Shewanella putrefactive bacteriophage SPX1 and its application

Abstract

This invention discloses a Shewanella putrefaciens bacteriophage SPX1 and its applications. The genomic and biological characteristics of a Shewanella putrefaciens bacteriophage SPX1 strain were analyzed, and it was named Shewanella putrefaciens bacteriophage SPX1, with accession number CCTCC NO: M 2024544. This invention provides a highly efficient, safe, and widely applicable kit for the control of Shewanella putrefaciens and biofilms at various stages in the seafood industry, promoting sustainable development in the entire seafood industry for controlling food spoilage bacteria.

Description

Technical Field

[0001] This invention relates to the field of food safety technology for controlling spoilage microorganisms, specifically to a Shewanella putrefactive bacteriophage SPX1 and its applications. Background Technology

[0002] Shewanella is a group of Gram-negative bacteria belonging to the phylum Proteobacteria and subphylum Gammaproteobacteria. They are widely distributed in nature and can be found in various environments, including marine, freshwater, and soil. Some of the major Shewanella species discovered to date include: *Shewanella oneidensis*, known for its redox activity against metals; *Shewanella algae*, which plays a role in marine ecosystems; and *Shewanella putrefaciens*, commonly found in food, especially seafood.

[0003] Seafood is rich in nutrients and delicious, but it is highly susceptible to microbial contamination and spoilage, resulting in nearly 30% of seafood losses annually. Shewanella putrefaction is a common specific spoilage organism (SSO) found in seafood. During spoilage, it destroys nutrients and degrades the flavor and quality of seafood such as fish and shrimp. Furthermore, Shewanella putrefaction can adhere to the surfaces of food and processing / transportation equipment, forming biofilms. Biofilms are composed of bacterial cells and extracellular polymeric substances (EPS). These EPS molecules enhance the stability of the biofilm, making it more difficult to remove than planktonic cells, thus causing subsequent seafood spoilage and cross-contamination in the processing industry. Biofilm removal methods include physical removal, chemical additives, and biocontrol. Commonly used physical methods include ultrasound and low-current methods. Chemical additives generally include inorganic and organic antimicrobial agents. Biocontrol methods focus on enzyme destruction, bacteriophages, and inhibition of quorum sensing systems. However, physical removal is not always effective in seafood and its processing facilities, and chemical additives may affect the sensory characteristics of food or the production environment. Therefore, biocontrol, as a novel strategy for removing biofilms, deserves further exploration.

[0004] Bacteriophages, a type of bacterial virus, are mainly composed of nucleic acid and a protein coat. They bind specifically to receptors on the surface of bacterial cells, injecting their genetic material into the host bacteria to begin multiplication, leading to bacterial cell lysis. They are the most abundant biological group in the biological world, exhibiting strict host specificity, highly efficient at infecting and killing bacteria, and are also simple to prepare and store. In recent years, bacteriophages have received widespread attention as novel bactericidal agents, and the U.S. Food and Drug Administration (FDA) permitted their use as antimicrobial agents in food in 2015. For example, formulations containing two Salmonella-specific bacterial phages (BP-63 and BP-12 Triumvirate) (Salmonella phage formulations) can be used as antimicrobial agents to control Salmonella on poultry products; Salmonella phage formulations (FO1a and S16) have obtained Generally Recognized as Safe (GRAS) through scientific procedures and can be used as antimicrobial agents to control Salmonella on beef and vegetables; Escherichia coli phage formulations can be used as antimicrobial agents in food, especially in beef carcasses, cut beef, and ground beef, to control E. coli O157.

[0005] Currently, bacteriophages are important bactericidal tools, capable of inhibiting bacterial growth and also suppressing and removing biofilms. In biocontrol of biofilms, they can effectively remove bacterial extracellular polymers, reducing bacterial numbers. As a biocontrol agent, bacteriophages are also highly efficient, safe, and do not affect the quality of seafood, showing promising application prospects in controlling food spoilage bacteria and their biofilms in seafood. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a Shewanella putrefaciens bacteriophage SPX1 and its applications.

[0007] To achieve the above objectives, the technical solution designed by the present invention is as follows:

[0008] This invention provides a strain of Shewanella putrefaciensbacteriophage SPX1, which is deposited at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2024544.

[0009] The aforementioned *Shewanella putrefaction* phages were isolated and screened using *Shewanella putrefaction* ATCC 49138 as the host bacterium. Morphological observation and genomic analysis were performed on the screened phages, providing new resources for the *Shewanella putrefaction* phage resource library. The *Shewanella putrefaction* phage SPX1 described herein possesses the following characteristics:

[0010] 1) Morphological characteristics: Observed by transmission electron microscopy, the size of bacteriophage (Shewanella putrefaciens) SPX1 is about 258.7 nm, the head has a polyhedral structure with a length of about 61.4 nm, and the tail can shrink to a length of about 190.1 nm.

[0011] 2) Nucleic acid type: Bacteriophage SPX1 is a dsDNA bacteriophage.

[0012] 3) Genomic characteristics: The full-length genome of bacteriophage SPX1 is 53,428 bp, with a GC content of 49.72%. It contains no tRNA or any virulence factors and includes 69 open reading frames (ORFs), 29 of which are predicted to be functional proteins. According to ICTV classification, it belongs to the domain Duplodnaviria, kingdom Heunggongvirae, phylum Uroviricota, class Caudoviricetes, subfamily Nefertitivirinae, and genus Yushanvirus. Whole-genome alignment with the NCBI database revealed an 80.15% similarity to the bacteriophage with the highest homology (Shewanella putrefactiveis bacteriophage Spp001), thus classifying it as a novel bacteriophage.

[0013] The aforementioned *Shewanella putrefactive* phage SPX1 is approximately 258.7 nm in size, possessing a polyhedral head with a diameter of 61.4 ± 0.01 nm and a retractable tail with a length of 190.1 ± 0.02 nm. According to ICTV classification, it belongs to the domain *Duplodnaviria*, kingdom *Heunggongvirae*, phylum *Uroviricota*, class *Caudoviricetes*, subfamily *Nefertitivirinae*, and genus *Yushanvirus*.

[0014] Shewanella putrefaciens bacteriophage SPX1, deposited at the China Center for Type Culture Collection (CCTCC) with accession number M 2024544, date of deposit March 25, 2024, address: Wuhan University, Wuhan, China.

[0015] The present invention also provides an application of the above-mentioned Shewanella putrefactive bacteriophage SPX1 in the control of Shewanella putrefactive bacteria.

[0016] The present invention also provides a kit for controlling Shewanella putrefactive bacteria, the kit comprising a lysate of the aforementioned bacteriophage SPX1, wherein the titer of the bacteriophage SPX1 lysate is 10. 9 PFU / mL.

[0017] The lysate of the above-mentioned bacteriophage SPX1 was obtained by activating bacteriophage SPX1 and culturing it to obtain a suspension (the suspension was used to lyse Shewanella putrefactive ATCC 49138).

[0018] Furthermore, the kit includes a lysis buffer for phage SPX1, a buffer solution, activation solution 1, and activation solution 2; wherein,

[0019] The buffer solution is sterile 1×PBS buffer.

[0020] Activation solution 1 is a 2% sterile LB broth medium.

[0021] Activation solution 2 has a concentration of 10. 9 Shewanella putrefactive bacteria ATCC 49138 at CFU / mL.

[0022] The activation solution 1 of the above-mentioned reagent kit can be stored at 4°C, and the lysis buffer and activation solution 2 can be stored at -20°C for 2 years. If it has not been used for more than 1 month, the lysis buffer should be activated before use. The steps are as follows:

[0023] 1) Mix 100 μL of lysis buffer and 100 μL of activation buffer 2 with 10 mL of activation buffer 1, and incubate on a shaker;

[0024] 2) After the culture is completed, centrifuge and filter. The supernatant is the activated lysis solution. The culture conditions are: temperature 28℃, speed 120rpm, culture time 12 hours; centrifugation conditions are: temperature 4℃, speed 8000r / min, centrifugation time 20min.

[0025] The application range is a temperature not exceeding 50℃ and an environmental pH value of 3-11. It can be used successively with ultraviolet sterilization but not simultaneously.

[0026] The present invention also provides an application of the above-mentioned reagent kit in the biocontrol of Shewanella putrefactive bacteria.

[0027] The present invention also provides an application of the above-mentioned kit in inhibiting or reducing infection or quality deterioration caused by Shewanella putrefactive in seafood / meat products.

[0028] Furthermore, the seafood is a shrimp-related substrate.

[0029] The aforementioned kit can effectively inhibit or reduce infection or quality deterioration caused by Shewanella putrefactive bacteria in seafood, with a sterilization rate of 99%-99.99%.

[0030] The present invention also provides the application of the above-mentioned reagent kit in inhibiting the growth of biofilm in Shewanella putrefactive bacteria and removing mature biofilm.

[0031] The aforementioned kit can effectively inhibit and remove Shewanella putrefactive biofilm, with an inhibition rate of 61.42% and a removal rate of 63.78%.

[0032] Furthermore, the biofilm is attached to the surface of the article, wherein the article is selected from seafood, meat products and their processing environment, processing equipment and transport packaging materials.

[0033] The aforementioned equipment and transport packaging materials are made of stainless steel, glass, and polyethylene. The reagent kit effectively prevents and removes biofilms in the aforementioned scenarios, reducing biofilm formation by approximately 2 log on the surfaces of seafood, meat products and their processing environments, processing equipment, and seafood transport packaging materials. 10 CFU / cm 2 The mature biofilm reduces subsequent cross-contamination by bacteria. This invention also provides an application of the above-mentioned reagent kit in food biocontrol.

[0034] The aforementioned kit will not change the color or texture of the shrimp meat and can be safely used in food for biocontrol.

[0035] The inventive principle of this invention:

[0036] Bacteriophages can specifically bind to receptors on the surface of bacterial cells, inject their genetic material into the host bacteria, and begin to multiply, causing bacterial cell lysis. In addition to inhibiting bacterial growth, they also have the ability to inhibit and remove biofilms. EPS in biofilms can act as secondary receptors for bacteriophages, while acting as primary receptors when bacteriophages cannot directly contact bacterial surface receptors. Therefore, bacteriophages can effectively remove bacterial extracellular polymeric substances and reduce the number of bacteria.

[0037] The beneficial effects of this invention are:

[0038] This invention analyzes the genome and biological characteristics of a bacteriophage SPX1 of Shewanella putrefactive bacteria. This invention provides a highly efficient, safe and widely applicable biological kit for the control of Shewanella putrefactive bacteria and biofilms at various stages in the seafood industry, promoting the sustainable development of the entire seafood industry in controlling food spoilage bacteria. Attached Figure Description

[0039] Figure 1 This image shows the morphology of plaques formed by bacteriophage SPX1 on a double-layer plate.

[0040] Figure 2 This is a morphological image of bacteriophage SPX1 under a transmission electron microscope.

[0041] Figure 3 This is a diagram illustrating the biological characteristics of bacteriophage SPX1.

[0042] In the figure, A is a schematic diagram of the optimal multiplicity of infection for bacteriophage SPX1.

[0043] B is a schematic diagram showing the adsorption rate of bacteriophage SPX1.

[0044] C is the one-step growth curve of bacteriophage SPX1.

[0045] D is a schematic diagram illustrating the ability of bacteriophage SPX1 to lyse host bacteria under different MOIs.

[0046] Figure 4 This is a schematic diagram illustrating the environmental stability of bacteriophage SPX1.

[0047] In the figure, A is a schematic diagram of the temperature stability of bacteriophage SPX1.

[0048] B is a schematic diagram of the pH stability of bacteriophage SPX1.

[0049] C is a schematic diagram of the UV tolerance of bacteriophage SPX1.

[0050] Figure 5 This is a schematic diagram illustrating the inhibitory effect of bacteriophage SPX1 on host bacteria in shrimp matrix.

[0051] In the figure, A is a schematic diagram of the inhibitory effect of bacteriophage SPX1 on host bacteria in shrimp juice, and B is a schematic diagram of the inhibitory effect of bacteriophage SPX1 on host bacteria in shrimp pieces.

[0052] Figure 6 This is a schematic diagram illustrating the inhibitory and scavenging effects of bacteriophage SPX1 on biofilms.

[0053] In the figure, A is a schematic diagram illustrating the inhibitory effect of bacteriophage SPX1 on developing biofilms in LB.

[0054] B is a schematic diagram illustrating the scavenging effect of bacteriophage SPX1 on mature biofilms in LB.

[0055] Figure 7 This is a schematic diagram illustrating the biofilm removal effect of bacteriophage SPX1 in practical applications.

[0056] In the figure, A is a schematic diagram illustrating the scavenging effect of bacteriophage SPX1 on mature biofilms in shrimp substrate.

[0057] B is a schematic diagram illustrating the removal effect of bacteriophage SPX1 on mature biofilms on food contact surfaces. Detailed Implementation

[0058] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0059] Example 1: Isolation, purification, and analysis of Shewanella putrefactive bacteriophage SPX1

[0060] 1. Isolation and purification of bacteriophages

[0061] Samples taken from the water used for farming whiteleg shrimp in a seafood market were cultured overnight with Shewanella putrefactive bacteria in the logarithmic growth phase for 12 hours. The mixture was then filtered through a 0.22 μm microporous membrane. The filtrate was enriched once using the same method to obtain the phage stock solution. The phage was purified using the double-layer plate method, repeated 5-10 times until plaques of uniform size and transparency appeared on the double-layer plates. This indicated purified phage. The number of plaques was observed to determine the phage titer. Phage titer (PFU / mL) = number of plaques × dilution factor × 10. The results showed that the phage solution titer reached 10. 9 PFU / mL.

[0062] 2. Morphological analysis of bacteriophages

[0063] The phage suspension was centrifuged at 40,000 r / min for 1 hour at 4℃ to precipitate phage particles. The precipitate was resuspended in 0.1 mol / L ammonium acetate. Using the phosphotungstic acid negative staining method, a copper mesh was immersed in the phage resuspension. After 10 minutes, the excess liquid was aspirated, and the mesh was stained with 2% (v / v) phosphotungstic acid dye at pH=7 for 10 minutes. The mesh was then air-dried until completely dry. The morphology of the phages was observed under a transmission electron microscope, and their size was measured using the software Digital Micrograph Demo 3.9.1.

[0064] The above results show that this bacteriophage can form clear and transparent plaques of approximately 1.0 mm in size on agar plates. Figure 1 ), observed under a transmission electron microscope ( Figure 2 The phage is approximately 258.7 nm in size, with a polyhedral head of approximately 61.4 ± 0.01 nm in diameter and a retractable tail of approximately 190.1 ± 0.02 nm in length.

[0065] 3. Phage SPX1 Genome Analysis

[0066] 1) Extracting bacteriophage DNA

[0067] a. Take 1 mL of high-titer phage suspension, add 3 μL of DNase I and RNase A, incubate at 37°C for 1 hour, then add 24 μL of 0.5% EDTA (pH = 8.0), mix well, and heat in an 80°C water bath for 15 minutes.

[0068] b. Add 1.5 μL of 20 μg / mL proteinase K and 30 μL of 10% SDS solution to the enzyme-treated phage solution, mix by inverting, and incubate at 56°C for 1 hour.

[0069] c. After naturally cooling to room temperature, add an equal volume of equilibrated phenol solution, centrifuge at 12000 r / min for 10 minutes, add an equal volume of phenol / chloroform / isoamyl alcohol (volume ratio of 25:24:1) to the aspirated upper layer solution, and centrifuge at 12000 r / min for 10 minutes.

[0070] d. Add an equal volume of chloroform to the aspirated supernatant, centrifuge at 12000 r / min for 10 minutes, add an equal volume of isopropanol to the aspirated supernatant, and incubate at -20℃ on ice for 3 hours to precipitate DNA.

[0071] e. Finally, centrifuge at 13000 r / min for 20 minutes, add 1 mL of 75% pre-cooled ethanol to the precipitate, let stand for 10 minutes, centrifuge at 12000 r / min for 10 minutes, discard the ethanol and place it in a clean bench to evaporate the ethanol; dissolve the DNA with TE (pH 7.6) to obtain the genomic DNA sample, and store at -20℃.

[0072] 2) Phage whole genome sequencing

[0073] After the extracted phage DNA passed the test using an ultra-micro spectrophotometer, it was sent to Wuhan Boyue Zhihe Biotechnology Co., Ltd. for whole-genome sequencing.

[0074] 3) Genome analysis and whole-genome comparative analysis

[0075] All open reading frames (ORFs) were predicted using Gene Mark S and RAST, followed by functional analysis of the ORFs using BLASTp and a conserved domain database. Two closely related bacteriophages were selected from the NCBI database using BLASTn according to the classification of the International Committee on Taxonomy of Viruses, and whole-genome comparisons of the three bacteriophages were performed using BRIG and Easyfig 2.2.5.

[0076] The results showed that the SPX1 phage genome is a double-stranded DNA, without tRNAs or any virulence factor genes. The entire genome size is 53428 bp, the GC content is 49.72%, and it has 69 open reading frames, of which 29 are predicted to be functional proteins. According to ICTV classification, it belongs to the domain Duplodnaviria, kingdom Heunggongvirae, phylum Uroviricota, class Caudoviricetes, subfamily Nefertitivirinae, and genus Yushanvirus. Genome-wide comparison with the NCBI database revealed that the two closest bacteriophages to SPX1 are bacteriophage Spp001 (80.15% homology) and bacteriophage SppYZU05 (77.02% homology). Based on the genomic data, it can be determined that this is a novel bacteriophage; namely, Shewanella putrefaciens bacteriophage SPX1, deposited at the China Center for Type Culture Collection (CCTCC) with accession number M2024544, deposited on March 25, 2024, at Wuhan University, Wuhan, China.

[0077] Example 2: Biological characteristics analysis of Shewanella putrefactive bacteriophage SPX1

[0078] 1. Detection of the host range of bacteriophage SPX1

[0079] A total of 15 bacterial strains were selected for testing, including 2 Shewanella putrefaciens strains, 2 Vibrio parahaemolyticus strains, 3 Salmonella strains, 2 Listeria monocytogenes strains, 2 Staphylococcus aureus strains, 2 Escherichia coli strains, and 2 Pseudomonas strains. After activation, 100 μL of the bacterial suspension cultured to the logarithmic development phase was poured into a double-layer plate. After solidification, 10 μL of a titer of 10 was added. 7 PFU / mL phage SPX1 was placed on the surface of the upper plate, dried, and then inverted in an incubator at 37℃ or 28℃ for 4-6 hours. The lysis was observed. The experiment was repeated 3 times, with 2 replicates each time.

[0080] 2. Detection of the optimal multiple of infection (MOI) for bacteriophage SPX1

[0081] The multiplicity of infection (MOI) is the ratio of the number of bacteriophages to the number of host bacteria at the time of initial infection. Bacteriophages were mixed with host bacteria in the logarithmic growth phase at MOIs of 0.0001, 0.001, 0.01, 0.1, 1, 10, 100, and 1000, incubated at 28°C for 4 hours, centrifuged at 8000 rpm for 20 minutes, and the phage titer in the supernatant of samples with different MOI values ​​was determined using the double-layer plate method. The experiment was repeated three times, with two replicates each time.

[0082] 3. Detection of SPX1 phage adsorption rate

[0083] Phage fluid was mixed with host bacteria cultured to the logarithmic growth phase in centrifuge tubes at the optimal MOI of 0.001 and incubated on a shaker at 28°C. Starting from 0 minutes, the titer of phage in the supernatant was determined every 3 minutes using the double-layer plate method. The experiment was repeated three times, with two replicates each time. Adsorption rate = 1 - (Title of unadsorbed phage at each time point / Phage titer at 0 minutes) × 100%

[0084] 4. Detection of the one-step growth curve of bacteriophage SPX1

[0085] Equal volumes of bacteriophage and host bacteria were mixed at an MOI of 0.001, incubated at 28°C for 3 minutes, centrifuged at 8000 rpm for 2 minutes, and the supernatant was discarded. The precipitate was washed with 1 mL of LB medium and centrifuged twice. The resuspension was added to LB liquid medium and incubated on a shaker at 28°C. Samples were taken every 10 minutes and centrifuged at 8000 rpm for 2 minutes. The titer of bacteriophage in the supernatant was determined using the double-layer plate method. The experiment was repeated three times, with two replicates each time. Lysis rate = phage titer at the end of lysis / host bacteria concentration at the initial stage of infection.

[0086] 5. Detection of SPX1 phage lysis curve

[0087] Add 100 μL of 10 to the 96-well plate 5 CFU / mL host bacterial culture medium was used to add 100 μL of bacteriophage to each well at MOI values ​​of 100, 10, 1, 0.1, 0.01, 0.001, and 0.0001. A negative control was added to 200 μL of LB medium, and a positive control was added to 100 μL of LB medium. 5 The host bacterial culture was prepared at CFU / mL and 100 μL of LB liquid medium. The absorbance was measured at 600 nm every hour.

[0088] The results above show that, as shown in Table 1, bacteriophage SPX1 only has a lytic effect on the host bacteria; Figure 3 As shown in Figure A, the phage titer reaches its maximum at MOI = 0.001, approximately 1.6 × 10⁻⁶.9 PFU / mL indicates that a phage titer to host bacterial concentration ratio of 0.001 is optimal for phage proliferation, resulting in more progeny phages. Figure 3 As shown in Figure B, the bacteriophage reached its optimal adsorption rate of 85.69% at 3 minutes, and adsorption was completed within 15-18 minutes; Figure 3 As shown in C, the phage latency period was 10 minutes, the outbreak period was 150 minutes, and the lysis rate was 43.81 PFU / cell; Figure 3 As shown in D, the phage significantly inhibited the growth of the host bacteria within 13 hours.

[0089] Table 1. Lysis spectrum of bacteriophage SPX1

[0090]

[0091] Note: a++ indicates that the phage plaque is clear and transparent, and — indicates that it has no lysis ability.

[0092] b LC,Lab Conservation.ATCC,American Type Culture Collection.

[0093] Example 3

[0094] The kit for controlling Shewanella putrefactive bacteria includes the aforementioned phage SPX1 lysis buffer, buffer solution, activation solution 1, and activation solution 2; wherein,

[0095] The titer of the lysate of bacteriophage SPX1 was 10. 9 PFU / mL;

[0096] The lysate of the above-mentioned bacteriophage SPX1 was obtained by activating bacteriophage SPX1 and culturing it to obtain a suspension (the suspension was used to lyse Shewanella putrefactive ATCC 49138).

[0097] The buffer solution is sterile 1×PBS buffer.

[0098] Activation solution 1 is a 2% sterile LB broth medium.

[0099] Activation solution 2 has a concentration of 10. 9 Shewanella putrefactive bacteria ATCC 49138 at CFU / mL.

[0100] The activation solution 1 of the above-mentioned reagent kit can be stored at 4°C, and the lysis buffer and activation solution 2 can be stored at -20°C for 2 years. If it has not been used for more than 1 month, the lysis buffer should be activated before use. The steps are as follows:

[0101] 1) Mix 100 μL of lysis buffer and 100 μL of activation buffer 2 with 10 mL of activation buffer 1, and incubate on a shaker;

[0102] 2) After culture, centrifuge and filter. The supernatant is the activated lysate, and the titer reaches 10. 9 PFU / mL indicates activation is complete; the shaking conditions for cultivation are: temperature 28℃, rotation speed 120 rpm, cultivation time 12 hours; the centrifugation conditions are: temperature 4℃, speed 8000 r / min, centrifugation time 20 minutes.

[0103] The application range is a temperature not exceeding 50℃ and an environmental pH value of 3-11. It can be used successively with ultraviolet sterilization but not simultaneously.

[0104] Example 4: Stability of lysate of bacteriophage SPX1

[0105] 1. Stability of SPX1 lysate under different temperature conditions

[0106] The suspension obtained by activating bacteriophage SPX1 and culturing it is a solution with a titer of 10. 8 The lysate of phage SPX1 was prepared by dividing it into multiple 1 mL portions and placing them in constant temperature water baths at 30°C, 40°C, 50°C, 60°C, 70°C, and 80°C. The titer of the phage in each tube was determined every 20 minutes using the double-layer plate method.

[0107] 2. Stability of SPX1 lysate under different pH conditions

[0108] The suspension obtained by activating bacteriophage SPX1 and culturing it is a solution with a titer of 10. 6 PFU / mL phage SPX1 lysis buffer; take 100 μL of phage SPX1 lysis buffer with a titer of 10 6 Phage solution at a concentration of PFU / mL was added to 900 μL of PBS buffer at different pH values ​​(2–13) and incubated at 28°C for 2 hours. The titer of the phage in each centrifuge tube was then determined using the double-layer plate method. 5 mL of phage solution with a titer of 10... 6 PFU / mL phage solution was poured into a 90mm culture dish to form a uniform thin layer. The dish was then irradiated with a 40W UV lamp at a height of 30cm in a clean bench. The titer was determined every 15 minutes using the double-layer plate method. The experiment was repeated three times, with two replicates each time.

[0109] The results showed that bacteriophage SPX1 was effective at temperatures between 30-50℃. Figure 4 A) and pH 3-11 ( Figure 4 B) exhibits good stability, but its activity decreases in a gradient under ultraviolet light. Figure 4 C).

[0110] Example 5: Inhibition and removal effect of the reagent kit on Shewanella putrefactive bacteria and its biofilm.

[0111] 1. Inhibitory ability against Shewanella putrefactive bacteria

[0112] Purchase fresh whiteleg shrimp from a local supermarket. Thoroughly wash the shrimp several times with sterile distilled water. Select undamaged shrimp, remove the shells, and mince the shrimp meat and sterile water in a 1:3 ratio using a food processor. Centrifuge at 5000 rpm for 15 minutes. Filter the shrimp juice sequentially through sterile filter paper and a 0.22 μm filter membrane, and store at -20°C for later use. Similarly, cut the undamaged shrimp into uniformly sized pieces (approximately 2 × 2 cm). 2 Place the shrimp pieces in a sterile petri dish and wipe the surface with alcohol. Then, irradiate both sides of the shrimp pieces with ultraviolet light for 30 minutes each. Add sterile shrimp juice and shrimp pieces containing PBS buffer to sterile EP tubes, then add bacterial suspension (10... 5 After adding the lysis buffer (CFU / mL) from the kit, the positive control group used PBS buffer instead. The cells were incubated at 4°C and 25°C, and bacterial counts were determined using a live cell counting method.

[0113] The results show that: Figure 5 As shown in Figure A, after reacting at 25℃ for 9 hours, the bacterial count in the shrimp juice increased from 10 log... 10 CFU / mL decreased to 4 log 10 CFU / mL (>99.99%) Figure 5 In B, the bacterial count in the shrimp pieces increased from 9 log... 10 CFU / mL decreased to 4 log 10 CFU / mL (>99.99%). After reacting at 4°C for 12 hours, the colony count in both systems decreased to 4 log₂. 10 CFU / mL.

[0114] 2. Inhibitory effect on the formation of immature biofilms

[0115] 100 μL each of the host bacteria and the phage lysate from the kit were mixed in a 96-well plate. No phage was added to the negative control group. The plates were incubated at 28°C for 24 hours. After incubation, the supernatant from each well was collected and washed twice with sterile PBS buffer. Subsequently, each well was stained with 1% crystal violet for 15 minutes at room temperature. After washing off the stain and air-drying, 200 μL of 95% ethanol was added to each well, and the absorbance was measured at 595 nm. Similarly, after removing airborne bacteria, the wells were wiped with sterile cotton swabs and then placed in centrifuge tubes containing sterile glass beads and PBS buffer. The biofilm was vortexed in PBS buffer for viable cell counting. Biofilm inhibition rate = (control group OD) / (control group OD) ... 595 -Experimental group OD 595 ) / Control group OD595 ×100%.

[0116] The results show that: Figure 6 As shown in Figure A, in LB, the lysate of bacteriophage SPX1 from this kit, co-cultured with Shewanella putrefactive bacteria for 24 hours, showed a significant inhibitory effect on biofilm formation at any time point, with the highest inhibition rate reaching 61.42% at 12 hours, and the viable cell count decreased by approximately 2 log. 10 CFU / mL (>99%).

[0117] 3. Scavenging effect on mature biofilms

[0118] The host bacterium SPX1 was mixed with LB broth and cultured in 96-well plates for 24 hours until the biofilm matured. The suspension was then aspirated, and the wells were washed three times with PBS buffer. The experimental group received lysis buffer of phage SPX1 from the kit, while the negative control group used PBS buffer instead of phage. Samples were then treated at 28°C for 3, 5, and 7 hours. Results were subsequently determined using viable cell counting and crystal violet staining. Biofilm clearance rate = (control group OD...) / ( ... 595 -Experimental group OD 595 ) / Control group OD 595 ×100%.

[0119] Bacterial suspension and sterile shrimp juice were added to 6-well plates, and shrimp pieces were immersed in the bacterial suspension for 120 seconds. The plates were then air-dried in a biosafety cabinet for 20 minutes and sealed in sterile petri dishes. All petri dishes were incubated at 28°C for 24 hours until the biofilm matured. After incubation, the wells and shrimp surfaces were rinsed three times with sterile distilled water to remove any unattached cells. The experimental group received phage lysis buffer from the kit for 10 minutes, while the control group used PBS buffer instead of the phage solution. After 5 hours of incubation at 28°C, the amount of residual biofilm was determined using a cotton swab method.

[0120] After inoculating the bacterial suspension onto shrimp pieces and air-drying them, sterile stainless steel, glass, and PE plastic (approximately 2 x 2 cm) were used. 2 The phages were attached to the surface of shrimp shells and cultured at 28°C until the biofilm matured. Subsequently, phages were treated using the method described above, and viable cell counts were performed to assess the clearance effect.

[0121] The above results indicate that: Figure 6 B showed that the kit also had a good clearance effect on the mature SPX1 biofilm in LB, with a clearance rate of approximately 63.78% after 7 hours and a reduction in viable cell count of approximately 3 log. 10 CFU / mL (>99.9%). In two substrates for shrimp ( Figure 7 A) After 5 hours, the number of bacteria in the biofilm of shrimp juice and shrimp pieces decreased by approximately 2.456 log₂ / ₃.10 CFU / cm 2 (>99%) and 1.838 log 10 CFU / cm 2 (>98.5%). On the surfaces of the three contact materials ( Figure 7 B) The kit also exhibits good biofilm removal performance, with a glass thickness of approximately 1.975 log₂. 10 CFU / cm 2 Stainless steel is approximately 1.962 log. 10 CFU / cm 2 PE is approximately 1.382 log 10 CFU / cm 2 .

[0122] Example 7: Evaluation of shrimp meat quality after using the reagent kit

[0123] The sterile shrimp meat pieces of the same size were divided into four groups. The first group was left untreated, and the second group was inoculated with Shewanella putrefaction (10). 5 After incubating for 5 hours following a 2-minute inoculation with CFU / mL, the third group was inoculated with lysate of phage SPX1 from the kit (10 CFU / mL). 9 After incubating for 10 minutes with PFU / mL, the fourth group was inoculated with bacteria from the second group and phages from the third group and incubated for 5 hours. The quality of the shrimp meat in each group was tested using a colorimeter and a texture analyzer.

[0124] The results showed that, as shown in Tables 2 and 3, the color of the shrimp meat surface did not change significantly after treatment with the reagent kit, and its elasticity, adhesion and resilience were also unchanged.

[0125] Table 2. Color of shrimp meat surface before and after treatment.

[0126]

[0127] Note: L represents black and white brightness, a represents red and green value, and b represents yellow and blue value.

[0128] Table 3. Texture of shrimp meat before and after treatment

[0129]

[0130] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Shewanella putrefactive bacteriophage ( Shewanella putrefaciens (bacteriophage) SPX1, deposited at Wuhan University, Wuhan, China, accession number CCTCC NO: M 2024544. It belongs to the Yushanvirus genus.

2. The use of the Shewanella putrefactive bacteriophage SPX1 as described in claim 1 in the preparation of a kit for controlling Shewanella putrefactive bacteria.

3. A kit for controlling Shewanella putrefactive bacteria, characterized in that: The kit includes the Shewanella putrefactive bacteriophage SPX1 as described in claim 1.

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