A lysozyme of a bacteriophage yrd1 capable of lysing salmonella pullorum, a biological preparation comprising the same and use thereof

Abstract

The application belongs to the technical field of biological prevention and control and food safety, and discloses a bacteriophage Yrd1 capable of lysing chicken white diarrhea Salmonella, a biological preparation containing the bacteriophage Yrd1 and application. Salmonella pullorum The bacteriophage Yrd1 is isolated by taking chicken white diarrhea Salmonella as host bacteria and is named as bacteriophage Yrd1, which is preserved in China Center for Type Culture Collection on April 22, 2026, has a preservation number of CCTCC NO: M 2026766 and has good lytic activity and biological stability. The biological preparation containing the bacteriophage can be prepared into a liquid preparation, a freeze-dried powder preparation, a spray preparation, a preparation for water drinking, a preparation for feedstuff addition or a food surface treatment preparation, is used for biological prevention and control of chicken white diarrhea Salmonella and is especially suitable for inhibiting or reducing the pollution of chicken white diarrhea Salmonella in chicken juice, chicken and other chicken related substrates.

Description

Technical Field

[0001] This invention belongs to the field of biocontrol and food safety technology, specifically relating to a bacteriophage Yrd1 capable of lysing Salmonella pullorum, a biological agent containing the phage, and its application in the control of Salmonella pullorum. More specifically, it relates to a bacteriophage Yrd1 isolated from Salmonella pullorum as the host bacterium, and the application of liquid formulations, freeze-dried powder formulations, spray formulations, or food surface treatment formulations containing the phage in inhibiting or reducing Salmonella pullorum contamination in chicken-related matrices. Background Technology

[0002] Salmonella pullorum is one of the important pathogens in poultry production, causing high mortality rates, stunted growth, transmission of the bacteria, and decreased breeding performance in chicks, resulting in significant economic losses to the poultry industry. Furthermore, this pathogen can contaminate poultry meat and related products during breeding, slaughtering, processing, and distribution, affecting product quality and biosafety.

[0003] Currently, control measures for Salmonella pullorum mainly include biosafety management, disinfection, and antimicrobial intervention. However, chemical or antibiotic control methods have problems such as bacterial imbalance, increased risk of drug resistance, residues, and limited application scenarios. In contrast, bacteriophages have the advantages of strong host specificity, targeted killing, and less impact on non-target microorganisms, making them a promising candidate for precise control of pathogenic bacteria.

[0004] However, several limitations remain in the practical application of natural phage formulations. For example, the stability of liquid phage formulations is easily affected by temperature, pH, and external environment during storage and transportation, and their ease of use and shelf life are also constrained. Therefore, further developing specific phages with good lytic activity into lyophilized powder formulations that are easy to store, transport, and use in the field is an important direction for promoting their practical application.

[0005] In the existing technology, research and product development on lyophilized phage powder formulations specifically targeting Salmonella pullorum and their application in chicken-related matrices are still relatively limited. Therefore, it is necessary to provide a phage formulation with good lytic activity against Salmonella pullorum, which can be prepared as a lyophilized powder and is suitable for antibacterial activity in chicken-related matrices, in order to meet the actual needs of livestock farming and meat product safety control. Summary of the Invention

[0006] The purpose of this invention is to overcome the problems of insufficient targeting, inconvenience of use, or poor stability of existing technologies for the prevention and control of Salmonella pullorum, and to provide a bacteriophage Yrd1 that can lyse Salmonella pullorum, a biological agent containing it, and its application.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: As a first aspect of the present invention, the present invention provides a Salmonella pullorum bacteriophage Yrd1, which was isolated and screened using Salmonella pullorum ATCC 13036 as the host bacterium, named Salmonella pullorum bacteriophage Yrd1, and deposited at the China Center for Type Culture Collection (CCTCC) on April 22, 2026, with accession number CCTCC NO: M 2026766.

[0008] The above-mentioned Salmonella pullorum phage Yrd1 has the following characteristics: (1) Morphological characteristics: The bacteriophage formed well-defined plaques on a double-layer plate; according to transmission electron microscopy, the bacteriophage Yrd1 showed a typical tailed bacteriophage morphology under the transmission electron microscope, with an icosahedral head that appeared as a near-hexagonal projection under the electron microscope, and a relatively short tail. According to the scale bar, the head diameter of bacteriophage Yrd1 was approximately 75–80 nm, and the tail length was approximately 40–50 nm. (2) Nucleic acid type: DNA; (3) Genomic characteristics: The full-length genome of phage Yrd1 is 87,890 bp, with a GC content of 37.97%. A total of 131 open reading frames (ORFs) were predicted, and 18 tRNA genes were detected. Functional annotation results showed that the phage genome contains multiple functional genes related to phage assembly, nucleic acid metabolism, and host lysis, including tailfiber protein, tailspike protein, terminase large subunit, head protein, tail protein, DNA polymerase I, RNA polymerase, holin, lysozyme / endolysin, and DNA-binding protein. Further homology comparison results showed that multiple ORFs had high similarity to Salmonella phage FelixO1 proteins, suggesting that this phage is closely related to FelixO1-like phages.

[0009] As a second aspect of the present invention, the present invention provides a biological agent for the prevention and control of Salmonella pullorum in chickens, said biological agent comprising bacteriophage Yrd1.

[0010] The biological agent may be a liquid preparation, a freeze-dried powder preparation, a spray preparation, a soaking treatment preparation, a feed additive preparation, a drinking water additive preparation, an environmental treatment preparation, or a food surface treatment preparation.

[0011] Preferably, the biological agent is a lyophilized powder formulation, which includes a biologically active bacteriophage Yrd1, a protectant, an excipient, and / or an adjuvant.

[0012] Preferably, the protective agent, excipient and / or auxiliaries are selected from one or more of trehalose, sucrose, mannitol, skim milk powder, gelatin, whey protein, sorbitol, starch, dextrin, lactose, microcrystalline cellulose, and maltodextrin.

[0013] As a third aspect of the invention, the invention also provides the use of the above-mentioned bacteriophage Yrd1 or biological agents containing it in the preparation of products for the prevention and control of Salmonella pullorum in chickens.

[0014] As a fourth aspect of the present invention, the present invention also provides the application of the above-mentioned bacteriophage Yrd1 or a biological agent containing it in inhibiting or reducing Salmonella pullorum contamination in chicken-related matrices, specifically: phage Yrd1 is administered at a concentration of 1.0 × 10⁻⁶... 7 ~1.0×10 8 Add the chicken-related matrix to a final concentration of PFU / mL or at an MOI of 100.

[0015] Preferably, the chicken-related matrix is ​​chicken broth, chicken chunks, chicken homogenate, or other poultry product matrix.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. The bacteriophage Yrd1 provided by this invention can specifically lyse Salmonella pullorum, providing a new bacteriophage resource for the prevention and control of Salmonella pullorum.

[0017] 2. The biological agent containing bacteriophage Yrd1 described in this invention can be prepared into liquid formulations, lyophilized powder formulations, spray formulations, or food surface treatment formulations according to application requirements. Among them, the lyophilized powder formulation has better storage convenience, transportation stability, and on-site application operability.

[0018] 3. The biological agent containing bacteriophage Yrd1 described in this invention can be used to inhibit or reduce the contamination of Salmonella pullorum in chicken broth, chicken meat and other chicken-related matrices, and has good application prospects.

[0019] 4. The bacteriophage Yrd1 and the biological agents containing it described in this invention have the advantages of strong targeting, high safety and minimal impact on chicken quality, and can provide technical support for the biocontrol of Salmonella pullorum in poultry farming and related meat processing. Attached Figure Description

[0020] Figure 1 This is a diagram showing the morphology of plaques formed by bacteriophage Yrd1 on a double-layer plate.

[0021] Figure 2 This is a transmission electron microscope image of bacteriophage Yrd1, showing its icosahedral head and short tail structure. The scale bar is 100 nm.

[0022] Figure 3 This is a diagram illustrating the biological characteristics of bacteriophage Yrd1, where A represents the optimal multiplicity of infection for bacteriophage Yrd1. B is a one-step growth curve of phage Yrd1; C is a schematic diagram of the adsorption rate of phage Yrd1.

[0023] Figure 4 This is a schematic diagram of the environmental stability of bacteriophage Yrd1, where A is a schematic diagram of the temperature stability of bacteriophage Yrd1; and B is a schematic diagram of the pH stability of bacteriophage Yrd1.

[0024] Figure 5 This is a schematic diagram illustrating the inhibitory effect of bacteriophage Yrd1 on host bacteria in a meat matrix. In this diagram, A represents the inhibitory effect of bacteriophage Yrd1 on host bacteria in chicken broth, and B represents the inhibitory effect of bacteriophage Yrd1 on host bacteria in chicken meat.

[0025] Figure 6 This is a schematic diagram of the quality evaluation results of chicken after treatment with bacteriophage Yrd1, where A is the L* value of the chicken surface color parameter and B is the chicken firmness. Detailed Implementation

[0026] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments. In the following embodiments, unless otherwise specified, the microbial culture, detection and analysis methods used are all conventional experimental operations recognized in the art, and the strains, reagents, culture media and other consumables involved can all be obtained commercially.

[0027] This invention screened and obtained a strain of Salmonella pullorum phage Yrd1, which was deposited at the China Center for Type Culture Collection (CCTCC) on April 22, 2026, with accession number CCTCC NO: M 2026766.

[0028] Example 1: Isolation, purification, and identification of Salmonella pullorum phage Yrd1 1. Isolation and purification of bacteriophages Supernatant from chicken farm feces samples was cultured to the logarithmic growth phase (OD).600 The host bacterial suspensions of Salmonella pullorum (ATCC 13036) with a concentration of 0.6-0.8 μg / mL were mixed at a volume ratio of 10:1 and enriched at 37°C for 6 h. After centrifugation to remove bacterial residue, the supernatant was filtered through a 0.22 μm microporous membrane to obtain the phage stock solution. The phage stock solution was purified using the double-layer plate method, repeated 5-10 times until a single phage plaque with consistent morphology, size, and transparency was obtained, which was the purified phage Yrd1. The morphology of the phage plaques formed on the double-layer plate is shown in the figure below. Figure 1 As shown.

[0029] The titer of phage Yrd1 was determined using the double-layer plate method. 0.1 mL of phage dilution was used for plate testing, the number of plaques formed on the plates was counted, and the phage titer was calculated using the following formula: Phage titer (PFU / mL) = Number of phage plaques × Dilution factor × 10.

[0030] 2. Morphological analysis of bacteriophages The phage suspension was ultracentrifuged at 4℃ and 40,000 r / min for 1 h, and the phage pellet was collected and resuspended in 0.1 mol / L ammonium acetate. Negative staining was performed using 2% (v / v) phosphotungstic acid (pH 7.0). A copper mesh was immersed in the phage resuspension for 10 min, excess liquid was aspirated, and phosphotungstic acid staining solution was added for 10 min. The phages were then air-dried. Transmission electron microscopy was used to observe the phage morphology, and the head and tail dimensions were measured using image analysis software.

[0031] like Figure 2 As shown in the electron microscope images, bacteriophage Yrd1 exhibits a typical tailed bacteriophage morphology under a transmission electron microscope. The head is a regular icosahedron, appearing as a nearly hexagonal outline in two-dimensional projection, while the tail is relatively short and thick. Preliminary measurements based on the scale bar in the image indicate that the head diameter of bacteriophage Yrd1 is approximately 75–80 nm, and the tail length is approximately 40–50 nm. These results demonstrate that bacteriophage Yrd1 is a short-tailed bacteriophage with a complete morphology and clear structure.

[0032] 3. Phage DNA extraction and genome sequencing High-titer phage lysis buffer was treated with DNase I and RNase A to remove free nucleic acids. Then, proteinase K and SDS were added to lyse the phage particles. Phage DNA was extracted using the phenol / chloroform extraction method or a commercial viral DNA extraction kit. The quality and concentration of the extracted DNA were measured using an ultra-micro spectrophotometer. Samples meeting the requirements were sent for whole-genome sequencing.

[0033] Preferably, the sequencing platform is Illumina NovaSeq 6000, and the sequencing service provider is Shandong Inset Biotechnology Co., Ltd.

[0034] 4. Genome annotation and comparative analysis The results showed that the full-length genome of phage Yrd1 was 87,890 bp, with a GC content of 37.97%. A total of 131 open reading frames (ORFs) were predicted, and 18 tRNA genes were detected. Gene functional annotation indicated that this phage contains multiple protein-coding genes related to phage replication, packaging, structural assembly, and host lysis, including RNA polymerase, DNA polymerase I, terminase large subunit, head protein, tail protein, tail fiber protein, tail spike protein, holin, and lysozyme / endolysin. Some ORFs were also annotated as hypothetical proteins. Further homology comparison revealed that several encoded products showed high similarity to proteins derived from Salmonella phage FelixO1, suggesting a certain phylogenetic relationship between phage Yrd1 and FelixO1-like phages.

[0035] Example 2: Biological characteristics analysis of Salmonella pullorum phage Yrd1 1. Detection of the host range of bacteriophage Yrd1 Twelve strains were selected for host range determination. The tested strains included *Salmonella pullorum* and different serotypes of *Enterobacter*. Specific strain numbers, sources, serotypes, and lysis results of phage Yrd1 are shown in Table 1. Each tested strain was activated and cultured to the logarithmic growth phase (OD2). 600 =0.6–0.8), take 100 μL of bacterial suspension and mix it with the upper semi-solid culture medium, then invert it onto the surface of an LB agar plate. After solidification, add 10 μL of a solution with a titer of 1.0 × 10⁻⁶. 7 Phage Yrd1 suspension at PFU / mL was incubated at 37℃ for 4–6 h, and lysis was observed. The experiment was repeated 3 times, with 2 replicates each time.

[0036] Table 1. Information on tested strains and lysis results of bacteriophage Yrd1 Note: "++" indicates obvious lysis, "+" indicates lysis, and "-" indicates no obvious lysis. In the table, CICC 0604P, CICC 01095P, and CICC numerical codes are strain numbers provided by the China Industrial Microbial Culture Collection Center (CICC); PU519 and PU534 are Salmonella strains of pullorum disease provided by Wang Xiaohong's research group at Huazhong Agricultural University.

[0037] The results are shown in Table 1. Phage Yrd1 exhibited significant lytic activity against PU, PU519, PU534, AG, and TY, and against GA, HE, EN, IN, and KE, but no significant lytic activity was observed against HA and NE. These results indicate that phage Yrd1 has a certain host range and can be used for lytic control of Salmonella pullorum and related susceptible Salmonella.

[0038] 2. Detection of the optimal multiple of infection (MOI) for phage Yrd1. The multiplicity of infection (MOI) is the ratio of the number of bacteriophages to the number of host bacteria at the time of initial infection. Bacteriophage Yrd1 was mixed with logarithmically growing host bacteria (Salmonella pullorum ATCC 13036) at MOIs of 0.0001, 0.01, 0.1, 1, and 10, and incubated at 37°C for 4 h. After centrifugation at 8000 r / min for 20 min, the phage titer in the supernatant under different MOI conditions was determined using the double-layer plate method. The experiment was repeated three times, with two replicates each time.

[0039] The results are as follows Figure 3 A shows that, under the set MOI conditions, phage Yrd1 has the highest titer after amplification at an MOI of 0.1, therefore its optimal multiplicity of infection is determined to be 0.1.

[0040] 3. Detection of the one-step growth curve of bacteriophage Yrd1 Bacteriophage Yrd1 was mixed with host bacteria at the optimal MOI of 0.1, incubated at 37°C, and then centrifuged to remove unadsorbed phage. The precipitate was washed with LB medium and resuspended in fresh LB liquid medium, and cultured at 37°C. Samples were taken every 10 min until 120 min, and the phage titer in the samples was determined using the double-layer plate method, and a one-step growth curve was plotted. The experiment was repeated three times, with two replicates each time.

[0041] The results are as follows Figure 3B shows that the incubation period of phage Yrd1 is about 25 min, the burst period is about 60 min, and the lysis rate is about 127 ± 22 PFU / CFU, indicating that the phage has a fast proliferation rate and high amplification capacity.

[0042] 4. Detection of the adsorption rate of bacteriophage Yrd1 Bacteriophage Yrd1 was mixed with host bacteria (Salmonella pullorum ATCC 13036) cultured to the logarithmic growth phase at an optimal MOI of 0.1 and adsorbed at 37°C. Starting from 0 min, samples were taken every 4 min, and the titer of unadsorbed phage in the supernatant was determined using the double-layer plate method. The adsorption rate was calculated using the following formula: Adsorption rate (%) = [1 - (unadsorbed phage titer at each time point / phage titer at 0 min)] × 100%; The experiment was repeated 3 times, with 2 parallel experiments each time.

[0043] The results are as follows Figure 3 C showed that phage Yrd1 had a rapid adsorption capacity for the host bacteria. The average adsorption rate was 6.67% at 4 min post-infection, rising to 68.03% at 8 min, reaching 82.39% at 12 min, and still maintained a high level at 16 min, indicating that its main adsorption process could be basically completed within 12 min.

[0044] Example 3: Preparation of freeze-dried powder formulation of Salmonella pullorum phage Yrd1 1. Preparation of high-titer bacteriophage suspensions The purified phage Yrd1 was inoculated into a culture system of Salmonella pullorum ATCC 13036, a host bacterium in the logarithmic growth phase, at a ratio of 0.1 for the multiple of infection (MOI). The culture was incubated at 37°C and 180 rpm with shaking for 4–6 h. After the bacterial suspension had largely lysed, it was centrifuged at 8000 rpm for 15 min, and the supernatant was collected. The supernatant was then filtered through a 0.22 μm microporous membrane to obtain a high-titer phage suspension with an initial titer of 1.0 × 10⁻⁶. 9 PFU / mL.

[0045] 2. Preparation of lyophilization protection solution Weigh trehalose, skim milk powder and mannitol, add them to sterile SM buffer and dissolve them thoroughly to prepare a lyophilization protection solution, wherein the final concentrations of each component are: trehalose 10% (w / v), skim milk powder 5% (w / v) and mannitol 1% (w / v).

[0046] The above high-titer phage suspension and lyophilization protection solution were mixed evenly at a volume ratio of 1:1 to obtain the phage mixture to be lyophilized.

[0047] 3. Pre-freezing and freeze-drying The phage mixture to be lyophilized was dispensed into sterile lyophilization bottles at a rate of 1.0 mL per bottle, pre-frozen at -80℃ for 6 h, and then placed in a freeze dryer for lyophilization under the following conditions: cold trap temperature -50℃, vacuum degree ≤20Pa, and lyophilization time 24 h.

[0048] After freeze-drying, the initial freeze-dried powder of phage Yrd1 was obtained.

[0049] 4. Preparation of lyophilized powder formulations The above-mentioned lyophilized initial powder was taken out under sterile conditions and directly sealed to obtain the lyophilized powder preparation of bacteriophage Yrd1; preferably, the obtained preparation was stored at 4°C in the dark for later use.

[0050] 5. Reconstitution and Activity Assay Take an appropriate amount of lyophilized phage Yrd1 powder, reconstitute it with sterile PBS buffer at the original volume, mix gently, and then determine the titer of the reconstituted phage using the double-layer plate method. Compare the titer with that of the phage before lyophilization to evaluate the effect of the lyophilization process on phage activity.

[0051] 6. How to use After reconstituted the above-mentioned Yrd1 lyophilized phage powder, add it according to the target system volume to make a final concentration of 1.0 × 10⁻⁶. 7 ~1.0×10 8 PFU / mL is used for the biocontrol of Salmonella pullorum contamination systems in chickens.

[0052] The results showed that the lyophilized powder of phage Yrd1 prepared using the above protection system and lyophilization process had a loose and uniform appearance, good resolubility, and maintained high phage activity after resolubility, indicating that the lyophilization process is suitable for the preparation and preservation of lyophilized powder of phage Yrd1.

[0053] Example 4: Stability evaluation of bacteriophage Yrd1 and its lyophilized powder formulation 1. Stability of phage Yrd1 lysate under different temperature conditions With a valence of 1.0 × 10 10Phage Yrd1 lysis buffer at approximately PFU / mL was aliquoted into sterile centrifuge tubes and incubated at 30℃, 40℃, 50℃, 60℃, and 70℃ for a specified time. The residual phage titer in each sample was then determined using the double-layer plate method. The experiment was repeated three times, with two replicates per run.

[0054] The results are as follows Figure 4 A shows that phage Yrd1 exhibits good thermal stability at temperatures ranging from 30 to 60°C, with the phage titer remaining at a relatively high level. However, its activity significantly decreases when the temperature rises to 70°C. These results indicate that phage Yrd1 has good stability under medium and low temperature conditions, but is easily inactivated at higher temperatures.

[0055] 2. Stability of Yrd1 phage lysate under different pH conditions Take 100 μL of the drug with a potency of 1.0 × 10⁻⁶. 10 Phage Yrd1 lysis buffer at approximately PFU / mL was added to 900 μL of sterile buffer at different pH values ​​to achieve final pH values ​​of 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. After incubation at 37°C for 2 h, the residual phage titer in each treated sample was determined using the double-layer plate method. The experiment was repeated three times, with two replicates per experiment.

[0056] The results are as follows Figure 4 B shows that phage Yrd1 exhibits significantly different stability under different pH conditions. Its activity is highest at pH 6–8; it maintains good stability within the pH range of 5–10; however, its activity decreases significantly under strongly acidic or alkaline conditions. This indicates that phage Yrd1 is suitable for maintaining high lytic activity in a near-neutral environment.

[0057] 3. Storage stability of freeze-dried powder The Yrd1 lyophilized phage powder obtained in Example 3 was sealed and stored at -20℃, 4℃, and 25℃. Samples were taken at 0, 7, 14, 30, 60, 90, and 180 days of storage. The phage titer was determined by reconstitution with sterile PBS buffer and then by the bilayer plate method. The results showed that the Yrd1 lyophilized phage powder maintained high lytic activity after 90 days of storage at 4℃, and the titer decreased by no more than 1 log PFU / mL.

[0058] 4. Freeze-thaw stability The lysate of phage Yrd1 was repeatedly frozen and thawed at -80℃ for 3 and 5 times, respectively, after each complete freezing and thawing. The phage titer was determined by the double-layer plate method. The results showed that after 3 and 5 freeze-thaw cycles, the lysate of phage Yrd1 still maintained high activity, and the titer decreased by no more than 1 log PFU / mL.

[0059] The above results indicate that the Yrd1 phage lysate maintains high activity under medium and low temperature conditions and has good stability in neutral to weakly alkaline environments; its lyophilized powder formulation can maintain phage lysing activity well under low temperature storage conditions, showing good storage stability and application potential.

[0060] Example 5: Antibacterial effect of phage Yrd1 lyophilized powder formulation in chicken-related matrices. According to Example 2, the optimal multiplicity of infection (MOI) for phage Yrd1 amplification was determined to be 0.1. To evaluate its rapid lysis ability, the antibacterial experiment in this example was conducted with a high MOI of 100.

[0061] 1. Chicken gravy mold Purchase fresh chicken from the local market, rinse with sterile distilled water, prepare chicken homogenate by mixing chicken and sterile water at a mass-to-volume ratio of 1:3, centrifuge at 5000 r / min for 15 min, and filter successively through sterile filter paper and 0.22 μm filter membrane to obtain sterile chicken juice, which is stored at -20℃ for later use.

[0062] Take sterile chicken broth into a sterile container, add Salmonella pullorum ATCC 13036 suspension to make the initial bacterial concentration 1.0 × 10⁻⁶. 5 CFU / mL, then add the reconstituted lyophilized phage Yrd1 powder to make a final concentration of 1.0 × 10⁻⁶ CFU / mL. 7 PFU / mL or MOI of 100. A positive control group (Control) was established, consisting of bacteria only without bacteriophages, and a negative control group was established, consisting of sterile chicken broth. The cultures were incubated at 37°C, and samples were taken at 0, 2, 4, 6, and 8 hours. The number of surviving bacteria was determined using the plate count method.

[0063] 2. Chicken nugget model Cut undamaged chicken meat into pieces approximately 2 cm × 2 cm in size, place them in sterile petri dishes, wipe the surface with alcohol, and then sterilize by irradiating both sides with UV light for 30 min each. Add the chicken pieces to sterile EP tubes or petri dishes, and inoculate with a 1.0 × 10⁻⁶ Salmonella pullorum ATCC 13036 suspension. 5 The initial contamination level of the chicken pieces was approximately 1.0 × 10⁻⁶ CFU / mL.5 CFU / g; then add the reconstituted lyophilized phage Yrd1 powder to make a final concentration of 1.0 × 10⁻⁶ CFU / g. 7 PFU / mL or MOI of 100. A positive control group was defined as the group inoculated with bacteria only. After incubation at 37℃, samples were taken at 0, 2, 4, 6, and 8 hours, and the viable bacterial count was determined using the elution-plate counting method.

[0064] The results are as follows Figure 5 As shown, in both the chicken broth and chicken nugget models, the bacterial count in the lyophilized Yrd1 phage formulation treatment group was significantly lower than that in the positive control group. Specifically, the phage treatment group achieved the highest inhibitory effect compared to the positive control group after treatment at 37℃ for 6 h; in the chicken broth model, the bacterial count decreased by approximately 4.85 log CFU / mL; and in the chicken nugget model, the bacterial count decreased by approximately 3.85 log CFU / g. These results indicate that this lyophilized powder formulation can effectively inhibit or reduce Salmonella pullorum contamination in chicken-related matrices.

[0065] Example 6: Evaluation of chicken quality after treatment with bacteriophage Yrd1 The aseptic chicken pieces described in Example 5 were divided into four groups: the first group was a blank control group, which received no treatment; the second group was a bacterial treatment group, which was inoculated with a suspension of Salmonella pullorum (concentration of 1.0 × 10⁻⁶). 5 CFU / mL or 1.0×10 5 After incubation for 5 h, the third group was the phage treatment group, inoculated with phage Yrd1 lysis buffer or reconstituted lyophilized powder (final concentration 1.0 × 10⁻⁶ CFU / g); 7 After incubation for 5 h, the bacteria were treated with PFU / mL; the fourth group was the bacteria + phage treatment group, which was simultaneously inoculated with Salmonella pullorum (concentration of 1.0 × 10⁻⁶ PFU / mL). 5 CFU / mL or 1.0×10 5 CFU / g) and bacteriophage (final concentration 1.0 × 10⁻⁶) 7 After incubation for 5 h (PFU / mL), the culture was completed.

[0066] The color parameter L* of the chicken surface was measured using a colorimeter, and the hardness index was measured using a texture analyzer.

[0067] The results are as follows Figure 6 As shown, compared with the blank control group, treatment with phage Yrd1 alone had no significant adverse effects on the L* value of chicken surface color and the hardness index; compared with the bacterial treatment group, the bacterial + phage treatment group was able to maintain the chicken quality index better while inhibiting bacteria, indicating that the phage preparation has good application compatibility.

[0068] The embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A bacteriophage Yrd1 capable of lysing Salmonella pullorum, characterized in that, The bacteriophage was named Salmonella pullorum bacteriophage Yrd1 was deposited at the China Center for Type Culture Collection (CCTCC) on April 22, 2026, with accession number CCTCC NO: M 2026766.

2. The phage Yrd1 capable of lysing Salmonella pullorum according to claim 1, characterized in that, The bacteriophage was isolated using Salmonella pullorum ATCC 13036 as the host bacterium.

3. A biological agent for the prevention and control of Salmonella pullorum in chickens, characterized in that, The biological agent comprises the bacteriophage Yrd1 as described in claim 1 or 2.

4. The biological agent according to claim 3, characterized in that, The biological agent is a liquid preparation, a freeze-dried powder preparation, a spray preparation, a soaking treatment preparation, a feed additive preparation, a drinking water additive preparation, an environmental treatment preparation, or a food surface treatment preparation.

5. The biological agent according to claim 3 or 4, characterized in that, The biological agent also includes protectants, excipients, stabilizers, carriers and / or adjuvants.

6. The biological agent according to claim 5, characterized in that, The protective agent, excipient, stabilizer, carrier and / or auxiliaries are selected from one or more of trehalose, sucrose, mannitol, skim milk powder, gelatin, whey protein, sorbitol, starch, dextrin, lactose, microcrystalline cellulose and maltodextrin.

7. The use of the bacteriophage Yrd1 according to claim 1 or 2 or the biological agent according to any one of claims 3 to 6 in the preparation of products for the prevention and control of Salmonella pullorum in chickens.

8. The use of the bacteriophage Yrd1 as described in claim 1 or 2, or the biological agent as described in any one of claims 3 to 6, in inhibiting or reducing Salmonella pullorum contamination in chicken-related matrices.

9. The application according to claim 8, characterized in that, The chicken-related matrix includes chicken broth, chicken chunks, chicken homogenate, or poultry products.

10. The application according to claim 8 or 9, characterized in that, The application is specifically: the bacteriophage Yrd1 is added into the chicken related matrix at a final concentration of 1.0×10 7 ~1.0×10 8 PFU / mL or at an MOI of 100.

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