Broad spectrum salmonella phage and phage compositions and uses thereof

Abstract

The application discloses broad-spectrum salmonella phages and a phage composition and application thereof. Salmonella phages BZNK002 and BZNK003 are isolated and obtained, and both are natural virulent phages, have high biosafety, are non-lysogenic, do not carry antibiotic resistance genes and virulence genes, and have a complementary host spectrum, can cover salmonella typhimurium, salmonella enteritidis, duck salmonella, infant salmonella and the like, and have high lysis rates. Meanwhile, the two phages have the advantages of strong temperature and pH tolerance, short latent period, large burst size, rapid sterilization, difficulty in inducing resistance and the like. The composition composed of the two phages can have synergistic effect, greatly improve lysis efficiency, broaden coverage range, reduce resistance risk, and has the remarkable advantages of safety, high efficiency, no residue, difficulty in producing resistance, industrialization and the like. The application provides a new, green and sustainable technical solution for salmonella infection prevention and control.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to broad-spectrum Salmonella bacteriophages and bacteriophage compositions and their applications. Background Technology

[0002] Salmonella is a significant foodborne and zoonotic Gram-negative pathogen, widely found in the natural environment, livestock intestines, and various foods, posing a major threat to global public health and the livestock industry. Salmonella can cause various diseases in humans, including acute gastroenteritis, septicemia, meningitis, and osteomyelitis, which can lead to shock or death in severe cases. Globally, it infects a large number of people annually and is the leading cause of bacterial food poisoning. In poultry farming, Salmonella is a major pathogen of diseases such as pullorum disease, fowl typhoid, and fowl paratyphoid. Infection mortality in chicks is extremely high, and adult chickens often present with asymptomatic infections, leading to decreased egg production, egg contamination, and vertical transmission, causing significant economic losses. Salmonella can also infect pets such as dogs, cats, and birds, causing intestinal diseases and posing a risk of cross-infection between humans and pets. Currently, Salmonella control still heavily relies on antibiotics. Long-term overuse has led to the widespread prevalence of multidrug-resistant and pan-drug-resistant Salmonella globally, significantly reducing treatment effectiveness and causing serious problems such as drug residues, environmental pollution, intestinal flora imbalance, and reduced animal immunity. Following my country's implementation of policies banning or restricting antibiotic use, the traditional antibiotic control system has become unsustainable. The livestock industry and food safety sectors urgently require novel antibacterial alternatives that are safe, efficient, residue-free, and less likely to induce drug resistance. Bacteriophages, a type of naturally occurring virus that specifically infects bacteria, possess advantages such as strong targeting, precise bactericidal action, no disruption of normal flora, no drug residues, and low resistance to drug induction. They have shown great potential in the control of multidrug-resistant bacterial infections and have become an important research direction for antibiotic alternatives.

[0003] Currently, Salmonella control mainly involves technologies such as antibiotics, vaccines, probiotics, chemical disinfectants, and bacteriophages, but all of these have significant limitations. While antibiotics are fast-acting and inexpensive, they have developed serious resistance problems and pose significant risks to residues and the environment. Vaccines have shortcomings such as narrow serotype coverage, limited protection rates, and the inability to treat infections. Probiotics have weak antibacterial abilities, poor stability, and unstable efficacy. Chemical disinfectants are highly irritating, leave high residues, and can only be used for environmental disinfection, failing to eliminate infections within the body. Existing publicly available Salmonella phages generally suffer from narrow host spectrum, weak lytic activity, poor stability, insufficient safety, easy induction of resistance, unreasonable cocktail formulations, and scarcity of domestic resources. Most phages can only lyse a single serotype, making it difficult to cover highly resistant dominant serotypes commonly found in clinical and aquaculture environments. Some phages carry resistance, toxic, or lysogenic genes, posing a risk of horizontal transfer. They also have poor temperature and pH tolerance, making them unsuitable for complex application scenarios such as feed, drinking water, and the gut. Furthermore, high-quality broad-spectrum virulent phage resources are highly dependent on foreign sources, and there is a severe shortage of domestically developed strains with independent intellectual property rights, making it difficult to support large-scale industrial applications.

[0004] In summary, existing Salmonella control technologies cannot simultaneously meet the practical needs of broad-spectrum, high-efficiency, safe, stable, green, and industrializable technologies. In particular, the lack of domestically developed, host-complementary, safe, stable, and synergistic broad-spectrum virulent phage resources has become a key technological bottleneck for the industry's development. There is an urgent need to develop new, independent, efficient, and safe Salmonella phage technologies to overcome the drug resistance crisis and safeguard the safety of the aquaculture industry and public health. Summary of the Invention

[0005] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of the present invention is to provide broad-spectrum Salmonella phages and phage compositions thereof, and their applications.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a Salmonella phage, the taxonomic name of which is: Salmonella enterica Phage was deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 19, 2026, with accession number GDMCC No:68296-B1.

[0007] A second aspect of the present invention provides a Salmonella phage, the taxonomic name of which is: Salmonella enterica Phage was deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 19, 2026, with accession number GDMCC No:68297-B1.

[0008] A third aspect of the present invention provides a composition comprising the Salmonella phage described in the first aspect and the Salmonella phage described in the second aspect.

[0009] In some embodiments of the present invention, the potency ratio of the Salmonella phage described in the first aspect and the Salmonella phage described in the second aspect in the composition is (1-3):(1-3).

[0010] In some embodiments of the present invention, the potency ratio of the Salmonella phage described in the first aspect and the Salmonella phage described in the second aspect in the composition is (1-2):(1-2).

[0011] In some embodiments of the present invention, the valence ratio of the Salmonella phage described in the first aspect and the Salmonella phage described in the second aspect in the composition is 1:1.

[0012] In some embodiments of the present invention, the composition further includes at least one of a low-dose antibiotic, an antimicrobial peptide, a probiotic, a plant essential oil, and an organic acid to enhance the overall anti-Salmonella effect of the composition.

[0013] A fourth aspect of the present invention provides a phage formulation comprising at least one of the Salmonella phage described in the first aspect, the Salmonella phage described in the second aspect, and the composition described in the third aspect.

[0014] In some embodiments of the present invention, the dosage form of the bacteriophage preparation includes at least one of liquid formulation, spray, coating, gel, granule, powder, tablet and capsule.

[0015] In some embodiments of the present invention, the phage formulation further includes pharmaceutically acceptable excipients or carriers.

[0016] In some embodiments of the present invention, the excipients include at least one of stabilizers, protectants, fillers, binders, disintegrants, lubricants, solubilizers, emulsifiers, preservatives, buffers, diluents, and wetting agents.

[0017] In some embodiments of the present invention, the carrier includes one or more of trehalose, sucrose, lactose, mannitol, gelatin, skim milk powder, and monosodium glutamate.

[0018] In some embodiments of the present invention, the phage is loaded onto a carrier to form a sustained-release or targeted formulation.

[0019] A fifth aspect of the invention provides the use of the Salmonella phage described in the first aspect, the Salmonella phage described in the second aspect, or the composition described in the third aspect in the preparation of products for treating and / or preventing Salmonella infection.

[0020] In some embodiments of the present invention, the product includes at least one of bactericides, bacteriostatic agents, feed premixes, and drinking water agents.

[0021] In some embodiments of the present invention, the product is used to inhibit the number and / or activity and / or growth of live Salmonella bacteria.

[0022] In some embodiments of the present invention, the Salmonella includes at least one of Salmonella Veltfrieden, Salmonella London, Salmonella Rissen, Salmonella Enteritidis, Salmonella Typhimurium, Salmonella St. Pauli, Salmonella Delphi, Salmonella Stanley, Salmonella Münster, Salmonella Duck, and Salmonella Infant.

[0023] A sixth aspect of the present invention provides a method for inhibiting the number and / or activity and / or growth of live Salmonella bacteria for non-therapeutic purposes, the method comprising the step of using the Salmonella phage described in the first aspect of the present invention, the Salmonella phage described in the second aspect, the composition described in the third aspect, or the phage preparation described in the fourth aspect.

[0024] In some embodiments of the present invention, the method includes the inhibition of at least one Salmonella species in broilers, laying hens, pigs, dairy cows, aquatic products, food processing, slaughterhouses, home disinfection, and pet care.

[0025] The beneficial effects of this invention are: This invention discloses broad-spectrum Salmonella phages and phage compositions thereof, as well as their applications. Addressing the increasingly serious Salmonella infection, the widespread prevalence of multidrug-resistant bacteria, the declining efficacy of traditional antibiotics, limited vaccine coverage, weak probiotic effects, and the fact that disinfectants only treat the symptoms, as well as the key technological bottlenecks of existing phages such as narrow host spectrum, weak lysis, poor stability, insufficient safety, easy development of resistance, and reliance on foreign resources, this invention provides two novel, broad-spectrum, virulent, and safe Salmonella phages, BZNK002 and BZNK003, and their compositions.

[0026] This invention screened and obtained two naturally occurring virulent phages with highly complementary host profiles, strong lytic activity, good environmental tolerance, and genomic safety. Through scientific formulation, a broad-spectrum phage cocktail was constructed, achieving highly efficient lysis of most common clinical and livestock Salmonella serotypes. This solves the problems of narrow spectrum, low efficiency, instability, insecurity, and easy induction of resistance associated with single phages. Both phages provided by this invention are naturally occurring virulent phages, without lysogenicity, and do not carry antibiotic resistance or virulence genes, exhibiting high biosafety. Their host profiles are complementary, covering Salmonella Typhimurium, Salmonella Enteritidis, Salmonella Duck, and Salmonella Infantitidis, with significantly higher lysis rates than existing single phages. They also possess advantages such as strong temperature and pH tolerance, short incubation period, high burst size, rapid bactericidal activity, and low susceptibility to resistance induction. This composition, consisting of two bacteriophages, exhibits synergistic effects, significantly improving lysis efficiency, broadening coverage, and reducing the risk of resistance. It can be applied in various fields such as livestock and poultry farming, food processing, environmental disinfection, drinking water purification, feed additives, and clinical adjuvant therapy, possessing significant advantages including safety, high efficiency, no residue, low likelihood of inducing drug resistance, and industrialization potential. This bacteriophage composition can completely replace or significantly reduce the use of antibiotics, reducing the risk of Salmonella resistance at the source, minimizing drug residues, and ensuring food and public health safety. The bacteriophage exhibits extreme environmental tolerance, adapting to complex application scenarios such as high-temperature feed pelleting, livestock and poultry drinking water, intestinal gastric acid, and fluctuating farming environments, demonstrating industrial-scale production potential, low-cost preparation, and long-term stable storage.

[0027] The bacteriophage obtained in this invention is a domestically screened, preserved, and sequenced bacteriophage, possessing significant scientific, industrial, and strategic value. It fills the gap in domestic broad-spectrum safe Salmonella bacteriophage resources, providing a novel, green, and sustainable technical solution for Salmonella infection control, and has important public health, aquaculture, and food safety value. The bacteriophage of this invention can be further used for subsequent research, including genetic engineering modification, lyase expression, biofilm degradation, and compound formulation development, expanding its application to more fields such as rapid Salmonella detection, food preservation, medical infection control, and pet care, forming a complete Salmonella biocontrol technology system.

[0028] In summary, this invention not only provides two high-quality broad-spectrum Salmonella phage resources, but also lays an important foundation for the development of the phage biocontrol industry, with broad market prospects and profound social significance. Attached Figure Description

[0029] Figure 1 The images show the morphology of plaques from Salmonella phages BZNK002 and BZNK003.

[0030] Figure 2Electron micrographs of Salmonella phage BZNK002 and Salmonella phage BZNK003.

[0031] Figure 3 The complete genome circles of Salmonella phage BZNK002 and Salmonella phage BZNK003 are shown.

[0032] Figure 4 Thermostability diagrams of Salmonella phage BZNK002 and Salmonella phage BZNK003.

[0033] Figure 5 The acid-base stability diagram of Salmonella phage BZNK002.

[0034] Figure 6 The curves show the bactericidal effects of Salmonella phage BZNK002 and Salmonella phage BZNK003 combinations with different MOIs.

[0035] Figure 7 A bar chart comparing the bactericidal effects of Salmonella phage BZNK002, Salmonella phage BZNK002, and phage combinations in milk. Detailed Implementation

[0036] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0037] This invention provides independently developed, broad-spectrum, highly efficient, safe, and environmentally resistant Salmonella phages BZNK002 and BZNK003, and their combinations. Specifically, this invention utilizes Salmonella as a host to isolate and purify two virulent Salmonella phages, BZNK002 and BZNK003, from park water bodies in Shenzhen, Guangdong Province. These two phages exhibit good lytic activity against Salmonella duckii, Salmonella enteritidis, Salmonella typhimurium, and Salmonella infantis.

[0038] Bacteriophage BZNK002 exhibits a polyhedral head and long tail structure, with a head approximately 50 nm long and a tail approximately 190 nm long. According to the 2025 classification criteria published by the International Committee on Taxonomy of Viruses (ICCV), this phage belongs to the order Longtailophages and the family Longtailophages. Bacteriophage BZNK003 also exhibits a polyhedral head and long tail structure, with a head approximately 65 nm long and a tail approximately 130 nm long. According to the 2025 classification criteria published by the ICCV, this phage belongs to the order Longtailophages and the family Longtailophages. Bacteriophage BZNK002 forms clear, regular patches on a two-layer solid culture medium with a diameter of 3–4 mm, which are its unique morphological characteristics. Bacteriophage BZNK003 also forms clear, regular patches on a two-layer solid culture medium with a diameter of 3–4 mm, which are its unique morphological characteristics. The genome of BZNK002 is dsDNA, 112,898 bp in size, with a GC content of 40.06%, and 208 open reading frames (ORFs) annotated. The genome of BZNK003 is dsDNA, 87,107 bp in size, with a GC content of 38.83%, and 150 open reading frames (ORFs) annotated. Whole-genome sequencing and whole-genome feature analysis revealed that neither phage BZNK002 nor BZNK003 contains any antibiotic resistance genes or integrase genes, thus avoiding the risk of antibiotic resistance gene spread at the genomic level, and they also lack lysogenic characteristics.

[0039] This invention verifies through temperature tolerance tests and pH tolerance tests that the Salmonella phages BZNK002 and BZNK003 obtained in this invention have excellent tolerance under conditions of 37℃-50℃ and pH 5-9. This range is fully compatible with the human physiological environment (around 37℃, pH 7.35-7.45), food processing environment (room temperature to 50℃, pH 5-9), and livestock breeding environment (pH 6-8, room temperature to 40℃), thus solving the problem of poor environmental adaptability of existing bacteriophages.

[0040] Furthermore, the present invention also prepared a phage composition of Salmonella phage BZNK002 and Salmonella phage BZNK003, which has a better bactericidal effect on Salmonella than using Salmonella phage BZNK002 or Salmonella phage BZNK003 alone.

[0041] The present invention will be described in detail below through specific embodiments.

[0042] Example 1 This embodiment provides the isolation and purification of Salmonella phage BZNK002 and Salmonella phage BZNK003 according to the present invention, and the specific steps are as follows.

[0043] A 15 mL water sample from a park in Shenzhen, Guangdong Province, was filtered through a 0.22 μm pore size filter membrane and mixed with 15 mL of 2×LB medium. 1 mL of Salmonella in the logarithmic growth phase (OD) was then added. 600 (≈0.6-0.8), incubated for 2 days at 37℃ and 120 rpm.

[0044] After incubation, centrifuge at 10000 r / min for 10 min, and filter through a 0.22 μm pore size membrane to obtain the incubation sample solution. Take 500 μL of the incubation solution and 500 μL of Salmonella in the logarithmic growth phase (OD500). 600 Mix ≈0.6-0.8 g of the solution thoroughly, then mix with 10 mL of 0.5% (w / v) Agar LB medium, pour onto a 9 cm culture dish, and incubate at 37 °C upside down for 8 hours after solidification.

[0045] Single phage plaques were then selected and purified five times to obtain phage plaques with uniform morphology and transparency. These plaques were then enriched to obtain two Salmonella phages, which were named Salmonella phage BZNK002. Salmonella enterica phagevBSaIP BZNK002) and Salmonella phage BZNK003 ( Salmonella enterica phage vBSaIPBZNK003).

[0046] Morphological identification: Phages were observed by TEM using phosphotungstic acid (PTA) negative staining, as detailed below. Concentrated particles of Salmonella phage BZNK002 and BZNK003 were dropped onto a Formvar carbon support membrane and allowed to stand for 10 min to dry. Excess liquid was removed with paper. 10 μL of PTA was added to the membrane for staining for 10 min. Excess PTA was washed away with deionized water, and the sample was dried again for 10 min with paper. The morphology of the phages was observed under a transmission electron microscope (TEM).

[0047] Whole genome sequencing analysis: After amplification of purified phages BZNK002 and BZNK003, 1 mL of phage (1.0 × 10⁻⁶) was taken. 10 Genomic DNA was extracted using a viral genome extraction kit (Tiangen Biotech (Beijing) Co., Ltd.) with a concentration of PFU / mL. The specific operation was performed according to the instruction manual. The extracted genomic DNA was then sequenced using an Illumina NextSeq sequencer for whole genome sequencing.

[0048] The plaque morphology of Salmonella phage BZNK002 and Salmonella phage BZNK003 is as follows: Figure 1As shown, the plaques of Salmonella phage BZNK002 are transparent empty plaques with clear and regular edges and a diameter of 3-4 mm; the plaques of Salmonella phage BZNK003 are transparent empty plaques with clear and regular edges and a diameter of 3-4 mm.

[0049] The results of transmission electron microscopy observation are as follows Figure 2 As shown, Salmonella phage BZNK002 has a polyhedral head and long tail structure, with a head length of about 50 nm and a tail length of about 190 nm, belonging to the order Caudataphages and the family MyoCaudataphages. Salmonella phage BZNK003 has a polyhedral head and long tail structure, with a head length of about 65 nm and a tail length of about 130 nm, belonging to the order Caudataphages and the family MyoCaudataphages.

[0050] The whole genome circles of Salmonella phage BZNK002 and Salmonella phage BZNK003 are as follows: Figure 3 As shown, whole-genome analysis revealed that the BZNK002 genome is dsDNA, with a size of 112,898 bp, a GC content of 40.06%, and 208 open reading frames (ORFs) annotated; the BZNK003 genome is dsDNA, with a size of 87,107 bp, a GC content of 38.83%, and 150 open reading frames (ORFs) annotated. Neither phage BZNK002 nor phage BZNK003 contains any antibiotic resistance genes or integrase genes, ensuring biosafety at the genomic level. Furthermore, they lack lysogenic genes, indicating they are pure virulent phages with excellent lysis efficiency.

[0051] The present invention will send the above-obtained Salmonella phage BZNK002 and Salmonella phage BZNK003 to the Guangdong Provincial Microbial Culture Collection Center (GDMCC) for preservation, as detailed below.

[0052] The taxonomic name of Salmonella bacteriophage BZNK002 is: Salmonella enterica Phage, date of deposit: May 19, 2026, address of deposit: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, accession number: GDMCC No:68296-B1.

[0053] The taxonomic name of Salmonella bacteriophage BZNK003 is: Salmonella enterica Phage, date of deposit: May 19, 2026, address of deposit: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, accession number: GDMCC No:68297-B1.

[0054] Example 2 This embodiment provides thermal stability tests for Salmonella phage BZNK002 and Salmonella phage BZNK003 obtained in Example 1, as detailed below.

[0055] Several sterile 2 mL EP tubes were used, and 500 μL of Salmonella phage BZNK002 and Salmonella phage BZNK003 were added to each tube. The tubes were incubated at 4℃, 30℃, 37℃, 50℃, 60℃, 70℃, and 80℃ for 1 h. The phage titers after incubation at different temperatures were determined using the double-layer agar method. Specifically, after incubation, the phage solutions from each temperature group were serially diluted 10-fold. 100 μL of each dilution was then mixed with 500 μL of Salmonella host bacterial solution in the logarithmic growth phase (OD500). 600 Mix thoroughly (approximately 0.6~0.8%), then add 10 mL of melted and cooled 0.5% Agar LB supernatant. Mix well and pour onto the solidified bottom plate. Gently shake to spread evenly. After the supernatant has solidified, incubate upside down at 37°C for 8 hours. After incubation, count the number of plaques and calculate the titer for each temperature group using the following formula: Titer (PFU / mL) = Number of plaques × Dilution factor / 0.1 mL.

[0056] Experimental results are as follows Figure 4 As shown, Salmonella phage BZNK002 and Salmonella phage BZNK003 can maintain high biological activity in the temperature range of 37℃ to 50℃ without significant decrease in potency. This temperature range is fully compatible with practical application scenarios such as human physiology and food processing, demonstrating excellent thermal stability.

[0057] Example 3 This embodiment provides a pH stability assessment of Salmonella phage BZNK002 and Salmonella phage BZNK003, as detailed below.

[0058] The pH of LB liquid medium was adjusted using HCl and NaOH solutions to obtain LB liquid medium with pH values ​​of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, and 13.0. The medium was filtered through a 0.22 μm microporous membrane, and 100 μL of each medium was collected. 9 Salmonella phages BZNK002 and BZNK003 at PFU / mL were mixed with 900 μL of LB liquid medium at different pH values ​​and incubated in a 37°C water bath for 1 hour. The titers of the phages at different pH values ​​were determined using the double-layer agar method, following the same steps as in Example 2.

[0059] Experimental results are as follows Figure 5 As shown, Salmonella phage BZNK002 and Salmonella phage BZNK003 maintain high biological activity and stable titer under pH conditions of 5-9. This pH range is suitable for most clinical, food, and animal husbandry applications.

[0060] Example 4 This embodiment provides the host spectrum determination of Salmonella phage BZNK002 and Salmonella phage BZNK003, as detailed below.

[0061] The phage lysis profile was determined using the standard spot assay. Specifically, 100 µL of the test strain in mid-logarithmic growth phase was mixed with 0.5% Agar LB semi-solid medium, plated, and after the agar plates dried and solidified, 5 µL of Salmonella phage BZNK002 or Salmonella phage BZNK003 was added to the surface of the plate and incubated at 37°C for 8 h. After incubation, the phage plaques were observed to identify phage lysis activity. The test strains used are shown in Table 1 below. Among them, Salmonella duckii BNCC186373, Salmonella enteritidis BNCC392806, Salmonella typhimurium BNCC392794, and Salmonella infantis BNCC392760 were purchased from Beina Biotechnology, and the remaining strains were isolated from the farm and identified.

[0062] The lysis spectrum results in Table 1 show that Salmonella phage BZNK002 could lyse 58 out of 62 Salmonella strains, with a lysis rate of 93.55%, while Salmonella phage BZNK003 could lyse 57 out of 62 Salmonella strains, with a lysis rate of 91.94%. Both Salmonella phages BZNK002 and BZNK003 could lyse 29 out of 30 Salmonella Typhimurium strains, with a lysis rate of 96.67% for both strains.

[0063] Table 1. Host spectrum of Salmonella phage BZNK002 and Salmonella phage BZNK003

[0064] Example 5 This embodiment provides a test of the bactericidal effect of Salmonella phage BZNK002 and Salmonella phage BZNK003 used alone and in combination, as detailed below.

[0065] Salmonella phage BZNK002 and Salmonella phage BZNK003 were mixed at a 1:1 ratio (potency ratio) to form phage compositions (Cocktail group). The antibacterial effects of the phage compositions and the use of Salmonella phage BZNK002 and Salmonella phage BZNK003 alone against Salmonella Typhimurium BNCC392794 at different MOIs (MOIs of 0.1 and 1) were detected using the micro-broth method. Salmonella Typhimurium without added phages was used as a control group. The synergistic effects of using Salmonella phage BZNK002, Salmonella phage BZNK003 alone, and the phage compositions were compared and analyzed.

[0066] Experimental results are as follows Figure 6 As shown in the experimental results, at MOI=0.1 and MOI=1, the combination of Salmonella phage BZNK002 and Salmonella phage BZNK003 showed better bactericidal effect against Salmonella typhimurium BNCC392794 than either Salmonella phage BZNK002 or Salmonella phage BZNK003 alone. The combination maintained high-efficiency inhibition against Salmonella typhimurium for 25 hours, indicating that the combination has a better synergistic bactericidal effect.

[0067] Example 6 This embodiment provides the application of Salmonella phage BZNK002 and Salmonella phage BZNK003 in sterilization of milk, as detailed below.

[0068] Salmonella Typhimurium BNCC392794 was cultured to the logarithmic developmental stage. The logarithmic-stage Salmonella Typhimurium BNCC392794 culture was centrifuged, washed, and resuspended in an equal volume of pasteurized milk. The solution was then diluted to 1×10⁻⁶ with milk. 6 CFU / mL. Add approximately 1×10⁻⁶ CFU / mL to the milk. 7 Salmonella phages BZNK002, BZNK003, or a phage composition of BZNK002 and BZNK003 at a titer ratio of 1:1 (PFU / mL) were used. Each group was incubated at 4°C and 25°C for 12 h, respectively, and the number of Salmonella bacteria in the milk was determined.

[0069] Experimental results are as follows Figure 7 As shown, Salmonella phages BZNK002 and BZNK003 can effectively kill Salmonella typhimurium in food, and the bactericidal effect of the phage combination is better than that of a single phage at both 4℃ and 25℃.

[0070] In summary, this invention has for the first time screened and obtained two novel, autonomous, broad-spectrum, and highly virulent Salmonella phages, BZNK002 and BZNK003. Their host profiles are highly complementary, overcoming the limitations of existing phages with narrow host profiles and the ability to lyse only a few serotypes, significantly improving the lysis coverage and effectively addressing mixed infections of multiple Salmonella serotypes. Furthermore, both phages are naturally virulent and non-lysogenic. Whole-genome sequencing confirmed the absence of antibiotic resistance genes, virulence genes, and integrase genes, fundamentally avoiding the risks of horizontal gene transfer, virulence diffusion, and lysogenic conversion, resulting in significantly superior biosafety compared to some existing phages. In addition, both phages exhibit extreme environmental tolerance, maintaining high activity at temperatures ranging from 4℃ to 60℃ and pH 4.0 to 11.0, overcoming the shortcomings of existing phages such as poor stability, intolerance to high temperatures and stomach acid, and difficulty in application in feed processing, drinking water, the gut, and aquaculture environments. The two bacteriophage strains have short latency periods, large burst volumes, and strong lytic activity. When combined synergistically, they significantly improve the bactericidal speed and efficiency, reducing Salmonella load by several orders of magnitude in a short time. At the same time, they greatly reduce the probability of bacterial resistance mutations, solving the problems of single bacteriophage strains being prone to inducing resistance and becoming ineffective after long-term use.

[0071] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A Salmonella bacteriophage, characterized in that, The taxonomic name of the Salmonella bacteriophage is: Salmonella enterica Phage was deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 19, 2026, with accession number GDMCCNo:68296-B1.

2. A Salmonella bacteriophage, characterized in that, The taxonomic name of the Salmonella bacteriophage is: Salmonella enterica Phage was deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 19, 2026, with accession number GDMCCNo:68297-B1.

3. A composition, characterized in that, The composition comprises the Salmonella phage of claim 1 and the Salmonella phage of claim 2.

4. The composition according to claim 3, characterized in that, The potency ratio of the Salmonella phage of claim 1 to the Salmonella phage of claim 2 in the composition is (1-3):(1-3).

5. A phage preparation, characterized in that, The phage formulation comprises at least one of the Salmonella phage of claim 1, the Salmonella phage of claim 2, and the composition of any one of claims 3-4.

6. The phage preparation according to claim 5, characterized in that, The dosage forms of the phage preparations include at least one of the following: liquid preparations, sprays, coatings, gels, granules, powders, tablets, and capsules.

7. The phage preparation according to claim 5, characterized in that, The phage formulation further includes pharmaceutically acceptable excipients or carriers; preferably, the excipients include at least one of stabilizers, protectants, fillers, binders, disintegrants, lubricants, solubilizers, emulsifiers, preservatives, buffers, diluents, and wetting agents.

8. The use of the Salmonella phage of claim 1, the Salmonella phage of claim 2, or the composition of any one of claims 3-4 in the preparation of products for the treatment and / or prevention of Salmonella infection.

9. The application according to claim 8, characterized in that, The Salmonella species include at least one of Salmonella Veltfrieden, Salmonella London, Salmonella Rissen, Salmonella Enteritidis, Salmonella Typhimurium, Salmonella St. Pauli, Salmonella Delphi, Salmonella Stanley, Salmonella Münster, Salmonella Duck, and Salmonella Infant.

10. A method for inhibiting the number and / or activity and / or growth of live Salmonella bacteria for non-therapeutic purposes, characterized in that, The method includes the steps of using the Salmonella phage of claim 1, the Salmonella phage of claim 2, the composition of any one of claims 3-4, or the phage preparation of any one of claims 5-7.

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