Phytobacterium plantarum and application thereof in improving intestinal inflammation and repairing intestinal mucosa injury

Abstract

The invention relates to the technical field of functional microorganism screening and application, and particularly provides a strain of lactobacillus plantarum and an application of the lactobacillus plantarum in improvement of intestinal inflammation and repair of intestinal mucosa injury. The plant lactobacillus is preserved in the China General Microbiological Culture Collection Center (CGMCC for short) on August 1, 2024, the address is No.3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number of the plant lactobacillus is CGMCC No.31521. The strain has remarkable antioxidant, bacteriostatic and anti-inflammatory effects, can repair intestinal barrier injury of mice with colitis, promote renewal of intestinal stem cells, promote growth of intestinal beneficial bacteria and inhibit proliferation of harmful bacteria, can be widely applied to prevention and treatment of enteritis, and has a wide market prospect.

Description

Technical Field

[0001] The invention relates to the technical field of microbial screening and application, and specifically provides a strain of Lactobacillus plantarum and application thereof in improving intestinal inflammation and repairing intestinal mucosal barrier damage. Background Art

[0002] The mucosal barrier is an important barrier in the intestine, which is mainly composed of intestinal epithelial-related cells. Among them, tight junction proteins are the most important connection mode and play a role in the intestine. In addition, changes in the composition of intestinal microorganisms are an important cause of impaired intestinal epithelial barrier function and the occurrence and recurrence of enteritis. The destruction of the integrity of the intestinal epithelial barrier can cause the intestinal symbiotic flora and pathogenic microorganisms to transfer to the intestinal lamina propria, and ultimately induce intestinal inflammation. Its main manifestations are: increased intestinal permeability, lipopolysaccharide entering the systemic circulation, triggering an inflammatory response; affecting the expression and distribution of tight junctions between intestinal epithelial cells; inducing epithelial cell oxidative stress and apoptosis, increasing the levels of barrier-damaging cytokines such as TNF-α, IL-1β, IL-6, and reducing the levels of barrier-forming cytokines such as IL-10, IL-17, IL-22; and increasing the number of pathogenic bacteria in the intestinal flora. At present, drugs for the treatment of enteritis focus on controlling inflammation, but have limited effects on restoring the intestinal epithelial barrier function, and there is still a lack of drugs that can cure enteritis. Biologic drugs can be used for treatment but are generally expensive, and their efficacy and safety are greatly reduced due to immunogenicity.

[0003] Studies have found that probiotic strains can regulate intestinal flora and are highly safe, and are expected to become an important means of preventing or assisting in the treatment of intestinal barrier damage. Lactobacillus is commonly found in the human digestive tract and is closely related to human diet. Therefore, it is necessary to screen out a lactic acid bacteria that can regulate intestinal barrier damage. Summary of the invention

[0004] The present invention aims to provide a plant lactobacillus (Lactiplantibacillus plantarum) and its application in improving intestinal inflammation and repairing intestinal mucosal damage. The plant lactobacillus has significant antioxidant and pathogenic bacteria inhibition capabilities, can effectively alleviate lipopolysaccharide-induced cell oxidative stress damage, reduce intestinal inflammation, enhance intestinal tight junctions, and repair damaged intestinal mucosal barriers.

[0005] On the one hand, the present invention provides a plant lactobacillus, which is the plant lactobacillus W8 strain, which has been deposited in the General Microbiology Center of the China Culture Collection Administration (CGMCC) on August 1, 2024, with an address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and its deposit number is CGMCC No. 31521.

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

[0007] The present invention also provides application of the Lactobacillus plantarum W8 strain in preparing medicines for preventing or treating enteritis.

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

[0009] The probiotic preparation further comprises any one or more combinations of Bifidobacterium longum, Lactobacillus casei, Bacillus coagulans, Tetragenococcus halophilus, Lactobacillus fermentum, Lactobacillus paracasei, Bifidobacterium infantis, Bifidobacterium adolescentis, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus bulgaricus, Pediococcus acidilactici and Leuconostoc lactis.

[0010] The amount of live bacteria of Lactobacillus plantarum W8 strain in the probiotic preparation is not less than 10 8 CFU / g.

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

[0012] The present invention also provides application of the probiotic preparation in preparing medicines for preventing or treating enteritis.

[0013] Beneficial Effects

[0014] (1) Lactobacillus plantarum W8 has strong acid and bile salt resistance. After being treated at pH 3.0 for 3 hours, its survival rate can still reach 92.54%; after being treated in the presence of 0.2% bile salt for 4 hours, its survival rate reaches 85.76%. Lactobacillus plantarum W8 has strong tolerance to the digestive tract environment and can achieve effective colonization in the intestine. After being treated with simulated gastric juice for 2 hours and simulated intestinal juice for 3 hours, its survival rate can still reach 90.35%. After being treated with simulated intestinal juice for 24 hours, the survival rate is still as high as 41.18%.

[0015] (2) Lactobacillus plantarum W8 has significant antioxidant and antibacterial abilities. The scavenging rates of its fermentation supernatant for DPPH free radicals and ABST+ free radicals are 96.92% and 94.31%, respectively. It has an outstanding inhibitory ability against pathogenic bacteria Salmonella and Enterobacter sakazakii, with the diameters of the inhibition zones reaching 23.47 mm and 19.82 mm, respectively, which 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 effects.

[0017] (4) Lactobacillus plantarum W8 can effectively alleviate lipopolysaccharide (LPS)-induced oxidative stress and has a significant improvement on 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 injury model group, the lipid peroxidation damage index MDA content in the Lactobacillus plantarum W8 intervention group was significantly reduced, and the antioxidant indexes GSH-PX, SOD and CAT activities were significantly increased. The effect was very significant and close to the level of the normal group. Intervention with fermentation supernatant of Lactobacillus plantarum W8 significantly increased the epithelial resistance of Caco2 cells and improved the increased permeability caused by LPS.

[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 intestinal stem cell recovery and renewal, regulate the composition of the intestinal flora of colitis hosts, and balance the homeostasis of intestinal flora.

[0019] Intervention with Lactobacillus plantarum W8 can significantly reverse the DSS-induced increase in serum D-lactic acid (D-LA) and diamine oxidase (DAO) levels, reduce the secretion of pro-inflammatory factors to varying degrees, achieve the purpose of alleviating intestinal inflammation in mice, and reduce the degree of colon damage.

[0020] After intervention with Lactobacillus plantarum W8, the expression levels of intestinal tight junction genes ZO-1, Occludin, Claudin-1, and Claudin-2 in colitis mice increased significantly, approaching the levels of the normal group; the expression of intestinal antimicrobial peptides (AMPs) genes was downregulated; the expression and distribution of the intestinal stem cell marker LGR5, the LGR5 protein level was twice that of the colitis model group, which was equivalent to the expression level of the normal group; the abundance of short-chain fatty acid-producing bacteria Allobaculum, Muribaculaceae, and Bifidobacterium in the intestinal contents of mice increased, while the abundance of flora related to colon inflammation, 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 the present invention has significant antioxidant, antibacterial and anti-inflammatory effects, can repair intestinal barrier damage in colitis mice, promote intestinal stem cell renewal, promote the growth of beneficial intestinal bacteria, and inhibit the proliferation of harmful bacteria. It can be widely used in the prevention and treatment of enteritis and has broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0023] Figure 2 This is the colony morphology of Lactobacillus plantarum W8 on the MRS plate;

[0024] Figure 3 This is the result of Lactobacillus plantarum inhibiting intestinal pathogens (A is Salmonella; B is Enterobacter sakazakii);

[0025] Figure 4 The results of MTT cell toxicity assay;

[0026] Figure 5 The results of the determination of antioxidant enzymes and oxidative stress products in Caco2 cells;

[0027] Figure 6 The results of Caco2 cell permeability assay;

[0028] Figure 7 is the serum diamine oxidase (DAO) and D-lactic acid (D-LA) content of mice in each group;

[0029] Figure 8 is the cytokine level in colon tissue of mice in each group;

[0030] Fig. 9 HE staining images of colon tissues of mice in each group;

[0031] Fig.10 Tight junction gene expression in colon tissue of mice in each group;

[0032] Fig.11 The antimicrobial peptide gene expression in colon tissue of mice in each group;

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

[0034] Fig.13 The microbial composition of intestinal contents at the genus level in each group of mice. DETAILED DESCRIPTION

[0035] In order to more clearly describe the technical solution of the present invention, it is further described below in conjunction with specific embodiments, but it cannot be used to limit the present invention. This is only a partial embodiment of the present invention.

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

[0037] 1.1 Isolation and screening of lactobacilli

[0038] 0.1g of naturally fermented soybean paste from Northeast China was mixed with 900μL of sterile saline and gradiently diluted. Three consecutive gradient dilutions were spread on MRS solid medium and cultured at 37℃ for 48h. Single colonies were picked and further isolated and purified by plate streaking. Finally, 12 strains of lactobacillus were isolated and named W1, W2, W3, ..., W8.

[0039] 1.2 Rescreening of Antioxidant Lactobacillus

[0040] The 12 strains of lactobacilli obtained from the initial screening were subcultured three times in MRS liquid medium, centrifuged at 4°C, 4000r / min for 10min, washed three times with PBS, and the bacterial solution concentration was adjusted to 1.0×109CFU / mL. Ultrasonic disruption was performed at 240W power for 10min (5s per ultrasonic treatment and 5s interval in an ice bath). Subsequently, centrifuged at 4°C, 6000r / min for 10min, and the supernatant was filtered through a 0.22μm filter to obtain a cell-free supernatant.

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

[0042] (1) DPPH free radical scavenging test

[0043] After equal amounts of cell-free supernatant and 0.1 mol / L DPPH solution (dissolved in 95% ethanol) were shaken and mixed for 1 min, the mixture was allowed to stand at room temperature in the dark for 30 min, and then the supernatant was centrifuged 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] Take the cell-free supernatant and ABTS working solution and mix them in a ratio of 1:7. After incubation at 25°C for 10 min, 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 3 of the 12 lactobacilli obtained in the initial screening of the present invention had strong scavenging abilities for both DPPH and ABTS free radicals, among which the W8 strain had the highest scavenging rates for DPPH and ABTS free radicals, reaching 96.92% and 94.31% respectively, and had a significant antioxidant effect.

[0049] 1.3 Identification of strains

[0050] Using the W8 single colony as a template, bacterial universal primers 27F and 1492R as primers, a certain amount of 2×Taq PCRmix was added for 16S rDNA PCR amplification. The reaction conditions were: 98C 5min; 94℃30s, 55℃30s, 72℃90s; 72℃5min, 30 cycles. After verification by agarose gel electrophoresis, the PCR product was sent to Shanghai Bioengineering Co., Ltd. for sequence analysis. The measured 16S rDNA sequence was BLAST aligned in the National Center for Biotechnology Information (NCBI) database. After alignment, the sequence similarity between the W8 strain and Lactiplantibacillus plantarum was the highest; further through Figure 1 The phylogenetic tree shown shows that the W8 strain 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 obvious frankincense characteristics. The Gram staining of the bacteria is blue-purple, and the morphology is single-rod, double-rod, or chain-rod arrangement.

[0052] In summary, combined with the colony morphology, physiological and biochemical characteristics and molecular biological identification results of the W8 strain, the applicant determined that the screened W8 strain is a new strain of Lactobacillus plantarum, and named it Lactobacillus plantarum W8 (Lactiplantibacillus plantarumW8), which was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration (CGMCC for short) on August 1, 2024, with the address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and its deposit number is CGMCC No. 31521.

[0053] Example 2 Analysis of acid and bile resistance of Lactobacillus plantarum W8

[0054] 2.1 Acid resistance determination

[0055] The culture medium was 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 at a ratio of 2% (V / V) into liquid MRS medium with a pH value of 6.7;

[0057] (2) Experimental group: The activated Lactobacillus plantarum W8 culture 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°C for 3 h, and the absorbance (OD) was measured at 600 nm. 600 ).

[0058] The survival rate of the strains was calculated according to the following formula.

[0059] Survival rate (%) = (test OD 600 / Control OD 600 )×100%.

[0060] The results showed that the Lactobacillus plantarum W8 provided by the present invention has a strong acid resistance. After being treated at pH 3.0 for 3 hours, its survival rate can still reach 92.54%, indicating that the strain can enter the intestine through gastric juice and has the prerequisite for entering the intestine to play a probiotic role.

[0061] 2.2 Bile salt tolerance test

[0062] The inoculum was inoculated with 2% of the culture medium containing 0.10%, 0.20%, 0.30%, and 0.40% of sodium taurocholate, respectively, and cultured at 37°C for 4 h. The absorbance (OD) was measured at 600 nm. 600 ).

[0063] The results showed that the survival rates of Lactobacillus plantarum W8 provided by the present invention reached 85.76% and 66.11% respectively after being treated for 4 hours in the presence of 0.2% and 0.3% bile salts, indicating that the strain has a strong bile salt tolerance and is capable of entering the intestine to exert a probiotic effect.

[0064] 2.3 Simulated gastrointestinal tolerance test

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

[0066] Survival rate (%) = (number of live bacteria after passing through intestinal juice / number of live bacteria before adding gastric juice) × 100%.

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

[0068] time 3h 6h 12h 24h 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] From the results in Table 1, it can be seen that after being treated with simulated gastric juice for 2 hours and simulated intestinal juice for 3 hours, the survival rate of Lactobacillus plantarum W8 reached 90.35%, and after being treated with simulated intestinal juice for 24 hours, the survival rate was still as high as 41.18%. This shows that the Lactobacillus plantarum W8 provided by the present invention has strong tolerance to the digestive tract environment and can achieve effective colonization in the intestine, which is conducive to exerting its probiotic effect.

[0070] Example 3 Determination of antibacterial ability of Lactobacillus plantarum W8

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

[0072] The fresh bacterial liquid of Lactobacillus plantarum W8 was centrifuged at 4°C and 12000 rpm for 15 min to obtain the supernatant. The concentration of pathogenic bacteria Salmonella and Enterobacter sakazakii was adjusted to 1×10 8 CFU / mL, spread it on the surface of the culture medium, and punch holes (aperture ± 8mm, depth ± 4.5mm). The culture medium was covered with 1% agar, 100 μL of filtrate was drawn into each hole, and it was placed at room temperature for 2 hours and then cultured at 37°C. After about 18 hours of culture, a clear inhibition zone appeared, and the diameter of the inhibition zone was observed and measured. At the same time, the standard plant lactobacillus ATCC8014 was used as a control.

[0073] The results are as follows Figure 3 As shown, Lactobacillus plantarum W8 has significant antibacterial ability against Salmonella and Enterobacter sakazakii, with antibacterial diameters of 23.47 mm and 19.82 mm, respectively. The antibacterial effect is significant, 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 lipid-lowering ability of Lactobacillus plantarum

[0075] 4.1 Experimental methods

[0076] (1) Determination of cholesterol removal rate

[0077] The suspension of Lactobacillus plantarum W8 was added to the cholesterol culture medium, the cholesterol content was determined by the o-phthalaldehyde method, and the cholesterol removal rate was calculated.

[0078] (2) Determination of triglyceride degradation rate

[0079] The plant lactobacillus W8 bacterial suspension was added to the triglyceride medium for cultivation, and the degradation rate of triglyceride by plant lactobacillus W8 was determined according to the instructions of the triglyceride kit. The reagent addition method is shown in Table 2. The degradation rate of triglyceride by plant lactobacillus was calculated according to the following formula.

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

[0081] Table 2 Table of reagent addition for in vitro triglyceride lowering test of Lactobacillus plantarum W8

[0082]

[0083] 4.2 Results Analysis

[0084] The in vitro lipid-lowering test showed that the Lactobacillus plantarum W8 provided by the present invention has a strong lipid-lowering function, with a cholesterol removal rate of 89.05% and a triglyceride degradation rate of 72.74%, achieving an unexpected technical effect.

[0085] Example 5 Regulatory effect of Lactobacillus plantarum W8 on oxidative stress in Caco2 cells

[0086] 5.1 Strain activation

[0087] The glycerol-preserved Lactobacillus plantarum W8 was activated in liquid MRS medium, cultured at 37°C for 18 h, and obtained the experimental strain after three generations.

[0088] 5.2 Recovery, culture and subculture of Caco2 cells

[0089] Human colorectal cancer cell line Caco-2 was taken out of liquid nitrogen, thawed in a 37°C water bath within 3 minutes, then centrifuged quickly, the culture medium was discarded, 1 mL of DMEM complete culture medium was added and dispersed, and then transferred to a culture flask for culture. When the cells grew to about 80% of the surface of the culture flask, the cells were digested with trypsin-EDTA for passage, and the cells were used for subsequent experiments when they were cultured for 5 generations.

[0090] 5.3 Cytotoxicity assay

[0091] Caco-2 cells were cultured at 2 × 10 per ml. 5 The cells were inoculated into 96-well plates at a concentration of 10 cells / mL. The cells were washed with phosphate buffered saline (PBS). Then, 100 μL of bacterial suspension (at a concentration of 10 7 CFU / mL, 10 8 CFU / mL, 10 9 CFU / mL, 10 10CFU / mL), and the cells were incubated for 24 hours, with dimethyl sulfoxide (DMSO) without antibiotics as a control. Then, 20 microliters of 0.5 mg / mL thiazolyl blue (MTT, diluted with PBS buffer) were incubated at 37°C for 4 hours in the dark. The supernatant was removed, 150 microliters of dimethyl sulfoxide were added, and the mixture was shaken for 10 minutes. The absorbance was read at a wavelength of 490nm using an ELISA reader. The results are expressed as the percentage of live cells compared to the control. The absorbance of the control is considered to represent 100% cell viability.

[0092] 5.4 Caco2 cell grouping and modeling

[0093] Caco2 cells were treated as follows:

[0094] (1) Blank group: no intervention;

[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 treated with the 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 ultrasonicated 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 produced 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 concentrations were non-toxic to Caco2 cells, so the middle value of bacterial concentration was 10 8 CFU / mL for further experiments. Figure 5 As shown, compared with the normal group, LPS can induce Caco2 cell oxidative stress, thereby causing Caco2 cell MDA content to significantly increase, and the activity of GSH-PX, SOD and CAT to significantly reduce. After the intervention of plant lactobacillus W8 cell-free supernatant, the levels of MDA, GSH, SOD and CAT induced by LPS were reversed, and the effect was very significant, close to the normal group level. Thus illustrating, plant lactobacillus W8 provided by the invention can effectively alleviate the oxidative stress induced by LPS.

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

[0102] 6.1 Activation of strains, cell culture, grouping and modeling as in Example 5

[0103] 6.2 Determination of epithelial resistance

[0104] Logarithmically growing Caco-2 cells were plated at 1×10 9 The cells were inoculated into a 6-well Transwell plate at a concentration of 1.5 mL / mL. 1.5 mL and 2.5 mL of culture medium were added to the apical side (AP) and the basal side (BL), respectively. The medium was changed every other day in the first week, and the medium was changed every day from the second week onwards. The cells were cultured for about 21 days until the cells were completely differentiated. The cell grouping and modeling methods were the same as in Example 5. After the modeling was completed, the epithelial resistance value was measured using an epithelial resistance meter.

[0105] 6.3 Fluorescein isothiocyanate-dextran (FITC-Dextran) permeability assay

[0106] After modeling, wash the monolayer membrane three times with HBSS, then add 500 μL of DMEM medium containing 1 mg / mL FITC-Dextran to the AP side, and add 1.5 mL of DMEM medium to the BL side. After 6 hours of treatment, aspirate 0.1 mL of culture medium from the BL side and add it to a light-proof black 96-well plate. Use an ELISA reader to measure the fluorescence intensity at 490-520 nm, and repeat each test three times. Calculate the concentration of FITC-Dextran on the outside and the apparent permeability coefficient Papp based on the standard curve.

[0107] 6.4 Results Analysis

[0108] Depend on Figure 6 It can be seen that compared with the normal group, the epithelial resistance of Caco2 cells induced by LPS was significantly reduced, and intervention with the supernatant of Lactobacillus plantarum W8 fermentation significantly increased the epithelial resistance of Caco2 cells. This shows that the supernatant of Lactobacillus plantarum W8 fermentation has the potential to increase the tightness of intestinal epithelial cells and improve the intestinal barrier.

[0109] Compared with the normal group, the FITC permeability of Caco2 cells in the LPS group was significantly increased, 1.5 times that of the normal group; the Lactobacillus plantarum supernatant group significantly improved the increase in permeability caused by LPS, approaching the level of the normal group. The results once again show that the supernatant of Lactobacillus plantarum W8 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 bacterial suspension

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

[0113] 7.2 Animal grouping, modeling and drug administration

[0114] Experimental animals: 5-week-old healthy male C57BL / 6J mice (20 g ± 2 g) were purchased from Liaoning Changsheng Biotechnology Co., Ltd., license number: SVXK (Liao) 2020-0001, and were kept in the SPF-level laboratory of the Experimental Animal Center of Shenyang Agricultural University. The animal experiment was approved by the Ethics Committee of the Experimental Animal Center of Shenyang Agricultural University, ethics approval number: NO.2023102702. 24 mice were randomly divided into 5 groups: control group (Con), model group (DSS), negative control group (NC, Lactobacillus plantarum standard bacteria ATCC8014), positive control group (PC, mesalazine), Lactobacillus plantarum intervention group (W8);

[0115] Acute colitis was induced using a 2.5% (w / v) dose of dextran sulfate sodium (DSS). During the 7-day experiment, mice in the control group received only water. Mice in the DSS group were gavaged with 2.5% DSS for 5 consecutive days. Mice in the normal group were orally administered with a standard bacterial suspension (10 mg / kg) once a day, mice in the W8 group were orally administered with a Lactobacillus plantarum W8 bacterial suspension (10 mg / kg) once a day, and mice in the PC group were gavaged with 200 mg / kg mesalazine / day for 7 consecutive days, and supplemented with DSS for 5 days. Body weight was recorded once a day. Mice were killed on the 7th day. The colon was removed and the length was measured. The colon specimens were fixed in 10% formalin and stained with hematoxylin-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 colon homogenate (100 mg) was prepared with phosphate buffered saline (PBS) (w / v 1 / 9). In colon homogenate and cell samples, IL-10, IL-1β and TNF-α cytokines and serum diamine oxidase (DAO) and D-lactic acid (D-LA) levels were detected according to the manufacturer's instructions (Nanjing Jiancheng, Nanjing, China).

[0118] 7.4 H&E staining

[0119] Fresh colon tissue was rapidly fixed in 4% paraformaldehyde for 48 h. Subsequently, tissue samples were dehydrated, embedded in paraffin, and sectioned at 4 μm. After dewaxing, tissue samples were stained with H&E and analyzed using a light 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 M5-Super-qPCR RT Kit with gDNA removal reagent. The mRNA expression was determined by the instrument. The primer sequences are listed in Table 5 below. The levels of the target genes were quantified relative to the levels of the Gapdh gene, and the results obtained were analyzed using the 2-ΔΔCt analysis method.

[0122] Table 5 Primer sequences

[0123]

[0124] 7.6 Expression of intestinal stem cell marker LGR5 in colon tissue

[0125] (1) Mouse colon tissue was obtained, quickly frozen in liquid nitrogen, and ground to extract total protein.

[0126] (2) Add an appropriate volume of cell tissue lysis buffer to the tissue to be lysed, lyse on ice for 10 min, centrifuge at 12,000 rpm for 10 min, and then collect the supernatant.

[0127] (3) Measure protein concentration using BCA kit.

[0128] (4) Protein denaturation: Add 5× SDS-PAGE loading buffer and cook at 100°C for 10 min.

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

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

[0131] (7) Blocking: Place the membrane in 5% skim milk and shake for blocking at 37°C for 1 h.

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

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

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

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

[0136] 7.7 Determination of intestinal flora composition

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

[0138] 7.9 Result Analysis:

[0139] like Figure 7 As shown in the figure, compared with the normal group, the levels of D-LA and DAO in the colitis model group (DSS) and the negative control group (NC) were significantly increased, 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 of the positive drug group (PC). This shows that Lactobacillus plantarum W8 can significantly reduce intestinal permeability and alleviate DSS-induced intestinal damage.

[0140] like Figure 8 As shown in the figure, compared with the DSS group, the inflammatory factor content of mice in the W8 intervention group was significantly reduced, the IL-10 level was extremely significantly increased, and there was no significant difference between the W8 intervention effect and the PC group. This shows that Lactobacillus plantarum W8 intervention in mice can reduce the secretion of pro-inflammatory factors to varying degrees, thereby alleviating intestinal inflammation in mice and improving the intestinal barrier defense ability of mice.

[0141] like Fig. 9As shown in the figure, the colon mucosa of the normal group mice was intact and continuous, the glands were neatly arranged, the crypts were normal, and there was no inflammatory cell infiltration. The colon damage of the DSS group mice was serious, with a large number of inflammatory cells infiltrating the mucosa and submucosa. The NC group had lymph node hyperplasia in the colon tissue, accompanied by a small amount of inflammatory infiltration, while the W8 intervention group and the PC group had neatly arranged colon glands and normal crypts. It is further illustrated that the plant lactobacillus W8 provided by the present invention has the effect of relieving intestinal inflammation, reducing intestinal damage and repairing intestinal mucosa.

[0142] like Fig.10 The results showed that after intervention with Lactobacillus plantarum W8, the expression of intestinal tight junction genes ZO-1, Occludin, Claudin-1, and Claudin-2 in colitis mice increased significantly, reaching the level 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 Fig.11 As shown in the results, antimicrobial peptides (AMPs) play an important role in the establishment and maintenance of mucosal barriers. Therefore, the expression of AMPs genes Reg3a, Reg3g and Defa5 was detected. Compared with the normal group, the expression of AMPs genes in the DSS group and the NC group was significantly reduced, and the intervention of Lactobacillus plantarum W8 significantly inhibited the downregulation of AMPs gene expression, and the effect was slightly better than that of the PC group. This indicates that Lactobacillus plantarum W8 can help the intestine resist pathogenic bacteria invasion by regulating the expression of antimicrobial peptides.

[0144] like Fig.12 As shown, Lactobacillus plantarum W8 intervention 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 was equivalent to the expression level of the normal group. This shows that Lactobacillus plantarum W8 can effectively promote the recovery and renewal of intestinal stem cells.

[0145] like Fig.13 As shown in the figure, the changes in the intestinal flora 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 Allobaculum, Muribaculaceae, and Bifidobacterium in DSS-induced colitis mice was significantly reduced, while the abundance of bacteria associated with colon inflammation, such as Faecalibaculum and Candidatus Saccharimonas, increased. However, Lactobacillus plantarum intervention significantly reversed this trend, and the abundance and composition of intestinal flora were closer to those of the normal group. The above results indicate that Lactobacillus plantarum W8 can regulate the composition of intestinal flora in colitis hosts, promote the growth of beneficial bacteria, and inhibit the proliferation of harmful bacteria, thereby balancing the homeostasis of intestinal flora.

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

Claims

1. A strain of Lactobacillus plantarum, characterized in that The deposit number of the plant lactobacillus is CGMCC NO.31521.

2. Use of the plant lactobacillus according to claim 1 in preparing food.

3. Use of the plant lactobacillus according to claim 1 in the preparation of medicines for preventing or treating enteritis.

4. A probiotic preparation, characterized in that The probiotic preparation comprises the Lactobacillus plantarum according to claim 1.

5. The probiotic preparation according to claim 4, characterized in that The probiotic preparation further comprises any one or more combinations of Bifidobacterium longum, Lactobacillus casei, Bacillus coagulans, Tetragenococcus halophilus, Lactobacillus fermentum, Lactobacillus paracasei, Bifidobacterium infantis, Bifidobacterium adolescentis, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus bulgaricus, Pediococcus acidilactici and Leuconostoc lactis.

6. The probiotic preparation according to claim 4, characterized in that The amount of live bacteria of Lactobacillus plantarum in the probiotic preparation is not less than 10 8 CFU / g.

7. Use of the probiotic preparation according to any one of claims 4 to 6 in preparing food.

8. Use of Lactobacillus plantarum according to any one of claims 4 to 6 in the preparation of a medicine for preventing or treating enteritis.

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