CN115725456A

CN115725456A - Lactobacillus salivarius and application thereof

Info

Publication number: CN115725456A
Application number: CN202211242871.5A
Authority: CN
Inventors: 李静; 尚可; 范振鑫; 岳碧松
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2022-10-11
Filing date: 2022-10-11
Publication date: 2023-03-03

Abstract

The invention provides lactobacillus salivarius and application thereof, belonging to the technical field of microorganisms, wherein the lactobacillus salivarius is preserved in the common microorganism center of the China Committee for culture Collection of microorganisms at 2022, 9, 2 months, addresses: the lactobacillus salivarius is helpful for recovering weight, relieving inflammation level, protecting intestinal tract tissue and regulating intestinal tract flora structure, and can be used as probiotic candidate strain for preventing and treating animal enteritis.

Description

Lactobacillus salivarius and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly discloses lactobacillus salivarius and application thereof.

Background

Inflammatory enteritis is an important factor influencing and threatening the health of various animals such as macaques and the like, and the occurrence reasons of inflammatory enteritis are comprehensive and complex, so that the inflammatory enteritis is difficult to prevent and radically cure. Clinically, the sick macaques are usually treated by broad-spectrum antibiotics, but the normal microbial structures of the intestinal tracts of the macaques are destroyed by long-term use of the antibiotics, so that the dysbacteriosis is caused. As an alternative to antibiotics, probiotics have attracted considerable attention in recent years in the field of human and animal health, and are thought to have the effects of enhancing immunity, relieving inflammation, increasing nutrients, and regulating intestinal flora.

However, there are few related probiotic strains that are disclosed to regulate the intestinal flora and improve the intestinal environment, and the disclosed lactobacillus salivarius cannot be effectively used as a drug for relieving inflammatory enteritis.

Disclosure of Invention

The first objective of the present invention is to provide a Lactobacillus salivarius (deposited in the china general microbiological culture collection center on 9-2/2022, address: the collection number of the microorganism research institute of the Chinese academy of sciences, no.3 Xilu No.1 of Beijing, chaoyang, and China is CGMCC NO.25641, which can solve the technical problem that related probiotic strains for relieving inflammatory enteritis are few at present.

The second purpose of the invention is to provide an application of lactobacillus salivarius in preparing medicines and health products for preventing and treating colitis, protecting intestinal tissues and regulating intestinal flora structure.

The third purpose of the invention is to provide a microbial inoculum containing the lactobacillus salivarius, which can be used as a raw material for preparing a medicament for relieving inflammatory enteritis of animals or directly used as a medicament, and provides a novel probiotic medicament for relieving inflammatory enteritis.

The fourth purpose of the invention is to provide a medicament containing the lactobacillus salivarius, which can be directly used clinically to relieve inflammatory enteritis of animals.

The fifth purpose of the invention is to provide a health care product for protecting intestinal tissues, which can protect the internal environment of the intestinal tract, so that the intestinal tract can maintain normal metabolic function, and a certain prevention effect on colitis and other diseases can be achieved.

Compared with the prior art, the invention at least has the following advantages and positive effects: the lactobacillus salivarius is beneficial to restoring weight, relieving inflammation level, protecting intestinal tissues and regulating the structure of intestinal flora, can be used as a probiotic candidate strain and is used for preventing and treating enteritis of animals.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 shows the colony morphology of Lactobacillus salivarius MK0901 on MRS agar medium in the present invention;

FIG. 2 is a tree of evolution of Lactobacillus salivarius MK0901 in the invention;

FIG. 3 is a graph showing the results of measuring the hemolytic activity of Lactobacillus salivarius MK0901 in Experimental example 1 in accordance with the present invention;

FIG. 4 is a graph showing the body weight and spleen index changes of a mouse in Experimental example 7, wherein A is the body weight change trend of the mouse during the experiment and B is the spleen index of the mouse at the end of the experiment;

FIG. 5 is a graph showing HE staining of mouse colon in Experimental example 7 of the present invention, wherein A is CK group, B is DSS group, C is LGG group, and D is MK0901 group;

FIG. 6 is a graph showing the levels of inflammatory factors in mice in Experimental example 7 of the present invention, wherein A is TNF-. Alpha.and B is IL-1. Beta., and C is IL-6, and the significance of the difference compared with the CK group is shown by "#" and the significance of the difference compared with the DSS group is shown by "#";

FIG. 7 is a diagram showing the differences in the bacterial flora structures among the groups of samples according to Experimental example 7 of the present invention, wherein A is a Venn diagram, B is a PCoA analysis, C is a phylum-level species stacking diagram, and D is a genus-level species stacking diagram;

FIG. 8 is a GC-MS analysis chart of a fermentation liquid of Lactobacillus salivarius MK0901 in Experimental example 5 of the invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are conventional products which are not indicated by manufacturers and are commercially available.

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail below with reference to specific examples.

Examples

1. Separation, purification and identification of the strain:

reagents and consumables:

the main reagents include: MRS agar medium, MRS broth medium, nutrient Agar (NA) medium, trypticase Soy Broth (TSB) medium, and gram stain solution were purchased from Kyowa Kai Microbiol technologies, inc.; pig bile salt is purchased from Beijing Hongbaoshun technology Limited; the blood plate was purchased from Chengdu Qi medical science and technology, inc.; artificial gastric juice, artificial intestinal juice and antibiotic drug sensitive paper sheets are purchased from Shanghai Yuan leaf Biotech limited company; trypsin-EDTA solution, DMEM (Dulbecco's Modified Eagle Medium) Medium purchased from Saimer Feishell science and technology (China) Co., ltd; PCR Premix was purchased from Dow Oakbio, inc.; 16S rRNA gene primers (27F: SEQ ID NO.2, 1492R: SEQ ID NO. 3) were synthesized by Biotechnology engineering (Chengdu) Ltd; HT-29 cells are available from other laboratories of the college. The main consumables include: centrifuge tubes, cell culture flasks, cell culture plates, disposable pipettes, disposable plastic culture dishes, disposable cell coating rods, strain preservation tubes, slides, and the like.

An instrument device:

the main instrumentation consists of: an ultra-clean workbench, a constant-temperature incubator, a vibration incubator, a vortex oscillator, a vertical autoclave, a PCR instrument, a centrifuge, a micropipette, an optical microscope, a pH meter, a vernier caliper, an electronic balance and the like.

Collecting a healthy macaque excrement sample:

fresh excrement of 10 healthy young macaques is collected in a macaque culture base in Meishan City, sichuan province. These young macaques have not experienced a diarrheal condition and have not been used with antibiotics for a period of one year. Collecting fresh macaque feces in a sterile sampling tube by using a sterile sampler, storing the sterile macaque feces in an incubator with an ice bag, and finally transferring the sterile macaque feces to a laboratory for immediate treatment.

Strain separation and purification:

taking about 5 g of fresh macaque manure, conveniently adding 5mL of sterile normal saline into a 10mL sterile centrifuge tube, fully oscillating the fresh macaque manure into uniform manure liquid by using a vortex oscillator, diluting the manure liquid by using a 10-fold dilution method, and selecting 10 ³ -10 ⁵ The diluted liquid was spread on MRS agar plates and cultured at 37 ℃ for 24 hours. According to the colony morphology difference on the culture medium, a single colony is selected by using an inoculating loop to be inoculated on an MRS agar plate, the culture is carried out for 24 hours at 37 ℃, and then the single colony is selected for secondary purification. The purified single colony was dipped with an inoculating loop and shaken in a strain storage tube containing 600. Mu.L of MRS broth, cultured overnight at 37 ℃ with shaking, added with 400. Mu.L of 50% glycerol, and stored at-80 ℃ for further use. Gram staining of bacteria:

and sucking proper sterile normal saline on the glass slide by using a pipettor, selecting a single bacterial colony in the normal saline by using an inoculating loop, and uniformly coating the bacterial liquid. The slide was quickly baked using an alcohol burner until the broth was dry. And (3) dripping 2 drops of crystal violet dye solution on the fixed and dried glass slide, and washing with sterile water until the color is colorless after the initial dyeing is carried out for 1 min. 2 drops of iodine solution are added dropwise, after 1min of mordant dyeing, the mixture is washed by sterile water until colorless. After elution with 95% alcohol for 30s, the column was rinsed with sterile water. 2 drops of safranine dye solution are dripped on the glass slide, and after 1min of re-dyeing, the glass slide is washed by sterile water until colorless. Excess water was blotted dry with absorbent paper, and after the slide was dried, the slide was observed with an oil mirror (x 100 times) to record the form and color of the cells. Gram-positive bacteria appear purple and gram-negative bacteria appear red.

16S rRNA gene identification:

the bacterial frozen stock solution is dipped by an inoculating loop and streaked on an MRS agar plate, the bacterial frozen stock solution is cultured for 24h at 37 ℃, a single colony is selected to be cultured in an MRS broth culture medium for 24h at 37 ℃, and then the bacterial DNA is extracted by using a CTAB/NaCl method. The 16S rRNA gene of the bacteria is amplified by using a PCR method, wherein the PCR system comprises the following steps: 12.5. Mu.L Taq MasterMix, 1. Mu.L upstream primer, 1. Mu.L downstream primer, 1. Mu.L DNA template, 9.5. Mu.L ddH ₂ O, final total volume 25. Mu.L. The reaction procedure for PCR was: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing at 54 ℃ for 30s, and extension at 72 ℃ for 45s, wherein 30 cycles are performed, and final extension at 72 ℃ is 10min. And (3) carrying out electrophoresis detection on the PCR product by adopting 1.0% agarose gel, and sending a qualified sample to a biological engineering (Chengdu) limited company for sequencing. The sequenced sequences were aligned using BLAST and the 16SrRNA sequence is shown in SEQ ID NO. 1.

Primary screening of strains:

the frozen stock solution of the strain to be detected is dipped by using an inoculating loop, streaked on an MRS agar plate, and cultured for 18h at 37 ℃. Preparing MRS broth, adjusting pH to 3 with hydrochloric acid, sucking 3mL of the adjusted MRS broth, culturing in 5mL sterile centrifuge tube, dipping 4 single colonies in the centrifuge tube with sterile cotton swab, shaking thoroughly, eluting the colonies, adjusting the concentration of the bacteria solution to about 1 × 10 ⁸ CFU/mL, at 37 degrees C shaking culture for 18h, then suction 1 u L bacterial liquid spot in MRS agar plate, 37 degrees C culture for 24h.

Then, the frozen stock solution of the strain to be detected is dipped by using an inoculating loop, streaked on an MRS agar plate, and cultured for 18h at 37 ℃. Preparing MRS broth culture medium, preparing MRS broth culture medium containing 0.3% bile salt by using pig bile salt, sucking 3mL of prepared MRS broth culture medium into a 5mL sterile centrifuge tube, dipping 4 single bacterial colonies into the centrifuge tube by using a sterile cotton swab, fully oscillating, eluting the bacterial colonies, and adjusting the concentration of the bacterial liquid to about 1 × 10 ⁸ CFU/mL, at 37 degrees C shaking culture for 18h, then suction 1 u L bacterial liquid spot in MRS agar plate, 37 degrees C culture for 24h, observed growth (details are shown in figure 1).

A total of 69 Lactobacillus salivarius (Lactobacillus salivarius) strains were isolated from macaque feces by gram staining and identification of the 16S rRNA gene. The acid resistance and cholate resistance of all separated strains are preliminarily screened to obtain 6 strains of lactobacillus salivarius. A phylogenetic tree (see FIG. 2 for details) was constructed by downloading the reference genome in NCBI, and the distribution of strains on the phylogenetic tree was consistent with the 16S rRNA identification results.

The target strain was successfully selected and named lactobacillus salivarius MK0901.

Experimental example 1

1. Hemolytic assay of lactobacillus salivarius MK0901 strain:

and (3) after the frozen stock solution of the strain to be detected is streaked on an MRS agar plate by using an inoculating loop, selecting a single colony, streaking on a blood plate, culturing for 24 hours, and observing the hemolysis condition. Hemolysis is mainly classified into alpha hemolysis (grass green hemolysis ring appears around bacterial colony), beta hemolysis (clear hemolysis ring appears around bacterial colony), and gamma hemolysis (no hemolysis ring appears around bacterial colony), and each strain is repeated three times. The results obtained using Staphylococcus haemolyticus ATCC29970 (Staphylococcus haemolyticus ATCC 29970) as a positive control are shown in FIG. 3, and the Lactobacillus salivarius is nonhemolytic as shown by the results in FIG. 3.

Experimental example 2

1. Drug susceptibility testing of lactobacillus salivarius MK0901 strain:

the strains were subjected to a drug susceptibility test using the paper agar diffusion method (K-B) (Cockerrill, 2015). After the frozen stock solution of the strain to be detected is streaked on an MRS agar plate by using an inoculating loop, the strain is cultured for 24 hours at 37 ℃.3mL of sterile normal saline is sucked into a 5mL centrifuge tube, 5 single colonies are dipped by using a sterile cotton swab and sufficiently oscillated in the centrifuge tube, and the concentration of the bacteria liquid is adjusted to about 1.5 multiplied by 10 ⁸ And (5) CFU/mL, dipping bacteria liquid by using sterile cotton, extruding redundant bacteria liquid, and uniformly coating the bacteria liquid on an MRS agar plate. And (3) pasting antibiotic drug sensitive paper sheets on MRS agar plates, performing three repetitions for each antibiotic, culturing the MRS agar plates at 37 ℃ for 24h, and measuring the diameter of the inhibition zone by using a vernier caliper, wherein the diameter of the inhibition zone of each strain is the average value of the diameters of the three inhibition zones. The antibiotics tested included: fourthlyThe detection results of the tetracycline, ampicillin, ceftriaxone, clarithromycin, chloramphenicol, clindamycin and ciprofloxacin are shown in table 1.

TABLE 1 resistance of Lactobacillus salivarius MK0901 to antibiotics

Table 1: r: resistance (Resistant); i, intermediaries (intermediaries); s, sensitive (Susceptible)

The results in table 1 show that the strain is sensitive to 7 antibiotics including tetracycline, ampicillin, ciprofloxacin, ceftriaxone, clindamycin, clarithromycin and chloramphenicol, has poor drug resistance, does not cause the body to have drug resistance effect after long-term use, and has excellent probiotic properties.

Experimental example 3

1. Bacteriostatic activity of lactobacillus salivarius MK0901

Measurement of bacterial inhibition Escherichia coli CMCC (B) 44102 (Escherichia coli CMCC (B) 44102), salmonella Paratyphi B CMCC (B) 50094 (Salmonella enterica serotyphi B CMCC (B) 50094), staphylococcus aureus CMCC (B) 26003 (Staphylococcus aureus CMCC (B) 26003), pseudomonas aeruginosa CMCC (B) 10104 (Pseudomonas aeruginosa CMCC (B) 10104) were selected as pathogenic bacteria, and the pathogenic bacteria were cultured with TSB overnight at 37 ℃. Sterilizing 400mL of NA culture medium by using a conical flask, gradually cooling to 45 ℃, sucking 1mL of pathogenic bacteria liquid into the NA culture medium, uniformly mixing the culture medium, pouring the culture medium into a flat plate, and vertically punching 6 holes on the flat plate by using a sterile 6mm puncher after the flat plate is fully solidified. After the strain to be tested is cultured for 24 hours at 37 ℃ by using MRS broth culture medium, the strain broth culture medium to be tested is centrifuged for 10 minutes at 10000r/min, 50 mu L of supernatant is absorbed and added into the hole, three holes are selected for each strain to be tested as a repetition, after the plate is cultured for 24 hours at 37 ℃, the diameter of an inhibition zone is measured by using a vernier caliper, and the diameter of the inhibition zone of each strain is the average value of the diameters of the three inhibition zones. The results obtained using Lactobacillus rhamnosus LGG (Lactobacillus rhamnosus GG, LGG) as a control are shown in Table 2.

TABLE 2 inhibition zone diameter of Lactobacillus salivarius MK0901 against Escherichia coli, salmonella, staphylococcus aureus, and Pseudomonas aeruginosa

The results in the table 2 show that the lactobacillus salivarius strain has better inhibition effect on 4 pathogenic bacteria of escherichia coli, salmonella, staphylococcus aureus and pseudomonas aeruginosa, and the inhibition effect is equivalent to or superior to that of the LGG standard strain of lactobacillus rhamnosus.

Experimental example 4

1. Tolerance of lactobacillus salivarius MK0901 strain to artificial gastric juice and artificial intestinal juice is determined:

the frozen stock solution of the strain to be tested is dipped by using an inoculating loop, streaked on an MRS agar plate, and then cultured for 18h at 37 ℃. Sucking 3mL artificial gastric juice into 5mL aseptic centrifuge tube, dipping 3 single colonies in artificial gastric juice with aseptic cotton swab, oscillating sufficiently, eluting the colonies, adjusting the concentration of bacterial liquid to about 1 × 10 ⁸ CFU/mL, viable count was performed using 10-fold dilution. After culturing the artificial gastric juice at 37 ℃ for 3 hours, viable cell counting was performed again by using a 10-fold dilution method, and simultaneously 1mL of the cultured artificial gastric juice was taken out into 9mL of the artificial intestinal juice, and after culturing for 3 hours, viable cell counting was performed by using a 10-fold dilution method. The survival rate of the strain in the artificial gastric juice and the artificial intestinal juice is calculated by the formula: the survival rate = viable count after culture/viable count before culture × 100%, the frozen stock solution of the strain to be tested is streaked on an MRS agar plate by using an inoculating loop, a single colony is selected and inoculated in an MRS broth culture medium, the culture is carried out at 37 ℃ for 24h, the centrifugation is carried out at 5000r/min for 10min, the sterile PBS is used for washing, and the concentration of the bacterial solution is adjusted to about 1 × 10 ⁶ CFU/mL, viable bacteria count of the bacterial suspension was performed by 10-fold dilution.

Adhesion assay of strains to HT-29 cells:

subculturing the cultured HT-29 cells, and adjusting the cell concentration to 5 × 10 ⁶ Per mL, 1mL of the cell suspension was added to each cell culture plate, and the concentration of CO was 5% at 37 ℃% ₂ Culturing in an incubator, pouring out DMEM culture solution when the cells form a monolayer, washing with sterile PBS, pouring out BPS, adding 1mL of bacterial solution into each hole, repeating each sample for three times, and culturing at 37 ℃ for 2h. After the culture was completed, the bacterial solution was removed, washed with sterile PBS, the non-adherent strains were removed, 0.7mL of 0.25% trypsin-EDTA was used for digestion for 10min, 0.3mL of DMEM was added to stop the digestion, and the culture was counted for viable cells using a 10-fold dilution method. The adhesion rate of the cells was calculated as: the adhesion rate = bacterial liquid concentration after culture/bacterial liquid concentration before culture × 100%. The results of viable cell count and adhesion rate are shown in Table 3.

TABLE 3 survival rate and cell adhesion rate of Lactobacillus salivarius MK0901 in artificial gastric juice and artificial intestinal juice

Sample numbering	Gastric juice survival rate (%)	Intestinal juice survival rate (%)	Cell adhesion Rate (%)
				Lactobacillus salivarius	94.3	92.7	87.5

As shown in the results of Table 3, the Lactobacillus salivarius has high survival rate in gastric juice and intestinal juice, has excellent adhesion rate, and can protect and improve the environment of the stomach and intestinal tract.

Experimental example 5

1. Determination of short-chain fatty acid content of fermentation liquor of lactobacillus salivarius MK0901 strain:

preparing fermentation liquor: after 24 hours of activation culture of the lactobacillus salivarius MK0901 preserved strain, 4uL of the bacterial liquid is sucked and added into 4mL of broth culture medium, and the mixture is cultured for 24 hours at 37 ℃ for later use.

Detection of short chain fatty acids: the detection instrument is a gas chromatograph-mass spectrometer (GCMS-QP 2010 Plus) of Shimadzu corporation, japan, and the chromatographic column is an Rtx-5 fused silica capillary column (30 m × 0.25mm × 0.25 μm) of RESTEK corporation, USA. The GC ramp was initiated at 40 ℃ for 5min, 5 ℃ to 150 ℃ per minute, 10 ℃ to 280 ℃ per minute for 2min. The carrier gas was high purity helium (purity > 99.999%), flow rate: 1.0mL/min. MS conditions: the ionization mode is EI; the temperature is 200 ℃, the interface temperature is 220 ℃, and the mass scanning range m/z is 33-500. Taking 4mL of fermentation liquor, adding 10 μ L of 2-ethylbutyric acid internal standard solution with the concentration of 200 μ g/mL, injecting 1 μ L of sample in a split mode of 1. The concentrations of 5 kinds of short-chain fatty acids (acetic acid, n-butyric acid, isobutyric acid, isovaleric acid, isocaproic acid) were calculated by the internal standard method, and the results are shown in table 4 and fig. 8.

TABLE 4 Lactobacillus salivarius MK0901 fermentation broth short chain fatty acid content (μ g/mL)

According to the results in Table 4, the fermentation liquid of Lactobacillus salivarius MK0901 contains a plurality of short-chain fatty acids, wherein the content of acetic acid is the highest and reaches 10.876 mu g/mL, and is followed by isovaleric acid and isocaproic acid, and 3 kinds of butyric acid are detected (Table 4).

Experimental example 6

1. Ability of lactobacillus salivarius MK0901 to regulate mouse intestinal microorganisms:

selecting 15 SPF male Kunming mice of about 20g, randomly dividing the mice into 3 groups after adapting for one week, dividing the mice into a blank control group, a natural recovery group and a lactobacillus salivarius experimental group, wherein each group comprises 5 mice, and simultaneously labeling each mouse. All mice live in a similar environment, providing the same food and water, and were guaranteed to be free to eat and drink.

After the experiment begins, except that the blank control group of mice is intragastrically filled with 0.2mL of sterile normal saline every day, the other two groups of mice are intragastrically filled with 0.2mL of ceftriaxone antibiotic solution each daily at the concentration of 200mg/mL for 10 days continuously. Then, the experimental groups are irrigated with the lactobacillus salivarius for 12 days every day, each of which is 0.2mL, and the concentration of the bacterial liquid is 1 multiplied by 10 ⁸ CFU/mL. The control group and the natural recovery group mice were gavaged with 0.2mL of sterile physiological saline, and the mouse feces were collected once every 3 days and stored at-80 ℃ for later use. Sequencing of 16S rRNA gene amplicon was performed using Illumina Miseq PE250 platform, and V3-V4 region of bacterial 16S rRNA gene was determined with 338F: see SEQ ID NO.4 and 806R: see SEQ ID NO.5. The Shannon index and Simpson index of intestinal microorganisms of each group of mice were calculated and compared. The results obtained are shown in Table 5.

TABLE 5 modulation of the intestinal microbial flora in mice by Lactobacillus salivarius MK0901

According to the results shown in table 5, the microbial diversity of the intestinal tract of the mice is remarkably reduced after the antibiotic is perfused, and the Shannon index and Simpson index are greatly lower than those of the blank control group; after feeding lactobacillus salivarius MK0901 for three days, the diversity is obviously increased, and the normal level is basically recovered on 9 th to 12 th days, which is very close to that of a blank control group. And the Shannon index and Simpson index of the natural recovery group after the antibiotic gavage are still obviously lower than those of a blank control group and a lactobacillus salivarius treatment group on day 12, which shows that the lactobacillus salivarius MK0901 has a good regulating effect on the intestinal flora of mice.

Experimental example 7

Experimental animals:

60 SPF male Kunming mice of 4 weeks old were provided by Woodhootsu laboratory animals Co., ltd and were housed in the laboratory animal room. Carrying out adaptive feeding for 7d before formal experiments, wherein the feeding conditions are as follows: the humidity is about 50%, the temperature is about 24 ℃, the light and the dark are respectively 12 hours every day, and the feed and the drinking water are added in time to enable the food to drink freely.

Experimental strains:

the strain Lactobacillus salivarius MK0901 (Lactobacillus salivarius MK 0901) provided by the invention is separated from stool samples of healthy macaques in the subject group previously, and is stored in a laboratory bacterial bank under the storage condition of-80 ℃. Lactobacillus rhamnosus ATCC 53103 LGG was purchased from GmbH.

The main reagents are as follows:

sterile DPBS buffer, 4% paraformaldehyde fixing solution, sodium chloride (analytical grade): a white shark organism; MRS broth, MRS agar medium: circulant organisms; dextran Sodium Sulfate (DSS): next saint creatures; tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) detect the kit: jiangsu Jingmei biology; excrement DNA extraction kit: tiangen Biochemical technology.

Main apparatus and equipment:

high-pressure steam sterilization pot: shanghai Shenan; a biochemical incubator: a Sanyo motor; an aseptic operation table: zhejiang Jiangsu Jing; a multifunctional microplate reader: bio Tek; an ultra-low temperature refrigerator: haier.

1. Preparing a gastric lavage agent:

the strain provided by the invention and a control strain LGG are recovered and activated. Centrifuging at-4 deg.C and 6000r/min for 5min, removing supernatant, and collecting bacterial sludge. Resuspending the bacteria with sterile PBS buffer solution, and adjusting the concentration of the bacteria solution to 10 ⁹ CFU/mL。

2. Animal grouping and treatment:

after adaptive feeding was completed, 60 Kunming male mice were randomly divided into 4 groups of 12 cages, 1 group per 3 cages, blank group, model group, LGG group and Lactobacillus salivarius treatment group. The official experiment totals 16d and is divided into a DSS molding stage and a recovery stage (see Table 6 for details). The DSS solution is replaced every other day in the molding period, the spirit and the activity state of the mice are observed every day in the experimental period, the weight of the mice is measured every day, the excrement of the mice is collected according to groups, and the collected excrement sample is frozen in an ultra-low temperature refrigerator at minus 80 ℃.

TABLE 6 animal Experimental design protocol

3. Mouse weight change and immune organ index determination:

the mice were weighed daily and their change curves were plotted. At the end of the experiment, the mice were sacrificed by cervical dislocation, their spleens were rapidly removed and washed with normal saline, and were weighed after the water was absorbed by filter paper. The spleen index = spleen mass (g)/mouse body weight (g) × 100%, and the obtained results are shown in fig. 4A and fig. 4B, and as can be seen from the trend of the average body weight change of each group of mice (fig. 4A), the average body weights of each group of mice treated with the DSS solution for 8 days during the molding period are very close to each other, but are obviously lower than those of the blank control group of mice, which indicates that the DSS solution negatively affects the intestinal structure of the mice, reduces the absorption function of the mice on food, and causes the weight increase speed to be slow. After the recovery period, the average body weight of the mice of the lactobacillus salivarius and LGG treatment group is rapidly recovered, and the average body weight at the end of the 8-day recovery period is obviously higher than that of the mice of the natural recovery group, which indicates that the intervention of probiotics is helpful for the mice to recover the body weight. The spleen is the most important immune organ, and the spleen is enlarged due to inflammatory reaction, so that the spleen index is increased. At the end of the experiment, the spleen index comparison results of mice in each group show (fig. 4B), the spleen index of the mice in the lactobacillus salivarius treatment group and the LGG treatment group is not significantly different from that of the mice in the blank control group, but is significantly lower than that of the mice in the DSS natural recovery group, which indicates that the probiotics may have the efficacy of relieving splenomegaly and inflammatory reaction.

4. Histopathological manifestations of the colon:

the mouse colorectal was removed with forceps and scissors and its length was measured using the cecum as an identifier. 1cm of colon was dissected and fixed with 4% paraformaldehyde fixative for 24h. The preparation method comprises the following main steps of slicing with paraffin, dewaxing, dyeing, dehydrating and mounting. Pathological manifestations of colon tissue in mice were observed with a light microscope. The results are shown in fig. 5A-D, and pathological analysis shows that the intestinal mucosa tissue of the mice (fig. 5A) in the blank control group is intact, no ulcer is found, and lymphatic follicular formation is found in the mucosa; in contrast, in the mice of the DSS-treated group (fig. 5B), the colonic mucosa and muscular layer were thinned, the glandular layer was decreased, the intestinal cavity was dilated, local ulcer was formed, no glandular epithelial coating was seen on the surface, bleeding and small blood vessel hyperplasia occurred below the surface; compared with CK, the LGG group (figure 5C) and the Lactobacillus salivarius group (figure 5D) have slightly thinned mucous membrane and muscular layer, reduced glandular layer and expanded intestinal cavity, but compared with DSS group, the mucosa has no obvious ulcer and has only a little chronic inflammatory cell infiltration. This indicates that the probiotics has protective effect on colon tissues of mice, counteracts the destructive effect of DSS solution on intestinal mucosa to a certain extent, and slows down inflammatory reaction.

5. Serum inflammatory factor assay:

a fresh blood sample of the mouse is obtained by adopting an orbital bleeding method, the blood sample is placed at 3000r/min for centrifugation for 10min, and serum is collected. Three inflammatory factor indicators of serum samples were determined using TNF- α, IL-1 β and IL-6 detection kits, following strictly the protocol. The results are shown in FIGS. 6A-C, and it is clear from the results that the stimulation with DSS solution significantly upregulated IL-1 β levels (p < 0.05) in mouse serum and very significantly upregulated TNF-. Alpha.and IL-6 levels (p < 0.01). The TNF-alpha and IL-6 levels in the Lactobacillus salivarius and LGG groups were significantly lower than in the DSS group (p < 0.01). The LGG group was significantly higher than the CK group (p < 0.05), while the Lactobacillus salivarius group was not significantly different from the CK group in IL-1 β level (p > 0.05), but significantly lower than the LGG and DSS groups. In general, the Lactobacillus salivarius MK0901 provided by the invention has the effect of reducing the expression of inflammatory factors induced by DSS solution, the efficacy of regulating the levels of TNF-alpha and IL-6 is similar to that of LGG, and the efficacy of regulating the level of IL-1 beta is better than that of LGG.

6. And (3) analyzing intestinal flora:

samples of mice feces at the end of the experiment (16 d) were taken from the ultra-low temperature refrigerator and total fecal DNA was extracted using a fecal genomic DNA kit. The bacterial 16S rRNA gene V3-V4 hypervariable region was amplified using PCR with primers 338F and 806R. 16S rRNA gene amplicon sequencing was completed by using an Illumina MiSeq PE250 platform, and noise reduction and subsequent analysis of raw data were completed based on QIIME2, and the obtained results are shown in FIGS. 7A-D, as shown in the figure, the OTU numbers of the four groups of samples were 523, 513, 556 and 617, respectively, wherein the DSS group had the lowest OTU number, and the Lactobacillus salivarius group had the highest OTU number. In addition, the unique OTU numbers of the CK group, the DSS group, the LGG group and the Lactobacillus salivarius group are 135, 115, 129 and 174 respectively, which shows that the flora compositions of different samples have obvious difference, and the unique flora type of the Lactobacillus salivarius group is most abundant.

The principal axis analysis (PCoA) can visually reflect the structural difference among different samples, the PCoA analysis results of four groups of samples are shown in FIG. 7B, and the sum of PC1 and PC2 reaches 53.98% (> 50%), which indicates that the better grouping relationship exists. The structure position of the flora of the DSS group is far away from the rest three groups, which shows that the DSS destroys the intestinal flora structure of the mice and the natural recovery is slow, while the structure positions of the flora of the LGG group and the Lactobacillus salivarius group are close to the CK group, which shows that the intragastric probiotics are helpful for the intestinal microbial structure of the mice to be recovered to the normal level.

The species stacking chart can compare the types of flora contained in different samples and their abundance differences. The stacking plots of species at the phylum level for the four groups of samples showed (fig. 7C) that bacteroidetes (bacteroidata) and Firmicutes (Firmicutes) occupy higher abundance in each group, being the major phylum of flora. In the DSS group, bacteroidetes predominates significantly (P = 70.98%), while the firmicutes contained in the lowest proportion (P = 25.24%) compared to the other groups, the composition structure differed most from the CK group. Further analysis of the constitutional structure of microorganisms at the genus level (fig. 7D) revealed that the difference in the flora structure was large between the groups, but the abundance of muria (muribacteriaceae) and Lactobacillus (Lactobacillus) was the highest, and particularly, the LGG group was rich in Lactobacillus up to 41.56%, and also, bacteroides (Bacteroides) were abundant. Compared with the other 3 groups, the lactobacillus salivarius treatment group has higher abundance and balance of intestinal microorganisms at the genus level, and further shows that lactobacillus salivarius MK0901 has better regulation effect on the pairs of intestinal flora of mice.

TABLE 7 sequence Listing

In summary, the following steps:

the lactobacillus salivarius is beneficial to restoring body weight, relieving inflammation level, protecting intestinal tissues and regulating intestinal flora structure, can be used as a probiotic candidate strain, and is used for preventing and treating enteritis of animals.

The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Claims

1. Lactobacillus salivarius is preserved in China general microbiological culture Collection center at 9 months and 2 days 2022, addresses: the collection number of the microorganism research institute of Chinese academy of sciences, no.3 Xilu No.1 of Beijing, chaoyang, and the institute of microorganisms is CGMCC NO.25641.

2. Use of Lactobacillus salivarius (Lactobacillus salivarius) as claimed in claim 1 in the preparation of medicaments and health products for preventing and treating colitis, protecting intestinal tract tissue and regulating intestinal flora structure.

3. A bacterial agent comprising the Lactobacillus salivarius (Lactobacillus salivarius) as claimed in claim 1.

4. A medicament for alleviating inflammatory bowel disease comprising Lactobacillus salivarius (Lactobacillus salivarius) as claimed in claim 1.

5. A health product for protecting intestinal tissues, comprising Lactobacillus salivarius according to claim 1.