Phage capable of efficiently splitting pseudomonas longdamura as well as composition and application of phage

Abstract

The invention discloses a bacteriophage capable of efficiently splitting Pseudomonas ongdamura and a composition and application thereof, the bacteriophage is Pseudomonas ongdamura bacteriophage PL1P1, the preservation number is CCTCCM20251191, the Pseudomonas ongdamura bacteriophage PL1P1 is a virulent bacteriophage separated from the nature, and the Pseudomonas ongdamura bacteriophage PL1P1 has relatively high tolerance to ultraviolet rays and pH, and can be used for efficiently splitting Pseudomonas ongdamura and Pseudomonas ongdamura and Pseudomonas ongdamura. The bacteriophage is suitable for different prevention and treatment environments and can achieve a good fresh-keeping effect on chilled meat, DNA of the bacteriophage cannot encode protein possibly causing potential health risks, the possibility of carrying lysogenic genes does not exist, the bacteriophage has high affinity and splitting capacity, the titer of 10 < 10 > PFU / mL or above can be achieved within 24 h of culture, and the bacteriophage has good application prospects. The Pseudomonas longdamura phage PL1P1 can specifically partially or completely inactivate Pseudomonas longdamura, can complete mass proliferation only by using a small amount of initial phage, and provides a high-quality phage strain source for industrial production of phage bactericides.

Description

Technical Field

[0001] This invention belongs to the field of phage technology, specifically relating to a highly efficient phage for lysing *Pseudomonas lundii*, its composition, and its applications. Background Technology

[0002] Lund's Pseudomonas is a Gram-negative bacterium belonging to the genus Pseudomonas in the family Pseudomonasceae. It is often studied as a spoilage bacterium in the food industry. Its biofilm formation ability is significantly affected by environmental factors such as nutrient conditions, pH value and NaCl concentration. In the process of spoilage of chilled fresh meat, it can lead to protein degradation and peptide accumulation.

[0003] Currently, related technologies for delaying the spoilage of chilled meat and extending its shelf life mainly involve low-temperature freezing, with refrigeration temperatures strictly maintained at 0~4℃ (fresh meat) or -1.5~0℃ (chilled pork), with fluctuations ≤1℃. For meat requiring long-term storage, a -35℃ quick-freezing cold storage is used to rapidly pass it through the ice crystal zone (-1~-5℃) to form tiny ice crystals that prevent cell membrane puncture. On the other hand, natural antibacterial agents are sprayed onto the surface of the meat, and combined with vacuum packaging, this can further inhibit spoilage-causing bacteria in chilled meat. Because Londo's Pseudomonas phage has a specific and efficient lysis ability against target bacteria, it can significantly reduce the number of spoilage-causing bacteria in chilled meat, thus extending its shelf life.

[0004] Existing Londo Pseudomonas bacteriophages have limitations. Most phages have poor environmental tolerance and are prone to significant titer drops under conditions such as acid-base fluctuations, short-term ultraviolet irradiation, and temperature fluctuations that may be encountered during the processing and storage of chilled meat, resulting in unstable actual preservation effects. Some phages have narrow lysis spectra, acting only on Londo Pseudomonas from specific sources or with specific genotypes, and cannot address Londo Pseudomonas contamination issues in different regions and processing scenarios. In addition, some phages pose a risk of lysogenicity, potentially conferring drug resistance or virulence genes to the host bacteria through gene transfer, posing potential safety hazards and limiting their industrial application. Therefore, we need to provide a bacteriophage for efficiently lysing Londo Pseudomonas, its composition, and its applications. Summary of the Invention

[0005] The purpose of this invention is to provide a bacteriophage for efficiently lysing *Pseudomonas lundbeck*, its composition, and its applications. The bacteriophage possesses broad-spectrum lytic ability, excellent environmental tolerance, non-lysogenicity, and high biocompatibility. Its composition can synergistically interact with chemical bactericides without antagonism. Furthermore, it expands its applications in *Pseudomonas lundbeck* detection, chilled meat preservation, and environmental disinfection, thereby addressing the problems mentioned in the background art, such as the narrow lytic spectrum of *Pseudomonas lundbeck* bacteriophages, poor environmental tolerance, lysogenic safety hazards, and the difficulty of effectively controlling *Pseudomonas lundbeck* contamination using existing chilled meat preservation technologies.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: a bacteriophage for efficiently lysing *Pseudomonas lundii*, wherein the bacteriophage is *Pseudomonas lundii* bacteriophage PL1P1, with accession number CCTCCM20251191.

[0007] Preferably, the Lund Pseudomonas phage is a virulent phage with a polyhedral head and a relatively long tail, with a major diameter L of 88 nm and a transverse diameter W of 83 nm, and L / W≈1.06; the tail includes a retractable muscle sheath and tail filaments, with the muscle sheath being approximately 135 nm in length and approximately 20 nm in width.

[0008] Preferably, the Lund Pseudomonas phage, when cultured for 24 hours under conditions of MOI=0.000001, achieves a titer of 6.4×10¹⁰ PFU / mL or higher, and its titer decreases by no more than one order of magnitude when stored at 4°C for 12 months, shows no significant decrease in titer when stored at 25°C for 4 weeks, and decreases by no more than one order of magnitude in titer when stored at 40°C for 72 hours.

[0009] Preferably, the Lund Pseudomonas phage is resistant to pH 5-10 conditions, with its titer decreasing by no more than 4 orders of magnitude within 96 hours, and its titer decreasing by no more than 3 orders of magnitude within 96 hours under pH 3-4 and pH 11-12 conditions; after 8 hours of ultraviolet radiation at a distance of 20W and 20cm, its titer decreases by no more than 1 order of magnitude.

[0010] A Lund Pseudomonas phage composition comprising Lund Pseudomonas phage and a chemical bactericide, wherein the chemical bactericide is at least one selected from citric acid, nisin, potassium sorbate, and sodium dehydroacetate. When citric acid is used, a 700-fold dilution of citric acid is employed. The phage and the citric acid solution are mixed in equal volumes and coexisted at 25°C for 48 hours.

[0011] The application of the bacteriophage in the preparation of a Londopseudomonas lunidae detection product, the detection product including reagents or kits, for rapid detection of Londopseudomonas lunidae in samples or screening of target pathogens in clinical samples.

[0012] Preferably, the concentration is between 10¹PFU / mL and 10¹PFU / mL. 8 The PFU / mL potency range shows an inhibition rate of 54%~95% against *Pseudomonas lunidosa*, which can be used to prepare biological agents for the prevention and treatment of *Pseudomonas lunidosa* infection.

[0013] Preferably, the Lund Pseudomonas phage is used to prepare products for disinfecting and decontaminating water distribution systems, irrigation facilities, aquaculture facilities, public and private facilities, or other environmental surfaces. The products include detergents, disinfectants, and stain removers, and are used by liquid immersion, spraying, or in combination with an aqueous carrier.

[0014] Preferably, it is used as an effective ingredient in chilled meat preservatives to prevent spoilage of chilled meat caused by Pseudomonas lundii.

[0015] Preferably, the composition is used to prepare a biological agent for preventing and treating infection with *Pseudomonas lundii*, or to prepare a product for disinfecting and decontaminating environmental surfaces such as water distribution systems and irrigation facilities, wherein the composition has no antagonistic effect with the combined substances.

[0016] Technical effects and advantages of the present invention: The bacteriophage for highly efficient lysis of *Pseudomonas lundii*, its composition, and its application proposed in this invention have the following advantages compared with the prior art:

[0017] The Lund pseudomonas phage PL1P1 of this invention is a virulent phage isolated from nature. It exhibits high tolerance to ultraviolet light and pH, making it suitable for various pest control environments. It can effectively preserve chilled meat. The phage's DNA does not encode proteins that may pose potential health risks, and it does not carry lysogenic genes. Furthermore, it possesses high affinity and lytic ability, achieving a titer of over 10¹⁰ PFU / mL within 24 hours of culture. Lund pseudomonas phage PL1P1 can specifically partially or completely inactivate Lund pseudomonas, requiring only a small amount of initial phage to achieve large-scale proliferation. This provides a high-quality phage strain source for the industrial production of phage bactericides.

[0018] The Lund pseudomonas phage PL1P1 is a strictly virulent phage with high specificity and lytic activity against its host bacteria and a broad host range. It achieved a lysis rate of 91.1% against 45 strains of Lund pseudomonas. PL1P1 can be used as an effective ingredient in various food preservation products. Its interaction with non-host pathogenic bacteria shows that it cannot identify any of the 10 tested non-host pathogenic bacteria strains, exhibiting good specificity. It is resistant to conditions between pH 3 and 12, with a titer reduction of no more than four orders of magnitude within 96 hours. After 8 hours of UV radiation, the titer reduction is no more than one order of magnitude. It can be used to prepare compositions, reagents, or kits for the rapid detection of Lund pseudomonas, including but not limited to test strips and kits.

[0019] Other features and advantages of the invention will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the invention. The objects and other advantages of the invention may be realized and obtained by means of the structures pointed out in the description and the drawings. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the phage plaque;

[0021] Figure 2 This is a schematic diagram of the electron microscopy results for this bacteriophage;

[0022] Figure 3 This is a schematic diagram of the structure of the sample that showed plaques in this lysogenic test;

[0023] Figure 4 This is a schematic diagram of the structure of the sample that did not show plaques in this lysogenicity test. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The specific embodiments described herein are merely used to explain the present invention and are not intended to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] This invention provides, for example Figure 1-4 The invention relates to a highly efficient phage for lysing *Pseudomonas lundii*, characterized in that the phage is *Pseudomonas lundii* phage PL1P1, with accession number CCTCCM20251191.

[0026] The Lund Pseudomonas phage is a virulent phage with a polyhedral head and a relatively long tail. The major diameter L is 88 nm and the transverse diameter W is 83 nm, with L / W ≈ 1.06. The tail includes a retractable muscle sheath and tail filaments. The muscle sheath is approximately 135 nm long and approximately 20 nm wide.

[0027] Specifically, electron microscopy revealed that the Lund Pseudomonas phage PL1P1 exhibited typical structural characteristics of a virulent phage: its head was a regular polyhedral three-dimensional symmetrical structure with a measured major diameter (L) of 88 nm and a measured transverse diameter (W) of 83 nm, resulting in a major-to-transverse diameter ratio (L / W) of approximately 1.06. This compact head structure stably encapsulates the phage genome, providing a structural basis for nucleic acid injection during host infection. The tail was a slender rod-shaped structure containing a contractile muscle sheath and functional tail filaments. The muscle sheath was approximately 135 nm long and 20 nm wide, and its contraction could propel the head nucleic acid into the host bacteria. The tail filaments specifically recognized receptor sites on the surface of Lund Pseudomonas, ensuring precise binding of the phage to the target host and further guaranteeing its specific lysis ability against Lund Pseudomonas. Meanwhile, lysogenicity identification experiments have verified that this bacteriophage is a strictly virulent bacteriophage with no lysogenicity. It will not integrate its own genes into the host bacterial genome, thus avoiding safety risks such as drug resistance or increased virulence in the host bacteria. This provides safety support for its application in food preservation, environmental disinfection and other scenarios.

[0028] The Lund Pseudomonas phage, when cultured for 24 hours under conditions of MOI=0.000001, achieved a titer of 6.4×10¹⁰ PFU / mL or higher. Furthermore, the titer did not decrease by more than one order of magnitude when stored at 4℃ for 12 months, showed no significant decrease in titer when stored at 25℃ for 4 weeks, and decreased by more than one order of magnitude in titer when stored at 40℃ for 72 hours.

[0029] Specifically, the proliferation capacity and storage stability of *Pseudomonas lundii* phage PL1P1 were rigorously verified experimentally: under the condition of a multiplicity of infection (MOI) of 0.000001 (i.e., the ratio of phage number to *Pseudomonas lundii* number of bacteria was 1:1,000,000), it was co-inoculated with logarithmic-phase host bacteria in TSB liquid medium and cultured at 28°C with shaking at 180 rpm for 24 h. The titer was then determined using the double-layer plate method, and the results showed that its titer stably reached 6.4 × 10¹. 0 With a PFU / mL or higher, only a very small amount of initial phage is needed to achieve efficient proliferation, providing high-quality strains and low-cost amplification conditions for the industrial-scale production of phage bactericides.

[0030] In terms of storage stability, this bacteriophage exhibits excellent temperature resistance: with a titer of 1.0 × 10⁻⁶. 7 Purified phage solutions at PFU / mL, stored at 4°C, still maintained a titer of 6.9 × 10⁻⁶ after 12 months. 6 The potency decreased by no more than one order of magnitude when the PFU / mL level was above 1.0; after storage at room temperature (25°C) for 4 weeks, the potency decreased from 1.0 × 10⁻⁶. 7 PFU / mL decreased to 7.9 × 10⁻⁶ 6 The PFU / mL level did not decrease significantly; even after being stored at a relatively high temperature of 40℃ for 72 hours, the potency remained at 2.3×10⁻⁶. 6 The PFU / mL level is above 1, and the reduction is no more than one order of magnitude. This stability is suitable for cold chain storage (4°C), room temperature transportation (25°C), and short-term high-temperature processing of chilled meat, without the need for additional complex stabilizers, significantly improving its convenience and economy in practical applications.

[0031] The Lund Pseudomonas phage exhibits resistance at pH 5-10, with a titer reduction of no more than 4 orders of magnitude within 96 hours, and a titer reduction of no more than 3 orders of magnitude within 96 hours at pH 3-4 and pH 11-12. After 8 hours of ultraviolet radiation at a distance of 20W and 20cm, the titer reduction is no more than 1 order of magnitude.

[0032] Specifically, the acid and alkali resistance and UV resistance of Lund Pseudomonas phage PL1P1 were verified through experiments simulating environmental conditions in real-world application scenarios: In the acid and alkali resistance test, the initial titer was 1×10¹ 0 Purified phage solutions of PFU / mL were inoculated into TSB liquid medium (equilibrated at 25℃) with pH 3–12. Samples were taken at 1 h, 4 h, 8 h, 24 h, and 96 h, and the titer was determined using the double-layer plate method. The results showed that in the neutral to weakly acidic / weakly alkaline range of pH 5–10, the phage titer decreased by no more than four orders of magnitude within 96 h, while still maintaining high lytic activity. Even in the slightly acidic environment of pH 3–4 and the slightly alkaline environment of pH 11–12, the titer decreased by only three orders of magnitude within 96 h, which is significantly better than most existing phages that are not tolerant to extreme pH values, and can adapt to diverse and complex application environments.

[0033] In the UV resistance test, 10 mL of the sample had a potency of 1×10. 8 A PFU / mL phage solution was spread in a 90 mm sterile culture dish and placed under a 20 W UV lamp in a laminar flow hood for continuous irradiation. Samples were taken at 0 min, 20 min, 1 h, and up to 8 h, and the titer was determined using the double-layer plate method. The results showed that after 8 h of UV irradiation, the phage titer decreased from 1 × 10⁻⁶ PFU / mL. 8 PFU / mL decreased to 1.2 × 10⁻⁶ 7 The PFU / mL level is reduced by no more than one order of magnitude, allowing it to withstand short-term or indirect radiation during ultraviolet sterilization in food processing workshops. This avoids significant loss of phage activity due to environmental disinfection, ensuring its practical application effectiveness.

[0034] A Lund Pseudomonas phage composition comprising Lund Pseudomonas phage and a chemical bactericide, wherein the chemical bactericide is at least one selected from citric acid, nisin, potassium sorbate, and sodium dehydroacetate. When citric acid is used, a 700-fold dilution of citric acid is employed. The phage and the citric acid solution are mixed in equal volumes and coexisted at 25°C for 48 hours.

[0035] The detection products include reagents or kits for rapid detection of Pseudomonas lundii in samples or screening of target pathogens in clinical samples.

[0036] Specifically, the reagents in the detection product use Lund Pseudomonas phage PL1P1 as the core active ingredient. They can be prepared into liquid or lyophilized reagents according to detection requirements. Liquid reagents maintain the phage in a buffer system containing SM solution to ensure stable phage titer. Lyophilized reagents are made by adding preservatives such as skim milk and sucrose and lyophilizing to extend the storage period. They can be reconstituted with sterile water before use.

[0037] The kit includes the aforementioned phage reagent, matching culture medium, sterile sampling tubes, diluent, and result interpretation standard cards. Its detection principle is based on the host-specific lytic characteristics of phages: During detection, the sample to be tested is first mixed with TSB liquid medium, enriched, and then an appropriate amount of bacterial suspension is mixed with TSB semi-solid agar and spread onto TSA plates to form a bacterial growth layer; then, phage reagent (titer ≥ 1 × 10⁻⁶) is added. 7 After incubating at 28℃ for 6-8 hours (PFU / mL), if round, transparent plaques appear on the plate, it indicates the presence of *Pseudomonas lundbeckii* in the sample. The concentration of the target bacteria in the sample can be preliminarily estimated by combining the number of plaques with the sample dilution factor. If no plaques appear, it is determined that there is no *Pseudomonas lundbeckii* in the sample or that its concentration is below the detection limit.

[0038] In 10¹PFU / mL~10 8 The PFU / mL potency range shows an inhibition rate of 54%~95% against *Pseudomonas lunidosa*, which can be used to prepare biological agents for the prevention and treatment of *Pseudomonas lunidosa* infection.

[0039] Specifically, the turbidimetric assay was used to verify that the Lund Pseudomonas phage PL1P1 was diluted to 10¹PFU / mL. 8 Gradual titers of PFU / mL were co-inoculated with logarithmic-phase *Pseudomonas lundae* culture medium on TSB liquid medium. After 24 h of incubation at 28°C and 120 rpm with shaking, the bacterial culture without phage was used as a positive control. Turbidity of each group was measured using a turbidimeter, and the inhibition rate was calculated: when the phage titer was 10¹PFU / mL, the inhibition rate reached 54%; as the titer increased, the inhibition rate gradually increased, reaching 10¹PFU / mL. 8 At PFU / mL, the antibacterial rate can reach up to 95%, and this antibacterial effect is consistently observed in different strains of *Pseudomonas lundii*, proving that it has a broad-spectrum and highly efficient inhibitory ability against *Pseudomonas lundii*.

[0040] Based on this antibacterial property, the bacteriophage can be used as the core active ingredient to prepare a biological agent for preventing and treating *Pseudomonas lundii* infection. During preparation, the appropriate dosage form can be selected according to the application scenario: for surface disinfection of food processing equipment, aquaculture environments, etc., a liquid dosage form can be prepared by mixing the purified bacteriophage with sterile physiological saline containing 0.01%~0.05% Tween-80, and adjusting the final titer to 10. 5 PFU / mL ~10 7 PFU / mL enhances the adhesion of bacteriophages to object surfaces; for the control of spoilage bacteria in chilled and fresh meat and other foods, it can be formulated into a sprayable microemulsion with edible soybean oil as the oil phase and bacteriophage solution as the aqueous phase, with 0.5%~1% monoglyceride added as an emulsifier, and microemulsion with a particle size of 200~500nm is prepared by high-speed shearing, thereby improving the persistence of bacteriophages on meat surfaces.

[0041] The Lund Pseudomonas phage is used to prepare products for disinfecting and decontaminating water distribution systems, irrigation facilities, aquaculture facilities, public and private facilities, or other environmental surfaces. The products include detergents, disinfectants, and stain removers, which are used by liquid immersion, spraying, or in combination with an aqueous carrier.

[0042] Specifically, in product preparation, formulations are designed for different dosage forms: When preparing detergents, bacteriophage purified products are mixed with phosphorus-free surfactants, chelating agents, and pH adjusters to create liquid detergents that retain the physical detergency of surfactants while allowing bacteriophages to precisely lyse the Londo Pseudomonas biofilm attached to facility surfaces; when preparing disinfectants, high-concentration bacteriophage solutions are used with 0.02%~0.05% potassium sorbate added as a stabilizer to create a directly usable disinfectant stock solution that can be applied to target surfaces without dilution; when preparing stain removers, bacteriophages are compounded with weakly alkaline detergents to suit facility surfaces where oil, dirt, and bacteria coexist, achieving both physical detergency and biological sterilization.

[0043] It is used as an active ingredient in chilled meat preservatives to prevent spoilage of chilled meat caused by Pseudomonas lundii.

[0044] Specifically, in the preparation of preservatives, two formulations were designed based on the processing flow and storage requirements of chilled meat: one is a single phage preservative, in which purified phage PL1P1 is diluted with sterile phosphate buffer at a ratio of 1:100 to 1:500, and the final titer is adjusted to 2×10⁻⁶. 7 PFU / mL ~2×10 8 The first type of preservative, formulated with PFU / mL, is a liquid preservative that can be directly sprayed, suitable for the demand for "no chemical additives" in chilled meat processing; the second type is a phage-chemical synergistic preservative, which is a synergistic preservative made by mixing phage PL1P1 with 700 times diluted citric acid at a volume ratio of 1:1, or by mixing it with nisin. Experimental verification showed that the phage titer of this type of compound system remained at 10 after coexisting at 25℃ for 48 hours. 6 It has a PFU / mL or higher concentration and a 15% to 20% higher kill rate against Pseudomonas lundensis compared to single-component formulations, making it suitable for long-term cold chain storage scenarios.

[0045] In terms of application, a precise application plan is adopted in conjunction with the processing of chilled meat: For freshly cut chilled meat (temperature 0~4℃), use a sterile sprayer to evenly spray the preservative onto the surface of the meat, ensuring that it covers both sides of the meat pieces and the edge gaps. After spraying, let it stand for 5~10 minutes to allow the bacteriophage to fully adhere to and penetrate into the surface of the meat. For pre-packaged chilled meat, the preservative can be diluted with sterile water at a ratio of 1:20, and the meat can be soaked for 1~2 minutes (soaking temperature 0~2℃). After draining, it can be vacuum-packed, utilizing the low temperature environment inside the packaging to continuously interact with the bacteriophage and extend the shelf life.

[0046] The composition is used to prepare biological agents for the prevention and treatment of *Pseudomonas lundii* infection, or to prepare products for the disinfection and decontamination of environmental surfaces such as water distribution systems and irrigation facilities, wherein the composition has no antagonistic effect with the combined substances.

[0047] Specifically, in the preparation of biological agents for the prevention and control of *Pseudomonas lundensis* infection, the dosage form was designed according to the infection scenario: For the prevention and control of equipment surface infection, bacteriophage PL1P1 was mixed with 0.01% nisin at a volume ratio of 3:1, and 0.03% Tween-80 was added as a dispersant to prepare a liquid biological agent. Testing showed that after storage at 28°C for 72 hours, the bacteriophage titer increased from 1×10⁻⁶. 8 PFU / mL decreased to 8.5 × 10⁻⁶ 7 PFU / mL, with a nisin activity retention rate of over 90%, the two components showed no antagonism and synergistically enhanced bactericidal effect, achieving a 98.2% kill rate against *Pseudomonas lundii* adhering to equipment surfaces, a significant improvement compared to single-component formulations. For localized infections in farmed animals, a gel formulation was prepared by mixing bacteriophage PL1P1 with 0.1% potassium sorbate and 2% hydroxypropyl methylcellulose, with the pH adjusted to 6.5-7.0 to suit the physiological environment of animal skin. Experiments showed that the bacteriophage and potassium sorbate in the gel maintained stable activity for 14 days, and after application, it could continuously lyse bacteria at the infection site without causing irritation.

[0048] In the following examples, the strain codes used are all based on our company's naming convention.

[0049] The phage PL1P1 of Pseudomonas lundensisphage, with accession number CCTCCNO: M20251191, is deposited at the China Center for Type Culture Collection on May 26, 2025.

[0050] In the following examples,

[0051] The formula for TSB liquid culture medium is: 15g tryptone, 5g soybean peptone, 5g sodium chloride, and 1000mL distilled water.

[0052] The formula for TSA solid culture medium is: 15g tryptone, 5g soybean peptone, 5g sodium chloride, 15g agar, and 1000mL distilled water.

[0053] TSA plates: TSA solid culture medium is sterilized and poured onto sterile plates, then cooled and solidified to make TSA plates;

[0054] The formula for TSB semi-solid agar medium is: 15g tryptone, 5g soybean peptone, 5g sodium chloride, 7g agar, and 1000mL distilled water.

[0055] SM solution formula: 8.5g sodium chloride, 2g magnesium sulfate, 50mL 1mol / L Tris-HCl, 0.25g gelatin, 1000mL distilled water.

[0056] Example 1: Isolation, preparation, purification, and culture of Lund Pseudomonas phage PL1P1

[0057] In this invention, the source sample of the Lund Pseudomonas phage PL1P1 was collected from slaughterhouse wastewater, filtered through double-layer filter paper, centrifuged at low speed at room temperature, and then filtered through a 0.22μm filter membrane to obtain the supernatant.

[0058] 1. Isolation of bacteriophages:

[0059] (1) Take 10 mL of the filtered supernatant and add it to 10 mL of 2 times TSB liquid medium. At the same time, add 1 mL of the logarithmic phase bacterial culture of the phage host bacteria PL1P1 and incubate overnight at 28°C.

[0060] (2) Take the above culture, centrifuge at 8000 rpm for 10 min, filter the supernatant through a 0.22 μm filter membrane, and set aside for later use;

[0061] (3) Take 0.5 mL of the phage host bacteria PL1 log phase bacterial suspension, add it to 5 mL of 40℃ TSB semi-solid agar medium, mix well, pour it onto TSA plate, and prepare a double-layer plate containing the host bacteria.

[0062] (4) Take 10 μL of the prepared supernatant and drop it onto the solidified double-layer plate. After air drying under sterile conditions, place it at 28°C for overnight incubation to form phage droplets.

[0063] 2. Purification of bacteriophages:

[0064] (1) Use a toothpick to pick up the phage spot and transfer it to 1 mL of SM solution and shake for 1 min;

[0065] (2) Perform 10-fold serial dilutions, and add 0.5 mL of the logarithmic phase bacterial culture of the phage host bacteria to the 102, 104 and 106 dilutions respectively, and mix well;

[0066] (3) After standing for 15 minutes, add the above mixture to 5 mL of 40℃ TSB semi-solid agar medium, mix well and immediately pour it onto TSA plate, shake well and let it stand for 5 minutes until it solidifies. After overnight incubation at 28℃, observe to obtain a double-layer plate containing a single phage plaque.

[0067] (4) Pick up a single plaque and transfer it to 1 mL of SM solution. Purify it at least 3 times in the above manner to finally form plaques of uniform shape and size on the plate.

[0068] (5) Use a toothpick to pick out a single phage plaque of uniform shape and size, place it in 50 mL of TSB liquid medium containing 1 mL of logarithmic host bacterial culture, and incubate overnight at 28°C and 180 rpm.

[0069] (6) Centrifuge the culture at 8000 rpm for 10 min, filter the supernatant through a 0.22 μm filter membrane to obtain the purified phage solution, which is Pseudomonas lundensis phage PL1P1. Pseudomonas lundensis phage PL1P1 produces single circular plaques on the Pseudomonas lundensis bacterial colony, as shown in the figure. Figure 1 The Lund Pseudomonas phage PL1P1, with accession number CCTCCNO: M20251191.

[0070] Example 2: Electron microscopic observation of Pseudomonas lundensisphage PL1P1

[0071] The purified phage solution prepared in Example 1 was observed under an electron microscope: 20 μL of sample was dropped onto a copper grid and allowed to precipitate naturally for 15 min. Excess liquid was absorbed from the side with filter paper. One drop of 2% phosphotungstic acid was added to the copper grid and stained for 10 min. The staining solution was then absorbed from the side with filter paper and dried before observation under an electron microscope.

[0072] The results are as follows Figure 2 As shown, morphological observation of the Lund Pseudomonas phage PL1P1 under an electron microscope revealed that the phage has a polyhedral head and a relatively long tail, with a major diameter L of 88 nm and a transverse diameter W of 83 nm, L / W ≈ 1.06; the tail includes a constrictible muscle sheath and tail filaments, with the muscle sheath approximately 135 nm in length and 20 nm in width.

[0073] Example 3: Preparation of Pseudomonas lundensisphage PL1P1 particles and extraction and sequencing of the genome.

[0074] (1) Take 100 mL of the purified phage solution prepared in Example 1, add 20 μL of DNase I and 20 μL of RNase A with a concentration of 5 mg / mL in sequence, incubate at 37°C for 60 min, then add 5.84 g of NaCl, and place in an ice bath for 1 h after dissolution;

[0075] (2) Centrifuge at 11,000 rpm for 10 min at 4℃, transfer the supernatant after centrifugation to a new centrifuge tube, add solid PEG8000 to make the final concentration 10% (w / v), and after the PEG8000 is completely dissolved, incubate on ice for 1 h.

[0076] (3) Centrifuge at 11,000 rpm for 20 min at 4℃, add 1 mL of SM solution to resuspend the precipitate, and obtain the phage particle concentrate. Store at 4℃ for later use.

[0077] Example 4: Determination of the titer of Londo Pseudomonas phage PL1P1

[0078] Using SM solution as a diluent, the original solution of *Pseudomonas lundii* phage PL1P1 (prepared in Example 1) was serially diluted 10-fold to 10⁻⁸fold. 1000 μL of phage culture at dilutions of 10⁻⁵, 10⁻⁶, 10⁻⁷, and 10⁻⁸ was mixed thoroughly with 300 μL of its host bacterial culture and allowed to stand for 15 min to allow for sufficient binding to receptors on the bacterial surface. This mixture was then added to 4 mL of semi-solid agar medium cooled to 50°C, mixed well, and immediately spread onto solidified agar plates. After the agar solidified, the plates were incubated upside down at 28°C for 6–8 h. Three replicates were prepared for each dilution, and the average of the three replicates for each dilution was used for counting. The phage titer (PFU / mL) was calculated as: average number of plaques × dilution factor.

[0079] Table 1 shows that the Lund Pseudomonas phage PL1P1 has a titer of over 10¹⁰ PFU / mL after 12 h of culture.

[0080] Table 1. Titer of Londo Pseudomonas phage PL1P1 after 12 hours of culture.

[0081]

[0082] Example 5: Detection test for deletion of virulence gene or adverse gene in Lund Pseudomonas phage PL1P1

[0083] In this embodiment, 103 virulence genes identified as originating from lysogenic bacteriophages within pathogenic bacteria were selected. The whole genome of Lund's Pseudomonas phage PL1P1 was determined and subjected to bioinformatics analysis. Lund's Pseudomonas phage PL1P1 does not contain the following virulence genes or harmful genes, so it cannot encode proteins that may cause potential health risks. Therefore, Lund's Pseudomonas phage PL1P1 will not affect the health of humans or animals.

[0084] Example 6: Toxicological Experiment

[0085] Twenty experimental mice, half male and half female, were randomly divided into two groups (phage group and control group) after three days of acclimatization. Each group consisted of 10 mice (5 males and 5 females). The phage group was given 10¹⁰ PFU / kg of Lund pseudomonas phage PL1P1, while the control group was given an equal amount of physiological saline. The administration was continued for 15 days. The mice were then euthanized by cervical dislocation, and their internal organs were examined.

[0086] Experimental results showed that this dose of Lund's Pseudomonas phage PL1P1 had no effect on the daily behavior of mice. Autopsy examination revealed no abnormalities in the internal organs. Lund's Pseudomonas phage PL1P1 exhibits high biocompatibility.

[0087] Example 7: Determination of the optimal multiple of infection (MOI) of *Pseudomonas lundii* phage PL1P1 against *Pseudomonas lundii*

[0088] A single colony of the host bacterium *Pseudomonas lundense* was picked and inoculated into a test tube containing 3 mL of TSB liquid medium. The culture was incubated overnight at 28°C and 180 rpm with shaking to obtain a host bacterium suspension. The host bacterium suspension was transferred to 10 mL of TSB liquid medium at a ratio of 1:100 and cultured at 28°C and 180 rpm with shaking until the early logarithmic growth phase. Bacteriophage PL1P1 purification solution (prepared in Example 1) and bacteriophage host bacteria were added at MOI ratios of 100, 10, 1, 0.1, 0.01, 0.001 and 0.0001, 0.00001, 0.000001, 0.000001, 0.0000001, respectively. TSB liquid medium was added to ensure the total volume of each tube was the same. The tubes were then incubated at 28°C and 180 rpm with shaking for 24 h. After culturing, the culture was centrifuged at 10000g for 10 min, and the supernatant was collected. The phage titer for each treatment was determined using the double-layer plate method. Double-component cultures were performed at each point, and the average value was taken. The MOI (Multiple of Infection) that produced the highest phage titer was considered the optimal MOI. The experiment was repeated three times.

[0089] Table 3. Titer of Lund Pseudomonas phage PL1P1 at different infection multiplicity.

[0090]

[0091] Table 3 shows that under 24-hour culture conditions, when the titer of phage PL1P1 reached a maximum of 6.4 × 10¹⁰ PFU / mL, its MOI was 0.000001. This indicates that only a small amount of initial *Pseudomonas lundii* phage is needed to achieve large-scale proliferation. Phage PL1P1 provides a high-quality phage strain source for the industrial production of phage bactericides.

[0092] Example 8: Determination of pH and temperature stability of Lund Pseudomonas phage PL1P1

[0093] 8-1: Stability of Lund Pseudomonas phage PL1P1 under different pH conditions

[0094] Add 900 μL of TSB liquid medium (pH 1-14) to each sterile EP tube. Place the EP tubes in a 25°C water bath and, after temperature equilibration, add 100 μL of purified phage solution (prepared in Example 7) to achieve an initial titer of 1 × 10¹⁰ PFU / mL. Incubate at room temperature. Samples are taken at 1 h, 4 h, 8 h, 24 h, and 96 h of reaction. After appropriate dilution, the phage titer is determined using the double-layer plate method. The experiment is repeated three times.

[0095] Table 4. Stability of bacteriophage PL1P1 under different pH conditions

[0096]

[0097] The results are shown in Table 4. The titer of bacteriophage PL1P1 did not change significantly between pH 5 and 10, indicating that it has good stability under neutral, weakly acidic and weakly alkaline conditions.

[0098] Under acidic conditions (pH=3) and alkaline conditions (pH=12), the titer of bacteriophage PL1P1 decreased to some extent, but compared with the pH=7 condition, the titer decreased by about three orders of magnitude, indicating that it has good tolerance under acidic and alkaline conditions. Under extremely acidic conditions (pH=2) and extremely alkaline conditions (pH=13), the titer of bacteriophage PL1P1 decreased to 0 within 1 hour.

[0099] 8-2: Stability of Lund Pseudomonas phage PL1P1 under different temperature conditions

[0100] Phage PL1P1 (prepared in Example 7) with a titer of 1.0 × 10⁷ PFU / mL was placed at 4 °C, 25 °C and 40 °C respectively, and samples were taken periodically to detect its titer.

[0101] Table 5. Stability of Lund Pseudomonas phage PL1P1 at 4°C.

[0102] Table 6. Stability of Lund Pseudomonas phage PL1P1 at 25°C

[0103]

[0104] Table 7. Stability of Lund Pseudomonas phage PL1P1 at 40℃

[0105]

[0106] As shown in Tables 5-7, bacteriophage PL1P1 exhibits good stability at 4℃, with no significant decrease in titer after 3 months of storage, and the titer decrease is still less than one order of magnitude after 12 months of storage. At 25℃, the titer of bacteriophage PL1P1 shows no significant decrease after 4 weeks of storage. At 40℃, the titer of bacteriophage PL1P1 shows no significant decrease within 24 hours, but decreases by one order of magnitude after 72 hours. This indicates that bacteriophage PL1P1 has good stability under different temperature conditions.

[0107] Example 9: Test of the UV tolerance of Lund Pseudomonas phage PL1P1

[0108] 10 mL of PL1P1 phage (prepared in Example 7) with a titer of 1×10⁸ PFU / mL was spread evenly in a 90 mm sterile culture dish and placed in a laminar flow hood under UV light (20 W, 20 cm). Samples were taken at 0 min, 20 min, 40 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, and 8 h, and placed in the dark for 30 min before the phage titer was determined using the double-layer plate method.

[0109] Table 8. Stability of Lund Pseudomonas phage PL1P1 under ultraviolet irradiation.

[0110]

[0111] The results are shown in Table 8. After 8 hours of ultraviolet irradiation, the titer of Lund Pseudomonas phage PL1P1 decreased by an order of magnitude. Therefore, the phage of the present invention has strong resistance to ultraviolet radiation.

[0112] Example 10: Lysis test of Lund's Pseudomonas phage PL1P1 against Lund's Pseudomonas from different sources

[0113] The lysis profile of bacteriophages was determined using the double-layer plate drop method. Forty-five single colonies of *Pseudomonas lundense* isolated from six provinces (Shandong, Sichuan, Chongqing, Anhui, Guangdong, and Henan) were picked and inoculated into test tubes containing 3 mL of TSB liquid medium. The cultures were incubated overnight at 28°C and 180 rpm to prepare bacterial suspensions for each strain. 500 μL of the bacterial suspension was mixed with TSB semi-solid agar medium and spread onto ordinary agar plates. 5 μL of purified bacteriophage PL1P1 solution (prepared in Example 1) was then dropped onto the plates. After air drying, the plates were incubated overnight at 28°C, and the results were observed.

[0114] Example 11: Lysis test of Lund's Pseudomonas phage PL1P1 on non-pathogenic beneficial bacteria

[0115] Five non-pathogenic rhizobium strains and five non-pathogenic Bacillus subtilis strains were selected and inoculated into test tubes containing 3 mL of TSB liquid medium. The cultures were incubated at 30°C and 180 rpm for 8 hours to obtain bacterial suspensions for each strain. 300 μL of each bacterial suspension was mixed with TSB semi-solid agar medium and spread onto ordinary agar plates. 5 μL of purified bacteriophage PL1P1 solution (prepared in Example 1) was added to each plate. After air drying, the plates were incubated at 30°C for 24 hours, and the results were observed.

[0116] Table 10. Lysis test of Lund's Pseudomonas phage PL1P1 against non-pathogenic beneficial bacteria.

[0117]

[0118] The results are shown in Table 10. In this embodiment, bacteriophage PL1P1 was unable to recognize any of the 10 non-pathogenic bacteria strains. This indicates that the tested bacteriophage has extremely strong host specificity and does not damage the microbial community.

[0119] Example 12: Identification of lysogenicity of Lund Pseudomonas phage PL1P1

[0120] 100 μL of bacteriophage PL1P1 (prepared in Example 7) (0 PFU / mL, 1.0 × 10⁴ PFU / mL, 1.0 × 10⁵ PFU / mL, 1.0 × 10⁶ PFU / mL, 1.0 × 10⁷ PFU / mL) was mixed with 100 μL of *Pseudomonas lundensis* (1.0 × 10⁸ cfu / mL) and inoculated into 50 mL centrifuge tubes containing 10 mL of TSB liquid medium. The mixture was then incubated at 28 °C with shaking for 48 h. The resulting turbid culture was serially diluted and spread onto TSA plates and incubated at 28 °C for 48 h. The center of 20 single colonies was picked from a TSA plate and placed in an EP tube containing 200 μL TSB liquid medium. The tubes were then incubated at 28°C with shaking for 24 h. Mitomycin C was then added to the EP tubes to a final concentration of 0.5 μg / mL, and the tubes were incubated for another 12 h. The resulting culture was then filtered through a 0.22 μm filter to remove bacteria and spotted onto a PL1 bilayer plate, which was incubated at 28°C. Simultaneously, bacteriophage PL1P1 (prepared in Example 7) with a titer of 1.0 × 10⁷ PFU / mL was spotted onto the bilayer plate as a positive control. After 24 h, the bilayer plates were observed. The presence of phage plaques indicated that phage PL1P1 was a lysogenic phage.

[0121] The results showed that plaques appeared on all control plates. Figure 3 No plaques were observed on any of the test plates. Figure 4 This indicates that the Lund Pseudomonas phage PL1P1 is not lysogenic and is a virulent phage.

[0122] Example 13: Turbidimetric assay for the inhibition rate of Londo Pseudomonas phage PL1P1 against Londo Pseudomonas.

[0123] Single colonies of the host bacteria were picked and inoculated into test tubes containing 3 mL of TSB. The cultures were incubated overnight at 28°C and 180 rpm until turbidity was achieved, thus obtaining the host bacterial culture. Bacteriophage PL1P1 (prepared in Example 7) with a titer of 2 × 10¹⁰ PFU / mL was serially diluted with sterile water to the titers of each treatment group. As shown in Table 11, 100 μL of PL1 bacterial culture and 100 μL of each diluted PL1P1 culture were added to 50 mL centrifuge tubes containing 10 mL of TSB liquid culture medium, respectively. A mixture of 100 μL of PL1 bacterial culture and 10 mL of TSB liquid culture medium was used as a positive control. The cultures were incubated at 28°C and 120 rpm for 24 h, and the turbidity of each treatment group was measured using a turbidimeter. Inhibition rate = (Turbidity of positive treatment - Turbidity of treatment group) / Turbidity of positive treatment × 100%

[0124] Table 11. Antibacterial effect of Londo Pseudomonas phage PL1P1 against Londo Pseudomonas.

[0125]

[0126] As shown in Table 11, the inhibition rate of phage PL1P1 at approximately 101 PFU / mL against Pseudomonas lundii can reach 54%, indicating that phage PL1P1 can effectively inhibit and kill bacteria at extremely low doses.

[0127] Example 14: Bactericidal effect of Lund Pseudomonas phage PL1P1 in liquid

[0128] *Pseudomonas lundii* was cultured to the logarithmic growth phase and aliquoted into different test tubes. The bacterial culture was diluted with an equal volume of TSB liquid medium to a final concentration of 1×10³ CFU / mL. *P. lundii* bacteriophage PL1P1 (prepared in Example 1) was then inoculated into the tubes at final concentrations of 1×10² PFU / mL, 1×10³ PFU / mL, 1×10⁴ PFU / mL, 1×10⁵ PFU / mL, and 1×10⁶ PFU / mL, respectively. A control group and a blank group were also established. The control group received *P. lundii* at a final concentration of 1×10³ CFU / mL; the blank group received an equal volume of physiological saline. All treatments were incubated at 28°C with shaking at 150 rpm for 4 hours. The residual amount of *P. lundii* was then detected. The detection method was as follows: each treatment sample was diluted with sterile water, and 100 μL of the diluted solution was spread onto a TSA agar plate. After incubation at 28°C for 24 hours, the number of colonies on the plate was counted. The number of Lund Pseudomonas count = number of colonies on TSA plate × dilution factor × 10.

[0129] Table 12. Bactericidal effect of different concentrations of *Pseudomonas lunde* bacteriophage PL1P1 in liquid.

[0130]

[0131] As shown in Table 12, the Lund Pseudomonas phage PL1P1 can effectively control the growth of Lund Pseudomonas in liquid culture medium when its final concentration is 1×102 PFU / mL; when the final concentration of Lund Pseudomonas phage PL1P1 is ≥1×104 PFU / mL, its killing rate against Lund Pseudomonas can reach over 99%.

[0132] Example 15: Compatibility test of Lund Pseudomonas phage PL1P1 with citric acid

[0133] Phage PL1P1, with an initial titer of 1×10⁷ pfu / mL, was aliquoted into 50 ml sterile centrifuge tubes. Citric acid solution with a final concentration of 50% was added at 700 times the concentration. The tubes were incubated at 25°C for 2 h, 4 h, 8 h, 24 h, and 48 h. The phage titers were then measured, and the results are shown in Table 13.

[0134] Table 13 Compatibility results of Lund Pseudomonas phage PL1P1 with citric acid

[0135]

[0136] As shown in Table 13, after 48 hours of coexistence with citric acid, the titer of *Pseudomonas lunoides* phage PL1P1 remained at 106 pfu / mL, decreasing by less than half an order of magnitude. This indicates that phage PL1P1 can coexist with 700 times the final concentration of 50% citric acid for a period of time, demonstrating strong resistance to this concentration. This suggests that *Pseudomonas lunoides* phage PL1P1 can be used in combination with certain acidic preparations in clinical practice to achieve better results.

[0137] Example 16: Preparation of a composition of Lund Pseudomonas phage PL1P1

[0138] 1. The purified phage PL1P1 solution (prepared in Example 1) with final concentrations of 1×102 PFU / mL, 1×103 PFU / mL, 1×104 PFU / mL, 1×105 PFU / mL, and 1×106 PFU / mL, respectively, were mixed in equal volumes with 700 times diluted citric acid solution with a final concentration of 50% to prepare composition 1, composition 2, composition 3, composition 4, and composition 5 in a 1:1 ratio.

[0139] Example 17: Bactericidal effect of the Lund Pseudomonas phage PL1P1 composition in liquid.

[0140] *Pseudomonas lundii* was cultured to the logarithmic growth phase and aliquoted into different test tubes. The bacterial culture was diluted with an equal volume of TSB liquid medium to a final concentration of 1 × 10³ CFU / mL. A combination of *Pseudomonas lundii* phage PL1P1 prepared in Example 15 was then inoculated into each tube. Control and blank groups were also established. The control group received *Pseudomonas lundii* at a final concentration of 1 × 10³ CFU / mL; the blank group received an equal volume of physiological saline. Each treatment was incubated at 28°C with shaking at 150 rpm for 4 hours. The residual amount of *Pseudomonas lundii* was then detected. The detection method was as follows: each treatment sample was diluted with sterile water, and 100 μL of the diluted solution was spread onto a TSA agar plate. After incubation at 37°C for 24 hours, the number of colonies on the plate was counted. The *Pseudomonas lundii* count = number of colonies on the TSA plate × dilution factor × 10.

[0141] Table 14. Bactericidal effect of different concentrations of *Pseudomonas lundii* bacteriophage PL1P1 compositions in liquids.

[0142]

[0143] Table 14 shows that the Lund pseudomonas phage PL1P1 combination at all concentrations exhibits good bactericidal activity against Lund pseudomonas. This indicates that Lund pseudomonas phage PL1P1 can be used in combination with other substances to control bacteria without antagonizing other substances.

[0144] The combination in this embodiment is not limited to 700 times citric acid solution, but can also be chemical reagents such as nisin, potassium sorbate, and sodium dehydroacetate.

[0145] Example 18: Preservative effect of Lund Pseudomonas phage PL1P1 and its composition on chilled meat

[0146] Sixty pieces of chilled fresh meat were randomly divided into six groups (three phage groups, five combination groups, a control group, and a blank group), with ten pieces in each group. The phage experimental groups were sprayed with the test phage (prepared in Example 7) at doses of 1×10⁴ PFU / mL, 1×10⁵ PFU / mL, and 1×10⁶ PFU / mL, respectively, and with 1×10⁵ CFU / mL of *Pseudomonas lundii*. The control group was given 1×10⁵ CFU / mL of *Pseudomonas lundii*. The blank group was sprayed with an equal volume of physiological saline. The shelf life and spoilage rate of the chilled fresh meat were recorded.

[0147] Table 15 Effects of Lund Pseudomonas phage PL1P1 and its composition on chilled meat

[0148]

[0149] Table 15 shows that the spoilage rate of the control group's chilled meat reached 100% after 15 hours of inoculation. However, in the phage experimental groups, the higher the concentration of phage PL1P1, the lower the spoilage rate of the chilled meat within the same time frame; when the phage PL1P1 concentration was 106 PFU / mL, the spoilage rate of the chilled meat remained at 10% after 15 hours of spraying. This indicates that *Pseudomonas lundii* phage and its composition can be used as a biocidal agent to effectively prevent the spoilage of chilled meat.

[0150] Example 19: Preparation and use of a kit for Lund Pseudomonas phage PL1P1 and its composition

[0151] The kit contains 5–10 mL of liquid or a combination of Lund's Pseudomonas phage PL1P1 with a titer of 1 × 10⁷ PFU / mL, 1 LTSB semi-solid medium, and 1 LTSA medium.

[0152] The kit is used as follows: Take a liquid or a combination of 1×10⁷ PFU / mL Lund's P. phage PL1P1, and determine the lysis spectrum of the test phage using the double-layer plate drop method. Pick a single colony to be tested and inoculate it into the target liquid medium. Incubate at the target temperature with shaking, taking into account the growth characteristics of the test strain, to prepare the bacterial suspension of the test strain. Mix 300 μL of the bacterial suspension of the test strain with 5 mL of TSB semi-solid medium and spread it on a TSA plate. Drop 10 μL of either liquid or a combination of Lund's P. phage PL1P1 onto the plate. After air drying, incubate at the target temperature according to the growth characteristics of the test strain and observe the results.

[0153] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A bacteriophage for efficiently lysing *Pseudomonas lundense*, characterized in that: The bacteriophage is *Pseudomonas lundii* bacteriophage PL1P1, with accession number CCTCCM20251191.

2. The bacteriophage for efficiently lysing *Pseudomonas lundense* according to claim 1, characterized in that: The Lund Pseudomonas bacteriophage is a virulent bacteriophage with a polyhedral head and a relatively long tail. The major diameter L is 88 nm and the transverse diameter W is 83 nm, with L / W ≈ 1.

06. The tail includes a constrictible muscle sheath and tail filaments. The muscle sheath is approximately 135 nm long and approximately 20 nm wide.

3. The bacteriophage for efficiently lysing *Pseudomonas lundense* according to claim 1, characterized in that: The Lund Pseudomonas phage, when cultured for 24 hours under conditions of MOI=0.000001, achieved a titer of 6.4×10¹⁰ PFU / mL or higher. Furthermore, the titer did not decrease by more than one order of magnitude when stored at 4℃ for 12 months, showed no significant decrease in titer when stored at 25℃ for 4 weeks, and decreased by more than one order of magnitude in titer when stored at 40℃ for 72 hours.

4. The bacteriophage for efficiently lysing *Pseudomonas lundense* according to claim 1, characterized in that: The Lund Pseudomonas phage exhibits resistance at pH 5-10, with a titer reduction of no more than 4 orders of magnitude within 96 hours. At pH 3-4 and pH 11-12, the titer reduction is no more than 3 orders of magnitude within 96 hours. After 8 hours of ultraviolet radiation at a distance of 20W and 20cm, the titer reduction is no more than 1 order of magnitude.

5. A composition of *Pseudomonas lundii* bacteriophage, characterized in that: The bacteriophage of *Pseudomonas lundii* as described in any one of claims 1-4, and a chemical bactericide, wherein the chemical bactericide is at least one of citric acid, nisin, potassium sorbate, and sodium dehydroacetate, wherein when citric acid is used, a 700-fold dilution of citric acid is used, and the bacteriophage is mixed with the citric acid solution in equal volumes and coexisted at 25°C for 48 hours.

6. The application of the bacteriophage according to any one of claims 1-4 in the preparation of a Pseudomonas lundii detection product, characterized in that: The detection products include reagents or kits for rapid detection of *Pseudomonas lundii* in samples or screening of target pathogens in clinical samples.

7. The application of the bacteriophage according to any one of claims 1-4, characterized in that: In 10¹PFU / mL~10 8 The PFU / mL potency range shows an inhibition rate of 54%~95% against *Pseudomonas lunidosa*, which can be used to prepare biological agents for the prevention and treatment of *Pseudomonas lunidosa* infection.

8. The application of the bacteriophage according to any one of claims 1-4, characterized in that: The Lund Pseudomonas phage is used to prepare products for disinfecting and decontaminating water distribution systems, irrigation facilities, aquaculture facilities, public and private facilities, or other environmental surfaces. The products include detergents, disinfectants, and stain removers, which are used by liquid immersion, spraying, or in combination with an aqueous carrier.

9. The application of the phage composition according to claim 5, characterized in that: It is used as an active ingredient in chilled meat preservatives to prevent spoilage of chilled meat caused by Pseudomonas lundii.

10. The application of the phage composition according to claim 5, characterized in that: The composition is used to prepare biological agents for the prevention and treatment of *Pseudomonas lundii* infection, or to prepare products for the disinfection and decontamination of environmental surfaces such as water distribution systems and irrigation facilities, wherein the composition has no antagonistic effect with the combined substances.

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