A strain of Enterococcus faecalis YL-EF32 and its application

Abstract

The present invention discloses a strain of Enterococcus faecalis YL‑EF32 and an application thereof, and belongs to the field of microbial technology. The deposit number of the Enterococcus faecalis YL‑EF32 provided by the present invention is CCTCC NO: M 20242241. The strain can effectively inhibit the proliferation of Clostridium perfringens from chickens, has good tolerance to bile salts, artificial gastric juice, and phenol, and can metabolize dietary fibers such as pectin, glucan, and cellobiose. Adding Enterococcus faecalis YL‑EF32 to broiler diets can significantly improve necrotic enteritis induced by coccidia and Clostridium perfringens by enhancing the intestinal barrier, balancing the stability of intestinal flora, and alleviating inflammatory responses, and can be used to resist Clostridium perfringens infection in livestock and poultry farming.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a strain of Enterococcus faecalis YL-EF32 and an application thereof. Background Art

[0002] Clostridium perfringens ( Clostridium perfringens ) is a Gram-positive anaerobic bacterium that can produce spores and is widely found in the natural environment, including soil, animal intestines and decaying organic matter. Clostridium perfringens can produce a variety of toxins, which can damage the intestinal barrier and induce intestinal inflammation, leading to severe intestinal damage and symptoms of necrotic enteritis. Necrotic enteritis is common in chickens in broiler and laying hen farms, seriously affecting the breeding efficiency. At present, the use of antibiotics to prevent and treat necrotic enteritis in chickens has a good effect, but long-term use of antibiotics will lead to the development of drug resistance, which will affect the treatment effect. The abuse of antibiotics may also cause intestinal microbial imbalance and induce other diseases. In order to deal with the problem of antibiotic resistance, alternative antibiotic strategies have received increasing attention. Studies have found that certain probiotics can regulate the balance of intestinal microorganisms, inhibit the growth of pathogenic bacteria, and improve intestinal health. At present, there is a lack of effective treatment for necrotic enteritis in chickens caused by Clostridium perfringens, the traditional use of antibiotics is limited, and new alternative therapies are still in the exploratory stage.

[0003] Probiotic enterococci are widely present in the intestines of healthy chickens and have multiple probiotic effects. Among them, Enterococcus faecalis can inhibit the growth of Clostridium perfringens in the intestine through competitive inhibition and the production of antibacterial substances, promote the growth of beneficial bacteria, and maintain the ecological balance of intestinal microorganisms. Enterococcus faecalis can also help enhance intestinal barrier function, reduce intestinal inflammatory response, and improve intestinal health by promoting the proliferation and repair of intestinal epithelial cells. By introducing Enterococcus faecalis, it is possible to effectively replace or reduce dependence on traditional antibiotics and reduce the risk of the development of antibiotic resistance. As a natural microbial preparation, chicken-derived Enterococcus faecalis is relatively safe, does not significantly interfere with the endogenous microbiome of chickens, and is not prone to toxic side effects. This makes it more sustainable in poultry farming.

[0004] Although Enterococcus faecalis has good probiotic effects, there are still differences in the antibacterial ability and biochemical characteristics of different strains. For example, Enterococcus faecalis LD33 did not show obvious inhibitory effect on the growth of Salmonella and Shigella, but showed strong inhibitory ability on Listeria and Staphylococcus aureus. Enterococcus faecalis NPL-493 can not only inhibit the growth of Escherichia coli and Salmonella, but has no inhibitory effect on Staphylococcus aureus. Therefore, screening for better probiotic strains for the prevention and treatment of necrotic enteritis in chickens in nature is still a technical problem that needs to be solved urgently in this field. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a strain of Enterococcus faecalis ( Enterococcus faecalis )YL-EF32 and its application. The strain can effectively inhibit the chicken-derived Clostridium perfringens ( Clostridium perfringens ) and has good tolerance to bile salts, artificial gastric juice, and phenol, and can metabolize dietary fiber such as pectin, glucan, and cellobiose. Adding YL-EF32 bacteria to broiler diets significantly improves necrotic enteritis induced by coccidia and Clostridium perfringens by strengthening the intestinal barrier, balancing the stability of intestinal flora, and alleviating inflammatory responses. It can be used to resist Clostridium perfringens infection in livestock and poultry farming.

[0006] In order to achieve the above technical objectives, the technical solution implemented by the present invention is:

[0007] The present invention provides a strain of Enterococcus faecalis YL-EF32, which was deposited in China Center for Type Culture Collection on October 17, 2024, with a deposit address of Wuhan, China, a deposit number of CCTCC NO: M 20242241, and a classification name of Enterococcus faecalis .

[0008] The present invention also provides the use of the Enterococcus faecalis YL-EF32 in preparing products for resisting Clostridium perfringens infection.

[0009] The present invention also provides the use of the Enterococcus faecalis YL-EF32 in preparing products for preventing and treating necrotic enteritis in poultry.

[0010] Preferably, the necrotic enteritis in poultry is induced by Clostridium perfringens.

[0011] Furthermore, the poultry necrotic enteritis is induced by Clostridium perfringens and coccidia.

[0012] Furthermore, the poultry is laying hens or broiler chickens.

[0013] Preferably, the product is selected from any one of medicine, feed or feed additive.

[0014] Furthermore, the product is feed, and its preparation method is that Enterococcus faecalis YL-EF32 is inoculated into a culture medium for fermentation, and the fermentation liquid is sprayed onto the feed for mixing.

[0015] In one embodiment of the present invention, the feed contains Enterococcus faecalis YL-EF32.

[0016] The present invention also provides a microbial agent, which uses the Enterococcus faecalis YL-EF32 as an active ingredient.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) The present invention obtains a strain of Enterococcus faecalis YL-EF32 having strong inhibitory activity against Clostridium perfringens by isolating, purifying, identifying and analyzing the inhibitory ability of the feces of healthy wild birds with strong disease resistance. The strain has a strong inhibitory effect on the biofilm formed by Clostridium perfringens.

[0019] (2) The Enterococcus faecalis YL-EF32 provided by the present invention has a strong tolerance to bile salts, artificial gastric juice, and phenol, and can metabolize dietary fiber such as pectin, glucan, and cellobiose to help maintain intestinal health; it can significantly inhibit the proliferation activity of Clostridium perfringens in vitro. Therefore, the Enterococcus faecalis YL-EF32 can be used to prepare products for resisting Clostridium perfringens infection.

[0020] (3) Using the Enterococcus faecalis YL-EF32 provided by the present invention as a feed additive to feed broilers can significantly reduce intestinal inflammatory response, support the balance of intestinal flora, and improve intestinal barrier function, thereby improving the characteristic symptoms of necrotic enteritis in chickens induced by Clostridium perfringens. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0022] Figure 1 These are microscopic images of Enterococcus faecalis YL-EF32, YL-EF7 and YL-EF28 in Example 1 of the present invention.

[0023] Figure 2 This is a graph showing the hemolytic activity of Enterococcus faecalis YL-EF32, YL-EF7 and YL-EF28 in Example 2 of the present invention.

[0024] Figure 3 This is a graph showing the growth characteristics of Enterococcus faecalis YL-EF32, YL-EF7 and YL-EF28 in Example 2 of the present invention.

[0025] Figure 4 This is a graph showing the acid production capacity of Enterococcus faecalis YL-EF32, YL-EF7 and YL-EF28 in Example 2 of the present invention.

[0026] Figure 5 This is a graph of the co-culture inhibition test of Enterococcus faecalis YL-EF32, YL-EF7 and YL-EF28 with Clostridium perfringens in Example 2 of the present invention.

[0027] Figure 6This is a graph showing the inhibition test of Clostridium perfringens biofilm on Enterococcus faecalis YL-EF32, YL-EF7 and YL-EF28 in Example 2 of the present invention.

[0028] Figure 7 This is a graph of the relative abundance of Lactobacillus in the intestinal flora of chickens after necrotic enteritis infection in Example 3 of the present invention.

[0029] Figure 8 This is a graph showing the relative expression levels of mRNA of tight junction proteins in chickens challenged with necrotic enteritis in Example 3 of the present invention.

[0030] Fig. 9 This is a graph of the relative expression levels of mRNA of inflammatory cytokines in chickens after necrotic enteritis infection in Example 3 of the present invention.

[0031] Fig.10 This is a diagram showing the results of the lymphocyte proliferation test in Example 3 of the present invention. DETAILED DESCRIPTION

[0032] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0033] Blister meat culture medium: Weigh 48.0 g of cooked meat culture medium base (Haibo Biological, Qingdao), heat and dissolve in 1000 mL of distilled water. Take a few pieces of cooked beef cubes (Haibo Biological, Qingdao) and add them to a test tube about 1 cm high, autoclave at 121℃ for 15 minutes, and set aside.

[0034] MRS medium: Weigh 52.24 g of MRS broth (Haibo Biotechnology, Qingdao), dissolve it in 1000 mL of distilled water, sterilize it by high pressure at 118°C for 15 min, and set aside.

[0035] Example 1 Screening and identification of Enterococcus faecalis YL-EF32

[0036] Several strains of bacteria were isolated from the feces of healthy and disease-resistant wild birds. The inhibitory ability of these strains against Clostridium perfringens was tested on blood plates by the Oxford cup method. Finally, three strains of Enterococcus faecalis were selected. The colony morphology was white, smooth and round colonies. Figure 1 .

[0037] Antagonistic ability test of Clostridium perfringens: Clostridium perfringens and 33 isolated strains were cultured anaerobically in blister medium and MRS medium at 37 °C for 24 hours. The lactic acid bacteria fermentation broth was collected aseptically and placed in a 1 mL centrifuge tube. Centrifuged at 12000r / min for 5 min, the supernatant was taken, and sterilized by 0.22 μm filter membrane for later use. The Clostridium perfringens broth was diluted with sterile PBS to an optical density of OD 600 =0.8. Under a sterile environment, 100 μL of the diluted Clostridium perfringens solution was evenly spread on the surface of a blood plate containing 5% sheep blood, and then the Oxford cup was placed vertically on the blood plate, 200 μL of the fermentation supernatant of the isolated strain was added to the Oxford cup, and the cup was placed in an anaerobic workstation at 37 °C for 48 hours. The diameter of the inhibition zone was measured, and the results are shown in Table 1.

[0038] Table 1 Measurement of antibacterial activity of isolated lactic acid bacteria against Clostridium perfringens

[0039]

[0040] +: low antibacterial activity; ++: relatively strong antibacterial activity; +++: strong antibacterial activity; -: no inhibition zone.

[0041] Purification and enrichment of colonies: Use the plate streak method to pick a single colony on an MRS medium plate and invert it for culture at 37°C for one day. Continue to pick a single colony on the MRS plate and repeat the purification 2-3 times. Pick a single colony in a 5 mL MRS medium tube and culture it in a shaking incubator at 37°C for two days. After sufficient enrichment, perform 16S rDNA identification. Add glycerol to the remaining bacterial solution so that the final glycerol concentration in the bacterial solution is in the range of 15%-25%, and store it in a -80°C refrigerator.

[0042] 16S rDNA identification: The genomic DNA of the bacterial solution was extracted, and the bacterial universal sequencing primer 27F was used for colony PCR amplification and 16S rDNA sequencing identification. The obtained 16S sequence was compared and analyzed in the NCBI database. The comparison results showed that the sequence similarity with Enterococcus faecalis was 100%. It was determined that these three strains were all Enterococcus faecalis ( Enterococcus faecalis ), and formally named them as YL-EF7, YL-EF28, and YL-EF32. The complete 16S rDNA sequences of the above-mentioned Enterococcus faecalis YL-EF7, YL-EF28, and YL-EF32 are shown in the sequence table SEQ ID NO.1.

[0043]

[0044] Example 2 Enterococcus faecalis ( Enterococcus faecalis ) characteristic detection

[0045] Hemolytic test: Aseptically take the Enterococcus faecalis liquid and streak it on the blood plate, incubate it anaerobically for 24 hours, and the hemolytic activity is Figure 2 ,In the figure, 7, 32 and 28 are YL-EF7, YL-EF32 and YL-EF28 respectively.

[0046] Growth curve and acid production curve: Streak and activate Enterococcus faecalis on MRS plates, inoculate single colonies in 5 mL MRS medium tubes and culture overnight in a shaker at 37 °C, inoculate Enterococcus faecalis culture at a ratio of 1:100 in 200 mL MRS medium and culture in a shaker at 37 °C, take 5 mL at the 2nd hour, 4th hour and every 6 hours to measure pH and OD of the culture 600 Value, MRS liquid culture medium as blank control, by Figure 3 Figure 4 It can be seen that the growth characteristics and acid production capacity of the three strains of Enterococcus faecalis are basically the same. They enter the logarithmic growth phase from the second hour, and enter the stable phase around the sixth hour. Until the 36th hour, the OD 600 The value has been maintained between 1.2-1.3. The acid production capacity test found that the pH value of the three strains dropped to 4.8 after the 6th hour of culture. When entering the plateau period (12-18 hours), the pH value of the bacterial solution became about 4.6, and at the 24th-36th hour, the pH value of the bacterial solution dropped to about 4.4. Figure 3 and Figure 4 Among them, E.F7, E.F32 and E.F28 are YL-EF7, YL-EF32 and YL-EF28 respectively.

[0047] Artificial gastric juice resistance and bile salt resistance test: Enterococcus faecalis was streaked on an MRS plate for activation, and a single colony was inoculated into a 5 mL MRS medium tube and cultured overnight at 37 °C in a shaker. The Enterococcus faecalis culture solution was inoculated into 200 mL MRS medium at a ratio of 1:100 and cultured at 37 °C in a shaker. The resulting culture solution was adjusted to a concentration of OD 600 The lower is 0.8 (about 10 8). Take 1 mL of the adjusted concentration of Enterococcus faecalis into EP tubes and centrifuge at 5000 rpm for 10 minutes. During the centrifugation, prepare bile salts (0.2%, 0.25%, dissolved in PBS) and artificial gastric fluid MRS culture medium tubes (artificial gastric fluid: 0.32% pepsin, 1.6μg / ml lysozyme, pH=2), and take 1 mL of each into EP tubes for later use. Discard the supernatant of the culture medium after centrifugation, resuspend the bacterial precipitate with 1 mL of bile salt solution and artificial gastric fluid MRS culture medium under the corresponding conditions, incubate in a 37 ℃ incubator for 3 hours, and use PBS to gradiently dilute the incubated strains to 10 -9 , drop the plate and count in the corresponding area. Dry the plate on the clean bench, and then place it in the incubator for culture. At the same time, use PBS to dilute the three bacterial strains to 10 -9 , plate 100 μL of each gradient dilution onto an MRS plate. Dry the plate on a clean bench, and then place it in an incubator for overnight culture to calculate the initial number of viable bacteria. Record the data, calculate the mean, standard deviation, and survival rate, see Table 2, Table 3.

[0048] Table 2 Bile salt tolerance test

[0049]

[0050] Table 3 Artificial gastric fluid resistance test

[0051]

[0052] Phenol resistance test: Enterococcus faecalis was streaked and activated on an MRS plate, and a single colony was inoculated into a 5 mL MRS medium tube and cultured overnight at 37°C in a shaker as the strain. MRS medium tubes containing 0.1%, 0.2%, 0.3%, and 0.4% phenol were prepared, and the strain was added to the MRS tubes with the corresponding phenol concentration at a ratio of 1:100, and cultured at 37°C and 220rpm for 24 hours. The OD of each group of bacterial liquid was measured in 3 replicates per group. 600 Values, MRS liquid culture medium was used as blank control, see Table 4.

[0053] Table 4 Artificial phenol resistance test

[0054]

[0055] Antibiotic resistance test: Enterococcus faecalis was streaked on an MRS plate for activation, and a single colony was inoculated into a 5 mL MRS medium tube and cultured overnight at 37 °C in a shaker. The resulting bacterial solution was adjusted to a concentration of OD 600 The lower is 0.8 (about 10 8). Spread 100 μL of the diluted bacterial solution on the surface of the MRS medium plate, stick the antibiotic tablet on the MRS plate, place it in a bacterial incubator at 37 ℃ for 24 hours, and measure the diameter of the inhibition zone, see Table 5.

[0056] Table 5 Common antibiotic resistance tests

[0057]

[0058] Biofilm inhibition test of Clostridium perfringens: Streak and activate Enterococcus faecalis on MRS plates, inoculate single colonies in 5 mL MRS medium tubes, and culture overnight at 37 °C in a shaker. Inoculate Clostridium perfringens in TSB medium at a ratio of 1:100, and culture overnight at 37 °C in an anaerobic workstation. Prepare the cultured Clostridium perfringens (CP) and Enterococcus faecalis (EF) solutions, and filter the supernatant of Enterococcus faecalis after centrifugation (4000 rpm, 5 minutes) through a 0.22 μm filter membrane to obtain the supernatant of Enterococcus faecalis (EF supernatant). Dilute the Clostridium perfringens and Enterococcus faecalis solutions with TSB medium until the concentration of the solutions is between OD 600 The concentration of the above reagents was 0.1, and the mixture was mixed in a 96-well plate according to the following grouping (Table 6), with 6 replicates in each group. The 96-well plate was sealed with a sealing film to isolate oxygen, and the plates were incubated at room temperature for 5 days.

[0059] Table 6 Grouping of Clostridium perfringens biofilm inhibition test

[0060]

[0061] Note: In Table 6, CP stands for Clostridium perfringens, and EF stands for YL-EF32, YL-EF7 or YL-EF28.

[0062] After the culture was completed, the suspension in each well was transferred and the supernatant OD was measured. 600 The suspension of each group was diluted to 10 with sterile PBS. -9 , drop plate count on TSN medium plate. Gently rinse the culture wells after transferring the supernatant twice with PBS, add 300 μL of crystal violet stain and leave at room temperature for 30 minutes. After removing the crystal violet, gently rinse twice with PBS again. Add 300 μL of methanol solution to each well and incubate at room temperature for 30 minutes. Transfer the liquid in each well to a new 96-well plate and measure the OD of each well 570 Value, denoted as A 570 . Calculate the relative biofilm measurement value = A 570 / OD 600 .Depend on Figure 6 It can be seen that the three strains of Enterococcus faecalis and the supernatant of sterile Enterococcus faecalis all inhibited the biofilm formation of Clostridium perfringens. Figure 5 Figure 6 The analysis showed that the ability of the three strains to inhibit Clostridium perfringens activity was ranked from strong to weak as follows: YL-EF32, YL-EF7, and YL-EF28. YL-EF32, which had the strongest activity in inhibiting the biofilm protection formed by Clostridium perfringens, was selected for animal experiments.

[0063] Microbial metabolism analysis: Enterococcus faecalis was streaked and activated in an MRS plate, and a single colony was streaked and activated again in an MRS plate. According to the method provided by the automatic microbial identification system of BIOLOG, the activated colony was picked and placed in 200 μL IF-B medium to make an inoculum. The inoculum was used to adjust the IF-B medium (BIOLOG, USA) to an appropriate transmittance to make a bacterial suspension. 100 μL of bacterial suspension was added to each well of the GEN III plate and placed in a microbial metabolism identification incubator for 36 hours. The data was analyzed. As shown in Table 7, YL-EF32 showed the typical metabolic characteristics of Enterococcus faecalis strains, confirming that Enterococcus faecalis YL-EF32 has a strong metabolic capacity for dietary fibers such as glucan, cellobiose, and pectin, thereby producing short-chain fatty acids and promoting intestinal health.

[0064] Table 7 Carbon source metabolism analysis

[0065]

[0066] +: metabolic activity positive, -: metabolic activity negative, w + : Metabolic activity is weakly positive.

[0067] Example 3 Protective effect of Enterococcus faecalis YL-EF32 on necrotic enteritis in chickens

[0068] One-day-old broilers with similar body weight were randomly divided into four treatment groups, with 18 chickens in each group. (1) The control group was fed a basal diet (NC group); (2) the necrotic enteritis group was challenged with coccidia and Clostridium perfringens (4×10 8 CFU / mL), fed with basal diet (PC group); (3) antibiotic group challenged with coccidia and Clostridium perfringens (4×10 8 CFU / mL), and fed a basal diet containing 20 mg / kg of enramycin (PT group); (4) YL-EF32 experimental group challenged with coccidia and Clostridium perfringens (4×10 8 CFU / mL), fed with 1×10 9 CFU / kg of Enterococcus faecalis YL-EF32 in the basic diet (E.F32 group). The experimental period was 25 days.

[0069] In the present embodiment, the NC group represents the control group, the PC group represents the enteritis group, the PT group represents the antibiotic group, and the E.F32 group represents the YL-EF32 group.

[0070] On the 15th day, all groups except the control group were orally inoculated with 2×10 4 From day 21 to day 24, all groups except the control group were challenged with Clostridium perfringens (4×10 8 CFU / mL) was used to induce necrotic enteritis in chickens. On the 25th day, the lesions of all groups of chickens were observed and scored, and samples were taken. The scoring criteria were: 0 points (no obvious damage); 1 point (the intestinal wall became thinner and brittle, the white pseudomembrane fell off, and there were bleeding spots on the intestinal wall); 2 points (1-5 necrotic foci); 3 points (6-15 necrotic foci); 4 points (more than 15 necrotic foci); 5 points (2-3 cm long flake necrosis), as summarized in Table 8.

[0071] Table 8 Effect of adding Enterococcus faecalis YL-EF32 on intestinal lesions in chickens challenged with necrotic enteritis

[0072]

[0073] Note: Different superscript lowercase letters indicate significant differences (P<0.05), while the same superscript letters indicate no significant differences (P>0.05).

[0074] As shown in Table 8, compared with the control group, the difference in intestinal pathological changes in the enteritis group was significantly increased (P < 0.05). Compared with the enteritis group, the intestinal lesion scores in the antibiotic group and the YL-EF32 group were reduced, among which the difference in intestinal pathological changes in the antibiotic group and the YL-EF32 group was significantly reduced (P < 0.05).

[0075] Table 9 Effect of adding Enterococcus faecalis YL-EF32 on chicken production performance

[0076]

[0077] As shown in Table 9, on days 1-14, compared with the control group, the weight gain of the Enterococcus faecalis YL-EF32 group increased and the feed-to-weight ratio was improved, while there was no significant difference in the feed-to-weight ratio between the enteritis group and the antibiotic group and the control group, indicating that adding Enterococcus faecalis YL-EF32 to the diet can promote growth and development.

[0078] Depend on Figure 7 It can be seen that compared with the control group, after the virus attack, the relative abundance of Lactobacillus in the intestinal flora of the enteritis group and the antibiotic group decreased, and the relative abundance of bacteria of other genera increased, indicating that the balance of the intestinal flora was destroyed. In the YL-EF32 group, the relative abundance of Enterococcus increased, the relative abundance of the beneficial bacteria Lactobacillus salivarius increased, and other flora were relatively stable, showing a balancing effect on the disorder of intestinal flora after the virus attack.

[0079] Depend on Figure 8It can be seen that compared with the control group (NC group), the relative mRNA expression levels of tight junction proteins OLCN and ZO-1 in the enteritis group (PC group) were downregulated, and the relative mRNA expression level of myosin light chain kinase (MLCK) was upregulated; the antibiotic group (PT group) could reverse the downregulation of tight junction protein OLCN, but the reversal of the downregulation of tight junction protein ZO-1 was not obvious, and the relative mRNA expression level of myosin light chain kinase (MLCK) was upregulated; the relative mRNA expression levels of tight junction proteins OLCN and ZO-1 in the YL-EF32 group (E.F32 group) were upregulated, among which the relative expression level of OLCN in the YL-EF32 group (E.F32 group) was significantly higher than that in the enteritis group (P < 0.05), and the relative mRNA expression level of myosin light chain kinase (MLCK) in the YL-EF32 group was downregulated. Fig. 9 As shown in the data, compared with the control group (NC group), the relative mRNA expression levels of inflammatory cytokines (TNF-α, IFN-γ, IL-10) in the enteritis group (PC group) were significantly increased; compared with the enteritis group (PC group), the relative mRNA expression levels of inflammatory cytokines (TNF-α, IFN-γ, IL-10) in the antibiotic group (PT group) and the YL-EF32 group (E.F32 group) were decreased, among which the reduction effect of the YL-EF32 group (E.F32 group) was more significant (P < 0.05), indicating that the addition of Enterococcus faecalis YL-EF32 to the diet showed a strong anti-inflammatory effect and improvement of intestinal barrier function after necrotic enteritis in chickens.

[0080] Lymphocyte proliferation test: Peripheral blood lymphocytes of 5 chickens in the control group (NC group) and YL-EF32 group (E.F32 group) were isolated and counted. 1×10 lymphocytes in each group were counted. 6 / well plate, add concanavalin to stimulate cell proliferation, the final concentration is 10 μg / ml, as the experimental group, and set up a control group without concanavalin stimulation. Set up a blank well (blank group; PBS+CCK8) in a cell culture incubator for 48 hours, add CCK8 to measure absorbance, and the lymphocyte proliferation index conversion formula is (OD 试验 -OD 空白) / (OD 对照 -OD 空白 ). OD of NC group and EF32 group was detected 试验 and OD 对照 The average values ​​were 0.40, 0.42, 0.37, 0.37, respectively. The OD of the blank group 空白 The average value is 0.34. Fig.10 It can be seen that compared with the control group, there was no significant difference in the proliferation ability of chicken peripheral blood lymphocytes in the YL-EF32 group.

[0081] After analysis, the isolated Enterococcus faecalis YL-EF32 of the present invention has good probiotic properties and can inhibit the growth of Clostridium perfringens in vitro. Adding Enterococcus faecalis YL-EF32 to broiler diets can effectively enhance the intestinal barrier, balance the stability of intestinal flora, and alleviate inflammatory responses, which significantly improves the coccidia and Clostridium perfringens ( Clostridium perfringens )-induced necrotic enteritis and can be used to prevent Clostridium perfringens infection in livestock and poultry farming.

[0082] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A strain of Enterococcus faecalis ( Enterococcus faecalis )YL-EF32, characterized by, The Enterococcus faecalis YL-EF32 was deposited in the China Center for Type Culture Collection on October 17, 2024, with the deposit address in Wuhan, China, with the deposit number being CCTCC NO: M 20242241, and the classification name being Enterococcus faecalis .

2. The Enterococcus faecalis according to claim 1 ( Enterococcus faecalis ) Application of YL-EF32 in the preparation of products for resisting Clostridium perfringens infection.

3. The Enterococcus faecalis according to claim 1 ( Enterococcus faecalis ) Use of YL-EF32 in the preparation of products for preventing and treating necrotic enteritis in poultry; the poultry are laying hens or broilers.

4. The use according to claim 3, characterized in that: The necrotic enteritis in birds is induced by Clostridium perfringens.

5. The use according to claim 3, characterized in that: The necrotic enteritis in poultry is induced by Clostridium perfringens and coccidia.

6. The use according to any one of claims 2 or 3, characterized in that: The product is selected from any one of medicine, feed or feed additive.

7. The use according to claim 6, characterized in that: The product is a feed, and the preparation method thereof is as follows: Enterococcus faecalis ( Enterococcus faecalis )YL-EF32 is inoculated into the culture medium for fermentation, and the fermentation liquid is sprayed onto the feed for mixing.

8. The use according to claim 7, characterized in that: The feed contains Enterococcus faecalis ( Enterococcus faecalis )YL-EF32.

9. A microbial agent, characterized in that: The Enterococcus faecalis according to claim 1 ( Enterococcus faecalis )YL-EF32 is the active ingredient.

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