A thermostable algae Shewanella phage PKP-SH23021 and its application

Abstract

The present invention provides a high-temperature-resistant seaweed Shewanella phage PKP‑SH23021 and its application. The seaweed Shewanella phage was deposited in the General Microbiology Center of the China Microbiological Culture Collection Committee on November 29, 2023, with a deposit number of CGMCC No.45781. The present invention also provides a phage composition, which includes the high-temperature-resistant seaweed Shewanella phage PKP‑SH23021 as described above. The present invention also provides a phage pharmaceutical preparation, a water environment disinfectant, and a biological antibacterial agent for treating fresh or frozen aquatic products. The seaweed Shewanella phage PKP‑SH23021 provided by the present invention is sprayed with powder by a spray dryer to make a phage powder, and the potency can still be maintained at 10 8 PFU / g. The algae Shewanella phage PKP-SH23021 powder can not only withstand short-term high temperature, but also can be stored for a long time. The phage provided by the invention is obtained from nature, can be stably inherited, is suitable for large-scale industrial production, and is safe to use and environmentally friendly.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a high-temperature resistant algae Shewanella phage PKP-SH23021 and an application thereof. Background Art

[0002] Shewanella algae Shewanella alga ) belongs to the genus Shewanella and is widely distributed in nature, especially in the marine environment. Shewanella algae is a photosynthetic bacterium that usually coexists with seaweed, can decompose organic matter in seaweed, and provide nutrition for seaweed. At the same time, Shewanella algae can also be used as a biofertilizer and bioremediation agent, etc., which plays an important role in the balance and stability of the marine ecosystem. However, if the number of Shewanella algae is too large, it will consume too much oxygen, making it impossible for other organisms in the marine ecosystem to survive. It is also an important conditional pathogen of human-fish comorbidity, causing serious economic losses to aquaculture. This bacterium can cause fish infection, and there are more and more reports of its infection. At the same time, it is extremely harmful to human health. Shewanella algae can cause sepsis, bacterial infection, etc.

[0003] At present, antibiotics are mainly used to control Shewanella algae in aquaculture. Due to the irregular use of antibiotics, bacterial resistance is becoming more and more common. In response to the national policy of reducing and replacing antibiotics, it is necessary to find antibiotic substitutes that can control Shewanella algae, reduce the use of antibiotics, and protect human health and safety.

[0004] Bacteriophage is a general term for viruses that infect microorganisms such as bacteria, fungi, actinomycetes or spirochetes. They are extremely small in size, have no complete cell structure, and are composed of nucleic acids and proteins. They cannot grow and reproduce on their own without a host, so they must grow in living cells. Bacteriophages must parasitize in living bacteria and have strict host specificity, which depends on the molecular structure and complementarity of the phage adsorption organs and the receptors on the surface of the receptor bacteria. Generally, a bacterium can often be infected by multiple phages, but a phage can only infect one bacterium and bacteria similar to it. Bacteriophages are the most common and widely distributed group of viruses. They can usually be found in places full of bacterial communities, such as soil and the intestines of animals.

[0005] The main advantages of phage therapy include:

[0006] ① High specificity: Phages are highly specific to bacterial infection and will not kill bacteria other than the target bacteria, thus avoiding the occurrence of dysbacteriosis-related diseases caused by broad-spectrum antibiotics.

[0007] ② Automatically disappear after eliminating the target bacteria: After the bacteriophage eliminates the target bacteria in the body, it will "automatically disappear" in the body due to the loss of the host bacteria and cannot continue to replicate, and no cumulative poisoning will occur.

[0008] ③Does not disrupt animal metabolism: Phages only infect bacteria, will not disrupt animal metabolism, and will not cause infectious diseases in animals. So far, phage therapy has not been found to cause serious toxic side effects.

[0009] ④ It can reduce the use of antibiotics: The application of phage therapy can reduce the use of antibiotics and provide protection for food safety.

[0010] ⑤ Can identify specific pathogenic bacteria: bacteriophages can identify specific pathogenic bacteria and have little impact on the environment.

[0011] ⑥ Can use the host to proliferate and enter the bacteria: bacteriophages can use the host to proliferate and enter the bacteria, efficiently lysing pathogenic bacteria.

[0012] Therefore, given that phage therapy has many advantages, it is a green ecological technology with great potential and has been widely used in clinical medicine, animal husbandry, aquaculture, and planting industries.

[0013] Normally, most algae Shewanella phages cannot tolerate high temperatures, because high temperatures will destroy their protein shells and nucleic acid structures, causing the phages to lose their infection activity. In the production process of algae Shewanella phage products, there is a "dusting" process, which will cause the phage titer to decrease and affect the performance of the phage. Although a small number of algae Shewanella phages can withstand short-term high temperatures and powder, their titers will drop significantly after 1-3 months of storage. To solve this problem, in addition to optimizing phage protective agents, it is crucial to screen phages that are resistant to high temperatures, have stable titers, and can be stored for a long time. Summary of the invention

[0014] The present invention aims to provide a heat-resistant algae Shewanella phage PKP-SH23021 and its application in view of the deficiencies of the prior art. The heat-resistant algae Shewanella phage PKP-SH23021 provided by the present invention has strong lysis and a wide lysis spectrum against algae Shewanella, and is heat-resistant in the process of preparing phage powder, has good long-term storage stability, and is conducive to the industrial production and preparation of phage products.

[0015] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0016] In a first aspect, the present invention provides a Shewanella algae phage ( Shewanella algae Bacteriophage)PKP-SH23021, the phage was isolated from a shrimp farm in Zhangzhou, Fujian. The algae Shewanella phage was deposited in the General Microbiology Center of China Committee for Microbiological Culture Collection on November 29, 2023. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.45781.

[0017] Electron microscopy showed that the phage has a polyhedral, three-dimensionally symmetrical head that encloses nucleic acid, with a diameter of about 62nm, a tail about 65nm long, and a neck connecting the head and tail. According to the classification standards of the International Committee on Taxonomy of Viruses (ICTV), the phage is classified as the Myoviridae family of the order Caudovirales.

[0018] In a second aspect, the present invention provides a phage composition, wherein the phage composition comprises the above-mentioned thermostable algae Shewanella phage PKP-SH23021.

[0019] In the third aspect, the present invention provides the use of the above-mentioned thermostable algae Shewanella phage PKP-SH23021 or the above-mentioned phage composition in the preparation of a drug for preventing and treating diseases infected by algae Shewanella, a water environment disinfectant, or a bio-bacteriostatic agent for treating fresh or frozen aquatic products. The term "prevention" herein refers to "prevention and treatment". "Prevention" refers to all actions including suppressing or delaying the disease by administering the phage. The term "treatment" herein refers to all actions including improving or improving the disease by administering the phage. The disease includes aquatic animal diseases caused by algae Shewanella infection, etc. For example, when aquatic animals such as fish, shrimps, shellfish, etc. are infected by algae Shewanella and cause diseases, the algae Shewanella phage PKP-SH23021 specifically infects the algae Shewanella, reduces its number by lysing host cells, thereby preventing or treating diseases caused by the bacteria and improving the survival rate and growth performance of aquatic animals.

[0020] In a fourth aspect, the present invention provides a phage pharmaceutical preparation, the active ingredient of which includes the above-mentioned high-temperature resistant algae Shewanella phage PKP-SH23021 or the above-mentioned phage composition. Preferably, the phage pharmaceutical preparation also includes a pharmaceutically acceptable carrier, and its dosage form is a powder, a solution, an emulsion, a gel, a granule or a lyophilized agent. More preferably, the dosage form of the phage pharmaceutical preparation is a powder. After the phage stock solution prepared by the present invention is sprayed with a spray dryer to prepare a phage powder, the potency can still be maintained at 10 8 PFU / g, and can be stored for a long time. After 3 months of storage, it remains basically unchanged compared with the initial phage powder. This shows that the phage can not only withstand short-term high temperatures, but also has stable titer and can be stored for a long time.

[0021] The bacteriophage powder is prepared by mixing the bacteriophage PKP-SH23021 stock solution and auxiliary materials (starch, β-cyclodextrin, mannitol) in a ratio of 100:5:8:3 (v / m). The bacteriophage powder can maintain a certain biological activity after long-term storage, thereby extending the shelf life of the bacteriophage product; and the bacteriophage powder is easy to package, transport and distribute.

[0022] Preferably, the titer of the phage in the phage pharmaceutical preparation is 1×10 8 PFU / g or above.

[0023] In a fifth aspect, the present invention provides a water environment disinfectant, wherein the active ingredient of the water environment disinfectant includes the above-mentioned thermostable algae Shewanella phage PKP-SH23021 or the above-mentioned phage composition. The titer of the phage in the water environment disinfectant is 1×10 8 PFU / g or above.

[0024] In a sixth aspect, the present invention provides a biological antibacterial agent for treating fresh or frozen aquatic products, wherein the active ingredient of the biological antibacterial agent comprises the above-mentioned thermostable algae Shewanella phage PKP-SH23021 or the above-mentioned phage composition. The titer of the phage in the biological antibacterial agent is 1×10 8 In the aquatic product processing, bacteriophage PKP-SH23021 can be used to treat fresh or frozen aquatic products to reduce the number of pathogenic bacteria Shewanella algae and reduce the risk of foodborne diseases.

[0025] The beneficial effects of the present invention are:

[0026] 1. The present invention provides a self-isolated algae Shewanella phage PKP-SH23021, which has the following physiological characteristics and beneficial effects in terms of phage titer, optimal infection multiplicity (MOI), optimal growth temperature, and the effect of pH stability on phage survival stability: (1) It has a high titer, which can reach 10 10 PFU / mL; (2) The optimal infection multiplicity is 0.01, corresponding to the highest titer of 10 10 PFU / mL; (3) The optimal growth temperature is 30°C; (4) The titer is highest at pH = 7, and its titer can be maintained at 10 within the range of pH 5-10. 8 -10 10 PFU / mL higher level.

[0027] 2. The algae Shewanella phage PKP-SH23021 provided by the present invention has good high temperature resistance. The phage stock solution is placed in a water bath at 60°C, 70°C, 80°C, and 90°C for 10 min. The titer is 10 at 60°C. 10 PFU / mL; titer 10 at 70℃ 9 PFU / mL, only one order of magnitude lower than the stock solution; the titer at 80°C is 10 8 PFU / mL, only two orders of magnitude lower than the stock solution; the titer at 90°C is 10 6 PFU / mL, which was only four orders of magnitude lower than that of the original solution.

[0028] The high temperature resistance makes the algae Shewanella phage PKP-SH23021 have the following advantages:

[0029] (1) Processing stability: When preparing bacteriophage products, such as preparing bacteriophage powder, the bacteriophage can still maintain biological activity even when the powder spraying temperature is as high as 80°C, indicating that the bacteriophage can adapt to the process parameter range of higher temperature, which is conducive to the processing of stable bacteriophage products;

[0030] (2) Convenient storage and transportation: For long-term storage of bacteriophage products, they can remain stable at higher storage temperatures without having to rely strictly on low-temperature freezing, which reduces storage costs and improves the convenience of logistics and transportation;

[0031] (3) Enhanced environmental adaptability: When used in natural environments or industrial applications, such as as a water environment disinfectant or a bio-inhibitor for treating fresh or frozen aquatic products, the ambient temperature may fluctuate, but the phage can function in a wider temperature range;

[0032] (4) Improving processing efficiency: In the process of aquatic product processing and disinfection, it may be necessary to subject materials to high-temperature treatment to ensure the food safety of aquatic products. The bacteriophage can remain effective in high-temperature processes and help kill the algae-producing Shewanella in the high-temperature treatment process.

[0033] 3. The algae Shewanella phage PKP-SH23021 provided by the present invention has strong lytic ability and a wide host range. It can lyse 90 out of 100 different algae Shewanella strains, with a lysis rate of 90%.

[0034] 4. The algae Shewanella phage PKP-SH23021 provided by the present invention can still maintain a potency of 10 after being sprayed with a spray dryer to make a phage powder. 8 PFU / g.

[0035] 5. The algae Shewanella phage PKP-SH23021 powder provided by the present invention can not only withstand short-term high temperature, but also can be stored for a long time.

[0036] 6. The bacteriophage provided by the present invention is obtained from nature, can be stably inherited, is suitable for large-scale industrial production, and is safe to use and environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The colony morphology of Shewanella algae SH23021 on TCBS plate

[0038] Figure 2 This is a plaque image of Shewanella algae phage PKP-SH23021;

[0039] Figure 3 This is an electron microscope photo of Shewanella algae phage PKP-SH23021;

[0040] Figure 4 is the optimal multiplicity of infection of Shewanella algae phage PKP-SH23021;

[0041] Figure 5 is the one-step growth curve of Shewanella algae phage PKP-SH23021;

[0042] Figure 6 is the optimal growth temperature of Shewanella algae bacteriophage PKP-SH23021;

[0043] Figure 7 This is a pH stability test result diagram of Shewanella algae bacteriophage PKP-SH23021;

[0044] Figure 8 This is a diagram showing the genetic stability test results of Shewanella algae bacteriophage PKP-SH23021;

[0045] Fig. 9 The results of the high temperature resistance experiment of Shewanella algae bacteriophage PKP-SH23021;

[0046] Fig.10 This is a comparison chart of the original solution and powder of Shewanella algae bacteriophage PKP-SH23021;

[0047] Fig.11 These are the results of the long-term storage experiment of Shewanella algae bacteriophage PKP-SH23021 powder. DETAILED DESCRIPTION

[0048] The following will combine the contents in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0049] In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments, unless otherwise specified, are conventional methods in the art. Unless otherwise defined, all technical and scientific terms used in this specification have the same meanings as those commonly understood by technicians in the technical field of the present invention. In the present invention, Shewanella algae SH23021 may be referred to as "SH23021", and Shewanella algae bacteriophage PKP-SH23021 may be referred to as "bacteriophage PKP-SH23021" or "PKP-SH23021".

[0050] Example 1 Isolation and identification of Shewanella algae SH23021

[0051] (1) Take samples from a shrimp farm in Zhangzhou, Fujian, and inoculate them on TCBS solid culture medium. Place the sample in a 37°C constant temperature incubator for 18-24 hours and observe the morphology and color of the colonies on the plate. Select colonies with a smooth surface and green color, such as Figure 1 shown.

[0052] (2) Streak the culture on TCBS medium and subculture and purify for 3-5 times until uniform colonies are obtained. Pick a single colony and streak it on LB agar medium. Pick the colony and identify it by 16S rRNA gene sequencing technology. It is determined to be Shewanella algae. One of the strains is named SH23021. After identification, scrape the bacterial moss and place it in 30% glycerol broth and store it in a refrigerator at -80℃.

[0053] Example 2 Isolation and purification of Shewanella algae phage

[0054] (1) Treatment of aquaculture water samples: Take sludge water samples from the aquaculture farm, weigh 5 g, add them to 10 mL SM buffer and soak overnight, then centrifuge the overnight extract at 10,000 rpm for 5 min, take the supernatant and filter it with a 0.22 μm filter, and store the filtrate at 4°C for later use.

[0055] (2) Phage enrichment: Take 0.1 mL of Shewanella algae suspension and 1 mL of filtrate and add them to 5 mL of LB liquid. Incubate at 37°C on a shaking incubator overnight, then centrifuge at 10,000 rpm for 10 min. Filter the supernatant with a 0.22 μm filter and keep the filtrate for later use.

[0056] (3) Phage isolation: The double plate method was used to isolate phages. 0.1 mL of Shewanella algae suspension was mixed with 0.6% LB soft agar and spread on an LB solid plate. After the soft agar solidified, 0.1 mL of the filtrate was spotted on the plate and allowed to stand until the mixture was absorbed. After incubation at 37°C for 6-8 h, transparent plaques were picked up with an inoculation loop and placed in 2 mL of SM buffer. The plaques were stored at 4°C overnight to allow the phages to be completely released.

[0057] (4) Phage purification: Use the double plate method to separate phages. Take 0.1 mL of the mixed bacterial suspension filtrate and 0.2 mL of the host algae Shewanella SH23021 bacterial suspension, mix them evenly, add soft agar cooled to about 50°C and mix them evenly, then spread on two plates, place them in a 37°C incubator for 6-8 hours, pick out a single transparent spot with smooth edges and put it in 1 mL SM buffer, and store it at 4°C overnight. Take 0.1 mL of the overnight stored extract and 0.1 mL of the bacterial suspension, mix them, add soft agar cooled to about 50°C and mix them evenly, spread on two plates, and culture them in a 37°C incubator for 4-6 hours. Follow these steps to purify until the phage plaques are uniform in size and have smooth edges, such as Figure 2 As shown, the purified phage is obtained.

[0058] (5) Preparation of bacteriophage: 0.2 mL of Shewanella algae suspension and 1 mL of purified phage suspension were added to 50 mL of LB broth, incubated overnight at 37 °C in a shaking incubator, and then centrifuged at 10,000 rpm for 10 min. The supernatant was filtered through a 0.22 μm filter and the phage filtrate was set aside.

[0059] (6) Preservation of bacteriophages: Add 60% glycerol to the phage suspension in a 1:1 ratio and store in a -80°C refrigerator.

[0060] Example 3 Electron microscopic observation of Shewanella algae bacteriophage PKP-SH23021

[0061] Take 20 μL of liquid containing crude phage particles and drop it on the copper mesh. Let it settle naturally for 15 minutes, and use filter paper to absorb excess liquid from the side. Add a drop of 2% phosphotungstic acid (PTA) on the copper mesh to stain the phage for 10 minutes, then use filter paper to absorb the staining liquid from the side. After the sample is dry, use an electron microscope to observe the phage morphology, such as Figure 3 shown.

[0062] Electron microscopy showed that the phage has a polyhedral, three-dimensionally symmetrical head that encloses nucleic acid, with a diameter of about 62nm, a tail about 65nm long, and a neck connecting the head and tail. According to the classification standards of the International Committee on Taxonomy of Viruses (ICTV), the phage is classified as the Myoviridae family of the order Caudovirales.

[0063] The phage was isolated from a shrimp farm in Zhangzhou, Fujian. The algae Shewanella phage was deposited in the General Microbiology Center of the China Culture Collection Committee on November 29, 2023. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 45781, and it is named PKP-SH23021.

[0064] Example 4 Whole genome sequencing and analysis of Shewanella algae phage PKP-SH23021

[0065] The library was constructed using the Illumina TruSeq™ Nano DNA Sample Prep Kit method; the specific steps are as follows:

[0066] (1) Construct the library with 1 μg of phage genomic DNA as the starting amount;

[0067] (2) Covaris M220 ultrasonically breaks DNA into 300-500 bp;

[0068] (3) Fill in the 3' end, add A, and connect the index adapter (TruSeq™ Nano DNA Sample Prep Kit);

[0069] (4) Library enrichment, PCR amplification for 8 cycles;

[0070] (5) Recover the target band using 2% agarose gel (Certified Low Range Ultra Agarose);

[0071] (6) TBS380 (Picogreen) quantification, mixing according to the data ratio;

[0072] (7) Bridge PCR amplification is performed on the cBot solid phase carrier to generate clusters;

[0073] (8) Illumina Hiseq sequencing platform, performing 2×150 bp sequencing.

[0074] The whole genome of the phage PKP-SH23021 is 43907bp in size, and its sequence is shown in SEQ ID NO.1. It contains 60 genes with an average gene length of 654bp and a G+C% content of 54.0%. According to the prediction analysis, the genome of the phage PKP-SH23021 contains 60 open reading frames (ORFs) with an average length of 217bp, containing 25 known functional proteins, which are divided into two parts according to protein function: 13 structural proteins, 12 functional proteins, of which ORF33 is its lytic enzyme, and 35 hypothetical proteins.

[0075] Example 5 Determination of the potency of Shewanella algae bacteriophage PKP-SH23021

[0076] 1. Experimental methods:

[0077] Take 100 μL of host bacterial solution cultured to the exponential phase, mix it into a 5mL centrifuge tube, shake it up and down, place it on LB semi-solid agar, and completely cover it on a 20mL LB solid agar plate (9cm diameter). After the semi-solid is dried, take 100 μL of the purified phage concentrate and place it on the plate. Spread 3 plates for one phage, culture it at 37℃ overnight, pick out the plaques and put them in 20 mL SM buffer, put it in 4℃, take the supernatant the next day, filter it through a 0.22μm filter membrane, take 100 μL of the filtrate and dilute it 10 times with sterile water, then mix 100 μL of the bacterial solution and phage solution on two plates, and culture the plates in a 37℃ incubator overnight.

[0078] The above method was used to determine the phage titer of PKP-SH23021. Select a countable plate, count the phage plaques, and calculate the phage titer (PFU / mL). The phage titer (PFU / mL) is the number of plaques × dilution factor / sample volume (mL).

[0079] 2. Experimental results:

[0080] The titer of phage PKP-SH23021 was determined to be 10 10 PFU / mL.

[0081] Example 6 Determination of the Optimal Multiplicity of Infection (MOI) of Shewanella algae Phage PKP-SH23021

[0082] 1. Experimental methods:

[0083] The multiplicity of infection (MOI) refers to the ratio of the number of phages to the number of host bacteria at the initial infection. The concentration of the host bacteria cultured to the exponential phase was adjusted to 10 8CFU / mL. The MOI was set to 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100, respectively. The phage solution with the determined titer was added to the bacterial solution in proportion, 500 μL of the bacterial solution and phage solution respectively, mixed, and cultured at 37°C, 200 r / min and shaking for 5 hours. The mixed culture was centrifuged at 10000 r / min for 10 minutes, and the phage titer was measured. The multiplicity of infection with the highest titer is the optimal multiplicity of infection. The optimal multiplicity of infection of bacteriophage PKP-SH23021 was determined by the above method. The results are as follows Figure 4 shown.

[0084] 2. Experimental results:

[0085] Depend on Figure 4 It can be seen that the phage titer is highest when the infection multiplicity of the phage is 0.01, so the optimal infection multiplicity of the phage PKP-SH23021 is 0.01, corresponding to the highest titer of 10 10 PFU / mL.

[0086] Example 7 One-step growth curve of Shewanella algae phage PKP-SH23021

[0087] 1. Experimental methods:

[0088] Mix 500 μL of phage liquid and host bacterial liquid at the optimal infection multiplicity ratio, incubate at 37°C for 15 min, centrifuge at 10,000 r / min for 1 min, and wash the precipitate three times with LB liquid. Add 10 mL of LB liquid culture medium preheated at 37°C, mix well and quickly place in a 37°C shaker for shaking culture. Take samples every 10 minutes to measure the phage titer, make 3 parallels, and take the average value of the results. Draw a one-step growth curve with the infection time as the horizontal axis and the logarithm of the phage titer in the infection system as the vertical axis to obtain the incubation period and outbreak period of phage PKP-SH23021, and calculate the outbreak volume. The results are as follows Figure 5 shown.

[0089] Burst volume = total number of phages at the end of the outbreak / total number of bacteria at the beginning of the outbreak

[0090] 2. Experimental results:

[0091] from Figure 5 It can be seen that within 40 minutes after phage PKP-SH23021 infected the host bacteria, the number of phages did not increase, and this period was called the latent period; 40 to 120 minutes after infecting the host bacteria, the number of phages increased rapidly, and this period was the outbreak period of phages, that is, the outbreak period of phages was about 80 minutes, and the outbreak amount was 10 3 PFU / mL.

[0092] Example 8 Determination of the Optimal Growth Temperature of Shewanella algae Phage PKP-SH23021

[0093] 1. Experimental methods:

[0094] The phage stock solution was gradiented tenfold to a suitable concentration of 10 8 PFU / mL, take 100μL and mix with an equal amount of bacterial solution, pour into double-layer plates, and culture at 15℃, 20℃, 25℃, 30℃, 37℃, 40℃, 45℃ and 50℃ for 4 hours to detect the phage titer. With temperature as the horizontal axis and the logarithm of phage titer as the vertical axis, the phage growth temperature curves were drawn respectively. The results are shown in Figure 6 shown.

[0095] 2. Experimental results:

[0096] from Figure 6 It can be seen that the titer of bacteriophage PKP-SH23021 is highest at 30°C, and its optimal growth temperature is 30°C.

[0097] Example 9 Determination of pH stability of Shewanella algae bacteriophage PKP-SH23021

[0098] 1. Experimental methods:

[0099] The pH value of LB liquid medium was adjusted to 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and 100 μL of each was mixed with an equal volume of phage stock solution. After being placed at 37°C for 2 hours, the titer was determined by the double-layer plate method, and 3 replicates were set for each pH. The pH stability curves of phages were plotted with pH as the horizontal axis and the logarithm of phage titer as the vertical axis. The results are shown in Figure 2. Figure 7 shown.

[0100] 2. Experimental results:

[0101] from Figure 7 It can be seen that the titer of bacteriophage PKP-SH23021 is highest when pH=7, and its titer can be maintained at 10 in the range of pH5-10. 8 -10 10 PFU / mL higher level.

[0102] Example 10 Genetic experiment on the stability of Shewanella algae phage PKP-SH23021

[0103] 1. Experimental methods:

[0104] The phage was subcultured 30 times, with equal amounts of phage and host bacteria added to the culture medium for each generation. Each generation was cultured for 6 hours, and then double plates were spread. After the phage was subcultured for 29 generations, its titer was determined. Figure 8 shown.

[0105] 2. Experimental results:

[0106] from Figure 8 It can be seen that the overall titer of the phage is stable during the culture process, indicating that the phage has good biological genetic stability and is an excellent choice for the production process.

[0107] Example 11 Analysis of the lysis spectrum of Shewanella algae phage PKP-SH23021

[0108] 1. Experimental methods:

[0109] The double-layer plate method was used to measure the lysis effect of bacteriophage PKP-SH23021 on 100 strains of algae Shewanella preserved in the strain library. Prepare different algae Shewanella bacterial liquids, take 200μL of the test bacterial liquid cultured for 6 hours, add it to 0.6% LB semi-solid medium, mix well, pour it into a plate, and let it stand to solidify. Take 20μL of phage suspension and drop it on the soft agar plate, culture it at 37℃ for about 6-8 hours, and observe whether plaques appear. The results are shown in Table 1.

[0110] 2. Experimental results:

[0111] As can be seen from Table 1, the bacteriophage PKP-SH23021 can lyse 90 of the 100 different strains of Shewanella algae, with a lysis rate of 90%.

[0112] Table 1 The lysis spectrum of Shewanella algae phage PKP-SH23021

[0113]

[0114] Note: +: cleaved; -: not cleaved.

[0115] Example 12 Experiment on the high temperature resistance of Shewanella algae bacteriophage PKP-SH23021

[0116] 1. Experimental methods:

[0117] The phage PKP-SH23021 stock solution was centrifuged at 10,000 revolutions per minute for 10 minutes to precipitate the host bacteria, and the supernatant was transferred to a new centrifuge tube. The residual bacteria were filtered out using a filter with a pore size of 0.22 microns. In this way, the phage stock solution without host bacteria was obtained. The phage stock solution was then placed in a water bath at 60°C, 70°C, 80°C, and 90°C. After a 10-minute water bath, the treated solution was graded diluted and its titer was measured. The results are as follows: Fig. 9 shown.

[0118] 2. Experimental results:

[0119] from Fig. 9 It can be seen that the phage PKP-SH23021 was placed in a water bath at 60℃, 70℃, 80℃, and 90℃ for 10 minutes. 10 PFU / mL; titer 10 at 70℃ 9 PFU / mL, only one order of magnitude lower than the stock solution; the titer at 80°C is 10 8 PFU / mL, only two orders of magnitude lower than the stock solution; the titer at 90°C is 10 6 PFU / mL was only four orders of magnitude lower than that of the original solution, indicating that the phage can tolerate higher temperatures.

[0120] Example 13 Preparation of Shewanella algae bacteriophage PKP-SH23021 powder

[0121] 1. Experimental methods:

[0122] The prepared bacteriophage PKP-SH23021 stock solution and excipients (starch, β-cyclodextrin, mannitol) were mixed, wherein the ratio of stock solution, starch, β-cyclodextrin, and mannitol was 100:5:8:3 (v / m), placed in a spray dryer, set the inlet temperature to 80°C, and the outlet temperature to 58°C, collected the bacteriophage powder, and determined its potency. The results are as follows Fig.10 shown.

[0123] 2. Experimental results:

[0124] from Fig.10 It can be seen that the titer of phage PKP-SH23021 stock solution can still be maintained at 10 after being sprayed with powder by a spray dryer to make phage powder. 8 PFU / g.

[0125] Example 14 Storage stability test of Shewanella algae bacteriophage PKP-SH23021 powder

[0126] 1. Experimental methods:

[0127] The phage powder prepared in Example 13 was stored in a room temperature environment, and samples were taken at 1 day, 15 days, 30 days, 45 days, 60 days, 75 days, and 90 days to measure the phage titer. The results are shown in FIG. Fig.11 shown.

[0128] 2. Experimental results:

[0129] from Fig.11 It can be seen that at 90 days, the potency of the phage powder can still be maintained at 10 8PFU / g or above, the potency remains basically unchanged compared with the initial phage powder, which shows that the phage does not have to strictly rely on low-temperature freezing and can be stored for a long time at room temperature, thus reducing storage costs and improving the convenience of logistics and transportation, and has a good prospect for actual production application.

[0130] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A thermostable algae Shewanella phage PKP-SH23021, characterized in that: The algae Shewanella phage was deposited in the General Microbiology Center of China Culture Collection Committee on November 29, 2023, with the deposit number CGMCC No.45781.

2. A bacteriophage composition, characterized in that: The phage composition comprises the thermostable Shewanella algae phage PKP-SH23021 as claimed in claim 1.

3. Use of the thermostable algae Shewanella phage PKP-SH23021 as claimed in claim 1 or the phage composition as claimed in claim 2 in the preparation of a drug for preventing and treating diseases caused by algae Shewanella infection, a water environment disinfectant, or a bio-inhibitor for treating fresh or frozen aquatic products.

4. A bacteriophage pharmaceutical preparation, characterized in that: The active ingredient of the phage pharmaceutical preparation includes the high-temperature-resistant algae Shewanella phage PKP-SH23021 as claimed in claim 1 or the phage composition as claimed in claim 2.

5. The bacteriophage pharmaceutical preparation according to claim 4, characterized in that The bacteriophage drug preparation further comprises a pharmaceutically acceptable carrier, and the dosage form thereof is powder, solution, emulsion, gel, granule or lyophilized agent.

6. The bacteriophage pharmaceutical preparation according to claim 5, characterized in that The dosage form of the bacteriophage pharmaceutical preparation is powder.

7. The bacteriophage pharmaceutical preparation according to claim 6, characterized in that The titer of the bacteriophage in the bacteriophage pharmaceutical preparation is 1×10 8 PFU / g or above.

8. A water environment disinfectant, characterized in that: The active ingredient of the water environment disinfectant includes the high-temperature-resistant algae Shewanella phage PKP-SH23021 as described in claim 1 or the phage composition as described in claim 2.

9. A biological antibacterial agent for treating fresh or frozen aquatic products, characterized in that: The active ingredient of the biological antibacterial agent includes the high-temperature-resistant algae Shewanella phage PKP-SH23021 as claimed in claim 1 or the phage composition as claimed in claim 2.

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