Application of lactobacillus salivarius LS08 in preparation of medicine for preventing or treating intestinal inflammation

Abstract

The invention discloses application of lactobacillus salivarius LS08 in preparation of a medicine for preventing or treating intestinal inflammation, and belongs to the technical field of microorganisms. The preservation number of the lactobacillus salivarius LS08 disclosed by the invention is CGMCC (China General Microbiological Culture Collection Center) No.22988. The lactobacillus salivarius LS08 can inhibit the DSS from inducing neutrophil to gather towards the intestinal tract of the zebra fish, so that a theoretical reference and a guidance basis are provided for developing the medicine for preventing or treating the intestinal inflammation by utilizing the lactobacillus salivarius LS08.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically to the use of Lactobacillus salivarius LS08 in the preparation of drugs for the prevention or treatment of intestinal inflammation. Background Technology

[0002] Enteritis is generally caused by bacteria, viruses, fungi, or parasites. Symptoms mainly include abdominal pain, diarrhea, and fever. Enteritis includes chronic and acute enteritis. Chronic enteritis is characterized by intermittent abdominal pain, bloating, increased bowel movements, and diarrhea. Symptoms are often related to exposure to cold, consumption of spicy or greasy foods, lack of sleep, or fatigue. Acute enteritis is characterized by nausea, vomiting, abdominal pain, diarrhea, and fever. The abdominal pain is paroxysmal and spasmodic, relieved by defecation. Bowel movements are frequent, with stools being watery or containing mucus and blood; some patients experience a feeling of incomplete evacuation or tenesmus. If acute enteritis patients experience diarrhea and vomiting without effective treatment, dehydration, electrolyte imbalance, and even acidosis may occur.

[0003] Traditional treatments for enteritis can be divided into modern medical treatments and traditional Chinese medicine treatments. Probiotics, as an endogenous and immune defense barrier in the gut, can antagonize pathogenic bacteria and are characterized by safety, controllability, effectiveness, and minimal side effects, making them an ideal method for treating enteritis. However, the lack of strong scientific research evidence regarding the functions of probiotics has seriously hindered the promotion of probiotics and their products. Therefore, in-depth exploration of the functions of bacterial strains and the screening of novel probiotic strains with independent intellectual property rights, specific functional properties, and suitability for the physiological characteristics of the Chinese population are crucial for enhancing the core competitiveness of Chinese probiotic manufacturers and promoting the development of probiotic products in my country.

[0004] Therefore, providing the application of Lactobacillus salivarius LS08 in the preparation of drugs for the prevention or treatment of intestinal inflammation is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides the use of Lactobacillus salivarius LS08 in the preparation of drugs for the prevention or treatment of intestinal inflammation.

[0006] Inflammation is an immune response to tissue damage and infection, characterized by the aggregation of white blood cells (granulocytes and macrophages) around infected tissue. The zebrafish immune system is very similar to that of mammals; when trauma occurs, neutrophils and macrophages respond to traumatic inflammation almost simultaneously. Neutrophils migrate rapidly, recruiting to the site of injury first, followed by macrophages. Several hours later, the inflammation begins to subside, and macrophages and neutrophils leave the site of injury. Inducing intestinal inflammation in zebrafish with sodium dextran sulfate (DSS) stimulates an immune response in zebrafish neutrophils.

[0007] Using transgenic fluorescent neutrophil fish (green in color), the number of neutrophils in the intestine of DSS-induced zebrafish was significantly increased compared to that of normal zebrafish under a fluorescence microscope.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A strain of *Lactobacillus salivarius* LS08, with accession number CGMCC No. 22988, has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit date was July 30, 2021, and the strain is classified as *Lactobacillus salivarius*.

[0010] Furthermore, the application of the aforementioned Lactobacillus salivarius LS08 in the preparation of drugs for the prevention or treatment of intestinal inflammation.

[0011] Furthermore, the application of the aforementioned Lactobacillus salivarius LS08 in the preparation of a drug that inhibits DSS-induced neutrophil aggregation in the zebrafish intestine.

[0012] Furthermore, the Lactobacillus salivarius LS08 is a bacterial suspension.

[0013] As can be seen from the above technical solution, compared with the prior art, the present invention discloses the application of Lactobacillus salivarius LS08 in the preparation of drugs for the prevention or treatment of intestinal inflammation. Lactobacillus salivarius LS08 was isolated and screened from the feces of long-lived elderly people in Jiaoling County, Meizhou City, Guangdong Province. Lactobacillus salivarius LS08 can inhibit DSS-induced neutrophil aggregation in the zebrafish intestine. This provides a theoretical reference and guiding basis for the development of drugs for the prevention or treatment of intestinal inflammation using Lactobacillus salivarius LS08. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0015] Figure 1 The attached figure shows the colony morphology of Lactobacillus salivarius LS08 of the present invention on MRS agar plates;

[0016] Figure 2 The attached figure is a visual representation of how Lactobacillus salivarius LS08 of the present invention inhibits DSS-induced neutrophil aggregation in the zebrafish intestine.

[0017] Among them, A: normal group; B: model group; C: positive control group; D: Lactobacillus salivarius 11741; E: Lactobacillus salivarius LS08;

[0018] Figure 3 The attached figure is a statistical graph showing the inhibition of DSS-induced neutrophil aggregation in the zebrafish intestine by Lactobacillus salivarius LS08 of the present invention. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] The zebrafish were Tg(mpx:EGFP) centriole cell green fluorescent zebrafish and AB line zebrafish.

[0021] Sodium dextran sulfate (DSS) (MedChemExpress); mesalazine (MedChemExpress); and Lactobacillus salivarius 11741 (ATCC11741) were purchased from Beijing Bio-Bio Biotechnology Co., Ltd.

[0022] Example 1: Isolation, identification and preservation of Lactobacillus salivarius LS08

[0023] (1) Separation:

[0024] 1) Dissolve the feces of the long-lived elderly (about 0.1g) in a 1.5mL centrifuge tube containing 1mL of sterile physiological saline, and mix thoroughly by pipetting with a 1mL sterile pipette tip.

[0025] 2) Add 900 μL of sterile physiological saline to each of six sterile 1.5 mL centrifuge tubes.

[0026] 3) Use a serial dilution method to dilute the sample to 10. -2 10 -3 10 -4 10 -5 10 -6 10 -7 .

[0027] 4) From the container containing 10 -4 Pipe 100 μL of the sample dilution from centrifuge tubes and inoculate it onto MRS, BHI, and BS solid media. Spread the 100 μL of bacterial culture evenly and quickly, being careful to use a gentle and rapid spreading technique, and ensuring the process is performed near an alcohol lamp flame. After spreading, label the side of the petri dish with information including the name, sample number, culture medium name, incubation time, dilution gradient, and incubation conditions (anaerobic / aerobic).

[0028] 5) Repeat the previous step to complete 10. -5 10 -6 10 -7 Dilution gradient dilution coating.

[0029] 6) After coating, place the petri dishes in anaerobic conditions at 37°C and incubate for 48 hours. Observation and recording can be carried out after 48 hours.

[0030] 7) Use an inoculation loop to pick up a single colony from the plate and streak it onto MRS solid medium. Incubate anaerobically at 37°C for 48 hours to obtain pure colonies.

[0031] 8) Inoculate the pure colonies on the plate into MRS liquid medium, anaerobic culture at 37°C for 12-16 h, add 20% glycerol, and store in a -80°C refrigerator.

[0032] (2) Molecular biological identification of the strain: Genomic DNA was extracted from the obtained strain, and the full-length 16S rDNA fragment was amplified by PCR using the universal 16S rDNA primers 27F and 1492R. Then, sequencing was performed to identify the species of the strain.

[0033] The primer sequences for universal primers 27F and 1492R are as follows:

[0034] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; SEQ ID NO.1;

[0035] 1492R: 5'-GGTTACCTTGTTACGACTT-3'; SEQ ID NO. 2.

[0036] Experimental results: The strains screened from the feces of centenarians in Jiaoling County, Meizhou City, Guangdong Province were identified as Lactobacillus salivarius by morphological observation and 16S rDNA identification. The 16S rDNA sequence of strain LS08 is shown in SEQ ID NO.3.

[0037]

[0038] When a single colony of strain LS08 was inoculated onto MRS solid medium, it grew well anaerobically at 37°C. The colonies were milky white, spherical, and smooth. Figure 1 Strain LS08 has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit date was July 30, 2021. The strain is classified as Lactobacillus salivarius, and the accession number is CGMCC No. 22988.

[0039] Example 2: Preparation of Lactobacillus salivarius LS08 bacterial suspension (cells)

[0040] Lactobacillus salivarius LS08 was activated and cultured, then inoculated into MRS liquid medium and cultured at 37°C for 24 h. The culture was then centrifuged at 4°C and 6000 rpm for 10 min to obtain a bacterial pellet. The bacterial pellet was washed twice with PBS, and the cells were resuspended in PBS to adjust the cell concentration to 1 × 10⁻⁶. 6 A bacterial suspension (bacterial cells) was obtained at CFU / mL.

[0041] Example 3: Preparation of Lactobacillus salivarius 11741 bacterial suspension (cells)

[0042] Lactobacillus salivarius 11741 was activated and cultured, then inoculated into MRS liquid medium and cultured at 37°C for 24 h. The culture was then centrifuged at 4°C and 6000 rpm for 10 min to obtain a bacterial pellet. The bacterial pellet was washed twice with PBS, and the cells were resuspended in PBS to adjust the cell concentration to 1 × 10⁻⁶ cells / mL. 6 A bacterial suspension (bacterial cells) was obtained at CFU / mL.

[0043] Example 4: Effect of Lactobacillus salivarius LS08 on DSS-induced neutrophil aggregation in zebrafish intestine.

[0044] Healthy Tg(mpx:EGFP) zebrafish (3dpf) were selected and placed in 6-well cell culture plates, one well per group, with 15 fish per well (15 fish per well, neutrophil count in the intestine of each fish). 5 mL of PBS was added to the normal group, and 1 × 10⁻⁶ cells were added to the model group, positive control group (mesalazine), and Lactobacillus salivarius 11741 intervention group (1 × 10⁻⁶ cells per well). 6 CFU / mL), Lactobacillus salivarius LS08 intervention group (1×10 6All zebrafish (CFU / mL) were added to 5 mL of 0.5% DSS solution (w / w) and incubated at 28°C for 72 h. Each group of zebrafish was then washed three times with PBS. The normal and model groups were then added to 5 mL of PBS; the positive control group was added to 5 mL of 50 μg / mL mesalazine solution; and the Lactobacillus salivarius 11741 intervention group was added to 1×10⁻⁶ CFU / mL solution. 6 CFU / mL Lactobacillus salivarius 11741, 5 mL per well; Lactobacillus salivarius LS08 intervention group added 1×10 6 CFU / mL Lactobacillus salivarius LS08, 5 mL per well, incubated in a biochemical incubator at 28℃ for 48 h, then observed the aggregation of neutrophils in the zebrafish intestine under a fluorescence microscope and photographed.

[0045] Data were statistically processed using SPSS 19.0 software. Experimental data are expressed as mean ± SEM values. T-tests were used for analysis, comparing the results with the normal group: #### p<0.001, compared with the model group: **P<0.01, ***P<0.005.

[0046] The experimental results of Lactobacillus salivarius LS08 inhibiting DSS-induced neutrophil aggregation in the zebrafish intestine are as follows: Figure 2 and Figure 3 As shown.

[0047] Depend on Figure 2 and Figure 3 It was found that a small number of neutrophils (15.27±1.41) were aggregated in the intestine of zebrafish in the normal group, while a large number of neutrophils were aggregated in the intestine of zebrafish in the model group. Furthermore, the number of neutrophils in the intestine of zebrafish in the model group was 28.87±2.59, which was significantly different from that in the normal group (15.27±1.41) (P<0.001), indicating that the DSS-induced zebrafish intestinal inflammation model was successfully established.

[0048] Depend on Figure 2 and Figure 3 It was found that only a small number of neutrophils were aggregated in the intestines of zebrafish in the positive control group (methalazine). Simultaneously, the number of neutrophils in the intestines of zebrafish in the positive control group (methalazine) was 17.53±1.09, which was significantly different from that in the model group (28.87±2.59) (P<0.005). Therefore, methalazine has an effect in alleviating intestinal inflammation, consistent with clinical results, indicating that this trial evaluating its efficacy in alleviating intestinal inflammation is effective. Lactobacillus salivarius 11741 intervention group (1×10⁻⁶) 6 The zebrafish gut containing CFU / mL showed a large accumulation of neutrophils, similar to the model group; meanwhile, the Lactobacillus salivarius 11741 intervention group (1×10⁻⁶ CFU / mL) showed similar activity. 6The number of neutrophils in the zebrafish intestine (CFU / mL) was 25.67±2.15, which was not significantly different from that in the model group (28.87±2.59) (P>0.05). Furthermore, only a small number of neutrophils were aggregated in the zebrafish intestine of the *Lactobacillus salivarius* LS08 group, similar to the positive control group (mesalazine); meanwhile, the *Lactobacillus salivarius* LS08 intervention group (1×10⁻⁶ CFU / mL) showed similar neutrophil aggregation. 6 The number of neutrophils in the zebrafish intestine (CFU / mL) was 19.73±1.45, which was significantly different from that in the model group (28.87±2.59) (P<0.01). Therefore, the above results indicate that at the same concentration, *Lactobacillus salivarius* LS08 has a stronger inhibitory effect on DSS-induced neutrophil aggregation in the zebrafish intestine than *Lactobacillus salivarius* 11741, demonstrating a good potential to alleviate intestinal inflammation.

[0049] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of Lactobacillus salivarius LS08 in the preparation of drugs for the prevention or treatment of intestinal inflammation, characterized in that, The preservation number of the Lactobacillus salivarius LS08 is CGMCC No. 22988.

2. The application of Lactobacillus salivarius LS08 according to claim 1 in the preparation of drugs for the prevention or treatment of intestinal inflammation, characterized in that, The Lactobacillus salivarius LS08 is a bacterial suspension.

3. The use of Lactobacillus salivarius LS08 as described in claim 1 in the preparation of a drug for inhibiting DSS-induced neutrophil aggregation in the zebrafish intestine.

4. The application of Lactobacillus salivarius LS08 according to claim 3 in the preparation of a drug for inhibiting DSS-induced neutrophil aggregation in the zebrafish intestine, characterized in that, The Lactobacillus salivarius LS08 is a bacterial suspension.

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