Shewanella putrefaciens phage SpuP2 and application thereof

Abstract

The invention discloses shewanella putrefaciens bacteriophage SpuP2 and application thereof. The preservation number of the shewanella putrefaciens bacteriophage SpuP2 is CCTCC (China Center For Type Culture Collection) NO: M 20251492. The method belongs to the brachycauda family. The titer of a lysate prepared based on the bacteriophage is 108-9PFU / mL, and the lysate can be stably applied under the conditions that the temperature is less than or equal to 50 DEG C and the pH is 5-10. The bacteriophage and the preparation thereof can efficiently inhibit the growth of shewanella putrefaciens, significantly inhibit the formation of a biofilm, remove a mature biofilm, and achieve a sterilization rate of 99-99.99% in aquatic products such as grass carp fillets and the like. The preparation is safe and residue-free, can replace a chemical antibacterial agent to be used for rot prevention and control in the food storage, transportation and processing processes, and has good safety and application prospects.

Description

Technical Field

[0001] This invention relates to the field of food safety, specifically to a Shewanella putrefactive bacteriophage SPUP2 and its applications. Background Technology

[0002] Aquatic products, as an important source of high-quality animal protein in the human diet, are rich in amino acids, unsaturated fatty acids, and trace elements, and are highly favored by consumers. However, aquatic products are also highly susceptible to spoilage microorganisms, posing serious risks of spoilage and food safety during storage, transportation, and processing. Shewanella putrefactive bacteria (Shewanella putrefactive bacteria) Shewanella putrefaciens Shewanella putrefactive bacteria (S. putrefactive) is one of the important specific spoilage bacteria in aquatic products. This strain is a Gram-negative, motile, psychrophilic bacillus, widely distributed in nature, especially in marine and freshwater environments. Its putrefactive ability stems from its ability to reduce trimethylamine oxide (TMAO) to trimethylamine (TMA), producing other amines and volatile gases. Even under refrigeration conditions, it can damage food quality and safety, posing a significant spoilage and food safety risk. Particularly challenging is Shewanella putrefactive's strong biofilm-forming ability, allowing it to adhere to the surfaces of food and its processing and transportation equipment. Biofilms are composed of extracellular polysaccharides, extracellular DNA (eDNA), proteins, and lipids, forming a three-dimensional protective structure on biological or non-biological surfaces. Compared to planktonic bacteria, bacteria within biofilms exhibit higher activity and significantly enhanced resistance to antimicrobial agents, forming a barrier that is difficult to remove. Traditional biofilm control methods (such as physical removal and chemical antimicrobial agents) often face problems such as poor effectiveness and easy induction of resistance during actual food processing and storage. Therefore, biocontrol, as a novel strategy for controlling spoilage bacteria and their biofilms, deserves further exploration. Bacteriophages are a class of viruses that obligately infect and lyse bacteria, and are the most numerous and widely distributed biological entities on Earth. Their high targeting specificity allows them to effectively eliminate target pathogens while avoiding disruption of beneficial food microbiota, giving them a unique advantage in the field of biocontrol. As early as 2006, the US FDA approved them for the control of Listeria monocytogenes in ready-to-eat foods and poultry products; this application was subsequently approved in Europe, Australia, and New Zealand. In 2013, bacteriophages were further approved for the control of Salmonella in poultry and other foods. In recent years, bacteriophages, as novel bactericides, have shown significant potential in the fields of food antimicrobial and biofilm control. For example, researchers isolated specific bacteriophages from wastewater samples and applied them to antimicrobial experiments on chicken and beef. The results showed that under conditions of storage at 4°C for 72 hours, the number of E. coli in beef samples decreased by 0.43 to 3.98 log. 10CFU / g. In addition, phage cocktails (a combination of multiple phages) can effectively control multiple drug-resistant bacterial biofilms on food contact surfaces, with a removal rate of up to 66.67% on stainless steel surfaces; another research team found that phages can inhibit the growth of Vibrio parahaemolyticus on food contact surfaces and the formation of biofilms, with an inhibition rate of more than 50%.

[0003] Current studies have shown that phages not only inhibit bacterial growth, but also have the effect of inhibiting and removing biofilms. In the fight against biofilms, phages can effectively reduce the number of bacteria by degrading the extracellular polymeric matrix of bacteria. As a biological control agent, phages have the significant advantages of high efficiency, safety, and no adverse effects on product quality, and have broad application prospects in controlling food spoilage bacteria and their biofilms.

[0004] However, the existing technology still has certain deficiencies in the application process: 1. Limitations of traditional prevention and control methods: Physical cleaning and chemical disinfection can reduce spoilage bacteria to some extent, but the effect is limited in the biofilm environment, and long-term use of chemical antibacterial agents can easily lead to the emergence of drug-resistant strains, and there is a risk of adverse effects on food quality and the environment.

[0005] 2. Challenges in the application of phages: Although phages have been proven to lyse a variety of pathogenic bacteria and spoilage bacteria, a single phage in application is easily affected by the limited host range, bacterial mutation to produce phage-resistant bacteria, and other problems, resulting in unstable bactericidal effect.

[0006] 3. Insufficient biofilm removal effect: There have been reports that phages can partially degrade bacterial biofilms, but in complex food environments, their removal efficiency for high-density or mature biofilms is still insufficient, making it difficult to meet the actual needs of aquatic product processing and storage.

[0007] Therefore, the existing technology urgently needs a new prevention and control method that is efficient, safe, and can target both planktonic and biofilm states of Shewanella putrefaciens, to effectively delay the spoilage process of aquatic products, improve food safety, and extend the shelf life. SUMMARY

[0008] The purpose of the present application is to overcome the deficiencies of the prior art and provide a Shewanella putrefaciens phage SPuP2 and its application. The Shewanella putrefaciens phage SPuP2 is screened in the present application, which can be used for biofilm prevention and food biological control, achieving the following purposes: 1. Efficiently inhibiting the growth of spoilage bacteria: using the high specificity of phages to host bacteria, rapidly identifying and lysing Shewanella putrefaciens, and reducing the survival number of Shewanella putrefaciens in aquatic products.

[0009] 2. Effective control of biofilm formation and removal: by directly lysing cells or synergizing with the lytic enzymes secreted by the bacteriophage, the Shewanella putrefaciens biofilm matrix structure is destroyed, improving the inhibition of biofilm formation and the removal efficiency of mature biofilm.

[0010] 3. Delaying the process of food spoilage: during the storage and transportation of aquatic products, by inhibiting the growth of Shewanella putrefaciens, reducing the trimethylamine (TMA) and other volatile spoilage products produced by Shewanella putrefaciens metabolism, and maintaining the sensory quality and nutritional value of aquatic products.

[0011] 4. Safety and applicability advantages: avoid the residue risk and drug resistance problem caused by chemical antibacterial agents, and at the same time, do not affect the beneficial food microbial community, and ensure the food safety of aquatic products.

[0012] To achieve the above purposes, the technical solutions designed by the present application are as follows: The present application provides a Shewanella putrefaciens bacteriophage (SPuP2) with a preservation number of CCTCC NO: M 20251492. Shewanella putrefaciens The Shewanella putrefaciens bacteriophage (SPuP2) has a preservation number of CCTCC NO: M 20251492, and is preserved in the China Center for Type Culture Collection (CCTCC) on June 30, 2025, and the address is Wuhan University, Wuhan, China.

[0013] The Shewanella putrefaciens bacteriophage (SPuP2) has a preservation number of CCTCC NO: M 20251492, and is preserved in the China Center for Type Culture Collection (CCTCC) on June 30, 2025, and the address is Wuhan University, Wuhan, China. Shewanella putrefaciens The Shewanella putrefaciens bacteriophage (SPuP2) has a preservation number of CCTCC NO: M 20251492, and is preserved in the China Center for Type Culture Collection (CCTCC) on June 30, 2025, and the address is Wuhan University, Wuhan, China.

[0014] The Shewanella putrefaciens bacteriophage (SPuP2) has a preservation number of CCTCC NO: M 20251492, and is preserved in the China Center for Type Culture Collection (CCTCC) on June 30, 2025, and the address is Wuhan University, Wuhan, China. Shewanella putrefaciens The Shewanella putrefaciens bacteriophage (SPuP2) has a preservation number of CCTCC NO: M 20251492, and is preserved in the China Center for Type Culture Collection (CCTCC) on June 30, 2025, and the address is Wuhan University, Wuhan, China.

[0015] The Shewanella putrefaciens bacteriophage (SPuP2) has a preservation number of CCTCC NO: M 20251492, and is preserved in the China Center for Type Culture Collection (CCTCC) on June 30, 2025, and the address is Wuhan University, Wuhan, China. Shewanella putrefaciens The Shewanella putrefaciens bacteriophage (SPuP2) has a preservation number of CCTCC NO: M 20251492, and is preserved in the China Center for Type Culture Collection (CCTCC) on June 30, 2025, and the address is Wuhan University, Wuhan, China. Podoviridae The Shewanella putrefaciens bacteriophage (SPuP2) has a preservation number of CCTCC NO: M 20251492, and is preserved in the China Center for Type Culture Collection (CCTCC) on June 30, 2025, and the address is Wuhan University, Wuhan, China.

[0016] The present application also provides a Shewanella putrefaciens bacteriophage SPuP2 for preventing and controlling Shewanella putrefaciens.

[0017] The application further provides a bacteriophage preparation for preventing and controlling Shewanella putrefaciens, which comprises a lysate of the bacteriophage SPuP2 in claim 1, wherein the titer of the lysate of the bacteriophage SPuP2 is 10 8-9 PFU / mL.

[0018] The lysate of the bacteriophage SPuP2 described above can be directly used for preventing and controlling Shewanella putrefaciens, or used after being activated.

[0019] Further, the preparation further comprises an activation solution; wherein, the activation solution is 10 9 CFU / mL of Shewanella putrefaciens CICC 22940.

[0020] Further, the lysate of the bacteriophage SPuP2 comprises the following steps: 1) 100 μL of the bacteriophage SPuP2 (glycerol content 20%) stored in a glycerol tube is mixed with 100 μL of Shewanella putrefaciens CICC 22940 bacterial solution in a logarithmic growth phase, and 15 mL of LB broth medium is added; under the condition of a temperature of 28℃ and a rotation speed of 120 rpm, the mixture is cultured for 12 hours in a shaking table to obtain a bacteriophage-containing suspension; 2) the suspension is centrifuged at a rotation speed of 8500 rpm and a temperature of 4℃ for 15 min, the supernatant is taken and filtered through a 0.22 μm sterile filter membrane, and the obtained supernatant is the lysate of the activated bacteriophage SPuP2.

[0021] The bacteriophage lysate can be stored at 20℃ for 2 years. If it is not used after being stored for more than 1 month, the bacteriophage lysate can be activated according to the above steps and then used, specifically as follows: 1) 100 μL of the bacteriophage SPuP2 (glycerol content 20%) stored in a glycerol tube is mixed with 100 μL of Shewanella putrefaciens CICC 22940 bacterial solution in a logarithmic growth phase, and 15 mL of LB broth medium is added; under the condition of a temperature of 28℃ and a rotation speed of 120 rpm, the mixture is cultured for 12 hours in a shaking table to obtain a bacteriophage-containing suspension; 2) the suspension is centrifuged at a rotation speed of 8500 rpm and a temperature of 4℃ for 15 min, the supernatant is taken and filtered through a 0.22 μm sterile filter membrane, and the obtained supernatant is the lysate of the activated bacteriophage SPuP2.

[0022] The bacteriophage preparation is suitable for use under the condition of a temperature not higher than 50℃ and an environmental pH of 5-10.

[0023] The application further provides the use of the above-mentioned preparation in the biological prevention and control of Shewanella putrefaciens.

[0024] The application also provides application of the preparation in inhibiting growth of Shewanella putrefaciens biofilm and removing mature biofilm.

[0025] The liquid preparation can effectively inhibit formation of the Shewanella putrefaciens biofilm, and the inhibition rate is 87.08%. Meanwhile, the removal rate of the mature biofilm is 75.56%.

[0026] Further, the bacteriophage preparation can effectively inhibit growth and metabolism of the Shewanella putrefaciens in the biofilm, and the sterilization rate can reach 99%-99.99%; the inhibition rates of the extracellular polysaccharide and the extracellular protein are 51.60% and 70.78% respectively.

[0027] The application also provides application of the preparation in biological control of the Shewanella putrefaciens in food.

[0028] Further, the food is aquatic products.

[0029] The aquatic product is a fish slice related substrate.

[0030] The fish slice related substrate is dorsal fish meat, the dorsal fish meat is divided into fish slices with uniform size and thickness, the fish skin and red meat part are removed, and only the white fish meat part is reserved; the application preferably uses grass carp fish slices.

[0031] The application evaluates the biological control effect of the bacteriophage preparation through relevant spoilage indicators.

[0032] The preparation can effectively inhibit or reduce the Shewanella putrefaciens in the aquatic product, and the sterilization rate can reach 99%-99.99%.

[0033] The application also provides application of the preparation in biological control of food spoilage.

[0034] Principle of the application: The core principle of bacteriophage in preventing spoilage bacteria and biofilm lies in its specific recognition and infection mechanism. The bacteriophage surface has highly specific adsorption proteins that can accurately recognize specific receptors on the surface of spoilage bacteria cells and specifically bind to them. After binding, the bacteriophage injects its genetic material into the host bacteria through the tail tube, uses the host bacteria's metabolic system for nucleic acid replication, protein synthesis, and progeny bacteriophage assembly, ultimately leading to bacterial cell lysis and death, effectively inhibiting the growth and reproduction of spoilage bacteria. In terms of biofilm prevention, extracellular polymeric substances (EPS) widely exist in biofilm and play a key role. When the bacteriophage cannot directly contact the receptor on the surface of spoilage bacteria cells, EPS can be recognized by the bacteriophage as a secondary receptor, acting as a primary binding site. The bacteriophage penetrates the biofilm barrier by binding to EPS, and then contacts and infects the internal spoilage bacteria cells. In this process, the bacteriophage not only can lyse spoilage bacteria cells, but also can effectively degrade bacterial extracellular polymeric substances, disintegrate the biofilm structure, and ultimately achieve inhibition and removal of biofilm, significantly reducing the number of spoilage bacteria in the biofilm.

[0035] The beneficial effects of the present application are: The genome and biological characteristics of a bacteriophage SPuP2 of a spoilage Shewanella are analyzed, the bacteriophage SPuP2 can complete the maximum adsorption of spoilage Shewanella in about 10 minutes, and the inhibition rate and removal rate of spoilage Shewanella biofilm can reach 87.08% and 75.56%. In addition, the bacteriophage can be used as a biological control agent for the biological control of spoilage Shewanella in aquatic products.

[0036] In summary, the present application provides a highly efficient, safe and widely applicable bacteriophage preparation for the prevention and control of spoilage Shewanella and biofilm in aquatic products, promoting the sustainable development of the aquatic product industry in food spoilage control. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 The picture of bacteriophage plaque in the double-layer plate of bacteriophage SPuP2, Figure 2 The transmission electron microscope photo of bacteriophage SPuP2, Figure 3 The biological characteristics graph of bacteriophage SPuP2, In the figure, A is the optimal infection multiple graph of bacteriophage SPuP2, B is the adsorption rate curve graph of bacteriophage SPuP2, C is the one-step growth curve graph of bacteriophage SPuP2, D is the temperature stability graph of bacteriophage SPuP2, E is the pH stability graph of bacteriophage SPuP2, F is a schematic diagram of the tolerance of phage SPuP2 to UV irradiation, G is a schematic diagram of the tolerance of phage SPuP2 to chloroform solution; Figure 4 A is a genome analysis diagram of phage SPuP2, B is a genome circle diagram of phage SPuP2, C is a phylogenetic tree diagram of phage SPuP2.

[0038] Figure 5 D is a diagram of the lysis ability of phage SPuP2 to Shewanella putrefaciens under different MOI conditions; E is a diagram of the inhibition of biofilm formation by phage SPuP2 and the removal of mature biofilm, Figure 6 F is a diagram of the inhibition of biofilm formation by phage SPuP2 to Shewanella putrefaciens, G is a diagram of the removal of mature biofilm by phage SPuP2, H is a diagram of the inhibition of the content of extracellular polysaccharides in the biofilm by phage SPuP2, I is a diagram of the inhibition of the content of extracellular proteins in the biofilm by phage SPuP2; J is a diagram of the inhibition of biofilm by phage SPuP2 and the removal of mature biofilm by phage SPuP2. K is a diagram of the inhibition of biofilm by phage SPuP2 and the removal of mature biofilm by phage SPuP2. Figure 7 L is a diagram of the inhibition of biofilm by phage SPuP2 and the removal of mature biofilm by phage SPuP2. M is a diagram of the preservation effect of phage SPuP2 on grass carp fillets,

[0039] N is a diagram of the inhibition of the growth of Shewanella putrefaciens in fish fillets stored at 4°C by phage SPuP2, Figure 8 O is a diagram of the change in pH of fish meat during storage at 4°C after treatment with phage SPuP2, P is a diagram of the change in TVB-N value of fish meat during storage at 4°C after treatment with phage SPuP2, Q is a diagram of the change in TBARS value of fish meat during storage at 4°C after treatment with phage SPuP2. DETAILED DESCRIPTION

[0040] The present application will be further described in conjunction with specific embodiments so as to be understood by those skilled in the art.

[0041] Example 1: Phage isolation and purification and morphological analysis 1. Phage isolation and purification Clam culture water from a Wuhan aquaculture market was incubated overnight with Shewanella putrefactive bacteria (CICC22940) cultured to the logarithmic growth phase for 12 hours. The solution was filtered through a 0.22 μm microporous membrane, and the filtrate was repeated once using the same method to screen for phage stock solution containing lysing activity of Shewanella putrefactive bacteria (CICC22940). The phage was purified using a double-layer agar plate method, repeated at least 10 times until the phage plaques on the double-layer plates were of uniform size and transparency; this was considered purified phage. Figure 1 ).

[0042] 2. Phage morphological analysis After staining the bacteriophages with phosphotungstic acid, their morphology was observed under a transmission electron microscope. The specific steps were as follows: A copper mesh was immersed in the bacteriophage solution for 10 minutes, then excess liquid was absorbed with filter paper. The copper mesh was then stained with phosphotungstic acid dye and allowed to air dry completely. The prepared copper mesh was then observed under a transmission electron microscope to observe the morphology of the bacteriophages, and their size was measured using Digital Micrograph Demo 3.9.1 software. Figure 2 ).

[0043] The results showed that bacteriophage SPUP2 has an icosahedral head and a short, retractable tail. The head diameter is 52.27±1.19 nm and the tail length is 8.81±1.53 nm. It belongs to the Podophyllaceae family.

[0044] The above-mentioned putrefactive Shewanella phage ( Shewanella putrefaciens bacteriophage) SPUP2, deposited at the China Center for Type Culture Collection, accession number: CCTCC M 20251492, deposit date: June 30, 2025, address: Wuhan University, Wuhan, China.

[0045] Example 2: Biological characteristics analysis of bacteriophage SPUP2 1. Host lineage analysis of bacteriophage SPUP2 The host spectrum of bacteriophages in various foodborne pathogens and foodborne spoilage bacteria was examined by spot sampling assays. 100 µL of the bacterial culture to be tested, cultured to the logarithmic growth phase, was added to warm semi-solid culture medium, mixed well, and poured onto a pre-prepared LB agar plate. After solidification, 5 µL of the plate with a titer of 10 was taken. 9 PFU / mL phage was added to the surface of the upper plate, dried, and then incubated upside down in a 28℃ incubator for 4-6 hours. The lysis was observed, and the results are shown in Table 1.

[0046] Table 1 Host spectrum of bacteriophage T102 Note: a +++ strong intensity (Clear plaque); - no lytic activity (No plaque). b ATCC, American Type Culture Collection. CMCC, Center for Medical Culture Collections. CICC, China Center of Industrial Culture Collection. NTCC, National Collection of Type Cultures. 2. Optimal multiplicity of infection (MOI) of phage Multiplicity of infection (MOI) refers to the ratio of the number of phages to the number of host bacteria at the initial infection, which is defined by the ratio of phages to host bacteria at the time of infection. Phages and host bacteria were mixed at a certain MOI value (0.001, 0.01, 0.1, 1, 10, 100, 1000), incubated at 28°C for 4h, centrifuged at 8500r / min for 10min, and then the phage titer was determined by double-layer plate method to determine the optimal MOI of phage. The test was set in triplicate.

[0047] The results are shown in Figure 3 A: The phage titer reached a maximum at MOI = 0.01, i.e. the optimal MOI of phage was 0.01, indicating that when the phage titer and host bacteria were mixed at a ratio of 0.01, more phages could be proliferated.

[0048] 3. Adsorption rate Adsorption rate reflects the efficiency of specific binding of phage to host. Fresh phage solution and host bacteria suspension were mixed in a centrifuge tube at the optimal MOI value and incubated on a 28°C shaker. Starting from 0min, the phage titer in the supernatant was determined every 5min using double-layer plate method. The test was set in triplicate.

[0049] Adsorption rate = 1 - (unadsorbed phage titer at each time point / phage titer at 0min) x 100%, The results of the adsorption rate of phage to host bacteria are shown in Figure 3 B: The adsorption period of phage was about 27min. The maximum adsorption rate was reached 10min after co-incubation of phage and host bacteria, and the adsorption rate was 95.73±0.86%.

[0050] 4. One-step growth curve One-step growth curve of phage SPuP2 was determined by the following method. The phage and host bacteria were mixed at the optimal MOI value, and incubated at 28℃ for 10 min to allow the phage to better adsorb to the bacteria. Then, the mixture was centrifuged at 8000 r / min for 2 min at 4℃, and the supernatant was discarded. The phage was resuspended in an equal volume of LB medium, and the process was repeated twice to remove the unadsorbed phage. The above-mentioned liquid was added to 9 mL of LB liquid medium, and 300 μL of the mixture was sampled every 10 min, centrifuged at 8000 r / min for 2 min, and the titer of the phage in the supernatant was determined by the double-layer plate method. The test was performed in triplicate. The lysis amount = the titer of the phage at the end of lysis / the concentration of the host bacteria at the beginning of infection. The results are shown in Figure 3 C, the latent period and the lysis period of the phage can be seen from the one-step growth curve. The latent period of the phage SPuP2 was 10 min, the lysis period was 80 min, and the lysis amount was 49.95 ± 11.53 PFU / CFU.

[0051] 5. Stability analysis of phage SPuP2 The phage stock solution was diluted to 10 6 PFU / mL and divided into 1 mL sterile centrifuge tubes. The centrifuge tubes were placed in a constant-temperature water bath at 30℃, 40℃, 50℃, 60℃ and 70℃, respectively, and the titer of the phage in each tube was determined at 0 min, 15 min, 30 min, 45 min and 60 min. The test was performed in triplicate. As can be seen from Figure 3 D, the activity of the phage SPuP2 between 30℃ and 40℃ was not significantly different from the initial titer of the phage, and the titer of the phage gradually decreased with the increase of the temperature. However, the phage was completely inactivated after being incubated at 70℃ for 30 min, indicating that SPuP2 has moderate heat resistance and is unstable at high temperature.

[0052] 100 μL of the phage suspension with a known titer (10 7 PFU / mL) was added to 900 μL of PBS buffer with different pH values (4-11), and the titer of the phage in each centrifuge tube was determined after being placed in a water bath at 28℃ for 2 h. The test was performed in triplicate. As can be seen from Figure 3 E, the phage remained stable at a titer of 5-10, i.e., high activity, and was almost completely inactivated at pH = 4 and pH = 11, indicating that strong acid and strong base directly destroy the activity of the phage.

[0053] The phage suspension with a known titer (10 6 PFU / mL) was exposed to ultraviolet light (40w, 15cm) for irradiation, and the titer of the phage in each tube was determined at 0 min, 15 min, 30 min, 45 min and 60 min. The test was performed in triplicate. As can be seen from Figure 3FIt is known that under UV irradiation, the phage titer rapidly decreases to 1.56±0.07 Log 10 PFU / mL within 1 h, indicating that UV radiation with a wavelength between 240 and 280 nm can induce damage to the genetic material, structural components and infective function of the phage.

[0054] Take the known titer of phage suspension (10 7 PFU / mL) 100 μL respectively into 900 μL of 5% and 10% chloroform solution, and place it in a 28°C water bath. Measure the titer of the phage in each centrifuge tube at 0 min, 30 min, 60 min, 90 min and 120 min. The test has 3 parallels. From Figure 3 GIt is known that the phage shows a significant decrease in activity after exposure to 5% and 10% chloroform for 30 minutes.

[0055] Example 3 Genome bioinformatics analysis of phage SPuP2 1. Extract phage DNA 1) Take 1 mL of high-titer phage solution, add 5 μL of DNase I and RNase A enzyme in turn, and incubate at 37°C for 1 h to remove other DNA and RNA interference. Add 24 μL of 50 mM EDTA (pH=8.0) and place in a 80°C water bath to inactivate the enzyme for 15 min; 2) Add 1.5 μL of 20 μg / mL proteinase K and 10% SDS solution to the treated phage solution, mix well and incubate at 56°C for 1 h; 3) After natural cooling to room temperature, add an equal volume of phenol / chloroform / isoamyl alcohol (volume ratio 25:24:1) to extract the DNA in the liquid, mix well and centrifuge at 12000 r / min for 10 min.

[0056] 4) Absorb the upper solution and add an equal volume of chloroform, mix well and centrifuge at 12000 r / min for 10 min. Absorb the upper solution again, add an equal volume of isopropanol and precipitate the DNA in a -20°C ice bath for 2 h; 5) After the precipitation is completed, centrifuge at 12000 r / min for 10 min, discard the upper liquid, add 1 mL of 75% pre-cooled ethanol to the precipitate and stand for 10 min, then centrifuge at 12000 r / min for 10 min. After the ethanol is discarded, dry it, dissolve the genomic DNA with TE (pH=7.8) and store it at -20°C.

[0057] 2. Whole genome sequencing of phage The extracted phage DNA is detected by ultramicro spectrophotometer and sent to Wuhan Boyuezhihe Biotechnology Co., Ltd. for whole genome sequencing.

[0058] 3. Genome analysis and whole-genome comparative analysis De novo assembly of qualified sequence reads yielded the complete genome sequence of bacteriophage SPUP2. GeneMarkS, ORF finder, and RAST were used to predict all open reading frames (ORFs). BLASTp and InterProscan from the NCBI database were used to predict protein function. Genes encoding tRNA were screened using tRNAScan-SE. A genome map was constructed using BRIG, and BLASTn alignments were performed against the NCBI database. Two highly homologous genome sequences were selected for comparative genomic analysis.

[0059] The results are as follows Figure 4 As shown: The genome of bacteriophage SPUP2 is a double-stranded DNA, containing no genes encoding tRNA or any virulence factors. The entire genome is 45,785 bp in size, with a GC content of 45.90%. The genome encodes 51 open reading frames (ORFs), of which 21 are annotated as having specific functions. According to ICTV classification, bacteriophage SPUP2 belongs to... Duplodnaviria domain, Heunggongvirae boundary, Uroviricota Door, Caudoviricetes Outline, Autographivirales Science. A genome-wide comparison using the NCBI database revealed that the two bacteriophages most closely related to SPUP2 are... Vibrio phage vB_VpaP_MGD2 (homology 75.97%) Escherichia Phage PSK8 (70.36% homology), however, the coverage of similar gene regions was only 1%. This further confirms that phage SPUP2 represents a newly discovered novel Shewanella phage; namely, a putrefactive Shewanella phage. Shewanella putrefaciens bacteriophage) SPUP2, deposited at the China Center for Type Culture Collection, accession number: CCTCC M20251492, deposit date: June 30, 2025, address: Wuhan University, Wuhan, China.

[0060] Example 4: Phage preparations for controlling Shewanella putrefactive bacteria Phage preparations for controlling Shewan's putrefaction include lysis and activation buffers of Shewan's putrefaction phage SPUP2; among which, The activation solution has a concentration of 10. 9 Shewanella putrefactive bacteria CICC 22940 at CFU / mL; The lysis buffer of the above-mentioned bacteriophage SPUP2 includes the following steps: 1) Take 100 μL of bacteriophage SPuP2 stored in a glycerol tube (glycerol content 20%) and mix with 100 μL of Shewanella putrefaciens CICC22940 bacterial liquid in the logarithmic growth phase, add 15 mL of LB broth medium; under the condition of temperature 28℃ and rotation speed 120 rpm, shake culture for 12 h to obtain a bacteriophage-containing suspension; 2) Centrifuge the suspension at 8500 rpm and 4℃ for 15 min, take the supernatant and filter through a 0.22 μm sterile filter membrane, and the obtained supernatant is the lysate of activated bacteriophage SPuP2, with a titer of 10 8-9 PFU / mL.

[0061] The application scope is under the condition of temperature not higher than 50℃ and environmental pH value of 5-10.

[0062] The bacteriophage lysate can be stored at 20℃ for 2 years. If stored for more than 1 month and not used, activate the lysate before use, including the following steps: 1) Mix 100 μL of lysate and 100 μL of activation liquid with 15 mL of LB broth medium, and shake culture at 28℃ and 120 rpm for 12 h; 2) Centrifuge the obtained suspension at 4℃ and 8500 rpm for 15 min, filter through a 0.22 μm sterile filter membrane, and the supernatant is the activated lysate, with a titer of 10 8-9 PFU / mL.

[0063] Example 5 Analysis of bacteriophage SPuP2 lysis ability The lysis ability of bacteriophage SPuP2 was determined by measuring the optical density (OD 600nm ) value. The above-mentioned bacteriophage SPuP2 lysate and host bacteria were added to a 96-well plate at different MOIs (ranging from 100 to 0.001), and incubated at 28℃ for 15 hours. The positive control consisted of an equal volume of host strain suspension and LB broth mixture, while the negative control only contained LB broth. Subsequently, the OD 600nm value was measured using a microplate reader.

[0064] The results are shown in Figure 5 : at all MOIs, except MOI=0.001, the growth of host cells was significantly inhibited (OD 600nm <0.10) within 12 hours. In addition, a slow recovery of bacterial growth was observed after 12 hours of incubation. This indicates that bacteriophage SPuP2 has strong lysis ability to host Shewanella putrefaciens CICC 22940 within 12 h, and can significantly inhibit the growth of host bacteria.

[0065] Example 6: Inhibition and removal effects of bacteriophage preparations on Shewanella putrefactive biofilms 1. Inhibitory effect on biofilm formation 100 μL of host bacterial sample (Shewanella putrefaction CICC22940) and 100 μL of phage SPUP2 lysis buffer were mixed in a 96-well plate at the optimal MOI (MOI = 0.01); 100 μL of LB broth was used as a control instead of the phage solution. The 96-well plate was incubated at 28°C for 24 hours. Then, the culture was gently removed, and each well was washed twice with sterile PBS buffer. Each well was stained with 200 μL of 1% crystal violet dye for 15 minutes at room temperature, followed by washing three times with sterile PBS buffer to remove residual crystal violet. Finally, the stained biofilm was extracted with 200 μL of 95% ethanol and analyzed at OD... 595nm Quantitative quantity.

[0066] Biomembrane inhibition rate = (control group OD) 595 -Experimental group OD 595 ) / Control group OD 595 ×100%.

[0067] Similarly, after removing airborne bacteria, the biofilm on the well wall was scraped off with a sterile cotton swab and placed into a sterile centrifuge tube containing PBS buffer. The biofilm was then dispersed by shaking to count live cells.

[0068] The results are as follows Figure 6 As shown in Figure A, when the phage SPUP2 lysate in this formulation was co-cultured with Shewanella putrefactive bacteria for 24 hours, the lysate significantly inhibited biofilm formation during this period. The inhibition rate was highest at 12 hours, reaching 87.08%, at which point the number of viable cells in the biofilm decreased by approximately 1.09 ± 0.26 Log. 10 CFU / mL (99.99%).

[0069] 2. Scavenging effect of mature biofilms 100 μL of Shewanella putrefaction bacterial culture incubated overnight was added to 10 6 CFU / mL was mixed with 100 μL LB broth and added to a 96-well plate. The plate was incubated at 28°C for 24 hours to obtain a mature biofilm. After removing the culture medium, the cells were gently washed twice with PBS buffer. The experimental group was given the above-mentioned phage SPUP2 lysis buffer, while the negative control group was given PBS buffer instead of the phage lysis buffer. The cells were incubated at 28°C for 6 hours, and the results were measured using viable cell counting and crystal violet staining at 1, 2, 4, and 6 hours. Biofilm clearance rate = (control group OD...) / ( ... 595 -Experimental group OD 595 ) / Control group OD 595 ×100%.

[0070] Results table Figure 6 B shows: the preparation also has significant clearing effect on the mature biofilm, the clearing rate of biofilm reaches 75.56% after 6h treatment.

[0071] 3. Inhibition of biofilm extracellular substance production Take 100 μL host bacteria sample (Shewanella putrefaciens CICC22940) and 100 μL phage SPuP2 lysate to a 96-well plate to mix at the optimal MOI (MOI=0.01); use 100 μL LB broth instead of phage solution as the control group. Place the 96-well plate in a constant temperature culture environment at 28°C and continue to culture for 24 hours. Wash the biofilm formed in the well and resuspend in 1 mL PBS buffer, and ultrasonicate (53kHZ, 224W) the resuspended sample in an ice bath for 10 minutes. Centrifuge the mixture at 8500 rpm for 3 minutes at 4°C, and then filter the supernatant with a 0.22 μm membrane filter. Analyze the amount of biofilm extracellular polysaccharide by phenol-sulfuric acid method, and the result is expressed as OD 490nm . Determine the quantity of extracellular protein by Bradford protein concentration determination kit, and the result is expressed as OD 595nm .

[0072] Results Figure 6 C and Figure 6 D show: the phage preparation has obvious inhibitory effect on biofilm production, and can significantly inhibit the secretion of biofilm-related substances such as extracellular polysaccharide and extracellular protein, and after 12 hours of lysate treatment, the inhibition rate of extracellular polysaccharide is 51.60%, and the inhibition rate of extracellular protein is 70.78%.

[0073] 4. Observation of biofilm structure Observe the inhibitory effect of the phage preparation on biofilm formation and the clearing effect on the formed biofilm by CLSM measurement. The biofilm sample is prepared on a 24mm×24mm glass coverslip, and then the biofilm is fixed with 2.5% glutaraldehyde at 4°C for 1 hour, and then washed with 0.85% NaCl solution to ensure the removal of any glutaraldehyde residues. Subsequently, the biofilm is stained with BBcellProbe N01 dye in the dark for 30 minutes. Rinse the plate with 0.85% NaCl solution to remove excess dye and dry. Use a 100x objective to observe the green fluorescence excited at 488nm and emitted at 515nm.

[0074] As Figure 7 shown: the lysate of phage SPuP2 can collapse the biofilm structure and significantly reduce the green fluorescence produced by the living cells in the biofilm. It intuitively shows the inhibitory effect of the lysate in the phage preparation on the formation of Shewanella putrefaciens biofilm and the clearing effect on the mature biofilm.

[0075] Example 7 Bacteriophage preparation use in grass carp fillet samples for bacteriostatic effect Grass carp dorsal fillets were divided into uniform pieces (5 g ± 0.1 g) and drained. All cutting and weighing steps were performed in a sterile environment. The front and back surfaces of the fish were subjected to ultraviolet irradiation for 30 minutes, with the aim of ensuring the sterility of the samples. The uniform-sized and -thick fillets were divided into two groups, one group was inoculated with Shewanella putrefaciens (10 6 CFU / g), and the other group was inoculated with both Shewanella putrefaciens (10 6 CFU / g) and an equal volume of bacteriophage SPuP2 lysate, and the fillets were stored at 4°C for 7 days, and the viable bacteria count was determined on day 0, day 1, day 3, day 5, and day 7, respectively.

[0076] As shown in Figure Figure 8 A, under the condition of 4°C refrigerated storage, the number of Shewanella putrefaciens in the fillets was significantly inhibited after treatment with the liquid preparation, and the fillet spoilage was delayed. After 3 days of bacteriophage preparation treatment, S.putrefaciens the growth was significantly inhibited, and the viable bacteria count was determined to be 6.70 ± 0.02 Log 10 CFU / g, which was reduced by 1.93 ± 0.07 Log 10 CFU / g. S. putrefaciens Shewanella putrefaciens is a psychrophilic microorganism. Although there is a short lag phase at the initial stage of growth under low temperature conditions, the number of viable bacteria in fish meat continues to increase, showing a linear growth trend, and reaches 10.30 ± 0.13 Log 10 CFU / g after 7 days of storage. After treatment with the bacteriophage liquid preparation, it is 8.26 ± 0.11 Log 10 CFU / g.

[0077] Example 8 Evaluation of the physicochemical quality of grass carp fillets after the use of bacteriophage preparation The grass carp fillets treated in Example 7 were randomly divided into three groups as follows: The first group was inoculated with Shewanella putrefaciens (10 6 CFU / g), the second group was inoculated with both Shewanella putrefaciens (10 6 CFU / g) and an equal volume of bacteriophage SPuP2 lysate, and the third group was not treated.

[0078] After 7 days of storage at 4°C, samples were taken on day 0, day 1, day 3, day 5, and day 7, respectively, and pH meter, colorimeter, texture analyzer, etc. were used to detect the changes in physicochemical indicators of the fillets in each group during storage, including (pH, TVB-N value, TBARS value, color difference, texture).

[0079] Results as Figure 8 As shown in Table 2 and 3, after the liquid treatment, the color index of the fish fillets changed relatively gently,

[0080] Meanwhile, as shown in Table 2 and 3, after the liquid treatment, the color index of the fish fillets changed relatively gently, L the value decreased at a significantly lower rate than the control group, and b the value increased at a smaller amplitude, and the color of the fish fillets could be maintained for a longer time. Similarly, the hardness, elasticity, adhesion and resilience of the fish fillets decreased at a significantly lower amplitude than the control group, indicating that the liquid effectively delayed the deterioration of the color and texture of the fish fillets.

[0081] Table 2 Change in color difference on the surface of fish fillets during storage at 4℃ Table 3 Change in texture of fish fillets during storage at 4℃ Note: "G1" is the control group inoculated with only Shewanella putrefaciens, "G2" is the phage liquid treatment group, and "G3" is the blank group without any treatment.

[0082] Other parts not specifically described are prior art. Although the above examples make a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiments without creativity, which all belong to the protection scope of the present application.

Claims

1. A strain of Shewanella putrefactive bacteriophage ( Shewanella putrefaciens The bacteriophage) SPUP2 has the accession number CCTCC NO: M 20251492.

2. Use of the bacteriophage SPuP2 of Shewanella putrefaciens of claim 1 in the prevention and control of Shewanella putrefaciens.

3. A bacteriophage preparation against Shewanella putrefaciens, characterized by: The preparation comprises the lysate of the phage SPuP2 according to claim 1, wherein the titer of the lysate of the phage SPuP2 is 10 8-9 PFU / mL.

4. The bacteriophage preparation of claim 3, wherein: The preparation also comprises an activation liquid; wherein the activation liquid is a Shewanella putrefaciens CICC 22940 with a concentration of 10 9 CFU / mL. The preparation also comprises an activation liquid; wherein the activation liquid is a Shewanella putrefaciens CICC 22940 with a concentration of 10 9 CFU / mL.

5. The bacteriophage preparation of claim 3, wherein: The preparation method of the lysate of the bacteriophage SPuP2 is as follows: 1) Take the bacteriophage SPuP2 of Shewanella putrefaciens stored in a glycerol tube and mix with the bacterial liquid of Shewanella putrefaciens CICC22940 in the logarithmic growth phase, add broth medium for culture, and obtain a suspension liquid containing the bacteriophage; 2) Centrifuge the suspension liquid, take the supernatant and filter through a sterile filter membrane, and the obtained supernatant is the lysate of the bacteriophage SPuP2.

6. Use of the preparation of claim 3 in the biological prevention and control of Shewanella putrefaciens.

7. Use of the preparation of claim 3 in the inhibition of the growth of the biofilm of Shewanella putrefaciens and the removal of the mature biofilm.

8. Use of the preparation of claim 3 in the biological prevention and control of Shewanella putrefaciens of food.

9. Use according to claim 8, characterized in that: The food is aquatic products.

10. Use of the preparation of claim 3 in the biological prevention and control of food spoilage.

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