A strain of Enterococcus monteri and its application

Abstract

The present invention discloses a strain of Enterococcus mundtii and its application. It belongs to the technical field of probiotics. A strain of Enterococcus mundtii is separated and identified by traditional identification methods and molecular biological methods, and its probiotic performance and in vivo safety evaluation are performed. At the same time, microbiology is combined with pathology, etc., and it is found that oral administration of the bacteria can reduce organ lesions in mice caused by Salmonella, and can produce antagonistic effects on Salmonella in mice. The present invention can not only expand the research on the genus Enterococcus, but also provide a reference basis for the future development of probiotic microecological preparations for promoting digestion and growth in animals, antagonizing pathogens, and regulating intestinal microecological environments.

Description

Technical Field

[0001] The present invention relates to the technical field of probiotics, and more particularly to a strain of Enterococcus monteroni and application thereof. Background Art

[0002] The intestinal microbiota plays a key role in many metabolic, physiological, nutritional and immune processes in animals. Different dietary patterns, obesity, disease and the use of antibiotics can affect the abundance and structure of the intestinal microbiota. In addition, probiotics can also repair inflammation and metabolic disorders caused by intestinal diseases. In recent years, there are two main motivations for the use of probiotics. First, normal people use probiotics and probiotic products to maintain their own health, while enhancing immunity and reducing the risk of disease. Secondly, for specific diseases, the use of probiotics can also be used as an effective treatment, but the probiotic effects produced by different strains may also be very different.

[0003] Lactic acid bacteria, including Enterococcus, can inhibit intestinal inflammatory responses by enhancing the immune function of the intestinal mucosa and can antagonize foodborne pathogens. Enterococci can be used as an effective alternative to antibiotics. After dietary supplementation with Enterococci, the changes in the microbial community in the pig intestine are similar to those after taking antibiotics. Probiotic Enterococci have been shown to stimulate and enhance host immunity, reduce multiple related indicators of patients with high cholesterol, and alleviate discomfort symptoms caused by diarrhea and irritable bowel syndrome.

[0004] Although some species of Enterococci are currently used as probiotics, finding and introducing new potential probiotic strains that are safe for humans and animals remains one of the most important topics in microbiology and animal husbandry.

[0005] In summary, how to provide an enterococcus and study its probiotic properties is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0006] In view of this, the present invention provides a strain of Enterococcus monteroni and applications thereof.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A strain of Enterococcus mundtii, named HCD01, was classified as Enterococcus mundtii and was deposited at the General Microbiology Center of China Culture Collection on July 24, 2024, with the deposit number CGMCC No.31447. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0009] Further, it is Enterococcus monteri of bovine origin.

[0010] Furthermore, the Enterococcus monteri can tolerate a pH of 2 and a salt concentration of 10%.

[0011] The application of the above-mentioned Enterococcus monteri in inhibiting Salmonella and Streptococcus dysgalactiae.

[0012] The use of the above-mentioned Enterococcus monteri in the preparation of probiotics.

[0013] Furthermore, the probiotics are used to alleviate symptoms of Salmonella infection.

[0014] Furthermore, it is used to increase the abundance of beneficial bacteria in the intestinal flora, alleviate intestinal damage caused by Salmonella infection, and reduce lung and liver lesions caused by Salmonella.

[0015] A probiotic agent comprises the above-mentioned Enterococcus monteri.

[0016] It can be seen from the above technical solution that, compared with the prior art, the beneficial effects achieved by the present invention are:

[0017] A strain of Enterococcus mundtii was isolated and identified by traditional identification methods and molecular biology methods, and its probiotic performance and safety in animals were evaluated. At the same time, microbiology and pathology were combined, and it was found that oral administration of the bacteria could reduce organ lesions in mice caused by Salmonella, and could produce antagonistic effects on Salmonella in mice. The present invention can not only expand the research on the genus Enterococcus, but also provide a reference for the future development of probiotic microecological preparations for promoting digestion and growth in animals, antagonizing pathogens, and regulating intestinal microecological environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0019] Figure 1 It is the technical roadmap of the present invention;

[0020] Figure 2 The colony morphology of strain HCD01 on MRS solid medium in Example 1 of the present invention;

[0021] Figure 3 The Gram staining microscopic examination result of strain HCD01 in Example 1 of the present invention (10×100);

[0022] Figure 4 is the NJ phylogenetic tree of strain HCD01 in Example 1 of the present invention;

[0023] Figure 5 The results of functional gene and virulence gene detection of strain HCD01 in Example 1 of the present invention, wherein 1 represents EC; 2 represents vanA; 3 represents tetM; 4 represents tetK; 5 represents ermB; 6 represents CylA; 7 represents Esp; 8 represents asa1; 9 represents EfaAfm; 10 represents GelE; 11 represents Agg; 12 represents Ace; 13 represents negative control; M represents 2000 bp Marker;

[0024] Figure 6 This is the hemolytic test result of Enterococcus monteri HCD01 in Example 2 of the present invention;

[0025] Figure 7 1 is the growth curve and acid production curve of Enterococcus monteri HCD01 in Example 2 of the present invention;

[0026] Figure 8 The weight gain test results of the Enterococcus mongoliae group in Example 3 of the present invention;

[0027] Fig. 9 The test results of the feed-to-weight ratio (F / G) in Example 3 of the present invention, wherein the same and different lowercase letters represent no significant difference (P>0.05) and significant difference (P<0.05), respectively;

[0028] Fig.10 The organ coefficient result in Example 3 of the present invention;

[0029] Fig.11 This is a mouse autopsy diagram in Example 3 of the present invention, wherein a is a control group; b is a pathogenic bacteria group; and c is a protective group (i.e., an antagonistic Salmonella group);

[0030] Fig.12 The isolated strain in Example 3 of the present invention antagonizes the effect of Salmonella on the intestinal morphology of mice;

[0031] Fig.13 The isolated strain in Example 3 of the present invention antagonizes the effect of Salmonella on the liver and lung tissues of mice;

[0032] Fig.14 The OTU annotation Venn diagram in Example 3 of the present invention, wherein CK is the control group; Emun1 is the group that was gavaged with Enterococcus monteri for 7 consecutive days; Emun2 is the group that was gavaged with Enterococcus monteri for 14 consecutive days; EmunP1 is the group that was gavaged with Enterococcus monteri for 7 consecutive days and then attacked with Salmonella; EmunP2 is the second stage of Enterococcus monteri antagonizing Salmonella (the same below);

[0033] Fig.15 The Chao index and Shannon index analysis of fecal flora in Example 3 of the present invention;

[0034] Fig.16 is the rarefaction curve of each group of samples in Example 3 of the present invention;

[0035] Fig.17 : is the grade abundance curve of each group of samples in Example 3 of the present invention, wherein the control group CKAn is not administered with the test bacteria; the probiotic group Emun1: the test probiotic (Enterococcus monteri) was administered with the test probiotic for 7 consecutive days; the probiotic group Emun2: the test probiotic was administered with the test probiotic for 14 consecutive days; the antagonistic group EmunP1: the test probiotic was administered with the test probiotic for 7 consecutive days and then attacked with Salmonella, i.e., the 7th day of antagonism (Enterococcus monteri antagonistic Salmonella group); the antagonistic group EmunP2: the second stage after the attack with Salmonella, i.e., the 14th day of antagonism;

[0036] Fig.18 The results of the LEfSe-based phylogenetic analysis of the fecal flora of mice in Example 3 of the present invention, including: (a) comparison of the dominant bacterial groups in the feces of mice between the probiotic Emun group and the control CKAn group; (b) comparison of the dominant bacterial groups between the probiotic antagonistic Salmonella group EmunP and the control CKAn group;

[0037] Fig.19 The PCoA analysis result of the species similarity of the fecal flora of mice in Example 3 of the present invention is shown, wherein CKAn is the control group; Emun is the probiotic group (the group of oral administration of Enterococcus monteri); and EmunP is the antagonistic group (the group of Enterococcus monteri antagonizing Salmonella);

[0038] Fig. 20 This is a PCA diagram of the characteristic genera / differential genera in the feces of mice in the control CKAn group, the probiotic Emun group, and the antagonistic EmunP group in Example 3 of the present invention. DETAILED DESCRIPTION

[0039] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] The drugs required for the present invention are conventional experimental drugs, which are purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods and will not be described in detail here.

[0041] Example 1

[0042] Isolation, identification and preservation of strains

[0043] (1) Isolation and purification of bacterial strains

[0044] Fecal samples: The present invention selects the grassland of Simmental dairy cows in Xianghuang Banner, Xilin Gol League, Inner Mongolia Autonomous Region (41°56′~42°45′ N, 113°30′~114°45′ E) to collect dairy cow fecal samples, and the cow fecal samples are placed in sterile bags and stored at low temperature for subsequent separation and screening of potential probiotics.

[0045] Use a sterile cotton swab to dip an appropriate amount of cow dung on the solid culture medium, dilute it with physiological saline with a pipette, and spread it evenly with a spreader, and culture it at a constant temperature overnight at 37°C. The next day, use a sterile inoculation loop to pick colonies with consistent morphology and absolute dominance on TSB solid culture medium for streaking purification. After a single colony appears on the culture medium, perform Gram staining on it, and repeat this process until the staining results have consistent bacterial morphology and color. Pick a fresh bacterial lawn of a single bacterium and expand it in TSB liquid culture medium. Use a pipette to draw 1 mL of bacterial solution into a storage tube, add 1 mL of 30% glycerol, and finally freeze it in a -40°C freezer and store it. It is named HCD01.

[0046] (2) Morphological identification

[0047] The isolated and purified strains were cultured under appropriate conditions, the phenotypic characteristics of the colonies were observed with the naked eye, and the morphology, color and size were recorded; the isolated strains were Gram-stained and then observed under an optical microscope for cell morphological characteristics.

[0048] Depend on Figure 2 It can be seen that strain HCD01 is a round, grayish-white colony with neat edges and smooth surface on MRS medium. Figure 3 As shown, the screened strain HCD01 is spherical and is a Gram-positive bacterium arranged alone or in pairs.

[0049] (3) Bacterial 16S rRNA gene sequencing and identification

[0050] The gene was extracted according to the instructions of the bacterial gene extraction kit (TIANamp Bacteria DNA Kit), and the extracted isolated bacterial genome was used as a template to perform PCR amplification using universal primers for bacterial 16S rRNA gene. The upstream and downstream primers were 27 F: 5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO.1 and 1492 R: 5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO.2.

[0051] The amplified products were sequenced, and after sequencing, BLAST homology comparison was performed, and the NJ (Neighbor junction) phylogenetic tree was constructed using MEGA X software.

[0052] The sequencing results of the amplified 16S rRNA gene of strain HCD01 are as follows, with a sequence length of 1436 bp.

[0053]

[0054] The 16S rRNA gene sequence of strain HCD01 was compared by BLAST sequence comparison, and the NJ (Neighbor-Joining) phylogenetic tree was constructed using MEGA X software. Figure 4 As shown, the homology between strain HCD01 and Enterococcus mundtii RP1C1, CECT972T, and XA3 were 99.85%, 99.65%, and 99.79%, respectively. The results showed that strain HCD01 was Enterococcus mundtii.

[0055] (4) PCR detection of functional genes and virulence genes

[0056] The primer sequences involved are shown in Table 1. The primers used were all synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0057]

[0058] Results Figure 5 Only a bright band of about 250-300 bp was produced at the EC gene, and the band size was consistent with the expected result (282 bp), indicating that strain HCD01 contained the EC gene; no bright band was produced at the vanA, tetM, tetK, and ermB genes related to drug resistance; the test results showed that no bright band was produced at the virulence genes CylA, Esp, asa1, EfaAfm, GelE, Agg, and Ace, indicating that strain HCD01 did not carry the above virulence genes.

[0059] (5) Biochemical identification

[0060] The biochemical tests of isolated strains were carried out with reference to the Common Bacteria Identification Manual and the instructions for microbiochemical identification tubes. The main tests included glycolysis test, urea test, lysine decarboxylase test, hydrogen sulfide test, Simon's citrate test, hydrogen peroxide gas production test, glucose gas production test and MR-VP test. The strains were further identified based on the test results.

[0061]

[0062] Strain HCD01 can utilize most sugars including glucose, but does not produce glucose gas; can utilize mannitol, but not sorbitol; can decompose urea but does not react with urease, does not decompose arginine dihydrolase; produces lysine decarboxylase; does not produce hydrogen sulfide or Simon's citrate; hydrogen peroxide gas production test result is positive; MR-VP test result is negative. The biochemical characteristics of strain HCD01 are basically consistent with enterococci, and the results are shown in Table 2.

[0063] (6) Preservation

[0064] The Enterococcus mundtii is named HCD01, and its classification name is Enterococcus mundtii. It was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 24, 2024, with the deposit number CGMCC No.31447, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0065] Example 2

[0066] Evaluation of probiotic properties of strains in vitro

[0067] (1) Hemolysis test

[0068] A single colony of Enterococcus monteri HCD01 was selected, and fresh bacterial moss was picked up with a sterile inoculation loop on the fresh blood agar medium and streaked on the plate. The plate was inverted and cultured in a constant temperature incubator (37°C) for one day (24 h), and then observed for hemolysis.

[0069] The morphology of Enterococcus monteri HCD01 on fresh blood agar medium is as follows Figure 6 As shown, the absence of hemolytic zone indicates that Enterococcus monteri HCD01 is not hemolytic.

[0070] (2) Drug sensitivity test

[0071] The drug sensitivity test was conducted by the Kirly-Baue method, and antibacterial drugs such as β-lactams, chloramphenicol, quinolones, sulfonamides, and tetracyclines were selected. 200 μL of bacterial culture solution was evenly spread on the solid culture medium and dried at room temperature for 3-5 min until the bacterial solution was fully absorbed and then the drug sensitivity sheet was pasted with tweezers. Three parallel drug sensitivity sheets were set for each drug, and the diameter of the inhibition zone was measured with a vernier caliper after inverted culture in a 37°C constant temperature incubator for one day (24 h). The test results were compared with the "Standards for Determining the Diameter of the Inhibition Zone in the Paper Disk Method Drug Susceptibility Test", and the results were divided into sensitive (S), intermediate (I), and resistant (R).

[0072]

[0073] The results of drug sensitivity test of Enterococcus monteri HCD01 are shown in Table 3. Enterococcus monteri HCD01 is sensitive to azithromycin, midecamycin, tetracycline, doxycycline and vancomycin; intermediate to cefotaxime, ciprofloxacin, novobiocin and clindamycin; resistant to penicillin G, chloramphenicol, norfloxacin, ofloxacin, cotrimoxazole, tobramycin, amikacin, streptomycin, neomycin, kanamycin, gentamicin and polymyxin B. According to the results, Enterococcus monteri HCD01 is resistant to all aminoglycosides used.

[0074] (3) Antibacterial test

[0075] The antibacterial test was carried out by the Oxford cup double-layer plate diffusion method. Prepare solid agar medium, sterilize at high temperature and take it out. The amount poured into each culture dish is controlled to be about 10 mL. After cooling and solidification, make the lower plate and place the Oxford cup at a certain interval. Repeat the above steps to prepare the upper plate and pour it on the lower plate. After it solidifies, pull out the Oxford cup. Salmonella (Salmonella) CVCC2184, Streptococcus dysgalactiaeequisimilis (Streptococcus dysgalactiaeequisimilis) BNCC337480 and Staphylococcus aureus (Staphylococcus aureus) ATCC29213 were selected as pathogenic indicator bacteria. 50 μL of bacterial solution was taken from each and evenly spread on the culture medium; 200 μL of the culture solution of the isolated strain was added to the small hole, and it was placed in a 37℃ constant temperature incubator for one day (24 h). Observe whether the inhibition zone appears and measure the diameter of the inhibition zone.

[0076]

[0077] The Oxford cup double-layer plate diffusion method was used to perform an in vitro antibacterial test on the isolated bacteria. The results are shown in Table 4. Enterococcus monteri HCD01 had no inhibitory effect on Staphylococcus aureus, but had an inhibitory effect on Salmonella and a certain inhibitory effect on Streptococcus dysgalactiae.

[0078] (4) Growth curve and acid production curve

[0079] The isolated bacteria were inoculated into the corresponding liquid culture medium and cultured with constant temperature shaking (37°C, 200 rpm). The bacterial solution was collected every 2 hours (i.e., after 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, and 16 h), and the absorbance at a wavelength of 600 nm (OD600) was measured using an enzyme reader. The pH value of the bacterial solution was also measured to draw the growth curve and acid production curve.

[0080] Depend on Figure 7It can be seen that Enterococcus monteri HCD01 entered the logarithmic growth phase after 2 hours and gradually entered the stable phase after 8 hours; the pH value of the bacterial solution dropped from 6.2 to 5.5 within 16 hours, and the acid production trend line decreased relatively slowly, indicating that Enterococcus monteri HCD01 has the ability to produce acid, but the acid production rate is slow.

[0081] (5) Acid resistance test

[0082] The isolated bacteria cultured overnight were inoculated into liquid culture media with different pH values ​​(i.e., 2, 3, and 4) at a 1% inoculation rate, and cultured at a constant temperature with shaking (37°C, 200 rpm) for 24 h to observe whether the culture medium became turbid.

[0083]

[0084] The results of the acid resistance test of the strain are shown in Table 5. After overnight culture in liquid culture medium with pH values ​​of 2, 3, and 4, turbidity can be produced, indicating that the strain has a certain degree of acid resistance.

[0085] (6) Salt tolerance test

[0086] MRS liquid culture medium containing NaCl was prepared with a concentration gradient of 2%, 4%, 6%, 8% and 10%, and sterilized by high pressure steam (121°C, 15 min). The isolated bacteria were inoculated into the corresponding culture medium at a 1% inoculation rate, and the OD600 value of the bacterial solution was measured with an ELISA instrument after constant temperature culture (37°C, 200 rpm) for 24 h.

[0087]

[0088] According to the results in Table 6, as the NaCl concentration increased, the OD600 value of Enterococcus monteri HCD01 showed a trend of first decreasing and then increasing. It grew most slowly at a concentration of 4% and grew best at a concentration of 10%, indicating that Enterococcus monteri HCD01 has a strong tolerance to high salinity.

[0089] (7) Coagulation test

[0090] The isolated bacteria were inoculated into liquid culture medium at a 1% inoculation rate and cultured overnight with constant temperature shaking (37°C, 200 rpm). Then, a mixed solution was prepared according to the bacterial liquid / skim milk ratio of 1:5, 1:10, 1:15, 1:20, and 1:25, and placed in a 37°C constant temperature incubator. After 24 h, the occurrence of condensation was observed and the pH value of the mixed solution was measured.

[0091]

[0092] The results of the coagulation test and the measured pH value are shown in Table 7. When the ratio of Enterococcus monteroni HCD01 bacterial solution to skim milk was 1:15, the pH value reached the lowest, but coagulation still did not occur, indicating that Enterococcus monteroni HCD01 did not have the ability to coagulate milk.

[0093] Example 3

[0094] Evaluation of the probiotic and safety performance of the strain in vivo

[0095] Experimental animals: 4-6 week-old SPF Kunming mice were selected. All the experimental mice were male and purchased from Inner Mongolia Medical University.

[0096] Preparation of bacterial solution: Inoculate the isolated bacteria into MRS liquid culture medium at a ratio of 1%, culture overnight at 37°C and 200 rpm, and adjust the bacterial solution concentration to 1×10 8 CFU / mL. In the same way, Salmonella was inoculated into TSB medium and cultured overnight with constant temperature shaking. The concentration of the bacterial solution was adjusted to 1×10 7 CFU / mL, for future use.

[0097] (1) Mouse weight gain and food intake test

[0098] Ten male mice were randomly divided into two groups, 5 mice in each group, and the experiment was conducted after one week of adaptive breeding. The mice in the experimental group (Enterococcus monteri group) were gavaged with Enterococcus monteri liquid once a day, and the mice in the control group were gavaged with the same volume of sterile saline, with the gavage volume of 0.2 mL / mouse, and the experimental period was 14 days.

[0099] During the experiment, the mice's mental state, appetite, coat color, feces, etc. were observed. Each mouse was weighed once a day for 14 days, and the feed intake of the mice was weighed daily to calculate the average daily weight gain (ADG), average daily feed intake (ADFI), and feed-to-weight ratio (F / G).

[0100]

[0101]

[0102]

[0103]

[0104] In the mouse weight gain test, within 14 days of gavage, the mice in each group had no adverse reactions, their mental state was normal, their fur and feces were normal, and there was no diarrhea. The measured and recorded test data were statistically organized using Excel software, and the results were expressed as "mean ± standard deviation". According to Table 8, all groups showed a trend of weight growth within 14 days of gavage, and the average daily weight gain of each group of mice within 7 days (half cycle) was greater than that within 14 days (one cycle), which shows that the weight gain of mice was faster in a short period, and then the growth condition gradually stabilized.

[0105] Graphpad Prism Version 9.5.1 software was used to perform T test on the average daily weight gain of mice in each group. In the same stage, "ns" indicates no significant difference (P>0.05), "*" indicates significant difference (P<0.05); the same letters indicate no significant difference (P>0.05), and different letters indicate significant difference (P<0.05). Figure 8 It can be seen that within 7 days and 14 days, the average daily weight gain of mice administered Enterococcus mondeloni was higher than that of the control group, indicating that the bacteria is beneficial to the growth and development of mice, but there was no significant difference compared with the control group within half a cycle and a whole cycle (P>0.05).

[0106]

[0107] The results of the average daily feed intake of mice are shown in Table 9, and the feed-to-weight ratio results are shown in Fig. 9 During the 7th and 14th days, the feed-to-weight ratio of the group administered with Enterococcus monteroi was lower than that of the control group, indicating that the bacteria can promote the growth and development of mice. As the experimental period becomes longer, the effect of Enterococcus monteroi HCD01 on mice is still beneficial.

[0108] (2) Mouse organ coefficient determination test

[0109] Ten male mice were randomly divided into two groups and gavage once a day. One group was gavaged with Enterococcus monteroni solution, and the remaining group was gavaged with sterile saline as a blank control. The mice were autopsied on the 7th day, and the hearts, livers, kidneys, and lungs of the mice were removed. At the same time, the tissues were observed for visible lesions, and the organ coefficients were measured.

[0110]

[0111] During the mouse organ coefficient test, it was found that the organ color and size of each group of mice were normal, and there were no visible lesions. The organ coefficient results of each group of mice are as follows Fig.10As shown, there was no significant difference in the organ coefficient of mice orally administered Enterococcus monteri HCD01 compared with the control group (P>0.05), indicating that the bacteria did not cause edema or hyperplasia or other pathological changes in the immune organs of mice, nor did it cause degenerative changes such as atrophy.

[0112] (3) Antagonism test in mice

[0113] Fifteen mice were randomly divided into three groups, with 5 mice in each group. The protective group was the Enterococcus monteri group (i.e., the antagonistic Salmonella group). The mice in the protective group were gavaged with 0.2 mL of Enterococcus monteri daily; the pathogenic bacteria group and the control group were gavaged with the same volume of sterile saline daily. After 7 consecutive days of gavage, according to the results of the preliminary test, the Salmonella was gavaged and the control group was not treated. After 96 hours, the mice were randomly dissected to observe whether the organs had lesions. The lungs, livers, small intestines, and colons of the mice were collected and fixed with a universal tissue fixative and sent to Wuhan Seville Biological Company for HE staining and pathological tissue section preparation.

[0114] After being challenged with Salmonella, the mice became listless and showed symptoms such as shortness of breath and diarrhea. There was no death in the control group and the protection group, and their mental state and defecation were normal. Fig.11 As shown, there were no abnormalities in the liver and intestines of the mice in the control group; the mice in the pathogenic bacteria group had visible lesions such as bleeding and bloating in the intestines; after Enterococcus mondeloni antagonized Salmonella, the symptoms of tissue lesions in the mice were alleviated.

[0115] After in vivo antagonism between Enterococcus monteri and Salmonella, the intestinal morphology of mice was Fig.12 As shown. Compared with the control group, the mice in the pathogenic bacteria group had shorter intestinal villi and a large number of intestinal villus epithelial cells fell off; the mucosal layer of the colon tissue produced wrinkles and focal erosions, uneven thickness, necrosis and shedding of mucosal epithelial cells, a decrease in the number of intestinal glands, a very small amount of connective tissue hyperplasia, and a very small amount of granulocyte infiltration. The mice in the protection group had abundant intestinal glands in the lamina propria of the small intestine, and compared with the control group, the length of the intestinal villi became shorter, and a large number of epithelial cells fell off; and the goblet cells in the colon tissue were abundant and had no wrinkles. Therefore, Enterococcus monteroni HCD01 can alleviate the intestinal damage caused by Salmonella infection.

[0116] like Fig.13As shown in the figure, after Salmonella challenged mice, compared with the control group, the lung tissue of the mice showed granulocyte infiltration of alveolar walls, mild thickening of focal alveolar walls, widening of alveolar septa, alveolar hemorrhage, irregular arrangement of bronchiolar epithelium, and a small amount of eosinophilic substances in the cavity. In the protection group, there was less congestion of blood vessels and alveolar wall capillaries, and no alveolar wall thickening and inflammatory cell infiltration. In the liver tissue of mice challenged with Salmonella, it can be seen that the boundaries of liver lobules were not obvious, and symptoms such as hydropic degeneration of hepatocytes and venous congestion appeared. Although the liver of the mice in the protection group was damaged, the symptoms were alleviated compared with those in the pathogenic bacteria group. Therefore, after oral administration of Enterococcus monteri HCD01 for in vivo Salmonella antagonism test, the lung and liver lesions caused by Salmonella can be alleviated.

[0117] (4) High-throughput sequencing of 16S rRNA gene of mouse fecal bacterial community

[0118] The above-mentioned fresh mouse fecal samples were collected on the 7th and 14th days, respectively, and the fecal samples were packaged in sterile cryopreservation tubes, with a sample volume of approximately 0.5-2.0 g in each tube. After packaging, they were immediately transported on dry ice and sent to Nanjing Paisonno Gene Technology Co., Ltd. for sequencing. The sequencing results were analyzed using the Paisonno Gene Cloud Platform.

[0119] Cluster analysis was performed on the species sequences of each sample, and bioinformatics statistical analysis was performed at a 97% similarity level. The distribution of the number of OTUs between different groups was compared based on the Venn diagram. The clustering and species annotation results are shown in Fig.14 As shown. The number of OTUs in the Emun1, EmunP1, Emun2, and EmunP2 groups of Enterococcus monteri were 146, 410, 122, and 147, respectively. The number of OTUs after oral administration of Enterococcus monteri and antagonism of Salmonella was different from that of the corresponding control group.

[0120] like Fig.15 As shown, the Chao1 index and Shannon index of the Enterococcus monnieri group were slightly lower than those of the control group.

[0121] The rarefaction curve can reflect the impact of changes in sequencing depth on the diversity of each group of samples. Fig.16 With the increase of sampling depth, the rarefaction curve of each group of samples gradually reaches a flat state, indicating that the sequencing results can reflect the diversity contained in the current samples and the Alpha diversity has reached saturation.

[0122] The rank abundance curve can reflect the uniformity of community composition. Fig.17 The grade abundance curve shown has gradually flattened, indicating that the abundance differences between ASV / OTUs in the sample communities detected in this experiment are gradually decreasing, the number of new species is no longer increasing with continuous sampling, the sequencing depth is sufficient, and the amount of sequencing data is reasonable.

[0123]

[0124]

[0125] The structure of the fecal microbial community of mice in different groups was analyzed at the phylum and genus levels, and the composition abundance is shown in Tables 10 and 11. There are two main phylum levels: one is the Bacteroidetes, which is one of the absolute dominant bacterial communities in mouse feces, among which the genus Bacteroides has a high abundance at the genus level. The relative abundance of Bacteroidetes in the Emun2 group (75.950%) was higher than that in the control CKAn2 group (67.145%). The genus Bacteroides was higher in Emun2 (6.257%) and EmunP2 (10.792%) than in the control CKAn2 group (2.745%). The second was Firmicutes, mainly including Lactobacillus. The relative abundance of Firmicutes was the highest in the Emun1 group, reaching 62.923%. The relative abundance of Lactobacillus in the collected fecal samples was higher in each group than in the control CKAn1 group (1.719%). At 14 days, the EmunP2 group (38.389%) was higher than the control CKAn2 group (18.193%). The results showed that oral administration of Enterococcus monteri HCD01 could increase the abundance of beneficial bacteria in the intestines of mice, and after antagonizing Salmonella, the bacterial flora gradually restored balance compared with the control group.

[0126] The LDA threshold was set to 2, and the comparison strategy was selected as one-against-all to draw the LEfSe-based phylogenetic map of mouse fecal flora. Fig.18 (a) shows that the relative abundance of intestinal flora in the control group CKAn and the probiotic group Emun mice at the class (c), order (o), family (f) and genus (g) levels were significantly different (P<0.05). Seven dominant groups and two dominant groups (Enterococcaceae and Enterococcus) were screened out in the control CKAn group (green fan ring) and the probiotic Emun group (yellow fan ring), respectively.

[0127] Depend on Fig.18 As can be seen from (b), there was a significant difference in the relative abundance of the intestinal flora of the mice in the control group CKAn and the antagonistic Salmonella group EmunP at the genus (g) level (P<0.05); two dominant genera were screened out in both the control group CKAn (green fan ring) and the antagonistic Salmonella group EmunP (yellow fan ring).

[0128] Fig.19 The bray_curtis distance algorithm was used for principal coordinate analysis (PCoA) to characterize the level of β diversity. The distances of the control group CKAn samples on the first axis (PCo1=21.4%) and the second axis (PCo2=18.5%) were the smallest, and the degree of aggregation was high, indicating that the β diversity of the control group CKAn was low; the distances of the probiotic group Emun samples on the first axis (PCo1=21.4%) were the largest, and the distances on the second axis (PCo2=18.5%) were large, and the degree of dispersion was high, indicating that the β diversity of the probiotic group Emun was high; the distances of the antagonistic Salmonella group EmunP on the first axis (PCo1=21.4%) were the smallest, and the distances on the second axis (PCo2=18.5%) were the largest, and the degree of dispersion was moderate, indicating that the β diversity of the antagonistic Salmonella group EmunP was moderate.

[0129] Fig. 20 Principal coordinate analysis (PCA) was used to screen the iconic / differential genus. The explanatory power of the first axis (PCA1) was as high as 92.1%, and the explanatory power of the second axis (PCA2) was 3.6%, indicating that the explanatory power of the first axis played a dominant role. The distance of the control group CKAn samples on the first axis (PCA1=92.1%) was the smallest, and the degree of aggregation was high, indicating that the control group CKAn had the fewest iconic / differential genus; the distance of the probiotic group Emun samples on the first axis (PCA1=92.1%) was the largest, and the degree of dispersion was the highest, indicating that the probiotic group Emun had the most iconic / differential genus; the aggregation and dispersion of the antagonistic Salmonella group EmunP samples on the first axis (PCA1=92.1%) were in the middle, indicating that the iconic / differential genus of the antagonistic Salmonella group EmunP was in the middle.

[0130] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0131] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one 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 present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A strain of Enterococcus monteri, characterized in that Named HCD01, its classification name is Enterococcus monteri Enterococcus mundtii , deposited in the General Microbiology Center of China Microorganism Culture Collection on July 24, 2024, with the deposit number CGMCC No.31447, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. The Enterococcus montegrioides according to claim 1, characterized in that It is Enterococcus monteri from cattle.

3. The Enterococcus montegrioides according to claim 1, characterized in that The Enterococcus monteri can tolerate a pH of 2 and a salt concentration of 10%.

4. Use of the Enterococcus monteri according to claim 1 in the preparation of a probiotic; The probiotic is used to alleviate the symptoms of Salmonella infection.

5. The use according to claim 4, characterized in that Used to increase the abundance of beneficial bacteria in the intestinal flora, alleviate intestinal damage caused by Salmonella infection, and reduce lung and liver lesions caused by Salmonella.

6. A probiotic, characterized in that: The method comprises the Enterococcus montegrioides described in claim 1.

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