CN119242534B

CN119242534B - Enterococcus faecalis YL-EF25 and application thereof

Info

Publication number: CN119242534B
Application number: CN202411729783.7A
Authority: CN
Inventors: 姜延龙; 张格瑞; 王占楠; 张宇航; 卢新; 徐瑶; 王春凤
Original assignee: Jilin Agricultural University
Current assignee: Jilin Agricultural University
Priority date: 2024-11-29
Filing date: 2024-11-29
Publication date: 2025-03-04
Anticipated expiration: 2044-11-29
Also published as: CN119242534A

Abstract

The present invention discloses a strain of Enterococcus faecalis YL‑EF25 and its application, and belongs to the field of microbial technology. The deposit number of the Enterococcus faecalis YL‑EF25 provided by the present invention is CCTCC NO: M 20242269. The strain has a strong inhibitory ability against Clostridium perfringens, and can also regulate the immune response of poultry and improve feed conversion rate. The weight gain of chickens in the group taking Enterococcus faecalis YL‑EF25 increased, and the feed-to-weight ratio improved, indicating that adding Enterococcus faecalis YL‑EF25 to the diet is beneficial to the growth and development of chickens; the Enterococcus faecalis YL‑EF25 provided by the present invention also helps to maintain the balance of the immune system, and can produce more immune cells faster when pathogens invade, thereby improving the vaccination effect.

Description

Enterococcus faecalis YL-EF25 and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to enterococcus faecalis YL-EF25 and application thereof.

Background

The necrotic enteritis of the poultry is mainly caused by clostridium perfringens, the death rate of different poultry after the occurrence of the disease is greatly different, typical symptoms are not represented, the best treatment time is easy to lose, once the microbial environment in the intestinal tract of the poultry is destroyed, good proliferation conditions are created for the clostridium perfringens, a large amount of endotoxin and exotoxin are generated along with the increase of the gas in the intestinal tract and the clostridium perfringens, and finally the necrotic enteritis of the poultry is caused. In addition, a large number of cases prove that the poultry flock infected with necrotic enteritis often has a relation with coccidiosis, which generally occurs first or together with necrotic enteritis, and the occurrence rate of the situation is high in the management of the flat-raising broiler breeder. After the poultry is infected with coccidiosis, gastrointestinal epithelial cells are destroyed, and clostridium perfringens is easier to access the damaged mucous membrane, thereby improving proliferation speed and hazard degree.

The probiotics enterococcus are widely existing in intestinal tracts of healthy chickens and have various probiotics. Wherein enterococcus faecalis can inhibit and produce antibacterial substances by competition, inhibit clostridium perfringens growth in intestinal tract, promote beneficial bacteria growth, and maintain ecological balance of intestinal microorganisms. Enterococcus faecalis also helps to enhance intestinal barrier function, reduce inflammatory reaction of intestinal tract, and improve intestinal health by promoting proliferation and repair of intestinal epithelial cells. By introducing enterococcus faecalis, the dependency on traditional antibiotics can be effectively replaced or reduced, and the risk of development of antibiotic resistance is reduced.

Although enterococcus faecalis has good probiotics, the bacteria inhibition capability and biochemical characteristics of different strains still have differences. Screening out strains with antibacterial activity, which can regulate the immunity of the poultry and promote the growth of the poultry, will contribute to the prevention and treatment of necrotic enteritis of the poultry.

Disclosure of Invention

In view of the shortcomings of the prior art, the invention provides enterococcus faecalis (Enterococcus faecalis) YL-EF25 and application thereof, and the strain has the functions of immunoregulation and growth promotion for poultry. The technical scheme provided by the invention is as follows:

The invention provides enterococcus faecalis YL-EF25 which is preserved in China center for type culture collection, wherein the preservation address is Wuhan in China, the preservation time is 2024, 10 and 21 days, the preservation number is CCTCC NO: M20242269, and the classification and naming are Enterococcus faecalis.

The invention also provides application of the enterococcus faecalis YL-EF25 in preparing a product for resisting clostridium perfringens infection.

The invention also provides application of the enterococcus faecalis YL-EF25 in preparing a product for preventing and treating necrotic enteritis of poultry.

Preferably, the avian necrotic enteritis is induced by clostridium perfringens.

Further, the avian necrotic enteritis is co-induced by clostridium perfringens and coccidium.

Further, the poultry is a layer chicken or a broiler chicken.

Preferably, the product is selected from any one of a medicament, a feed or a feed additive.

Furthermore, the product is feed, and the preparation method comprises the steps of inoculating enterococcus faecalis YL-EF25 into a culture medium for fermentation, spraying the fermentation liquor into the feed, and mixing.

In one embodiment of the invention, the feed comprisesEnterococcus faecalis YL-EF25.

The invention also provides a microbial agent, which takes the enterococcus faecalis YL-EF25 as an active ingredient.

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

(1) The invention provides enterococcus faecalis YL-EF25, which has strong inhibition capability on clostridium perfringens, can regulate poultry immune response, improves feed conversion rate, and experiments prove that the weight gain of chickens taking enterococcus faecalis YL-EF25 groups is increased, the feed weight ratio is improved, and the addition of enterococcus faecalis YL-EF25 in daily ration is beneficial to the growth and development of chickens.

(2) The enterococcus faecalis YL-EF25 provided by the invention is beneficial to maintaining the balance state of an immune system, and can generate more immune cells more quickly when pathogens invade, so that the vaccination effect is improved.

(3) The enterococcus faecalis YL-EF25 provided by the invention has strong tolerance to bile salts, artificial gastric juice and phenol, can metabolize dietary fibers such as pectin, glucan and cellobiose, and is beneficial to intestinal health of poultry.

(4) The enterococcus faecalis YL-EF25 is used as a feed additive for feeding broiler chickens, is beneficial to enhancing the intestinal barrier function, can obviously relieve the characteristic symptoms of necrotic enteritis of chickens, effectively reduces the inflammatory reaction of intestinal tracts, and supports the balance of intestinal flora.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.

FIG. 1 is a microscopic image of enterococcus faecalis YL-EF25, YL-EF7 and YL-EF28 in example 1 of the present invention.

FIG. 2 is a graph showing the hemolytic activity of enterococcus faecalis YL-EF25, YL-EF7 and YL-EF28 in example 2 of the present invention.

FIG. 3 is a graph showing growth characteristics of enterococcus faecalis YL-EF25, YL-EF7 and YL-EF28 in example 2 of the present invention.

FIG. 4 is an acid generating capacity profile of enterococcus faecalis YL-EF25, YL-EF7 and YL-EF28 in example 2 of the present invention.

FIG. 5 is a graph of Clostridium perfringens co-culture inhibition assay of enterococcus faecalis YL-EF25, YL-EF7 and YL-EF28 in example 2 of the present invention.

FIG. 6 is a graph of clostridium perfringens biofilm inhibition assays of enterococcus faecalis YL-EF25, YL-EF7 and YL-EF28 in example 2 of the present invention.

FIG. 7 is a graph showing the relative abundance of lactic acid bacteria in intestinal flora after challenge with necrotic enteritis in example 3 of the present invention.

FIG. 8 is a graph showing the proliferation capacity of peripheral blood lymphocytes of a chicken after challenge with necrotic enteritis in example 3 of the present invention.

FIG. 9 is a graph showing the relative expression level of mRNA of claudin after challenge in example 3 of the present invention.

FIG. 10 is a graph showing the relative expression levels of mRNA of inflammatory cytokines after challenge in example 3 of the present invention.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The preparation method comprises weighing Paeonia suffruticosa culture medium base (Haibo organism, qingdao) 48.0 g, and dissolving in 1000: 1000 mL distilled water under heating. Several pieces of the meat of the Paodia (Haibo organism, qingdao) were added to a test tube at about 1 cm gauge, and autoclaved at 121℃for 15 minutes for use.

MRS culture medium is prepared by weighing MRS broth 52.24 g, dissolving in 1000 mL distilled water under heating, and autoclaving at 118 deg.C for 15 min.

Example 1 screening and identification of enterococcus faecalis YL-EF25

Separating and screening a plurality of isolated strains from the feces of healthy wild birds with strong disease resistance, testing the inhibition capability of the strains on clostridium perfringens on a blood plate by an oxford cup method, and finally screening three enterococcus faecalis, wherein the colony forms are off-white circular colonies with smooth surfaces, and the figure 1 is shown.

Antagonism of clostridium perfringens capability test clostridium perfringens and 33 isolated strains were anaerobically cultured in bulla and MRS media at 37 ℃ for 24 hours, respectively, for use. The lactic acid bacteria fermentation broth is collected aseptically and placed in a 1mL centrifuge tube, centrifuged for 5 minutes at 12000 r/min, the supernatant is taken, and a 0.22 μm filter membrane is used for filtration sterilization. Clostridium perfringens bacteria fluid was diluted with sterile PBS to an optical density of OD ₆₀₀ = 0.8. Under the aseptic environment, 100 mu L of diluted clostridium perfringens bacteria liquid is absorbed and evenly coated on the surface of a blood plate containing 5% sheep blood, then an oxford cup is vertically placed on the blood plate, 200 mu L of separated strain fermentation supernatant is added into the oxford cup, the mixture is placed in an anaerobic working station for culturing for 48 hours at 37 ℃, and the diameter of a bacteriostasis ring is measured, and the result is shown in table 1.

TABLE 1 measurement of bacteriostatic Activity of isolated lactic acid bacteria against Clostridium perfringens

Low bacteriostatic activity, strong bacteriostatic activity, jiang Yijun activity, no zone of inhibition.

And (3) colony purification and enrichment, namely picking single colony on an MRS culture medium plate by adopting a plate streaking method, culturing for one day at 37 ℃ in an inversion mode, continuously picking single colony on the MRS culture medium plate, repeating purification for 2-3 times, picking single colony, culturing for two days in a 5 mL MRS culture medium test tube at 37 ℃ in a shaking mode, carrying out 16S rDNA identification after full enrichment, adding glycerol into the rest bacterial liquid, enabling the final concentration range of the glycerol in the bacterial liquid to be 15-25%, and storing in a-80 ℃ refrigerator.

And (3) 16S rDNA identification, namely extracting genome DNA of bacterial liquid, performing colony PCR amplification by using a bacterial universal sequencing primer 27F, performing 16S rDNA sequencing identification, and performing sequence comparison analysis on the obtained 16S sequence in an NCBI (NCBI) database, wherein the sequence similarity of the comparison result and enterococcus faecalis is 100%. These three strains were identified as enterococcus faecalis (Enterococcus faecalis) and formally designated YL-EF7, YL-EF25, YL-EF28. The 16S rDNA complete sequences of the enterococcus faecalis YL-EF7, YL-EF25 and YL-EF28 are shown in a sequence table SEQ ID NO. 1.

GGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGCTTCTTTCCTCCCGAGTGCTTGCACTCAATTGGAAAGAGGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTACCCATCAGAGGGGGATAACACTTGGAAACAGGTGCTAATACCGCATAACAGTTTATGCCGCATGGCATAAGAGTGAAAGGCGCTTTCGGGTGTCGCTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCCACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGAGAAGAACAAGGACGTTAGTAACTGAACGTCCCCTGACGGTATCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCAAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGACCACTCTAGAGATAGAGCTTTCCCTTCGGGGACAAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTGTTAGTTGCCATCATTTAGTTGGGCACTCTAGCGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGAAGTACAACGAGTCGCTAGACCGCGAGGTCATGCAAATCTCTTAAAGCTTCTCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTTGGAGCCAGCCGCCTAAGGTGGGATAGATGATTGGGGTGAAGTCGT.

Example 2 detection of the Properties of enterococcus faecalis (Enterococcus faecalis)

Haemolytic test. A sterile staining of the enterococcus faecalis solution was streaked on the platelets and incubated anaerobically for 24 hours, the haemolytic activity being shown in FIG. 2, wherein 7,25 and 28 are YL-EF7, YL-EF25 and YL-EF28 respectively.

Growth curve and acid production curve, namely marking and activating enterococcus faecalis strains in an MRS flat plate, inoculating single colony to a culture medium test tube of 5mL MRS, culturing overnight at a shaking table of 37 ℃, inoculating enterococcus faecalis bacterial liquid to 200mL of MRS culture medium according to a ratio of 1:100, culturing at 37 ℃ in the shaking table, taking 5mL to measure pH value and bacterial liquid OD ₆₀₀ value at 2 hours, 4 hours and every 6 hours, taking the MRS liquid culture medium as blank contrast, and taking the bacterial liquid OD ₆₀₀ value from 1.2 to 1.3 from 2 hours to about 6 hours as shown in figure 3 and 4, wherein the growth characteristics and acid production capacity of three enterococcus faecalis strains are basically the same, and starting to enter the logarithmic phase from 2 hours to about 6 hours, and starting to enter the stationary phase. The acid production capacity detection shows that after the three strains are cultured for 6 hours, the pH value of the bacterial liquid is reduced to 4.8. The pH value of the bacterial liquid is changed to about 4.6 when the bacterial liquid enters a platform stage (12-18 hours), and the pH value of the bacterial liquid is reduced to about 4.4 when the bacterial liquid enters 24-36 hours. In FIGS. 3 and 4, E.F7, E.F25 and E.F28 are YL-EF7, YL-EF25 and YL-EF28, respectively.

Artificial gastric juice resistance and cholate resistance test, namely marking and activating enterococcus faecalis strains in an MRS flat plate, inoculating single colony to a 5mL MRS culture medium test tube, culturing overnight at a shaking table of 37 ℃, inoculating enterococcus faecalis bacterial liquid into 200mL of MRS culture medium according to a ratio of 1:100, culturing at the temperature of 37 ℃ in the shaking table, and regulating the bacterial liquid concentration of the bacterial liquid to 0.8 (about 10 ⁸) at OD ₆₀₀ by using a cuvette. Enterococcus faecalis with adjusted concentration of 1 mL is taken into an EP tube, and centrifuged for 10 minutes at 5000: 5000 rpm. During centrifugation, bile salts (0.2%, 0.25%, PBS dissolved) and artificial gastric juice MRS medium tubes (artificial gastric juice: 0.32% pepsin, 1.6 μg/ml lysozyme, ph=2) were prepared, 1 mL each to EP tubes for use. The supernatant of the culture medium after centrifugation is discarded, bacterial sediment is resuspended by using a bile salt solution and an artificial gastric juice MRS culture medium under the corresponding conditions of 1 mL, the culture is placed in a 37 ℃ incubator for incubation for 3 hours, the incubated strain to be detected is diluted to 10 ^-9 by PBS gradient, and the plates are dripped in the corresponding areas. And airing the plates on an ultra-clean bench, culturing in a incubator after airing, simultaneously carrying out gradient dilution on three bacterial solutions to 10 ^-9 by using PBS, and placing 100 mu L of each gradient dilution bacterial solution drop plate on an MRS flat plate. And airing the plate on an ultra-clean bench, putting the plate in a incubator for culturing overnight after airing, and calculating the initial viable count. Data were recorded and mean, standard deviation, survival were calculated as shown in table 2, table 3.

TABLE 2 bile salt resistance test

TABLE 3 Artificial gastric juice resistance test

Phenol resistance test enterococcus faecalis strains were streaked and activated in MRS plates, inoculated with single colonies and cultured in 5 mL MRS medium tubes overnight at 37℃on a shaker as the strain. MRS culture medium test tubes containing 0.1%, 0.2%, 0.3% and 0.4% phenol are prepared, strains are added into the MRS culture tubes with corresponding phenol concentrations according to the proportion of 1:100, the strains are cultured for 24 hours at the temperature of 37 ℃ 220rpm, OD ₆₀₀ values of bacterial solutions of each group are measured in 3 repeated tests, and the MRS liquid culture medium is used as a blank control, and is shown in table 4.

TABLE 4 Artificial phenol resistance test

Antibiotic resistance test by streaking E.faecalis strains on MRS plates, inoculating single colonies on 5 mL MRS medium tubes, culturing overnight on shaker 37℃and adjusting the concentration of the resulting bacterial solution to 0.8 (about 10 ⁸) at OD ₆₀₀ with a cuvette. 100 mu L of diluted bacterial liquid is coated on the surface of an MRS culture medium flat plate, an antibiotic tablet is stuck on the MRS flat plate, and the MRS flat plate is placed in a bacteria incubator for culturing for 24 hours at 37 ℃, and the diameter of a bacteria inhibition zone is measured, which is shown in Table 5.

Table 5 test for resistance to common antibiotics

Clostridium perfringens biofilm inhibition test comprising streaking and activating enterococcus faecalis strain in MRS plate, inoculating single colony in 5mL MRS culture medium tube, culturing overnight in shaking table 37 ℃, inoculating Clostridium perfringens strain in TSB culture medium at ratio of 1:100, culturing overnight at anaerobic workstation 37 ℃, preparing cultured clostridium perfringens bacterial solution (C.P) and enterococcus faecalis bacterial solution (E.F), filtering centrifuged enterococcus faecalis (4000 rpm, 5 min) supernatant with 0.22 μm filter membrane to obtain enterococcus faecalis supernatant (E.F supernatant). The clostridium perfringens bacterial liquid and the enterococcus faecalis bacterial liquid are diluted by a TSB culture medium until the bacterial liquid concentration is 0.1 at OD ₆₀₀, and are mixed in 96-well plates according to the following grouping (table 6) mixing method, 6 repetitions are carried out on each group, the 96-well plates are sealed by a sealing film to isolate oxygen, and the culture is carried out for 5 days at room temperature.

TABLE 6 Clostridium perfringens biofilm inhibition assay group

Note that C.P in table 6 refers to clostridium perfringens, E.F refers to YL-EF25, YL-EF7 or YL-EF28, respectively.

After the incubation, the suspensions in each well were transferred, the OD ₆₀₀ value of the supernatant was measured, and each set of suspensions was diluted to 10 ^-9 with sterile PBS gradient and drop plates were counted on TSN medium plates. The culture wells after transferring the supernatant twice were gently rinsed with PBS, and then stained with 300. Mu.L of crystal violet and left at room temperature for 30 minutes. After removal of crystal violet, the solution was gently rinsed twice with PBS again. 300. Mu.L of methanol solution was added to each well and incubated at room temperature for 30 minutes, the liquid in each well was transferred to a new 96-well plate, and the OD ₅₇₀ value per well was measured and designated A ₅₇₀. Calculate biofilm relative measurement = a ₅₇₀/OD₆₀₀. As can be seen from FIG. 6, both the three enterococcus faecalis bacterial solutions and the sterile enterococcus faecalis supernatant exert an inhibitory effect on the biofilm formation of Clostridium perfringens. Analysis according to FIGS. 5 and 6 shows that the ability of the three strains to inhibit Clostridium perfringens activity is ranked from strong to weak as YL-EF25, YL-EF7 and YL-EF28. Animal experiments were performed with YL-EF25 with the strongest biofilm protective activity inhibiting clostridium perfringens formation.

Microorganism metabolism analysis, namely streaking and activating enterococcus faecalis strains in an MRS plate, inoculating single colonies, streaking and activating again in the MRS plate, and picking the activated colonies to prepare an inoculating liquid in 200 mu L of IF-B culture medium according to a method provided by an automatic microorganism identification system of BIOLOG company. The IF-B medium (BIOLOG, USA) was adjusted to a suitable light transmittance with an inoculum to prepare a bacterial suspension. 100 mu L of bacterial suspension is added to each hole of a GEN III plate, and the plate is placed in a microorganism metabolism identification incubator for 36 hours, and analysis data show that YL-EF25 shows the metabolism characteristics of typical enterococcus faecalis strains, and the fact that the enterococcus faecalis YL-EF25 has strong metabolism capability on dietary fibers such as glucan, cellobiose, pectin and the like is proved, so that short-chain fatty acid is generated, and intestinal health is promoted.

TABLE 7 analysis of carbon source metabolism

Note that +: metabolic activity is positive, -: metabolic activity is negative, w ⁺: metabolic activity is weakly positive.

EXAMPLE 3 protective Effect of enterococcus faecalis YL-EF25 on necrotic enteritis in chickens

1 Day-old broiler chickens with similar weights are selected and randomly divided into 4 treatment groups, and 18 chickens in each group. (1) control group fed basal diet (NC group), (2) necrotic enteritis group (4X 10 ⁸ CFU/mL) against coccidium and clostridium perfringens, (PC group), (3) antibiotic group (4X 10 ⁸ CFU/mL) against coccidium and clostridium perfringens, (PT group, antibiotic group) against enramycin containing 20 mg/kg, (4) YL-EF25 test group (4X 10 ⁸ CFU/mL) against coccidium and clostridium perfringens, (E.F25 group) against basal diet containing 1X 10 ⁹ CFU/kg of enterococcus faecalis YL-EF 25.

In the present embodiment, NC represents control group, PC represents enteritis group, PT represents antibiotic group, E.F25 represents YL-EF25 group.

The production performance of the chickens comprises parameters such as egg yield, growth speed, feed conversion rate, feed intake and the like, the invention records parameters such as weight gain, feed intake, feed weight ratio and the like, and the parameters are summarized as shown in table 8:

TABLE 8 Effect of enterococcus faecalis YL-EF25 addition on chicken production Performance

As can be seen from Table 8, on days 1-14, the weight gain of enterococcus faecalis YL-EF25 group was increased compared with that of the control group, the feed weight ratio was improved, and the feed weight ratio between the enteritis group and the antibiotic group was not significantly different from that of the control group, which indicates that the addition of enterococcus faecalis YL-EF25 in the daily ration is beneficial to the growth and development of chickens.

On day 15, all but the control groups were inoculated with 2×10 ⁴ chicken coccidian oocysts by oral administration. On days 21 to 24, all but the control groups induced necrotic enteritis in chickens by oral daily challenge with clostridium perfringens (4 x 10 ⁸ CFU/mL). All groups of chickens were scored for lesion observations on day 25 and sampled. The scoring criteria were 0 score (no obvious injury), 1 score (thinning, embrittling, peeling off of white pseudo film, bleeding spot of intestinal wall), 2 scores (1-5 necrosis foci), 3 scores (6-15 necrosis foci), 4 scores (more than 15 necrosis foci), 5 scores (flaky necrosis of 2-3cm length). The results are shown in Table 9.

TABLE 9 Effect of enterococcus faecalis YL-EF25 addition on intestinal lesions after toxicity attack by necrotic enteritis in chickens

Note that the superscript lower case letter indicates that the difference is significant (P < 0.05), and the superscript letter indicates that the difference is not significant (P > 0.05).

As can be seen from table 9, the difference in intestinal pathology was significantly increased in the enteritis group (P < 0.05) compared to the control group, and the intestinal pathology scores were significantly decreased in the antibiotic group and YL-EF25 group (P < 0.05) compared to the enteritis group.

As can be seen from fig. 7, the relative abundance of lactic acid bacteria in the intestinal flora of the enteritis group and the antibiotic group is decreased after challenge, the relative abundance of bacteria of other genera is increased, showing that the balance of intestinal flora is destroyed, while the relative abundance of enterococcus is increased, the relative abundance of beneficial bacteria lactobacillus salivarius is increased, and the other flora is relatively stable in the YL-EF25 group, showing a balance effect on intestinal flora disorder after challenge, compared to the control group.

Lymphocyte proliferation assay lymphocyte proliferation and differentiation is an important stage in the immune response process of the body. Thus, detection of lymphocyte proliferation levels is a common method of cellular immune studies and clinical immune function detection. First, peripheral blood lymphocytes of 5 chickens each of the control group (NC group) and YL-EF25 group (e.f25 group) were isolated, and the isolated cells were counted. The lymphocytes of each group were plated at 1X 10 ⁶/well, proliferation of cells was stimulated with the addition of Canavalia gladiata protein at a final concentration of 10. Mu.g/ml, as a test group, and a control group without Canavalia gladiata protein stimulation was set. Blank wells (blank; PBS+CCK8) were set up and stimulated for 48 hours in the cell incubator, and then CCK8 was added to measure absorbance, and the lymphocyte proliferation index conversion formula was (OD _Test-OD_{Blank space )}/(OD_Control-OD_{Blank space}). The average values of OD _Test and OD _Control in NC and e.f25 groups were detected as 0.40, 0.51 and 0.37, 0.4, respectively, and the average value of OD _{Blank space} in blank group was 0.34. From the calculation, as shown in fig. 8, the proliferation capacity of the peripheral blood lymphocytes of the chickens in the YL-EF25 group (E.F25) is obviously higher than that of the chickens in the control group (P < 0.05), which indicates that the addition of enterococcus faecalis YL-EF25 in daily ration is helpful for maintaining the balance state of the immune system, and more immune cells can be generated more quickly when pathogens invade, so that the vaccination effect is improved.

As can be seen from FIG. 9, the relative expression levels of the mRNAs of the zonal proteins OLCN and ZO-1 in the enteritis group (PC group) were down-regulated, the relative expression levels of the mRNAs of the Myosin Light Chain Kinase (MLCK) were up-regulated, the down-regulation of the zonal protein OLCN was reversed but the reversal of the down-regulation of the zonal protein ZO-1 was not obvious, the relative expression levels of the mRNAs of the Myosin Light Chain Kinase (MLCK) were up-regulated, the relative expression levels of the mRNAs of the zonal proteins OLCN and ZO-1 in the YL-EF25 group (E.F25 group) were up-regulated, and the relative expression levels of the mRNAs of the Myosin Light Chain Kinase (MLCK) in the YL-EF25 group (E.F25 group) were down-regulated, as compared with the control group (NC group). As shown in FIG. 10, the relative expression level of mRNA of inflammatory cytokines (TNF-alpha, IFN-gamma, IL-10) was significantly increased in the enteritis group (PC group) compared to the control group (NC group), and the relative expression level of mRNA of inflammatory cytokines (TNF-alpha, IFN-gamma, IL-10) was decreased in the antibiotic group (PT group) and YL-EF25 group (E.F25 group) compared to the enteritis group (PC group), wherein the YL-EF25 group (E.F25 group) was significantly decreased (P < 0.05), indicating that the E.faecalis YL-EF25 added in daily ration showed a stronger anti-inflammatory effect and an improving effect on intestinal barrier function after necrotic enteritis challenge in chickens.

According to analysis, the isolated enterococcus faecalis YL-EF25 has good probiotics characteristics and can inhibit the growth of clostridium perfringens in vitro. The enterococcus faecalis YL-EF25 is added into daily ration of the broiler chickens, so that the growth and development can be effectively improved, the intestinal barrier is enhanced, the stable intestinal flora is balanced, the necrotic enteritis induced by coccidium and clostridium perfringens (Clostridium perfringens) is obviously improved in the modes of relieving inflammatory reaction and the like, and the composition can be used for resisting clostridium perfringens infection in livestock and poultry cultivation.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The enterococcus faecalis (Enterococcus faecalis) YL-EF25 is characterized in that the enterococcus faecalis YL-EF25 is preserved in China center for type culture Collection (China center for type culture Collection), the preservation address is China WU, the preservation number is CCTCC NO: M20242269, and the classification is Enterococcus faecalis.

2. Use of enterococcus faecalis (Enterococcus faecalis) YL-EF25 according to claim 1 for the preparation of a product against clostridium perfringens infection.

3. The use of enterococcus faecalis (Enterococcus faecalis) YL-EF25 according to claim 1 for the preparation of a product for the control of necrotic enteritis in poultry, said poultry being laying hen or broiler chicken.

4. Use according to claim 3, wherein the avian necrotic enteritis is induced by clostridium perfringens.

5. The use according to claim 3, wherein the avian necrotic enteritis is co-induced by clostridium perfringens and coccidium.

6. A use according to any one of claims 2 or 3, wherein the product is selected from any one of a medicament, a feed or a feed additive.

7. The use according to claim 6, wherein the product is a feed and is prepared by inoculating enterococcus faecalis (Enterococcus faecalis) YL-EF25 in a culture medium for fermentation and spraying the fermentation broth onto the feed for mixing.

8. The use according to claim 7, wherein the feed comprisesEnterococcus faecalis (Enterococcus faecalis) YL-EF25.

9. A microbial agent comprising the enterococcus faecalis (Enterococcus faecalis) YL-EF25 according to claim 1 as an active ingredient.