Cluster sports enterobacter chinense and application thereof

Abstract

The invention belongs to the technical field of new application of microorganisms, and particularly relates to enterobacter chinense and application thereof to improvement of inflammatory bowel diseases. The name of the strain is Enterobacter huxiensis LS-21, the strain is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation date is October 28, 2024, and the preservation number is CGMCC NO.32366. The invention further discloses a preparation method of the enterobacter huxiensis LS-21. The strain has a cluster movement capability, has relatively strong adaptive capacity to the intestinal environment, can well tolerate the acidity of gastric juice, is sensitive to various common antibiotics, and has a good improvement effect on colitis.

Description

Technical Field

[0001] The present invention belongs to the field of microorganisms, and in particular relates to a swarming motile Enterobacter szechwanensis and a use thereof. Background Art

[0002] Inflammatory bowel disease (IBD) is a group of chronic, relapsing intestinal inflammatory diseases that primarily affect the intestinal mucosa and include two major subtypes: Crohn's disease (UC) and ulcerative colitis (CD). The symptoms of IBD may vary from person to person, but common symptoms include abdominal pain and diarrhea, bloody stools, weight loss, fatigue, malnutrition, etc., which affect the patient's quality of life. In addition, the chronic nature and unpredictable course of IBD impose long-term psychological and economic burdens on patients and their families. The exact cause of IBD is not yet fully understood, but it is generally believed to be the result of a combination of factors such as host genetic susceptibility, intestinal flora disorders, intestinal mucosal barrier disruption, and intestinal mucosal immune abnormalities. IBD itself is not fatal, but it may increase the risk of other diseases such as colon cancer, blood clots, and liver disease.

[0003] Treatment for IBD primarily relies on medication, primarily for symptom relief, and a complete cure is currently unavailable. The main clinical treatments include salicylates, glucocorticoids, and immunosuppressants. Salicylates exert anti-inflammatory effects by reducing the release of proinflammatory cytokines in the intestine, but they can have severe gastrointestinal side effects and can even cause drug-induced kidney injury. Glucocorticoids alleviate inflammation by inhibiting the release of inflammatory substances and are commonly used in patients with moderate to severe enteritis. However, long-term or high-dose use can lead to drug resistance and multiple adverse reactions. Immunosuppressants inhibit the proliferation of inflammatory cells, but they can easily cause adverse reactions such as nausea, vomiting, and diarrhea. In summary, although salicylates, glucocorticoids, or immunosuppressants can alleviate the clinical symptoms of IBD, they all have drawbacks, such as relapse upon discontinuation and low patient compliance with long-term use.

[0004] Compared with healthy individuals, the gut microbiota of patients with active IBD exhibits altered bacterial diversity, composition, and / or abundance, while similar microbial composition patterns are observed across IBD patients, such as decreased microbial diversity, reduced abundance of Firmicutes, and increased abundance of Proteobacteria. Gut commensal bacteria have demonstrated significant positive effects in the prevention and treatment of IBD. The protective effects of these microbes, such as regulation of intestinal microbial repair, immunomodulation, enhanced anti-inflammatory effects, and intestinal barrier repair, appear to be strain-specific. Therefore, screening for strains that ameliorate inflammatory bowel disease is crucial for the development of new therapies for inflammatory bowel disease.

[0005] The patent of this invention adopts a method of isolating and purifying symbiotic clustering bacteria from the feces of patients with colon cancer based on the clustering motility of the strains. A strain of Enterobacter sinensis was screened out, which came from the preoperative feces of a 37-year-old female patient with colon cancer and had the ability to cluster. Summary of the Invention

[0006] Based on the above existing technology,

[0007] The first aspect of the present invention provides a strain of Enterobacter serrata LS-21, which is deposited in the China General Microbiological Culture Collection Center with a deposit date of October 28, 2024 and a deposit number of CGMCC NO.32366.

[0008] The strain has the ability of swarming and motility, specifically, it can grow monoclonal colonies that spread like thin films.

[0009] The strain has a short rod-like morphology; further, it is a white dot-like clone.

[0010] This strain is a Gram-negative bacterium.

[0011] The 16S rDNA gene sequence of the strain is shown in SEQ ID NO.3.

[0012] A second aspect of the present invention provides a composition comprising the Enterobacter szechuanensis strain LS-21, or provides a bacterial agent comprising the Enterobacter szechuanensis strain LS-21.

[0013] The present invention further provides a microbial preparation product comprising the Enterobacter szechwanensis strain LS-21, which can be a food, a medicine, or a health product.

[0014] The number of viable bacteria is not less than 1.5E+09CFU / g.

[0015] The preferred route of administration is oral;

[0016] The dosage form can be tablets, capsules, granules, pills or oral liquids;

[0017] In some specific embodiments, the drug further comprises a pharmaceutical carrier and / or a pharmaceutical excipient. The pharmaceutical carrier comprises microcapsules, microspheres, nanoparticles, and liposomes; the pharmaceutical excipient comprises one or more of a solvent, a propellant, a solubilizer, a cosolvent, an emulsifier, a colorant, an adhesive, a disintegrant, a filler, a lubricant, a wetting agent, an osmotic pressure regulator, a stabilizer, a glidant, a flavoring agent, a preservative, a suspending agent, a coating material, a fragrance, an anti-adhesive agent, an integrator, a penetration enhancer, a pH regulator, a buffer, a plasticizer, a surfactant, a foaming agent, a defoaming agent, a thickener, a clathrate, a humectant, an absorbent, a diluent, a flocculant and a deflocculant, a filter aid, an excipient, an additive, and a release retardant.

[0018] In a specific embodiment, the health food further comprises food additives, and the food additives are selected from: acidity regulators, anticaking agents, defoaming agents, antioxidants, bleaching agents, leavening agents, colorants, color preservatives, enzyme preparations, flavor enhancers, nutritional enhancers, preservatives, sweeteners, thickeners, and spices.

[0019] The type of bacterial agent can be an inactivated bacterial agent or a live bacterial agent, but in order to retain the bacterial activity to the greatest extent, the bacterial agent is preferably a live bacterial agent.

[0020] The bacterial agent further comprises fermentation products and non-fermentation products of Enterobacter szechwanensis, wherein the non-fermentation products mainly include metabolites.

[0021] The pharmaceutical composition contains the above-mentioned Enterobacter serrata strain LS-21, a pharmaceutical carrier and / or pharmaceutical excipients.

[0022] In one embodiment, the present invention provides a food composition capable of improving intestinal health, comprising the Enterobacter szechwanensis strain LS-21. The food composition may be a functional food and may comprise additives commonly used in food compositions. The food composition may be a dietary supplement.

[0023] In a specific embodiment, the present invention provides a health care composition capable of improving intestinal health, wherein the health care composition comprises the Enterobacter szechwanensis strain LS-21.

[0024] In a specific embodiment, the present invention provides a pharmaceutical composition comprising the Enterobacter szechwanensis strain LS-21.

[0025] The third aspect of the present invention provides the use of Enterobacter sinoecidioides strain LS-21

[0026] Specifically, the invention relates to the use of the Enterobacter huaxiensis strain LS-21 in treating, alleviating colitis and / or improving intestinal health, and its use in preparing a medicine for treating, alleviating colitis and / or improving intestinal health;

[0027] Use of Enterobacter serrata strain LS-21 in preparing a food composition for improving intestinal health.

[0028] The application includes at least one of the following functions

[0029] (1) Stabilize the subject's weight and, further, significantly improve the subject's weight loss;

[0030] (2) Improve the subject's symptoms of sticky stool;

[0031] (3) Improve the patient's bloody stool symptoms;

[0032] Furthermore, the E. coli strain LS-21 can reduce the disease activity index (DAI).

[0033] (4) Improved the pathological changes of colon tissue in the subjects with acute colitis.

[0034] (5) Repair colon inflammation.

[0035] The fourth aspect of the present invention provides a method for isolating bacteria

[0036] The present invention provides a method for isolating bacteria, which is based on screening strains based on their swarming motility.

[0037] Furthermore, in the isolation method, the strain screening criteria are to dilute the isolated sample and apply it to a solid culture medium, then observe whether a thin film-like monoclonal colony is formed, thereby determining whether the target strain has swarming motility. Furthermore, in the isolation method, the strains screened are derived from stool samples, and the stool samples are obtained from patients with colon cancer.

[0038] Furthermore, the isolation method includes homogenizing fecal samples from patients with colon cancer, inoculating them into solid culture medium after dilution, and culturing them at 37°C and 20% humidity. The colonies show wavy diffusion and multi-layer diffusion, and thin film diffusion-like colonies are observed to grow. The edges of the colonies are further inoculated and streaked for purification until the purified single strain grows thin film diffusion-like monoclonal colonies, indicating that the strain is a clustered bacterium.

[0039] The inoculation culture medium is a 0.8%-1.5% LB agar plate; the homogenization treatment refers to homogenizing the feces sample to a concentration of 200 mg / mL.

[0040] Swarming motility refers to a form of bacterial swarming, in which bacteria move rapidly and collectively on a surface by driving their flagella. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Culture of swarming motile bacteria from homogenized fecal samples

[0042] Figure 2 Swarming movement of Enterobacter huaxiensis LS-21

[0043] Figure 3 Gram staining of Enterobacter huaxiensis LS-21

[0044] Figure 4 Growth curve of Enterobacter huaxiensis LS-21

[0045] Figure 5 The total number of viable bacteria in the original bacterial solution treated with PBS at different pH values

[0046] Figure 6 Body weight changes in mice with DSS-induced colitis

[0047] Figure 7 Body weight changes in different groups after treatment (The results are expressed as "mean ± standard error". The experimental data were analyzed by one-way analysis of variance (one-ANOVA) using GraphPad Prism 10.1.2. * indicates P < 0.05.)

[0048] Figure 8 Disease Activity Index (DAI) Assessment in Different Groups (Results are expressed as mean ± standard error. Experimental data were analyzed using GraphPad Prism 10.1.2 with Tukey's test. ** represents P < 0.01, and **** represents P < 0.0001.)

[0049] Figure 9 Spleen index of different groups (The results are expressed as "mean ± standard error". The experimental data were analyzed by one-way analysis of variance (one-ANOVA) using GraphPad Prism 10.1.2. ** represents P < 0.01.)

[0050] Figure 10 Comparison of tissue section staining of mice treated in different groups

[0051] Figure 11 Changes in MPO levels in mice treated with different methods (Results are expressed as mean ± standard error. Experimental data were analyzed using GraphPad Prism 10.1.2 with Tukey's test. ** represents P < 0.01, and **** represents P < 0.0001.)

[0052] Figure 12 Changes in inflammatory factors in mice treated with different methods (The results are expressed as "mean ± standard error". The experimental data were analyzed by one-way analysis of variance (one-ANOVA) using GraphPad Prism 10.1.2.) DETAILED DESCRIPTION

[0053] 1. Isolation, Screening and Identification of Enterobacter huaxiensis LS-21

[0054] 1. Sample Processing

[0055] Stool samples from patients with colon cancer were thawed gradually as follows: the sample was removed from a -80°C freezer, placed in a -25°C freezer for 1 hour, and then thawed on ice for 1 hour. On a scale, a sterile microcentrifuge tube was weighed and tare set. In a sterile operating table, the sample was removed with a sterile pipette tip and placed in a microcentrifuge tube. The stool sample was weighed using the same scale. Sufficient sterile phosphate-buffered saline (PBS: pH = 7.4, room temperature) was added to the microcentrifuge tube to achieve a final stool concentration of 200 mg / mL. The sterile pestle was rotated in the microcentrifuge tube approximately 20 times to completely homogenize the stool pellet.

[0056] 2. Bacterial plate culture

[0057] Vortex the microcentrifuge tube containing the stool sample for about 10 seconds, then inoculate 5 μL of the homogenized stool solution on the center surface of a 0.8% LB agar plate. Place the plate in an incubator at 37°C and 20% humidity. After 16 hours of incubation (i.e., swarming conditions), thin film-like colonies are observed, such as Figure 1 As shown, the bacterial film has grown all over the culture dish, and is a wave-like layered diffusion growth.

[0058] 3. Screening of Monoclonal Strains Based on Swarming Ability

[0059] On plates with swarm-expanding colonies, gently scrape the edges of the colonies at four different points with a sterile inoculating loop and streak them onto 1.5% LB agar plates. Incubate the streaked plates in a 37°C incubator overnight.

[0060] After the streaked plates have grown sufficiently, take two single colonies from each plate and place them in 5 ml of LB broth. Incubate the sample on a shaker at 200 rpm and 37°C for 16 hours. Inoculate 5 μL of the bacterial suspension in the center of the surface of a 0.8% LB agar plate and incubate at 37°C and 20% humidity for 16 hours to confirm again whether the bacteria have the ability to colonize. Figure 2 As shown, the inoculated bacterial liquid grew monoclonal colonies in the form of thin film diffusion, indicating that the strain was a swarming bacterium (i.e., "swarming culture verification").

[0061] At the same time, the bacterial suspension was streaked on a 1.5% LB agar plate and cultured at 37°C to observe whether it was a pure culture.

[0062] For each sample, two tubes of bacteria were randomly selected for PCR. The bacterial solution after 16 hours of shaking was diluted 50 times and the PCR amplification reaction was performed according to Table 1 below. Prepare 1% agarose gel (1g agarose plus 100mL deionized water), heat and boil 2-3 times, add 10μL 4S Green buffer when the temperature drops to about 55°C, mix well, and pour into the gel tank. After solidification, add 1μL Loading buffer and 5μL PCR product mixture into the gel wells, and perform gel electrophoresis at 220V for 30min. If a band appears at the target band (1500bp), send the sample for 16S rRNA sequencing. The sequencing results were compared on the NCBI website.

[0063] Table 1 PCR amplification reaction system

[0064]

[0065] 4. Purification and Identification of Strain

[0066] Morphological identification:

[0067] Use Gram staining to stain LS-21 bacteria and observe its morphology under a 100x oil microscope. The specific steps are as follows: Inoculate LS-21 bacteria into 5mL of LB broth medium and shake the bacteria at 37℃ and 200rpm for 16h. Streak the bacterial liquid on a 1.5% LB agar plate and culture it overnight at 37℃ and 40% humidity. Take a small amount of LS-21 single colony, dip it in 2μL of physiological saline, and spread it clockwise to about 1cm 2 Shape the slide into a uniform, thin, round shape and allow it to dry at room temperature. Fix the slide by passing it through the flame of an alcohol burner 1-2 times, being careful not to overheat; the slide should be warm to the touch. Add a drop of crystal violet stain to the bacteria, stain for 1 minute, and rinse with water. Add a drop of iodine solution, stain for 1 minute, and rinse with water. Add a drop of decolorizer, shake the slide, decolorize for 30 seconds, rinse with water, and remove moisture by blotting. Add a drop of safranin stain, stain for 1 minute, and rinse with water. Remove excess moisture with filter paper and examine under a 100x oil immersion lens.

[0068] Gram staining microscopy results showed that the bacteria were Gram-negative (see Figure 3 ), short rod-shaped.

[0069] Molecular biology identification:

[0070] Universal primer sequences for 16S rDNA amplification:

[0071] 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO: 1)

[0072] 1492R:5'-TACGGYTACCTTGTTACGACTT-3'(SEQ ID NO:2)

[0073] (Y represents any base here, which can be A, T, C, or G)

[0074] PCR product sequencing results:

[0075]

[0076] 5. Growth Curve Determination of Strain

[0077] The growth curve of the bacteria was determined by turbidimetry. The OD600 value indirectly measured using a UV-visible spectrophotometer was used to infer the growth trend of the bacterial species in the sample. Inoculation and culture of the bacterial species: Pick an appropriate amount of glycerol or monoclonal bacterial species on agar and inoculate it into 5 mL of culture medium for culture. After culture reaches the plateau phase, take a sample and streak to verify the purity of the bacterial solution. Amplify the 5 mL bacterial solution to a volume of 25-30 mL. After culture reaches the plateau phase, take a sample and streak to verify the purity of the bacterial solution. Detect the OD600 value of the bacterial solution, dilute it by an appropriate multiple so that the OD600 value of the bacterial solution is between 0.02 and 0.1, and inoculate the seed solution into an appropriate amount of liquid culture medium according to the dilution multiple. Set the number of test groups according to the characteristics of the bacterial species. After mixing the bacterial solution, dispense it into 15 mL centrifuge tubes, dispense 4-5 mL into each tube, make 3 parallels for each group, and keep the culture medium as a blank control. Detect the initial OD600 value of the mixed bacterial solution, and place the blank control and the dispensed bacterial solution together in a shaker for culture. Determine OD600 values: Set the UV-Vis spectrophotometer to a wavelength of 600 nm. Measure the OD600 values ​​of the bacterial culture at different incubation times using a blank culture medium as a control. Record the incubation time and OD600 values. When setting sampling points, shorten the intervals during the exponential growth phase (measurements can be shortened to 15-30 minutes). Data processing: Use Origin or Prism software for graphing and data analysis (using the logistic nonlinear fitting equation).

[0078] The growth curve of E. coli LS-21 was determined (see Figure 4 ), it entered the logarithmic growth phase after about 1 hour, and after 5-6 hours of culture, the growth entered the stationary phase.

[0079] 2. Acid tolerance evaluation of Enterobacter huaxiensis LS-21

[0080] The acid tolerance of the LS-21 strain is expressed as the ratio of the total viable bacterial count after treatment with PBS at different pH values ​​to the initial total viable bacterial count. The pH values ​​were determined based on the pH values ​​of fasting simulated gastric fluid, standard simulated gastric fluid, and full simulated gastric fluid, which were 2.0, 3.0, and 4.0, respectively.

[0081] Inoculate LS-21 strain into 5 mL of LB broth and incubate in a constant temperature shaker at 37°C, 200 rpm for 16 hours. Place 30 mL of PBS (pH = 7.4) buffer in a centrifuge tube and slowly add 1 mol / L HCl dropwise, measuring with a pH meter as the pH is added, until the pH reaches just 2.0, 3.0, and 4.0. Mix 5 mL of PBS (pH = 7.4) and 5 mL of PBS at different pH values ​​with equal volumes of the bacterial suspension and incubate according to Table 1. Prepare the PBS buffer and bacterial suspension freshly before use.

[0082] After the culture was completed, the mixture was diluted 10 3 , 10 5 and 10 7 Pipette 100 μL of the dilution solution into a sterile petri dish, and make two sterile petri dishes for each dilution; at the same time, pipette 100 μL of PBS (pH = 2.0, pH = 3.0, and pH = 4.0) into a sterile petri dish as a blank control. Pour about 15 mL of 1.5% LB agar medium cooled to 50°C into the sterile petri dish, rotate the sterile petri dish clockwise or counterclockwise at least 20 times to mix it evenly, and after the agar solidifies, turn the plate over and culture it in a 37°C, 40% humidity incubator overnight. If there is no growth or the growth is small, you can choose to culture it for 48 to 72 hours. After culture, count all the colonies on the plate. The process from sample dilution to plate pouring should be completed within 15 minutes.

[0083] The total viable count of the sample solution treated with PBS (pH = 7.4) is recorded as N1, and the total viable count of the sample solution treated with PBS at pH = 2.0, pH = 3.0, or pH = 4.0 is recorded as N1'. Repeat the above steps to complete the acid tolerance test twice, where the total viable count of the sample solution treated with PBS (pH = 7.4) is recorded as N2 and N3, and the total viable count of the sample solution treated with PBS at pH = 2.0, pH = 3.0, or pH = 4.0 is recorded as N2' and N3'. Calculate the relative average deviation of the viable counts of N1, N2, and N3 from those of N1', N2', and N3'. The relative average deviation of the two sets of data should be no greater than 15%, otherwise the acid tolerance test must be repeated.

[0084] Table 2 Main parameters of the acid tolerance test method for Enterobacter sinoecii LS-21

[0085]

[0086] Acid tolerance is calculated according to the following formula:

[0087]

[0088] Where:

[0089] A--Acid tolerance, expressed as a percentage (%)

[0090] N1 - the total number of viable bacteria at the beginning of the first acid tolerance test, in CFU / mL;

[0091] N2--the total number of viable bacteria at the beginning of the second acid tolerance test, in CFU / mL;

[0092] N3 - the total number of viable bacteria at the beginning of the third acid tolerance test, in CFU / mL;

[0093] N1'--The total number of viable bacteria after the first acid tolerance test, in CFU / mL;

[0094] N2'--The total number of viable bacteria after the second acid tolerance test, in CFU / mL;

[0095] N3'--The total number of viable bacteria after the third acid tolerance test, in CFU / mL;

[0096] 1 / 3--the average value of three parallel data;

[0097] The calculation result is expressed as an integer.

[0098] according to Figure 5 The results of "Total number of viable bacteria in the original bacterial solution treated with PBS at different pH values" shown in the figure show that the tolerance of LS-21 bacteria to pH = 4.0 is 102%, the tolerance to pH = 3.0 is 105%, and the tolerance to pH = 2.0 is 74%. This indicates that LS-21 has good tolerance to acid and can well withstand the acidity of gastric juice, making it suitable for making oral live bacterial products.

[0099] 3. Antibiotic Susceptibility Testing of Enterobacter sinoecii LS-21

[0100] Antibiotic susceptibility testing is used to determine whether bacteria can be inhibited by the concentrations achieved by conventional doses of antimicrobial drugs, which is used to demonstrate their safety.

[0101] Experimental method: LS-21 bacteria were cultured overnight in TSA broth at 37°C and 200 rpm, and then streaked onto 1.5% TSA agar plates. Aseptically, 3 to 4 pure colonies cultured on the TSA plates for 18 to 24 hours were inoculated into 5 mL of trypticase soy broth (TSB), and cultured at 37°C and 200 rpm for 2 hours. The OD values ​​were calibrated to 0.5 McFarland units (OD) using a standard McFarland turbidimetric tube. 625 =0.08~0.10), which is equivalent to a bacterial concentration of 1.5×10 8 CFU / mL.

[0102] The prepared bacterial solution should be used within 15 minutes. Add 300 μL of the bacterial solution to the surface of 1.5% MHA agar and spread evenly with a sterile L-stick. Allow the plate to stand at room temperature for 3–5 minutes until the moisture on the plate is completely absorbed by the agar.

[0103] Use sterile tweezers to apply standard antimicrobial drug paper strips one by one as needed, gently pressing them firmly against the agar surface. Once the strips touch the plate, they should not be moved. The center-to-center distance between the strips should be greater than 24 mm, and the distance from the inner edge of the plate should be greater than 15 mm. Generally, no more than five strips should be placed on a 90 mm diameter plate. Within 15 minutes of applying the strips, flip the agar plate over and incubate at 37°C, 40% CO₂ for 17 h ± 1 h. During the incubation process, the plates should be placed individually, with no more than two stacked on top of each other.

[0104] Take out the plate and measure the diameter of the inhibition ring of each paper piece (including the diameter of the paper piece) on the back of the plate with a vernier caliper under transmitted light. The edge of the inhibition ring is limited to the point where no obvious bacterial growth can be seen with the naked eye. The growth of tiny colonies at the edge of the inhibition ring that can only be observed under a magnifying glass can be ignored.

[0105] Table 3 Interpretation of the inhibition zone diameter of Enterobacter huaxiensis LS-21

[0106]

[0107]

[0108] The results, as shown in Table 3, showed that E. coli LS-21 was sensitive to multiple antibiotics, including gentamicin (10 μg / tablet), tetracycline (30 μg / tablet), ciprofloxacin (5 μg / tablet), streptomycin (10 μg / tablet), polymyxin B (300 IU), and kanamycin (30 μg / tablet). (Bacterial antimicrobial resistance refers to the gradual weakening of bacterial sensitivity to antimicrobial drugs after repeated exposure, or even the ability to resist and not be inhibited or killed. This characteristic of bacteria is called drug resistance. It is usually measured through antimicrobial susceptibility testing (AST), and the results can be expressed as sensitive (S), intermediate (I), and resistant (R). Resistant (R) refers to bacterial growth and reproduction being inhibited by achievable antimicrobial concentrations at standard doses. Intermediate (I) refers to bacterial strains whose efficacy at standard doses of antimicrobial drugs is lower than that of susceptible strains, whose efficacy is at physiologically concentrated sites, or whose treatment can be achieved with higher-than-normal doses.)

[0109] The results of the antibacterial experiments showed that the diameter of the inhibition zone fell within the range of certain bacterial resistance mechanisms, and the bacteria could not be inhibited by the concentrations achieved by conventional doses of antimicrobial drugs. This indicates that LS-21 is sensitive to most antibiotics, indicating that this strain is relatively safe for use in biological preparations or dietary supplements.

[0110] Effects of Enterobacter huaxiensis LS-21 on growth performance and organ indices in mice with DSS-induced colitis

[0111] 1. Source and Treatment of Mice

[0112] The experiment used 8-week-old, female C57BL / 6 mice, SPF grade, from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd. After arrival, the animals were first acclimated to the institution for 1 week, with approximately 12 h / 12 ​​h alternating light / dark lighting per day. The ambient temperature and relative humidity of the animal room were controlled at 22-26°C and 40-70%, respectively.

[0113] 2. Grouping of Mice

[0114] Fifteen healthy C57BL / 6 mice were randomly divided into three groups according to their body weight, with 5 mice in each group, namely the No DSS group / blank control group, the DSS model group, and the DSS+LS-21 group.

[0115] Table 4 Grouping and treatment of experimental mice

[0116]

[0117] 3.DSS-induced colitis model mice

[0118] All mice (except the No DSS group) were given drinking water containing 3% DSS (Dextran Sulfate Sodium Salt) (ad libitum). The mice were gavaged daily from the beginning of the experiment and weighed before gavage until the weight loss rate of the DSS group mice was >20%, indicating successful model establishment. The weight loss rate formula for mice is as follows:

[0119] Mouse weight loss rate (%) = (initial weight - current weight) / initial weight * 100%

[0120] See the results Figure 6 shown.

[0121] 4. Treatment of Control and Experimental Group Mice

[0122] The experimental group mice were given 3% DSS in their drinking water, and the control group mice were gavaged with 200 μL of fresh LS-21 culture medium every day. Figure 7 The weight changes of different groups after treatment are shown. On the 11th day of the experiment, the weight of mice in the DSS+LS-21 group was significantly higher than that in the DSS group, indicating that LS-21 can significantly improve the weight loss of mice induced by DSS.

[0123] 5. Disease Activity Index (DAI) Assessment

[0124] During the trial, the clinical progression of colitis was assessed daily. DAI is a comprehensive score based on the animal's weight loss rate, stool consistency, and blood in stool. DAI = (weight change score + stool consistency score + blood in stool score) / 3. Figure 8 As shown in the figure, on the 9th day of the experiment, the DAI score of mice in the DSS+LS-21 group was significantly lower than that in the DSS group, indicating that oral administration of LS-21 improved the decline in disease index caused by DSS induction.

[0125] Fecal occult blood detection in mice: Testing was performed using a Fecal Occult Blood Qualitative Detection Kit (o-Tolidine Method, Leigen Biotechnology) (see the instructions for detailed procedures). Results were interpreted in the Fecal Occult Blood Interpretation Chart. Visible blood in the stool was assigned a score of 4.

[0126] Table 5 Scoring criteria for the disease activity index (DAI) of mice

[0127]

[0128] *Normal stool is formed; loose stool is loose or soft stool that does not stick to the anus; and loose stool is unformed stool that sticks to the anus.

[0129] **The degree of blood in stool is assessed according to Table 4. If there is visible blood in the stool, it is directly scored as 4 points.

[0130] Table 6 Fecal occult blood interpretation table

[0131]

[0132] 6. Spleen Index

[0133] Methods: After euthanasia, the spleens of mice were taken out and weighed for the determination of spleen index.

[0134] Spleen index = spleen weight (mg) / mouse body weight (g)

[0135] like Figure 9 As shown in the figure, the spleen index of mice in the DSS+LS-21 group was significantly lower than that in the DSS model group, indicating that the degree of inflammation in mice gavage with LS-21 was significantly reduced.

[0136] 5. Effects of Enterobacter huaxiensis LS-21 on biochemical parameters in mice with DSS-induced colitis

[0137] 1. Tissue Section Staining

[0138] After measuring the length of the colon, the cecum was cut off and the colon was twisted into a spiral on a horizontal plane and fixed in 4% tissue fixative. The intestinal tissue was stained with hematoxylin-eosin (HE) and sent to Wuhan Saiweier Biotechnology Co., Ltd. for preparation.

[0139] like Figure 10 As shown, the DSS group showed extensive colonic ulceration, with loss of mucosal epithelium at the ulcer site. Intestinal glandular structures disappeared and were replaced by proliferating connective tissue, accompanied by abundant lymphocyte and granulocyte infiltration. Irregular arrangement of intestinal glands was observed around the ulcer site. The submucosa was slightly edematous, with loosely arranged connective tissue and a small infiltration of lymphocytes and granulocytes. The muscularis layer was severely thickened. In the DSS+LS-21 group, the colonic surface was characterized by a single layer of columnar epithelium with normal morphology and structure. Intestinal glands were abundant in the lamina propria, with goblet cells interspersed between epithelial cells. A double layer of smooth muscle cells, the muscularis mucosa, separated the intestinal crypts from the submucosa. The submucosa consisted of connective tissue, while the remaining intestinal wall consisted of a muscularis layer and a serosa, both composed of smooth muscle cells. This suggests that oral administration of LS-21 significantly ameliorated the pathological changes in the colon of mice with acute colitis induced by DSS.

[0140] 2. MPO detection

[0141] Myeloperoxidase (MPO) is a marker of neutrophil activation, and its level and activity represent neutrophil function and status. Physiologically, MPO is part of the innate immune system, combating the invasion of pathogens such as bacteria and fungi. Under specific conditions, MPO can catalyze reactions to generate excessive amounts of oxidants. When these exceed the body's antioxidant defenses, they can lead to oxidative stress and oxidative tissue damage, contributing to the development of numerous diseases, including inflammation, vasculitis, tumors, nephritis, and atherosclerosis.

[0142] Four colon samples were randomly selected from each experimental group for MPO testing. Colonic MPO was measured using a kit purchased from Nanjing Jiancheng Bioengineering Institute. MPO in the colon was quantitatively analyzed using an ultraviolet spectrophotometer. Specific procedures were followed according to the kit instructions.

[0143] like Figure 11 As shown, the MPO of the middle and distal segments of the colon of mice in the DSS+LS-57 group were significantly lower than those in the DSS group, and the MPO of the distal segment of the colon of mice in the DSS+LS-57 group was extremely significantly lower than that in the DSS group, indicating that the E. coli LS-21 significantly repaired the inflammation in the colon of mice.

[0144] 3. Expression of pro-inflammatory / anti-inflammatory factors

[0145] The expression levels of pro-inflammatory factors TNF-α, IL-6 and TNFR-2 genes in the colon were analyzed by qPCR.

[0146] Colon tissue stored at -80°C was lysed with TriZol lysis buffer, and then total RNA was extracted by treating with chloroform, isopropanol, and 75% ethanol. The absorbance values ​​at A260 / A280 and A260 / A230 were measured using an ultra-micro spectrophotometer to detect RNA concentration and purity. Qualified RNA was reverse transcribed into cDNA and purified using the DNA sequence of Beijing Quanshijin Biotechnology Co., Ltd. One-Step gDNA Removal and cDNA Synthesis SuperMix Kit was used. The specific operation steps were carried out according to the kit instructions. The reaction system was 20μl, containing 2×TS Reaction Mix 10μl, Random Primer 1μl, RT / RI Enzyme Mix 1μl, gDNA Remover 1μl, RNase-free water to 20μl, total RNA 500ng. Reaction conditions: incubate at 25℃ for 10min, incubate at 42℃ for 15min, and inactivate by heating at 85℃ for 5s. RT / RI and gDNA Remover.

[0147] Real-time fluorescence quantitative PCR (qPCR) primer sequences are detailed in Table 6. 7 μL of ddH₂O was used. PCR amplification conditions included 39 cycles of pre-denaturation at 95°C for 3 min, followed by denaturation at 95°C for 10 s and annealing at 60°C for 30 s. TBP was used as an internal reference. The 2-ΔΔCt method was used for analysis of gene expression. The formula for calculating gene expression was: 2Ct target (control) – Ct target (treatment) / 2Ct TBP (control) – Ct TBP (treatment).

[0148] like Figure 12 The results showed that the expression levels of pro-inflammatory factors TNF-α, IL-6 and TNFR-2 genes in the colon tissue of mice in the DSS+LS-21 group were lower than those in the DSS model group, showing a downward trend, indicating that LS-21 has a good therapeutic effect on DSS-induced acute colitis in mice.

Claims

1. A strain of Enterobacter huaxiensis LS-21, deposited with the China General Microbiological Culture Collection Center on October 28, 2024, with the deposit number CGMCC NO.32366.

2. The Enterobacter szechwanensis according to claim 1, characterized in that: The 16S rDNA gene sequence thereof comprises the sequence shown in SEQ ID NO.

3.

3. The Enterobacter sphenotype according to claim 1, wherein the bacterium has the ability to move in clusters; further, the bacterium can grow into monoclonal colonies that appear to diffuse in a thin film.

4. The Enterobacter sphenanthera according to claim 1, wherein the bacterium is a Gram-negative bacterium with a short rod-shaped morphology, and further, is a white-like clone.

5. A composition comprising the Enterobacter szechuanensis according to any one of claims 1 to 4, wherein the Enterobacter szechuanensis composition can be a food, a medicine, or a health product.

6. Use of the Enterobacter schensisensis according to claims 1-4 and the composition according to claims 5-6 in the preparation of medicines or foods for treating or alleviating colitis and / or improving intestinal health.

7. A food composition capable of improving intestinal health, comprising the Enterobacter huaxiensis strain LS-21.

8. The composition according to claim 5, which is an oral bacterial agent; preferably, the oral bacterial agent is a viable bacterial agent, and further the viable bacterial count is not less than 1.5E+09 CFU / g.

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