Lactobacillus paracasei e10 for modulating intestinal inflammatory response

Abstract

The present application relates to a kind of Lactobacillus paracasei E10 that adjusts intestinal inflammatory response, the strain is preserved in China Microorganism Strain Preservation Management Committee on June 21, 2021, and the preservation address is Beijing Chaoyang District Beichen West Road No. 1 Yard No. 3, and the preservation number is CGMCC No.22744.This application provides the strain Lactobacillus paracasei E10 can effectively protect in vivo inflammatory response and intestinal flora disorder, significantly reduce the abundance of in vivo sulfate reducing bacteria.Therefore, Lactobacillus paracasei E10 in the direction of food or microecological preparation has the prospect of wide application for colitis.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a Lactobacillus paracasei E10 that regulates intestinal inflammatory responses. Background Technology

[0002] Inflammatory bowel disease (IBD) is a chronic, nonspecific inflammatory bowel disease that primarily affects the colonic mucosa and submucosa, manifesting as recurrent episodes of chronic intestinal inflammation. IBD is divided into two subtypes: Crohn's disease and ulcerative colitis. Recent data indicate that the global incidence and prevalence of IBD continue to rise, with approximately 0.2% of the European population suffering from it, making it a significant global health burden. However, drug treatment for IBD often involves certain side effects. With advancements in research, alternative treatments that supplement the shortcomings of traditional drug therapies, such as microbial preparations and probiotics, are gradually gaining attention.

[0003] *Lactobacillus paracasei* is a probiotic with functions such as regulating intestinal flora balance, anti-tumor activity, and enhancing human immunity, and has attracted widespread attention both domestically and internationally in recent years. *Lactobacillus paracasei* is a Gram-positive heterofermentative lactic acid bacterium that can be isolated from the human intestine, oral cavity, and various dairy products. It is a facultative anaerobic, non-motile, non-spore-forming bacillus or long rod-shaped organism. Studies have shown that *Lactobacillus paracasei* can protect human health by inhibiting the secretion of pro-inflammatory cytokines, improving the intestinal epithelial cell barrier, increasing the total antioxidant capacity in colonic tissue, and regulating intestinal flora balance. Furthermore, research has found that the metabolites of *Lactobacillus paracasei*, such as organic acids, bacteriocins, and phenyllactic acid, can effectively inhibit the growth of pathogenic and putrefactive bacteria.

[0004] Patent CN201811008074.4 discloses a strain of *Lactobacillus paracasei* N1115, which can be used to prevent colitis; Patent CN202011369708.6 discloses the application of *Lactobacillus paracasei* ET-22 in enhancing intestinal bacterial infection resistance and intestinal immunity, which can inhibit the adhesion of intestinal pathogens and the production of intestinal inflammatory factors, and alleviate the inflammatory response caused by ETECH10407; Patent CN201911139707.X discloses the application of *Lactobacillus paracasei* K56 in alleviating intestinal inflammation, which can reduce inflammatory factors IL-6 and / or TNF-α, promote the anti-inflammatory factor IL-10, and reduce colitis tissue damage; CN202111386682.0 discloses *Lactobacillus paracasei* Jn1 and its application, which has a survival rate of 82% in simulated digestion in gastrointestinal fluid and strong bile salt tolerance, with an ABS of 14.08 when the bile salt concentration reaches 0.6%. However, the currently disclosed strains that have the effect of relieving enteritis are not ideal in actual treatment, so this invention is proposed. Summary of the Invention

[0005] To address the aforementioned technical problems, the strain Lactobacillus paracasei E10 provided by this invention has broad application prospects in the preparation of foods and microecological preparations that alleviate inflammatory diseases.

[0006] The first objective of this invention is to provide a Lactobacillus paracasei E10 that regulates intestinal inflammatory responses, which was deposited on June 21, 2021, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 22744.

[0007] A second objective of this invention is to provide the application of the aforementioned Lactobacillus paracasei E10 in the preparation of products that alleviate intestinal inflammatory responses.

[0008] Furthermore, in the product for alleviating intestinal inflammatory response, the viable count of Lactobacillus paracasei E10 is not less than 1 × 10⁻⁶. 6 CFU / mL or not less than 1×10 6 CFU / g.

[0009] Furthermore, the product for relieving intestinal inflammatory response is a food, medicine, or health product.

[0010] Furthermore, the food is fermented fruits and vegetables, fermented milk, cheese, milk-containing beverages, or milk powder.

[0011] Furthermore, the drug contains a drug carrier and / or pharmaceutical excipients.

[0012] Furthermore, the drug carrier is a microcapsule, microsphere, nanoparticle, and / or liposome.

[0013] Furthermore, the pharmaceutical excipients include excipients and / or additives.

[0014] Furthermore, the excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, absorbents, diluents, flocculants, anti-flocculators, filter aids, and / or release inhibitors.

[0015] Furthermore, the additives comprise microcrystalline cellulose, hydroxypropyl methylcellulose, and / or refined lecithin.

[0016] Furthermore, the dosage form of the medicine is powder, granules, capsules, tablets, pills, or oral liquid.

[0017] Furthermore, the product for relieving intestinal inflammation is a drug for the prevention or treatment of inflammatory bowel disease.

[0018] A third objective of this invention is to provide a bacterial agent containing the aforementioned Lactobacillus paracasei E10.

[0019] Furthermore, the bacterial agent is a solid bacterial agent, which may be in powder or granule form.

[0020] Furthermore, the bacterial agent is a liquid bacterial agent.

[0021] By means of the above-described solution, the present invention has at least the following advantages:

[0022] (1) The strain Lactobacillus paracasei E10 of the present invention significantly restored the weight loss, disease activity index score, shortened colon length, and colonic histopathological damage in mice caused by sodium dextran sulfate (DSS) infection to near-normal levels. Therefore, the strain E10 of the present invention, which has the ability to alleviate ulcerative colitis, has broad application prospects in the fields of food and microecological preparations.

[0023] (2) The strain Lactobacillus paracasei E10 provided by this invention can significantly reduce the increase in the levels of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α caused by DSS infection and colitis aggravated by a high-sulfur diet. Compared with the Lgg and Jn1 bacterial intervention groups, the E10 group has a better effect on reducing the levels of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α.

[0024] (3) The strain Lactobacillus paracasei E10 provided by the present invention can significantly reduce the increase in the proportion of sulfate-reducing bacteria (SRB) caused by sodium dextran sulfate (DSS) infection and supplementation with a high-sulfur diet, and reduce the content of hydrogen sulfide metabolites produced by sulfate-reducing bacteria after metabolizing sulfate.

[0025] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described below. Attached Figure Description

[0026] Figure 1 The effects of strain Lactobacillus paracasei E10 on the body weight of mice with colitis and the disease activity index (DAI) scores of the mice were evaluated. ** indicates P < 0.01, and * indicates P < 0.05.

[0027] Figure 2 The effect of strain Lactobacillus paracasei E10 on colon length in mice with colitis is shown in the figure. ** indicates P < 0.01, and * indicates P < 0.05.

[0028] Figure 3 For the morphological observation and pathological scoring of mouse colon tissue, ** indicates P<0.01, * indicates P<0.05;

[0029] Figure 4 The effect of strain Lactobacillus paracasei E10 on the levels of cytokines IL-6, IL-1β, and TNF-α in mouse serum was investigated. ** indicates P < 0.01, and * indicates P < 0.05.

[0030] Figure 5 The effect of strain Lactobacillus paracasei E10 on the proportion of sulfate-reducing bacteria (SRB) in the mouse gut microbiota and its metabolite hydrogen sulfide is given by: ** indicating P < 0.01, * indicating P < 0.05.

[0031] Preservation of biological materials

[0032] Lactobacillus paracasei E10 was deposited on June 21, 2021, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 22744. Detailed Implementation

[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0034] The strains involved in the following examples are as follows:

[0035] Lactobacillus rhamnosus (Lgg), purchased from ATCC, product number ATCC53103.

[0036] Lactobacillus paracasei Jn1 was deposited on June 21, 2021, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 22745, and is described in patent CN202111386682.0.

[0037] The culture media involved in the following examples are as follows:

[0038] MRS culture medium formula: Dissolve the following substances in 1L of distilled water.

[0039] peptone 10g Beef extract 10g yeast powder 5g Glucose (glucose monohydrate) 20g diamine citrate 2g (Anhydrous) Sodium acetate 5g Dipotassium hydrogen phosphate (trihydrate) 2.6g Magnesium sulfate heptahydrate 0.2g manganese sulfate 0.05g Twain 80 1mL

[0040] MRS solid medium: Add 15g of agar to 1L of MRS liquid medium, heat to sterilize, and allow it to solidify.

[0041] The preparation method of the Lactobacillus paracasei E10 bacterial suspension involved in the following examples is as follows:

[0042] Strawberry strain Lactobacillus paracasei E10 was streaked onto MRS solid medium and cultured at 37°C for 24 h to obtain single colonies. Single colonies were picked and inoculated into MRS liquid medium and cultured at 37°C for 24 h for activation. This activation process was repeated for two generations to obtain the activated solution. The activated solution was inoculated into MRS liquid medium at a 2% (v / v) inoculation rate and cultured at 37°C for 24 h to obtain the bacterial suspension. The bacterial suspension was centrifuged at 5000g for 10 min to obtain Lactobacillus paracasei E10 cells. The Lactobacillus paracasei E10 cells were washed with PBS and resuspended in 50% glycerol solution to a final bacterial concentration of 1×10⁻⁶. 10 The bacterial suspension was obtained by dispersing CFU / mL and stored at -80℃ for later use.

[0043] The preparation method of the Lactobacillus paracasei Jn1 bacterial suspension involved in the following examples is as follows:

[0044] Strawberry strain Lactobacillus paracasei Jn1 was streaked onto MRS solid medium and cultured at 37°C for 24 h to obtain single colonies. Single colonies were picked and inoculated into MRS liquid medium and cultured at 37°C for 24 h for activation. This activation process was repeated for two generations to obtain the activated solution. The activated solution was inoculated into MRS liquid medium at a rate of 2% (v / v) and cultured at 37°C for 24 h to obtain the bacterial suspension. The bacterial suspension was centrifuged at 5000g for 10 min to obtain Lactobacillus paracasei Jn1 cells. The Lactobacillus paracasei Jn1 cells were washed with PBS and resuspended in 50% glycerol solution to a final bacterial concentration of 1×10⁻⁶. 10 The bacterial suspension was obtained by dispersing CFU / mL and stored at -80℃ for later use.

[0045] The preparation method of the Lactobacillus rhamnosus Lgg bacterial suspension involved in the following examples is as follows:

[0046] The strain *Lactobacillus rhamnosus* Lgg was streaked onto MRS solid medium and cultured at 37°C for 24 h to obtain single colonies. Single colonies were picked and inoculated into MRS liquid medium and cultured at 37°C for 24 h for activation. This activation process was repeated for two generations to obtain the activated solution. The activated solution was inoculated into MRS liquid medium at a rate of 2% (v / v) and cultured at 37°C for 24 h to obtain the bacterial suspension. The bacterial suspension was centrifuged at 5000g for 10 min to obtain *Lactobacillus rhamnosus* Lgg cells. The *Lactobacillus rhamnosus* Lgg cells were washed with PBS and resuspended in 50% glycerol solution to a final bacterial concentration of 1×10⁻⁶. 10 The bacterial suspension was obtained by dispersing CFU / mL and stored at -80℃ for later use.

[0047] The detection methods involved in the following embodiments are as follows:

[0048] Methods for detecting the Disease Activity Index (DAI):

[0049] The disease activity index combines the percentage of weight loss in the affected animal, fecal shape, and fecal occult blood / gross blood score. The scoring criteria are shown in the table below:

[0050]

[0051] Normal stool = well-formed pellets; loose stool = pasty or semi-formed stool that does not stick to the anus; abdominal...

[0052] Loose stool = watery stool that sticks to the anus

[0053] The DAI score is calculated by combining the following three factors: ① weight loss (0 for no change, 1-5 for 1 point, 5-10 for 2 points, 10-15 for 3 points, and greater than 15 for 4 points); ② stool shape (0 for normal, 2 for loose stool, and 4 for diarrhea); and ③ rectal bleeding (0 for normal, 2 for occult blood, and 4 for visible blood in stool). The total score is then divided by 3 to obtain the DAI value. Therefore, DAI = (Body Mass Index + Stool Shape + Bleeding) / 3.

[0054] Methods for measuring colon length:

[0055] After successful modeling, the mice were euthanized and dissected. Colon tissue was taken from the mice, and the total length of the colon of each mouse was measured with a ruler (unit: cm).

[0056] Methods for detecting histopathological features of colon tissue and scoring criteria for tissue damage: The degree of tissue damage is scored based on the degree of inflammatory cell infiltration (0-3 points) and tissue damage (0-3 points).

[0057] For the detection methods and tissue damage scoring criteria for colonic histopathological features, please refer to the paper: Propolis from Different Geographic Origins Decreases Intestinal Inflammation and Bacteroides spp. Populations in a Model of DSS-Induced Colitis.

[0058] Determination of biochemical parameters in colon tissue:

[0059] Mouse blood was collected, allowed to stand at room temperature for half an hour, and then centrifuged at 1500g for 15 minutes at 4°C to obtain serum. The levels of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α in the serum were measured using an ELISA kit (Thermo Fisher Scientific Inc, Waltham, USA).

[0060] 16S rDNA sequencing: After the mouse modeling was completed, the feces were collected in a 1.5mL centrifuge tube and sent to BGI Genomics for 16S rDNA sequencing.

[0061] H2S in mouse fecal samples was determined using an endogenous hydrogen sulfide (H2S) assay kit (Nanjing Jiancheng Biotechnology, Nanjing, China) according to the manufacturer's instructions.

[0062] Example 1: Isolation and screening of strain Lactobacillus paracasei E10

[0063] Isolation of strains

[0064] The strain Lactobacillus paracasei E10 was a purified strain isolated from vinegar mash. It was stored at -80°C with 750 μL of bacterial culture in 750 μL of 50% glycerol.

[0065] strain preservation and identification

[0066] (1) Preservation of bacterial strain: Mix 750 μL of purified Lactobacillus paracasei E10 culture medium, add 750 μL of 50% glycerol, and store in a -80℃ freezer. Make a backup tube for bacterial identification, centrifuge at 5000 r / min for 3 min, discard the supernatant to obtain bacterial cells.

[0067] (2) Strain identification: Take the preservation tube used for strain identification in step (1) and operate according to the instructions of the bacterial genomic DNA extraction kit provided by the reagent supplier (Shanghai Generay Biotech Co., Ltd.). The specific operation is as follows:

[0068] Take the preservation tube used for bacterial identification in step (1), centrifuge at 8000 rpm / min for 1 min, discard the supernatant to obtain bacterial cells, add 150 μL TE (pH 8.0) to suspend the bacteria, add 5 μL lysozyme to the above 150 μL TE solution, and incubate at room temperature for 5-10 min. Then add 300 μL of digestion solution and mix well; then add 4 μL RNase A and mix well, incubate at 55℃ for 10 min; then add 4 μL proteinase K and incubate at 55℃ for 10-30 min. Then add 300 μL PB solution, shake well to mix, centrifuge at 12000 rpm at room temperature for 5 min to obtain supernatant. Prepare the gDNA recovery column, add 200 μL Buffer CBS to the column, centrifuge at 10000 rpm for 1 min, discard the permeate, and set aside. Transfer the supernatant obtained above to the gDNA recovery column placed in a 2 mL collection tube. Centrifuge at 8000 rpm for 1 min at room temperature, discarding the waste liquid in the collection tube. Place the gDNA recovery column back into the collection tube, add 500 μL of washing buffer, centrifuge at 8000 rpm for 1 min, remove the gDNA recovery column, discard the waste liquid in the collection tube, and repeat this step once. Place the gDNA recovery column back into the collection tube, centrifuge at 12000 rpm for 1 min to remove residual washing buffer. Place the gDNA recovery column into a new, clean 1.5 mL centrifuge tube, add 50-100 μL of Elution buffer to the center of the gDNA recovery column, incubate at room temperature or 37°C for 2 min, centrifuge at 12000 rpm for 1 min at room temperature, and the liquid in the centrifuge tube is the bacterial genome DNA.

[0069] The concentration of the extracted genome and its OD260 / 280 ratio were determined for subsequent PCR reactions.

[0070] PCR reaction system (20 μL):

[0071] Reverse primer 1μL 2X PCR 10μL DNA template <100ng <![CDATA[ddH2O]]> Add to 20μL

[0072] The PCR template was 27F (5'-AGAGTTTGATCCTGGCCTCA-3'), 1492R (GGTTACCTTGTTACGACTT); the reaction conditions were: 95℃, 3 min, 1 cycle; 95℃, 30 s, 55℃, 30 s, 72℃, 90 s, 35 cycles; 72℃, 5 min.

[0073] After the PCR product was confirmed by nucleic acid electrophoresis analysis, it was sent to BGI for sequencing. The sequenced product is being confirmed for species identification by NCBI. By comparing with the NCBI database, the results showed that strain Lactobacillus paracasei E10 belongs to the genus Lactobacillus, and it was named strain Lactobacillus paracasei E10.

[0074] Example 2: Effect of strain Lactobacillus paracasei E10 on DSS-induced ulcerative colitis

[0075] Thirty-six 7-week-old male C57BL / 6J mice were divided into six groups of six according to their average body weight, as shown in Table 1. These groups were: ① Control group, ② Model group (DSS group), ③ DSS + high-sulfur diet group (abbreviated as high-sulfur group, i.e., DSS+GL group), ④ DSS + high-sulfur diet + strain Lgg group (abbreviated as Lgg group, i.e., DSS+GL+Lgg group), ⑤ DSS + high-sulfur diet + strain E10 group (abbreviated as E10 group, i.e., DSS+GL+E10 group), and ⑥ DSS + high-sulfur diet + strain Jn1 group (abbreviated as Jn1 group, i.e., DSS+GL+Jn1 group).

[0076] After a week of acclimatization, mice in the Lgg, E10, and Jn1 groups were pre-administered by gavage with Lgg, E10, and Jn1 bacterial suspensions for 14 consecutive days. Simultaneously, the control group, model group, and high-sulfur group were administered sterile PBS buffer by gavage. Starting from day 8, the model group, high-sulfur group, Lgg group, E10 group, and Jn1 group were given 2.5% DSS solution for free drinking.

[0077] The preparation method for the 2.5% DSS aqueous solution is as follows: Weigh 2.5g of DSS solid powder and dissolve it in 100mL of sterile water, gently shake until completely dissolved, and replace with fresh 2.5% DSS solution daily.

[0078] Normal control group: From day 1 to day 14, the animals were continuously administered 200 μL of sterile PBS buffer by gavage for 14 days, with free access to water and ordinary purified feed.

[0079] Model group: From day 1 to day 14, the animals were continuously gavaged with 200 μL of sterile PBS buffer for 14 days. From day 8 to day 14, the animals had free access to an aqueous solution containing 2.5% DSS and were fed ordinary purified feed.

[0080] High-sulfur group: From day 1 to day 14, 200 μL of sterile PBS buffer was continuously administered by gavage for 14 days. From day 8 to day 14, the group had free access to an aqueous solution containing 2.5% DSS and was fed high-sulfur feed.

[0081] Lgg group: From day 1 to day 14, 200 μL of Lgg bacterial solution was continuously administered by gavage for 14 days. From day 8 to day 14, the group had free access to an aqueous solution containing 2.5% DSS and was fed a high-sulfur feed.

[0082] Group E10: From day 1 to day 14, 200 μL of E10 bacterial solution was continuously administered by gavage for 14 days. From day 8 to day 14, the group had free access to an aqueous solution containing 2.5% DSS and was fed a high-sulfur feed.

[0083] Jn1 group: From day 1 to day 14, 200 μL of Jn1 bacterial solution was continuously administered by gavage for 14 days. From day 8 to day 14, the group had free access to drinking water containing 2.5% DSS and was fed high-sulfur feed.

[0084] During the colitis modeling process, the mice were weighed every other day to calculate the percentage change in body weight. On day 15, the mice were sacrificed and samples were taken for subsequent experiments.

[0085] Table 1 shows the animal experimental design as follows:

[0086]

[0087] The specific composition tables for ordinary purified feed and high-sulfur feed are shown below:

[0088]

[0089]

[0090] Note: The daily oral dose of sterile PBS and bacterial suspension is 200 μL, and the viable count of the bacterial suspension is 1 × 10⁻⁶. 9 CFU / mL.

[0091] Example 3: Changes in body weight and colon length in mice with DSS-induced ulcerative colitis caused by strain Lactobacillus paracasei E10.

[0092] DSS-induced colitis in mice leads to weight loss and colon shortening; therefore, body weight and colon length are important indicators for evaluating the severity of inflammation in colitis-affected mice. After successful model establishment, mice were sacrificed and dissected to obtain colon tissue. The total length of the colon in each mouse was measured using a ruler.

[0093] Weight changes such as Figure 1As shown in Figure A, compared with the normal group, the DSS group mice showed a significant decrease in body weight, indicating the successful establishment of the ulcerative colitis model. Compared with the DSS group, the weight loss of mice was exacerbated after supplementation with a high-sulfur diet. However, under the dual intervention model of DSS and a high-sulfur diet, supplementation with E10 bacterial solution significantly alleviated the weight loss of mice, and the slowing effect of E10 bacterial solution on weight loss was better than that of the LGg intervention group. After supplementation with another strain of Lactobacillus paracasei Jn1, the trend of weight loss was significantly different from that of the DSS+GL group, and the E10 group showed better relief of weight loss in mice than after supplementation with Jn1 bacterial solution. In the Disease Activity Index (DAI) score, as shown... Figure 1 As shown in Figure B, compared to the DSS group, the disease activity index (DIA) score of mice increased after supplementation with a high-sulfur diet. Supplementation with E10 probiotics on top of DSS and a high-sulfur diet significantly reduced the DIA score. Furthermore, supplementation with Lgg and Jn1 probiotics also reduced the DIA score, but their scores were higher than those of the E10 group. The Jn1 probiotic supplementation group had the highest pathological disease score among the three probiotic supplementation groups. This suggests that E10 probiotics may be more effective than Lgg or Jn1 probiotics in restoring clinical function in mice.

[0094] Changes in colon length, such as Figure 2 As shown, the normal group of mice had the longest colon, while the colon length of other groups of mice decreased to varying degrees. Compared with the control group, the DSS group of mice had a significantly shorter colon, indicating inflammation in the mice. Under the combined effects of DSS and high sulfur, supplementation with E10 bacteria could significantly restore the colon length of the mice. Supplementation with Lgg or Jn1 bacteria could also restore part of the colon length in mice, but the improvement effect was not as obvious as that of supplementation with E10 bacteria.

[0095] A 1 cm section of distal mouse colon was fixed in 4% paraformaldehyde solution for 12 hours. Sections were then prepared through washing, gradient dehydration with ethanol solution, clearing, paraffin embedding, embedding, and sectioning. Hematoxylin and eosin (H&E) staining was performed on the sections. The H&E-stained sections were then scanned, observed, and photographed using a Pannoracic MIDI digital slide scanner. Histopathological scoring of the mouse colon tissue was performed based on inflammatory cell infiltration (0-3 points) and tissue damage (0-3 points). The histopathological images and scores of the mouse colon tissue are shown below. Figure 3 As shown.

[0096] The H&E staining results showed that the colonic tissue of mice in the blank control group had clear structures in all layers, intact mucosal epithelium, no obvious epithelial cell degeneration, necrosis, or shedding, and abundant, neatly arranged glands in the lamina propria without obvious inflammatory cell infiltration. In contrast, the colonic tissue of mice in the DSS and DSS+GL groups showed mucosal damage, loss of mucosal epithelium and glandular structures, a significant reduction in goblet cells, edema in the submucosa and lamina propria, and extensive infiltration of lymphocytes and neutrophils. This indicates that DSS and high sulfur content caused severe damage to the colonic tissue of mice, resulting in a significantly elevated colonic tissue pathological score (e.g., ...). Figure 3 B). Compared with the DSS+GL group mice, E10 significantly increased the integrity of colonic crypt structure and reduced goblet cell consumption. Therefore, the E10 strain significantly reduced the histopathological score of colonic tissue. In conclusion, DSS and high sulfur caused severe damage to mouse colonic tissue, while E10 has a certain protective effect on colonic tissue.

[0097] Colonic tissue sections and histopathological scores of mice with a dual intervention of DSS and a high-sulfur diet, induced by strain Lactobacillus paracasei E10, were observed. In the DSS-induced colitis model, the colonic mucosal barrier of mice showed significant damage and numerous inflammatory cell infiltration foci. Similar histopathological manifestations were observed under the combined influence of DSS and a high-sulfur diet, with the histopathological score of the DSS+GL group being higher than that of the DSS group. However, after supplementation with E10, the colonic barrier of mice significantly recovered to a healthy phenotype, and its histopathological score was significantly lower than that of the DSS+GL group. Supplementation with Lgg or Jn1 also showed some recovery of intestinal barrier damage in mice, but their histopathological scores were higher than those of the E10 group, indicating that supplementation with E10 was more effective in restoring intestinal barrier damage than supplementation with Lgg or Jn1.

[0098] Example 4: Detection of cytokine levels in colonic tissue of mice with DSS-induced ulcerative colitis using strain Lactobacillus paracasei E10.

[0099] The levels of three pro-inflammatory cytokines, IL-6, IL-1β, and TNF-α, were detected in the obtained mouse serum. The results are as follows: Figure 4 As shown in AC:

[0100] Compared with the control group, the levels of pro-inflammatory factors IL-6, IL-1β and TNF-α in the colon of mice in the model group were significantly increased, indicating that DSS caused a severe intestinal inflammatory response in mice.

[0101] Compared with the DSS group, the high-sulfur diet promoted an increase in the levels of two cytokines, IL-1β and TNF-α.

[0102] Compared with the DSS+GL group, E10 supplementation downregulated three pro-inflammatory cytokines: IL-6, IL-1β, and TNF-α, indicating that E10 probiotics can regulate the immune response dysregulation in colitis mice, thereby reducing intestinal inflammatory damage.

[0103] Compared with the DSS+GL group, LGG supplementation significantly downregulated the three pro-inflammatory cytokines IL-6, IL-1β, and TNF-α.

[0104] Compared with the DSS+GL group, Jn1 supplementation reduced the levels of three pro-inflammatory cytokines: IL-6, IL-1β, and TNF-α.

[0105] A comparison of inflammatory cytokines in the serum of mice supplemented with E10 and those supplemented with Lgg bacteria revealed that E10 bacteria had a better downregulation effect on three pro-inflammatory cytokines: IL-6, IL-1β, and TNF-α than Lgg bacteria.

[0106] A comparison of inflammatory cytokines in the serum of mice supplemented with E10 and Jn1 bacteria revealed that E10 bacteria had a better downregulation effect on three pro-inflammatory cytokines: IL-6, IL-1β, and TNF-α than Jn1 bacteria.

[0107] Example 5: Effect of strain Lactobacillus paracasei E10 on the proportion of sulfate-reducing bacteria in the colonic contents of mice with DSS-induced ulcerative colitis.

[0108] like Figure 5 As shown in Figure A, the results indicate that supplementing with E10 probiotics significantly reduced the proportion of sulfate-reducing bacteria in the mouse intestines, despite the increased proportion caused by the combined effects of DSS and a high-sulfur diet. Supplementation with either Lgg or Jn1 probiotics also reduced the proportion of sulfate-reducing bacteria, but the reduction was not as significant as with E10 supplementation. Furthermore, there was no significant difference in sulfate-reducing bacteria content between the Lgg and DSS+GL groups, while significant differences were observed between the E10 and Lgg groups, and between the E10 and Jn1 groups.

[0109] Example 6: Detection of hydrogen sulfide content in feces of mice with DSS-induced ulcerative colitis using strain Lactobacillus paracasei E10

[0110] Mouse feces were collected and weighed. The tissue mass (g) was homogenized in an ice bath at a ratio of 1:5-10 to the extraction liquid volume (mL) (it is recommended to weigh approximately 0.1g of tissue and add 1mL of extraction liquid). The homogenate was then centrifuged at 12000rpm and 4℃ for 10min. The supernatant was collected, placed on ice to dry, and reagents were added according to the reagent manufacturer's instructions. The results were then incorporated into a standard curve for calculation. The results are as follows: Figure 5 As shown in B. Figure 5 As shown in Figure B, the hydrogen sulfide content in mouse feces was significantly increased in both the model group and the high-sulfur group, while it significantly decreased after E10 gavage treatment. Compared with the DSS+GL group, the hydrogen sulfide content in mice administered the Lgg bacterial agent by gavage showed no significant change; compared with the Lgg group, the hydrogen sulfide content in mice administered the E10 bacterial agent by gavage was significantly decreased; compared with the DSS+GL group, the hydrogen sulfide content in mice administered the Jn1 bacterial agent by gavage decreased, but its effect on reducing hydrogen sulfide was not as significant as that of E10 bacterial agent supplementation.

[0111] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A type of Lactobacillus paracasei Lactobacillus paracasei E10, characterized in that, The Lactobacillus paracasei Lactobacillus paracasei E10 was deposited on June 21, 2021, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 22744.

2. The Lactobacillus paracasei as described in claim 1 Lactobacillus paracasei Application of E10 in the preparation of drugs to relieve ulcerative colitis.

3. The application according to claim 2, characterized in that: The aforementioned medication for relieving ulcerative colitis contains Lactobacillus paracasei. Lactobacillus paracasei E10 has a viable bacterial count of not less than 1×10⁻⁶. 6 CFU / mL or not less than 1×10 6 CFU / g.

4. The application according to claim 2, characterized in that: The drug contains a drug carrier and / or pharmaceutical excipients.

5. A strain containing the Lactobacillus paracasei as described in claim 1 Lactobacillus paracasei E10 bacterial agent.

6. The microbial agent according to claim 5, characterized in that: The bacterial agent is a solid bacterial agent.

7. The microbial agent according to claim 5, characterized in that: The bacterial agent is a liquid bacterial agent.

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