Phage Csp-TJDF02 and application thereof in controlling Cronobacter sakazakii

Abstract

The invention discloses a Cronobacter sakazakii bacteriophage Csp-TJDF02 and an application of the Cronobacter sakazakii bacteriophage The phage is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 20252611. The bacteriophage belongs to the muscular tail bacteriophage family, the whole length of a genome is 143, 084 bp, and the bacteriophage does not contain virulence genes and drug-resistant genes and has high biological safety. The Csp-TJDF02 has excellent lysis performance, the optimal infection complex number is as low as 0.001, the incubation period is short (10 min), and the Csp-TJDF02 has good pH stability and heat resistance. Particularly, the bacteriophage can still keep efficient antibacterial activity in a high-protein milk powder matrix and can remarkably inhibit formation of a bacterial biofilm. The bacteriophage provided by the invention can be prepared into a cleaning solution, a spraying agent or a biological bacteriostatic agent, is used for preventing and controlling cronobacter sakazakii in a medical environment, a food processing environment and a food matrix, and effectively reduces infection risk and quality deterioration.

Description

Technical Field

[0001] This application belongs to the fields of bioengineering and food safety technology, specifically relating to a novel lytic Cronobacter sakazakii bacteriophage Csp-TJDF02, and the application of this bacteriophage in inhibiting the proliferation of Cronobacter sakazakii, inhibiting biofilm formation, and in the preparation of disinfection compositions. Background Technology

[0002] *Cronobacter sakazakii* is an important opportunistic foodborne pathogen and the dominant species in the *Cronobacter* genus, exhibiting stronger pathogenicity than other species in the same genus. This bacterium can infect people of all ages, but it can cause severe diseases in newborns, such as meningitis, bacteremia, and necrotizing colitis. *Cronobacter sakazakii* can spread through medical environments, food processing environments, or by attaching to high-risk contact surfaces such as medical devices and food processing equipment, as well as through contaminated food substrates such as powdered infant formula (PIF) or reconstituted high-protein milk powder, thus causing medical and food safety incidents. Therefore, there is an urgent need to develop new technologies for the effective prevention and control of *Cronobacter sakazakii*.

[0003] Currently, chemical disinfectants such as sodium hypochlorite and peracetic acid are commonly used to inactivate Cronobacter sakazakii on the production environment and equipment surfaces during infant formula processing. However, studies have shown that long-term or improper use of these disinfectants can easily lead to equipment corrosion, increased bacterial resistance, and chemical residues, adversely affecting the operating cycle of production equipment and product safety. In high-risk exposure areas such as medical environments and medical devices, although antibiotics are used for preventative disinfection of Cronobacter sakazakii, the continuous increase in bacterial resistance significantly reduces their antibacterial effect, weakens the effectiveness of pollution control, and thus exacerbates the risk of infection in infants and young children, seriously threatening the health of susceptible populations. Therefore, developing green and safe new technologies for the control of Cronobacter sakazakii is of significant practical importance.

[0004] Bacteriophages are a class of viruses that specifically infect and replicate within bacterial hosts. As natural biological disinfectants, they can highly specifically target and lyse foodborne pathogens. These viruses are widely distributed in the natural environment and have advantages such as abundant resources, convenient isolation and purification, low preparation costs, high biosafety, and strong host specificity. In recent years, bacteriophage biological agents have been approved by the U.S. Food and Drug Administration (FDA) for use as food additives in the control of pathogens in poultry and ready-to-eat meat products. However, there are few reports on the research and application of bacteriophages in the remediation of Cronobacter sakazakii contamination in dairy products, especially high-protein infant formula.

[0005] It is worth noting that existing bacteriophages often become significantly inactive in complex food matrices, especially under high-protein, high-osmotic-pressure conditions, due to protein adsorption or interference from environmental factors, making it difficult to exert effective antibacterial effects. Therefore, developing a novel bacteriophage bio-killer with strong targeting, high disinfection efficiency, and the ability to maintain activity in high-protein matrices has become one of the key research directions for solving the problem of Cronobacter sakazakii contamination in dairy products such as milk powder. Summary of the Invention

[0006] The purpose of this application is to provide a bacteriophage Csp-TJDF02 and its application in controlling Cronobacter sakazakii, so as to solve the problems mentioned in the background art.

[0007] This application discloses a strain of Cronobacter sakazakii bacteriophage Csp-TJDF02, which has been deposited at the China Center for Type Culture Collection on November 20, 2025, with accession number CCTCC NO: M 20252611.

[0008] This application discloses a Cronobacter sakazakii disinfection composition comprising bacteriophage Csp-TJDF02 as described above.

[0009] In a preferred embodiment, the disinfectant composition is in the form of a cleaning solution, a spray, or a bio-inhibitory agent.

[0010] In a preferred embodiment, the bacteriophage Csp-TJDF02 in the disinfection composition is used alone or in combination with other Cronobacter sakazakii bacteriophages.

[0011] In a preferred embodiment, the isolate or culture of the bacteriophage Csp-TJDF02 is used as the active ingredient in the disinfection composition.

[0012] This application discloses the use of the bacteriophage Csp-TJDF02 described above or the disinfection composition described above in inhibiting Cronobacter sakazakii for non-disease diagnosis and treatment purposes.

[0013] In a preferred embodiment, the application is to inhibit the proliferation, metabolism, and / or formation of *Cronobacter sakazakii* biofilm.

[0014] In a preferred embodiment, the application scenarios include medical environments, food processing environments, medical device surfaces, food processing equipment surfaces, or food matrices; preferably, the application is used to reduce or inhibit contamination or quality deterioration caused by Cronobacter sakazakii in the scenarios.

[0015] In a preferred embodiment, the application method includes rinsing or spraying the scene with purified bacteriophage Csp-TJDF02 or the disinfectant composition.

[0016] In a preferred embodiment, the food matrix is ​​high-protein milk powder.

[0017] In a preferred embodiment, the high-protein milk powder is selected from one or more of the following: high-protein infant formula, premature / low birth weight infant formula, children's high-protein milk powder, adult high-protein milk powder, elderly milk powder, sports nutrition milk powder, and high-protein milk powder for special medical purposes; wherein, the high-protein milk powder for special medical purposes includes high-fiber protein milk powder or low-sugar high-protein milk powder.

[0018] This application discloses the use of the bacteriophage Csp-TJDF02 described above in the preparation of a Cronobacter sakazakii disinfectant.

[0019] This application discloses the use of the bacteriophage Csp-TJDF02 described above in the preparation of a medicament for the prevention or treatment of infectious diseases caused by Cronobacter sakazakii.

[0020] Compared with the prior art, this application has the following beneficial effects: (1) A novel bacteriophage with a unique genome structure The bacteriophage Csp-TJDF02 described in this application is a novel lytic Cronobacter sakazakii bacteriophage, deposited at the China Center for Type Culture Collection (CCTCC NO: M 20252611). It possesses a unique genome structure—143,084 bp in length, with a GC content of 46.70% and containing 243 open reading frames. Comparative analysis with professional databases revealed no lysogenic genes, drug resistance genes, or virulence genes, indicating good biocompatibility and suitability for use as a Cronobacter sakazakii disinfectant.

[0021] (2) Excellent lysis performance and extremely low infection multiple. The bacteriophage Csp-TJDF02 of this application exhibits excellent lysis performance—an optimal multiple of infection (MOI) of only 0.001, a short latency period (approximately 10 min), and a high burst rate (approximately 185 PFU / cell). These characteristics indicate that only a very small amount of bacteriophage is needed to efficiently lyse a large number of host bacteria in a short time, resulting in a significant cost advantage in practical applications.

[0022] (3) Good environmental tolerance The bacteriophage Csp-TJDF02 of this application exhibits excellent environmental tolerance—maintaining a survival rate of over 60% within a pH range of 6 to 10, demonstrating good acid and alkali resistance; it also retains high biological activity after treatment at 60℃ for 60 min, making it particularly suitable for application conditions in food processing environments. Simultaneously, treatment at 80℃ for 60 min completely inactivates the bacteriophage, facilitating heat treatment when necessary to ensure no active bacteriophage residue remains in the final product, thus guaranteeing product safety.

[0023] (4) It maintains highly effective antibacterial activity in high-protein milk powder matrix. Of particular importance is that the bacteriophage Csp-TJDF02 of this application maintains highly efficient antibacterial activity in high-protein milk powder matrices. Experimental results show that in high-protein infant formula solutions and mixed high-protein milk powder solutions, the bacterial density of the bacteriophage-treated group was consistently significantly lower than that of the control group throughout the entire storage period. This demonstrates that the bacteriophage of this application overcomes the technical challenge of ordinary bacteriophages being easily adsorbed or inactivated by proteins in complex food matrices. This characteristic makes it particularly suitable for the contamination control of Cronobacter sakazakii in high-protein dairy products.

[0024] (5) Lysis ability against mixed strains The bacteriophage Csp-TJDF02 of this application can not only effectively lyse wild-type Cronobacter sakazakii D17 isolated from milk powder samples, but also simultaneously lyse mixed bacterial cultures of multiple ATCC standard strains (ATCC 21545, ATCC 21569, ATCC 22919, ATCC 29544), showing a broad host spectrum within the tested strain range, and can cope with the diversity of Cronobacter sakazakii in actual contamination situations.

[0025] (6) Inhibition ability on biofilm Cronobacter sakazakii can form biofilms on the surfaces of food processing equipment and medical devices, which are often difficult to remove effectively with chemical disinfectants and antibiotics. The bacteriophage Csp-TJDF02 described in this application can significantly inhibit the formation of Cronobacter sakazakii biofilms on solid surfaces, providing a new approach for contamination control in medical and food processing environments.

[0026] (7) Multiple dosage forms and application methods The bacteriophage Csp-TJDF02 of this application can be prepared into disinfection compositions in various dosage forms such as cleaning solutions, sprays, or biological antibacterial agents. It can be applied to medical environments, food processing environments, medical device surfaces, food processing equipment surfaces, and food matrices by rinsing or spraying, with a wide range of applications and flexible and convenient use.

[0027] Safety Statement The bacteriophage Csp-TJDF02 of this application, as a natural biological material, has the following safety characteristics: Genome safety Whole-genome sequencing and database comparison analysis revealed no lysogen-related genes (screened by the PHASTER database), virulence genes (screened by the VFDB database (2024 update)), or drug resistance genes (screened by the CARD database (version 3.2.5)) in the Csp-TJDF02 genome, nor were any tRNA genes detected. These results indicate that this phage is a strictly lytic phage, lacking the ability to integrate into the host genome and posing no risk of transferring harmful genes to the host bacteria or human gut microbiota.

[0028] Host specificity Bacteriophages are highly host-specific, capable of infecting and lysing only their specific host bacterium—Cronobacter sakazakii—without affecting the normal human gut microbiota or other beneficial microorganisms, nor do they have any adverse effects on non-target organisms.

[0029] International Recognition Trends Bacteriophages, as natural biological disinfectants, have been approved by the U.S. Food and Drug Administration (FDA) for use as food additives in the control of pathogens in ready-to-eat poultry and meat products. The European Food Safety Authority (EFSA) has also conducted safety assessments on various bacteriophage products and given positive feedback. These international regulatory body approvals provide important references for the application of bacteriophages in the field of food safety.

[0030] Controllable thermal deactivation According to the thermal stability test results of this application, bacteriophage Csp-TJDF02 was completely inactivated after treatment at 80℃ for 60 min, indicating that if necessary, appropriate heating processes can be used to ensure that there are no active bacteriophage residues in the final product, thus ensuring product safety.

[0031] Application security The bacteriophage Csp-TJDF02 disclosed in this application is primarily used as a disinfectant for pollution control in medical and food processing environments, as well as for disinfection of medical device surfaces and food processing equipment surfaces, or for processing food substrates. Appropriate processing techniques after treatment ensure product safety.

[0032] Information on the preservation of biological materials The bacteriophage Csp-TJDF02 of this application was deposited at the China Center for Type Culture Collection (CCTCC) on November 20, 2025.

[0033] Address of the depository: Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, China Classification and nomenclature: Cronobacter sakazakii phage Csp-TJDF02 Accession number: CCTCC NO: M 20252611 The specification of this application contains numerous technical features distributed across various technical solutions. Listing all possible combinations of these technical features (i.e., technical solutions) would make the specification excessively lengthy. To avoid this problem, the various technical features disclosed in the above-described invention, the various technical features disclosed in the following embodiments and examples, and the various technical features disclosed in the accompanying drawings can be freely combined to form various new technical solutions (all of which are considered to have been described in this specification), unless such a combination of technical features is technically infeasible. For example, one example discloses feature A+B+C, and another example discloses feature A+B+D+E. Features C and D are equivalent technical means that serve the same function, and technically only one needs to be used; they cannot be used simultaneously. Feature E can technically be combined with feature C. Therefore, the solution A+B+C+D should not be considered as described because it is technically infeasible, while the solution A+B+C+E should be considered as described. Attached Figure Description

[0034] Figure 1 The image shows the morphology of phage Csp-TJDF02, the bacteriophage of this application, on a double-layer nutrient broth plate. As shown in the figure, the phage plaques are circular with neat edges, uniform size, and clear, indicating that the isolated phage is a typical lytic phage.

[0035] Figure 2 The image shows the microscopic morphology of bacteriophage Csp-TJDF02 of this application under a transmission electron microscope (scale bar: 100 nm). As shown in the figure, bacteriophage Csp-TJDF02 has typical myotail bacteriophage morphological characteristics, with an icosahedral symmetry head, a diameter of approximately 75.09 nm, and a tail length of approximately 86.16 nm, possessing a retractable tail sheath structure.

[0036] Figure 3 The figure shows the pH stability test results of the bacteriophage Csp-TJDF02 of this application. As shown in the figure, the bacteriophage maintains a survival rate of over 60% in the pH range of pH 6 to pH 10, demonstrating good pH tolerance.

[0037] Figure 4The figure shows the thermal stability test results of bacteriophage Csp-TJDF02 of this application. As shown in the figure, the bacteriophage maintained high biological activity after treatment at 50℃ and 60℃ for 60 min, but was completely inactivated after treatment at 80℃ for 60 min.

[0038] Figure 5 This diagram illustrates the ability of the bacteriophage Csp-TJDF02 of this application to lyse the host bacteria under different multiples of infection (MOI) conditions. As shown in the figure, the progeny bacteriophage titer is highest when MOI=0.001, therefore the optimal MOI is determined to be 0.001.

[0039] Figure 6 This is a one-step growth curve of the bacteriophage Csp-TJDF02 of this application. As shown in the figure, the phage has a latency period of approximately 10 min, a burst period of approximately 60 min, and a lysis rate of approximately 185 PFU / cell.

[0040] Figure 7 The image shows a complete genome sequence diagram of phage Csp-TJDF02 from this application. As shown, the full-length phage genome is 143,084 bp, with a GC content of 46.70% and 243 open reading frames (ORFs).

[0041] Figure 8 This diagram illustrates the inhibitory effect of the bacteriophage Csp-TJDF02 of this application on the biofilm of mixed Cronobacter sakazakii on a solid surface. As shown in the figure, the amount of biofilm formed in each bacteriophage treatment group was significantly lower than that in the control group, indicating that the bacteriophage can effectively inhibit biofilm formation.

[0042] Figure 9 This diagram illustrates the inhibitory effect of phage Csp-TJDF02 (as described in this application) on mixed Cronobacter sakazakii in high-protein infant formula. As shown, each phage treatment group effectively inhibited bacterial growth throughout the entire storage period, with bacterial density consistently significantly lower than the control group.

[0043] Figure 10 This diagram illustrates the inhibitory effect of the bacteriophage Csp-TJDF02 of this application on mixed Cronobacter sakazakii in high-protein mixed milk powder. As shown in the figure, the bacteriophage exhibits good antibacterial effects on various types of high-protein milk powder products, demonstrating broad product applicability. Detailed Implementation

[0044] In the following description, many technical details are presented to help the reader better understand this application. However, those skilled in the art will understand that the technical solutions claimed in this application can be implemented even without these technical details and various variations and modifications based on the following embodiments.

[0045] Explanation of some concepts: Cronobacter sakazakii is an important opportunistic foodborne pathogen in the genus Cronobacter, and is the dominant species in the genus. It is more pathogenic than other species in the genus, and can infect people of all ages, especially causing severe diseases such as meningitis, bacteremia, and necrotizing colitis in newborns. It often contaminates infant formula products through medical environments, food processing environments, or by adhering to the surfaces of medical instruments and food substrates.

[0046] Bacteriophages are a class of viruses that can specifically infect bacteria and replicate within the bacterial host cell. As natural biological disinfectants, bacteriophages have advantages such as abundant resources, convenient isolation and purification, low preparation cost, high biosafety, and strong host specificity, and can specifically target and lyse foodborne pathogens.

[0047] Lytic phages are a type of bacteriophage that infects host bacteria, directly enters the lytic cycle, causes host cell lysis, and releases progeny phages. Unlike lysogenic phages, which can integrate into the host genome, lytic phages have higher biosafety because they do not carry lysogen-related genes, making them more suitable for use as biocides.

[0048] Lysogenic phages (also known as temperate phages) are types of bacteriophages that, after infecting host bacteria, integrate their genetic material into the host genome and are passed down as prophages along with the host cell during replication. Under specific conditions, lysogenic phages can be induced to enter the lysis cycle.

[0049] Myoviridae is a family within the order Caudovirales, characterized by its retractable tail sheath and typically icosahedral head. The bacteriophage Csp-TJDF02 in this application belongs to this family based on its morphological characteristics.

[0050] dsDNA (double-stranded DNA) is a double-helix structure formed by two complementary deoxyribonucleic acid strands through base pairing. The nucleic acid type of the bacteriophage Csp-TJDF02 in this application is dsDNA.

[0051] An open reading frame (ORF) is a segment of nucleotide sequence in a genomic DNA sequence that may encode a protein, running from the start codon to the stop codon. It is the basic unit for genome annotation and functional analysis. The bacteriophage genome in this application contains 243 ORFs.

[0052] High-protein milk powder refers to formula milk powder products with a relatively high protein content. In this application, it specifically includes high-protein infant formula (including formula for premature / low birth weight infants), high-protein children's formula, high-protein adult formula (including formula for the elderly and sports nutrition formula), and high-protein formula for special medical purposes (including high-fiber protein type and low-sugar high-protein type).

[0053] The multiplicity of infection (MOI) is the ratio of the number of bacteriophages (PFU) to the number of host bacteria (CFU) in a bacteriophage infection experiment. MOI is an important parameter affecting the lysis efficiency of bacteriophages. This application achieves the technical effect of efficiently lysing a large number of host bacteria with a very small number of bacteriophages by optimizing MOI.

[0054] The latency period refers to the time required from the time a bacteriophage infects a host cell to the release of the first progeny bacteriophages. A shorter latency period indicates a faster phage lysis rate. The latency period of the bacteriophage in this application is approximately 10 minutes.

[0055] The burst period, also known as the lysis period, refers to the time when progeny phages are released in large quantities after a bacteriophage infects a host cell. In a one-step growth curve, this is characterized by a rapid increase in phage titer.

[0056] Burst size refers to the average number of progeny phages released from each infected host cell after lysis, and is an important indicator of phage proliferation capacity. A higher burst size indicates higher phage proliferation efficiency. The burst size of the phage in this application is approximately 185 PFU / cell.

[0057] Biofilms are three-dimensional microbial communities formed by bacteria attaching to solid surfaces such as medical devices and food processing equipment, or within food matrices, and secreting matrix substances such as extracellular polysaccharides and proteins. Biofilm formation significantly enhances bacterial resistance to chemical disinfectants and antibiotics; the bacteriophages described in this application can significantly inhibit its formation.

[0058] A disinfection composition refers to a composition containing the bacteriophage Csp-TJDF02 of this application as an active ingredient, used to inhibit or eliminate Cronobacter sakazakii. Its dosage form includes, but is not limited to, cleaning solutions, sprays, or bio-inhibitory agents, and may further contain suitable carriers or stabilizers.

[0059] Disinfectant is a preparation used to kill or inhibit pathogenic microorganisms. In this application, it specifically refers to a preparation made with bacteriophage Csp-TJDF02 as the active ingredient, used for environmental disinfection or surface treatment.

[0060] The double-layer agar method is a classic microbiological method used for the isolation, purification, and titer determination of bacteriophages. It involves mixing bacteriophages and host bacteria in a semi-solid culture medium and then spreading the mixture onto the surface of a solid culture medium to form a double-layer structure. After incubation, the formation of plaques is observed.

[0061] A plaque is a transparent or translucent area formed on a double-layered agar plate after a single bacteriophage particle infects and lyses the surrounding host bacteria. The plaques formed by the bacteriophages in this application are circular, transparent, and have well-defined edges.

[0062] Titer refers to the number of infectious phage particles per unit volume of phage suspension, usually expressed as PFU / mL, and is an important indicator for measuring phage concentration and activity.

[0063] Plaque forming unit (PFU) refers to the number of phages that can form a clear plaque on a bilayer plate, and is used to quantitatively represent the active concentration of phages.

[0064] A colony-forming unit (CFU) is a unit of bacteria that can form a visible colony on a solid culture medium. It is used to quantitatively express the concentration of bacteria.

[0065] SM buffer is a buffer commonly used for the dilution, preservation, and handling of bacteriophages. Its main components include sodium chloride, magnesium sulfate, and Tris-HCl, which can maintain the stability and activity of bacteriophages.

[0066] Survival rate refers to the degree to which a bacteriophage retains its activity under specific conditions (such as different pH and temperature) relative to control conditions (such as neutral pH and room temperature). It is usually expressed as a percentage and is used to evaluate the environmental tolerance of bacteriophages.

[0067] The present application will be further described in detail below with reference to the accompanying drawings and embodiments. Unless otherwise specified, all raw materials and reagents used in this application are commercially available products. It should be noted that the following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of this application. After reading this specification, those skilled in the art can make various modifications and equivalent substitutions to this application without departing from the technical concept of this application, and all such modifications and equivalent substitutions fall within the scope of protection of this application.

[0068] The term "high-protein milk powder" or "high-protein formula milk powder" as used in this application refers to formula milk powder products with a relatively high protein content. Specifically, the high-protein milk powder includes, but is not limited to, the following types: high-protein infant formula, premature / low birth weight infant formula, children's high-protein milk powder, adult high-protein milk powder (including elderly milk powder and sports nutrition milk powder), and special medical purpose high-protein milk powder (including high-fiber protein milk powder and low-sugar high-protein milk powder). Typically, the protein content is ≥15g / 100g (dry basis), or meets the protein content requirements stipulated in national standards such as GB 10765-2021 "National Food Safety Standard for Infant Formula" and GB10767-2021 "National Food Safety Standard for Toddler Formula." The "MOI" mentioned in this application refers to the Multiplicity of Infection, which is the ratio of the number of bacteriophages to the number of host bacteria. It is calculated by dividing the number of bacteriophages (PFU) by the number of bacteria (CFU). The MOI is an important parameter affecting the lysis efficiency of bacteriophages. This application achieves the technical effect of efficiently lysing a large number of host bacteria with a very small number of bacteriophages by optimizing the MOI.

[0069] The "lytic phage" described in this application refers to a type of bacteriophage that, after infecting host bacteria, directly enters the lytic cycle, causing host cell lysis and releasing progeny phages, as opposed to lysogenic phages that can integrate into the host genome. Because lytic phages do not carry lysogen-related genes, they possess higher biosafety and are more suitable for use as biological disinfectants.

[0070] The "biofilm" described in this application refers to a three-dimensional microbial community formed by bacteria attaching to a solid surface and secreting matrix substances such as extracellular polysaccharides and proteins. Biofilm formation is a major reason why *Cronobacter sakazakii* persists on the surfaces of medical devices and food processing equipment, causing contamination, and chemical disinfectants are often ineffective at removing it.

[0071] The "disinfecting composition" described in this application refers to a composition containing the bacteriophage Csp-TJDF02 of this application as an active ingredient, used to inhibit or eliminate Cronobacter sakazakii. The disinfecting composition may further contain suitable carriers, stabilizers, or other auxiliary ingredients, and its dosage form includes, but is not limited to, cleaning solutions, sprays, or bio-inhibitors.

[0072] Statistical analysis methods The experimental data in this application embodiment were statistically analyzed using GraphPad Prism software. Specifically, in some embodiments, the data are expressed as mean ± standard deviation (Mean ± SD), with parallel samples set for each experiment and repeated measurements for each parallel. Differences between groups were compared using one-way ANOVA or t-tests. p < 0.05 indicated a statistically significant difference, p < 0.01 indicated a statistically significant difference, and p < 0.001 indicated a highly statistically significant difference.

[0073] Unless otherwise stated, "suitable temperature" or "constant temperature incubation" in the embodiments of this application generally refers to 37°C; "shaking incubation" generally refers to incubation on a shaker at an appropriate speed (e.g., 150-200 rpm); "centrifugation" generally refers to centrifugation on a conventional benchtop centrifuge at a speed of approximately 8000 rpm; "sterile filter membrane" refers to a microporous filter membrane with a pore size of 0.22 μm. In the double-layer plate method, the agar concentration of the upper semi-solid culture medium is generally 0.5%-0.7%, and the agar concentration of the lower solid culture medium is generally 1.2%-1.5%. Phage titer is expressed in PFU / mL and is calculated by multiplying the plaque count by the dilution factor and then dividing by the inoculation volume. The above experimental conditions are conventional operating conditions in the art, and those skilled in the art can adjust them within a reasonable range according to actual conditions.

[0074] Materials and reagents The bacteriophage host bacterium used in this application was *Cronobacter sakazakii* (strain number: D17), isolated from a milk powder sample using conventional methods and preserved by the Nutrition Department of Shanghai East Hospital. It was a wild-type *Cronobacter sakazakii* strain. In addition, this application also used *Cronobacter sakazakii* ATCC 21545, ATCC 21569, ATCC22919, and ATCC 29544 as standard strains, all purchased from Beijing BioBio Biotechnology Co., Ltd.

[0075] The formulation of the SM buffer used in this application is as follows: NaCl 5.8 g, MgSO4·7H2O 2.0 g, 1 M Tris-HCl (pH 7.4) 50 mL, and deionized water to a final volume of 1 L.

[0076] Example 1: Isolation and purification of bacteriophage Csp-TJDF02 This embodiment details the methods for isolating, screening, purifying, and preserving bacteriophage Csp-TJDF02, providing high-purity, high-activity bacteriophage preparations for subsequent applications.

[0077] Step 100: Sample Collection and Preprocessing Wastewater samples were collected at Shanghai Oriental Hospital in October 2025. After thorough resuspending, the collected wastewater samples were centrifuged to remove large particulate matter and microorganisms such as bacteria. The supernatant was then filtered through a sterile membrane for sterilization before use. The purpose of this pretreatment step was to remove bacteria and impurities from the samples while retaining any potentially present bacteriophage particles.

[0078] Step 200: Phage enrichment The filtrate obtained in step 100 was mixed with the host bacterium D17 and nutrient broth liquid medium (Guangdong Huankai Microbial Technology Co., Ltd., optionally, nutrient broth (NB) medium (catalog number: 022010P1) with the following formulation: peptone 10 g / L, beef extract 3 g / L, NaCl 5 g / L, pH 7.2±0.2), and cultured overnight with shaking at a suitable temperature. After culture, the host bacterial cells were removed by centrifugation and filtration, and the resulting filtrate was the phage enrichment solution. The purpose of enrichment culture is to allow any phages that may be present in the sample to proliferate in large quantities in the presence of the host bacteria, thereby improving the success rate of subsequent screening.

[0079] Step 300: Preliminary phage screening A double-layer plate method was used for preliminary screening of bacteriophages. Specifically, the host bacterium D17 was mixed with a semi-solid nutrient broth medium and poured onto a pre-solidified nutrient broth plate to prepare a double-layer plate. After the upper layer of medium solidified, the bacteriophage enrichment solution obtained in step 200 was added dropwise to the surface of the double-layer plate. After allowing the droplets to dry, the plate was incubated at a suitable temperature. The appearance of transparent phage plaques on the plate was observed; the presence of phage plaques indicated the presence of bacteriophages capable of lysing the host bacterium D17 in the sample.

[0080] Step 400: Phage isolation and purification To obtain a purified single phage line, multiple rounds of isolation and purification are required for the phages obtained from the initial screening. The phage enrichment solution is serially diluted with SM buffer. An appropriately diluted phage suspension is mixed with the host bacteria and then plated using the double-layer agar plate method. A single, well-isolated phage plaque is picked and placed in SM buffer to release the phage. This serial dilution and plaque-picking process is repeated several times until all plaques on the plate exhibit uniform morphology, indicating that a purified single phage line has been obtained.

[0081] Step 500: Phage amplification and titer determination The purified phage was mixed with the logarithmic growth phase host bacterium D17 in nutrient broth liquid medium and cultured with shaking at a suitable temperature. After culture, bacterial residue was removed by centrifugation, and the supernatant was filtered through a sterile filter membrane to obtain the phage amplification solution. The phage titer was determined using the double-layer plate method, and the results showed that the titer of the amplified phage could reach 10. 9 PFU / mL or higher.

[0082] Step 600: Phage Preservation For short-term preservation, the phage amplification solution can be stored at low temperature. For long-term preservation, the phage amplification solution should be mixed with an appropriate amount of sterile glycerol and stored at ultra-low temperature. The phage Csp-TJDF02 of this application was deposited on November 20, 2025, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, with accession number CCTCC NO: M 20252611.

[0083] like Figure 1 As shown, the plaques formed by bacteriophage Csp-TJDF02 on a double-layer nutrient broth plate are circular, with neat edges, uniform size, and clear appearance. These plaque morphological characteristics indicate that the isolated bacteriophage is a typical lytic bacteriophage, capable of efficiently lysing the host bacteria and forming clear, transparent areas.

[0084] Example 2: Morphological characteristics of bacteriophage Csp-TJDF02 This embodiment uses transmission electron microscopy to observe the microscopic morphology of bacteriophage Csp-TJDF02, providing a basis for its taxonomic classification.

[0085] Step 100: Preparation of phage concentrate To obtain a high-purity, high-concentration phage suspension suitable for electron microscopy observation, the phage amplification solution needs to be concentrated and purified. First, nucleases (DNase I and RNase A) are added to the phage amplification solution to remove free nucleic acid substances. Then, NaCl is added and the solution is subjected to an ice bath. After removing impurities by centrifugation, PEG-8000 is added to the supernatant for precipitation and concentration to obtain the concentrated phage solution.

[0086] Step 200: Density gradient ultracentrifugation purification The phages were further purified using cesium chloride density gradient ultracentrifugation. Cesium chloride solutions of different densities were added sequentially to ultracentrifuge tubes, with phage concentrate added to the top layer. After ultracentrifugation, the phage particles formed visible bands at their isodense positions. The target bands were carefully aspirated to obtain a high-purity purified phage concentrate.

[0087] Step 300: Transmission electron microscopy observation Phages were stained using phosphotungstic acid negative staining. The phosphotungstic acid solution was mixed with the concentrated purified phage solution for negative staining. After staining, excess stain was blotted with filter paper and the phages were dried under an infrared lamp. The microscopic morphology of the phages was observed and photographed using a transmission electron microscope.

[0088] like Figure 2 As shown, transmission electron microscopy reveals that bacteriophage Csp-TJDF02 exhibits typical tailed bacteriophage morphology: the head is icosahedral in shape, with a diameter of approximately 75.09 nm; the tail is approximately 86.16 nm long and possesses a retractable tail sheath structure. Based on the classification criteria of the International Committee on Taxonomy of Viruses (ICTV) and its morphological characteristics, bacteriophage Csp-TJDF02 belongs to the family Myoviridae. A characteristic of Myoviridae is the presence of a retractable tail sheath, a structure that facilitates the injection of genetic material into the host cell.

[0089] Example 3: pH stability of bacteriophage Csp-TJDF02 This embodiment investigated the stability of bacteriophage Csp-TJDF02 under different pH conditions, providing a reference for its use in different application scenarios. pH stability is an important indicator for evaluating the practical application potential of bacteriophages as disinfectants, because the pH values ​​of medical environments, food processing environments, and food matrices may differ.

[0090] Step 100: Incubation treatment under different pH conditions Phage Csp-TJDF02 suspensions were added to nutrient broth liquid culture media pre-adjusted to different pH values ​​(pH 2.0 to pH 12.0) and incubated at a suitable temperature for a certain period of time. After incubation, the phage titer of each treatment group was determined using the double-layer plate method.

[0091] Step 200: Survival Rate Calculation The formula for calculating bacteriophage survival rate is as follows: Survival rate (%) = (phage titer of treatment group ÷ phage titer of control group) × 100% …… Formula (1) Survival rate represents the degree to which bacteriophages retain their activity relative to neutral conditions after treatment under specific pH conditions.

[0092] like Figure 3As shown, bacteriophage Csp-TJDF02 is completely inactivated under extremely acidic conditions (pH 2); within the pH range of 6 to 10, the phage still maintains a survival rate of over 60%, demonstrating good pH tolerance; under pH 5 and pH 11 conditions, the survival rate can still be maintained at approximately 40%. These results indicate that bacteriophage Csp-TJDF02 has good acid and alkali resistance and can adapt to the pH ranges commonly found in food processing and medical environments, a characteristic that makes it promising for broad applications.

[0093] Example 4: Thermal stability of bacteriophage Csp-TJDF02 This embodiment investigated the stability of bacteriophage Csp-TJDF02 under different temperature conditions. Thermal stability is another important indicator for evaluating the practical application value of bacteriophages, and it is directly related to the storage stability and usage conditions of the bacteriophage disinfection composition.

[0094] Step 100: Heat treatment under different temperature conditions The phage Csp-TJDF02 suspension was heat-treated in water baths at different temperatures (50℃, 60℃, 70℃, and 80℃). During the heat treatment, samples were taken at different time points, and the phage titer was determined using the double-layer plate method to plot the heat inactivation curve.

[0095] like Figure 4 As shown, bacteriophage Csp-TJDF02 exhibits good thermal stability: after treatment at 50℃ and 60℃ for 60 min, the bacteriophage still retains 10% of its original temperature. 8 The phage exhibited high bioactivity at PFU / mL; after treatment at 70℃ for 60 min, the phage still retained 10 PFU / mL. 2 The bioactivity was measured at PFU / mL; however, after treatment at 80℃ for 60 min, the phage was completely inactivated.

[0096] The above results have important practical significance: on the one hand, bacteriophage Csp-TJDF02 has good stability under room temperature and moderate temperature conditions, which is convenient for storage and transportation; on the other hand, heat treatment at 80℃ can completely inactivate the bacteriophage, which means that if necessary, appropriate heating processes can be used to ensure that there are no active bacteriophage residues in the final product, thus ensuring product safety.

[0097] Example 5: Determination of the optimal multiple of infection for bacteriophage Csp-TJDF02 This embodiment determines the optimal conditions for phage Csp-TJDF02 to lyse the host bacteria by measuring the phage proliferation efficiency under different multiplicity of infection (MOI) conditions. MOI is a key parameter in phage application, directly affecting disinfection effectiveness and usage costs.

[0098] Step 100: Infection culture under different MOI conditions Different titers of bacteriophage Csp-TJDF02 were mixed with host bacterium D17 to achieve multiplicity of infection (MOIs) of 0.001, 0.01, 0.1, 1, and 10, respectively. The mixtures were then added to nutrient broth and incubated at a suitable temperature for a specified time. After incubation, bacteria were removed by centrifugation and filtration, and the titer of the progeny bacteriophages was determined using the double-layer plate method.

[0099] like Figure 5 As shown, the progeny phage titer was highest, reaching 10, when the multiplicity of infection was 0.001. 9 PFU / mL. Therefore, the optimal multiplicity of infection for phage Csp-TJDF02 was determined to be 0.001.

[0100] This result is significant: an optimal MOI of only 0.001 means that only a very small amount of phage is needed to achieve highly efficient lysis. Specifically, only one phage particle is required per 1000 bacterial cells to achieve maximum proliferation efficiency. This characteristic gives the phage of this application a significant cost advantage in practical applications, enabling highly efficient disinfection with a low dosage.

[0101] Example 6: One-step growth curve of bacteriophage Csp-TJDF02 This embodiment plots a one-step growth curve of bacteriophage Csp-TJDF02 and measures key kinetic parameters such as its latency and burst size, providing a quantitative basis for evaluating its lysis efficiency.

[0102] Step 100: Adsorption and Removal of Free Phages Phage Csp-TJDF02 was mixed with host bacteria D17 in logarithmic growth phase at the optimal multiplicity of infection (MOI=0.001) and incubated briefly at a suitable temperature to allow the phage to adsorb onto the surface of the host bacteria. Unadsorbed free phage was removed by centrifugation, and the precipitate was washed and resuspended with fresh nutrient broth liquid medium. This process was repeated several times to ensure thorough removal of free phage.

[0103] Step 200: Timed sampling and potency determination The resuspended bacterial solution was cultured at a suitable temperature, and samples were taken periodically during the culture process. The phage titer was determined using the double-layer plate method. A one-step growth curve was plotted with infection time on the x-axis and the logarithm of the phage titer (Log PFU / mL) on the y-axis.

[0104] Step 300: Calculation of Pyrolysis Amount The formula for calculating the burst size is as follows: Lysis rate (PFU / cell) = Average phage titer during outbreak period / Average phage titer during incubation period ... Formula (2) Among them, lysis rate represents the average number of progeny phages released by each infected host cell after lysis, and is an important indicator for measuring the phage's proliferative capacity.

[0105] like Figure 6 As shown, the latency period of bacteriophage Csp-TJDF02 is approximately 10 min, the burst period is approximately 60 min, and the lysis rate is approximately 185 PFU / cell. The latency period refers to the time required from bacteriophage infection of the host cell to the release of the first batch of progeny bacteriophages; the burst period refers to the time stage during which a large number of progeny bacteriophages are released.

[0106] The short latency period (10 min) and high lysis rate (185 PFU / cell) exhibited by bacteriophage Csp-TJDF02 indicate that it synthesizes progeny bacteriophages rapidly and efficiently, capable of lysing large numbers of host bacteria in a short time. These excellent kinetic characteristics give the bacteriophage of this application great potential for use as a biological disinfectant.

[0107] Example 7: Whole genome sequencing and safety analysis of bacteriophage Csp-TJDF02 This embodiment performs whole-genome sequencing and bioinformatics analysis on bacteriophage Csp-TJDF02 to clarify its genomic characteristics and assess its biosafety. Safety is the primary prerequisite for the application of bacteriophages as disinfectants in medical environments and food safety fields.

[0108] The complete genome sequence of this bacteriophage has been submitted to GenBank, accession number: PX930468.

[0109] Step 100: Genomic DNA Extraction Genomic DNA was extracted from the purified concentrate of bacteriophage Csp-TJDF02 using a commercially available bacteriophage genomic DNA extraction kit. The extracted DNA, after passing quality testing, was used for subsequent sequencing analysis.

[0110] Step 200: High-throughput sequencing The phage genomic DNA was paired-end sequenced using the Illumina high-throughput sequencing platform (sequencing depth not less than 100×). Optionally, in some embodiments, the sequencing was outsourced to Shanghai Lingen Biotechnology Co., Ltd., with a sequencing depth ≥100×.

[0111] Step 300: Genome Assembly and Annotation After quality control processing of the raw sequencing data, the sequence was assembled using bioinformatics software to obtain the complete genome sequence of bacteriophage Csp-TJDF02. Open reading frames (ORFs) were predicted and functionally annotated using gene prediction software.

[0112] Step 400: Safe Genetic Screening To assess the biosafety of bacteriophage Csp-TJDF02, its whole genome sequence and predicted ORF protein sequences were compared with multiple professional databases, including: lysogen-associated gene screening (compared with the PHASTER database), drug resistance gene screening (compared with the CARD database), virulence gene screening (compared with the VFDB database), and tRNA gene screening. Specifically, in some embodiments, CARD database version 3.2.5 and VFDB database version 2023 were used.

[0113] like Figure 7 As shown, the genome sequencing results of bacteriophage Csp-TJDF02 indicate that its nucleic acid type is double-stranded DNA (dsDNA); the full-length genome is 143,084 bp, of which the total length of coding genes is 130,080 bp; the GC content is 46.70%; a total of 243 open reading frames (ORFs) were predicted, including 163 hypothetical protein-coding sequences and 80 sequences with known protein functions.

[0114] Further safety analysis revealed no tRNA genes, lysogens, drug resistance genes, or virulence genes in the complete genome of phage Csp-TJDF02. These results indicate that this phage is a strictly lytic phage, lacking the ability to integrate into the host genome and carrying no known harmful genes, making it a safe disinfectant for *Cronobacter sakazakii*.

[0115] Example 8: Inhibitory effect of bacteriophage Csp-TJDF02 on biofilm formation on solid surfaces This embodiment verifies the inhibitory effect of bacteriophage Csp-TJDF02 on the formation of Cronobacter sakazakii biofilm. Biofilm formation is a significant reason for the persistent presence and contamination of Cronobacter sakazakii on the surfaces of medical devices and food processing equipment. The results of this embodiment provide experimental support for the "inhibition of Cronobacter sakazakii biofilm formation" and the "application scenarios including medical device surfaces and food processing equipment surfaces" described in this application.

[0116] Step 100: Preparation of mixed bacterial culture To simulate the diversity of *Cronobacter sakazakii* in real-world contamination scenarios, this embodiment uses a mixed bacterial suspension of multiple *Cronobacter sakazakii* strains. Five strains of *Cronobacter sakazakii* (D17, ATCC 21545, ATCC 21569, ATCC 22919, and ATCC 29544) were cultured to suitable bacterial concentrations, and equal volumes of each strain were mixed to prepare a mixed bacterial suspension of *Cronobacter sakazakii*. This experimental design using a mixed suspension of multiple strains more realistically reflects the effectiveness of bacteriophages in real-world applications when facing diverse host bacterial communities.

[0117] Step 200: Biofilm culture and phage treatment The mixed bacterial suspension was mixed with different titers of bacteriophage Csp-TJDF02 to adjust to different multiples of infection (MOIs) (100, 10, 1, 0.1, and 0.01), and the mixtures were transferred to 96-well plates for incubation. A negative control group (mixed bacterial suspension with an equal volume of SM buffer) and a blank control group (containing only culture medium) were also set up. The plates were incubated at a suitable temperature for a certain period to allow bacteria to form a biofilm on the plate surface.

[0118] Step 300: Quantitative analysis of biofilms After cultivation, the biofilm was quantitatively analyzed using crystal violet staining. The specific procedure included: removing the culture medium from the wells, washing with PBS buffer to thoroughly remove airborne bacteria, drying at room temperature, and then adding crystal violet solution for staining. After staining, washing with PBS buffer until the wash solution was colorless, drying, and then adding acetic acid as a destaining agent to dissolve the stained biofilm. The absorbance (OD) at 600 nm was measured using a microplate reader. 600 ) OD 600 The higher the value, the greater the amount of biofilm formed.

[0119] like Figure 8 As shown, the OD values ​​of each phage Csp-TJDF02 treatment group (MOI 100, 10, 1, 0.1, 0.01) are... 600 The values ​​were all significantly lower than those of the negative control group (p<0.001), indicating that phage Csp-TJDF02 can significantly inhibit the formation of mixed Cronobacter sakazakii biofilms on solid surfaces. Notably, even under low multiplicity of infection conditions (e.g., MOI 0.01), a significant inhibitory effect was still observed, which corresponds to the extremely low optimal MOI (0.001) determined in Example 5, further confirming the highly efficient lysis characteristics of the phage in this application.

[0120] The results of this embodiment show that bacteriophage Csp-TJDF02 can be used for disinfection treatment of medical device surfaces and food processing equipment surfaces, reducing the risk of Cronobacter sakazakii contamination by inhibiting the formation of biofilms.

[0121] Supplementary Experiment: Host Profile Determination of Bacteriophage Csp-TJDF02 To clarify the host specificity of bacteriophage Csp-TJDF02, its lytic ability against various bacteria was determined using the spot method.

[0122] The test strains include: (1) Cronobacter sakazakii: D17 (wild type), ATCC 21545, ATCC 21569, ATCC 22919, ATCC29544; (2) Other species of Cronobacter: C. malonaticus ATCC 51329, C. turicensis ATCC BAA-1500; (3) Non-target bacteria: Escherichia coli CICC 10664, Salmonella enteritidis CICC 21513, Staphylococcus aureus CICC 21600.

[0123] Table 1. Results of lysis spectrum determination of bacteriophage Csp-TJDF02 Note: +: Plaque present; -: Plaque absent.

[0124] Table 1 shows that phage Csp-TJDF02 has lytic activity against all five strains of Cronobacter sakazakii, but no lytic activity against other species of Cronobacter or non-target bacteria, demonstrating its high specificity against Cronobacter sakazakii.

[0125] Example 9: Antibacterial effect of bacteriophage Csp-TJDF02 in high-protein infant formula This embodiment verifies the inhibitory effect of bacteriophage Csp-TJDF02 on Cronobacter sakazakii in a high-protein infant formula matrix. This is one of the core inventive highlights of this application, because existing bacteriophages often suffer significant inactivation in complex food matrices (especially high-protein, high-osmotic-pressure environments) due to protein adsorption or interference from environmental factors. This embodiment provides direct experimental support for the "food matrix being high-protein milk powder" mentioned in this application, as well as the specific type of high-protein milk powder.

[0126] Step 100: Preparation of high-protein infant formula milk powder solution Commercially available high-protein infant formula was selected and dissolved in deionized water at a specific mass-to-volume ratio. The solution was then autoclaved and cooled before use. The purpose of sterilization was to ensure the sterility of the formula solution itself, thus avoiding interference with subsequent artificial contamination experiments.

[0127] Step 200: Artificial Contamination and Phage Treatment The *Cronobacter sakazakii* mixed bacterial suspension prepared in Example 8 was serially diluted to a suitable final concentration using high-protein infant formula milk powder solution to prepare artificially contaminated milk powder solutions. Different titers of bacteriophage Csp-TJDF02 were added to the artificially contaminated milk powder solutions to achieve multiplicity of infection (MOI) values ​​of 100, 10, 1, 0.1, and 0.01, respectively. A negative control group (containing only SM buffer, without bacteriophage) was also included.

[0128] Step 300: Constant Temperature Storage and Dynamic Monitoring Each group of samples was kept at a suitable temperature and the absorbance (OD) of the mixed Cronobacter sakazakii bacteria at a wavelength of 600 nm was measured periodically. 600 This was done to monitor bacterial growth. Each group had parallel samples, and each parallel assay was repeated.

[0129] like Figure 9 As shown, during constant temperature storage, the OD of the negative control group... 600 The value increased rapidly over time, indicating that *Cronobacter sakazakii* could proliferate rapidly in the high-protein milk powder matrix. Meanwhile, each phage Csp-TJDF02 treatment group effectively inhibited bacterial growth throughout the entire storage period, with OD... 600 The values ​​were consistently significantly lower than those in the negative control group (p<0.01).

[0130] The above results demonstrate that the bacteriophage Csp-TJDF02 of this application exhibits significant inhibitory activity against mixed Cronobacter sakazakii bacteria in a high-protein infant formula matrix. Of particular importance is that the bacteriophage maintains highly efficient antibacterial activity even in a high-protein environment, overcoming the technical challenge of ordinary bacteriophages being easily adsorbed or inactivated by proteins in complex food matrices. This characteristic makes the bacteriophage of this application particularly suitable for the contamination control of Cronobacter sakazakii in high-protein dairy products.

[0131] Example 10: Antibacterial effect of bacteriophage Csp-TJDF02 in mixed high-protein milk powder This embodiment further verifies the broad applicability of bacteriophage Csp-TJDF02 to various types of high-protein milk powder, providing experimental support for the various high-protein milk powder products listed in this application.

[0132] Step 100: Preparation of the mixed high-protein milk powder solution To comprehensively evaluate the applicability of bacteriophages, various types of high-protein milk powder were selected for testing, including: high-protein infant formula (including formula for premature / low birth weight infants), high-protein children's formula, high-protein adult formula (including formula for the elderly and sports nutrition formula), and high-protein formula for special medical purposes (including high-fiber and low-sugar high-protein types). Several products from each category were selected, mixed in equal proportions, dissolved in deionized water at a specific mass-volume ratio, and then autoclaved and cooled to prepare a mixed high-protein milk powder solution.

[0133] Step 200: Artificial Contamination and Phage Treatment Using the same method as in Example 9, the mixed bacterial suspension of *Cronobacter sakazakii* was diluted to a suitable final concentration with a mixed high-protein milk powder solution to prepare an artificially contaminated mixed high-protein milk powder solution. Different titers of bacteriophage Csp-TJDF02 were added for treatment, and a negative control group was set up.

[0134] Step 300: Constant Temperature Storage and Dynamic Monitoring Each group of samples was stored at a suitable temperature under constant temperature, and OD was measured periodically. 600 Values ​​are used to monitor bacterial growth.

[0135] like Figure 10 As shown, during constant temperature storage, the total bacterial count in the mixed high-protein milk powder solution of each phage Csp-TJDF02 treatment group was consistently significantly lower than that of the negative control group. At the end of storage, the OD values ​​of the treatment and control groups were significantly lower. 600 The difference was significant (p<0.01).

[0136] The above results demonstrate that the bacteriophage Csp-TJDF02 of this application exhibits excellent antibacterial effects against various types of high-protein milk powder products, showing broad product applicability. This characteristic makes the bacteriophage of this application suitable for the control of Cronobacter sakazakii contamination in various dairy products, including high-protein infant formula, children's high-protein milk powder, adult high-protein milk powder, senior milk powder, sports nutrition milk powder, and high-protein milk powder for special medical purposes.

[0137] Example 11: Composition for the control of Cronobacter sakazakii and its application method This embodiment illustrates the preparation and application of the disinfection composition containing the bacteriophage Csp-TJDF02 of this application, providing support for the technical solutions described in this application.

[0138] Step 100: Preparation of the disinfection composition The Cronobacter sakazakii disinfectant composition of this application uses an isolate or culture of bacteriophage Csp-TJDF02 as the active ingredient. Depending on different application requirements, purified bacteriophage Csp-TJDF02 can be prepared into disinfectant compositions of different formulations.

[0139] For example, phage amplification solution or purified concentrate can be mixed with a suitable carrier or diluent and adjusted to a suitable phage titer to prepare a disinfection composition in the form of a cleaning solution for rinsing surfaces of medical devices, food processing equipment, etc.

[0140] Optionally, the bacteriophage solution can be loaded into a spray device to prepare a disinfectant composition in the form of a spray, which can be used for spraying treatment in medical environments, food processing environments, and other places.

[0141] Furthermore, suitable stabilizers, preservatives, or other auxiliary ingredients can be added to the phage solution to prepare a disinfection composition in the form of a biological antibacterial agent, thereby extending the product's shelf life or improving its performance.

[0142] Step 200: Application method of the disinfection composition The application methods of the disinfection composition of this application include, but are not limited to, two main methods: rinsing and spraying.

[0143] For disinfection of medical device surfaces and food processing equipment surfaces, rinsing is the preferred method. Specifically, the prepared disinfectant composition in the form of a cleaning solution is directly applied to rinse the surface to be treated, allowing the bacteriophages to fully contact any potentially present *Cronobacter sakazakii*, thereby inhibiting bacterial proliferation and biofilm formation.

[0144] For disinfection of medical and food processing environments, spraying is the preferred method. Specifically, a spraying device is used to evenly spray the disinfectant composition onto the environmental surface, effectively controlling Cronobacter sakazakii in the environment.

[0145] Step 300: Use of bacteriophage alone or in combination The bacteriophage Csp-TJDF02 in this application's disinfection composition can be used alone or in combination with other Cronobacter sakazakii bacteriophages. When used alone, the disinfection effect is achieved by relying on the highly efficient lysis characteristics of bacteriophage Csp-TJDF02 itself. When used in combination, by combining multiple bacteriophages with different host spectra, the applicability of the disinfection composition can be further expanded, and the coverage ability against different sources or subtypes of Cronobacter sakazakii can be enhanced.

[0146] Example 12: Application of bacteriophage Csp-TJDF02 in the preparation of disinfectants This embodiment illustrates the application of bacteriophage Csp-TJDF02 in the preparation of Cronobacter sakazakii disinfectant, providing support for this application.

[0147] Bacteriophage Csp-TJDF02 can be used as an active ingredient in the preparation of a Cronobacter sakazakii disinfectant. This disinfectant can be applied in the following scenarios: reducing or inhibiting contamination caused by Cronobacter sakazakii in medical and food processing environments; disinfecting the surfaces of medical devices and food processing equipment to inhibit the proliferation and biofilm formation of Cronobacter sakazakii; and controlling Cronobacter sakazakii contamination during the processing or storage of food matrices (especially high-protein milk powder), reducing the risk of quality deterioration caused by Cronobacter sakazakii.

[0148] Compared with traditional chemical disinfectants, the bacteriophage disinfectant of this application has the following technical advantages: First, it has high host specificity, acting only against Cronobacter sakazakii without affecting other beneficial microorganisms or normal flora; Second, it does not produce chemical residues, is a natural biological material, and has high safety; Third, it is not easy to induce bacteria to develop drug resistance and can be used for a long time; Fourth, it has low preparation cost and abundant raw material sources.

[0149] The above embodiments detail the isolation and purification method, biological characteristics, safety, and various applications of the bacteriophage Csp-TJDF02 in inhibiting Cronobacter sakazakii. It should be understood that the above embodiments are merely illustrative of the technical solutions of this application and are not intended to limit the scope of protection of this application. Those skilled in the art, after reading this specification, can make various modifications and equivalent substitutions without departing from the technical concept of this application, and all such modifications and equivalent substitutions fall within the scope of protection defined by this application.

[0150] The above embodiments have the following technical effects: Examples 1 and 2 demonstrate the successful isolation and purification of bacteriophage Csp-TJDF02, and transmission electron microscopy confirms that it belongs to the typical myotail bacteriophage morphology type, with an icosahedral head (approximately 75.09 nm in diameter) and a retractable tail sheath structure (approximately 86.16 nm in length).

[0151] Examples 3 and 4 demonstrate that bacteriophage Csp-TJDF02 has good environmental tolerance, maintaining a survival rate of over 60% in the pH range of 6 to 10, retaining high activity after treatment at 60°C for 60 min, and being completely inactivated after treatment at 80°C for 60 min, which facilitates safe control.

[0152] Examples 5 and 6 demonstrate that phage Csp-TJDF02 has excellent lysis kinetics, with an optimal multiplicity of infection as low as 0.001, a latency period of only about 10 min, and a burst rate of about 185 PFU / cell, indicating that it has a highly efficient host bacterial lysis capability.

[0153] Example 7 demonstrates the genomic safety of bacteriophage Csp-TJDF02, which is 143,084 bp in length, has a GC content of 46.70%, contains 243 ORFs, and no lysogenic genes, drug resistance genes, virulence genes, or tRNA genes were found in the comparison and analysis of professional databases, confirming that it is a strictly lytic bacteriophage.

[0154] Example 8 demonstrates that bacteriophage Csp-TJDF02 can significantly inhibit the formation of Cronobacter sakazakii biofilm on solid surfaces, providing technical support for the disinfection of equipment surfaces.

[0155] Examples 9 and 10 demonstrate that bacteriophage Csp-TJDF02 can maintain high antibacterial activity in a high-protein milk powder matrix, overcoming the technical problem that ordinary bacteriophages are easily inactivated in complex food matrices.

[0156] Examples 11 and 12 illustrate the preparation method and various applications of the disinfection composition, demonstrating that the bacteriophage of this application can be prepared into various dosage forms such as cleaning solution, spray or biological antibacterial agent, and applied to the pollution control of medical environment, food processing environment and equipment surface.

[0157] The sequence listing indicates that the complete genome nucleotide sequence (SEQ ID NO: 1) of the bacteriophage Csp-TJDF02 involved in this application is 143,084 bp in length. Due to space limitations, this sequence has been submitted electronically or to the GenBank database, accession number: PX930468.

[0158] It should be noted that in this patent application, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one" does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. In this patent application, if it refers to performing an action according to an element, it means performing the action at least according to that element, including two cases: performing the action only according to that element, and performing the action according to that element and other elements. Expressions such as "multiple," "repeatedly," and "various" include two, two times, two kinds, and more than two, more than two times, and more than two kinds.

[0159] All documents mentioned in this application are considered to be incorporated in their entirety into the disclosure of this application so that they can serve as a basis for modifications if necessary. Furthermore, it should be understood that after reading the foregoing disclosure of this application, those skilled in the art can make various alterations or modifications to this application, and these equivalent forms also fall within the scope of protection claimed in this application.

Claims

1. A strain of Cronobacter sakazakii bacteriophage Csp-TJDF02, characterized in that, The phage has been deposited at the China Center for Type Culture Collection (CCTCC) on November 20, 2025, with accession number CCTCC NO: M 20252611.

2. A composition for eliminating Cronobacter sakazakii, characterized in that, It contains the bacteriophage Csp-TJDF02 as described in claim 1.

3. The disinfection composition according to claim 2, characterized in that, The disinfection composition is in the form of a cleaning solution, a spray, or a biological antibacterial agent.

4. The disinfection composition according to claim 2 or 3, characterized in that, The bacteriophage Csp-TJDF02 in the disinfection composition can be used alone or in combination with other Cronobacter sakazakii bacteriophages.

5. The disinfection composition according to claim 2 or 3, characterized in that, The isolate or culture of the bacteriophage Csp-TJDF02 is used as the active ingredient in the disinfection composition.

6. The use of the bacteriophage Csp-TJDF02 of claim 1 or the disinfection composition of claim 2 or 3 in inhibiting Cronobacter sakazakii for non-disease diagnosis and treatment purposes.

7. The application according to claim 6, characterized in that, The application is to inhibit the proliferation, metabolism and / or formation of biofilm of *Cronobacter sakazakii*.

8. The application according to claim 6, characterized in that, The application scenarios include medical environments, food processing environments, medical device surfaces, food processing equipment surfaces, or food matrices; preferably, the application is used to reduce or inhibit contamination or quality deterioration caused by Cronobacter sakazakii in the aforementioned scenarios.

9. The application according to claim 8, characterized in that, The application method includes rinsing or spraying the scene using purified bacteriophage Csp-TJDF02 or the disinfectant composition.

10. The application according to claim 8 or 9, characterized in that, The food base is high-protein milk powder.

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