A strain of Lactobacillus johnsonii N5 and its application in preventing and treating enteritis and diarrhea

Abstract

The present invention relates to a strain of Lactobacillus johnsonii N5 and its application in preventing and treating enteritis and diarrhea. Lactobacillus johnsonii N5), was deposited in the China Center for Type Culture Collection on February 10, 2023, with the deposit number CCTCC NO: M2023104. The Lactobacillus johnsonii N5 was isolated from the fecal microorganisms of piglets resistant to heat stress-induced diarrhea. This Lactobacillus johnsonii N5 has the effect of alleviating enteritis and diarrhea, which is specifically reflected in: significantly enhancing the expression of intestinal epithelial tight junction proteins and cytoprotective heat shock protein 70, improving the immune balance of animal intestinal T cells, enhancing immune tolerance, and reducing the degree of intestinal inflammation and the occurrence of diarrheal diseases in animals. Therefore, Lactobacillus johnsonii N5 has a significant effect in preventing and treating diarrhea and related intestinal inflammatory diseases.

Description

Technical Field

[0001] The present invention relates to a Lactobacillus johnsonii N5 and application thereof in preventing and treating enteritis and diarrhea, belonging to the technical field of microorganisms. Background Art

[0002] Antibiotics as feed additives have made great contributions to the rapid development of animal husbandry. However, excessive and irrational use of antibiotics can lead to veterinary drug residues, causing a significant increase in the number of drug-resistant genes in livestock and poultry and the probability of horizontal transmission of drug-resistant genes, posing a huge threat to human health.

[0003] Probiotics are considered one of the most promising alternatives to antibiotics. They can be taken orally or as food additives, without the risk of bacterial resistance or residual disease. Lactobacillus is a common probiotic. Long-term feeding of Lactobacillus to animals can improve the balance of the intestinal microbiome, enhance intestinal barrier function, and protect animals from pathogens and the diseases they cause. Therefore, it is urgent to find an animal-derived Lactobacillus strain with multiple probiotic properties to effectively replace antibiotics and prevent enteritis and diarrhea in animals. Summary of the Invention

[0004] The purpose of the present invention is to address the above-mentioned existing problems and overcome the shortcomings and deficiencies of the existing technology, and to provide a strain of Lactobacillus johnsonii N5 and its use in preventing and treating enteritis and diarrhea; the Lactobacillus johnsonii N5 can not only effectively inhibit intestinal pathogens, but also can stably exist in the digestive tract, effectively solving the problems of animal diarrhea and high mortality faced by the current livestock and poultry farming industry, replacing the use of antibiotics in farming, and promoting the green, healthy and sustainable development of the farming industry.

[0005] The object of the present invention is achieved as follows: A strain of Lactobacillus johnsonii N5 was deposited in the China Center for Type Culture Collection on February 10, 2023, with a deposit number of CCTCC NO: M2023104. The address is Wuhan University, Wuhan, China

[0006] The Lactobacillus johnsonii N5 is isolated from fecal microorganisms of piglets resistant to diarrhea induced by heat stress.

[0007] The minimum tolerable pH of the Lactobacillus johnsonii N5 is 2.5, and the bile salt tolerance is 3.0 g / L.

[0008] The Lactobacillus johnsonii N5 is used to prevent and treat intestinal inflammation and diarrhea in animals.

[0009] The invention relates to an application of Lactobacillus johnsonii N5 in preventing and treating intestinal inflammation and diarrhea in animals, wherein the viable cell count of Lactobacillus johnsonii N5 is not less than 1*10 9 CFU / mL.

[0010] The application can reduce the damage caused by 2.5% dextran sulfate sodium salt solution to the intestinal barrier of mice.

[0011] The application of reducing the damage to the intestinal barrier of mice caused by 2.5% dextran sulfate sodium salt solution includes administering no less than 1*10 mol / L of dextran sulfate sodium salt solution to mice 7 days before using 2.5% dextran sulfate sodium salt solution to simulate the intestinal barrier damage model. 9 CFU / mL of Lactobacillus johnsonii N5 solution was added to observe the preventive and therapeutic effects of Lactobacillus johnsonii N5 on intestinal barrier damage in mice induced by dextran sulfate sodium salt solution.

[0012] A microbial agent comprising the Lactobacillus johnsonii N5 according to claim 1.

[0013] The invention relates to an application of the microbial agent in preventing and treating enteritis and diarrhea diseases in animals.

[0014] The present method is advanced and scientific. A strain of Lactobacillus johnsonii N5 was identified in the feces of piglets resistant to heat stress-induced diarrhea. This strain, Lactobacillus johnsonii N5, alleviates enteritis and diarrhea by significantly enhancing the expression of tight junction proteins and cytoprotective heat shock protein 70 in the intestinal epithelium, improving T cell immune balance in the animal's intestines, enhancing immune tolerance, and reducing the severity of intestinal inflammation and the incidence of diarrhea. Therefore, Lactobacillus johnsonii N5 is significantly effective in preventing and treating diarrhea and related intestinal inflammatory diseases.

[0015] The beneficial effects of the present invention are that the Lactobacillus johnsonii N5 of the present invention has excellent acid and bile salt resistance and significant effects of preventing and treating diarrhea and relieving enteritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 These are the phenotypic results of the two groups of piglets in Example 1 of the present invention in terms of growth performance, inflammatory response, and intestinal physiology;

[0017] The components represent the diarrhea index (A) and weight change (B) of the stress diarrhea group and the anti-stress diarrhea group after heat stress treatment, the levels of proinflammatory cytokines (TNFɑ, IL-6, CXCL1, IL-10) in the blood (C), the expression levels of HSP27 and HSP70 proteins and genes in colon tissue, and the expression of the transcription factor HSF1 at the gene level (DF).

[0018] Figure 2 This is a diagram showing the differences in the intestinal microbial composition of piglets in different experimental groups in Example 1 of the present invention;

[0019] Among them, each part represents the (OTU) abundance rank curve (A) and cluster analysis (B) of the fecal microbiota in the stress diarrhea group and the anti-stress diarrhea group, the Shannon (C) and Inverse Simpson index (D) of the fecal microbiota, and the bar charts of the fecal microbiota composition at the phylum level (E) and family level (F).

[0020] Figure 3 This is a diagram showing the results of screening, isolating, and identifying Lactobacillus johnsonii strains with anti-stress diarrhea properties in Example 1 of the present invention;

[0021] Among them, each part represents the heat map of fecal microbial composition at the genus level in the stress diarrhea group and the anti-stress diarrhea group after heat stress treatment (A), the relative abundance of Lactobacillus johnsonii, Lactobacillus panis and Lactobacillus ultunensis (BD), the KEGG functional classification of microorganisms (E), the branch diagram generated by LEfSe analysis and the strain phylogenetic tree (F).

[0022] Figure 4 This is a graph showing the results of the ability of Lactobacillus johnsonii N5 to resist Salmonella typhimurium SL1344 and Escherichia coli XYEH3934 in Example 1 of the present invention.

[0023] Figure 5 This is a graph showing the protective effect of Lactobacillus johnsonii N5 on colitis induced by dextran sulfate sodium in Example 5 of the present invention;

[0024] Among them, each part represents the treatment time points and weight change trends of the Lactobacillus johnsonii N5 + dextran sodium sulfate group and the dextran sodium sulfate group (A), disease activity index (DAI) (B), colon length of mice (C), colon H&E staining sections and their scores (D E), TNFα, IL-6, IFNγ, and IL-10 levels in colon tissue (F), and IL-10 level in serum (G).

[0025] Figure 6 This is a graph showing the results of the improvement of the protein levels of intestinal barrier tight junction proteins and heat shock protein 70 by Lactobacillus johnsonii N5 in Example 5 of the present invention in colitis induced by dextran sulfate sodium;

[0026] Among them, each part represents the control group, Lactobacillus johnsonii N5 group, dextran sodium sulfate group and Lactobacillus johnsonii N5 + dextran sodium sulfate group, fluorescence staining of colon barrier proteins (ZO-1, Occludin, JAM-A) (A), average fluorescence intensity of ZO-1 (B), average fluorescence intensity of Occludin (C), average fluorescence intensity of JAM-A (D), fluorescence staining of HSP70 in colon tissue (E) and its average fluorescence intensity (F).

[0027] Figure 7 This is a graph showing the response of T cells in the intestinal Peyer's patches of mice with colitis induced by dextran sodium sulfate induced by Lactobacillus johnsonii N5 in Example 5 of the present invention;

[0028] Among them, each part represents the control group, Lactobacillus johnsonii N5 group, dextran sulfate sodium group and Lactobacillus johnsonii N5 + dextran sulfate sodium group, the flow cytometry spectrum of regulatory T cells (Treg) in small intestinal lymph nodes (PP nodes) (A), CD4 + The number of T cells (B) and their percentage in total T cells (C), the number of Treg cells (D), the expression level of FoxP3 (E), the flow cytometry profile of Th17 cells in PP nodes (F), the number of Th17 cells (G), the expression levels of RORγt and IL-17a in Th17 cells in PP nodes (HI), and the ratio of Treg / Th17 cells (J). DETAILED DESCRIPTION

[0029] The technical solutions of the present invention will be described clearly and completely below with reference to the accompanying drawings of the present invention.

[0030] Example 1: Screening, isolation and identification of Lactobacillus johnsonii N5;

[0031] 1. Identification and screening of Lactobacillus johnsonii N5;

[0032] 35-day-old weaned piglets were selected for the experiment and divided into a control group and a heat stress treatment group. The control group was raised at a room temperature of 26-28°C; the heat stress treatment group was subjected to heat stress treatment at 36°C from 12:00 to 16:00 every day using a heat lamp, and was raised in an environment of 26-28°C during other time periods. The experiment lasted for 8 weeks and the piglets were observed. The heat stress group was further divided into a stress diarrhea group (HS-SUS) and an anti-stress diarrhea group (HS-RES) based on the diarrhea situation. We selected six pigs from each of the control group, stress diarrhea group, and anti-stress diarrhea group for the experiment.

[0033] like Figure 1 As shown, the anti-stress diarrhea group performed better than the stress diarrhea group in terms of weight change, diarrhea index, serum inflammatory factor content, and HSP27 and HSP70 protein and gene expression levels in colon tissue.

[0034] like Figure 2 As shown in the figure, there were significant differences in the fecal microbiota between the anti-stress diarrhea and stress diarrhea groups in terms of microbial diversity index and microbial composition at the phylum and genus levels.

[0035] like Figure 3 As shown in the figure, the relative abundance of Lactobacillus johnsonii in the fecal flora of the anti-stress diarrhea group was significantly higher than that of the stress diarrhea group.

[0036] 2. Isolation of Lactobacillus johnsonii N5;

[0037] The feces sample of anti-stress diarrhea pig was used as the sample, and the sample was mixed with MRS broth at a ratio of 1:10, cultured at 37℃ for 24 hours, diluted and spread, and cultured at 37℃ for 24 hours. The colonies with obvious characteristics were selected and repeatedly streaked and separated, and purified until colonies with consistent morphology and size were isolated.

[0038] MRS broth is a universal culture medium for culturing lactobacilli. Based on the total volume of the culture medium, its formula is: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 20 g / L glucose, 1 mL / L Tween-80, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium acetate trihydrate, 2 g / L triammonium citrate, 0.58 g / L magnesium sulfate heptahydrate, 0.05 g / L manganese sulfate monohydrate, pH 6.2. The MRS culture medium used in the present invention can be a liquid, i.e., MRS broth, or a solid culture medium.

[0039] The colonies of strain N5 on MRS plates were hemispherical, smooth and moist, milky white in color, and approximately 1-2 mm in diameter.

[0040] 3. 16S rDNA sequence identification of Lactobacillus johnsonii N5;

[0041] Using DNA from strain N5 as a template, amplification was performed using universal 16S rDNA primers, and the amplified product was sequenced. Blast comparison revealed that the strain sequence had the highest homology with the 16S rDNA sequence of Lactobacillus johnsonii in the GenBank gene library, confirming that strain N5 was Lactobacillus johnsonii. The 16S rDNA sequence of Lactobacillus johnsonii N5 has been deposited in GenBank under the accession number OQ256705.

[0042] Example 2: Determination of tolerance of Lactobacillus johnsonii N5 to acid and bile salts;

[0043] 1. Acid tolerance test of Lactobacillus johnsonii N5

[0044] Lactobacillus johnsonii N5 was inoculated at a 5% inoculum into 5 mL of MRS broth (pH 2.5). The N5-containing medium was pipetted and mixed thoroughly, and then incubated at 37°C for 4 hours. Before incubation (0 hour) and after incubation (2 and 4 hours), 1 mL of the bacterial suspension was diluted to the appropriate gradient. After dilution to the appropriate gradient, 0.1 mL of the dilution was spread onto MRS agar plates. The plates were incubated at 37°C for 24 hours and counted. Three replicates were used for each sample. The survival rate of the strain was expressed as the percentage of viable cells after incubation to the number of viable cells inoculated.

[0045] As shown in Table 1, under the condition of pH 2.5, the survival rates of Lactobacillus johnsonii N5 were approximately 110.34% and 65.52% after 2 h and 4 h, respectively, indicating that it has good acid tolerance.

[0046] 2. Bile salt tolerance test of Lactobacillus johnsonii N5;

[0047] Lactobacillus johnsonii N5 was inoculated into MRS broth containing 3g / L sodium deoxycholate at a 5% inoculum volume, mixed by pipetting, and cultured at 37°C for 4 hours. 1mL of bacterial suspension was taken before (0h) and after (2, 4h) culture for gradient dilution. After dilution to an appropriate gradient, 0.1mL of the dilution was spread on an MRS agar plate. The agar plates were cultured at 37°C for 24h and counted. Three replicates were taken for each sample. The tolerance of the strain was expressed as the percentage of the number of viable bacteria after culture to the number of viable bacteria inoculated.

[0048] Table 1 Results of acid and bile salt tolerance of Lactobacillus johnsonii N5

[0049]

[0050] As shown in Table 1, the survival rates of Lactobacillus johnsonii N5 at 0.3% (w / v) bile salt concentration were 95.74% and 76.60% at 2 h and 4 h, respectively, indicating that Lactobacillus johnsonii N5 had good bile salt tolerance.

[0051] Example 3: In vitro antibacterial experiment of Lactobacillus johnsonii N5;

[0052] The in vitro antibacterial test was determined by the agar spot diffusion method. First, the strain N5 was inoculated into a 2 mL finger tube containing 1.5 mL of MRS broth and cultured in a 37 ° C constant temperature incubator for 24 hours. After shaking and mixing, 2 μL of bacterial liquid was dripped into the MRS solid culture medium and cultured in a 37 ° C constant temperature incubator for 24 hours. Use a sterile inoculation loop to pick up single colonies of Salmonella typhimurium SL1344 and Escherichia coli XYEH3934, respectively, and inoculated into 5 mL of LB liquid culture medium, and cultured in a 37 ° C constant temperature shaker for 12 hours. The cultured bacterial liquid was washed with PBS, and the OD600 value was adjusted to zero with PBS, and the OD600 value of Salmonella typhimurium SL1344 and Escherichia coli XYEH3934 was adjusted to 10 8 CFU / mL. Then, add it to LB semi-solid medium at a ratio of 1:100, mix well, and pour it into MRS solid medium that has been dripped with the test bacteria solution, so that the surface is evenly spread. After cooling and solidification, place it in a 37°C constant temperature incubator. After 12 hours, observe and measure the size of the inhibition zone.

[0053] Table 2 Antibacterial results of Lactobacillus johnsonii N5 against Salmonella typhimurium and Enterobacter

[0054]

[0055] As shown in Table 2, Lactobacillus johnsonii N5 has a high antibacterial ability against Salmonella typhimurium SL1344 and Escherichia coli XYEH3934, and the antibacterial diameters of Salmonella typhimurium SL1344 and Escherichia coli XYEH3934 are 4 mm and 22.83 mm, respectively. The above results indicate that Lactobacillus johnsonii N5 has a good inhibitory effect on intestinal pathogens Salmonella typhimurium SL1344 and Escherichia coli XYEH3934.

[0056] Example 4: Effects of Lactobacillus johnsonii N5 on blood biochemistry in mice;

[0057] Two groups of experiments were set up for verification. Twenty male mice were randomly divided into two groups. The control group was gavaged with 100 μL of normal saline; the experimental group was gavaged with 100 μL of no less than 1*10 9CFU / mL of Lactobacillus johnsonii N5 suspension, and the mice were observed after 7 days; a blood biochemical analyzer was used to analyze the serum biochemical parameters of the mice, including the concentration levels of albumin, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, cholesterol, high-density lipoprotein cholesterol (HDL-C), lactate dehydrogenase (LDH), low-density lipoprotein cholesterol (LDL-C), triglycerides, urease, glucose, total protein, uric acid, and iron.

[0058] Table 3 Effects of Lactobacillus johnsonii N5 on routine blood biochemical indicators in mice

[0059]

[0060]

[0061] As shown in Table 3, compared with the control group, treatment with porcine Lactobacillus johnsonii N5 increased the levels of albumin, aspartate aminotransferase, high-density lipoprotein cholesterol, lactate dehydrogenase, low-density lipoprotein cholesterol, urease, total protein, uric acid, and iron in the blood of mice, while decreasing the levels of alanine aminotransferase, alkaline phosphatase, cholesterol, triglycerides, and glucose. These results indicate that porcine Lactobacillus johnsonii N5 does not significantly affect animal circulatory metabolism and systemic immunity, nor does it have a negative impact on animal physiology. On the contrary, it may promote animal growth and intestinal development.

[0062] Example 5: Effect of Lactobacillus johnsonii N5 in preventing and treating enteritis and diarrhea in animals;

[0063] Four groups of experiments were set up for verification. The control group was fed with ordinary commercial feed and gavaged with 100 μL normal saline every day. The mice were observed after 7 days. The Lactobacillus johnsonii N5 pretreatment group was fed with ordinary commercial feed and gavaged with 1*10 9 CFU / mL of Lactobacillus johnsonii N5, and the mice were observed after 7 days; the sodium dextran sulfate treatment group: the mice were fed with ordinary commercial feed, and 2.5% sodium dextran sulfate aqueous solution was given to the mice (for 7 days) and the mice were observed; the Lactobacillus johnsonii N5 + sodium dextran sulfate group: the mice were fed with ordinary commercial feed, and the mice were subjected to at least 1*10 9The mice were treated by gavage with Lactobacillus johnsonii N5 at a concentration of 100 CFU / mL, and then 7 days later, they were given an aqueous solution containing 2.5% dextran sodium sulfate. The condition of the mice was observed after the end of the experiment.

[0064] like Figure 5 As shown, compared with the sodium dextran sulfate group, the treatment with Lactobacillus johnsonii N5 + sodium dextran sulfate group was better in alleviating the symptoms of weight loss, damage to normal colon morphology, and shortened colon length caused by intestinal inflammation. The levels of inflammatory factors in colon tissue and serum also showed that Lactobacillus johnsonii N5 pretreatment inhibited the systemic inflammatory response induced by sodium dextran sulfate.

[0065] like Figure 6 As shown in the results, compared with the sodium dextran sulfate group, pretreatment with Lactobacillus johnsonii N5 increased the expression of tight junction protein ZO-1, closed protein Occludin and heat shock protein HSP70 in the colon of healthy mice, thereby significantly reducing the degree of intestinal barrier damage in mice caused by sodium dextran sulfate, indicating that Lactobacillus johnsonii N5 can resist the damaging effects of sodium dextran sulfate on the intestinal barrier.

[0066] like Figure 7 As shown, compared to the control group, the number of Treg cells increased and the number of Th17 cells decreased significantly in the L. johnsonii N5-treated group. The Treg / Th17 ratio in the L. johnsonii N5 group was significantly higher than in the control group. Furthermore, the Treg / Th17 ratio in the L. johnsonii N5 + dextran sulfate sodium group was significantly higher than that in the dextran sulfate sodium group. This suggests that pretreatment with L. johnsonii N5 improves immune homeostasis in mice by altering the balance between the number and effector function of Treg and Th17 cells.

[0067] In summary, the addition of Lactobacillus johnsonii N5 to the diet alleviated the symptoms of colon shortening, reduced the expression of intestinal inflammatory factors, and increased the expression of tight junction proteins in the colon, thereby alleviating the damage to intestinal function caused by sodium dextran sulfate. Furthermore, Lactobacillus johnsonii N5 can regulate the balance between intestinal immune cell activation and tolerance in mice treated with sodium dextran sulfate, preventing excessive inflammatory responses.

[0068] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A strain of Lactobacillus johnsonii ( Lactobacillus johnsonii ) N5, deposited in the China Center for Type Culture Collection on February 10, 2023, with the deposit number CCTCC NO: M2023104.

2. Use of the Lactobacillus johnsonii N5 according to claim 1 in the preparation of a medicament for preventing animal colitis.

3. The use according to claim 2, characterized in that In the application, the viable count of Lactobacillus johnsonii N5 is not less than 1*10 9 CFU / mL.

4. A microbial agent, characterized in that: The microbial agent includes the Lactobacillus johnsonii N5 described in claim 1.

5. Use of the microbial agent according to claim 4 in the preparation of a medicament for preventing animal colitis.

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