Lactobacillus plantarum capable of relieving ulcerative colitis and application of exopolysaccharide of lactobacillus plantarum

Abstract

The invention relates to the technical field of microbiology, in particular to lactobacillus plantarum ZGS521 for relieving ulcerative colitis and application of exopolysaccharides of the lactobacillus plantarum ZGS521. The invention provides lactobacillus plantarum ZGS521, and the preservation number of the lactobacillus plantarum ZGS521 is CCTCC (China Center For Type Culture Collection) NO: M20241181. The invention also provides the lactobacillus plantarum extracellular polysaccharide and components thereof obtained by fermenting the lactobacillus plantarum ZGS521, and the application of the lactobacillus plantarum extracellular polysaccharide and the components thereof is as follows: preparing a medicine for regulating intestinal flora and preparing a medicine for relieving ulcerative colitis.

Description

Technical Field

[0001] The present invention relates to the technical field of microbiology, and in particular to Lactobacillus plantarum ZGS521 and its exopolysaccharide application for relieving ulcerative colitis. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic, relapsing gastrointestinal disease believed to result from the interaction of genetics, environment, microorganisms, and immune responses. Patients with UC often experience symptoms such as abdominal pain, diarrhea, bloody stools, fever, loss of appetite, and weight loss.

[0003] Currently, IBD treatments primarily alleviate clinical symptoms rather than directly targeting the cause. Treatment is tailored to the severity of the disease. Mild to moderate cases are treated with aminosalicylic acid drugs; moderate to severe, acute, and steroid-dependent patients are treated with glucocorticoids or combined immunosuppressants; refractory cases are treated with biologics; and in recent years, probiotics have been used as adjunctive therapy for mild to moderate cases. However, due to significant individual variability, significant steroid side effects, and the high cost of biologics, the development of precise treatments is urgently needed.

[0004] Lactic acid bacteria (LAB) are microorganisms that ferment sugars to produce lactic acid. They possess antioxidant, acid-resistant, bile-tolerant, and antibacterial properties and are widely used in the food and pharmaceutical industries. Exopolysaccharides (EPS), a secondary metabolite of LAB, possess unique functions and activities and have been extensively studied in recent years. Lactobacillus plantarum, a typical LAB, not only has strong intestinal colonization ability but also possesses a robust capacity to synthesize EPS. Literature reviews indicate that LAB and its EPS significantly alleviate intestinal inflammation and have become an important therapeutic strategy for intestinal inflammation. However, the current pool of known LAB strains with potential for preventing or treating colitis remains extremely limited, and strains that effectively alleviate intestinal oxidative stress and inflammation, improve the intestinal barrier, modify intestinal microbiota composition, or regulate intestinal epithelial proliferation and differentiation are lacking. Therefore, further screening and identification of LAB strains and their EPS with potential to regulate intestinal epithelial proliferation and differentiation and anti-colitis activities, and elucidating their potential anti-intestinal inflammatory effects and underlying molecular mechanisms, are critical scientific issues that urgently need to be addressed, with both significant academic significance and practical application value. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a Lactobacillus plantarum ZGS521 (Lactobacillus plantarum ZGS521) which can effectively alleviate ulcerative colitis and an exopolysaccharide derived therefrom.

[0006] In order to solve the above technical problems, the present invention provides a Lactobacillus plantarum ZGS521, whose preservation number is: CCTCC NO: M 20241181.

[0007] The present invention also provides a Lactobacillus plantarum extracellular polysaccharide, which is obtained by fermenting the Lactobacillus plantarum ZGS521.

[0008] The present invention also provides a method for preparing the above-mentioned Lactobacillus plantarum exopolysaccharide, comprising the following steps:

[0009] Lactobacillus plantarum ZGS521 is inoculated into an M-MRS liquid culture medium or an M-MRS liquid culture medium containing CaCl2 at an inoculum size of 1% to 5% (preferably 1% to 2%), and the culture is placed in a constant temperature incubator at 37±0.5°C for 12 to 32 hours (preferably 24 to 26 hours). After the culture is completed, the supernatant is collected by centrifugation, and the protein is removed (using trichloroacetic acid (TCA) with a final concentration of 4%). The crude polysaccharide is obtained after alcohol precipitation (ethanol) and post-processing.

[0010] When M-MRS liquid medium was used, the crude polysaccharide obtained was named LPE (Lactobacillus plantarum exopolysaccharide LPE);

[0011] When M-MRS liquid culture medium containing CaCl2 is used, the obtained crude polysaccharide is named C-LPE (Lactobacillus plantarum exopolysaccharide C-LPE).

[0012] As an improvement to the preparation method of Lactobacillus plantarum exopolysaccharide of the present invention:

[0013] M-MRS liquid medium (modified MRS fermentation medium) is prepared by dissolving 10 g of peptone, 5 g of yeast extract, and 10 g of beef extract powder in 100-150 mL of water, adding 400-600 mL of ethanol for precipitation (4°C for 24 h), collecting the supernatant and drying it, adding 0.2 g of magnesium sulfate heptahydrate, 0.05 g of manganese sulfate, 2 g of dipotassium hydrogen phosphate, 2 g of triammonium citrate, 5 g of anhydrous sodium acetate, 20-30 g of glucose, and 1 mL of Tween, and adding 5 g of amino-free yeast nitrogen base (YNB). The volume is then made up to 1 L with water.

[0014] The M-MRS liquid culture medium containing CaCl2 is prepared by adding CaCl2 to the M-MRS liquid culture medium to a final concentration of 1 to 5 mM.

[0015] As a further improvement of the method for preparing the Lactobacillus plantarum exopolysaccharide of the present invention:

[0016] The crude polysaccharide C-LPE was separated and purified by cellulose DEAE-52 column chromatography to obtain polysaccharide components C-LPE-0, C-LPE-3, and C-LPE-5, respectively.

[0017] The details are as follows:

[0018] The crude polysaccharide C-LPE was separated and purified by cellulose DEAE-52 column chromatography (the crude polysaccharide C-LPE was dissolved in deionized water and then centrifuged, and the supernatant was loaded onto the column), and eluted with deionized water and different concentrations of NaCl solution (0.1M, 0.3M, 0.5M, 0.7M and 0.9M) respectively. The polysaccharide component obtained by post-treatment of the eluate with deionized water as the eluent was named C-LPE-0, the polysaccharide component obtained by post-treatment of the eluate with 0.3M NaCl solution as the eluent was named C-LPE-3, and the polysaccharide component obtained by post-treatment of the eluate with 0.5M NaCl solution as the eluent was named C-LPE-5.

[0019] Note: The flow rate of the eluent is 1 mL / min, cellulose DEAE-52 is used for separation, the specifications of the chromatography column are 60 cm × Φ2.6 cm, and the effective volume of the column is 50 cm × Φ2.6 cm.

[0020] The post-treatment is as follows: the obtained eluate is concentrated by rotary evaporation (evaporation temperature of 50°C is concentrated to 25% of the original volume), dialyzed (retention volume 3500Da, so as to achieve desalination), and finally freeze-dried (-60°C drying for 48h) to obtain polysaccharides.

[0021] As a further improvement of the method for preparing the Lactobacillus plantarum exopolysaccharide of the present invention:

[0022] The polysaccharide components C-LPE-0, C-LPE-3, and C-LPE-5 were purified using Sephadex G-200 gel columns, respectively, to obtain the corresponding purified polysaccharide components C-LPE0, C-LPE3, and C-LPE5, respectively.

[0023] The details are as follows:

[0024] The polysaccharide components C-LPE-0, C-LPE-3, and C-LPE-5 were purified using a Sephadex G-200 gel column (the polysaccharide was dissolved in deionized water, passed through a 0.45 μm filter membrane, and then loaded onto the column); deionized water was used as the eluent for elution, and the eluate was concentrated by rotary evaporation and freeze-dried to obtain the corresponding purified polysaccharide components C-LPE0, C-LPE3, and C-LPE5.

[0025] Note: Sephadex G-200 gel chromatography column was used for purification. The column size was 60 cm × Φ1.2 cm and the effective length of the column was 50 cm.

[0026] The present invention also provides uses of Lactobacillus plantarum exopolysaccharide and its components for preparing medicines for regulating intestinal flora and for preparing medicines for relieving ulcerative colitis.

[0027] As an improvement of the use of the present invention: Lactobacillus plantarum exopolysaccharide and its components include LPE, C-LPE, C-LPE-0, C-LPE-3, C-LPE-5, C-LPE0, C-LPE3, and C-LPE5.

[0028] As a further improvement of the use of the present invention: Lactobacillus plantarum exopolysaccharide and its components have antioxidant and anti-inflammatory activities.

[0029] As a further improvement of the use of the present invention: regulating intestinal epithelial proliferation and differentiation, and increasing the content of arginine (relieving colitis by increasing the content of arginine).

[0030] The strain of the present invention was isolated from the fermentation broth of Northeastern kimchi, and the preservation information is as follows:

[0031] Deposit name: Lactobacillus plantarum ZGS521, deposit unit: China Center for Type Culture Collection, deposit address: Wuhan University, Wuhan, China, deposit number: CCTCC NO: M 20241181, deposit date: June 7, 2024.

[0032] The present invention provides a method for preparing exocellular polysaccharides, comprising: culturing the Lactobacillus plantarum ZGS521 strain to obtain a fermentation broth, and then extracting the exocellular polysaccharides from the fermentation broth. A preferred method is: culturing the Lactobacillus plantarum ZGS521 strain of the present invention in an M-MRS liquid culture medium at 37°C, extracting the fermentation broth to obtain crude polysaccharides, and subjecting the crude polysaccharides to DEAE-52 column chromatography and Sephadex G-200 gel column purification to obtain purified components. A more preferred method is as follows: Lactobacillus plantarum ZGS521 strain is added to M-MRS liquid culture medium at a 2% inoculum size and cultured at 37°C for 26 hours. After the culture is completed, the supernatant is centrifuged at 12000g for 20 minutes at 4°C to collect the supernatant, deproteinized by trichloroacetic acid (TCA) method, extracted by alcohol precipitation with ethanol (4 volumes), and dried, dialyzed, and freeze-dried to obtain a crude polysaccharide; the crude polysaccharide is purified by DEAE-52 column chromatography with the mobile phase being deionized water and 0.1M, 0.3M, 0.5M, 0.7M, and 0.9M NaCl solutions; the three eluted aqueous solutions are respectively concentrated by rotary evaporation, freeze-dried, redissolved through a 0.45μm filter membrane, and purified by a Sephadex G-200 gel column, concentrated by rotary evaporation, and freeze-dried to obtain the product. The strain is cultured in a medium with glucose as the carbon source, and the extracellular polysaccharide (LPE) yield can be as high as 294mg / L. Further under the stimulation of 3mM CaCl2, the extracellular polysaccharide (C-LPE) was further increased to 670mg / L.

[0033] The plant lactobacillus exopolysaccharide C-LPE of the present invention and its component are preferably prepared by the following method: plant lactobacillus ZGS521 is in M-MRS liquid culture medium, with glucose as carbon source, 2% inoculum size, and is kept at a constant temperature of 37°C for 26h; the fermented liquid is centrifuged at 4°C, 12000g for 20min, and the supernatant is collected; the protein in the supernatant is removed by TCA method; the ethanol precipitation is extracted; the supernatant is dialyzed with flowing water (cut-off 3500Da) for 72h; and the plant lactobacillus exopolysaccharide C-LPE is obtained after freeze drying for 36-48h; the crude polysaccharide is separated by cellulose DEAE-52 column chromatography (60cm×Φ2.6cm), and the eluent is deionized water and 0.1M, 0.3M, 0.5M, 0.7M and 0.9M NaCl solutions; the three eluted component aqueous solutions are concentrated by rotary evaporation, freeze dried, redissolved in deionized water and filtered through a 0.45μm membrane, and then filtered using a dextran Sephadex column. The three components of Lactobacillus plantarum exopolysaccharide C-LPE were obtained after purification by G-200 gel column (60 cm×Φ1.2 cm), rotary evaporation concentration and freeze drying.

[0034] The crude polysaccharide was separated by DEAE-52 column chromatography and purified by Sephadex G-200 gel column to obtain three extracellular polysaccharide components C-LPE0, C-LPE3, and C-LPE5, whose molecular weights were 899.72 kDa, 20.48 kDa, and 18.34 kDa, respectively. Its chemical composition and monosaccharide composition are shown in Table 2 and Table 3. In vitro experiments showed that the plant lactobacillus extracellular polysaccharide and its three components had strong free radical scavenging ability and Fe 3+ Reducing ability; the three components have significant cellular antioxidant capacity and strong cellular anti-inflammatory capacity. Experiments in a DSS colitis mouse model showed that Lactobacillus plantarum and its purified component C-LPE3 can alleviate inflammatory symptoms in mice with colitis and have a good regulatory effect on the mouse intestinal flora. These biological activities indicate that the invented Lactobacillus plantarum and its exopolysaccharides have broad application value in the preparation of products for alleviating colitis and regulating intestinal flora.

[0035] In summary, the present invention provides a Lactobacillus plantarum ZGS521 and its EPS with the efficacy of alleviating ulcerative colitis. The strain and its EPS can reduce the weight loss of colitis mice, inhibit the increase in DAI scores, and reduce the shortening of colon length. At the same time, they regulate the composition of intestinal microbiota, increase the abundance of beneficial bacteria, reduce the abundance of harmful bacteria, and increase the content of short-chain fatty acids such as acetate, propionate and butyrate, so as to achieve the purpose of alleviating ulcerative colitis.

[0036] The present invention is based on lactic acid bacteria isolated from Northeastern kimchi, from which Lactobacillus plantarum ZGS521, which is effective in alleviating ulcerative colitis, was screened. This strain has excellent EPS synthesis, gastrointestinal fluid tolerance, self-aggregation ability, and adhesion to Caco-2 cells. Furthermore, Lactobacillus plantarum ZGS521 and its EPS have excellent anti-inflammatory properties, regulate intestinal flora, and increase short-chain fatty acid production, making them useful for the prevention or adjunctive treatment of ulcerative colitis. The Lactobacillus plantarum and its EPS of the present invention provide a theoretical basis for the development of probiotic-related products.

[0037] In vitro antioxidant experiments showed that the plant lactobacillus exopolysaccharide (C-LPE) and its three components had strong antioxidant capacity (DPPH free radical scavenging capacity, ABTS free radical scavenging capacity, hydroxyl free radical scavenging capacity and Fe 3+ reducing power).

[0038] In vitro RAW264.7 cell oxidative damage model experiments show that the Lactobacillus plantarum exopolysaccharide C-LPE and its three components can increase the activity of antioxidant enzymes and reduce the level of oxidative factors, thereby exerting antioxidant activity.

[0039] In vitro RAW264.7 cell inflammation model experiments show that the Lactobacillus plantarum exopolysaccharide C-LPE and its three components of the present invention have the ability to regulate cytokine levels, thereby alleviating cellular inflammation.

[0040] The experiment of constructing a DSS colitis mouse model showed that the plantarum lactobacillus and the purified exopolysaccharide component C-LPE3 of the present invention can effectively alleviate intestinal inflammation and regulate intestinal flora and its metabolites. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The specific embodiments of the present invention are further described in detail below with reference to the accompanying drawings.

[0042] Figure 1 For the identification of strain ZGS521;

[0043] Figure 1 Middle: A is the growth status of strain ZGS521 on MRS; B is the Gram staining result; C is the evolutionary tree result of strain ZGS521.

[0044] Figure 2 To evaluate the biological characteristics and potential probiotic properties of Lactobacillus plantarum ZGS521;

[0045] Figure 2 Middle: A is the growth curve; B is the curve of extracellular polysaccharide production; C is the tolerance to simulated gastrointestinal fluid; D is the self-aggregation; E is the adhesion rate to Caco-2 cells.

[0046] Figure 3 Ion exchange and gel purification elution curves of C-LPEs (AD) and representative photos of C-LPEs (E: C-LPE0, F: C-LPE3, G: C-LPE5).

[0047] Figure 4 The effect of Lactobacillus plantarum ZGS521 on the symptoms of colitis in mice;

[0048] Figure 4 Middle: A is weight change; B is DAI score; C is colon length; D is colon mass / length ratio.

[0049] Figure 5 The effect of Lactobacillus plantarum ZGS521 on intestinal mucosal tissue of colitis mice;

[0050] Figure 5Middle: A is H&E staining; B is AB-PAS staining; C is IF staining of Muc2 in the colon (red represents the target protein, blue represents the cell nucleus); D is the colon injury score; E is the quantification of the number of goblet cells; F is the relative abundance statistics of Muc2 protein, G is the mRNA expression of Lgr5, H is the mRNA expression of SI, and I is the mRNA expression of Chga.

[0051] Figure 6 The effect of Lactobacillus plantarum ZGS521 on the content of short-chain fatty acids in colitis mice;

[0052] Figure 6 Middle: A to G are total short-chain fatty acids (SCFA), acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid and isovaleric acid.

[0053] Figure 7 Effects of Lactobacillus plantarum ZGS521 on the genus level of intestinal flora in mice with colitis (A) and the relative abundance of Lactobacillus, norank_f__Muribaculaceae, Lachnospiraceae_NK4A136_group, Dubosiella, Desulfovibrio and Escherichia-Shigella (BG).

[0054] Figure 8 The chemical antioxidant activity of Lactobacillus plantarum exopolysaccharide C-LPEs;

[0055] Figure 8 In the middle: A is DPPH free radical; B is ABTS free radical; C is hydroxyl free radical; D is Fe 3+ Restoration power.

[0056] Figure 9 Effects of C-LPEs on the viability of RAW264.7 cells (A) and screening of H2O2 concentration (B).

[0057] Figure 10 Effects of C-LPEs on ROS content (A), SOD activity (B), CAT activity (C) and MDA content (D) in RAW 264.7 cells induced by H2O2.

[0058] Figure 11 Effects of C-LPEs on cytokine expression in LPS-induced RAW264.7 cells;

[0059] Figure 11 Middle: A is IL-6; B is IL-1β; C is TNF-α; D is IL-10; E is TGF-β1.

[0060] Figure 12 The effect of C-LPE3 on the symptoms of colitis in mice;

[0061] Figure 12 Middle: A is the experimental plan; B is the change in body weight; C is the DAI score; D is the colon length; E is the colon mass / length ratio; F is a representative photo of the colon.

[0062] Figure 13 The effect of C-LPE3 on intestinal mucosal tissue of colitis mice;

[0063] Figure 13 Middle: A is H&E staining; B is DAI score; C is AB-PAS staining; D is goblet cell count.

[0064] Figure 14 Representative images of immunofluorescence staining of Muc-2 protein in mouse colon tissue by C-LPE3.

[0065] Figure 15 Representative images of immunofluorescence staining of occludin (A) and ZO-1 (B) proteins in mouse colon tissues by C-LPE3.

[0066] Figure 16 The effect of C-LPE3 on cytokine secretion;

[0067] Figure 16 Middle: A is IL-1β; B is IL-6; C is IL-10; D is TNF-α; E is TGF-β1.

[0068] Figure 17 The effect of C-LPE3 on oxidative stress in intestinal mucosal tissue;

[0069] Figure 17 Middle: A is SOD; B is CAT; C is GSH-Px; D is MDA.

[0070] Figure 18 Effects of C-LPE3 on intestinal microbial phylum levels (A), relative abundance of Firmicutes (B), relative abundance of Bacteroidetes (C), and ratio of Firmicutes / Bacteroidetes (D).

[0071] Figure 19 Effects of C-LPE3 on the genus level of intestinal flora in mice (A), relative abundance of g_norank_f_Muribaculaceae, g_Dubosiella, g_Bifidobacterium and g_Lachnospiraceae_NK4A136_group (BE).

[0072] Figure 20 The effect of C-LPE3 on the content of short-chain fatty acids in mouse feces.

[0073] Figure 21 Effects of C-LPE3 on metabolites (A) and metabolic pathways (B) in DSS-induced mouse feces. DETAILED DESCRIPTION

[0074] The present invention is further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto:

[0075] Example 1. Acquisition and identification of Lactobacillus plantarum strains:

[0076] Fermented kimchi purchased from a Northeast vegetable market was cultured according to the following method: 10.0 g of sample was mixed with 200 mL of sterile saline, and after beating with a beating homogenizer for 3 minutes, 200 μL of the homogenate was aspirated and applied to MRS primary screening solid medium. After incubation at 37°C for 48 hours, colonies that produced calcium dissolution circles and stringing phenomena were selected and streaked 3-4 times on MRS solid medium for strain isolation and purification. In addition, the purified strains were subjected to morphological observation, physiological and biochemical identification, and 16S rDNA identification. The results are shown in Table 1 and Figure 1 shown.

[0077] Table 1. Physiological and biochemical test results of strain ZGS521

[0078]

[0079] Note: + represents positive, - represents negative

[0080] The strain ZGS521 was observed to be milky white on the MRS plate, with smooth and neat edges and a diameter of about 1-2 mm. The surface of the colony was moist and smooth, with a uniform texture and was opaque. In addition, the colony was sticky and slightly raised ( Figure 1 A). Gram staining results showed that strain ZGS521 was purple rod-shaped, indicating that it was a Gram-positive bacterium ( Figure 1B). The results of physiological and biochemical identification showed that the esculin and sugar alcohol fermentation tests (cellobiose, maltose, mannose, glucose, sorbitol, lactose, galactose, sucrose and fructose) were all positive, while the gelatin liquefaction, starch hydrolysis, 1% sodium hippurate and H2O2 catalase tests were all negative. The results showed that strain ZGS521 may be Lactobacillus plantarum (Table 1). After the 16S rDNA gene sequence was determined, the BLAST tool on the NCBI website was first used to perform homology comparison in the nucleic acid sequence database to identify similar species. Then, MEGA7.0 was used to construct a phylogenetic tree based on the comparison results ( Figure 1 C) The results showed that strain ZGS521 and Lactobacillus plantarum AUSA004 had the highest homology, with a similarity of 99%. Combined with morphological observation and physiological and biochemical identification, strain ZGS521 was determined to be Lactobacillus plantarum. The strain was stored in 30% glycerol and placed in a -80°C refrigerator.

[0081] The deposit information of the strain of the present invention is as follows:

[0082] Deposit name: Lactobacillus plantarum ZGS521, deposit unit: China Center for Type Culture Collection, deposit address: Wuhan University, Wuhan, China, deposit number: CCTCC NO: M 20241181, deposit date: June 7, 2024.

[0083] Example 2. Evaluation of biological characteristics and potential probiotic properties of Lactobacillus plantarum ZGS521

[0084] 1. Determination of growth curve and extracellular sugar production curve:

[0085] The activated strain ZGS521 was inoculated into MRS liquid culture medium and modified fermentation medium (M-MRS), respectively. After static culture at 37°C for 24 h, it was re-inoculated into MRS and M-MRS liquid culture media at 1% (volume %), and the growth curve of strain ZGS521 was determined using a microbial growth curve analyzer.

[0086] At the same time, strain ZGS521 was inoculated into M-MRS at 1% and incubated at 37°C for a defined period (0-32 hours). The resulting culture fluid was used for EPS (exopolysaccharide) extraction, and EPS production was calculated for the strain at different time periods. The EPS (exopolysaccharide) extraction method for the culture fluid is detailed in Example 3 below.

[0087] Depend on Figure 2A shows that the growth process of strain ZGS521 can be divided into four stages: 0-8h is the lag phase, 8-28h is the logarithmic growth phase, 28-44h is the stationary phase, and after 44h it enters the decline phase. During the entire culture process, the growth trend of the strain in MRS and M-MRS medium is basically the same. Figure 2 B shows that the EPS production of the strain increased significantly with time, reaching a peak at 26 h (about 236.47 mg / L).

[0088] 2. Simulated gastrointestinal fluid tolerance test:

[0089] The preparation method of the bacterial suspension of strain ZGS521 / Lactobacillus rhamnosus GG is as follows: the strain cultured to the logarithmic phase is centrifuged at 5000g and 4℃ for 10min, the supernatant is removed and the strain is collected, physiological saline is added, the above centrifugation operation is repeated twice, the precipitate is collected, and the concentration of the bacterial suspension is adjusted to 10 with physiological saline. 8 CFU / mL.

[0090] Bacterial suspensions were placed in test tubes containing simulated gastric fluid (pH 2.0 and 3.0) and simulated intestinal fluid (bile salt concentrations of 0.3% and 0.5%) and incubated in an incubator at 37°C for 2 and 4 hours, respectively. After treatment, viable bacteria were determined by plate count and survival rates were calculated. Lactobacillus rhamnosus GG was used as a positive control. Lactobacillus rhamnosus GG was purchased from the American Type Culture Collection (ATCC 53103).

[0091] Survival rate (%) = (A / B) × 100 (1)

[0092] A and B represent the bacterial counts after treatment and the initial bacterial counts, respectively.

[0093] Depend on Figure 2C shows that the survival rate of ZGS521 increased with increasing pH value, but showed a downward trend over time. After 2 hours of treatment at pH 2.0, the survival rate of ZGS521 was 56.92% (LGG was 40.30%), but it dropped significantly to 45.93% after 4 hours of treatment (LGG was 29.61%, P < 0.05). In simulated intestinal fluid, when the bile salt concentration was 0.3% and 0.5%, the survival rate of ZGS521 decreased significantly with the extension of treatment time (P < 0.05). At the same time, the survival rate of ZGS521 gradually decreased with increasing bile salt concentration. However, after 4 hours of treatment with 0.5% bile salt, the survival rate of ZGS521 was still 53.90%, significantly higher than the 38.60% of LGG (P < 0.05). These results indicate that ZGS521 has good tolerance to gastrointestinal fluid and is superior to the commercial strain Lactobacillus rhamnosus GG.

[0094] 3. Determination of self-cohesion:

[0095] After the bacterial suspension is incubated at 37°C for a certain period of time (1 to 48 hours), the absorbance of the mixture at 600 nm is recorded. The self-aggregation capacity of the strain is calculated according to formula (2).

[0096]

[0097] A t : absorbance value of the solution after treatment; A0: absorbance value of the untreated solution.

[0098] Depend on Figure 2 The initial self-aggregation rate of ZGS521 was 13.46%, which significantly increased to 91.52% after 48 hours, approximately 18.91% higher than that of Lactobacillus rhamnosus GG (72.61%) (P < 0.05). This result suggests that ZGS521 exhibits greater persistence and stability in the gastrointestinal environment, potentially positively impacting its probiotic function.

[0099] 4. Determination of Caco-2 cell adhesion rate:

[0100] Adjust the Caco-2 cell suspension density to 2×10 5 After the cells reached 1×10 / mL, 12-well plates were plated (1 mL of Caco-2 cell suspension). After confluence for about 13 days, the supernatant was removed and 1 mL of Lactobacillus plantarum ZGS521 / Lactobacillus rhamnosus GG suspension (1×10 8 After incubation for 1 and 2 h, the Caco-2 cells were washed 2-3 times with PBS and the colonies were counted using an MRS plate. The adhesion rate of the strain to Caco-2 cells was calculated according to formula (3).

[0101]

[0102] N0: initial colony count; A0: colony count adhered to Caco-2 cells

[0103] Depend on Figure 2 E shows that the adhesion of Lactobacillus plantarum ZGS521 to Caco-2 cells is significantly higher than that of Lactobacillus rhamnosus GG.

[0104] Example 3: Extraction of Lactobacillus plantarum exopolysaccharides

[0105] 1. Prepare M-MRS medium;

[0106] Modified MRS fermentation medium (M-MRS) was prepared using a method reported in the literature. M-MRS was prepared as follows: To remove the interference of polysaccharides in the MRS medium, 10 g of peptone, 5 g of yeast extract, and 10 g of beef extract powder were dissolved in 100 mL of water. The mixture was then precipitated with 400 mL of ethanol at 4°C for 24 h. The supernatant was collected and dried by spin drying. The same amount of inorganic salts as in MRS (specifically, 0.2 g of magnesium sulfate heptahydrate, 0.05 g of manganese sulfate, 2 g of dipotassium hydrogen phosphate, 2 g of triammonium citrate, and 5 g of anhydrous sodium acetate), glucose (20 g), and Tween (1 mL) were added. 5 g of amino-free yeast nitrogen base (YNB) was also added, and the volume was adjusted to 1 L with water. Conventional high-temperature sterilization was performed (1.1 atmospheres, 115°C for 20 min).

[0107] The documents are: CHEN L, GU Q, ZHOU T. Statistical optimization of novel medium to maximize the yield of exopolysaccharide from Lacticaseibacillusrhamnosus ZFM216 and its immunomodulatory activity [J]. Frontiers in Nutrition, 2022, 9: 924495.

[0108] 2. Preparation of Lactobacillus plantarum fermentation broth

[0109] The strain was inoculated into a modified MRS fermentation medium (M-MRS) at 1%, and cultured at 37° C. for 24 hours to obtain a Lactobacillus plantarum fermentation liquid; the Lactobacillus plantarum fermentation liquid was used for extracting EPS.

[0110] 3. Extraction of Lactobacillus plantarum exopolysaccharides

[0111] Protein removal by TCA: Centrifuge the Lactobacillus plantarum fermentation broth at 12,000 g for 20 minutes at 4°C. Collect the supernatant and add TCA to a final concentration of 40 mg / mL (TCA stock solution concentration is 800 mg / mL). Incubate at 4°C for 12 hours. Centrifuge at 12,000 g for 20 minutes at 4°C and collect the supernatant.

[0112] Extraction of extracellular polysaccharides by water extraction and alcohol precipitation: The supernatant collected above was concentrated by rotary evaporation (evaporation temperature of 50°C) to about 25% of the original volume, and then 4 times the volume of ethanol of the concentrate was added. After standing at 4°C for 12 h, the precipitate was collected by centrifugation at 4°C and 12000g for 20 min. After drying the ethanol at 40°C, a small amount of deionized water (about 20 mL) was added to dissolve the precipitate to obtain a crude polysaccharide solution.

[0113] Dialysis: The crude polysaccharide solution was dialyzed with reflux water for 72 h (cut-off 3500 Da). After the dialysis, the solution in the dialysis bag was collected and concentrated by rotary evaporation (evaporation temperature of 50°C) to 25% of the original volume, and then freeze-dried (-60°C) for 24 h to obtain Lactobacillus plantarum exopolysaccharide LPE.

[0114] Note: The conditions of monosaccharide, glucose dosage and inoculum amount were optimized without CaCl2 stress, and the obtained polysaccharides were named extracellular polysaccharides LPE.

[0115] 4. Optimization of fermentation conditions for Lactobacillus plantarum exopolysaccharides

[0116] 4.1) Using the modified MRS fermentation medium (M-MRS) with a 1% inoculation amount, 254 mg of Lactobacillus plantarum exopolysaccharide LPE can be obtained from 1 L of fermentation liquid.

[0117] Optimize the following conditions:

[0118] 4.2) Replace 20g glucose in M-MRS with equal amounts of sucrose, maltose, lactose, galactose and fructose for fermentation. The rest of the steps are the same as above. The final results are as follows:

[0119] When sucrose is the carbon source, 216 mg of Lactobacillus plantarum exopolysaccharide LPE can be obtained from 1 L of fermentation broth.

[0120] When the carbon source is maltose, 1L of fermentation broth can ultimately produce 232mg of Lactobacillus plantarum exopolysaccharide LPE.

[0121] When the carbon source is lactose, 1L of fermentation broth can ultimately produce 201mg of Lactobacillus plantarum exopolysaccharide LPE.

[0122] When the carbon source is galactose, 1L of fermentation broth can ultimately produce 183mg of Lactobacillus plantarum exopolysaccharide LPE.

[0123] When the carbon source is fructose, 1L of fermentation broth can ultimately produce 198mg of Lactobacillus plantarum exopolysaccharide LPE.

[0124] 4.3) The amount of glucose in the M-MRS was changed for fermentation. The rest of the steps were the same as above. The final results were as follows:

[0125] When the glucose dosage is 10 g / L, 1 L of fermentation liquid can finally produce 182 mg of Lactobacillus plantarum exopolysaccharide LPE.

[0126] When the glucose dosage is 20 g / L, 1 L of fermentation liquid can finally produce 254 mg of Lactobacillus plantarum exopolysaccharide LPE.

[0127] When the glucose dosage is 30 g / L, 1 L of fermentation liquid can finally produce 286 mg of Lactobacillus plantarum exopolysaccharide LPE.

[0128] When the glucose dosage is 40 g / L, 1 L of fermentation liquid can finally produce 265 mg of Lactobacillus plantarum exopolysaccharide LPE.

[0129] When the glucose dosage is 50 g / L, 1 L of fermentation liquid can finally produce 217 mg of Lactobacillus plantarum exopolysaccharide LPE.

[0130] 4.4) Change the strain inoculum amount for strain fermentation. The rest of the steps are the same as above. The final results are as follows:

[0131] When the inoculation amount is 1%, 254 mg of Lactobacillus plantarum exopolysaccharide LPE can be obtained from 1 L of fermentation liquid.

[0132] When the inoculation amount is 2%, 294 mg of Lactobacillus plantarum exopolysaccharide LPE can be obtained from 1 L of fermentation liquid.

[0133] When the inoculation amount is 3%, 283 mg of Lactobacillus plantarum exopolysaccharide LPE can be obtained from 1 L of fermentation liquid.

[0134] When the inoculation amount is 4%, 265 mg of Lactobacillus plantarum exopolysaccharide LPE can be obtained from 1 L of fermentation liquid.

[0135] When the inoculation amount is 5%, 241 mg of Lactobacillus plantarum exopolysaccharide LPE can be obtained from 1 L of fermentation liquid.

[0136] 4.5) The best conditions are:

[0137] The culture temperature was 37°C, the inoculum size was 2%, and the glucose concentration was 30 g / L. The obtained LPE was approximately 451 mg / L.

[0138] 4.6) In the above fermentation conditions, M-MRS does not contain CaCl2. In order to further increase the EPS yield, different concentrations of CaCl2 were added to M-MRS for fermentation (the polysaccharide obtained after CaCl2 stress was named C-LPE). The rest of the steps were the same as above, that is, the inoculation amount was 1%, the culture temperature was 37°C, the culture was carried out for 24 hours, and the glucose dosage was 20 g / L. The final results are as follows:

[0139] When the final concentration of CaCl2 was 1 mM, 559 mg of Lactobacillus plantarum exopolysaccharide C-LPE could be obtained from 1 L of fermentation broth.

[0140] When the final concentration of CaCl2 was 2 mM, 608 mg of Lactobacillus plantarum exopolysaccharide C-LPE could be obtained from 1 L of fermentation broth.

[0141] When the final concentration of CaCl2 is 3 mM, 670 mg of Lactobacillus plantarum exopolysaccharide C-LPE can be obtained from 1 L of fermentation broth.

[0142] When the final concentration of CaCl2 was 4 mM, 665 mg of Lactobacillus plantarum exopolysaccharide C-LPE could be obtained from 1 L of fermentation broth.

[0143] When the final concentration of CaCl2 was 5 mM, 666 mg of Lactobacillus plantarum exopolysaccharide C-LPE could be obtained from 1 L of fermentation broth.

[0144] Example 4, Isolation and Purification of Lactobacillus plantarum C-LPE

[0145] When the final concentration of CaCl2 was 3 mM, the following experiment was performed on the corresponding Lactobacillus plantarum exopolysaccharide C-LPE:

[0146] 1. Separation of crude Lactobacillus plantarum polysaccharide C-LPE by DEAE-52 column chromatography

[0147] (1) Cellulose activation: Excess deionized water and DEAE-52 were mixed and allowed to stand at room temperature for 36 h. The cellulose was first treated with 0.5 M NaOH for 1 h, then washed with distilled water until the pH value was close to 7.0, then treated with 0.5 M HCl for 1 h, then washed with distilled water until the pH value was close to 7.0; then treated with 0.5 M NaOH for 1 h, then washed with distilled water until the pH value was close to 7.0, and the resultant was stored at 4°C as an activated filler for later use.

[0148] (2) Preparation of the chromatography column: Add an appropriate amount of deionized water to a vertical chromatography column and slowly add the activated filler obtained in step (1). After the filler settles, adjust the liquid level to be slightly above the filler surface and equilibrate at a flow rate of 1 mL / min for 24 h.

[0149] (3) Elution of C-LPE: NaCl solution gradient elution and sample collection were performed according to the method reported in the literature (WAN C, XU YY, CHEN L, et al. Anti-tumor and immunomodulatory activities of a novel polysaccharide from Grifola frondosa prepared by hydrogen peroxide / vitamin C-assisted extraction [J]. Journal of Food Measurement and Characterization, 2024, 18 (9): 7402-7417). Specifically, 200 mg of Lactobacillus plantarum exopolysaccharide C-LPE (the corresponding result when the final concentration of CaCl2 is 3 mM) was dissolved in 10 mL of deionized water to prepare a polysaccharide solution of appropriate concentration. The solution was centrifuged at 8000 g for 10 min and the supernatant was collected. The sample volume was 10 mL, and deionized water, 0.3 M and 0.5 M NaCl solutions were used for elution in sequence, with a volume of 1 L and a flow rate of 1 mL / min. The eluates obtained with the same eluent were combined and collected.

[0150] After the sugar solutions in the test tubes were combined, they were concentrated by rotary evaporation (50°C evaporation temperature to 25% of the original volume), dialyzed (cut-off 3500Da), and finally freeze-dried (-60°C drying for 48h) and weighed to obtain the isolated polysaccharide samples C-LPE-0, C-LPE-3 and C-LPE-5 for later use. Figure 3 A~D.

[0151] Figure 3 A is the DEAE elution curve of C-LPE. C-LPE was eluted with deionized water, 0.3M and 0.5M NaCl solutions. The elution curve ( Figure 3 BD) showed a single peak, indicating good purity. The yields of C-LPE-0, C-LPE-3, and C-LPE-5 were 23.72%, 35.29%, and 15.87%, respectively.

[0152] Yield (%) = (mass of each component / total amount of polysaccharide) × 100.

[0153] 2.Sephadex G-200 gel column purification

[0154] (1) Activation of Sephadex G-200: Add excess distilled water to Sephadex G-200, let it stand at room temperature for 36 hours to allow it to fully swell, and then transfer it to 4°C for storage until use.

[0155] (2) Construction of column chromatography system: Referring to the treatment method of DEAE-52 cellulose, the swollen G-200 was loaded into the chromatography column and equilibrated for 24 hours.

[0156] (3) Elution of C-LPEs: Gel chromatography elution and C-LPEs collection were performed according to the literature (WAN C, XU YY, CHEN L, et al. Anti-tumor and immunomodulatory activities of a novel polysaccharide from Grifola frondosa prepared by hydrogen peroxide / vitamin C-assisted extraction [J]. Journal of Food Measurement and Characterization, 2024, 18(9): 7402-7417.), specifically:

[0157] 200 mg of each EPS fraction (i.e., C-LPE-0, C-LPE-3, and C-LPE-5) separated by DEAE was dissolved in 10 mL of deionized water and centrifuged at 8000 g for 10 minutes. The supernatant was filtered through a 0.45 μm filter membrane. Elution was performed using deionized water at a flow rate of 1 mL / min, using a 1 L volume of deionized water (5 mL per tube). The collected sugar solution was concentrated, freeze-dried, and stored at -60°C.

[0158] After purification by G-200 gel, C-LPE-0, C-LPE-3, and C-LPE-5 were obtained as single fractions C-LPE0, C-LPE3, and C-LPE5, respectively, with recoveries of 84.15%, 89.15%, and 86.59%, respectively.

[0159] Recovery rate (%) = (recovered mass of the component / loaded mass of the component) × 100.

[0160] Depend on Figure 3 EG shows that C-LPEs (C-LPE0, C-LPE3 and C-LPE5) are all white, C-LPE0 has a higher density, while C-LPE3 and C-LPE5 are more fluffy.

[0161] Example 5. Chemical composition analysis of C-LPEs

[0162] 1. Determination of total sugar content

[0163] The total sugar content of C-LPEs was determined by the phenol-sulfuric acid method reported in the literature (Wang Fan. Isolation, purification and biological activity research of small molecule pectin from orange peel [D]; Zhejiang Gongshang University, 2021.).

[0164] 2. Determination of Protein Content

[0165] The protein content in C-LPEs was determined by the Bradford method (Zhu Yuzhu. Study on the extraction, purification, physicochemical properties and hypoglycemic activity of Panax notoginseng polysaccharide[D]; Zhejiang Gongshang University, 2023.).

[0166] 3. Determination of sulfate content

[0167] The sulfate content in C-LPEs was detected with reference to relevant literature (Wang Fan. Isolation, purification and biological activity research of orange peel small molecule pectin [D]; Zhejiang Gongshang University, 2021.).

[0168] 4. Determination of uronic acid content

[0169] The uronic acid content in C-LPEs was determined using the m-hydroxybiphenyl method (Zhou Jiamin. Purification of Lactobacillus rhamnosus exopolysaccharide and study on its lipid-lowering and intestinal flora-regulating activities [D]; Zhejiang Gongshang University, 2020.).

[0170] 5. Determination of Monosaccharide Composition

[0171] The monosaccharide composition of EPS was determined using the HPAEC-PAD method according to the method reported in reference (LIU W, ZHANG L, WEI X, et al. Structural characterization of aninulin neoseries-type fructan from Ophiopogonis Radix and the therapeutic effect on liver fibrosis in vivo [J]. Carbohydrate Polymers, 2024, 327: 121659).

[0172] The total sugar, protein, sulfate, uronic acid content of each purified fraction and the monosaccharide composition of each fraction are shown in Tables 2 and 3.

[0173] Table 2 Chemical composition of Lactobacillus plantarum exopolysaccharides and their purified components

[0174]

[0175] Table 3 Monosaccharide composition of Lactobacillus plantarum exopolysaccharide and its purified fractions

[0176]

[0177] Note: The LPE in the table is the LPE obtained under the above "4.5) optimal conditions".

[0178] Example 6: Determination of Molecular Weight of Lactobacillus Plantarum Exopolysaccharide C-LPE and Its Purified Fractions

[0179] High-performance gel filtration chromatography (HPGPC) was used to determine the molecular weights of C-LPE and its purified components, C-LPEs (C-LPE0, C-LPE3, and C-LPE5). C-LPEs were prepared at 1.00 mg / mL using ultrapure water and filtered through 0.22 μm. Dextran of different molecular weights was selected as a standard, and its relative molecular weight was calculated based on the retention time of EPS. Based on the molecular weight standard curve lgMw = -0.6232x + 9.5330, the molecular weights of LPE were calculated to be 962.30 and 20.04 kDa, corresponding to proportions of 45.38% and 54.62%, while the molecular weights of C-LPE were 933.74 kDa and 18.44 kDa, corresponding to proportions of 40.67% and 59.33%. The molecular weight of C-LPE0 is larger (899.72 kDa), while the molecular weights of C-LPE3 and C-LPE5 components are smaller, at 20.48 kDa and 18.34 kDa, respectively.

[0180] Efficacy tests and results of C-LPE and its purified components:

[0181] Experiment 1: Lactobacillus plantarum colitis mouse experiment

[0182] 1. Colitis Mouse Model

[0183] (1) Grouping of mice: The mice were randomly divided into 4 groups, with 10 mice in each group. Group I was the control group, which had free access to water throughout the whole process; Group II was the model group; and Group III was the Lactobacillus plantarum ZGS521 group (400 μL, 10 9 CFU / mL) and Lactobacillus rhamnosus GG group IV (400 μL, 10 9 CFU / mL), groups II, III and IV were given 3% DSS (w / v) solution instead of drinking water from the second week, and groups III and IV were given corresponding drugs by gavage once a day throughout the whole process.

[0184] The experimental time was 14 days. On the 14th day, the mice were killed and the colons were taken.

[0185] (2) Determination of clinical symptoms: During the 14 days of the experiment, the weight of the mice was measured daily using an electronic analytical balance to monitor changes in the weight of the mice. The fecal characteristics and occult blood levels of the mice were monitored daily, and the disease activity index (DAI) was evaluated using Table 4 according to the method described in the literature (WAN C, QIAN WW, LIU W, et al. Exopolysaccharide from Lactobacillus rhamnosus ZFM231 alleviates DSS-induced colitis in mice by regulating gut microbiota [J]. Journal of the Science of Food and Agriculture, 2022, 102 (15): 7087-7097.).

[0186] Table 4. DAI scoring criteria

[0187]

[0188]

[0189] The results are as follows Figure 4 shown.

[0190] Depend on Figure 4 It can be seen that compared with the DSS group, after ZGS521 intervention, the weight of mice increased significantly, the DAI score decreased significantly, the colon length increased significantly, and the colon mass / length ratio decreased significantly (P<0.01). These results indicate that ZGS521 can alleviate the clinical symptoms of DSS-induced colitis and relieve colitis, and its effect is basically equivalent to that of LGG.

[0191] (3) Colon histological examination: The mouse colon tissue was immersed in a fixative (4% paraformaldehyde) and fixed at 25°C for more than 24 hours. After dehydration (tissue dehydrator overnight), gradient ethanol dehydration, xylene transparentization, paraffin impregnation and embedding, 3-4 μm thick sections were continuously prepared using a paraffin slicer. The sections were transferred to a 42°C constant temperature water bath slide spreader for spreading. After the tissue folds were fully unfolded, anti-shedding slides were used to pick up the sections. The slides were placed in a 65°C constant temperature oven and baked for 2 hours. After the water was completely evaporated and the wax was moderately melted, they were transferred to a dark and dry environment for use.

[0192] (4) Hematoxylin & Eosin (H&E) staining: Soak the paraffin sections in xylene for 5-10 minutes, repeat twice, and then dehydrate the sections in 100%-70% ethanol in a gradient manner, with each level of dehydration for 3 minutes, and then rinse with deionized water for 3 minutes. Stain with hematoxylin for 10 minutes and rinse with deionized water for 5 minutes until the sections turn blue. Soak the sections in eosin stain for 2 minutes and rinse with running water for 5 minutes. After ethanol dehydration, clearing, drying and sealing, observe the H&E stained sections under a microscope and take pictures. Referring to the method reported in the literature (WAN C, QIAN WW, LIU W, et al. Exopolysaccharide from Lactobacillusrhamnosus ZFM231 alleviates DSS-induced colitis in mice by regulating gutmicrobiota[J]. Journal of the Science of Food and Agriculture, 2022, 102(15): 7087-7097.), the H&E sections were pathologically scored using Table 5.

[0193] Table 5 H&E scoring criteria

[0194]

[0195] The AB-PAS staining protocol was as follows: paraffin sections were dewaxed and hydrated, then soaked in distilled water for 5 minutes. The sections were then stained in Alcian blue for 30 minutes and rinsed in distilled water for 5 minutes. The sections were oxidized in 1% periodic acid solution for 10 minutes and rinsed in distilled water for 5 minutes. The sections were then stained in Scheffler's reagent for 30 minutes and rinsed in running water for 10 minutes. The sections were then soaked in 1% hydrochloric acid alcohol for several seconds and rinsed in running water for 10 minutes. AB-PAS-stained sections were then dehydrated with ethanol, cleared with xylene, and mounted. The sections were scanned and images were obtained using a digital slide scanner and 3DHISTECH software. The number and density of goblet cells were counted using Image-Pro Plus 6.0 software.

[0196] Muc-2 protein immunofluorescence analysis: First, paraffin sections were dewaxed and hydrated, followed by antigen retrieval using EDTA antigen retrieval solution (pH 8.0) and three 5-min washes with PBS. Tissue boundaries were marked with a histochemical pen and blocked with BSA. After discarding the blocking solution, the specific primary antibody (Muc-2 protein) was added. The sections were incubated at 4°C for 12-18 hours, then washed three times with PBS for 5 minutes each. The secondary antibody was added and incubated at room temperature for 50 minutes. The sections were then dried and stained with DAPI solution. The sections were incubated at room temperature in the dark for 10 minutes. After treatment with an autofluorescence quencher for 5 minutes, the sections were washed three times with PBS on a shaker for 5 minutes each. The sections were dried and mounted with anti-fluorescence mounting media. Fluorescence images were obtained by scanning the sections using a slide scanner.

[0197] The results are shown in Figure 5 .

[0198] from Figure 5 A shows that the colon tissue of the DSS-induced Model group mice showed obvious pathological changes: the integrity of the colon mucosal structure was destroyed, the epithelial cell layer was damaged, the crypt structure was significantly reduced, and a large number of goblet cells disappeared. At the same time, a large number of inflammatory cells were observed to infiltrate the submucosa. After treatment with ZGS521, the above symptoms were alleviated, indicating that ZGS521 can alleviate the colon tissue lesions of colitis mice ( Figure 5 D).

[0199] AB-PAS staining was performed to assess the integrity of the mucosal layer and to quantify the number of goblet cells ( Figure 5 B), compared with the control group, the DSS group showed a significant decrease in goblet cells and mucus secretion levels, but the number of goblet cells and mucus secretion function were significantly restored after ZGS521 intervention ( Figure 5 E).

[0200] Figure 5 G shows that ZGS521 significantly increased the expression level of Lgr5 compared with the DSS group. These findings suggest that ZGS521 may regulate intestinal epithelial cell regeneration by enhancing the proliferation of ISCs (intestinal stem cells).

[0201] Immunofluorescence analysis showed that DSS treatment reduced the secretion level of Muc-2 and disrupted the function of the intestinal barrier ( Figure 5 C), which is consistent with the results of AB-PAS staining. Compared with the DSS group, ZGS521 supplementation significantly enhanced the expression of Muc-2 in colon tissue ( Figure 5 F)(P<0.01), similar to the control group.

[0202] To further analyze the effect of ZGS521 on ISC differentiation, the expression levels of sucrase isomaltase (SI, a marker of intestinal epithelial cell differentiation) and chromogranin a (Chga, a marker of enteroendocrine cell differentiation) were measured. Figure 5 As shown by HI, the transcription levels of SI and Chga in the ZGS521 group were significantly increased, indicating that ZGS521 promoted the differentiation of ISCs into intestinal epithelial cells and enteroendocrine cells.

[0203] It should be noted that currently available Lactobacillus plantarum does not have the above-mentioned properties of regulating intestinal epithelial proliferation and differentiation.

[0204] (5) Determination of short-chain fatty acid content: The content of short-chain fatty acids in mouse feces was determined by gas chromatography. The sample pretreatment and analysis were based on the existing method (ZHU Y, WANG D, ZHOU S, et al. Hypoglycemic Effects of Gynura divaricata (L.) DC Polysaccharide and Action Mechanisms via Modulation of Gut Microbiota in Diabetic Mice [J]. Journal of Agricultural and Food Chemistry, 2024, 72(17): 9893-9905.). The fecal supernatant and crotonic acid mixture was filtered through a 0.22 μm filter membrane and loaded onto the gas chromatography machine. The chromatographic conditions and program temperature changes were based on existing research (DENG Z, PAN Y, CHEN W, et al. Effects of cultivar and growth region on the structural, emulsifying and rheological characteristics of mango peel pectin [J]. Food Hydrocolloids, 2020, 103: 105707.). Figure 6 .

[0205] Depend on Figure 6 It can be seen that compared with the Control group, the concentrations of total short-chain fatty acids, acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid and valeric acid in the DSS-treated group were significantly decreased. After treatment with ZGS521 and LGG, the levels of all short-chain fatty acids were alleviated, and the intervention effects of ZGS521 and LGG were similar.

[0206] (6) Intestinal microbial analysis: The total DNA of microorganisms in feces was extracted using a kit, and the extracted DNA samples were quantified and evaluated for purity using a NanoDrop2000 spectrophotometer, and the integrity of the DNA was detected by agarose gel electrophoresis (1%). Using DNA as a template, PCR technology was used to amplify 16SrRNA using primers 388F and 806R. The PCR products were then recovered, purified, quantified, and library constructed. Finally, the Illumina Nextseq2000 platform was used for sequencing. After the raw data were quality controlled and spliced ​​using fastp 0.19.6 and FLASH 1.2.11 software, UPARSE v7.1 software was used to perform OTU cluster analysis with a similarity threshold of greater than 97%. Data analysis was completed on the Major Bio Cloud Platform (https: / / cloud.majorbio.com). Results are shown in Figure 7 .

[0207] Depend on Figure 7 DSS treatment decreased the relative abundance of Lactobacillus, norank_f__Muribaculaceae, and Dubosiella in mouse feces, and increased the relative abundance of Lachnospiraceae_NK4A136_group, Desulfovibrio, and Escherichia-Shigella. This trend was reversed after oral administration of ZGS521. These results suggest that ZGS521 can improve the intestinal flora at the genus level in the feces of colitis mice, which may be beneficial for alleviating colitis symptoms.

[0208] Experiment 2: In vitro chemical antioxidant experiment

[0209] 1. DPPH scavenging ability determination: C-LPE, C-LPE0, C-LPE3 and C-LPE5 polysaccharide samples were prepared into aqueous solutions with concentration gradients (1, 2, 3, 4 and 5 mg / mL), ascorbic acid was used as a positive control, and the C-LPEs were mixed with the DPPH solution according to the sample addition scheme in Table 6. The mixture was incubated at 37°C in the dark for 25 minutes, and the absorbance value of the mixed system at 517 nm was measured. The DPPH free radical scavenging activity of the C-LPEs was calculated according to formula (4). The results are shown in Figure 8 A.

[0210] DPPH free radical scavenging rate (%) = [1-(A3-A2) / A1] × 100 (4)

[0211] Table 6 Reaction system for DPPH free radical scavenging activity experiment (volume in mL)

[0212] Group C-LPEs Anhydrous ethanol DPPH-ethanol solution Blank group A1 - 1 1 Control group A2 1 1 - Sample group A3 1 - 1

[0213] 2. ABTS free radical scavenging activity assay: C-LPEs aqueous solution was prepared according to the DPPH scavenging activity test, with Vc as a positive control. After adding the sample according to Table 7, the solution was allowed to stand at room temperature for 10 minutes. Then, the OD value of the solution at 734 nm was recorded, and the ABTS free radical scavenging activity of C-LPEs was calculated according to formula (5). The results are shown in Figure 8 B.

[0214] ABTS free radical scavenging rate (%) = (1-A2 / A1) × 100 (5)

[0215] Table 7 Sample addition scheme for ABTS free radical scavenging experiment

[0216] Group C-LPEs Deionized water ABTS solution Blank group A1 - 1 3 Sample group A2 1 - 3

[0217] 3. Determination of hydroxyl radical scavenging ability: The hydroxyl radical scavenging activity of C-LPEs was determined using the salicylic acid method. The C-LPEs solution was prepared according to the DPPH scavenging ability determination experiment, with Vc as a positive control. After mixing the samples according to Table 8, incubate at 37°C in the dark for 30 minutes, record the OD value of the mixed solution at 517nm, and calculate the scavenging rate of C-LPEs according to formula (6). The results are shown in Figure 8 C.

[0218] Hydroxyl radical scavenging rate (%) = [1-(A3-A2) / A1] × 100 (6)

[0219] Table 8 Reaction system of hydroxyl radical scavenging experiment

[0220] Group C-LPEs <![CDATA[H2O2]]> distilled water <![CDATA[FeSO4]]> Salicylic acid-ethanol solution Blank group A1 - 1 1 1 1 Control group A2 1 - 1 1 1 Sample group A3 1 1 1 1 1

[0221] 4. Determination of reducing power: C-LPEs solution was prepared according to the DPPH scavenging ability test. C-LPEs solution, PBS solution and potassium ferricyanide solution (1%, w / v) were mixed in a volume ratio of 1:1:1 and heated in a water bath at 50°C for 20 min. Then, an equal volume of TCA solution (10%, w / v) was mixed and vigorously shaken. 2 mL of supernatant was aspirated and 0.4 mL of FeCl3 solution (0.1%, w / v) was added. After vortexing and standing at room temperature for 15 min, the OD value of the mixed system at 700 nm was recorded. The results are shown in Table 1. Figure 8 D.

[0222] Depend on Figure 8 AC shows that with the increase of C-LPEs concentration, the scavenging effect of C-LPEs on the three free radicals gradually increased. The IC values ​​of C-LPE, C-LPE0, C-LPE3 and C-LPE5 for scavenging DPPH· 50The values ​​were: 4.44, 6.44, 2.49 and 3.73 mg / mL, and the IC50 values ​​for scavenging ABTS free radicals were: 2.81, 5.68, 1.31 and 1.68 mg / mL, respectively. The IC 50 The values ​​were 5.07, 7.02, 3.36 and 4.20 mg / mL respectively. In addition, the four C-LPEs also showed good Fe 3+ Reduction ability ( Figure 8 D). In general, the free radical scavenging activity of the four C-LPEs is in the order of C-LPE3 > C-LPE5 > C-LPE > C-LPE0.

[0223] Experiment 3: Evaluation of antioxidant activity in RAW264.7 cells induced by H2O2

[0224] (1) Effects of C-LPEs on RAW264.7 cell viability

[0225] Culture of RAW264.7 cells: When the cell confluency reaches about 80-90%, remove the cell supernatant, wash with PBS (1X) and soak for a few minutes, then remove the PBS and add DMEM medium (10% fetal bovine serum and 1% double antibody) preheated at 37°C in advance, then pipette evenly and culture in a constant temperature incubator (37°C, 5% CO2). Passage at a ratio of 1:2-3. Adjust the cell density in the logarithmic phase to 2×10 5 / mL, add 100μL to each well of a 96-well culture plate. When the cell density reaches 70-80%, discard the culture medium and add EPS solution (prepared with DMEM complete medium) with different concentration gradients (25-800μg / mL). After continuing to culture for 24 hours, the cell survival rate is determined according to the cytotoxicity detection kit (CCK-8 method). The results are shown in Figure 9 A.

[0226] Depend on Figure 9 As shown in Figure 1, the survival rate of macrophages treated with C-LPEs was greater than 80% within the 25-400 μg / mL dose range, indicating that it exhibited no cytotoxicity within this concentration range. Further analysis showed that cell survival rate was significantly positively correlated with EPS dose at a gradient of 25-200 μg / mL (P<0.05); however, when the dose was increased to 400-800 μg / mL, the survival rate showed a downward trend. Based on these findings, to further evaluate the antioxidant activity of C-LPEs, subsequent experiments were conducted using four concentrations within the 25-200 μg / mL concentration range.

[0227] (2) Establishment of H2O2-induced oxidative stress model in RAW264.7 cells

[0228] Different concentrations of H2O2 were used to induce macrophages to construct an oxidative stress model. When the cell density reached 80-90%, the culture medium was aspirated and H2O2 solutions with a concentration gradient of 0.5-1.0mM (0.5mM, 0.6mM, 0.7mM, 0.8mM, 0.9mM and 1.0mM) were added. After treatment in a cell culture incubator for 1 hour, the cell survival rate was determined using the CCK-8 method. The results are shown in Figure 9 B.

[0229] Previous studies have shown that when the cell survival rate after H2O2 treatment is between 50% and 70%, it can be considered that the oxidative stress state is successfully established. In this experiment, H2O2 concentrations ranging from 0.5mM to 1mM were used to stimulate cells. The increase in H2O2 concentration led to a significant decrease in the survival rate of RAW264.7 cells ( Figure 9 B) When the H2O2 concentration was 0.8 mM, the cell viability was between 50% and 70%, so 0.8 mM H2O2 concentration was selected as the experimental condition.

[0230] (3) Experimental groups

[0231] Normal group (normal cells), model group (0.8 mM H2O2 treatment for 1 hour), and sample groups (25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL). After 24 hours of C-LPEs treatment, the supernatant was removed from the cells, washed twice with PBS, and then treated with the same concentration of H2O2 for another 1 hour. Six replicates were performed for each sample and concentration.

[0232] (4) Effects of C-LPEs on ROS content, SOD, CAT enzyme activities, and MDA levels in RAW264.7 cells

[0233] Adjust the density of RAW264.7 cell suspension to 5×10 5After 24 h of C-LPEs and 1 h of 0.8 mM H2O2, 6-well plates were plated with 2 mL per well. After treatment, the macrophages were washed 3-4 times with pre-cooled PBS, and 100 μL of 10 μM DCFH-DA solution was added to each well and incubated at 37 ° C for 1 h. The probe solution was discarded and the cells were washed three times with PBS. Except for the control group, 100 μL of hydrogen peroxide solution (0.8 mM) was added to the other groups and incubated in the incubator for 1 h. After the culture was completed, the cells were washed once with PBS and 100 μL of PBS was added to each well. The fluorescence intensity of the solution was measured and the ROS content was calculated according to the kit instructions. In addition, after repeating the above steps of cell culture, C-LPEs treatment and PBS washing of RAW264.7 cells, cells were lysed with pre-cooled RIPA lysis buffer (containing 1% protease inhibitors, 400 μL / well) (ice bath for 30 minutes), and then centrifuged at 4°C and 12,000g for 20 minutes to collect soluble protein fractions. The protein concentration in the supernatant was determined by BCA method, and the SOD, CAT activity and MDA content were determined using kits. The results are shown in Figure 10 .

[0234] Depend on Figure 10 As can be seen from A, the ROS levels in the C-LPE, C-LPE0, C-LPE3 and C-LPE5 treatment groups decreased by 23.91%, 20.28%, 29.83% and 27.42% respectively compared with the Model group (P<0.05), and the effect order was C-LPE3>C-LPE5>C-LPE>C-LPE0. Figure 10 B shows that the treatment of C-LPEs can increase the intracellular SOD enzyme activity to varying degrees. Under the treatment of 200 μg / mL C-LPEs, compared with the Model group, C-LPE, C-LPE0, C-LPE3 and C-LPE5 increased the SOD enzyme activity by 125.33%, 72.60%, 147.15% and 117.44% respectively (P<0.05). Figure 10 C shows that after C-LPEs intervention, the CAT enzyme activity of each treatment group increased significantly (P<0.05). Under the treatment of 200μg / mL concentration of C-LPEs, the CAT enzyme activity of the C-LPE, C-LPE0, C-LPE3 and C-LPE5 groups increased to 3.63, 3.30, 4.13 and 3.84U / mgprot, respectively, among which the effect of C-LPE3 was the most significant (P<0.05). Figure 10As shown in Figure 3, under the intervention of C-LPEs at a concentration of 200 μg / mL, MDA levels in the C-LPE, C-LPE0, C-LPE3, and C-LPE5 treatment groups decreased by 54.70%, 43.37%, 59.28%, and 55.18% compared with the Model group, respectively (P < 0.05). These experimental data indicate that C-LPEs can significantly reduce the synthesis of cellular MDA under oxidative stress and maintain the homeostatic balance of the cellular redox system.

[0235] Experiment 4: Analysis of anti-inflammatory activity in RAW264.7 cells induced by LPS

[0236] (1) Experimental groups

[0237] Normal group (normal cells), model group (1 μg / mL LPS treatment for 24 hours), and sample groups (25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL). RAW264.7 cells treated with 1 μg / mL LPS for 24 hours were treated with supernatant removed, washed twice with PBS, and then treated with different concentrations of therapeutic drugs. Six replicates were plated for each sample and concentration for 24 hours.

[0238] (2) Effects of C-LPEs on cytokine secretion levels of LPS-stimulated macrophages

[0239] Dilute RAW264.7 cells to a density of 5 × 10 5 / mL, inoculated into 6-well culture plates (2mL per well), wait until the cell density reaches 70-80%, wash the cells twice with PBS, add 1μg / mL of conventional LPS solution to induce for 24h. Subsequently, discard the LPS solution, add 25-800μg / mL concentration gradient of C-LPEs solution, and continue to culture for 24h. After the treatment is completed, discard the culture medium, and after C-LPEs intervention, collect the cell supernatant, and refer to the kit instructions to detect the content of pro-inflammatory factors (IL-6, TNF-α, IL-1β) and anti-inflammatory factors (TGF-β1, IL-10) in the supernatant. The results are shown in Figure 11 .

[0240] Depend on Figure 11Compared with the Normal group, the Model group significantly increased the expression levels of IL-6, IL-1β, TNF-α, and IL-10 (P < 0.05), and significantly decreased the expression of TGF-β1 (P < 0.05). After intervention with C-LPEs, C-LPEs treatment significantly inhibited the production of proinflammatory cytokines (IL-6, IL-1β, and TNF-α) in RAW264.7 cells in a dose-dependent manner and significantly enhanced the expression of anti-inflammatory cytokines (IL-10 and TGF-β1), with C-LPE3 and C-LPE5 showing the best therapeutic effects. At 200 μg / mL, compared with the Model group, C-LPE5 treatment reduced the levels of IL-6, TNF-α, and IL-1β in RAW264.7 cells by 35.14%, 41.62%, and 35.14%, respectively, and increased the secretion of TGF-β1 and IL-10 by 78.02% and 93.41%. The data showed that C-LPE3 was superior to other C-LPEs in regulating cytokine expression. These results indicate that C-LPEs can regulate the secretion levels of relevant cytokines in RAW264.7 cells and improve the inflammatory response of RAW264.7 cells.

[0241] Experiment 5: Colitis mouse experiment with purified Lactobacillus plantarum exopolysaccharide fraction C-LPE3

[0242] (1) Grouping of mice: After acclimation for 7 days, the mice were randomly divided into six treatment groups (n=10). Group A was the control group, with free access to drinking water throughout the treatment period; Group B was the model group; Group C was the LC-LPE3 group (50 mg / kg / day, low dose); Group D was the MC-LPE3 group (100 mg / kg / day, medium dose); Group E was the HC-LPE3 group (200 mg / kg / day, high dose); and Group F was the mesalazine enteric-coated tablets group (Mesalazine, 100 mg / kg / day). Groups BF were given 3% DSS (w / v) solution instead of drinking water from the second week onwards, and Group CF was given the corresponding drugs by gavage throughout the treatment period.

[0243] The experimental time was 14 days. On the 14th day, the mice were killed and the colons were taken.

[0244] (2) Clinical symptoms (mouse weight change, DAI score, colon length, and colon mass / length ratio) were determined according to the methods described above. The results are shown in Figure 12 .

[0245] Experimental design such as Figure 12 As shown in A, mice in the C-LPE3 group were orally gavaged with C-LPE3 every day for 14 days, and were allowed to drink 3% DSS freely for 7 days to establish the model. Figure 12As shown in Figure B, the weight of mice in all groups showed an increasing trend. On day 14, the weight of mice in the Model group was significantly lower than that in the Control group (P<0.01), indicating that DSS had a negative impact on body weight. Compared with the Model group, the weight of mice in the C-LPE3 group increased significantly (P<0.01), indicating that C-LPE3 can alleviate the weight loss of mice caused by DSS modeling, among which HC-LPE3 had the best intervention effect. Figure 12 As shown in Figure C, compared with the Model group, the disease activity index score of mice in the C-LPE3 treatment group was significantly reduced (P<0.01), indicating that C-LPE3 can significantly alleviate the DSS-induced inflammatory response in mice, among which HC-LPE3 intervention had the most significant effect. Figure 12 DF showed that after treatment with C-LPE3, the colon length of mice in the LC-LPE3 group was significantly increased, and the colon mass / length ratio was significantly decreased compared with the model group (P<0.05). The therapeutic effect of the MC-LPE3 and HC-LPE3 groups was even better (P<0.01). C-LPE3 significantly reduced weight loss in colitis mice, inhibited the increase in DAI scores, and reduced the shortening of colon length, thereby effectively alleviating DSS-induced colitis symptoms. These results suggest that C-LPE3 may have potential therapeutic value in improving the symptoms of ulcerative colitis.

[0246] (3) Colon histological examination, hematoxylin & eosin (H&E) staining and AB-PAS staining were performed according to the methods described above. Figure 13 .

[0247] from Figure 13 A shows that the colon tissue of mice treated with C-LPE3 showed milder pathological changes, lower degree of mucosal damage, increased number of goblet cells and crypts, and significantly reduced inflammatory cell infiltration. The morphology was close to that of the Control group, and the histopathological score was significantly lower than that of the Model group ( Figure 13 B) The above results show that supplementation of C-LPE3 can improve the degree of colon tissue lesions in colitis mice. Figure 13 C shows that compared with the Control group, the colon tissue of the Model group showed a significant depletion of goblet cells, which was manifested by a significant decrease in cell number, decreased mucus secretion level and decreased mucosal layer thickness. In some areas, the mucosal layer was even lost, which is consistent with the pathological characteristics of colitis mice. After intervention with C-LPE3, the above pathological characteristics were significantly improved, and the number of goblet cells and mucus secretion function were significantly restored ( Figure 13 D) These results suggest that C-LPE3 treatment may help maintain intestinal homeostasis and effectively alleviate DSS-induced intestinal mucosal damage.

[0248] (4) Muc-2 protein immunofluorescence analysis refers to the previous research method. The operation method of Occludin and ZO-1 protein immunofluorescence analysis is to replace the primary antibody with Occludin and ZO-1, and the rest of the steps are similar. Figure 14 , Figure 15 .

[0249] like Figure 14 As shown in the results, the expression level of Muc-2 protein in the colon of mice in the Model group was significantly lower than that in the Control group, suggesting that DSS may weaken the protective effect of the intestinal barrier by reducing Muc-2 secretion. After C-LPE3 intervention, the expression level of Muc-2 protein was significantly increased. Moreover, the increase in Muc-2 protein expression level by C-LPE3 was positively correlated with the concentration of C-LPE3 treatment. High doses of C-LPE3 can more effectively protect the intestinal mucosa, reduce intestinal damage caused by DSS, and alleviate intestinal inflammation.

[0250] Depend on Figure 15 As shown in Figure AB, after intervention with different concentrations of C-LPE3, the expression levels of occludin and ZO-1 proteins increased significantly, and the increase was positively correlated with the concentration of C-LPE3. These results indicate that C-LPE3 improves intestinal barrier function and integrity by upregulating the levels of tight junction proteins. In summary, the therapeutic effect of C-LPE3 on colitis is not only manifested in its anti-inflammatory effects, but also in its protection of intestinal barrier function. Notably, high concentrations of C-LPE3 are more effective than low concentrations in improving the intestinal barrier.

[0251] (5) Determination of cytokine secretion level: Accurately weigh 50 mg of colon tissue in a 2 mL sterile centrifuge tube, add 0.5 mL of tissue lysis buffer, grind with a tissue grinder, centrifuge at 4 ° C, and collect the supernatant. Enzyme-linked immunosorbent assay (ELISA) was used to quantitatively analyze the secretion levels of various cytokines in colon tissue. The detection method was carried out according to the operating instructions of the corresponding ELISA kit. The results are shown in Figure 16 .

[0252] Depend on Figure 16 It can be seen that DSS treatment increased the secretion levels of pro-inflammatory factors IL-1β, IL-6, and TNF-α, and reduced the secretion expression of anti-inflammatory factors IL-10 and TGF-β1 ( Figure 16 AE). C-LPE3 intervention was able to significantly reverse this trend (P < 0.05). These results indicate that C-LPE3 intervention can regulate cytokine and gene expression levels to alleviate ulcerative colitis.

[0253] (6) Determination of oxidative stress-related enzymes: Colon tissue homogenate was prepared according to (5), and the total protein content in the supernatant was detected by BCA method. The content of SOD, CAT, GSH-Px and MDA in the supernatant was detected according to the kit instructions. Figure 17 .

[0254] Depend on Figure 17 It can be seen that the activities of SOD, CAT and GSH-Px in the intestinal mucosal tissue of mice in the Model group were significantly reduced compared with those in the Control group, and the MDA level was significantly increased ( Figure 17 AD). However, after C-LPE3 intervention, GSH-Px, CAT, and SOD enzyme activities increased significantly, and MDA concentration decreased significantly (P < 0.05). These results suggest that C-LPE3 can effectively inhibit excessive oxidative stress in the intestinal mucosa. High-dose C-LPE3 intervention was most effective. C-LPE3 may alleviate UC by reducing oxidative stress in colonic tissue.

[0255] (7) Intestinal microbial analysis and short-chain fatty acid content determination were carried out according to the methods described above. The results are shown in Figure 18 , Figure 19 , Figure 20 .

[0256] Effects of C-LPE3 on the levels of intestinal microorganisms in mice Figure 18 As shown in A, the relative abundances of Firmicutes in the Control, Model, LC-LPE3, MC-LPE3, HC-LPE3, and Mes groups were 45.69%, 74.00%, 62.95%, 60.65%, 41.23%, and 50.25%, respectively ( Figure 18 B). The relative abundances of Bacteroidetes were 39.30%, 7.54%, 27.67%, 32.56%, 51.98% and 29.04% ( Figure 18 C). Figure 18 As shown in D, the F / B value of mice in the Model group was significantly increased compared with that in the Control group (P<0.05), while C-LPE treatment reversed this trend.

[0257] Effects of C-LPE3 on the genus-level taxonomic distribution of intestinal microorganisms in mice Figure 19 As shown in A, Figure 19B shows that the relative abundance of g_norank_f_Muribaculaceae in the Model group (2.70%) was significantly lower than that in the Control group (33.84%) and the HC-LPE3 group (37.70%). A large number of studies have shown that Muribaculaceae is a common bacterium in the intestines of mice and other rodents, which can break down carbohydrates to produce prebiotics such as succinic acid, acetic acid and propionic acid. Studies have shown that Dubosiella can produce short-chain fatty acids (especially propionic acid) and lysine to regulate the balance of colonic Treg / Th17 immunity, thereby inhibiting colonic inflammatory response and improving mucosal barrier damage. Compared with the Control group (1.65%), the proportion of g_Dubosiella in the Model group was relatively low (0.10%). After C-LPE3 intervention, the proportion of g_Dubosiella in the LC-LPE3, MC-LPE3 and HC-LPE3 groups increased to 1.96%, 1.51% and 2.30%, respectively ( Figure 19 C) Short-chain fatty acids produced by Bifidobacterium can promote intestinal epithelial cell regeneration and strengthen intestinal mucosal barrier function. Figure 19 D shows that after C-LPE3 intervention, the relative abundance of g_Bifidobacterium increased significantly (P<0.05). Studies have reported that g_Lachnospiraceae_NK4A136_group appears to be more abundant in patients with colitis, further exacerbating the symptoms of colitis patients. This genus is positively correlated with the pathological characteristics of colitis. After C-LPE3 treatment, the relative abundance of g_Lachnospiraceae_NK4A136_group was significantly lower than that of the Model group ( Figure 19 Furthermore, Escherichia-Shigella causes intestinal inflammation and diarrhea through multiple mechanisms, including invasion of intestinal epithelial cells, production of toxins, and immune responses. After treatment, only the MC-LPE3 group showed a small number of Escherichia-Shigella, significantly lower than the Model group (P<0.05). In summary, C-LPE3 can improve colitis symptoms by modulating the composition of the intestinal microbiome.

[0258] from Figure 20It was found that DSS treatment significantly reduced the content of SCFAs, including total SCFAs, acetic acid, propionic acid, isobutyric acid, and butyric acid, in mouse feces (P<0.05). C-LPE3 intervention restored the levels of SCFAs. Among them, the concentrations of butyric acid and isovaleric acid in the HC-LPE3 group were higher than those in the Mes group. In addition, the SCFA-enhancing effect of C-LPE3 was positively correlated with the concentration of C-LPE3. These results indicate that supplementation with C-LPE3 can increase the concentration of SCFAs, which helps regulate intestinal immune responses, maintain the intestinal mucosal barrier, and reduce inflammatory responses.

[0259] (8) Non-targeted metabolomics analysis of feces: Weigh 50 mg of fecal sample into a 2 mL sterile centrifuge tube, add 2-3 grinding beads and 400 μL of extraction solution (methanol: water = 4:1), grind the tissue for 15 minutes, place the centrifuge tube on ice and ultrasonicate for 20 minutes, and then place the centrifuge tube in a -20°C refrigerator for 30 minutes. Finally, centrifuge the mixture at 12000g for 20 minutes at 4°C, and collect the supernatant for subsequent experiments. Metabolites were detected using LC-MS / MS. Data analysis was completed on the MajorBio cloud platform (https: / / cloud.majorbio.com). Results are shown in Figure 21 .

[0260] Depend on Figure 21 A shows that compared with the Model, six metabolites were significantly downregulated in the HC-LPE3 group, including 3-hydroxybenzoic acid (3-Hydroxybenzoic Acid), lysophosphatidylcholine (14:1(9Z) / 0:0) (LysoPC(14:1(9Z) / 0:0)), phosphatidylcholine (34:2) (PC(34:2)), phosphatidylethanolamine (36:2) (PE(36:2)), glycerophosphatidylcholine (20:4 / 16:0) (GPCho(20:4 / 16:0)) and phosphatidylserine (16:0 / 15:0) (PS(16:0 / 15:0)). 24 metabolites were significantly upregulated, including lysophosphatidylcholine (P-18:1(9Z) / 0:0)(LysoPC(P-18:1(9Z) / 0:0)), lysophosphatidylcholine (18:3(6Z,9Z,12Z) / 0:0)(LysoPC(18:3(6Z,9Z,12Z) / 0:0)), and lysophosphatidylcholine (15:0 / 0:0)(LySOPC(15:0 / 0:0)). Figure 21As shown in Figure 2, the pathway with the greatest difference between the HC-LPE3 group and the Model group was arginine biosynthesis, followed by arginine and proline metabolism, alanine, aspartate, and glutamate metabolism, glycerolipid metabolism, and glycerophospholipid metabolism. The most significantly affected pathway was arginine biosynthesis, and arginine was also found among the differentially expressed metabolites. These results suggest that C-LPE3 may alleviate colitis by increasing arginine levels.

[0261] Finally, it should be noted that the above examples are merely specific embodiments of the present invention. Obviously, the present invention is not limited to the above examples and is subject to numerous variations. All variations that can be directly derived or conceived by a person of ordinary skill in the art from the disclosure of the present invention are considered to be within the scope of protection of the present invention.

Claims

1. Lactobacillus plantarum ZGS521, characterized in that: The accession number is: CCTCCNO:M 20241181.

2. A Lactobacillus plantarum exopolysaccharide, characterized in that: The method is obtained by fermenting the Lactobacillus plantarum ZGS521 as claimed in claim 1.

3. the preparation method of plant lactobacillus exopolysaccharide as claimed in claim 2, is characterized in that The following steps are involved: The Lactobacillus plantarum ZGS521 was inoculated into an M-MRS liquid culture medium or an M-MRS liquid culture medium containing CaCl2 at an inoculum rate of 1% to 5%, and the culture was placed in a constant temperature incubator at 37±0.5°C for 12 to 32 hours. After the culture was completed, the supernatant was collected by centrifugation, the protein was removed, and the crude polysaccharide was obtained after alcohol precipitation and post-processing. When M-MRS liquid medium was used, the crude polysaccharide obtained was named LPE; When M-MRS liquid medium containing CaCl2 was used, the crude polysaccharide obtained was named C-LPE.

4. The method for preparing plant lactobacillus exopolysaccharide according to claim 3, wherein: M-MRS liquid medium is as follows: dissolve 10 g of peptone, 5 g of yeast extract, and 10 g of beef extract powder in 100-150 mL of water, add 400-600 mL of ethanol for precipitation, collect the supernatant, spin-dry, add 0.2 g of magnesium sulfate heptahydrate, 0.05 g of manganese sulfate, 2 g of dipotassium hydrogen phosphate, 2 g of triammonium citrate, 5 g of anhydrous sodium acetate, 20-30 g of glucose, and 1 mL of Tween, add 5 g of amino-free yeast nitrogen source, and dilute to 1 L with water; The M-MRS liquid culture medium containing CaCl2 is prepared by adding CaCl2 to the M-MRS liquid culture medium to a final concentration of 1 to 5 mM.

5. The method for preparing plant lactobacillus exopolysaccharide according to claim 4, wherein: The crude polysaccharide C-LPE was separated and purified by cellulose DEAE-52 column chromatography to obtain polysaccharide components C-LPE-0, C-LPE-3, and C-LPE-5, respectively.

6. The method for preparing plant lactobacillus exopolysaccharide according to claim 5, wherein: The polysaccharide components C-LPE-0, C-LPE-3, and C-LPE-5 were purified using Sephadex G-200 gel columns, respectively, to obtain the corresponding purified polysaccharide components C-LPE0, C-LPE3, and C-LPE5, respectively.

7. The use of Lactobacillus plantarum exopolysaccharide and its components, characterized in that: Preparation of drugs for regulating intestinal flora and preparation of drugs for relieving ulcerative colitis.

8. The method according to claim 7, wherein: Lactobacillus plantarum exopolysaccharides and their components include LPE, C-LPE, C-LPE-0, C-LPE-3, C-LPE-5, C-LPE0, C-LPE3, and C-LPE5.

9. The method according to claim 7 or 8, characterized in that: Lactobacillus plantarum exopolysaccharides and their components have antioxidant and anti-inflammatory activities.

10. The method according to claim 9, wherein: Regulates intestinal epithelial proliferation and differentiation and increases arginine content.

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