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

Abstract

The present invention belongs to the field of microbial technology, and specifically relates to a rhamnosus lactobacillus MBP01 and a composition thereof for preventing and treating irritable bowel syndrome. The deposit number of the rhamnosus lactobacillus MBP01 is GDMCC No: 64725. The MBP01 provided by the present invention can improve the weight loss trend of IBS model mice, increase food intake and fecal water content, alleviate damage to colon tissue, downregulate IL-1β, TNF-α, PAR-2, and serum corticosterone levels and increase IL-10 levels, while high concentrations of MBP01 can increase the expression of tight junction proteins. Therefore, MBP01 has a certain positive regulatory effect on the intestinal function of IBS model mice and can effectively prevent and treat irritable bowel syndrome.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a Lactobacillus rhamnosus MBP01 for preventing and treating irritable bowel syndrome and a composition thereof. Background Art

[0002] Irritable bowel syndrome (IBS) is a functional intestinal disease characterized by chronic or recurrent abdominal pain and changes in bowel habits, and lacks morphological and biochemical abnormalities. It is mainly manifested by the symptoms of abdominal pain and discomfort that can be relieved or alleviated after defecation, accompanied by changes in bowel habits and stool characteristics. This symptom persists or recurs intermittently, and has a complex pathogenesis and many influencing factors. IBS can damage the intestinal mucosa, induce inflammation, destroy the epithelial barrier, and cause 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, further aggravating inflammation in the intestine, causing intestinal mucosal erosion, and in turn aggravating intestinal damage.

[0004] Inflammation is a response of the body's immune system to damage. The infiltration of immune cells in the intestine will lead to the secretion of a large number of pro-inflammatory cytokines, further aggravating the disorder of the immune system 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 can promote the survival of intestinal TH1 and TH2 cells and prevent T cell apoptosis, thereby promoting the occurrence of intestinal inflammation. The overexpression of TNF-α can directly induce intestinal epithelial cell apoptosis and significantly weaken the intestinal barrier function. On the contrary, as an anti-inflammatory cytokine, IL-10 plays a vital role in controlling and preventing intestinal inflammation. IL-10 is believed to block the metabolism of macrophages and promote autophagy of damaged mitochondria, thereby alleviating inflammation.

[0005] Intestinal barrier function plays a vital role in the human body. Exposure of intestinal epithelial cells to proinflammatory cytokines (such as TNF-α) can lead to cell death, alter the production of secreted mucins, and damage the epithelial barrier. Occludin is a membrane protein and a representative protein of tight junction structures. Its function in the intestine is usually to maintain the stability of intestinal tight junctions and ensure normal intestinal barrier function. IBS-related studies have shown that low-grade intestinal inflammation in IBS is characterized by damaged 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 tryptase, which in turn activates PAR-2 ​​on the surface of intestinal nerve cells, ultimately causing a continuous state of neural excitement that feeds back to the gastrointestinal tract, resulting in gastrointestinal motility disorders.

[0007] Studies have shown that dietary intervention, fiber and probiotics are effective options for the treatment of IBS. Probiotics can improve intestinal microbial flora imbalance, regulate gastrointestinal motility, reduce visceral allergies, have a positive effect on epithelial permeability, and reduce immune mucosal activation. Lactobacillus rhamnosus GG, LGG) is an L-lactic acid-producing, non-spore-producing, anaerobic, acid-resistant Gram-positive intestinal symbiotic bacterium. LGG has high acid resistance, high bile resistance, strong adhesion and good growth characteristics. It is one of the most widely studied probiotic strains. Its functional characteristics mainly include regulating intestinal flora, preventing and treating diarrhea, excreting toxins and enhancing the body's immunity, and has high application value. Summary of the invention

[0008] In order to solve the above problems, the present invention provides a strain of Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ), which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 5, 2024, with the collection number GDMCCNo: 64725. This strain has shown good efficacy in preventing and / or treating irritable bowel syndrome.

[0009] In one aspect, the present invention provides a strain of Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ), the Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) is deposited with GDMCC No: 64725.

[0010] In another aspect, the present invention provides a culture, wherein the culture is obtained by culturing the aforementioned Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus )get.

[0011] In another aspect, the present invention provides the aforementioned Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) or culture in the preparation of products for preventing and treating irritable bowel syndrome.

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

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

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

[0015] Furthermore, the food includes but is not limited to compressed candies, yogurt, canned food, biscuits, chocolate, cakes, cream, cheese, sour cream, milk powder, ice cream, popsicles, jam, puree, candied fruit, preserved fruit, bread, egg rolls, protein drinks, lactic acid bacteria drinks, plant protein drinks, carbonated drinks, coffee, and puffed food.

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

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

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

[0019] Furthermore, the product is a medicine.

[0020] Specifically, the Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) has a viable count of no less than 1×10 7 CFU / g.

[0021] Preferably, the Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) can have a viable count of 1×10 7 CFU / g-1×10 12 CFU / g.

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

[0023] Preferably, the pharmaceutically acceptable excipient is selected from one or a combination of two or more of wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, lubricants, antibacterial agents, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners and flavors.

[0024] Further preferably, the pharmaceutically acceptable excipient is selected from at least one of lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, magnesium stearate and mineral oil.

[0025] Specifically, the dosage form of the drug includes, but is not limited to, tablets, liquids, capsules, powders, suppositories or granules.

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

[0027] In another aspect, the present invention provides a drug for preventing and treating irritable bowel syndrome, the drug comprising the aforementioned Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) or culture.

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

[0029] Specifically, the culturing of Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) includes a carbon source, a nitrogen source and / or a phosphorus source necessary for the proliferation, survival or growth of the microorganisms, and optionally, may also contain growth additives such as trace elements that are beneficial to the proliferation, survival or growth of the microorganisms.

[0030] Further specifically, those skilled in the art can use any standard or known static drying or liquid fermentation technology known in the art to prepare the "culture" or "microorganism culture" of the present invention for use in the compositions or products herein. The optimal conditions for microbial culture can depend on the specific strain. Those skilled in the art will be able to determine the appropriate nutrients and conditions. Microorganisms can be grown in aerobic, anaerobic or facultative anaerobic liquid cultures on a culture medium.

[0031] Preferably, the culturing of Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) culture medium includes but is not limited to: MRS medium or MRS modified medium.

[0032] The technical effects achieved by the present invention are:

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

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

[0035] (3) 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) 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] Collection Instructions

[0038] Strain name: MBP01;

[0039] Deposit number: GDMCC No: 64725;

[0040] Category naming: Lacticaseibacillus rhamnosus ;

[0041] Deposit date: June 5, 2024;

[0042] Depository: Guangdong Microbiological Culture Collection Center;

[0043] Abbreviation of depository unit: GDMCC;

[0044] Address of the preservation unit: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0046] Figure 2 represents the changes in fecal water content and the number of fecal particles of mice, where A represents the number of fecal particles of female mice; B represents the number of fecal particles 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 The following are staining images of mouse colon tissue sections, where A is the colon tissue of a female mouse and B is the colon tissue of a male mouse.

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

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

[0050] Figure 6The figure shows the gene expression level determination in mouse colon, wherein A is female mouse PAR-2; B is male mouse PAR-2; C is female mouse OCC; and D is male mouse OCC. DETAILED DESCRIPTION

[0051] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0052] Example 1

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

[0054] Table 1 Experimental materials and reagents

[0055]

[0056] The main instruments and equipment in the present invention are shown in Table 2:

[0057] Table 2 Instruments and equipment

[0058]

[0059] 1.1 Experimental methods

[0060] 1.1.1 Animal models

[0061] C57BL / 6J male and female mice (25 mice each) were randomly divided into 5 groups: normal group, model group, positive control group, MBP01 high-dose group (MBP01-H), and MBP01 low-dose group (MBP01-L). Each group had 5 male and female mice, which were recorded 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. 1-7 days after the start of the experiment, except for the normal group, the rest of the groups were fasted for 12 hours and then enema 0.1 mL of TNBS solution was performed to establish the model. The MBP01-H group and the MBP01-L group were gavaged with 0.2 mL of live bacteria solution (1×10 9 CFU / kg, 1×10 7 CFU / kg), the positive drug group was gavaged with linaclotide (100μg / g) every day after modeling. The experimental period was 29 days, the weight and food intake of mice were recorded every other day, the number of feces particles of mice was recorded every two days, and fresh feces were collected and weighed in a dry EP tube, the wet weight of feces was recorded, and then the feces were placed in a drying box for drying and weighing to determine the water content.

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

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

[0064] After the experimental period, whole blood was collected by orbital sampling. The blood was allowed to stand for half an hour at 4°C and then centrifuged (4°C, 14000r / min, 15min). After centrifugation, the upper serum was collected in a centrifuge tube and stored in a -80°C refrigerator. The mouse colon tissue was weighed and homogenized with sterile saline. After homogenization, the supernatant was collected by centrifugation for standby use. According to the instructions of the kit, ELISA reagents were used to determine the corticosterone content (CORT) in serum, interleukins (IL-1β, IL-6, IL-10), tumor necrosis factor (TNF-α), tight junction protein (occludin, OCC), protease-activated receptor 2 (PAR-2), and mast cell tryptase (MCT) levels in the colon.

[0065] 1.1.3 Colon tissue staining sections

[0066] About 1 cm of the proximal colon of the mouse was removed by laparotomy. The residues in the colon were cleaned with 4°C pre-cooled saline, and the colon was cut open along the longitudinal axis. It was fixed in 4% polymethanol solution for 48 h, dehydrated, embedded, sliced, HE stained, and finally observed under a microscope, and images were collected and analyzed.

[0067] 1.1.4 Determination of mRNA in mouse intestinal tissue

[0068] Weigh the colon tissue of the mouse, wash it with sterile saline, and put it into a homogenizer tube. Add Trizol for homogenization to extract RNA from the mouse colon tissue. Then use a spectrophotometer to measure the concentration and purity of the RNA extract. According to the instructions of the reverse transcription kit, reverse transcribe the RNA extract into cDNA. After reverse transcription, prepare the cDNA reaction system in an eight-tube strip, that is, 10μL of reaction mixture (Mastermix), 0.4μL of upstream and downstream primers, 1μL of template cDNA, and 8.2μL of sterile enzyme-free water. After the reaction system is prepared, place it on a real-time fluorescence quantitative PCR instrument for amplification detection.

[0069] Table 3 Primer sequences

[0070]

[0071] 1.1.5 Data processing

[0072] The results of this experiment were expressed as mean ± standard deviation (x ± S), and one-way analysis of variance was used for comparison between groups. The same lowercase letters in ae indicate no significant difference between the two groups, and different lowercase letters indicate significant difference between the two groups P <0.05.

[0073] 1.2 Results and Analysis

[0074] 1.2.1 Results of mouse weight changes

[0075] Depend on Figure 1 It can be seen that compared with the normal group, the weight and food intake of mice modeled by TNBS were significantly reduced; during the drug administration period, after drug treatment, the weight of mice in the positive group gradually increased, and the growth rate was faster, while the weight growth rate and food intake of the MBP01-H group and the MBP01-L group were significantly increased after the intervention of Lactobacillus rhamnosus MBP01 compared with the model group. This shows that irritable bowel syndrome can reduce animal diet, reduce animal digestive ability, and cause digestion and absorption disorders; and the administration of Lactobacillus rhamnosus MBP01 can improve this symptom.

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

[0077] Depend on Figure 2 It can be seen that after the experimental modeling, except for the normal group, the feces water content of mice in other groups decreased and the number of fecal particles decreased. After the intervention of drugs and MBP01, the feces water content increased and the number of fecal particles increased.

[0078] 1.2.3 Observation of mouse colon histopathology

[0079] like Figure 3 As shown in the figure, the results of colon tissue HE examination showed that the colon mucosa, submucosa and muscular layer of the normal group animals were intact, with clear texture, no inflammatory reaction and damage to the mucosal epithelial cells, and the goblet cells were neatly arranged. Inflammatory cell infiltration was observed in the colon of the modeled mice, the submucosal structure was loose and edematous, and the goblet cells were loosely arranged. Compared with the model group, the colon damage of the mice in the drug group and MBP01 group was alleviated, and the inflammatory infiltration was reduced, indicating that the treatment with MBP01 can alleviate the colon damage caused by IBS to a certain extent.

[0080] 1.2.4 Determination of inflammatory factor levels

[0081] like Figure 4 As shown, compared with the normal group, the levels of IL-10 and TNF-α in female mice in the model group increased significantly ( P <0.05), the levels of IL-1β in female mice and TNF-α in male mice decreased in the model group ( P<0.05), while the IL-1β level in male mice did not change; compared with the model group, the levels of IL-1β and TNF-α in the MBP01 group and IL-10 in female mice were significantly reduced ( P <0.05), the IL-10 level in the MBP01-L male group increased significantly ( P <0.05). IL-6 factor did not show obvious trend in this experimental study and was not statistically significant ( P >0.05).

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

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

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

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

[0086] 1.3 Analysis and Discussion

[0087] In the present invention, it can be found that the state of the model group mice has changed greatly compared with the normal group mice. The model group mice showed a manic state in the early stage of modeling, and turned into a sluggish state in the late stage of modeling, accompanied by weight loss, reduced diet, reduced number of fecal particles, and reduced water content; after intervention with drugs and MBP01, the overall signs of the animals were significantly improved, the animals were more active, the food intake increased significantly, and the weight of the mice increased, the number of fecal particles increased, and the water content increased. This shows 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, a large number of inflammatory cell infiltrations can be observed in the colon tissue, the goblet cells are loosely arranged, and the intercellular connections are not tight; after MBP01 intervention, the inflammatory infiltration in the colon tissue of mice was reduced, the intercellular connections were tight, and the goblet cells were arranged more neatly. This shows that Lactobacillus rhamnosus MBP01 can effectively alleviate the colon damage of IBS mice, reduce inflammatory infiltration, protect the structural integrity of colon tissue, and thus restore intestinal function.

[0089] In order to further explore the alleviating effect of MBP01 on low-grade inflammation in IBS, the expression of pro-inflammatory cytokines and anti-inflammatory cytokines in the colon of mice was investigated. The results showed that mice would develop autoimmune disorders after TNBS enema modeling, and the experiment found that the level of TNF-α in the colon of female mice increased significantly. This shows that the intestinal immune homeostasis of mice was affected and the mice had inflammatory symptoms in the intestine. However, after treatment with Lactobacillus rhamnosus MBP01, the expression of TNF-α and IL-1β in the colon of mice was significantly reduced, and the IL-10 level in the MBP01-L male group increased significantly. This shows that Lactobacillus rhamnosus MBP01 has the effect of alleviating the symptoms of low-grade inflammation in the intestine of IBS.

[0090] In the present invention, PCR was used to determine the expression of tight junction proteins in the mouse colon, and the results showed that compared with the normal group, the tight junction protein expression of males in the modeling group was significantly decreased. After oral administration of Lactobacillus rhamnosus MBP01, the tight junction protein level of male mice in the MBP01-H group was significantly increased, proving that a high dose of Lactobacillus rhamnosus MBP01 can restore the intestinal barrier function damage caused by intestinal inflammation.

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

[0092] Comparative Example 1

[0093] The strain used in comparative example 1 to study the effect on irritable bowel syndrome was Lactobacillus rhamnosus 4F225 with a preservation number of CGMCC No. 26437. The modeling process and dosage of irritable bowel syndrome (1×10 9 The CFU / kg) were the same as those in Example 1, except for the specific strains.

[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 with the collection number CGMCC No. 26437.

Claims

1. A strain of Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ), characterized in that, The Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) is deposited with GDMCC No: 64725.

2. A culture, characterized in that The culture is obtained by culturing the Lactobacillus rhamnosus according to claim 1 ( Lacticaseibacillus rhamnosus )get.

3. The Lactobacillus rhamnosus of claim 1 ( Lacticaseibacillus rhamnosus ) or the culture of claim 2 in the preparation of a drug for preventing and treating irritable bowel syndrome.

4. The use according to claim 3, characterized in that: The irritable bowel syndrome is of diarrhea type, constipation type, alternating type or mixed type.

5. The use according to claim 3, characterized in that: The drug contains Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) has a viable count of no less than 1×10 7 CFU / g.

6. The use according to claim 3, characterized in that: The drug also includes pharmaceutically acceptable excipients.

7. The use according to claim 3, characterized in that: The dosage form of the drug is tablet, liquid, capsule, powder, suppository or granule.

8. A drug for preventing and treating irritable bowel syndrome, characterized in that: The drug comprises the Lactobacillus rhamnosus described in claim 1 ( Lacticaseibacillus rhamnosus ) or the culture of claim 2.

9. The method for preparing the drug according to claim 8, characterized in that: The preparation method comprises culturing Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ).

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