showed that, compared with the normal group, the expression of tight junction protein in male mice in the model group was significantly decreased. After oral administration of Lactobacillus rhamnosus MBP01, the level of tight junction protein in male mice in the MBP01-H group was significantly increased, demonstrating that high doses of Lactobacillus rhamnosus MBP01 can restore the intestinal barrier function damage caused by intestinal inflammation.

[0091] In this invention, it was found that compared with the normal group, the levels of PAR-2 ​​in the model group mice and CORT in female mice were significantly increased in the model group. After treatment with MBP01, the levels of PAR-2 ​​and CORT in the MBP01 group were significantly decreased, which indicates that Lactobacillus rhamnosus MBP01 may have the effect of improving visceral allergic symptoms in mice.

[0092] Comparative Example 1

[0093] In Comparative Example 1, the strain used to study the effect on irritable bowel syndrome was *Lactobacillus rhamnosus* 4F225, with accession number CGMCC No: 26437. The modeling process and dosage for irritable bowel syndrome (1×10⁻⁶) were investigated. 9 The CFU / kg values ​​were the same as in Example 1, with the only difference being the specific bacterial strain.

[0094] Experimental results: Compared with the model group, the level of TNF-α in female mice did not change significantly after intervention with Lactobacillus rhamnosus (CGMCC No: 26437).

Claims

1. A Lacticaseibacillus rhamnosus strain, characterized in that, The Lacticaseibacillus rhamnosus described is accessed under the number GDMCC No: 64725.

2. A culture, characterized in that, The culture was obtained by culturing Lactobacillus rhamnosus as described in claim 1.

3. The use of Lacticaseibacillus rhamnosus as described in claim 1 or the culture as described in claim 2 in the preparation of products for the prevention and treatment of irritable bowel syndrome.

4. Use according to claim 3, characterized in that, The irritable bowel syndrome is classified as diarrhea-predominant, constipation-predominant, alternating, or mixed.

5. Use according to claim 4, characterized in that, The product is a medicine.

6. Use according to claim 5, characterized in that, The viable cell count of Lacticaseibacillus rhamnosus in the drug is not less than 1 x 10 7 CFU / g.

7. Use according to claim 5, characterized in that, The drug also includes pharmaceutically acceptable excipients.

8. Use according to claim 5, characterized in that, The dosage form of the drug is tablets, liquids, capsules, powders, suppositories, or granules.

9. A drug for preventing and treating irritable bowel syndrome, characterized in that, The drug comprises Lacticaseibacillus rhamnosus as described in claim 1 or the culture as described in claim 2.

10. The method for preparing the drug according to claim 9, characterized in that, The preparation method includes culturing Lactobacillus rhamnosus.

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