Lactobacillus amyloliquefaciens and probiotic agent and application thereof

Abstract

The invention discloses a lactobacillus amyloliquefaciens and probiotic microbial inoculum and application of the lactobacillus amyloliquefaciens and probiotic microbial inoculum. The lactobacillus amyloporus is named as lactobacillus amyloporus YI39, and the preservation number of the lactobacillus amyloporus YI39 in the China Center for Type Culture Collection is CCTCC (China Center for Type Culture Collection) NO: M2026104; the nucleotide sequence of 16SrDNA (deoxyribonucleic acid) of the gene is SEQ ID NO. 1. The lactobacillus amyloliquefaciens YI39 provided by the invention is separated from ileum contents of 28-day-old healthy and rapid yellow-feathered broilers, has strong probiotic characteristics, and has relatively good capabilities of inhibiting growth of salmonella pullorum, avian pathogenic escherichia coli, A-type clostridium perfringens and staphylococcus aureus; the body of the broiler chicken can be effectively regulated and controlled, so that the chicken necrotic enteritis caused by clostridium perfringens infection is prevented.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a Lactobacillus amyloliquefaciens and probiotic preparation and their applications. Background Technology

[0002] Poultry farming is an important part of my country's animal husbandry industry. Necrotic enteritis (NE), caused by Clostridium perfringens, is a common acute infectious disease that causes huge economic losses to the global poultry industry. This disease primarily affects the small intestine of chickens, leading to necrosis of the intestinal mucosa and severely impacting the growth performance and health of the birds.

[0003] For a long time, adding antibiotics to feed has been the main method for preventing and controlling necrotic enteritis in chickens. However, with the global restrictions and bans on antibiotic growth promoters in feed, the incidence of necrotic enteritis in chickens has rebounded, posing new challenges to its prevention and control. Therefore, developing safe and effective antibiotic alternatives, such as probiotics, has become a current research hotspot.

[0004] Probiotics, especially lactobacilli, are considered strong candidates for the prevention and control of intestinal diseases due to their ability to regulate gut microbiota balance, enhance intestinal barrier function, and modulate immune responses. Studies have shown that certain lactobacillus strains can alleviate intestinal damage caused by necrotizing colitis by competitively excluding pathogens, producing antibacterial substances, and regulating the expression of inflammatory factors.

[0005] For example, CN117448213A discloses a *Lactobacillus plantarum* strain that inhibits *Clostridium perfringens*, its postbiotic, and its applications. The *Lactobacillus plantarum* strain is named *Lactobacillus plantarum* BLCC2-0072, and was deposited at the China Center for Type Culture Collection on May 29, 2023, with accession number CCTCC NO: M2023842. This bacterium exhibits good antibacterial activity, especially excellent inhibitory effects against various *Clostridium perfringens* species.

[0006] Although the probiotic effects of lactobacilli are widely recognized, significant differences exist in the probiotic properties of different species and strains. While *Lactobacillus amylovorus* is a potential probiotic, its application in preventing necrotic enteritis in chickens has not been reported. Therefore, this invention aims to provide a novel *Lactobacillus amylovorus* strain that can effectively inhibit *Clostridium perfringens* and enhance the infection resistance of broilers. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a Lactobacillus amyloliquefaciens and probiotic agent and their application, which have strong probiotic properties, good antibacterial activity, and can effectively regulate the body of broilers to prevent necrotizing enteritis caused by Clostridium perfringens infection.

[0008] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a Lactobacillus amylovorus, named Lactobacillus amylovorus YI39, with accession number CCTCCNO: M2026104 at the China Center for Type Culture Collection; and its 16S rDNA nucleotide sequence is SEQ ID NO.1.

[0010] Secondly, the present invention provides a probiotic preparation, characterized in that the probiotic preparation contains Lactobacillus amyloliquefaciens as described above.

[0011] Preferably, the live bacteria concentration of *Lactobacillus amyloliquefaciens* in the probiotic preparation is 3 × 10⁻⁶. 8 CFU / kg ~ 3×10 8 CFU / kg.

[0012] Furthermore, the probiotic agent also includes excipients, namely maltodextrin.

[0013] Furthermore, probiotic preparations can be in the form of liquid, powder, or granules.

[0014] Furthermore, the probiotic preparation is in the form of lyophilized powder.

[0015] Thirdly, the present invention also provides the use of the Lactobacillus amyloliquefaciens or the probiotic agent described above in the preparation of feed or medicine for the prevention and / or treatment of necrotizing enteritis in poultry.

[0016] Furthermore, the poultry mentioned is broiler chicken.

[0017] Furthermore, necrotic enteritis is a type of necrotizing enteritis in chickens caused by Clostridium perfringens infection.

[0018] Furthermore, the feed or medicine has at least one of the following functions:

[0019] (1) Improve the average daily weight gain of broilers after challenge with Clostridium perfringens;

[0020] (2) Reduce the jejunal lesion score caused by viral challenge and increase the ratio of villus height to crypt depth (villus-crypt ratio).

[0021] (3) It simultaneously reduces the levels of diamine oxidase (DAO) and interleukin-1β (IL-1β) in serum and upregulates the mRNA expression level of the tight junction protein Claudin-1 in jejunal tissue.

[0022] (4) Antibacterial function.

[0023] Furthermore, the antibacterial function includes inhibition of at least one of Salmonella pullorum, avian pathogenic Escherichia coli, Clostridium perfringens type A, and Staphylococcus aureus.

[0024] The present invention has the following beneficial effects:

[0025] The *Lactobacillus amyloliquefaciens* YI39 provided by this invention exhibits good acid and bile salt resistance and good self-aggregation ability. It possesses strong probiotic properties and shows good inhibitory effects against *Salmonella pullorum*, pathogenic *Escherichia coli*, *Clostridium perfringens* type A, and *Staphylococcus aureus*, thus enhancing the resistance of broilers to *Clostridium perfringens* infection.

[0026] Adding *Lactobacillus amyloliquefaciens* YI39 to a broiler necrotic enteritis model infected with *Clostridium perfringens* significantly improved the average daily weight gain of broilers 14-28 days after challenge, thus enhancing their production performance. It also reduced the jejunal lesion score in broilers infected with *Clostridium perfringens* and significantly increased the villous-cryptotropic ratio. Simultaneously, serum DAO and IL-1β levels decreased significantly, while the expression level of the jejunal tight junction protein Claudin-1 increased. These results indicate that *Lactobacillus amyloliquefaciens* YI39 provided by this invention can effectively alleviate the negative impact of *Clostridium perfringens* infection on broiler growth performance, reduce intestinal inflammation, and enhance intestinal barrier function, thereby effectively preventing and controlling necrotic enteritis in chickens. Attached Figure Description

[0027] Figure 1 Colony morphology of Lactobacillus amyloliquefaciens YI39.

[0028] Figure 2 Gram staining image of Lactobacillus amyloliquefaciens YI39.

[0029] Figure 3 A comparison of the acid tolerance of different Lactobacillus amyloliquefaciens strains under different pH conditions.

[0030] Figure 4 A comparative graph showing the bile salt tolerance of different strains of Lactobacillus amyloliquefaciens at different bile salt concentrations.

[0031] Figure 5 A comparison of the self-aggregation abilities of different strains of Lactobacillus amyloliquefaciens.

[0032] Figure 6A comparative diagram showing the antibacterial activity of different strains of Lactobacillus amyloliquefaciens against Escherichia coli, Clostridium perfringens, Staphylococcus aureus, and Salmonella; where: (a) is whole bacterial culture; (b) is live bacterial cells; and (c) is fermentation supernatant.

[0033] Figure 7 The image shows a comparison of the effects of Clostridium perfringens infection on the intestinal morphology of broiler chickens in different treatment groups; where A is the control group; B is the infection group; C is the low-dose YI39 group; D is the medium-dose YI39 group; E is the high-dose YI39 group; and F is the ultra-high-dose YI39 group.

[0034] Figure 8 Comparison images of the jejunum in each treatment group, where (a) shows the comparison of villus height, (b) shows the comparison of crypt depth, and (c) shows the comparison of villus-crypt ratio.

[0035] Figure 9 A comparison chart of DAO content in broiler serum from different treatment groups.

[0036] Figure 10 A comparison chart of IL-1β levels in broiler serum from different treatment groups.

[0037] Figure 11 A comparison chart of IL-6 levels in broiler serum from different treatment groups.

[0038] Figure 12 A comparison of the relative expression levels of Claudin-1 mRNA in the jejunal tissues of broilers from different treatment groups. Detailed Implementation

[0039] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0040] Unless otherwise defined, 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 pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0041] The MRS culture medium used in the following examples of the present invention has the following formulation: 10 g / L peptone, 8 g / L beef extract, 4 g / L yeast extract, 20 g / L glucose, 2 g / L dipotassium hydrogen phosphate, 2 g / L diamine hydrogen citrate, 5 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.04 g / L manganese sulfate, 1 g / L Tween 80, pH 5.8~6.2, sterilized at 121℃ for 15 min.

[0042] Example 1: Isolation and screening of Lactobacillus amyloliquefaciens YI39

[0043] 1. Strains Isolation

[0044] Take 0.5 g of ileal contents from a 28-day-old healthy fast-growing yellow-feathered broiler (Xiangjia Yellow Chicken No. 2) in a county in Changde City, Hunan Province. Place the 0.5 g fecal sample in 50 ml of sterile PBS or physiological saline to prepare a sample suspension. After vortexing and mixing, transfer the suspension to a clean bench and dilute it to different concentration gradients (generally 10). -5 and 10 -6 After vortexing, 50-100 μl of the culture medium is spread onto an agar plate and then placed in an anaerobic bag (containing anaerobic indicator and anaerobic gas generator) for inverted incubation at 37°C. After 24-48 hours of incubation, the culture dishes are removed, and colonies are picked up with a sterile pipette tip and placed into centrifuge tubes pre-filled with 1 ml of broth. These tubes are then placed in a shaker for further incubation and propagation. The cultured bacterial solution is then purified twice and sent for sequencing.

[0045] The sequencing company was Shanghai Paisenno Biotechnology Co., Ltd. Bidirectional sequencing was performed using universal primers 27F / 1492R. The base sequences were then copied onto BLAST for alignment. Sequences with a similarity of over 97% were considered to be of the same species, over 95% to the same genus, and over 80% to the same phylum.

[0046] The applicant isolated a total of 4 strains of Lactobacillus amyloliquefaciens, numbered YI39, YI46, BC15, and BC04, respectively.

[0047] 2. Strain screening

[0048] (1) Determination of acid and bile salt tolerance: Activated suspensions of four strains of Lactobacillus amyloliquefaciens were inoculated at a 5% (v / v) in MRS broth with different pH values ​​(2.0, 3.0, 4.0) and MRS broth containing different concentrations of porcine bile salts (0.1%, 0.2%, 0.3%). After incubation at 37℃ for 12 h, the acid and bile salt tolerance was measured using an enzyme-linked immunosorbent assay (ELISA) reader. Values ​​are used to assess their growth.

[0049] The results are as follows Figure 3 and Figure 4 As shown, under three conditions of pH 2.0, 3.0, and 4.0, YI39... The absorbance values ​​were significantly higher than other strains (P<0.001), indicating the strongest acid tolerance; and it maintained a high absorbance even at pH 2.0, significantly better than Y146, BC15, and BC04. As the pH increased to 4.0, the growth ability of all strains improved, but YI39 still performed best. Under different bile salt concentrations, strain YI39... The value was significantly higher than that of other strains (P<0.05); under 0.3% bile salt conditions, YI39 still maintained high growth activity ( = 0.23 ± 0.004), while the growth of BC15 and BC04 was significantly inhibited ( <0.2).

[0050] Therefore, under pH 2.0, 3.0, 4.0 and 0.1%, 0.2%, and 0.3% bile salt conditions, strain YI39... The values ​​were significantly higher than those of the other three strains (P<0.05), indicating that strain YI39 has the strongest acid and bile salt resistance.

[0051] (2) Determination of self-aggregation ability: The suspensions of 4 strains of Lactobacillus amyloliquefaciens were centrifuged (5000 r / min, 15 min), washed with PBS and resuspended, and adjusted. The value was 1.0 (denoted as A0). The bacterial suspension was incubated at 37°C, and the supernatant was collected at 3 h, 6 h, and 24 h for determination. The value (repeated 3 times) is used to calculate the self-aggregation rate. The formula is:

[0052] In the formula: Atime is the absorbance at 3h, 6h and 24h of treatment; A0 is the initial absorbance of the bacterial suspension.

[0053] The results are as follows Figure 5 As shown, after 24 h of culture, the self-aggregation rate of YI39 (94.7%±0.05%) was significantly higher than that of other strains (P<0.05), indicating that it has a strong potential for intestinal colonization.

[0054] (3) Antibacterial activity determination: Four strains of *Lactobacillus amyloliquefaciens* were fully activated and inoculated into MRS medium at a 2% inoculum. The cultures were then anaerobic at 37°C for 24 h to obtain whole bacterial suspensions. Indicator bacteria preparation: *Salmonella*, *Escherichia coli*, and *Staphylococcus aureus* were inoculated into LB broth, and *Clostridium perfringens* was inoculated into liquid thioglycolate medium. The cultures were incubated at 37°C with shaking at 200 r / min for 18 h. The bacterial concentration was adjusted to 10⁻¹⁰ with sterile PBS. 6CFU / mL. The pre-added bacterial suspension was used for plate preparation. A 1mL pipette tip was used to punch a hole in the plate, and 150μl of the test solution was added to each well. The plates were incubated at 37℃ for 18 h. The diameter of the inhibition zone was measured with calipers, and the average value was calculated. Judgment criteria: inhibition zone diameter ≥20mm - extremely sensitive; 15~20mm - highly sensitive; 10~15mm - moderately sensitive; <10mm - low sensitive or ineffective.

[0055] The results are as follows Figure 6 As shown, strain YI39 exhibited significant antibacterial activity against all four pathogens, particularly against Clostridium perfringens, where the inhibition zone diameter reached 16.23±0.60 mm, significantly superior to other strains (P<0.05). Based on the above evaluation of probiotic properties, YI39 was selected as the target strain for subsequent experiments.

[0056] Example 2 Identification and preservation of Lactobacillus amyloliquefaciens YI39

[0057] 1. Morphological identification of strains

[0058] After activating strain YI39, it was inoculated into liquid culture medium at a rate of 2% with an initial pH of 6.5. The medium was then incubated at 37°C at a rotation speed of 120 r / min for 30 h. The strain was then inoculated onto agar plates and cultured anaerobically at 37°C for 24–48 h. Colony morphology was then observed, and colonies were picked for Gram staining. Bacterial morphology was observed under a microscope and recorded.

[0059] The results showed that the colonies of strain YI39 isolated on MRS agar were round, milky white, and had a smooth surface. Figure 1 Colonies were picked and Gram-stained. Observed under an oil immersion microscope, the bacteria were rod-shaped, purple (Gram-positive), and non-spore-forming. Figure 2 ).

[0060] 2. Molecular biological identification

[0061] The purified YI39 strain was activated, and 1 ml of bacterial culture was prepared and sent to a sequencing company for sequencing. The sequencing result is shown in SEQ ID NO.1, with a length of 1440 bp.

[0062] The sequence was BLAST-aligned in the NCBI database, and the results showed that it had 100% homology with Lactobacillus amylovorus.

[0063] Based on the colony morphology, microscopic characteristics, and 16S rRNA identification of strain YI39, strain YI39 was identified as *Lactobacillus amylovorus* and named *Lactobacillus amylovorus* YI39. The applicant deposited this strain at the China Center for Type Culture Collection (CCTCC) in Wuhan, China on January 14, 2026, with accession number CCTCC NO: M2026104.

[0064] Example 3: Effect of Lactobacillus amyloliquefaciens YI39 on the resistance of broilers to Clostridium perfringens infection.

[0065] 1. Preparation of freeze-dried bacterial powder

[0066] Take the frozen Lactobacillus amyloliquefaciens YI39 and perform resuscitation and propagation (50 ml centrifuge tube, 50 ml culture medium + 0.1 ml bacterial suspension; if a large batch to bacterial powder is needed, a 500 ml Erlenmeyer flask or a 1 L Erlenmeyer flask can be used). After 24 h, centrifuge the amplified Lactobacillus amyloliquefaciens (5000 rpm for 5 min), discard the supernatant, and wash three times with sterile 1× PBS. After washing, add 10% maltodextrin (1 g maltodextrin + 9 ml PBS) at a 1:1 volume ratio to the centrifuged bacterial slurry, mix well, and freeze at -80℃ (for at least 3 hours). Open the centrifuge tube cap and freeze-dry the frozen bacteria for at least 24 h, then grind into bacterial powder, aliquot (centrifuge tubes, 1 g or 5 g / tube), and store at -80℃ for long-term storage.

[0067] 2. Animal Experiment Design: 432 healthy 1-day-old female yellow-feathered broiler chickens were randomly divided into 6 treatment groups, with 6 replicates per treatment group and 12 chickens per replicate. Among them:

[0068] Control group: fed a basal diet; Challenge group: fed a basal diet;

[0069] YI39 low-dose group: basal diet + 3×10 8 CFU / kg YI39 bacterial powder;

[0070] YI39 medium-dose group: basal diet + 6×10 8 CFU / kg YI39 bacterial powder;

[0071] YI39 high-dose group: basal diet + 9×10 8 CFU / kg YI39 bacterial powder;

[0072] YI39 ultra-high dose group: basal diet + 12×10 8 CFU / kg YI39 bacterial powder.

[0073] Except for the control group, broilers in all other groups were challenged with a 30-fold dose of the tetravalent live attenuated vaccine for fowl coccidiosis on day 14, and were orally administered 1 mL of Clostridium perfringens broth (1×10⁻⁶) daily from day 15 to 17. 9 (CFU / mL). The experiment lasted for 56 days. All chickens had free access to feed and water during the experiment.

[0074] 3. Detection indicators and results

[0075] (1) Growth performance: After the start of the experiment, detailed records were kept of the chickens. Body weight was measured at 14, 28, and 56 days of age, using replicate groups as units. Feed intake and mortality were also recorded. The average daily feed intake, average daily weight gain, and feed conversion ratio were calculated for each group of broilers at ages 1-14, 15-28, and 29-56. The calculation formulas are as follows:

[0076] Average daily feed intake (ADFI) = Total feed weight consumed / (Number of birds × Number of days).

[0077] Average daily weight gain (ADG) = Final weight of individual chicken - Initial weight of individual chicken / (Number of chickens × Number of days).

[0078] Material weight ratio (F / G) = Total material consumption / Total weight gain.

[0079] The results are shown in Table 1 below. Compared with the challenge group, all dose groups with added YI39 significantly increased the average daily weight gain of broilers aged 15-28 days (14 days after challenge) (P<0.05), indicating that YI39 can effectively alleviate the growth inhibition caused by Clostridium perfringens infection.

[0080]

[0081] (2) Intestinal injury score:

[0082] At 28 days of age, one broiler chicken with a weight close to the average weight of the replicate was randomly selected from each replicate. The lesions in the duodenum, jejunum, and ileum were observed after separation. The intestinal mucosal damage was evaluated using a 0-6 scoring system: 0 points (no obvious damage), 1 point (thin and brittle intestinal wall), 2 points (1-5 necrotic foci), 3 points (6-15 necrotic foci), 4 points (16 or more necrotic foci), 5 points (2-3 cm long patchy necrosis), and 6 points (large areas of diffuse necrosis).

[0083] The results are as follows Figure 7 , 8 As shown, the jejunum of the challenged group showed obvious necrotic lesions, with a score significantly higher than that of the control group (P<0.05); while the jejunal lesion scores of the groups with added YI39, especially the medium and high dose groups, were significantly lower than those of the challenged group (P<0.05), indicating that YI39 can effectively reduce the damage of Clostridium perfringens to the intestine.

[0084] (3) Serum immune and intestinal permeability indicators:

[0085] Serum samples stored at -80℃ were thawed on ice, and serum inflammatory factors were detected using an ELISA kit containing DAO (D-amino acid oxidase), TNF-α (α-tumor necrosis factor), IFN-γ (interferon-γ), IL-6 (interleukin-6), IL-1β (interleukin-1β), and LPS (lipopolysaccharide).

[0086] Jejunal tissue was aseptically collected from 28-day-old broiler chickens after challenge, flash-frozen in liquid nitrogen, and stored at -80°C for the detection of mRNA expression levels of inflammatory factors and intestinal barrier-related genes. Total RNA was extracted using the SteadyPure Universal RNA Extraction Kit (Hunan Aikerui Biotechnology Co., Ltd.), following the kit's instructions. The concentration and purity of the RNA were determined using a BioPhotometer nucleic acid and protein analyzer. The qualified RNA was then used to synthesize cDNA using the Evo M-MLV reverse transcription kit (Hunan Aikerui Biotechnology Co., Ltd.), following the kit's instructions. The cDNA synthesis conditions were 37°C for 15 min and 85°C for 5 s. qPCR reactions were performed strictly according to the instructions for the SYBR GreenPro Taq HS premixed qPCR kit (Hunan Aikerui Biotechnology Co., Ltd.). Primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. β-actin was used as an internal reference gene, and the 2-ΔΔCt method was used to calculate the mRNA expression level of the target gene relative to the internal reference gene β-actin. The gene primer sequences are shown in Table 2 below.

[0087]

[0088] The results are as follows Figure 9-12 As shown, compared with the challenge group, serum DAO levels were significantly reduced in all YI39 dose groups (P<0.05), and serum IL-1β and IL-6 levels were also significantly reduced in all YI39 dose groups (P<0.05), indicating that YI39 can reduce intestinal permeability and alleviate systemic inflammatory response. Compared with the challenge group, the mRNA expression level of Claudin-1 in the jejunum was significantly upregulated in the low- and medium-dose YI39 groups (P<0.05), indicating that YI39 can enhance intestinal barrier function.

[0089] This invention sets 3×10 8 6×10 8 9×10 8 12×10 8A systematic evaluation of YI39 supplementation levels was conducted at four doses (CFU / kg), considering factors such as growth performance, intestinal lesions, morphology, barrier function, inflammatory response, and microbial regulation. Results showed that YI39 supplementation at 3×10⁻⁶ CFU / kg significantly improved growth performance, intestinal lesions, morphology, barrier function, inflammatory response, and microbial regulation. 8 -9×10 8 All CFU / kg levels showed good control of necrotizing enteritis. Specifically, 3×10 8 The CFU / kg group showed the best performance in microbial regulation and overall probiotic properties; 9×10 8 The CFU / kg group showed the best effect in improving intestinal morphology and suppressing inflammation; while the 12×10 group showed the best effect in improving intestinal morphology and suppressing inflammation. 8 The high-dose group (CFU / kg) showed a decreasing trend in some indicators, indicating that the dose-effect of YI39 is not a simple linear relationship, and excessively high doses may have a "saturation effect" or slight negative effects.

[0090] The above results indicate that the Lactobacillus amyloliquefaciens YI39 provided by this invention has good in vitro probiotic properties, and exhibits good inhibitory effects against Salmonella pullorum, avian pathogenic Escherichia coli, Clostridium perfringens type A, and Staphylococcus aureus. In vivo, it can effectively alleviate the decline in growth performance, intestinal damage, and inflammatory response caused by Clostridium perfringens infection in broilers, and prevent the occurrence of necrotizing enteritis in chickens by enhancing intestinal barrier function.

[0091] Although the present invention has been described above in conjunction with preferred embodiments, the present invention is not limited to the specific embodiments described above. Those skilled in the art can make various equivalent or alternative modifications to the present invention within the scope of the invention's concept. All such modifications should be covered within the protection scope of the present invention.

Claims

1. A type of Lactobacillus amyloliquefaciens for preventing necrotic enteritis in chickens caused by Clostridium perfringens infection, characterized in that, The Lactobacillus amylovorus was named Lactobacillus amylovorus YI39, and its accession number at the China Center for Type Culture Collection is CCTCC NO: M2026104; its 16S rDNA nucleotide sequence is SEQ ID NO.1; the Lactobacillus amylovorus was derived from broiler chickens.

2. A probiotic preparation, characterized in that, The probiotic agent contains Lactobacillus amyloliquefaciens as described in claim 1.

3. The probiotic agent according to claim 2, characterized in that, The probiotic agent also includes excipients, namely maltodextrin.

4. The probiotic agent according to claim 2 or 3, characterized in that, Probiotic preparations are available in liquid, powder, or granule forms.

5. The probiotic agent according to claim 4, characterized in that, The probiotic preparation is in the form of lyophilized powder.

6. The use of Lactobacillus amyloliquefaciens as described in claim 1 or the probiotic agent as described in any one of claims 2 to 5 in the preparation of feed or medicine for the prevention and / or treatment of necrotizing enteritis in broilers caused by Clostridium perfringens infection.

7. The application as described in claim 6, characterized in that, The feed or medicine has at least one of the following functions: (1) Improve the average daily weight gain of broilers after challenge with Clostridium perfringens; (2) Reduce the jejunal lesion score caused by viral attack and increase the ratio of villus height to crypt depth; (3) It simultaneously reduces the levels of diamine oxidase and interleukin-1β in serum and upregulates the mRNA expression level of tight junction protein Claudin-1 in jejunal tissue. (4) Antibacterial function.

8. The application as described in claim 7, characterized in that, The antibacterial function includes the inhibition of at least one of the following bacteria: Salmonella pullorum, avian pathogenic Escherichia coli, Clostridium perfringens type A, and Staphylococcus aureus.

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