CN119040228B - Enterococcus mundtii and application thereof - Google Patents

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

Enterococcus mundtii and application thereof

Technical Field

The invention relates to the technical field of probiotics, in particular to enterococcus mundtii and application thereof.

Background

Intestinal microbiota plays a key role in numerous metabolic, physiological, nutritional and immune processes of animals, and different dietary patterns, obesity, diseases, the application of antibiotics and the like can influence the abundance and structure of the intestinal microbiota, and in addition, probiotics can repair inflammation and metabolic disorders caused by intestinal diseases. In recent years, the use of probiotics has two main motivations, namely, the normal population maintains the health state of the normal population by using probiotics and probiotic products, and simultaneously, immunity is enhanced and the risk of illness is reduced, and the use of probiotics can also be an effective treatment mode aiming at specific diseases, but the probiotics produced by different strains can be quite different.

Lactic acid bacteria, including enterococcus, can inhibit inflammatory reactions in the intestinal tract by enhancing the immune function of the intestinal mucosa, and can antagonize food-borne pathogens. Enterococci can be used as one of effective substitutes for antibiotics, and after the enterococci are supplemented by diet, the microflora change in the intestinal tract of pigs is similar to the result after the antibiotics are ingested. Probiotic enterococci have proven useful for stimulating and enhancing the host's immunity, reducing multiple associated indicators in high cholesterol patients, and also reducing the discomfort symptoms caused by diarrhea and irritable bowel syndrome, etc.

Although some species of enterococci have been used as probiotics at present, the search and introduction of new potential probiotic strains with safety to humans and animals is still one of the most important subjects in microbiology and livestock breeding.

In summary, how to provide an enterococcus and to study its probiotics is a problem to be solved by the person skilled in the art.

Disclosure of Invention

In view of this, the present invention provides a strain of enterococcus mundtii and uses thereof.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

enterococcus mundtii, named HCD01, is classified and named as enterococcus mundtii Enterococcus mundtii, and is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, with the preservation number of CGMCC No.31447, and the preservation address of the enterococcus mundtii is China academy of microorganisms, national institute of sciences No.3, west 1, north Star, chat, beijing.

Further, the bacterium is Niu Yuanmeng enterococcus faecalis.

Further, the enterococcus mundane tolerates a pH of 2 and a tolerates salt concentration of 10%.

The enterococcus mundtii is applied to inhibiting salmonella and streptococcus agalactiae.

The application of the enterococcus mundane in preparing probiotics.

Further, the probiotics are used for alleviating salmonella infection symptoms.

Further, the preparation method is used for increasing the abundance of beneficial bacteria in intestinal flora, relieving intestinal injury caused by salmonella infection and alleviating lung and liver lesions caused by salmonella.

A probiotic comprising enterococcus mundane as described above.

Compared with the prior art, the invention has the beneficial effects that:

A strain of enterococcus mundtii (Enterococcus mundtii) is isolated and identified by a traditional identification method and a molecular biological method, and is subjected to probiotic performance evaluation and animal in-vivo safety evaluation. Meanwhile, microbiology and pathology are combined, and the fact that the salmonella can be taken orally can relieve organ lesions of mice caused by salmonella, and antagonism can be generated on the salmonella in the mice. The invention not only can expand the research on enterococcus, but also can provide reference for developing and developing probiotics microecologics aiming at aspects of promoting digestion and growth of animals, antagonizing pathogenic bacteria, regulating intestinal microecologics and the like in future.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.

FIG. 1 is a technical roadmap of the invention;

FIG. 2 shows colony morphology of the strain HCD01 of example 1 of the present invention on MRS solid medium;

FIG. 3 shows the result of gram-color detection (10X 100) of the strain HCD01 of example 1 of the present invention;

FIG. 4 is an NJ phylogenetic tree of strain HCD01 of example 1 of the invention;

FIG. 5 shows the results of detection of functional genes and virulence genes of the strain HCD01 of 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, and M represents 2000 bp Marker;

FIG. 6 shows the results of a hemolysis test of enterococcus mundtii HCD01 in example 2 of the present invention;

FIG. 7 shows the growth curve and the acid production curve of enterococcus mundtii HCD01 in example 2 according to the present invention;

FIG. 8 is a graph showing the results of a weight gain test of the enterococcus mundtii group in example 3 of the present invention;

FIG. 9 is a result of the feed weight ratio (F/G) test in example 3 of the present invention, wherein the same and different lower case letters represent no significant difference (P > 0.05) and significant difference (P < 0.05), respectively;

FIG. 10 is a graph showing the organ coefficient results in example 3 of the present invention;

FIG. 11 is a sectional view of a mouse according to 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., antagonistic Salmonella group);

FIG. 12 shows the effect of isolated strain antagonistic salmonella on intestinal morphology in mice in example 3 of the present invention;

FIG. 13 shows the effect of isolated strain Salmonella antagonism on liver and lung tissue in mice in example 3 of the present invention;

FIG. 14 is a drawing of an OTU annotation Venn in example 3 of the present invention, wherein CK is a control group, emun1 is a continuous 7 d-drenched enterococcus mundane group, emun2 is a continuous 14 d-drenched enterococcus mundane group, emunP1 is a continuous 7 d-drenched enterococcus mundane, and EmunP2 is a second stage of salmonella antagonism by enterococcus mundane (hereinafter);

FIG. 15 is a Chao index and Shannon index analysis of fecal flora in example 3 of the present invention;

FIG. 16 is a sparse curve for each set of samples in example 3 of the present invention;

FIG. 17 is a graph showing the level abundance curves of the samples of example 3 of the present invention, wherein the control group CKAn is not filled with the test bacteria, the probiotic group Emun1 is filled with the test probiotic (enterococcus mundtii) for 7 days continuously, the probiotic group Emun2 is filled with the test probiotic for 14 d continuously, the antagonistic group EmunP1 is filled with the test probiotic for 7 days continuously, namely, the salmonella is antagonized for 7d (enterococcus mundtii antagonistic salmonella group), and the antagonistic group EmunP2 is the second phase after the salmonella is antagonized for 14 th d;

FIG. 18 shows the results of a LEfSe-based lineage diagram analysis of a mouse fecal flora in example 3 of the present invention, wherein (a) a comparison of a dominant population of mouse fecal bacteria between the probiotic Emun group and the control CKAn group, (b) a comparison of a dominant population between the probiotic antagonistic salmonella EmunP group and the control CKAn group;

FIG. 19 is a graph showing the results of PCoA analysis of species similarity of fecal flora in mice in example 3 of the present invention, wherein CKAn is a control group, emun is a probiotic group (enterococcus mundane group) and EmunP is an antagonistic group (enterococcus mundane antagonistic salmonella group);

FIG. 20 is a PCA plot of the control CKAn, probiotic Emun, antagonistic EmunP mice faecal marker genus/differential genus of example 3 of the present invention.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The required medicament is a conventional experimental medicament purchased from a commercial channel, and the unrecited experimental method is a conventional experimental method and is not described in detail herein.

Example 1

Isolation, identification and preservation of strains

(1) Separation and purification of strains

The method comprises the steps of selecting a Siemens cow free-range grassland of which the inner Mongolian autonomous region tin Lin Guole allies with yellow flag (41 DEG 56-42 DEG 45 'N, 113 DEG 30-114 DEG 45' E), collecting cow manure samples, and storing the cow manure samples in a sterile bag at low temperature. For subsequent separation and screening of potential probiotics.

A proper amount of cow dung is dipped on a solid culture medium conveniently by using a sterile cotton swab, a liquid transfer gun is dripped with normal saline for dilution, the mixture is uniformly coated by a coater, and the mixture is cultured overnight at a constant temperature of 37 ℃. The bacterial colony with consistent form and absolute dominant quantity is picked by the sterile inoculating loop for the secondary use, and streaked and purified on a TSB solid culture medium. After single bacterial colonies appear on the culture medium, gram staining is carried out on the culture medium, the staining result is repeated until the bacterial forms and colors are consistent, fresh bacterial lawn of single bacteria is picked up to be amplified and cultured in TSB liquid culture medium, 1 mL bacterial liquid is sucked into a preservation tube by a pipetting gun, then 1 mL 30% glycerol is added, finally the culture medium is frozen and placed in a freezer at the temperature of minus 40 ℃ for preservation, and the culture medium is named as HCD01.

(2) Morphological identification

Culturing the separated and purified strain under proper condition, observing the phenotype characteristic of colony with naked eye, recording the shape, color and size, gram staining the separated strain, and observing the cell morphology characteristic under an optical microscope.

As can be seen from FIG. 2, the strain HCD01 was an off-white colony with round shape, neat edge and smooth surface on MRS medium. As shown in FIG. 3, the strain HCD01 obtained by screening is spherical and is a single gram positive bacterium or a gram positive bacterium arranged in pairs.

(3) Sequencing identification of bacterial 16S rRNA gene

Extracting genes according to the steps of the specification of a bacterial gene extraction kit (TIANamp Bacteria DNA Kit), and carrying out PCR amplification by using the extracted isolated bacterial genome as a template and using bacterial 16S rRNA gene universal primers. The upstream and downstream primers were 27 F:5 '-AGAGTTTGATCTGGCTCGTAG-3', SEQ ID NO.1 and 1492 R:5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO.2, respectively.

Sequencing the amplified product, performing BLAST homology comparison after sequencing, and constructing NJ (Neighbor jioning) phylogenetic tree by using MEGA X software.

The 16S rRNA gene sequencing result of the amplified strain HCD01 is shown below, and the sequence length is 1436 bp.

GCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGCTTCTTTTCCCACCGGAGCTTGCTCCACCGGGAAAAGAGGAGTGGCGAACGGGTGAGTAACACGTGGGTAACCTGCCCATCAGAAGGGGATAACACTTGGAAACAGGTGCTAATACCGTATAACAATCGAAACCGCATGGTTTCGTTTTGAAAGGCGCTTTACGGTGCCGCTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCCACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGAGAAGAACAAGGGTGAGAGTAACTGTTCACCCCTTGACGGTATCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGGACATCCTTTGACCACTCTAGAGATAGAGCTTCCCCTTCGGGGGCAAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTGTTAGTTGCCATCATTTAGTTGGGCACTCTAGCAAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGAAGTACAACGAGTCGCGAAGTCGCGAGGCTAAGCTAATCTCTTAAAGCTTCTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCG,SEQ ID NO.3.

BLAST sequence alignment was performed on the 16S rRNA gene sequence of the strain HCD01, and an NJ (Neighbor-Joining) phylogenetic tree was constructed using MEGA X software. As shown in FIG. 4, the homologies of the strain HCD01 with enterococcus mundane (Enterococcus mundtii) RP1C1 and CECT972T, XA3 were 99.85%, 99.65% and 99.79%, respectively. The results indicated that strain HCD01 was enterococcus mundane (Enterococcus mundtii).

(4) PCR detection of functional and virulence genes

The primer sequences are shown in Table 1, and all primers are synthesized by the division of biological engineering (Shanghai).

The results are shown in FIG. 5. The size of the band is consistent with the expected result (282 bp), which indicates that the strain HCD01 contains the EC gene, no band is produced at vanA, tetM, tetK, ermB gene related to drug resistance, and the detection result shows that no band is produced at virulence genes CylA, esp, asa1 and EfaAfm, gelE, agg, ace, which indicates that the strain HCD01 does not carry the virulence genes.

(5) Biochemical identification

The biochemical tests of the isolated strain are carried out by referring to a common bacteria identification manual and a trace biochemical identification tube instruction book, and the main tests comprise a glycolysis test, a urea test, a lysine decarboxylase test, a hydrogen sulfide test, a Simmons citrate test, a hydrogen peroxide gas production test, a glucose gas production test, an MR-VP test and the like, and the strain is further identified according to the detection result.

The strain HCD01 can utilize most sugar including glucose but does not produce glucose gas, can utilize mannitol but does not utilize sorbitol, can decompose urea but does not react with urease, does not decompose arginine bishydrolase, produces lysine decarboxylase, does not produce hydrogen sulfide and Simmons citrate, has positive hydrogen peroxide gas production test results and has negative MR-VP test results. The biochemical properties of strain HCD01 substantially match those of enterococci, and the results are shown in Table 2.

(6) Preserving

The enterococcus mundtii is named HCD01, the enterococcus mundtii is named as enterococcus mundtii Enterococcus mundtii, and is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, with a preservation number of CGMCC No.31447, and a preservation address of the enterococcus mundtii is China academy of microorganisms, national institute of sciences No.3, west 1, the area North Star of Chat in Beijing.

Example 2

Evaluation of in vitro probiotic Performance of Strain

(1) Hemolysis test

A single colony of enterococcus mundtii HCD01 is selected, fresh lawn is picked up by a sterile inoculating loop on a fresh blood agar culture medium, and is subjected to flat streaking, and after being inversely placed in a constant temperature incubator (37 ℃ C.) for one day (24 h), the hemolysis phenomenon is observed.

The morphology of enterococcus mundane HCD01 in fresh blood agar medium is shown in FIG. 6, and the appearance of a hemolytic circle indicates that enterococcus mundane HCD01 has no hemolysis.

(2) Drug susceptibility testing

The drug sensitivity test adopts a paper sheet diffusion method (Kirly-Baue method) to select antibacterial drugs such as beta-lactams, chloramphenicol, quinolones, sulfonamides, tetracyclines and the like. And sucking 200 mu L of bacterial culture solution, uniformly coating on the solid culture medium, and drying at room temperature for 3-5 min until the bacterial solution is observed to be fully absorbed, and then sticking the drug sensitive tablet by using tweezers. Three drug sensitive tablets of each drug are arranged in parallel, and after one day of inverted culture (24 h) in a 37 ℃ constant temperature incubator, the diameter of the inhibition zone is measured by a vernier caliper. And comparing the obtained test result with a 'paper-sheet-method drug sensitivity test bacteriostasis ring diameter judgment standard', and evaluating the result to obtain sensitivity (S), intermediate (I) and drug resistance (R).

The results of the drug susceptibility test of enterococcus mundtii HCD01 are shown in table 3. Enterococcus mundtii HCD01 is sensitive to azithromycin, midecamycin, tetracycline, doxycycline and vancomycin, intermediate to cefotaxime, ciprofloxacin, novobiocin and clindamycin, and resistant to penicillin G, chloramphenicol, norfloxacin, ofloxacin, compound neonomine, tobramycin, amikacin, streptomycin, neomycin, kanamycin, gentamicin and polymyxin B, wherein the result shows that enterococcus mundtii HCD01 is resistant to all aminoglycoside drugs used.

(3) Bacteriostasis test

And (3) performing an antibacterial test by adopting an oxford cup double-layer flat plate diffusion method. Preparing a solid agar culture medium, sterilizing at high temperature, taking out, controlling the pouring amount in each culture dish to be about 10mL, preparing a lower-layer flat plate after cooling and solidifying, and placing oxford cups at certain intervals. Repeating the above steps to prepare the upper plate and pouring the upper plate onto the lower plate, and pulling out the oxford cup after the upper plate is solidified. Selecting Salmonella CVCC2184, streptococcus agalactiae (Streptococcus dysgalactiae equisimilis) BNCC337480 and Staphylococcus aureus (Staphylococcus aureus) ATCC29213 as pathogenic indicator bacteria, uniformly coating 50 μl of each bacterial liquid on a culture medium, adding 200 μl of culture solution of the isolated strain into a small hole, placing in a 37 ℃ incubator, culturing for one day (24 h), observing whether a bacteriostasis ring appears, and measuring the diameter of the bacteriostasis ring.

In-vitro antibacterial tests for separating bacteria by adopting an oxford cup double-layer flat plate diffusion method show that enterococcus mundtii HCD01 has no inhibitory capacity on staphylococcus aureus, has antibacterial effect on salmonella, and has a certain inhibitory effect on streptococcus dysgalactiae.

(4) Growth curve and acid production curve

The isolated bacteria are inoculated into a corresponding liquid culture medium, the bacteria are cultured at constant temperature and shaking (37 ℃ C., 200 rpm), bacterial liquid is taken every 2 hours (namely after 2h,4 h,6 h,8 h,10 h,12 h,14 h,16 h), the absorbance value (OD 600) at the wavelength of 600 nm is measured by an enzyme-labeling instrument, the pH value of the bacterial liquid is measured, and the growth curve and the acidogenic curve are drawn.

As shown in FIG. 7, the enterococcus mundtii HCD01 enters the logarithmic phase after 2h and gradually enters the stationary phase after 8 h, and the pH value of the bacterial liquid in 16 h is reduced from 6.2 to 5.5, so that the trend line of acid production is gradually reduced, which indicates that the enterococcus mundtii HCD01 has acid production capability but has slower acid production speed.

(5) Acid resistance test

The isolated bacteria cultured overnight were inoculated into liquid media of different pH values (i.e., 2,3, 4) at an inoculum size of 1%, respectively, and after incubation at constant temperature (37 ℃ C., 200 rpm) for 24: 24h, the medium was observed for turbidity.

The results of the acid resistance test of the strain are shown in Table 5. Turbidity can be generated after overnight culture in liquid culture medium with pH of 2,3 and 4, which shows that the culture medium has certain acid resistance.

(6) Salt tolerance test

MRS liquid medium containing NaCl was prepared separately, and the concentration gradient was set to 2%,4%,6%,8% and 10%, and autoclaving was performed after the preparation was completed (121 ℃,15 min). The isolated bacteria were inoculated into the corresponding medium at an inoculum size of 1%, and after incubation at constant temperature (37 ℃ C., 200 rpm) for 24: 24h, the OD600 of the bacterial solution was measured by an ELISA reader.

From the results of Table 6, the E.mongolicus HCD01 shows a tendency of decreasing before increasing with increasing NaCl concentration, and the E.mongolicus HCD01 grows most slowly at 4% concentration and has the best growth effect at 10% concentration, which shows that the E.mongolicus HCD01 has stronger tolerance to high salinity.

(7) Curd test

The isolated bacteria were inoculated in a liquid medium at an inoculum size of 1%, cultured overnight with shaking at constant temperature (37 ℃ C., 200 rpm), and then a mixed solution was prepared according to a bacterial liquid/skim pure milk ratio of 1:5,1:10,1:15,1:20,1:25, placed in a 37 ℃ C. Incubator, and after 24: 24h, observed for the occurrence of curding phenomenon and the pH value of the mixed solution was measured.

The curd test results and the measured pH values are shown in Table 7. When the ratio of the bacterial liquid of the enterococcus mundtii HCD01 to the defatted pure milk is 1:15, the pH value reaches the minimum, but no coagulation phenomenon occurs, which indicates that the enterococcus mundtii HCD01 does not have the capability of curding.

Example 3

Evaluation of in vivo probiotics and safety performance of strain

The test animals are selected from SPF-class Kunming mice of 4-6 weeks old, and the test mice are male and purchased from university of Endoconcha medical science.

Bacterial liquid preparation, namely inoculating the separated bacteria into MRS liquid culture medium according to the proportion of 1%, culturing the bacteria overnight at 37 ℃ and 200 rpm, and adjusting the concentration of the bacterial liquid to be 1 multiplied by 10 ⁸ CFU/mL. The same procedure is to inoculate salmonella into TSB culture medium, shake overnight at constant temperature, adjust the concentration of bacterial liquid to 1X 10 ⁷ CFU/mL for standby.

(1) Weight gain and feed intake test of mice

10 Male mice were selected and randomly divided into 2 groups of 5 mice each, and the test was performed after one week of adaptive breeding. The mice are infused with the enterococcus mundtii bacteria liquid by using the mice gastric lavage needle for 1 time a day, the mice in the test group (enterococcus mundtii group) are infused with the enterococcus mundtii bacteria liquid, the control group is infused with the same volume of sterile physiological saline, the gastric lavage amount is 0.2 mL/mouse, and the test period is 14 d.

Mice were observed for mental state, eating desire, hair color, fecal status, etc. during the test. 14 The mice were weighed once daily, and feed intake was measured daily, and Average Daily Gain (ADG), average Daily Feed Intake (ADFI) and feed-to-weight ratio (F/G) were calculated.

In the weight gain test of mice, the mice in each group are in the stomach 14 d without adverse reaction, normal mental state, normal hair color and fecal color, and no diarrhea. The test data recorded by the measurement were statistically consolidated with Excel software, and the results are expressed as "mean ± standard deviation". As can be seen from table 8, all groups showed a tendency to increase body weight in the gastric lavage 14 d, and the average daily gain of mice in 7 d (half period) was greater than that in 14 d (one period), which indicated that the mice grew faster in a short period, after which the growth gradually tended to be smooth.

The average daily gain results of each group of mice were T-checked using GRAPHPAD PRISM Version 9.5.1 software, at the same stage, "ns" indicated that the difference was not significant (P > 0.05), "x" indicated that the difference was significant (P < 0.05), and the same letter indicated that the difference was not significant (P > 0.05), and the different letters indicated that the difference was significant (P < 0.05). As can be seen from fig. 8, in both 7 d and 14d, the average daily gain of mice perfused with enterococcus mundtii was higher than that of the control group, indicating that the bacteria are beneficial for the growth and development of mice, but the difference between the mice is insignificant (P > 0.05) in half period and in whole period compared with the control group.

The average daily feed intake results of the mice are shown in Table 9, and the results of the feed weight ratio are shown in FIG. 9. In 7 d and 14 d, the weight ratio of the components of the enterococcus mundane is lower than that of the control group, which indicates that the enterococcus mundane can promote the growth and development of mice, and the influence of enterococcus mundane HCD01 on the mice is still beneficial along with the longer test period.

(2) Test for measuring organ coefficients of mice

10 Male mice were randomly divided into 2 groups, each of which was filled once daily, one group was filled with enterococcus mundtii bacteria liquid, and the remaining group was filled with sterile physiological saline as a blank control, and the mice were dissected at the time of the d th, hearts, livers, kidneys, and lungs of the mice were picked up, and at the same time, whether or not each tissue had macroscopic lesions was observed, and organ coefficients were measured.

During the measurement test of the organ coefficients of the mice, the mice in each group were found to be normal in organ color, size, etc., and had no ocular lesions. The results of organ coefficients of the mice taken in each group are shown in fig. 10, and the organ coefficients of the mice perfused with enterococcus mundtii HCD01 are not significantly different (P > 0.05) from those of the control group, which indicates that the strain has no pathological changes such as edema or hyperplasia and no degenerative changes such as atrophy on immune organs of the mice.

(3) In vivo antagonism test for mice

15 Mice were selected and randomly divided into 3 groups of 5 mice each. The protective group is enterococcus mundane group (namely antagonistic salmonella group), the mice of the protective group are filled with 0.2 mL enterococcus mundane liquid every day, and the pathogenic bacteria group and the control group are filled with sterile physiological saline with the same volume every day. After 7d is continuously infused, salmonella is used for stomach infusion and toxin counteracting according to the pre-test result, and a control group is not treated. 96 h, randomly dissecting the mice, observing whether pathological changes exist in organs, collecting the lung, liver, small intestine and colon of the mice, fixing the mice by using a universal tissue fixing solution, and sending the mice to a Wuhan Seville biological company for HE staining and pathological tissue section preparation.

After the salmonella is attacked, the mice are listless and have symptoms of shortness of breath, diarrhea and the like, and the mice in the control group and the protection group have no death phenomenon, and the mental state and the defecation condition are normal. The results of the section are shown in FIG. 11, the liver, the intestinal tract and the like of the mice in the control group are not abnormal, the intestinal tract of the mice in the pathogenic bacteria group has eye lesions such as bleeding, tympanites and the like, and after the enterococcus mundane antagonizes salmonella, the tissue lesion symptoms of the mice are relieved.

The intestinal morphology of mice after in vivo antagonism of enterococcus mundtii with salmonella is shown in figure 12. Compared with a control group, the mice of the pathogenic bacteria group have short intestinal villi and a large amount of intestinal villi epithelial cells fall off, the mucous membrane layer of colon tissues generates fold and focal erosion, the mucous membrane epithelial cells have necrosis and fall off, the intestinal gland quantity is reduced, little connective tissue hyperplasia is carried out, and meanwhile, little granulocyte infiltration is carried out. The mice in the protective group have abundant intestinal glands of the intestinal lamina propria, the intestinal villus length is shortened compared with the control group, and a large amount of epithelial cells fall off, while cup-shaped cells in colon tissues are abundant and no fold exists. Thus, enterococcus mundtii HCD01 can alleviate intestinal damage caused by salmonella infection.

As shown in fig. 13, after salmonella challenge mice, lung tissue of mice was seen with alveolar wall granulocyte infiltration, focal alveolar wall was slightly thickened, alveolar space was widened, alveolar hemorrhage occurred, bronchiolar epithelium was irregularly arranged, and small amounts of eosinophils were seen in the lumen, as compared with control group. The protective group had little blood vessel and alveolar wall capillary blood stasis, and had no alveolar wall thickening and inflammatory cell infiltration. The liver tissue of the mice after the salmonella challenge is not obvious in liver lobule demarcation, symptoms such as hepatocyte water sample denaturation and venous stasis appear, and the symptoms are reduced compared with pathogenic bacteria groups although the liver of the mice in the group is protected from injury. Therefore, after the salmonella in vivo antagonism test is carried out by filling the enterococcus mundtii HCD01, the lung and liver lesions caused by salmonella can be relieved.

(4) High-throughput sequencing of 16S rRNA genes of mouse fecal bacterial community

And (3) collecting the fresh mouse faeces samples at the 7 th d th and the 14 th d th respectively, packaging the faeces samples in sterile freezing tubes, carrying out dry ice transportation immediately after packaging the faeces samples with the sample amount of about 0.5-2.0 g, sequencing the faeces samples to Nanjing Pakino gene technology Co., ltd, and carrying out information analysis on the sequencing result by utilizing a Pakino gene cloud platform.

The sample species sequences were subjected to a clustering analysis, and the bioinformatic analysis was performed at 97% similarity level, and the distribution of OTU numbers among the different groupings was compared according to Venn plot, and the clustering and species annotation results are shown in fig. 14. The number of OTUs in Emun, emunP, emun, emunP groups of enterococcus mundane was 146, 410, 122 and 147, respectively. The number of OTUs was different after the infusion of enterococcus mundtii and the antagonism of salmonella compared with the corresponding control group.

As shown in fig. 15, both the Chao1 index and Shannon index of the enterococcus mundane population were slightly lower than the control.

The sparse curve can reflect the effect of variations in sequencing depth on the diversity of each set of samples. As the depth of the draw increases in fig. 16, the sparse curve of each set of samples gradually becomes gentle, indicating that the sequencing result can reflect the diversity contained in the current sample, and Alpha diversity has reached saturation.

The class abundance curve can reflect the uniformity of community composition. The level abundance curve shown in fig. 17 has gradually flattened, which shows that the abundance difference between ASV/OTU in the sample community detected by the test gradually becomes smaller, the number of new species is not increased with continuous sampling, the sequencing depth is enough, and the sequencing data volume is reasonable.

The structure of the mouse fecal microbiota was analyzed at the phylum and genus level for different groups, and the composition abundance is shown in tables 10 and 11. There are two main classes of phylum, namely Bacteroides (Bacteroidetes), which is one of the absolute dominant flora in mouse feces, where Bacteroides (bacterioides) are more abundant at the genus level. The relative abundance of Bacteroides (Bacteroidetes) was higher in Emun (75.950%) than in control CKAn (67.145%), bacteroides (bacterioides) was higher in Emun (6.257%), emunP (10.792%) than in control CKAn (2.745%), the relative abundance of the firmicutes (Firmicutes), mainly comprising Lactobacillus (Lactobacillus), was highest in Emun (Firmicutes) and reached 62.923%, and Lactobacillus (Lactobacillus) was higher in the collected fecal samples than in control CKAn (1.719%), and EmunP (38.389%) was higher in 14 d than in control CKAn (18.193%). The results show that the enterococcus mundtii HCD01 can increase the abundance of beneficial bacteria in the intestinal tract of the mice, and the bacterial group gradually returns to equilibrium compared with the control group after the salmonella is antagonized.

The LDA threshold was set to 2, and the comparison strategy selected one-against-all, and the mice fecal flora was mapped on the LEfSe-based pedigree map. Fig. 18 (a) shows that there is a significant difference in the relative abundance of the intestinal flora of mice in control CKAn and probiotic Emun at the levels of class (c), order (o), family (f) and genus (g) (P < 0.05). 7 dominant species and 2 dominant species (Enterococcus Enterococcaceae and Enterococcus) were screened out in the control CKAn (green fanning) and probiotic Emun (yellow fanning) groups, respectively.

As can be seen from fig. 18 (b), the relative abundance of the intestinal flora of mice in control CKAn and antagonistic salmonella EmunP at the genus (g) level was significantly different (P < 0.05), and 2 dominant genera were screened in control CKAn (green fan ring) and antagonistic salmonella EmunP (yellow fan ring).

Fig. 19 uses bray _ curtis distance algorithm to perform principal coordinate analysis (PRINCIPAL COORDINATE ANALYSIS, PCoA) to characterize β diversity. The control CKAn samples were the smallest distance between the first axis (pco1=21.4%) and the second axis (pco2=18.5%) and had a higher aggregation level, indicating that the control CKAn had a low β -diversity, the probiotic Emun samples were the largest distance between the first axis (pco1=21.4%) and the second axis (pco2=18.5%) and had a higher dispersion level, indicating that the probiotic Emun had a high β -diversity, the antagonistic salmonella EmunP had the smallest distance between the first axis (pco1=21.4%) and the second axis (pco2=18.5%) and the antagonistic salmonella EmunP had the greatest β -diversity and the dispersion level was centered.

FIG. 20 screens for markers/differences (genus) by principal component analysis (PRINCIPAL COORDINATE ANALYSIS, PCA). The first axis (PCA 1) has an interpretation level as high as 92.1% and the second axis (PCA 2) has an interpretation level of 3.6%, indicating that the interpretation level of the first axis dominates. The control CKAn samples were the least distant on the first axis (pca1=92.1%) and had a higher degree of aggregation, indicating that the control CKAn had the least marked/differentiated genus (genus), the probiotic Emun samples were the most distant on the first axis (pca1=92.1%) and the most dispersed, indicating that the probiotic Emun had the most marked/differentiated genus (genus), and the antagonistic salmonella EmunP had the more marked/differentiated genus (genus) centered on the first axis (pca1=92.1%) and the antagonistic salmonella EmunP.

In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended 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 (6)

1. A strain of Enterococcus mundtii, characterized in that it is named HCD01, which is classified as Enterococcus mundtii , deposited in the General Microbiology Center of China Microorganism Culture Collection Committee on July 24, 2024, with a deposit number of CGMCC No.31447, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. 2. The Enterococcus monteroni according to claim 1, characterized in that it is bovine Enterococcus monteroni. 3. The Enterococcus monteroi according to claim 1, characterized in that the Enterococcus monteroi 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 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. 6. A probiotic, characterized in that it comprises the Enterococcus montegrioides according to claim 1.