Salivaria lactobacillus and application of exopolysaccharide produced by the same in relieving colitis

Abstract

The application discloses a strain of salivary lactobacillus and application of exopolysaccharide produced by the strain in relieving colitis, and belongs to the technical field of microorganisms and the technical field of medicines. The salivary lactobacillus (Ligilactobacillus salivarius) CCFM1334 provided by the application can efficiently produce exopolysaccharide, and the yield is 185.6 mg / L. After the exopolysaccharide of the salivary lactobacillus (Ligilactobacillus salivarius) CCFM1334 provided by the application is used on colitis mice, the colitis symptoms of the mice can be significantly relieved, which is specifically reflected in the following aspects: the disease activity score index is significantly reduced, the weight loss is relieved, the colonic histopathology is relieved, the levels of proinflammatory factors TNF-alpha, IL-1beta, IL-6, INF-gamma and IL-17 are reduced, the level of anti-inflammatory factor IL-10 is increased, and the mRNA expression amounts of tight junction proteins ZO-1 and occludin are increased. Therefore, the exopolysaccharide of the salivary lactobacillus (Ligilactobacillus salivarius) CCFM1334 has great application prospect in the preparation of products for preventing and / or treating colitis.

Description

TECHNICAL FIELD

[0001] The application discloses a lactobacillus salivarius and application of exopolysaccharide produced by the lactobacillus salivarius in relieving colitis, and belongs to the technical field of microorganisms and the technical field of medicines. BACKGROUND

[0002] Inflammatory bowel disease (IBD) is generally characterized by tissue damage and intestinal inflammation, and mainly includes ulcerative colitis (UC) and Crohn's disease (CD). The number of IBD cases is increasing globally, which brings health and economic burdens to countries around the world and greatly damages the quality of life of patients, especially children and young patients with increasing incidence of IBD. UC is characterized by diffuse and continuous lesions, and inflammation usually affects the mucosal layer in the colon. Currently available IBD drugs include aminosalicylic acid, immunosuppressants, antibiotics, corticosteroids and new biological agents targeting TNF-α. However, it is reported that most drug treatments have a series of adverse reactions. For example, 5-aminosalicylic acid (5-ASA) is considered to be a commonly used drug for mild and moderate UC, but in some cases it cannot achieve the expected therapeutic effect and can cause side effects such as headache and bloating. New biological agents aim to reduce these side effects, but also have limitations, such as poor efficacy for some patients and high cost. Therefore, it is urgent to develop new alternative strategies to prevent and treat UC.

[0003] Lactobacillus exopolysaccharides (EPSs) are macromolecular saccharides produced by lactobacillus during growth and secreted outside the cell wall, and finally exuded into the culture medium. According to the location of lactic acid bacteria exopolysaccharides in the cell, it is generally divided into two types: one is attached to the microbial cell wall to form a capsule, called capsular polysaccharide, and the other is into the culture medium to form mucus, called mucus polysaccharide. In recent years, the research of lactobacillus exopolysaccharides has attracted widespread attention, and due to the structural diversity of exopolysaccharides, it has good anti-tumor, antioxidant and anti-diabetic activities in physiological functions.

[0004] In the prior art, patent CN104099263B discloses a Lactobacillus salivarius strain producing EPSs, and discloses the application of the EPSs produced by the strain in anti-tumor. However, the yield of EPSs is low, only 23.54 mg / L. In addition, the prior art discloses the role of EPSs from other Lactobacillus in anti-inflammatory, for example, the document (Exopolysaccharide from Lactobacillus rhamnosus ZFM231 alleviates DSS-induced colitis in mice by regulating gut microbiota) discloses that the EPSs from Lactobacillus rhamnosus ZFM231 can alleviate colitis in mice by regulating gut microbiota, transforming TGF-β and TNF-α. The document (Alleviative Effects of Exopolysaccharide Produced by Lactobacillus helveticus KLDS1.8701 on Dextran Sulfate Sodium-Induced Colitis in Mice) discloses that the EPSs derived from Lactobacillus helveticus KLDS1.8701 can treat colitis by reducing intestinal inflammation, improving mucosal barrier function and regulating the composition of intestinal microbiota. The document (Isolation and characterization of a high molecular mass β-glucan from Lactobacillus fermentum Lf2 and evaluation of its immunomodulatory activity) discloses that the high molecular weight β-glucan from Lactobacillus fermentum Lf2 can provide immune tolerance for LPS-treated peripheral blood mononuclear cells (PBMC) by stimulating very low levels of TNF-α. The document (Exopolysaccharides from Lactobacillus plantarum NCU116 Facilitate Intestinal Homeostasis by Modulating Intestinal Epithelial Regeneration and Microbiota) discloses that the EPSs derived from Lactobacillus plantarum NCU116 can alleviate colitis by promoting intestinal epithelial regeneration and gut microbiota.Although the prior art discloses that EPS produced by Lactobacillus can reduce colitis in mice, as described above, there are differences in the anti-inflammatory effects of EPS produced by different inter-species Lactobacillus, and there is no related research on EPSs of Lactobacillus salivarius in the prior art. SUMMARY

[0005] [Technical problem]

[0006] The technical problem to be solved by the present application is that the ability of Lactobacillus salivarius to produce exopolysaccharide is low in the prior art, and there is no research on exopolysaccharide of Lactobacillus salivarius in relieving colitis.

[0007] [Technical solution]

[0008] To solve the above technical problems, the present application provides a Lactobacillus salivarius CCFM1334 which can efficiently produce exopolysaccharide. In addition, the present application also studies the exopolysaccharide of Lactobacillus salivarius CCFM1334 in relieving colitis.

[0009] The first object of the present application is to provide a Lactobacillus salivarius CCFM1334 which is deposited in the Guangdong Microbial Culture Collection Center, and the deposit number is GDMCC No: 63790.

[0010] In an embodiment of the present application, the Lactobacillus salivarius CCFM1334 is isolated from human feces.

[0011] In an embodiment of the present application, the growth characteristics of the Lactobacillus salivarius CCFM1334 are as follows: the strain is a facultative anaerobe, is inoculated into a culture medium, and is cultured at a culture temperature of 30-40℃ for 12-36h.

[0012] In an embodiment of the present application, the culture medium is MRS culture medium.

[0013] In an embodiment of the present application, the MRS medium is formulated as follows: peptone 5-20 g / L, beef extract 5-20 g / L, yeast extract 1-10 g / L, glucose 10-30 g / L, sodium acetate 1-5 g / L, magnesium sulfate (MgSO4·7H2O) 0.1-2 g / L, manganese sulfate (MnSO4·H2O) 0.1-1 g / L, diammonium hydrogen citrate 1-4 g / L, dipotassium hydrogen phosphate (K2HPO4·3H2O) 1-5 g / L, and Tween 80 0.5-2 mL / L; preferably, peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, glucose 20 g / L, sodium acetate 2 g / L, magnesium sulfate (MgSO4·7H2O) 0.5 g / L, manganese sulfate (MnSO4·H2O) 0.25 g / L, diammonium hydrogen citrate 2 g / L, dipotassium hydrogen phosphate (K2HPO4·3H2O) 2.6 g / L, and Tween 80 1 mL / L.

[0014] In an embodiment of the present application, the culture temperature is 37°C.

[0015] In an embodiment of the present application, the culture time is 24 h.

[0016] In an embodiment of the present application, the colony characteristics of the Ligilactobacillus salivarius CCFM1334 are as follows: white, round and convex, with smooth edges on MRS solid medium.

[0017] A second object of the present application is to provide a method for producing exopolysaccharide, which comprises inoculating the above-mentioned Ligilactobacillus salivarius into a fermentation medium, carrying out fermentation, and collecting the supernatant.

[0018] In an embodiment of the present application, the fermentation medium comprises MRS medium.

[0019] In an embodiment of the present application, the culture temperature for fermentation is 30-40°C, preferably 37°C.

[0020] In an embodiment of the present application, the culture time for fermentation is 12-36 h, preferably 24 h.

[0021] In an embodiment of the present application, the inoculation amount of the Ligilactobacillus salivarius is 2%-8% (v / v), preferably 4% (v / v).

[0022] In an embodiment of the present application, after the supernatant is collected, the supernatant is further purified to obtain exopolysaccharide, and the purification steps are as follows:

[0023] (1) removing proteins in the supernatant;

[0024] (2) precipitating polysaccharides from the supernatant obtained in step (1);

[0025] (3) dialyzing the polysaccharides obtained in step (2) to obtain the exopolysaccharide.

[0026] In one embodiment of the present application, trichloroacetic acid is used for removing proteins in step (1).

[0027] In one embodiment of the present application, anhydrous ethanol is used for precipitating polysaccharides in step (2).

[0028] In one embodiment of the present application, a dialysis bag with a molecular weight cut-off of 1000 Da is used for dialysis in step (3).

[0029] A third object of the present application is to provide a Lactobacillus salivarius exopolysaccharide obtained by the method for producing an exopolysaccharide as described above.

[0030] In one embodiment of the present application, the monosaccharide composition of the Lactobacillus salivarius exopolysaccharide is fucose: galactosamine: rhamnose: arabinose: glucosamine: galactose: glucose: xylose: mannose: fructose, with a molar ratio of (0.05-0.15):(1.2-1.8):(0.1-0.5):(0.5-1.50):(0.2-0.8):(0.5-1.5):(3-5):(0.1-0.3):(0.1-0.5):(1-2), preferably fucose: galactosamine: rhamnose: arabinose: glucosamine: galactose: glucose: xylose: mannose: fructose, with a molar ratio of 0.10:1.58:0.38:1.00:0.66:1.17:4.09:0.16:0.25:1.41.

[0031] A fourth object of the present application is to provide a pharmaceutical composition containing the Lactobacillus salivarius exopolysaccharide as described above.

[0032] The present application also provides use of the Lactobacillus salivarius or the exopolysaccharide as described above in the preparation of a product for preventing and / or treating colitis.

[0033] In one embodiment of the present application, the prevention and / or treatment of colitis includes at least one aspect of the following (a)-(f):

[0034] (a) reducing the intestinal disease activity index of a colitis subject;

[0035] (b) reducing the expression level of proinflammatory factors in the colonic tissue of a colitis subject, the proinflammatory factors including at least one of TNF-α, IL-1β, IL-6, IFN-γ or IL-17;

[0036] (c) increasing the expression level of anti-inflammatory factors in the colon tissue of the colitis subject, the anti-inflammatory factors including IL-10;

[0037] (d) increasing the expression level of tight junction proteins ZO-1 and / or occludin in the colon tissue of the colitis subject;

[0038] (e) reducing the body weight loss of the colitis subject;

[0039] (f) reducing the inflammatory cell infiltration in the colon tissue of the colitis subject, and / or alleviating the crypt loss, and / or alleviating the goblet cell loss.

[0040] Beneficial effects

[0041] Firstly, the strain Ligilactobacillus salivarius CCFM1334 provided by the present application can efficiently produce extracellular polysaccharide, and the yield reaches 185.6 mg / L.

[0042] Secondly, the present application provides a Ligilactobacillus salivarius CCFM1334 extracellular polysaccharide which can alleviate colitis. The Ligilactobacillus salivarius CCFM1334 extracellular polysaccharide provided by the present application can significantly alleviate the occurrence and development of DSS-induced colitis in mice, which is embodied in the following aspects compared with the model group and the blank group:

[0043] (1) The disease activity index score of the colitis mice is reduced from 8.63±1.06 to 4.71±0.49;

[0044] (2) The body weight loss of the colitis mice is reduced, and is increased from 80.34±3.17% to 89.38±2.69% on the last day of intervention;

[0045] (3) The colon length of the colitis mice is increased from 5.2±0.48 cm to 6.42±0.29 cm, and there is no significant difference compared with the colon length of the blank group mice;

[0046] (4) The inflammatory cell infiltration in the colon tissue of the colitis mice is significantly reduced, the crypt loss is alleviated, and the goblet cell loss is alleviated;

[0047] (5) The concentration of inflammatory factor TNF-α in the colon of the colitis mice is reduced from 98.77±8.51 pg / mL to 57.57±12.29 pg / mL;

[0048] (6) The concentration of inflammatory factor IL-1β in the colon of colitis mice was reduced from 1460.06 ± 101.94 pg / mL to 386.25 ± 67.84 pg / mL;

[0049] (7) The concentration of inflammatory factor IL-6 in the colon of colitis mice was reduced from 300.59 ± 60.11 pg / mL to 40.69 ± 16.15 pg / mL, and there was no significant difference with the concentration of inflammatory factor IL-6 in the blank group mice;

[0050] (8) The concentration of inflammatory factor IFN-γ in the colon of colitis mice was reduced from 66.13 ± 6.46 pg / mL to 30.18 ± 4.29 pg / mL, and there was no significant difference with the concentration of inflammatory factor IFN-γ in the blank group mice;

[0051] (9) The concentration of inflammatory factor IL-17 in the colon of colitis mice was reduced from 122.71 ± 35.9 pg / mL to 47.77 ± 7.98 pg / mL, and there was no significant difference with the concentration of inflammatory factor IL-17 in the blank group mice;

[0052] (10) The concentration of inflammatory factor IL-10 in the colon of colitis mice was increased from 87.68 ± 10.02 pg / mL to 117.81 ± 9.41 pg / mL, and there was no significant difference with the concentration of inflammatory factor IL-10 in the blank group mice;

[0053] (11) The mRNA expression of ZO-1 in the colon of colitis mice was increased from 0.46 to 1.08, and there was no significant difference with the mRNA expression of ZO-1 in the blank group mice; the mRNA expression of occludin in the colon of colitis mice was increased from 0.41 to 0.87, and there was no significant difference with the mRNA expression of occludin in the blank group mice;

[0054] Therefore, the exopolysaccharide of Ligilactobacillus salivarius CCFM1334 has great application prospects in the preparation of products for preventing and / or treating colitis.

[0055] Biological material preservation

[0056] Ligilactobacillus salivarius CCFM1334, taxonomically named as Ligilactobacillus salivarius, was preserved in Guangdong Microbial Culture Collection Center on September 12, 2023, with the preservation number GDMCC NO: 63790 and the preservation address being No. 59 Building, 5th Floor, Institute of Microbiology, Guangdong Academy of Sciences, 100 Middle Martyrs Road, Guangzhou, China. BRIEF DESCRIPTION OF DRAWINGS

[0057] Figure 1 : Flow chart of animal experiment.

[0058] Figure 2 : Molecular weight and monosaccharide composition of exopolysaccharide of Ligilactobacillus salivarius CCFM1334.

[0059] Figure 3 : Disease activity index score of experimental mice in different groups.

[0060] Figure 4 : Body weight of experimental mice in different groups on the last day.

[0061] Figure 5 : Colon length of experimental mice in different groups.

[0062] Figure 6 : H&E staining results of colon tissue of experimental mice in different groups.

[0063] Figure 7 : TNF-α level of colon tissue of experimental mice in different groups.

[0064] Figure 8 : IL-1β level of colon tissue of experimental mice in different groups.

[0065] Figure 9 : IL-6 level of colon tissue of experimental mice in different groups.

[0066] Figure 10 : IFN-γ level of colon tissue of experimental mice in different groups.

[0067] Figure 11 : IL-17 level of colon tissue of experimental mice in different groups.

[0068] Figure 12 : IL-10 level of colon tissue of experimental mice in different groups.

[0069] Figure 13 : mRNA expression of ZO-1 and occludin in colon tissue of experimental mice in different groups.

[0070] In the above pictures: *, **, ***, **** represent p value less than 0.05, 0.01, 0.001, 0.0001 compared with the blank group; #, ##, ###, #### represent p value less than 0.05, 0.01, 0.001, 0.0001 compared with the model group; ns represents no significant difference compared with the blank group; NS represents no significant difference compared with the model group. DETAILED DESCRIPTION

[0071] The C57BL / 6 male mice involved in the following examples were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. TNF-α, IL-1β, IL-6, IFN-γ, IL-17, IL-10 Elisa detection kit was purchased from RD.

[0072] Intestinal disease score index definition and scoring method: intestinal disease activity index combined with the percentage of body weight loss of patients (sick animals) (body weight unchanged is 0, 1-5 is 1 point, 5-10 is 2 points, 10-15 is 3 points, more than 15 is 4 points), stool consistency (normal is 0, loose stool is 2 points, diarrhea is 4 points) and stool bleeding (normal 0, occult blood positive is 2 points, overt bleeding is 4 points) three cases are comprehensively scored, and the total score of the three results is the DAI value.

[0073] The culture medium involved in the following examples is as follows:

[0074] MRS liquid medium ( / L): 10g of proteose peptone, 10g of beef extract, 5g of yeast powder, 20g of anhydrous glucose, 2g of anhydrous sodium acetate, 0.5g of magnesium sulfate (MgSO4·7H2O), 0.25g of manganese sulfate (MnSO4·H2O), 2g of diammonium hydrogen citrate, 2.6g of potassium hydrogen phosphate (K2HPO4·3H2O), 1mL of Tween 80, pH value is 6.2-6.4.

[0075] MRS solid medium ( / L): 10g of proteose peptone, 10g of beef extract, 5g of yeast powder, 20g of anhydrous glucose, 2g of anhydrous sodium acetate, 0.5g of magnesium sulfate (MgSO4·7H2O), 0.25g of manganese sulfate (MnSO4·H2O), 2g of diammonium hydrogen citrate, 2.6g of potassium hydrogen phosphate (K2HPO4·3H2O), 1mL of Tween 80, 15g of agar, pH value is 6.2-6.4.

[0076] Example 1: Screening and identification of Ligilactobacillus salivarius CCFM1334

[0077] (1) Screening of Ligilactobacillus salivarius CCFM1334:

[0078] Take a spoonful of fecal sample and add it to 5 mL of PBS (with 0.05% cysteine) and mix well for gradient dilution. Select 10 -5 ~10 -7 Gradient dilutions are coated on the above-mentioned MRS solid medium and incubated at 37°C for 48 hours. Typical colonies are picked and inoculated on MRS solid medium for streak purification and incubated at 37°C in an inverted incubator for 48 hours. Several single colonies are inoculated in 5 mL of MRS liquid medium and incubated at 37°C for 16-18 hours. 1.5 mL of bacterial solution is centrifuged at 6000 r / min for 3 minutes to remove the supernatant, and 1 mL of 30% sterile glycerol is added for preservation. At the same time, 1.5 mL of bacterial solution is centrifuged, the supernatant is removed, and the bacterial solution is resuspended with sterile water for strain identification.

[0079] (2) Strain species identification

[0080] The resuspended bacterial solution is extracted according to the FastDNA SPIN Kit for Feces instructions to obtain genomic DNA, which is used as a PCR template. The genomic DNA is amplified by 16S rDNA according to the system described in Table 1 below.

[0081] Table 1 16S rDNA amplification system

[0082]

[0083] Sequencing and analysis: After the PCR product is confirmed by nucleic acid electrophoresis analysis, it is sent to a sequencing company for sequencing. The obtained spliced sequence is subjected to species confirmation by BLAST (Basic Local Alignment Search Tool) of NCBI (National Center for Biotechnology Information). According to the results, the purified strains are named as Ligilactobacillus salivarius CCFM1334, Limosilactobacillus fermentum FSH6_5, and Lacticaseibacillus rhamnosus FXJSW24L2, respectively. https: / / blast.ncbi.nlm.nih.gov / Blast.cgi

[0084] Example 2: Culture of Ligilactobacillus salivarius CCFM1334 and isolation of its exopolysaccharide

[0085] The specific steps are as follows:

[0086] ​Ligilactobacillus salivarius CCFM1334, Limosilactobacillus fermentum FSH6_5, Lacticaseibacillus rhamnosus FXJSW24L2 were inoculated into MRS liquid medium and cultured at 37℃ for 24h, then the bacterial liquid was transferred into fresh MRS liquid medium at an inoculation amount of 4%(v / v), and cultured under the same conditions for 24h.

[0087] After the culture was completed, the bacterial bodies were removed by centrifugation at 8000r / min for 20min, and the fermentation supernatant was obtained. 4%(m / v) trichloroacetic acid was added to the supernatant, and after 24h reaction at 4℃, the supernatant was obtained by centrifugation at 10000r / min for 20min to remove the protein. Then 2.5 times of anhydrous ethanol was added to the supernatant, and the ethanol precipitation was carried out for 24h. The ethanol-precipitated exopolysaccharide was dialyzed using a 1000Da dialysis bag, and the water was changed every 6h for 3 days. After dialysis, the liquid in the dialysis bag was collected, and the dried exopolysaccharide was obtained by freeze-drying for 3 days.

[0088] The molecular weight of CCFM1334 exopolysaccharide, FSH6_5 exopolysaccharide and FXJSW24L2 exopolysaccharide was analyzed by HPSEC, and the monosaccharide composition of CCFM1334 exopolysaccharide was analyzed by HPIC. As shown in Figure 2 A, the molecular weight of CCFM1334 exopolysaccharide was divided into two parts, 122158Da(40.25%) and 5185Da(59.95%). As shown in Figure 2 C, the molecular weight of FSH6_5 exopolysaccharide was divided into two parts, 110656Da(30.21%) and 5650Da(69.79%). As shown in Figure 2 E, the molecular weight of FXJSW24L2 exopolysaccharide was divided into two parts, 135693Da(32.77%) and 5313Da(67.23%). Figure 2 B, the numbers 1-10 in B represent fucose, galactosamine, rhamnose, arabinose, glucosamine, galactose, glucose, xylose, mannose and fructose, respectively. As shown in Figure 2 B, the monosaccharide composition of CCFM1334 exopolysaccharide was fucose: galactosamine: rhamnose: arabinose: glucosamine: galactose: glucose: xylose: mannose: fructose, and the molar ratio was 0.10: 1.58: 0.38: 1.00: 0.66: 1.17: 4.09: 0.16: 0.25: 1.41. Figure 2 B, the numbers 1-10 in B represent fucose, galactosamine, rhamnose, arabinose, glucosamine, galactose, glucose, xylose, mannose and fructose, respectively. As shown inFigure 2 As shown in D, the monosaccharide composition of FSH6_5 exopolysaccharide is fucose: galactosamine: rhamnose: arabinose: glucosamine: galactose: glucose: xylose: mannose, and the molar ratio is 0.12: 1.53: 0.51: 1.00: 0.70: 1.25: 3.68: 0.22: 0.64. Figure 2 In F, the numbers 1-11 represent fucose, galactosamine, rhamnose, arabinose, glucosamine, galactose, glucose, xylose, mannose, fructose, and galacturonic acid, respectively. Figure 2 As shown in F, the monosaccharide composition of FXJSW24L2 exopolysaccharide is fucose: galactosamine: rhamnose: arabinose: glucosamine: galactose: glucose: xylose: mannose: fructose: galacturonic acid, and the molar ratio is 0.34: 5.92: 1.32: 1.00: 2.21: 2.42: 6.01: 17.80: 0.76: 0.92: 2.53.

[0089] The content of CCFM1334 exopolysaccharide, FSH6_5 exopolysaccharide, and FXJSW24L2 exopolysaccharide was determined by the phenol-sulfuric acid method using glucose as a standard. The glucose standard solution was prepared at concentrations of 0, 20, 40, 60, 80, 100, 120, 140, 160, 180, and 200 mg / L. The CCFM1334 exopolysaccharide sample solution and different concentrations of glucose standard solution were taken in 10 mL test tubes with a stopper, 1.0 mL of 6% phenol and 5.0 mL of concentrated sulfuric acid were added, mixed well, and then placed in a 100°C water bath for 20 min, and cooled to room temperature. Distilled water was used as a blank control, and the absorbance was measured at 490 nm. The absorbance of the CCFM1334 exopolysaccharide, FSH6_5 exopolysaccharide, and FXJSW24L2 exopolysaccharide sample solution was substituted into the standard curve, and the yield of CCFM1334 exopolysaccharide, FSH6_5 exopolysaccharide, and FXJSW24L2 exopolysaccharide was finally obtained as 185.6 mg / L, 231.67 mg / L, and 298.77 mg / L, respectively.

[0090] Example 3: Effect of Ligilactobacillus salivarius CCFM1334 exopolysaccharide on intestinal disease activity score index of colitis mice

[0091] The specific steps are as follows:

[0092] SPF grade 4-week-old C57BL / 6J male mice were divided into 3 groups, namely blank group, model group and experimental group, wherein the experimental group was gavaged with 200 mg / kg of extracellular polysaccharide of Ligilactobacillus salivarius CCFM1334, extracellular polysaccharide of Lactobacillus fermentum FSH6_5, and extracellular polysaccharide of Lacticaseibacillus rhamnosus FXJSW24L2, respectively. There were 8 mice in each group, which were raised in the Jiangnan University Experimental Animal Center, with constant temperature of 21-26℃, humidity of 40-70%, noise less than or equal to 60 dB, and animal illumination of 15-20LX (all animal experiment procedures were reviewed and approved by the Jiangnan University Animal Welfare and Ethics Management Committee).

[0093] The experimental period was 15 days in total:

[0094] The first 7 days were an adaptation period, during which the mice in each cage were free to drink water and were given ordinary growth and reproduction feed.

[0095] After the adaptation period, from the 8th to the 15th day:

[0096] Model group (i.e. DSS group): 3% DSS drinking water was given every day;

[0097] CCFM1334 extracellular polysaccharide group: 3% DSS drinking water was given every day + gavage of 0.2 mL of 200 mg / kg of extracellular polysaccharide of Ligilactobacillus salivarius CCFM1334;

[0098] FSH6_5 extracellular polysaccharide group: 3% DSS drinking water was given every day + gavage of 0.2 mL of 200 mg / kg of extracellular polysaccharide of Lactobacillus fermentum FSH6_5;

[0099] FXJSW24L2 extracellular polysaccharide group: 3% DSS drinking water was given every day + gavage of 0.2 mL of 200 mg / kg of extracellular polysaccharide of Lacticaseibacillus rhamnosus FXJSW24L2;

[0100] Blank group (i.e. Control group): normal drinking water was given every day.

[0101] The specific process of the animal experiment is shown in Figure 1 .

[0102] The intestinal disease activity score index of the mice was detected on the last day of the experiment, and the final intestinal disease activity score index was as shown inFigure 3 As shown.

[0103] like Figure 3 As shown, after 7 days of drinking water containing 3% DSS, the intestinal disease activity score of the model group increased to 8.63±1.06. In contrast, the CCFM1334 extracellular polysaccharide group, which also drank 3% DSS-containing water for 7 days, had an intestinal disease activity score of 4.71±0.49, a decrease of 45.42% compared to the model group. However, after intervention with FSH6_5 and FXJSW24L2 extracellular polysaccharides, the intestinal disease activity scores were 6.25±1.75 and 6.63±0.74, respectively, representing decreases of only 27.58% and 23.17% compared to the model group. Therefore, the CCFM1334 extracellular polysaccharide of *Lactobacillus salivarius* is more effective in reducing the intestinal disease activity score of colitis in mice.

[0104] Example 4: Effect of extracellular polysaccharide from *Ligilactobacillus salivarius* CCFM1334 on body weight in mice with colitis

[0105] The specific steps are as follows:

[0106] Four-week-old SPF-grade male C57BL / 6J mice were randomly divided into three groups: a control group, a model group, and an experimental group. The experimental groups received extracellular polysaccharides of *Ligilactobacillus salivarius* CCFM1334, *Limosilactobacillus fermentum* FSH6_5, and *Lactobacillus rhamnosus* FXJSW24L2 via gavage. Eight mice were housed in each group at the Experimental Animal Center of Jiangnan University, under a constant temperature of 21-26℃, humidity of 40-70%, noise level ≤60dB, and illumination of 15-20 LX (all animal experimental procedures were reviewed and approved by the Animal Welfare and Ethics Management Committee of Jiangnan University).

[0107] The experiment lasted a total of 15 days.

[0108] Days 1-7 are the adaptation period. During this period, each cage of mice has free access to water and is given a normal growth and reproduction diet.

[0109] After the adaptation period ends, days 8-15:

[0110] Model group (i.e., DSS group): given 3% DSS in drinking water daily;

[0111] CCFM1334 exopolysaccharide group: 3% DSS drinking water was given every day + 0.2 mL of 200 mg / kg of Ligilactobacillus salivarius CCFM1334 exopolysaccharide was administered by gavage;

[0112] FSH6_5 exopolysaccharide group: 3% DSS drinking water was given every day + 0.2 mL of 200 mg / kg of Limois lactobacillus fermentum FSH6_5 exopolysaccharide was administered by gavage;

[0113] FXJSW24L2 exopolysaccharide group: 3% DSS drinking water was given every day + 0.2 mL of 200 mg / kg of Lacticaseibacillus rhamnosus FXJSW24L2 exopolysaccharide was administered by gavage;

[0114] Control group (i.e. Control group): normal drinking water was given every day.

[0115] The specific process of the animal experiment is shown in Figure 1 .

[0116] The last day of the mice drinking 3% DSS drinking water for 7 consecutive days, the body weight value is shown in Figure 4 . Compared with the model group, the CCFM1334 exopolysaccharide group significantly slowed down the percentage of weight loss of the mice. The change value of body weight of the mice in the model group on the last day of intervention was 80.34±3.17%, while that of the CCFM1334 exopolysaccharide group was 89.38±2.69%, which was increased by 9.04% compared with the model group. After the intervention of FSH6_5 exopolysaccharide and FXJSW24L2 exopolysaccharide, the change value of body weight was 81.92±3.61% and 83.48±3.01%, respectively, which was only increased by 1.97% and 3.91% compared with the model group. Therefore, the CCFM1334 exopolysaccharide can more effectively slow down the percentage of weight loss of the mice.

[0117] Example 5: Effect of Ligilactobacillus salivarius CCFM1334 exopolysaccharide on colon length of colitis mice

[0118] The specific steps are as follows:

[0119] SPF level 4-week-old C57BL / 6J male mice were divided into 3 groups, namely blank group, model group and experimental group, wherein the experimental group was gavaged with 200 mg / kg of extracellular polysaccharide of Ligilactobacillus salivarius CCFM1334, extracellular polysaccharide of Lactobacillus fermentum FSH6_5, and extracellular polysaccharide of Lacticaseibacillus rhamnosus FXJSW24L2, respectively. There were 8 mice in each group, which were raised in the Jiangnan University Experimental Animal Center, with constant temperature of 21-26℃, humidity of 40-70%, noise less than or equal to 60dB, and animal illumination of 15-20LX (all animal experiment procedures were reviewed and approved by the Jiangnan University Animal Welfare and Ethics Management Committee).

[0120] The experimental period was 15 days in total:

[0121] The first 7 days were an adaptation period, during which the mice in each cage were free to drink water and were given ordinary growth and reproduction feed.

[0122] After the adaptation period, from the 8th to the 15th day:

[0123] Model group (i.e. DSS group): 3% DSS drinking water was given every day;

[0124] CCFM1334 extracellular polysaccharide group: 3% DSS drinking water was given every day + gavage of 0.2 mL of 200 mg / kg of extracellular polysaccharide of Ligilactobacillus salivarius CCFM1334;

[0125] FSH6_5 extracellular polysaccharide group: 3% DSS drinking water was given every day + gavage of 0.2 mL of 200 mg / kg of extracellular polysaccharide of Lactobacillus fermentum FSH6_5;

[0126] FXJSW24L2 extracellular polysaccharide group: 3% DSS drinking water was given every day + gavage of 0.2 mL of 200 mg / kg of extracellular polysaccharide of Lacticaseibacillus rhamnosus FXJSW24L2;

[0127] Blank group (i.e. Control group): normal drinking water was given every day.

[0128] The specific process of the animal experiment is shown in Figure 1 .

[0129] After the experiment, the mice were dissected according to the ethical requirements, and the colon was taken for length measurement.

[0130] Colon length as Figure 5 As shown, compared to the blank group (7.01±0.53 cm), the colon length in the model group was significantly reduced to 5.2±0.48 cm. With intervention from CCFM1334 extracellular polysaccharide, the colon length significantly increased to 6.42±0.29 cm, a 23.46% increase compared to the model group, and there was no significant difference in colon length between the model and blank groups. After intervention with FSH6_5 and FXJSW24L2 extracellular polysaccharides, the colon lengths were 5.12±0.44 cm and 5.89±0.68 cm, respectively, representing increases of only -1.54% and 13.27% compared to the model group. Therefore, CCFM1334 extracellular polysaccharide can more effectively slow down the shortening of colon length in mice.

[0131] Example 6: Effects of extracellular polysaccharides from *Ligilactobacillus salivarius* CCFM1334 on inflammatory cell infiltration and crypt and goblet cell morphology in colitis mice.

[0132] The specific steps are as follows:

[0133] Four-week-old SPF-grade male C57BL / 6J mice were randomly divided into three groups: a control group, a model group, and an experimental group. The experimental groups received extracellular polysaccharides of *Ligilactobacillus salivarius* CCFM1334, *Limosilactobacillus fermentum* FSH6_5, and *Lactobacillus rhamnosus* FXJSW24L2 via gavage. Eight mice were housed in each group at the Experimental Animal Center of Jiangnan University, under a constant temperature of 21-26℃, humidity of 40-70%, noise level ≤60dB, and illumination of 15-20 LX (all animal experimental procedures were reviewed and approved by the Animal Welfare and Ethics Management Committee of Jiangnan University).

[0134] The experiment lasted a total of 15 days.

[0135] Days 1-7 are the adaptation period. During this period, each cage of mice has free access to water and is given a normal growth and reproduction diet.

[0136] After the adaptation period ends, days 8-15:

[0137] Model group (i.e. DSS group): given 3% DSS in drinking water daily;

[0138] CCFM1334 exopolysaccharide group: 3% DSS drinking water was given every day + 0.2 mL of 200 mg / kg of Ligilactobacillus salivarius CCFM1334 exopolysaccharide was given by gavage;

[0139] FSH6_5 exopolysaccharide group: 3% DSS drinking water was given every day + 0.2 mL of 200 mg / kg of Limo si lactobacillus fermentum FSH6_5 exopolysaccharide was given by gavage;

[0140] FXJSW24L2 exopolysaccharide group: 3% DSS drinking water was given every day + 0.2 mL of 200 mg / kg of Lacticaseibacillus rhamnosus FXJSW24L2 exopolysaccharide was given by gavage;

[0141] Blank group (i.e. Control group): normal drinking water was given every day.

[0142] The specific process of the animal experiment is shown in Figure 1 .

[0143] After the experiment, the mice were dissected according to the ethical requirements, 1 cm of the distal colon was taken and fixed in 4% paraformaldehyde solution for 36 h, then dehydrated by ethanol gradient, transparentized by xylene, embedded in paraffin, sectioned and stained by H&E, and then the colon section was observed.

[0144] The colon section is shown in Figure 6 , the crypt of the blank group mice is normal, the goblet cells are uniformly distributed, and there is no inflammatory cell infiltration. The crypt of the model group mice is lost, the goblet cells are partially lost, the mucin is partially depleted, and the inflammatory cell infiltration is severe. Under the intervention of CCFM1334 exopolysaccharide, the number of goblet cells in the colon tissue is reduced to a certain extent compared with the control group, but is significantly more than that of the DSS group, the crypt structure is partially restored, and the inflammatory cell infiltration is reduced. After the intervention of FSH6_5 exopolysaccharide and FXJSW24L2 exopolysaccharide, the crypt loss, goblet cell loss, mucin depletion and inflammatory cell infiltration are not significantly improved. Therefore, CCFM1334 exopolysaccharide can more effectively slow down the severity of the histopathology of the colonitis mice.

[0145] Example 7: Effect of Ligilactobacillus salivarius CCFM1334 exopolysaccharide on the level of pro-inflammatory factor TNF-α in the colon of colonitis mice

[0146] The specific steps are as follows:

[0147] SPF level 4-week-old C57BL / 6J male mice were divided into 3 groups, namely blank group, model group and experimental group, wherein the experimental group was gavaged with 200 mg / kg of extracellular polysaccharide of Ligilactobacillus salivarius CCFM1334, extracellular polysaccharide of Lactobacillus fermentum FSH6_5, and extracellular polysaccharide of Lacticaseibacillus rhamnosus FXJSW24L2, respectively. There were 8 mice in each group, which were raised in the Jiangnan University Experimental Animal Center, with constant temperature of 21-26℃, humidity of 40-70%, noise less than or equal to 60 dB, and animal illumination of 15-20 LX (all animal experiment procedures were reviewed and approved by the Jiangnan University Animal Welfare and Ethics Management Committee).

[0148] The experimental period was 15 days in total:

[0149] The first 7 days were an adaptation period, during which the mice in each cage were free to drink water and were given ordinary growth and reproduction feed.

[0150] After the adaptation period, from the 8th to the 15th day:

[0151] Model group (i.e. DSS group): 3% DSS drinking water was given every day;

[0152] CCFM1334 extracellular polysaccharide group: 3% DSS drinking water was given every day + gavage of 0.2 mL of 200 mg / kg of extracellular polysaccharide of Ligilactobacillus salivarius CCFM1334;

[0153] FSH6_5 extracellular polysaccharide group: 3% DSS drinking water was given every day + gavage of 0.2 mL of 200 mg / kg of extracellular polysaccharide of Lactobacillus fermentum FSH6_5;

[0154] FXJSW24L2 extracellular polysaccharide group: 3% DSS drinking water was given every day + gavage of 0.2 mL of 200 mg / kg of extracellular polysaccharide of Lacticaseibacillus rhamnosus FXJSW24L2;

[0155] Blank group (i.e. Control group): normal drinking water was given every day.

[0156] The specific process of the animal experiment is shown in Figure 1 .

[0157] After the experiment, the mice were dissected in accordance with ethical requirements. Colonic tissue was weighed and added to an appropriate volume of a mixture of RIPA lysis buffer and a protease phosphatase inhibitor. The tissue was then ground, and the supernatant was extracted. The concentration of TNF-α in the colonic tissue was determined according to the ELISA kit instructions.

[0158] TNF-α concentration in colon tissue is shown in Figure 7 .Depend on Figure 7 Quantitative results showed that the TNF-α concentration in the blank group was 24.39±10.3 pg / mL, while the TNF-α concentration in the model group was 98.77±8.51 pg / mL. After intervention with CCFM1334 extracellular polysaccharide, the TNF-α concentration significantly decreased to 57.57±12.29 pg / mL, a reduction of 41.71% compared to the model group. After intervention with FSH6_5 extracellular polysaccharide and FXJSW24L2 extracellular polysaccharide, the TNF-α concentrations were 61.58±10.41 pg / mL and 108.23±26.03 pg / mL, respectively, representing reductions of only 37.65% and -9.58% compared to the model group. Therefore, CCFM1334 extracellular polysaccharide can more effectively inhibit TNF-α levels in colonic tissue and alleviate colonic inflammation.

[0159] Example 8: Effect of extracellular polysaccharide from *Ligilactobacillus salivarius* CCFM1334 on the level of IL-1β, a pro-inflammatory factor in colitis, in mice.

[0160] Four-week-old SPF-grade male C57BL / 6J mice were randomly divided into three groups: a control group, a model group, and an experimental group. The experimental groups received extracellular polysaccharides of *Ligilactobacillus salivarius* CCFM1334, *Limosilactobacillus fermentum* FSH6_5, and *Lactobacillus rhamnosus* FXJSW24L2 via gavage. Eight mice were housed in each group at the Experimental Animal Center of Jiangnan University, under a constant temperature of 21-26℃, humidity of 40-70%, noise level ≤60dB, and illumination of 15-20 LX (all animal experimental procedures were reviewed and approved by the Animal Welfare and Ethics Management Committee of Jiangnan University).

[0161] The experiment lasted a total of 15 days.

[0162] Days 1-7 are the adaptation period. During this period, each cage of mice has free access to water and is given a normal growth and reproduction diet.

[0163] After the adaptation period ends, days 8-15:

[0164] Model group (i.e. DSS group): 3% DSS drinking water was given every day;

[0165] CCFM1334 exopolysaccharide group: 3% DSS drinking water was given every day + 0.2 mL of 200 mg / kg CCFM1334 exopolysaccharide of Ligilactobacillus salivarius was administered by gavage;

[0166] FSH6_5 exopolysaccharide group: 3% DSS drinking water was given every day + 0.2 mL of 200 mg / kg FSH6_5 exopolysaccharide of Limosilactobacillus fermentum was administered by gavage;

[0167] FXJSW24L2 exopolysaccharide group: 3% DSS drinking water was given every day + 0.2 mL of 200 mg / kg FXJSW24L2 exopolysaccharide of Lacticaseibacillus rhamnosus was administered by gavage;

[0168] Blank group (i.e. Control group): normal drinking water was given every day.

[0169] The specific process of the animal experiment is shown in Figure 1 .

[0170] After the experiment, the mice were dissected according to the ethical requirements, the colon tissues of the mice were weighed, and the mixed solution of RIPA lysis buffer and protease phosphatase inhibitor mixture was added. After the tissue was ground, the supernatant was extracted, and the IL-1β concentration in the colon tissue was determined according to the ELISA kit instructions.

[0171] The IL-1β concentration in the colon tissue is shown in Figure 8 . According to the quantitative results of Figure 8 , the IL-1β concentration of the blank group was 34.4 ± 13.41 pg / mL, and the IL-1β concentration of the model group was 1460.06 ± 101.94 pg / mL. The IL-1β concentration was significantly reduced to 386.25 ± 67.84 pg / mL under the intervention of CCFM1334 exopolysaccharide, which was reduced by 74% compared with the model group. After the intervention of FSH6_5 exopolysaccharide and FXJSW24L2 exopolysaccharide, the IL-1β concentrations were 1222.81 ± 499.87 pg / mL and 1643.5 ± 427.97 pg / mL, respectively, which were only reduced by 16.25% and -12.56% compared with the model group. Therefore, CCFM1334 exopolysaccharide can effectively inhibit the level of IL-1β in the colon tissue.

[0172] Example 9: Effect of Ligilactobacillus salivarius CCFM1334 exopolysaccharide on the level of colonic pro-inflammatory factor IL-6 in mice with colitis

[0173] The specific steps are as follows:

[0174] SPF level 4-week-old C57BL / 6J male mice were divided into 3 groups, namely blank group, model group and experimental group, wherein the experimental group was 200 mg / kg Ligilactobacillus salivarius CCFM1334 exopolysaccharide group, Lactobacillus fermentum FSH6_5 exopolysaccharide group, Lacticaseibacillus rhamnosus FXJSW24L2 exopolysaccharide group. 8 in each group, fed in Jiangnan University Experimental Animal Center, constant temperature 21-26℃, humidity 40-70%, noise less than or equal to 60dB, animal illumination 15-20LX (all animal experiment procedures are reviewed and approved by Jiangnan University Animal Welfare and Ethics Management Committee).

[0175] The experimental period was 15 days:

[0176] The first 7 days were the adaptation period, and the mice in each cage were free to drink water, and were given ordinary growth and reproduction feed.

[0177] After the adaptation period, from the 8th to the 15th day:

[0178] Model group (i.e. DSS group): 3% DSS drinking water was given every day;

[0179] CCFM1334 exopolysaccharide group: 3% DSS drinking water was given every day + 0.2 mL of 200 mg / kg Ligilactobacillus salivarius CCFM1334 exopolysaccharide was given by gavage;

[0180] FSH6_5 exopolysaccharide group: 3% DSS drinking water was given every day + 0.2 mL of 200 mg / kg Lactobacillus fermentum FSH6_5 exopolysaccharide was given by gavage;

[0181] FXJSW24L2 exopolysaccharide group: 3% DSS drinking water was given every day + 0.2 mL of 200 mg / kg Lacticaseibacillus rhamnosus FXJSW24L2 exopolysaccharide was given by gavage;

[0182] Control group: Normal drinking water was provided daily.

[0183] For detailed procedures of animal experiments, please refer to Figure 1 As shown.

[0184] After the experiment, the mice were dissected in accordance with ethical requirements. Colonic tissue was weighed and mixed with an appropriate volume of RIPA lysis buffer and a mixture of protease phosphatase inhibitors. The tissue was then ground, and the supernatant was extracted. The IL-6 concentration in the colonic tissue was determined according to the ELISA kit instructions.

[0185] Colon tissue IL-6 concentrations are shown in Figure 9 .Depend on Figure 9 Quantitative results showed that the IL-6 concentration in the control group was 27.97±9.73 pg / mL, while the IL-6 concentration in the model group was 300.59±60.11 pg / mL. Under CCFM1334 extracellular polysaccharide intervention, the IL-6 concentration was significantly reduced to 40.69±16.15 pg / mL, a decrease of 86.46% compared to the model group, and there was no significant difference compared to the control group. Therefore, CCFM1334 extracellular polysaccharide can effectively inhibit IL-6 levels in colonic tissue.

[0186] Example 10: Effects of extracellular polysaccharides from *Ligilactobacillus salivarius* CCFM1334 on the pro-inflammatory factor IFN-γ in colitis mice.

[0187] The specific steps are as follows:

[0188] Four-week-old SPF-grade male C57BL / 6J mice were randomly divided into three groups: a control group, a model group, and an experimental group. The experimental groups received extracellular polysaccharides of *Ligilactobacillus salivarius* CCFM1334, *Limosilactobacillus fermentum* FSH6_5, and *Lactobacillus rhamnosus* FXJSW24L2 via gavage. Eight mice were housed in each group at the Experimental Animal Center of Jiangnan University, under a constant temperature of 21-26℃, humidity of 40-70%, noise level ≤60dB, and illumination of 15-20 LX (all animal experimental procedures were reviewed and approved by the Animal Welfare and Ethics Management Committee of Jiangnan University).

[0189] The experiment lasted a total of 15 days.

[0190] Days 1-7 are the adaptation period. During this period, each cage of mice has free access to water and is given a normal growth and reproduction diet.

[0191] After the adaptation period, from day 8 to day 15:

[0192] Model group (i.e. DSS group): 3% DSS drinking water was given every day;

[0193] CCFM1334 exopolysaccharide group: 3% DSS drinking water was given every day + 0.2 mL of 200 mg / kg of CCFM1334 exopolysaccharide of Ligilactobacillus salivarius was administered by gavage;

[0194] FSH6_5 exopolysaccharide group: 3% DSS drinking water was given every day + 0.2 mL of 200 mg / kg of FSH6_5 exopolysaccharide of Limosilactobacillus fermentum was administered by gavage;

[0195] FXJSW24L2 exopolysaccharide group: 3% DSS drinking water was given every day + 0.2 mL of 200 mg / kg of FXJSW24L2 exopolysaccharide of Lacticaseibacillus rhamnosus was administered by gavage;

[0196] Blank group (i.e. Control group): normal drinking water was given every day.

[0197] The specific process of the animal experiment is shown in Figure 1 .

[0198] After the experiment, the mice were dissected according to the ethical requirements, the colon tissues of the mice were weighed, and the mixed solution of RIPA lysis buffer and protease phosphatase inhibitor mixture was added. After the tissue was ground, the supernatant was extracted, and the concentration of IFN-γ in the colon tissue was determined according to the ELISA kit instruction.

[0199] The concentration of IFN-γ in the colon tissue is shown in Figure 10 . According to the quantitative results of Figure 10 , the IFN-γ concentration of the blank group was 25.77 ± 3.18 pg / mL, and the IFN-γ concentration of the model group was 66.13 ± 6.46 pg / mL. The IFN-γ concentration under the intervention of CCFM1334 exopolysaccharide was significantly reduced to 30.18 ± 4.29 pg / mL, which was reduced by 54.36% compared with the model group, and had no significant difference with the blank group. After the intervention of FSH6_5 exopolysaccharide and FXJSW24L2 exopolysaccharide, the IFN-γ concentrations were 38.91 ± 1.97 pg / mL and 58.18 ± 7.54 pg / mL, respectively, which were reduced by 41.16% and 12.02% compared with the model group. Therefore, CCFM1334 exopolysaccharide can more effectively inhibit the level of IFN-γ in the colon tissue.

[0200] Example 11: Effect of Ligilactobacillus salivarius CCFM1334 exopolysaccharide on colonic pro-inflammatory factor IL-17 in mice with colitis

[0201] The specific steps are as follows:

[0202] 4-week-old SPF C57BL / 6J male mice were divided into 3 groups, namely a blank group, a model group, and an experimental group. The experimental group was administered 200 mg / kg of Ligilactobacillus salivarius CCFM1334 exopolysaccharide, Lactobacillus fermentum FSH6_5 exopolysaccharide, and Lacticaseibacillus rhamnosus FXJSW24L2 exopolysaccharide by gavage, respectively. There were 8 mice in each group, which were raised in the Jiangnan University Experimental Animal Center, with a constant temperature of 21-26°C, humidity of 40-70%, noise less than or equal to 60 dB, and animal illumination of 15-20 LX (all animal experiment procedures were reviewed and approved by the Jiangnan University Animal Welfare and Ethics Management Committee).

[0203] The experimental period lasted for 15 days:

[0204] The first 7 days were an adaptation period, during which the mice in each cage were free to drink water and were given ordinary growth and reproduction feed.

[0205] After the adaptation period, from the 8th to the 15th day:

[0206] Model group (i.e., DSS group): 3% DSS drinking water was given every day;

[0207] CCFM1334 exopolysaccharide group: 3% DSS drinking water was given every day + 0.2 mL of 200 mg / kg Ligilactobacillus salivarius CCFM1334 exopolysaccharide was administered by gavage;

[0208] FSH6_5 exopolysaccharide group: 3% DSS drinking water was given every day + 0.2 mL of 200 mg / kg Lactobacillus fermentum FSH6_5 exopolysaccharide was administered by gavage;

[0209] FXJSW24L2 exopolysaccharide group: 3% DSS drinking water was given every day + 0.2 mL of 200 mg / kg Lacticaseibacillus rhamnosus FXJSW24L2 exopolysaccharide was administered by gavage;

[0210] Control group: Normal drinking water was provided daily.

[0211] For detailed procedures of animal experiments, please refer to Figure 1 As shown.

[0212] After the experiment, the mice were dissected in accordance with ethical requirements. Colonic tissue was weighed and mixed with an appropriate volume of RIPA lysis buffer and a mixture of protease phosphatase inhibitors. The tissue was then ground, and the supernatant was extracted. The concentration of IL-17 in the colonic tissue was determined according to the ELISA kit instructions.

[0213] Colon tissue IL-17 concentrations are shown in the figure. Figure 11 .Depend on Figure 11 Quantitative results showed that the IL-17 concentration in the control group was 41.68±6.74 pg / mL, while the IL-17 concentration in the model group was 122.71±35.9 pg / mL. The IL-17 concentration was significantly reduced by CCFM1334 extracellular polysaccharide intervention to 47.77±7.98 pg / mL, a decrease of 61.07% compared to the model group, with no significant difference from the control group. After intervention with FSH6_5 and FXJSW24L2 extracellular polysaccharides, the IL-17 concentrations were 81.06±12.58 pg / mL and 85.68±42.02 pg / mL, respectively, representing reductions of only 33.94% and 30.18% compared to the model group. Therefore, CCFM1334 extracellular polysaccharide can more effectively inhibit IL-17 levels in colonic tissue.

[0214] Example 12: Effect of extracellular polysaccharide from *Ligilactobacillus salivarius* CCFM1334 on the anti-inflammatory factor IL-10 in the colon of colitis-affected mice.

[0215] The specific steps are as follows:

[0216] SPF level 4-week-old C57BL / 6J male mice were divided into 3 groups, namely blank group, model group and experimental group, wherein the experimental group was gavaged with 200 mg / kg of extracellular polysaccharide of Ligilactobacillus salivarius CCFM1334, extracellular polysaccharide of Lactobacillus fermentum FSH6_5, and extracellular polysaccharide of Lacticaseibacillus rhamnosus FXJSW24L2, respectively. There were 8 mice in each group, which were raised in the Jiangnan University Experimental Animal Center, with constant temperature of 21-26℃, humidity of 40-70%, noise less than or equal to 60 dB, and animal illumination of 15-20 LX (all animal experiment procedures were reviewed and approved by the Jiangnan University Animal Welfare and Ethics Management Committee).

[0217] The experimental period was 15 days in total:

[0218] The first 7 days were an adaptation period, during which the mice in each cage were free to drink water and were given ordinary growth and reproduction feed.

[0219] After the adaptation period, from the 8th to the 15th day:

[0220] Model group (i.e. DSS group): 3% DSS drinking water was given every day;

[0221] CCFM1334 extracellular polysaccharide group: 3% DSS drinking water was given every day + gavage of 0.2 mL of 200 mg / kg of extracellular polysaccharide of Ligilactobacillus salivarius CCFM1334;

[0222] FSH6_5 extracellular polysaccharide group: 3% DSS drinking water was given every day + gavage of 0.2 mL of 200 mg / kg of extracellular polysaccharide of Lactobacillus fermentum FSH6_5;

[0223] FXJSW24L2 extracellular polysaccharide group: 3% DSS drinking water was given every day + gavage of 0.2 mL of 200 mg / kg of extracellular polysaccharide of Lacticaseibacillus rhamnosus FXJSW24L2;

[0224] Blank group (i.e. Control group): normal drinking water was given every day.

[0225] The specific process of the animal experiment is shown in Figure 1 .

[0226] After the experiment, the mice were dissected in accordance with ethical requirements. Colonic tissue was weighed and mixed with an appropriate volume of RIPA lysis buffer and a mixture of protease phosphatase inhibitors. The tissue was then ground, and the supernatant was extracted. The concentration of IL-10 in the colonic tissue was determined according to the ELISA kit instructions.

[0227] Colon tissue IL-10 concentrations are shown in the figure. Figure 12 .Depend on Figure 12 Quantitative results showed that the IL-10 concentration in the control group was 115.76±6.76 pg / mL, while the IL-10 concentration in the model group was 87.68±10.02 pg / mL. After intervention with CCFM1334 extracellular polysaccharide, the IL-10 concentration significantly increased to 117.81±9.41 pg / mL, an increase of 34.36% compared to the model group, and there was no significant difference compared to the control group. After intervention with FSH6_5 extracellular polysaccharide and FXJSW24L2 extracellular polysaccharide, the IL-10 concentrations were 90.44±10.87 pg / mL and 110.44±16.42 pg / mL, respectively, an increase of only 3.15% and 25.95% compared to the model group. Therefore, CCFM1334 extracellular polysaccharide can more effectively promote IL-10 levels in colonic tissue.

[0228] Example 13: Effects of extracellular polysaccharide from *Ligilactobacillus salivarius* CCFM1334 on tight junction proteins in colon tissue of colitis-affected mice

[0229] The specific steps are as follows:

[0230] Four-week-old SPF-grade male C57BL / 6J mice were randomly divided into three groups: a control group, a model group, and an experimental group. The experimental groups received extracellular polysaccharides of *Ligilactobacillus salivarius* CCFM1334, *Limosilactobacillus fermentum* FSH6_5, and *Lactobacillus rhamnosus* FXJSW24L2 via gavage. Eight mice were housed in each group at the Experimental Animal Center of Jiangnan University, under a constant temperature of 21-26℃, humidity of 40-70%, noise level ≤60dB, and illumination of 15-20 LX (all animal experimental procedures were reviewed and approved by the Animal Welfare and Ethics Management Committee of Jiangnan University).

[0231] The experiment lasted a total of 15 days.

[0232] Days 1-7 are the adaptation period. During this period, each cage of mice has free access to water and is given a normal growth and reproduction diet.

[0233] After the adaptation period, from day 8 to day 15:

[0234] Model group (i.e. DSS group): 3% DSS drinking water was given every day;

[0235] CCFM1334 exopolysaccharide group: 3% DSS drinking water was given every day + 0.2 mL of 200 mg / kg CCFM1334 exopolysaccharide of Ligilactobacillus salivarius was given by gavage;

[0236] FSH6_5 exopolysaccharide group: 3% DSS drinking water was given every day + 0.2 mL of 200 mg / kg FSH6_5 exopolysaccharide of Limosilactobacillus fermentum was given by gavage;

[0237] FXJSW24L2 exopolysaccharide group: 3% DSS drinking water was given every day + 0.2 mL of 200 mg / kg FXJSW24L2 exopolysaccharide of Lacticaseibacillus rhamnosus was given by gavage;

[0238] Blank group (i.e. Control group): normal drinking water was given every day.

[0239] The specific process of the animal experiment is shown in Figure 1 .

[0240] After the experiment, the mice were dissected according to the ethical requirements, the colon tissues of the mice were weighed, and total RNA was extracted from the colon tissues using FreeZol reagent for reverse transcription. The relative content of cDNA was analyzed by ChamQ SYBR qPCR Master Mix quantitative real-time PCR thermal cycler. 2 -ΔΔCT The contents of ZO-1 and occludin normalized by GAPDH were calculated.

[0241] Tight junction proteins are one of the important components of intestinal barrier, and the most studied tight junction proteins are ZO-1 and occludin. The mRNA expression levels of ZO-1 and occludin in colon tissues are shown in Figure 13 . Figure 13The quantitative results of A show that the mRNA expression amount of ZO-1 in the model group is 0.46, the mRNA expression amount of ZO-1 under the intervention of CCFM1334 exopolysaccharide is significantly increased to 1.08, which is increased by 134.78% compared with the model group, and has no significant difference with the blank group. After the intervention of FSH6_5 exopolysaccharide and FXJSW24L2 exopolysaccharide, the mRNA expression amount of ZO-1 is 0.52±0.36 and 0.62±0.31 respectively, which is only increased by 13.04% and 34.78% compared with the model group. Figure 13 The quantitative results of B show that the mRNA expression amount of occludin in the model is 0.41, the mRNA expression amount of occludin under the intervention of CCFM1334 exopolysaccharide is significantly increased to 0.87, which is increased by 112.20% compared with the model group, and has no significant difference with the blank group. After the intervention of FSH6_5 exopolysaccharide and FXJSW24L2 exopolysaccharide, the mRNA expression amount of occludin is 0.46±0.14 and 0.39±0.08 respectively, which is only increased by 12.20% and -9.76% compared with the model group. Therefore, CCFM1334 exopolysaccharide can more effectively promote the mRNA expression amount of tight junction proteins ZO-1 and occludin in colon tissue.

[0242] Although the present application has been disclosed with the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be defined by the claims.

Claims

1. A strain of Ligilactobacillus salivarius CCFM1334, characterized in that, Ligilactobacillus salivarius CCFM1334 and its exopolysaccharide for use in preventing and / or treating colitis.

2. A method for producing an exocellular polysaccharide, characterized by, The method comprises inoculating the Ligilactobacillus salivarius of claim 1 into a fermentation medium, performing fermentation, and collecting the supernatant.

3. The method of claim 2, wherein, The fermentation medium comprises MRS medium.

4. The method of claim 2, wherein, The fermentation is performed at a temperature of 30-40℃.

5. The method of claim 2, wherein, The fermentation is performed for 12-36 hours.

6. The method of claim 2, wherein, The inoculation amount of the Ligilactobacillus salivarius is 2%-8% v / v.

7. Use of Ligilactobacillus salivarius CCFM1334 of claim 1 and its exopolysaccharide in the preparation of a product for preventing and / or treating colitis.

8. Use according to claim 7, characterized in that, The prevention and / or treatment of colitis comprises at least one aspect of the following (a)-(f): (a) reducing the intestinal disease activity index of the individual with colitis; (b) reducing the expression level of pro-inflammatory factors in the colon tissue of the individual with colitis, the pro-inflammatory factors comprising at least one of TNF-α, IL-1β, IL-6, IFN-γ or IL-17; (c) increasing the expression level of anti-inflammatory factors in the colon tissue of the individual with colitis, the anti-inflammatory factors comprising IL-10; (d) increasing the expression amount of tight junction proteins ZO-1 and / or occludin in the colon tissue of the individual with colitis; (e) reducing the weight loss of the individual with colitis; (f) reducing the inflammatory cell infiltration in the colon tissue of the individual with colitis, and / or relieving the crypt loss, and / or relieving the goblet cell loss.

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