Conjugated linoleic acid-producing plant lactobacillus for relieving irritable bowel syndrome and application of conjugated linoleic acid-producing plant lactobacillus

Abstract

The invention belongs to the technical field of probiotics, and provides conjugated linoleic acid producing plant lactobacillus capable of relieving irritable bowel syndrome and application of the conjugated linoleic acid producing plant lactobacillus, the plant lactobacillus is preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.33685, and the preservation date is February 28, 2025. The plant lactobacillus M4 can promote generation of conjugated linoleic acid, can remarkably relieve visceral hypersensitivity of the irritable bowel syndrome, reduces the water content of excrement, relieves damage to colon tissue, down-regulates the levels of IL-6 and TNF-alpha and improves the level of IL-10; the expression of the ZO-1, the Claudin-15 and the MUC2 is obviously improved; the PAR-2 level and the NLRP3 level of the colon are obviously reduced; the colon SERT level is obviously improved. Therefore, the plant lactobacillus M4 has a certain positive regulation effect on the intestinal function of the IBS model mouse, and can effectively prevent and treat the irritable bowel syndrome.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotics, and particularly relates to a conjugated linoleic acid-producing Lactobacillus plantarum for alleviating irritable bowel syndrome and an application thereof. Background Art

[0002] Irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder with a global prevalence of approximately 10%-15%. Its main symptoms include abdominal pain, bloating, diarrhea, constipation, or alternating episodes of both. The pathogenesis of IBS is complex and influenced by numerous factors, including intestinal dysbiosis, impaired intestinal barrier function, immune system abnormalities, and brain-gut axis dysfunction, all of which may play a significant role. IBS can damage the intestinal mucosa, induce inflammation, disrupt the epithelial barrier, and lead to visceral hypersensitivity.

[0003] Intestinal mucosal damage can lead to the activation and infiltration of immune cells in the intestine, thereby releasing a large number of pro-inflammatory cytokines, further aggravating intestinal inflammation, leading to intestinal mucosal erosion, and in turn exacerbating intestinal damage. IL-6 and TNF-α are 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. Overexpression of TNF-α can directly induce intestinal epithelial cell apoptosis and significantly weaken the intestinal barrier function. The anti-inflammatory cytokine IL-10 plays a vital role in controlling and preventing intestinal inflammation. IL-10 can block the metabolism of macrophages and promote autophagy of damaged mitochondria, thereby reducing inflammation.

[0004] 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, altered mucin production, and damage to the epithelial barrier. The tight junction protein ZO-1 in the intestine can maintain the stability of intestinal tight junctions and ensure 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.

[0005] Visceral hypersensitivity is another typical clinical symptom of IBS, in addition to low-grade inflammation. Research has shown that visceral hypersensitivity is essentially a disorder of the peripheral and central nervous systems. In the gastrointestinal tract, activated mast cells release tryptase, which in turn activates PAR-2 on the surface of intestinal nerve cells, ultimately causing a persistent state of neural excitation that feeds back to the gastrointestinal tract, resulting in impaired gastrointestinal motility.

[0006] Currently, the treatment of irritable bowel syndrome (IBS) focuses primarily on symptom relief, with commonly used medications including antispasmodics, antidiarrheals, laxatives, and antidepressants. However, these medications typically only provide short-term symptom relief, and long-term use can lead to side effects such as drug dependence and further intestinal dysfunction. Studies have shown that dietary intervention, dietary fiber, and probiotics are effective strategies for treating IBS. Among these, probiotics can play a positive role by improving gut microbiota dysbiosis, regulating gastrointestinal motility, reducing visceral sensitivity, enhancing epithelial barrier function, and reducing immune mucosal activation. Among the many probiotics, Lactobacillus and Bifidobacterium are the most widely studied genera. In particular, Lactobacillus plantarum, a common lactobacillus, has been shown to have multiple probiotic functions, including improving intestinal health, enhancing immunity, and exerting antioxidant and anti-inflammatory effects. Furthermore, conjugated linoleic acid (CLA), a naturally occurring fatty acid found in foods like dairy and meat, exhibits multiple biological activities, including anti-inflammatory, antioxidant, and immunomodulatory activities. Studies have shown that CLA may improve intestinal health by reducing intestinal inflammation and regulating intestinal flora. Therefore, screening for strains capable of producing CLA may provide a new solution for alleviating irritable bowel syndrome. Summary of the Invention

[0007] In response to the problems existing in the prior art, the present invention aims to provide a conjugated linoleic acid-producing Lactobacillus plantarum strain for relieving irritable bowel syndrome and its application. The present invention uses conjugated linoleic acid (CLA) production as a core indicator, screens a high-CLA-producing Lactobacillus plantarum strain M4, and uses Citrobacter rodentium combined with water avoidance stress to construct an animal model to explore the new use of Lactobacillus plantarum M4 in relieving irritable bowel syndrome. The beneficial effects of Lactobacillus plantarum M4 are reflected in multiple aspects such as visceral sensitivity, fecal water content, intestinal barrier, inflammatory response, and intestinal flora.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A first aspect of the present invention provides a conjugated linoleic acid-producing Lactobacillus plantarum for relieving irritable bowel syndrome. The Lactobacillus plantarum M4 is deposited in the General Microbiology Center of the China Culture Collection Administration, with a deposit number of CGMCC No. 33685 and a deposit date of February 28, 2025.

[0010] Furthermore, the conjugated linoleic acid includes cis-9, trans-11-octadecadienoic acid and trans-10, cis-12-octadecadienoic acid.

[0011] The second aspect of the present invention provides a culture obtained by culturing the Lactobacillus plantarum described in the first aspect. Preferably, the culture is cultured by inoculating the Lactobacillus plantarum M4 into MRS culture medium and culturing at 37°C for 12 to 36 hours.

[0012] The third aspect of the present invention provides use of the Lactobacillus plantarum described in the first aspect or the culture described in the second aspect in preparing a product for preventing and treating irritable bowel syndrome.

[0013] The fourth aspect of the present invention provides use of the Lactobacillus plantarum described in the first aspect or the culture described in the second aspect in preparing conjugated linoleic acid.

[0014] Furthermore, the irritable bowel syndrome is diarrhea type, constipation type, indefinite type or mixed type.

[0015] Furthermore, the viable count of Lactobacillus plantarum M4 in the product is not less than 1×10 10 CFU / mL.

[0016] Furthermore, the product is one or more of the following:

[0017] (1) The product can alleviate visceral hypersensitivity, reduce fecal water content, and alleviate damage to colon tissue;

[0018] (2) The product can downregulate the expression levels of IL-6 and TNF-α and increase the expression level of IL-10;

[0019] (3) The product can increase the gene expression of ZO-1, Claudin-15, E-cadherin and MUC2;

[0020] (4) The product can reduce the gene expression levels of colon PAR-2 and NLRP3 and increase the gene expression level of colon SERT.

[0021] Preferably, the dosage form of the product includes tablets, liquids, capsules, powders, suppositories or granules.

[0022] A fifth aspect of the present invention provides a preparation for preventing and treating irritable bowel syndrome, wherein the preparation comprises the Lactobacillus plantarum described in the first aspect or the culture described in the second aspect.

[0023] Culture deposit information:

[0024] Lactobacillus plantarum M4 was isolated from fresh milk. After purification and identification, it was deposited at the General Microbiology Center of the China General Culture Collection (CGMCC); Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China; Deposit Date: February 28, 2025; Deposit Number: CGMCC No. 33685; Taxonomic Name: Lactiplantibacillus plantarum M4.

[0025] The beneficial effects of the present invention compared to the prior art are:

[0026] 1. The present invention provides a novel strain of Lactobacillus plantarum M4, which has the ability to produce high levels of conjugated linoleic acid (CLA) without genetic modification. Experiments have shown that Lactobacillus plantarum M4 can exert anti-inflammatory functions by producing CLA, thereby alleviating symptoms associated with irritable bowel syndrome.

[0027] 2. The Lactobacillus plantarum M4 of the present invention can relieve visceral hypersensitivity and diarrhea symptoms of diarrhea-type irritable bowel syndrome, and at the same time alleviate the phenomenon of colon shortening;

[0028] 3. The Lactobacillus plantarum M4 of the present invention can effectively alleviate colon damage in mice with irritable bowel syndrome, reduce inflammatory infiltration, protect the integrity of intestinal tissue structure, and thus restore intestinal function;

[0029] 4. The Lactobacillus plantarum M4 of the present invention can effectively downregulate the expression levels of IL-6, TNF-α and PAR-2 and increase the expression level of IL-10;

[0030] 5. The Lactobacillus plantarum M4 of the present invention can increase the expression of tight junction proteins, increase the expression of MUC2, restore the mucus barrier, and alleviate the intestinal barrier function damage caused by intestinal inflammation;

[0031] 6. The Lactobacillus plantarum M4 described in the present invention is a potential probiotic that can alleviate irritable bowel syndrome and can be used to prevent or treat irritable bowel syndrome and other related intestinal diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The present invention will be further described below with reference to the accompanying drawings and examples:

[0033] Figure 1 The evaluation of the ability of Lactobacillus plantarum to produce conjugated linoleic acid is shown, wherein A: UV detection of the ability of Lactobacillus plantarum to produce conjugated linoleic acid; B: GC detection of the content of conjugated linoleic acid produced by Lactobacillus plantarum M4; C: GC spectrum;

[0034] Figure 2The fecal water content and abdominal withdrawal reflex score of mice are shown, where A: animal experiment design; B: fecal water content; C: colon ratio; D: abdominal withdrawal reflex score;

[0035] Figure 3 Shown are the effects of Lactobacillus plantarum M4 intervention on intestinal tissue damage: A: H&E staining and AB-PAS staining of colon tissue; B: colon crypt depth; C: goblet cell count;

[0036] Figure 4 The effect of Lactobacillus plantarum M4 intervention on the expression of inflammatory factors in colon tissue is shown: A: relative expression of IL-6; B: relative expression of TNF-α; C: relative expression of IL-10;

[0037] Figure 5 Shown are the effects of Lactobacillus plantarum M4 intervention on the expression of intestinal barrier-related genes: A: relative expression of ZO-1; B: relative expression of Claudin-15; C: relative expression of E-cadherin; D: relative expression of MUC2;

[0038] Figure 6 The figure shows the effect of Lactobacillus plantarum M4 intervention on colon tissue-related genes: A: relative expression of PAR-2; B: relative expression of NLRP3; C: relative change of SERT. DETAILED DESCRIPTION

[0039] The examples are provided to better illustrate the present invention, but are not intended to limit the present invention to the examples. Therefore, non-essential improvements and adjustments to the embodiments made by those skilled in the art based on the above-mentioned invention still fall within the scope of protection of the present invention.

[0040] The endpoints of the ranges and any values disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0041] The present invention will be described in detail below through examples. It should be understood that the following examples are only used to further explain and illustrate the content of the present invention in detail, and are not intended to limit the present invention. Example 1: Determination of the conjugated linoleic acid content of Lactobacillus plantarum M4

[0042] In this example, ultraviolet spectroscopy (UV) was used to conduct a preliminary evaluation of the conjugated linoleic acid production capacity of five strains of Lactobacillus plantarum (M4, B119-A-05, A028-A-10, H001-A-12, and B127-B-04; see Table 1 for strain information). The laboratory listed in the table below is the Probiotics Basic and Applied Research Laboratory, Department of Nutrition and Health, China Agricultural University.

[0043] Table-1 Strain information

[0044]

[0045] The specific detection methods are:

[0046] Linoleic acid (LA) emulsion (20 mg / ml) was prepared by adding a predetermined amount of linoleic acid (LA) to Milli-Q water containing 1% (v / v) Tween 80. The solution was thoroughly vortexed and sterilized by filtration through a 0.22 μm cellulose acetate membrane. The appropriate amount was added to sterile MRS-LA to a final LA concentration of 1 mg / ml or 3 mg / ml. A 2% inoculation of the strain was performed in MRS-LA and cultured at 37°C for 24 hours. One ml of the culture was centrifuged at 5000 g for 5 minutes to separate the supernatant from the precipitate. The supernatant was vigorously mixed with 1 mL of isopropanol, followed by the addition of 1 mL of n-hexane. The mixture was vortexed for 2 minutes, then allowed to stand and the supernatant collected for absorbance measurement at 233 nm (A233).

[0047] Test results such as Figure 1 As shown in Figure A, after adding 1 mg / mL and 3 mg / mL of substrate LA to Lactobacillus plantarum M4, the fatty acids in the fermentation broth were extracted after 24 hours of fermentation, and the absorbance at 233 nm was measured, which were 0.327 and 1.119, respectively. The absorbance was the highest among the five strains of Lactobacillus plantarum, indicating that M4 has the potential to produce high conjugated linoleic acid among the five strains of Lactobacillus plantarum.

[0048] The specific type and concentration of conjugated linoleic acid produced by Lactobacillus plantarum M4 were further detected by gas chromatography (GC). The detection method is:

[0049] After 24 hours of fermentation with 2.5 mg / ml LA, the bacterial broth was centrifuged and the supernatant was collected. To minimize degradation, the sample was thawed in an ice bath. A 50 μl sample was transferred to a 1.5 ml centrifuge tube, 500 μl of isopropanol was added, and the tube was shaken at 1450 rpm for 15 min at 10°C (MSC-100, Allsheng Instruments Co., Ltd, China). The tube was centrifuged at 18,000 g for 25 min at 4°C (Microfuge 20R, Beckman Coulter, Inc., IN, USA). 400 μl of the supernatant was transferred to a 2 ml glass vial and vacuum concentrated to dryness (Labconco, Kansas City, MO, USA). 200 μl of 2% methanolic sulfuric acid was added, and the sample was shaken at 650 rpm for 30 min at 80°C. After the reaction, the sample was cooled to room temperature, 200 μl of n-hexane was added, sonicated for 1 min, and then shaken at 1400 rpm for 10 min. The sample was allowed to stand at 4°C for 20 minutes, and the upper hexane layer was transferred to a sample injection vial for analysis. A ThermoFisher GC TRACE 1610 equipped with an MSD Orbitrap Exploris 240 was used to detect the target compounds.

[0050] Test results such as Figure 1 As shown in B and C, it was shown that Lactobacillus plantarum M4 was able to produce cis-9, trans-11-octadecadienoic acid and trans-10, cis-12-octadecadienoic acid, with concentrations of 190.5383 μmol / L and 132.1422 μmol / L, respectively.

[0051] In summary, UV spectroscopy and gas chromatography revealed that Lactobacillus plantarum M4 can produce conjugated linoleic acid (CLA), which has been shown to have anti-inflammatory effects by regulating inflammatory mediators, inhibiting the NF-κB pathway, activating PPARs, and reducing oxidative stress. It can also alleviate intestinal inflammation (such as inflammatory bowel disease) by regulating intestinal immune responses and microbiota. Therefore, it is speculated that Lactobacillus plantarum M4 may exert its anti-inflammatory function by producing CLA, thereby alleviating symptoms associated with irritable bowel syndrome.

[0052] Example 2: Lactobacillus plantarum M4 can alleviate irritable bowel syndrome induced by Citrobacter rodentium combined with water avoidance stress

[0053] 1. Experimental Animals

[0054] SPF male C57BL / 6J mice (6 weeks old, SPF grade), weighing 18–20 g, were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. All animal procedures were approved by the Laboratory Animal Welfare and Ethical Review Committee of China Agricultural University (Aw31604202-4-2) and performed in accordance with the European Union's guidelines for the protection of experimental animals (2010 / 63 / EU). All mice were housed at 22–24°C, 40–70% humidity, and maintained on a 12-h light–dark cycle. They had free access to a commercial standard diet and distilled water.

[0055] 2. Experimental Methods

[0056] 2.1 Citrobacter rodentium DBS100 culture

[0057] Citrobacter rodentium DBS100 (ATCC 51459) was purchased from Ningbo Minghai Biotechnology Co., Ltd.

[0058] After dissolving the bacterial powder, streak the plate and incubate at 37°C until distinct colonies appear on the plate. Pick a single colony and inoculate it into LB liquid medium. Activate it three times at 37°C and 200 rpm before using it in experiments.

[0059] Citrobacter rodentium DBS100 was cultured and centrifuged at 8000 g for 10 min at 4 °C to collect the cells. The cells were resuspended in sterile PBS and the cell concentration was adjusted to 6 × 10 10 CFU / mL.

[0060] 2.2 Culture of Lactobacillus plantarum M4

[0061] Streak Lactobacillus plantarum M4 glycerol plates and incubate at 37°C until distinct colonies appear on the plates. Pick a single colony and inoculate it into MRS liquid medium, incubate and subculture at 37°C, and activate three generations for use in experiments.

[0062] The Lactobacillus plantarum was cultured and centrifuged at 8000 g for 10 min at 4 °C to collect the cells. The cells were resuspended in sterile PBS and the cell concentration was adjusted to 1 × 10 10 CFU / mL.

[0063] 2.3 Establishment of an IBS mouse model induced by Citrobacter rodentium combined with water avoidance stress

[0064] The experiment started after the mice had adapted for seven days, and the experimental duration was 30 days.

[0065] On the first day of the experiment, mice in the control group were gavaged with sterile PBS solution, and mice in the other groups were gavaged with Citrobacter rodentium DBS100 (1×10 10CFU / 0.2mL / mouse). On days 2-8 of the experiment, all mice were intraperitoneally injected with 0.5ml of lactated Ringer's solution to prevent diarrhea and dehydration. On days 18-30 of the experiment, mice in all groups except the control group were subjected to water avoidance stress (WAS). The WAS apparatus consisted of a bucket with a diameter of 27cm and a height of 33cm. A dry platform with a diameter of 4cm and a height of 9cm was installed at the bottom of the bucket. The bucket was filled with water until the water level was 1cm below the platform. Mice were placed on the platform for 1 hour every day (17:00-18:00). If a mouse fell into the water, it was immediately picked up and wiped dry.

[0066] On days 2-29 of the experiment, mice in the control and model groups were gavaged with sterile PBS (200 μl / mouse / day), while mice in the Lactobacillus plantarum M4 intervention group were gavaged with Lactobacillus plantarum M4 resuspended in sterile PBS (200 μl / mouse / day).

[0067] Table-2 Animal experiment design

[0068]

[0069] LR, lactated Ringer's solution; WAS, water avoidance stress

[0070] 2.4 Determination of fecal water content

[0071] To assess fecal moisture content (MCF), a fecal sample is collected and weighed (w1), then dried in a 60°C oven to a constant weight and weighed (w2). The MCF is the ratio of the difference between the two fecal weights to the original fecal weight, using the formula: MCF = (w1 - w2) / w1.

[0072] 2.5 Visceral sensitivity determination

[0073] Abdominal withdrawal reflex scores (AWRs) were used to assess the visceral sensitivity of mice (Table 3).

[0074] After fasting and anesthetizing each group of mice, a lubricated 6Fr, 2mm outer diameter urinary catheter with a balloon was inserted into the mouse anus, with the balloon 1 cm from the anus. The catheter was secured to the mouse's tail with tape and the mouse was placed in a breathable plastic box. Within the box, the mouse was only able to walk forward and backward, not turn around. After acclimating for 30 minutes, colorectal dilation was performed using a gradual inflating and pressurizing method.

[0075] Three researchers, unaware of the group assignments, scored the colonic distension response of each group of mice. Pressures were increased gradually from 0, 0.1, 0.2, to 0.3 mL, with each pressure increasing for 20 seconds. At the end of the assessment, the balloon was deflated and withdrawn.

[0076] Table-3 Abdominal withdrawal reflex scores

[0077]

[0078] 2.6 Collection of stool, serum, and tissue samples

[0079] After the experiment, fecal samples were collected and stored at −80°C. Mice were then anesthetized with isoflurane and sacrificed by cervical dislocation. Blood was collected, centrifuged, and serum was extracted and stored at −80°C until use.

[0080] The lengths of the colon and the entire intestine of the mice were measured, and the ratio of colon to total intestine length was recorded.

[0081] 2.7 Hematoxylin-eosin staining and immunofluorescence staining

[0082] Colon tissue was fixed in 4% paraformaldehyde for 24 hours and then embedded in paraffin. The embedded tissue was cut into 4 μm sections and stained with hematoxylin and eosin. The sections were then photographed under a microscope.

[0083] For immunofluorescence, colon tissue sections were antigen-retrieved using citrate antigen retrieval buffer after deparaffinization and hydration. Sections were then incubated with primary antibodies against ZO-1 (1:2000, Abcam) and MUC2 (1:300, Proteintech), followed by incubation with fluorescently conjugated secondary antibodies. Cell nuclei were counterstained with DAPI, and images were acquired using confocal laser scanning microscopy.

[0084] 2.8 mRNA determination in mouse intestinal tissue

[0085] Weigh 50 mg of mouse colon tissue and put it into a homogenizer tube. Add Trizol for homogenization to extract RNA from the mouse colon tissue. Then use nanodrop 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 a cDNA reaction system in an eight-tube strip, namely 10 μL of reaction mixture (Mastermix), 0.5 μL of upstream and downstream primers, 1 μL of template cDNA, and 8 μL of sterile enzyme-free water. After the reaction system is prepared, place it on a real-time fluorescence quantitative PCR instrument for amplification detection. Table 4 lists all the primers used in this example. According to 2 -ΔCT Methods were used to determine the relative quantification of target genes and β-actin was used as a reference.

[0086] Table-4 Primer sequences

[0087]

[0088] 2.9 Data Statistics and Analysis

[0089] Statistical analysis was performed using GraphPad Prism 10. All data are presented as mean ± standard deviation (SD). Results were analyzed using one-way analysis of variance (ANOVA), and statistical significance was further determined using Tukey's multiple comparison test. P values ≤ 0.05 were considered statistically significant.

[0090] 3. Experimental Results

[0091] 3.1 Determination of fecal water content and visceral sensitivity in mice

[0092] The design of animal experiments Figure 2 As shown in A.

[0093] The fecal water content of each group was measured. Figure 2 In Figure B, the fecal water content of mice in the model group was significantly higher than that in the control group. Lactobacillus plantarum M4 could reduce the fecal water content to normal levels. After the modeling was completed, the mice were killed by cervical dislocation. The colon length and total intestinal length were measured and it was found that the proportion of colon to total intestinal length in the model group was significantly lower than that in the control group. However, the proportion of colon length in the Lactobacillus plantarum M4 group increased significantly, indicating that it plays a potential role in alleviating intestinal inflammation ( Figure 2 Middle C).

[0094] In the abdominal withdrawal reflex test ( Figure 2 (D) When the expansion volume was 0.1ml, 0.2ml, and 0.3ml, the behavioral scores of the control group ranged from 0 to 1, while the scores of the model group ranged from 1 to 3, indicating that the model group mice exhibited symptoms of visceral hypersensitivity. Notably, when the expansion volume was 0.2ml and 0.3ml, the AWR scores of the Lactobacillus plantarum M4 group ranged from 1 to 2, significantly lower than those of the model group. In summary, Lactobacillus plantarum M4 can effectively alleviate the symptoms of visceral hypersensitivity in IBS.

[0095] 3.2 Histopathological observation of mouse colon

[0096] like Figure 3 The H&E staining results of the colon tissue shown in A and B show that the colon mucosa, submucosa and muscularis of the normal group animals are intact, with clear textures, no inflammatory reaction or damage to the mucosal epithelial cells, and the goblet cells are neatly arranged. Inflammatory cell infiltration can be seen in the colon of the modeled mice, the submucosa structure is loose and slightly edematous, and the goblet cells are loosely arranged. Compared with the model group, the colon damage of the mice treated with Lactobacillus plantarum M4 was alleviated and the inflammatory infiltration was reduced. According to the AB-PAS staining results of the colon tissue ( Figure 3 As shown in A and C, PAS in the crypts of the model group +The number of goblet cells decreased significantly. + The number of goblet cells was significantly increased, indicating that administration of Lactobacillus plantarum M4 can alleviate the colon damage caused by IBS to a certain extent.

[0097] 3.3 Determination of inflammatory factor-related gene expression in mouse colon tissue

[0098] RT-qPCR revealed that ( Figure 4 In the model group, expression of the pro-inflammatory cytokines IL-6 and TNF-α was upregulated, while expression of the anti-inflammatory cytokine IL-10 was downregulated compared to the control group. After intervention with Lactobacillus plantarum M4, expression of IL-6, TNF-α, and IL-10 returned to normal levels. This suggests that the IBS model induced by Citrobacter rodentium combined with water avoidance stress promotes intestinal inflammation, and Lactobacillus plantarum M4 can exert its anti-inflammatory effects to alleviate the inflammatory response and protect intestinal health.

[0099] 3.4 Determination of intestinal barrier-related gene expression in mouse colon tissue

[0100] RT-qPCR revealed that ( Figure 5 In the AC group, the expression levels of ZO-1, Claudin-15, and E-cadherin genes related to the intestinal mechanical barrier, as well as MUC2 gene related to the mucus barrier, also changed. In the model group, the expression levels of ZO-1, Claudin-15, E-cadherin, and MUC2 were all downregulated, but returned to normal levels after intervention with Lactobacillus plantarum M4. This suggests that Lactobacillus plantarum M4 has the function of maintaining the integrity of the intestinal mechanical barrier and repairing mucus barrier damage.

[0101] 3.5 Determination of related gene expression in mouse colon tissue

[0102] The expression levels of PAR-2, NLRP3 and SERT genes in colon tissue Figure 6 The results showed that compared with the control group, the expression of PAR-2 and NLRP3 in the model group was significantly increased, while the expression of SERT was significantly decreased. After intervention with Lactobacillus plantarum M4, the expression of PAR-2 and NLRP3 decreased, while the expression of SERT increased. This suggests that Lactobacillus plantarum M4 can improve the symptoms of visceral hypersensitivity in mice with irritable bowel syndrome.

[0103] 4. Discussion and Analysis

[0104] In the present invention, it was found that the fecal water content of the model group mice increased, and symptoms of visceral hypersensitivity were also present. However, after intervention with Lactobacillus plantarum M4, the animals' overall physical signs were significantly improved, the fecal water content decreased, and the symptoms of visceral hypersensitivity were alleviated. This suggests that Lactobacillus plantarum M4 can improve the diarrhea and abdominal pain in mice with irritable bowel syndrome and has a certain therapeutic effect.

[0105] In the colons of IBS model mice, a large number of inflammatory cell infiltrates were observed, with loosely arranged goblet cells and loose intercellular junctions. After treatment with Lactobacillus plantarum M4, the inflammatory infiltration in the colonic tissue of the mice was reduced, intercellular junctions became tighter, and goblet cells were more neatly arranged. This suggests that Lactobacillus plantarum M4 can effectively alleviate colon damage in mice with IBS, reduce inflammatory infiltration, protect the structural integrity of colonic tissue, and thus restore intestinal function.

[0106] To further explore the alleviating effect of Lactobacillus plantarum M4 on low-grade inflammation in IBS, this example study examined the expression of pro-inflammatory cytokines and anti-inflammatory cytokines in the mouse colon. The results showed that mice developed autoimmune disorders after being treated with Citrobacter rodentium combined with water avoidance stress modeling. The experiment found that the levels of IL-6 and TNF-α in the colon increased significantly. This indicates that the intestinal immune homeostasis of the mice was affected and the mice had inflammatory symptoms in the intestine. However, after treatment with Lactobacillus plantarum M4, the expression of IL-6 and TNF-α in the mouse colon was significantly reduced, and the IL-10 level increased significantly. This shows that Lactobacillus plantarum M4 has the effect of alleviating the symptoms of low-grade inflammation in the intestine of IBS.

[0107] In this study, PCR was used to measure the expression of tight junction proteins in the mouse colon. The results showed that compared with the normal control group, the expression of tight junction proteins in the modeling group was significantly reduced. After oral administration of Lactobacillus plantarum M4, the level of tight junction proteins was significantly increased, demonstrating that Lactobacillus plantarum M4 can restore intestinal barrier function damaged by intestinal inflammation.

[0108] In IBS patients, low-grade intestinal inflammation is an important cause of visceral hypersensitivity. PAR-2 and NLRP3 play a central role in visceral hypersensitivity by mediating inflammation, pain conduction, and neuro-immune interactions. Their activation leads to increased excitability of sensory neurons, release of inflammatory mediators, and disruption of intestinal barrier function, ultimately triggering visceral hypersensitivity. At the same time, abnormal SERT expression or function leads to reduced 5-HT reuptake, increasing the concentration of 5-HT in the intestine. Excessive 5-HT, by activating 5-HT receptors (such as 5-HT3 and 5-HT4), enhances the excitability of intestinal sensory neurons and also leads to visceral hypersensitivity. In the present invention, it was found that compared with the normal group, the levels of PAR-2 and NLRP3 in the model group of mice in the model group increased significantly, while the level of SERT decreased significantly. After treatment with Lactobacillus plantarum M4, PAR-2 and NLRP3 decreased significantly compared with the model group, while SERT increased significantly. This suggests that Lactobacillus plantarum M4 may have the effect of improving the symptoms of visceral hypersensitivity in mice.

[0109] Finally, it should be noted that the above is only used to illustrate the technical solution of the present invention and is not limiting. Although the present invention is described in detail with reference to the preferred arrangement scheme, ordinary technicians in this field should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A conjugated linoleic acid-producing Lactobacillus plantarum for relieving irritable bowel syndrome, characterized in that: The plant lactobacillus is Lactiplantibacillus plantarum M4, which is deposited in the General Microbiology Center of the China Culture Collection Administration, with a deposit number of CGMCC No. 33685 and a deposit date of February 28, 2025.

2. The plant lactobacillus according to claim 1, wherein The conjugated linoleic acid includes cis-9, trans-11-octadecadienoic acid and trans-10, cis-12-octadecadienoic acid.

3. A culture, characterized in that The culture is obtained by culturing the Lactobacillus plantarum according to claim 1 or 2.

4. Use of the Lactobacillus plantarum according to claim 1 or 2 or the culture according to claim 3 in the preparation of a product for preventing and treating irritable bowel syndrome.

5. Use of the Lactobacillus plantarum according to claim 1 or 2 or the culture according to claim 3 in the preparation of conjugated linoleic acid.

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

7. The use according to claim 4, characterized in that The number of viable bacteria of Lactobacillus plantarum M4 in the product is not less than 1×10 10 CFU / mL.

8. The use according to claim 7, characterized in that The product is one or more of the following: (1) The product can alleviate visceral hypersensitivity, reduce fecal water content, and alleviate damage to colon tissue; (2) The product can downregulate the expression levels of IL-6 and TNF-α and increase the expression level of IL-10; (3) The product can increase the gene expression of ZO-1, Claudin-15, E-cadherin and MUC2; (4) The product can reduce the gene expression levels of colon PAR-2 and NLRP3 and increase the gene expression level of colon SERT.

9. A preparation for preventing and treating irritable bowel syndrome, characterized in that: The preparation comprises the Lactobacillus plantarum of claim 1 or 2 or the culture of claim 3.

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