Plant lactobacillus and application thereof in improving intestinal inflammation and repairing intestinal mucosal injury

Abstract

This invention relates to the field of functional microbial screening and application technology, specifically providing a strain of *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum This study investigated the *Lactobacillus plantarum* strain and its application in improving intestinal inflammation and repairing intestinal mucosal damage. The strain was deposited on August 1, 2024, 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. 31521. This strain exhibits significant antioxidant, antibacterial, and anti-inflammatory effects, and can repair intestinal barrier damage in colitis-affected mice, promote intestinal stem cell regeneration, promote the growth of beneficial intestinal bacteria, and inhibit the proliferation of harmful bacteria. It can be widely used for the prevention and treatment of enteritis. field The prospects are promising.

Description

Technical Field

[0001] This invention relates to the field of microbial screening and application technology, specifically providing a strain of *Lactobacillus plantarum* and its application in improving intestinal inflammation and repairing damage to the intestinal mucosal barrier. Background Technology

[0002] The mucosal barrier is a crucial barrier in the intestine, primarily composed of interconnected intestinal epithelial cells. Tight junction proteins are the most important type of junction, playing a vital role in the gut. Furthermore, alterations in the composition of the gut microbiota are a significant contributing factor to impaired intestinal epithelial barrier function and the occurrence and recurrence of enteritis. Disruption of the intestinal epithelial barrier integrity leads to the translocation of symbiotic intestinal flora and pathogenic microorganisms to the lamina propria, ultimately inducing intestinal inflammation. This manifests primarily as: increased intestinal permeability, allowing lipopolysaccharides to enter the systemic circulation and triggering an inflammatory response; affecting the expression and distribution of tight junctions between intestinal epithelial cells; inducing oxidative stress and apoptosis in epithelial cells; increasing the levels of barrier-damaging cytokines such as TNF-α, IL-1β, and IL-6, while decreasing the levels of barrier-forming cytokines such as IL-10, IL-17, and IL-22; and increasing the number of pathogenic bacteria in the gut microbiota. Current treatments for enteritis focus on controlling inflammation, with limited effectiveness in restoring intestinal epithelial barrier function; and there is currently a lack of drugs that can cure enteritis. Biologics can be used to treat this condition, but they are generally expensive, and their efficacy and safety can be significantly reduced due to immunogenicity.

[0003] Studies have found that probiotic strains can regulate the gut microbiota and have high safety profiles, making them a promising means of preventing or adjuvant treatment for intestinal barrier damage. Lactic acid bacteria are widely present in the human digestive tract and are closely related to human diet. Therefore, screening for lactic acid bacteria capable of regulating intestinal barrier damage is essential. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of *Lactiplantibacillus plantarum* and its application in improving intestinal inflammation and repairing intestinal mucosal damage. This *Lactiplantibacillus plantarum* exhibits significant antioxidant and pathogenic bacteria-inhibiting capabilities, effectively alleviating lipopolysaccharide-induced cellular oxidative stress damage, reducing intestinal inflammation, strengthening intestinal tight junctions, and repairing damaged intestinal mucosal barriers.

[0005] This invention provides a strain of *Lactobacillus plantarum*, namely *Lactobacillus plantarum* W8, which was deposited on August 1, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo. 31521.

[0006] The present invention also provides the application of the *Lactobacillus plantarum* strain W8 in the preparation of food.

[0007] The present invention also provides the use of the aforementioned Lactobacillus plantarum strain W8 in the preparation of medicines for the prevention or treatment of enteritis.

[0008] The present invention also provides a probiotic preparation comprising the Lactobacillus plantarum strain W8.

[0009] The probiotic preparation also contains any one or more combinations of Bifidobacterium longum, Lactobacillus casei, Bacillus coagulans, Halophilic tetracoccus, Lactobacillus fermentum, Lactobacillus paracasei, Bifidobacterium infantis, Bifidobacterium adolescentis, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus bulgaricus, Pediococcus lactis, and Leuconostoc mesenteroides.

[0010] The probiotic preparation contains no less than 10 live bacteria of Lactobacillus plantarum W8 strain. 8 CFU / g.

[0011] The present invention also provides the application of the probiotic preparation in the preparation of food.

[0012] The present invention also provides the use of the probiotic preparation in the preparation of medicines for the prevention or treatment of enteritis.

[0013] Beneficial effects

[0014] (1) *Lactobacillus plantarum* W8 exhibits strong acid and bile salt tolerance. After treatment at pH 3.0 for 3 hours, its survival rate remained at 92.54%; after treatment with 0.2% bile salts for 4 hours, its survival rate reached 85.76%. *Lactobacillus plantarum* W8 demonstrates strong tolerance to the digestive tract environment and can effectively colonize the intestines. After treatment with simulated gastric juice for 2 hours and simulated intestinal juice for 3 hours, its survival rate remained at 90.35%, and after treatment with simulated intestinal juice for 24 hours, its survival rate remained as high as 41.18%.

[0015] (2) Lactobacillus plantarum W8 has significant antioxidant and antibacterial capabilities. Its fermentation supernatant has scavenging rates of 96.92% and 94.31% for DPPH free radicals and ABST+ free radicals, respectively. It has outstanding inhibitory capabilities against pathogenic bacteria Salmonella and Cronobacter sakazakii, with inhibition zone diameters of 23.47 mm and 19.82 mm, respectively. The inhibition zone diameter is more than twice that of the standard Lactobacillus plantarum ATCC8014.

[0016] (3) Lactobacillus plantarum W8 has a strong lipid-lowering function. Its cholesterol removal rate reaches 89.05% and its triglyceride degradation rate is 72.74%, achieving unexpected technical results.

[0017] (4) *Lactobacillus plantarum* W8 can effectively alleviate lipopolysaccharide (LPS)-induced oxidative stress and has a significant ability to improve the intestinal barrier. Intervention with cell-free supernatant of *Lactobacillus plantarum* W8 reversed the levels of malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT) induced by LPS. Compared with the LPS-induced Caco2 cell damage model group, the *Lactobacillus plantarum* W8 intervention group showed a significant decrease in MDA content (a lipid peroxidation damage indicator) and a significant increase in the activities of antioxidant indicators GSH-PX, SOD, and CAT, with very significant effects, approaching the levels of the normal group. Intervention with fermentation supernatant of *Lactobacillus plantarum* W8 significantly increased the epithelial resistance of Caco2 cells and improved the LPS-induced increase in permeability.

[0018] (5) Lactobacillus plantarum W8 can significantly reduce intestinal permeability, relieve intestinal inflammation, reduce intestinal damage and repair intestinal mucosa, significantly enhance intestinal tight junctions, maintain intestinal barrier function, effectively promote the recovery and renewal of intestinal stem cells, regulate the composition of intestinal flora in colitis hosts, and balance intestinal flora homeostasis.

[0019] Intervention with Lactobacillus plantarum W8 significantly reversed the DSS-induced increase in serum D-lactic acid (D-LA) and diamine oxidase (DAO) levels, reduced the secretion of pro-inflammatory factors to varying degrees, thereby alleviating intestinal inflammation in mice and reducing the degree of colonic damage.

[0020] After intervention with *Lactobacillus plantarum* W8, the expression levels of the intestinal tight junction genes ZO-1, Occludin, Claudin-1, and Claudin-2 in colitis mice were significantly increased, approaching the levels of the normal group; the expression of intestinal antimicrobial peptide (AMP) genes was downregulated; the expression and distribution of the intestinal stem cell marker LGR5 were improved, with the LGR5 protein level being twice that of the colitis model group and comparable to the expression level of the normal group; the abundance of short-chain fatty acid-producing bacteria *Allobaculum*, *Muribaculaceae*, and *Bifidobacterium* in the mouse intestinal contents increased, while the abundance of bacteria associated with colitis, such as *Faecalibaculum* and *Candidatus Saccharimonas*, decreased, and the abundance and composition of the intestinal flora were closer to those of the normal group.

[0021] The *Lactobacillus plantarum* W8 provided by this invention has significant antioxidant, antibacterial, and anti-inflammatory effects. It can repair intestinal barrier damage in mice with colitis, promote the renewal of intestinal stem cells, promote the growth of beneficial bacteria in the intestine, and inhibit the proliferation of harmful bacteria. It can be widely used for the prevention and treatment of enteritis and has broad market prospects. Attached image description:

[0022] Figure 1 A phylogenetic tree of *Lactobacillus plantarum* based on the 16S rDNA gene sequence;

[0023] Figure 2 Image of colony morphology of Lactobacillus plantarum W8 on MRS plate;

[0024] Figure 3 The results of *Lactobacillus plantarum* inhibiting intestinal pathogens are shown in the figure (A represents Salmonella; B represents *Cronobacter sakazakii*).

[0025] Figure 4 The results are from the MTT assay for cytotoxicity.

[0026] Figure 5 The results show the content of antioxidant enzymes and oxidative stress products in Caco2 cells.

[0027] Figure 6 The results are from the Caco2 cell permeability assay.

[0028] Figure 7 The serum diamine oxidase (DAO) and D-lactic acid (D-LA) levels in each group of mice were measured.

[0029] Figure 8 The levels of cytokines in the colon tissue of mice in each group;

[0030] Figure 9 HE staining images of colon tissue from each group of mice;

[0031] Figure 10 To determine the expression of tight junction genes in the colon tissue of mice in each group;

[0032] Figure 11 Expression of antimicrobial peptide genes in colon tissue of mice in each group;

[0033] Figure 12 The expression of LGR5 protein in the colon of mice in each group;

[0034] Figure 13 The composition of intestinal contents microbiota at the genus level in each group of mice. Detailed Implementation

[0035] To more clearly illustrate the technical solution of the present invention, the following detailed description is provided in conjunction with specific embodiments, but these descriptions should not be construed as limiting the present invention; they are merely some embodiments of the present invention.

[0036] Example 1: Isolation, screening and identification of Lactobacillus plantarum W8

[0037] 1.1 Isolation and screening of lactobacilli

[0038] 0.1 g of naturally fermented soybean paste from Northeast China was mixed with 900 μL of sterile physiological saline and serially diluted. Three consecutive dilutions were plated onto MRS solid medium and incubated at 37°C for 48 h. Single colonies were picked and further purified by streak plating. Twelve strains of Lactobacillus were finally isolated and named W1, W2, W3, ..., W8.

[0039] 1.2 Secondary screening of antioxidant lactobacilli

[0040] The 12 Lactobacillus strains obtained from the initial screening were passaged three times consecutively in MRS liquid medium, centrifuged at 4000 rpm for 10 min at 4°C, washed three times consecutively with PBS, and the bacterial concentration was adjusted to 1.0 × 10⁹ CFU / mL. The culture was then sonicated at 240 W for 10 min (with 5 s intervals between sonication sessions in an ice bath). Subsequently, the culture was centrifuged at 6000 rpm for 10 min at 4°C, and the supernatant was filtered through a 0.22 μm filter to obtain a cell-free supernatant.

[0041] The antioxidant capacity of the strain was demonstrated by measuring the scavenging ability of cell-free supernatant of the strain on DPPH and ABTS free radicals.

[0042] (1) DPPH free radical scavenging test

[0043] Equal volumes of cell-free supernatant were mixed with 0.1 mol / L DPPH solution (dissolved in 95% ethanol) by shaking for 1 min, and then allowed to stand at room temperature in the dark for 30 min. After centrifugation, the supernatant was collected and the absorbance was measured at a wavelength of 517 nm. An equal volume of ethanol was used as a blank control. The calculation formula is as follows:

[0044]

[0045] (2) ABTS free radical scavenging test

[0046] Mix cell-free supernatant with ABTS working solution at a ratio of 1:7, incubate at 25°C for 10 min, and then measure the absorbance at 734 nm. Use an equal volume of anhydrous ethanol as a blank control. The calculation formula is as follows:

[0047]

[0048] The results showed that among the 12 Lactobacillus strains obtained in the initial screening of this invention, 3 strains had strong scavenging ability against DPPH and ABTS free radicals. Among them, strain W8 had the highest scavenging rate against DPPH and ABTS free radicals, reaching 96.92% and 94.31% respectively, and its antioxidant effect was very significant.

[0049] 1.3 Identification of strains

[0050] Using a single colony of W8 as a template, and universal bacterial primers 27F and 1492R as primers, 16S rDNA PCR amplification was performed in a certain amount of 2×Taq PCR mix. The reaction conditions were: 98°C for 5 min; 94°C for 30 s, 55°C for 30 s, 72°C for 90 s; 72°C for 5 min, for 30 cycles. After verification by agarose gel electrophoresis, the PCR products were sent to Shanghai Bioengineering Co., Ltd. for sequence analysis. The determined 16S rDNA sequence was compared with the BLAST sequence in the National Center for Biotechnology Information (NCBI) database. The comparison showed that strain W8 had the highest sequence similarity to *Lactiplantibacillus plantarum*; further analysis was performed using... Figure 1 As can be seen from the phylogenetic tree shown, strain W8 is most closely related to Lactobacillus plantarum.

[0051] like Figure 2 As shown, the colonies of strain W8 are milky white, round, smooth, and have a distinct frankincense aroma. The cells stain blue-purple with Gram stain and are arranged in single rod, double rod, or chain rod formations.

[0052] In summary, based on the colony morphology, physiological and biochemical characteristics, and molecular biological identification results of strain W8, the applicant has determined that the screened strain W8 is a novel *Lactiplantibacillus plantarum*, which has been named *Lactiplantibacillus plantarum* W8. It was deposited on August 1, 2024, 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. 31521.

[0053] Example 2: Analysis of the acid and bile salt tolerance of Lactobacillus plantarum W8

[0054] 2.1 Acid resistance test

[0055] The bacteria were inoculated into MRS liquid culture medium and cultured at 37°C. Samples were taken every 2 hours, and the absorbance was measured at 600 nm to obtain the growth curve of Lactobacillus plantarum W8.

[0056] (1) Control group: The activated Lactobacillus plantarum W8 solution was inoculated into liquid MRS medium with a pH of 6.7 at a ratio of 2% (V / V);

[0057] (2) Experimental group: Activated *Lactobacillus plantarum* W8 bacterial suspension was inoculated into liquid MRS medium with pH values ​​of 2.0, 2.5, and 3.0 at a ratio of 2% (V / V); cultured at 37℃ for 3 h, and the absorbance (OD) was measured at 600 nm. 600 ).

[0058] The survival rate of the strain is calculated using the following formula.

[0059] Survival rate (%) = (Experimental OD) 600 / Comparison OD 600 )×100%.

[0060] The results showed that the *Lactobacillus plantarum* W8 provided by this invention has a strong acid resistance. After treatment at pH 3.0 for 3 hours, its survival rate still reached 92.54%, indicating that the strain can enter the intestine through gastric juice and has the prerequisite to enter the intestine and exert a probiotic effect.

[0061] 2.2 Test of bile salt tolerance

[0062] Inoculation was performed at a rate of 2% in MRS liquid medium containing sodium taurocholate at concentrations of 0.10%, 0.20%, 0.30%, and 0.40%, respectively. After incubation at 37°C for 4 hours, absorbance (OD) was measured at 600 nm. 600 ).

[0063] The results showed that the survival rates of *Lactobacillus plantarum* W8 provided by this invention reached 85.76% and 66.11% respectively after treatment with 0.2% and 0.3% bile salts for 4 hours. This indicates that the strain has strong bile salt tolerance and the ability to enter the intestine and exert a probiotic effect.

[0064] 2.3 Simulated Gastrointestinal Tolerance Test

[0065] A suspension of *Lactobacillus plantarum* W8 bacteria was mixed with simulated gastric fluid at a volume ratio of 1:9 and incubated at 37°C for 2 hours. The incubated bacterial suspension was then washed and mixed with simulated intestinal fluid in the same ratio, and incubated at 37°C for 24 hours. Samples were taken at 3 hours, 6 hours, 12 hours, and 24 hours, and the viable bacterial count was determined using the plate count method to calculate the survival rate of *Lactobacillus plantarum* W8. The calculation formula is as follows:

[0066] Survival rate (%) = (Number of viable bacteria after passing through intestinal fluid / Number of viable bacteria before adding gastric fluid) × 100%.

[0067] Table 1 Survival rate of *Lactobacillus plantarum* W8 after digestion with simulated gastrointestinal fluid.

[0068] Survival rate <![CDATA[90.35%±3.25 a ]]> <![CDATA[75.01%±3.26 c ]]> <![CDATA[62.56%±4.63 d ]]> <![CDATA[41.18%±4.60 d ]]>

[0069] As shown in Table 1, the survival rate of *Lactobacillus plantarum* W8 reached 90.35% after treatment with simulated gastric juice for 2 hours and simulated intestinal juice for 3 hours. Even after treatment with simulated intestinal juice for 24 hours, the survival rate remained as high as 41.18%. This demonstrates that the *Lactobacillus plantarum* W8 provided by this invention exhibits strong tolerance to the digestive tract environment and can effectively colonize the intestines, thus facilitating its probiotic effects.

[0070] Example 3: Determination of the antibacterial activity of Lactobacillus plantarum W8

[0071] The antibacterial activity of Lactobacillus plantarum W8 metabolites was tested using the perforation method.

[0072] Fresh bacterial culture of *Lactobacillus plantarum* W8 was centrifuged at 4°C and 12,000 rpm for 15 min to obtain the supernatant; the concentrations of pathogenic bacteria *Salmonella* and *Enterobacter sakazakii* were adjusted to 1 × 10⁻⁶ using sterile PBS. 8 CFU / mL was spread onto the surface of the culture medium and perforated (pore diameter ±8 mm, depth ±4.5 mm). The medium was sealed with 1% agar, and 100 μL of filtrate was drawn into each well. After incubation at room temperature for 2 hours, the medium was incubated at 37°C. Clear inhibition zones appeared after approximately 18 hours of incubation; the diameter of these inhibition zones was observed and measured. A standard *Lactobacillus plantarum* ATCC8014 was used as a control.

[0073] The results are as follows Figure 3 As shown, *Lactobacillus plantarum* W8 exhibits significant antibacterial activity against both Salmonella and *Enterobacter sakazakii*, with inhibition diameters of 23.47 mm and 19.82 mm, respectively. The antibacterial effect is remarkable, and the diameter of the inhibition zone is more than twice that of the standard *Lactobacillus plantarum* ATCC8014, achieving unexpected technical results.

[0074] Example 4: Determination of the lipid-lowering ability of Lactobacillus plantarum

[0075] 4.1 Experimental Methods

[0076] (1) Determination of cholesterol removal rate

[0077] A suspension of *Lactobacillus plantarum* W8 bacteria was added to a cholesterol culture medium, and the cholesterol content was determined by the o-phthalaldehyde method to calculate the cholesterol removal rate.

[0078] (2) Determination of triglyceride degradation rate

[0079] A suspension of *Lactobacillus plantarum* W8 was added to triglyceride medium and cultured. The degradation rate of triglycerides by *Lactobacillus plantarum* W8 was determined according to the triglyceride kit instructions. The reagent addition method is shown in Table 2. The degradation rate of triglycerides by *Lactobacillus plantarum* was calculated using the following formula.

[0080] Triglyceride degradation rate = (OD546nm of control tube - OD546nm of test tube) / OD546nm of control tube × 100%

[0081] Table 2. Reagent Addition Table for In Vitro Triglyceride Lowering Test of Lactobacillus plantarum W8

[0082]

[0083] 4.2 Results Analysis

[0084] In vitro lipid-lowering experiments show that the *Lactobacillus plantarum* W8 provided by this invention has a strong lipid-lowering function, with a cholesterol removal rate of 89.05% and a triglyceride degradation rate of 72.74%, achieving unexpected technical results.

[0085] Example 5: Regulation of oxidative stress in Caco2 cells by *Lactobacillus plantarum* W8

[0086] 5.1 Strain activation

[0087] The *Lactobacillus plantarum* W8 strain preserved in glycerol was activated in liquid MRS medium, cultured at 37°C for 18 hours, and after 3 generations, it became the experimental strain.

[0088] 5.2 Resuscitation, Culture and Passage of Caco2 Cells

[0089] The human colorectal cancer cell line Caco-2 was extracted from liquid nitrogen, thawed in a 37°C water bath within 3 minutes, then rapidly centrifuged, the culture medium was discarded, 1 mL of DMEM complete culture medium was added and the cells were dispersed and transferred to a culture flask for culture. When the cells reached approximately 80% confluence with the surface of the culture flask, they were digested with trypsin-EDTA and passaged. After 5 passages, the cells were used for subsequent experiments.

[0090] 5.3 Cytotoxicity assay

[0091] Caco-2 cells were injected at a rate of 2 × 10⁻⁶ per ml. 5 Cells were seeded at a concentration of [number] cells per well in 96-well plates and cultured. Cells were washed with phosphate-buffered saline (PBS). Then, 100 μL of bacterial suspension (concentration of 10 [units]) was added. 7 CFU / mL, 10 8 CFU / mL, 10 9 CFU / mL, 10 10Cells were incubated with antibiotic-free dimethyl sulfoxide (DMSO) for 24 hours (CFU / mL) as a control. Next, 20 μL of 0.5 mg / mL thiazolyl blue (MTT, diluted with PBS buffer) was incubated at 37°C in the dark for 4 hours. The supernatant was removed, 150 μL of DMSO was added, and the mixture was shaken for 10 minutes. The absorbance was read at 490 nm using a microplate reader. Results are expressed as the percentage of viable cells compared to the control. The absorbance of the control was considered to represent 100% cell viability.

[0092] 5.4 Caco2 cell grouping and modeling

[0093] Caco2 cells were treated using the following method:

[0094] (1) Blank group: No intervention was given;

[0095] (2) Model group: Caco2 cells were treated with 1 μg / mL LPS (lipopolysaccharide) for 6 h;

[0096] (3) Experimental group: Caco2 cells were treated with 1 μg / mL LPS for 6 h, and then Caco2 cells in the experimental group were treated with cell-free supernatant of Lactobacillus plantarum W8 for 24 h.

[0097] 5.5 Detection of Oxidative Stress Indicators

[0098] After the intervention, the cells were washed by centrifugation and then sonicated in cold PBS. The malondialdehyde (MDA) content and the activities of glutathione peroxidase (GSH-PX), superoxide dismutase (SOD), and catalase (CAT) in Caco2 cells were detected using commercial kits developed by Nanjing Jiancheng.

[0099] 5.6 Results Analysis

[0100] Depend on Figure 4 It can be seen that 10 7 -10 10 CFU / ml bacterial concentration was not toxic to Caco2 cells, therefore a median bacterial concentration of 10 was chosen. 8 Further experiments are needed to determine the CFU / mL concentration. For example... Figure 5 As shown, compared with the normal group, LPS can induce oxidative stress in Caco2 cells, leading to a significant increase in MDA content and a significant decrease in the activities of GSH-PX, SOD, and CAT. After intervention with cell-free supernatant of *Lactobacillus plantarum* W8, the levels of LPS-induced MDA, GSH, SOD, and CAT were reversed, with a very significant effect, approaching the levels of the normal group. This demonstrates that *Lactobacillus plantarum* W8 provided by this invention can effectively alleviate LPS-induced oxidative stress.

[0101] Example 6: The effect of Lactobacillus plantarum W8 on the repair of Caco-2 cell barrier damage

[0102] 6.1 Activation, cell culture, grouping, and modeling of the strain are as described in Example 5.

[0103] 6.2 Measurement of epithelial resistivity

[0104] Log-grown Caco-2 cells were used at a density of 1 × 10⁻⁶ cells per well. 9 Cells were seeded at a concentration of [cells / mL] into 6-well Transwell plates. 1.5 mL of culture medium was added to the top (AP) and 2.5 mL to the basal (BL) wells, respectively. The medium was changed every other day during the first week, and daily thereafter, for approximately 21 days until complete cell differentiation. Cell grouping and modeling methods were the same as in Example 5. After modeling, epithelial resistance was measured using an epithelial resistance meter.

[0105] 6.3 Permeability determination of FITC-Dextran isothiocyanate

[0106] After modeling, the monolayer membrane was washed three times with HBSS. Then, 500 μL of DMEM medium containing 1 mg / mL FITC-Dextran was added to the AP side, and 1.5 mL of DMEM medium was added to the BL side. After 6 hours of treatment, 0.1 mL of culture medium was aspirated from the BL side and added to an opaque black 96-well plate. The fluorescence intensity was measured using a microplate reader at 490–520 nm. Each experiment was repeated three times. The concentration of FITC-Dextran on the outer side and the apparent permeability coefficient (Papp) were calculated based on the standard curve.

[0107] 6.4 Results Analysis

[0108] Depend on Figure 6 It was found that, compared with the normal group, the epithelial resistance of Caco2 cells induced by LPS was significantly reduced, while the intervention of *Lactobacillus plantarum* W8 fermentation supernatant significantly increased the epithelial resistance of Caco2 cells. This indicates that *Lactobacillus plantarum* W8 fermentation supernatant has the potential to improve the compactness of intestinal epithelial cells and enhance the intestinal barrier.

[0109] Compared with the normal group, the FITC permeability of Caco2 cells in the LPS group was significantly increased, reaching 1.5 times that of the normal group; the supernatant of *Lactobacillus plantarum* significantly improved the LPS-induced increase in permeability, approaching the level of the normal group. These results further demonstrate that *Lactobacillus plantarum* W8 supernatant has a significant ability to improve the intestinal barrier.

[0110] Example 7: Application of Lactobacillus plantarum W8 in reducing intestinal permeability and improving intestinal inflammation in mice

[0111] 7.1 Preparation of Lactobacillus plantarum W8 suspension

[0112] Lactobacillus plantarum W8 was incubated in MRS liquid medium at 37°C for 18 hours and passaged three times. After centrifugation, the bacterial cells were collected and washed twice with physiological saline. The bacterial cells were then resuspended in physiological saline to achieve a bacterial concentration of 1 × 10⁻⁶. 9 CFU / mL.

[0113] 7.2 Animal grouping, modeling, and drug administration

[0114] Experimental animals: Healthy male C57BL / 6J mice (20g±2g) at 5 weeks of age were purchased from Liaoning Changsheng Biotechnology Co., Ltd., license number: SVXK(Liaoning)2020-0001, and housed in the SPF-grade laboratory of the Experimental Animal Center of Shenyang Agricultural University. Animal experiments were approved by the Ethics Committee of the Experimental Animal Center of Shenyang Agricultural University, ethics approval number: NO.2023102702. Twenty-four mice were randomly divided into five groups: control group (Con), model group (DSS), negative control group (NC, *Lactobacillus plantarum* standard strain ATCC8014), positive control group (PC, mesalazine), and *Lactobacillus plantarum* intervention group (W8).

[0115] Acute colitis was induced using 2.5% (w / v) sodium dextran sulfate (DSS). During the 7-day experiment, control mice received only water. DSS-treated mice were administered 2.5% DSS by gavage for 5 consecutive days. Mice in the normal control group were orally administered a standard bacterial suspension (10 mg / kg) once daily, mice in the W8 group were orally administered *Lactobacillus plantarum* W8 bacterial suspension (10 mg / kg) once daily, and mice in the PC group were orally administered 200 mg / kg mesalazine daily; all for 7 consecutive days, with DSS supplementation for 5 days. Body weight was recorded daily. Mice were sacrificed on day 7. The colon was removed and its length measured. Colon specimens were fixed in 10% formalin and stained with hematoxylin and eosin (HE).

[0116] 7.3 Enzyme-linked immunosorbent assay

[0117] 100 μL of serum and 100 mg of colon tissue were collected from each group of mice and a colon homogenate (100 mg) was prepared using phosphate-buffered saline (PBS) (w / v 1 / 9). The levels of IL-10, IL-1β, and TNF-α cytokines, as well as serum diamine oxidase (DAO) and D-lactate (D-LA) in the colon homogenate and cell samples were measured according to the manufacturer's (Nanjing Jiancheng, Nanjing, China) instructions.

[0118] 7.4H&E staining

[0119] Fresh colon tissue was rapidly fixed in 4% paraformaldehyde for 48 hours. Subsequently, the tissue samples were dehydrated, embedded in paraffin, and sectioned to a thickness of 4 μm. After dewaxing, the tissue samples were stained with H&E and analyzed using an optical microscope.

[0120] 7.5 Determination of intestinal barrier-related gene expression in colon tissue

[0121] Total RNA was extracted from colon tissue using Trizol reagent, and 1 μg of RNA was reverse transcribed to synthesize cDNA using the M5-Super-qPCR RT kit with gDNA removal agent. Light... mRNA expression was measured using a 96-element instrument. Primer sequences are listed in Table 5 below. The levels of the target gene were quantified relative to the Gapdh gene level, and the results were analyzed using a 2-ΔΔCt analysis method.

[0122] Table 5 Primer sequences

[0123]

[0124] 7.6 Determination of LGR5 expression, a marker of intestinal stem cells, in colon tissue

[0125] (1) Take mouse colon tissue, freeze it with liquid nitrogen and grind it to extract total protein.

[0126] (2) Add an appropriate amount of volumetric cell lysis buffer to the tissue to be lysed, lyse on ice for 10 min, centrifuge at 12000 rpm for 10 min, and then collect the supernatant.

[0127] (3) Protein concentration was measured using a BCA kit.

[0128] (4) Protein denaturation: Add 5×SDS-PAGE Loading buffer and boil at 100℃ for 10 min.

[0129] (5) Gel preparation and electrophoresis: Take 10 μg of protein solution for SDS-PAGE gel electrophoresis, adjust the voltage to 150V, and continue for 1 hour.

[0130] (6) Transfer: Pour in sufficient transfer buffer. Perform quantitative electrophoresis on the sample solution, transfer the protein to a 0.2 μm PVDF membrane at a constant voltage of 80V for 2 hours, and fix the protein on the membrane at a current of 200mA. The transfer time is determined according to the specific molecular weight of the protein, usually 40-90 minutes.

[0131] (7) Sealing: Place the membrane in 5% skim milk and shake to seal, 37°C, 1h.

[0132] (9) Incubation with primary antibody: Dilute LGR5 antibody at a ratio of 1:1000 and incubate overnight at 4°C on a shaker. After incubation with primary antibody, wash the bands three times with PBST buffer at room temperature, each time for 10 min.

[0133] (10) Incubation with HRP secondary antibody: Dilute HRP goat anti-mouse secondary antibody at a concentration of 1:5000 with 5% blocking buffer, place the band in the secondary antibody, and incubate on a shaker at room temperature for 1 hour. After the secondary antibody incubation is completed, rinse the band three times with PBST buffer solution at room temperature, each time for 10 minutes.

[0134] (11) Development: The signal was further displayed using HRP color imaging on the Amersham Imager 600 imaging system.

[0135] (12) Quantitative analysis: Western blot was performed using ImageJ software to quantitatively analyze LGR5 and the internal reference (β-actin), and the gray-scale ratio of the target protein internal reference was calculated.

[0136] 7.7 Determination of intestinal microbiota composition

[0137] After the mice were euthanized, their colon contents were quickly collected and sent to Shanghai Meiji Biotechnology Co., Ltd. for sequencing.

[0138] 7.9 Results Analysis:

[0139] like Figure 7 As shown, compared with the normal group, the levels of D-LA and DAO were significantly increased in the colitis model group (DSS) and the negative control group (NC), indicating that the intestinal mucosal barrier of mice was damaged. Compared with the DSS group, the levels of D-LA and DAO in the *Lactobacillus plantarum* W8 intervention group were significantly reduced, and the effect was close to that in the positive drug group (PC). This indicates that *Lactobacillus plantarum* W8 can significantly reduce intestinal permeability and alleviate DSS-induced intestinal damage.

[0140] like Figure 8 As shown, compared with the DSS group, the W8 intervention group mice showed significantly lower levels of inflammatory factors and a highly significantly higher level of IL-10, and the W8 intervention effect was not significantly different from that of the PC group. This indicates that *Lactobacillus plantarum* W8 intervention in mice can reduce the secretion of pro-inflammatory factors to varying degrees, thereby alleviating intestinal inflammation and improving the intestinal barrier defense capacity of mice.

[0141] like Figure 9As shown, the colonic mucosa of mice in the normal group was intact and continuous, with neatly arranged glands, normal crypts, and no inflammatory cell infiltration. Mice in the DSS group showed severe colonic damage, with extensive inflammatory cell infiltration in the mucosa and submucosa. The NC group showed colonic lymph node hyperplasia with a small amount of inflammatory infiltration, while the W8 intervention group and PC group showed neatly arranged colonic glands and normal crypts. This further demonstrates that the *Lactobacillus plantarum* W8 provided by this invention has the effect of alleviating intestinal inflammation, reducing intestinal damage, and repairing the intestinal mucosa.

[0142] like Figure 10 The results showed that after intervention with *Lactobacillus plantarum* W8, the expression levels of the intestinal tight junction genes ZO-1, Occludin, Claudin-1, and Claudin-2 in colitis mice were significantly increased, reaching the levels of the normal group. This is consistent with the results of in vitro experiments, indicating that *Lactobacillus plantarum* W8 can significantly enhance intestinal tight junctions and maintain intestinal barrier function.

[0143] like Figure 11 As shown, antimicrobial peptides (AMPs) play an important role in the establishment and maintenance of the mucosal barrier. Therefore, the expression of AMP genes Reg3a, Reg3g, and Defa5 was examined. Compared with the normal group, AMP gene expression was significantly reduced in the DSS and NC groups. *Lactobacillus plantarum* W8 intervention significantly inhibited the downregulation of AMP gene expression, with a slightly better effect than the PC group. This indicates that *Lactobacillus plantarum* W8 can help the intestine resist pathogen invasion by regulating antimicrobial peptide expression.

[0144] like Figure 12 As shown, intervention with *Lactobacillus plantarum* W8 significantly promoted the expression and distribution of LGR5, a marker of intestinal stem cells, in colitis mice. The LGR5 protein level was twice that of the colitis model group and comparable to that of the normal group. This indicates that *Lactobacillus plantarum* W8 can effectively promote the recovery and renewal of intestinal stem cells.

[0145] like Figure 13 As shown, the changes in the gut microbiota of mice were analyzed, and the changes at the genus level were as follows: Figure 7As shown in the figure, compared with the normal group, the abundance of short-chain fatty acid-producing bacteria such as Allobaculum, Muribactaceae, and Bifidobacterium was significantly reduced in DSS-induced colitis mice, while the abundance of colon inflammation-related bacteria such as Faecalibaculum and Candidatus Saccharimonas increased. However, intervention with Lactobacillus plantarum significantly reversed this trend, and the abundance and composition of the intestinal flora were closer to those of the normal group. These results indicate that Lactobacillus plantarum W8 can regulate the composition of the intestinal flora in colitis hosts, promote the growth of beneficial bacteria, inhibit the proliferation of harmful bacteria, and thus balance intestinal flora homeostasis.

[0146] In summary, the *Lactobacillus plantarum* W8 provided by this invention has significant antioxidant, antibacterial, and anti-inflammatory effects. It can repair intestinal barrier damage in mice with colitis, promote the renewal of intestinal stem cells, promote the growth of beneficial bacteria in the intestine, and inhibit the proliferation of harmful bacteria. It can be widely used for the prevention and treatment of enteritis.

Claims

1. A strain of *Lactobacillus plantarum*, characterized in that, The plant lactobacillus is *Lactobacillus plantarum* (… Lactiplantibacillus plantarum )W8, with accession number CGMCC NO. 31521.

2. The application of the *Lactobacillus plantarum* as described in claim 1 in the preparation of food.

3. The use of the *Lactobacillus plantarum* as described in claim 1 in the preparation of a medicament for treating colitis.

4. A probiotic preparation, characterized in that, The probiotic preparation comprises *Lactobacillus plantarum* as described in claim 1.

5. The probiotic preparation as described in claim 4, characterized in that, The probiotic preparation also contains any one or more combinations of Bifidobacterium longum, Lactobacillus casei, Bacillus coagulans, Lactobacillus fermentum, Lactobacillus paracasei, Bifidobacterium infantis, Bifidobacterium adolescentis, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus bulgaricus, Pediococcus lactis, and Leuconostoc mesenteroides.

6. The probiotic preparation according to claim 4, characterized in that, The probiotic preparation contains no less than 10 live Lactobacillus plantarum bacteria. 8 CFU / g.

7. The use of the probiotic preparation according to any one of claims 4-6 in the preparation of food.

8. The use of the probiotic preparation according to any one of claims 4-6 in the preparation of a medicament for treating colitis.

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