High-temperature-resistant vibrio alginolyticus bacteriophage as well as composition and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and discloses a high-temperature-resistant vibrio alginolyticus bacteriophage and a composition and application thereof, the high-temperature-resistant vibrio alginolyticus bacteriophage is named as vibrio alginolyticus bacteriophage PJ644 and is preserved in China General Microbiological Culture Collection Center on August 16, 2024, the preservation address is No.3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO.9624. The preservation number of the strain is CGMCC (China General Microbiological Culture Collection Center) No. The bacteriophage has excellent high temperature resistance, and the bacteriophage and the bacteriophage composition not only can effectively prevent and treat various vibrio infections, but also can be used as active ingredients for preparing bacteriophage pharmaceutical preparations, aquatic feed additives, water disinfectants and aquatic product preservatives, and are used for effectively preventing and treating diseases caused by vibrio infections in aquaculture farms. The use is safe, and the problems of antibiotic residues and pathogen drug resistance caused by use of antibiotics are effectively avoided.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more particularly to a thermoresistant Vibrio alginolyticus bacteriophage, its composition, and its applications. Background Technology

[0002] Vibrio ( Vibrio Vibrio bacteria are a type of halophilic, facultative heterotrophic, Gram-negative marine bacterium widely distributed in estuarine and coastal ecosystems. They are an important pathogenic bacterium in aquatic animals (including farmed animals), and their pathogenicity is greatly influenced by a combination of factors such as the host's physiological state and water quality conditions. Furthermore, they spread rapidly and have a short course, causing significant harm to aquaculture. In Asia, vibriosis caused by Vibrio is one of the most common diseases leading to mass mortality in shrimp, fish, and shellfish in aquaculture.

[0003] To date, more than 20 types of Vibrio pathogens have been reported worldwide, including Vibrio alginolyticus, Vibrio vulnificus, Vibrio fluvialis, Vibrio harveyi, and Vibrio parahaemolyticus. Among them, Vibrio alginolyticus is an opportunistic pathogenic Vibrio that can survive independently in water, or as part of a biofilm, or exist on the surface and in the intestines of aquatic animals. It can harm a variety of aquatic animals, including fish, arthropods, cephalopods, and echinoderms, and is pathogenic to humans.

[0004] Currently, the traditional method for controlling pathogenic bacteria like Vibrio alginolyticus is antibiotics. However, the overuse of antibiotics leads to the emergence of drug-resistant bacteria, which has adverse effects on the livestock industry and public health, becoming a pressing issue for the poultry industry. Against the backdrop of antibiotic prohibition, bacteriophages have attracted widespread attention due to their antibacterial activity. Bacteriophages are characterized by obligate parasitism and high specificity, infecting only specific host bacteria without affecting other bacterial groups. They also die as the host bacteria are eliminated, resulting in high safety. When bacteriophages infect host bacteria, their self-encoded depolymerases and lysins degrade polysaccharides and peptidoglycans, disrupting extracellular polymeric structures and lysing biofilms. The successful isolation and application of numerous lysing bacteriophages targeting pathogenic bacteria in aquatic animals demonstrate the great potential of using bacteriophages to replace antibiotics in the control of bacterial diseases in aquatic animals.

[0005] However, the available bacteriophage resources for the prevention and control of Vibrio alginolyticus are limited and have poor heat resistance. They are easily inactivated under the high-temperature production conditions of bacteriophage preparations, which greatly affects their application effect. Therefore, the existing technology needs further improvement. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a heat-resistant Vibrio alginolyticus phage PJ644, its composition, and its applications. The Vibrio alginolyticus phage PJ644 exhibits excellent heat resistance. This phage and its composition can not only effectively prevent and control Vibrio alginolyticus infection, but also be used as an active ingredient in the preparation of phage drug formulations, aquatic feed additives, water disinfectants, and aquatic product preservatives. These are used to effectively prevent and control diseases caused by Vibrio infection in aquaculture facilities, and also avoid the problems of antibiotic residues and pathogen resistance caused by the use of antibiotics.

[0007] To address the above problems, the present invention provides the following technical solution: Firstly, this application provides a thermoresistant Vibrio alginolyticus phage ( Vibrio alginolyticus phage It was named Vibrio alginolyticus phage PJ644, and its accession number is CGMCC No. 46165.

[0008] Vibrio alginolyticus bacteriophage PJ644 was isolated from a shrimp farm in Guangxi and deposited on August 16, 2024, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. It is classified and named Vibrio alginolyticus bacteriophage.

[0009] Electron microscopy revealed that the phage PJ644 has a polyhedral head structure and a contractile tail. The head is 74-86 nm wide and 110-120 nm long, and the tail is about 110-122 nm long, making it a myotail phage.

[0010] In this application, Vibrio alginolyticus phage PJ644 includes mutant strains with point mutations, deletion mutations, or addition mutations exhibiting homology higher than 98% or 99% and maintaining substantially the same bactericidal activity. Since phages are highly susceptible to mutation during replication, mutants of the aforementioned phages are also within the scope of protection claimed in this application. The sequence of phage PJ644 can be obtained by sequencing the biological material preserved according to this invention using known methods. For those skilled in the art, screening for mutants with extremely similar characteristics to the phages provided by this invention requires no inventive effort.

[0011] The Vibrio alginolyticus phage PJ644 exhibits excellent heat resistance; it retains its original activity after exposure to temperatures below 70°C for 60 minutes. After exposure to 80°C for 20 minutes, the phage titer is approximately 3.4 × 10⁻⁶. 8 PFU / mL, incubated at 80℃ for 60 min, potency 2.00×10⁻⁶ 8After incubation at 85℃ for 60 min, phage PJ644 still retained some activity, with a titer of 1.08 × 10⁻⁶ PFU / mL. 7 PFU / mL. This demonstrates that Vibrio alginolyticus phage PJ644 exhibits good high-temperature tolerance, can adapt to higher temperature environments, and maintains higher activity during high-temperature processing of phage products, making it more suitable for industrial production.

[0012] The Vibrio alginolyticus phage PJ644 exhibits good acid-base stability, maintaining stable activity within a pH range of 5.0–10.0. After 2 hours of treatment at pH 2.0, the titer remained at 10. 5 The phage has a pfu / mL concentration, enabling it to resist certain strongly acidic environments; after 3 hours of treatment at pH 11, the titer of this phage remains at 10. 7 PFU / mL, at pH 13, for 3 hours, with a potency not lower than 10. 3 With a concentration of pfu / mL, phage PJ644 exhibits strong stability in acidic environments and can adapt to a certain range of strongly acidic environments.

[0013] Secondly, the alginolytic phage PJ644 exhibits broad-spectrum lytic activity against various Vibrio species, effectively lysing *Vibrio alginolyticus*, *Vibrio parahaemolyticus*, and *Vibrio harveyi*. Experiments showed that this phage achieved a lysis rate of up to 87.5% against 35 out of 40 different types of Vibrio strains, with a lysis rate of 94.44% against *Vibrio alginolyticus*, 83.33% against *Vibrio parahaemolyticus*, and 90% against *Vibrio harveyi*. Therefore, the alginolytic phage PJ644 possesses a specific lytic advantage against *Vibrio alginolyticus*, *Vibrio parahaemolyticus*, and *Vibrio harveyi*.

[0014] Therefore, based on its excellent biological characteristics, the Vibrio alginolyticus phage PJ644 has great application prospects and can be used as an active ingredient in the preparation of various products for the prevention and treatment of diseases caused by Vibrio infection.

[0015] Secondly, this application also provides a phage composition comprising the aforementioned Vibrio alginolyticus phage. To further broaden the lysis spectrum of the phage formulation, fully utilize the differences in the lysis spectra of different phages, and achieve complementary advantages, the aforementioned Vibrio alginolyticus phage PJ644 can be used in combination with other phages.

[0016] Optionally, the phage composition further includes one or two strains of Vibrio alginolyticus phage vB_ValP_PJ115 and Vibrio alginolyticus phage vB_ValP_PJ32. Vibrio alginolyticus phage PJ644 can be used in combination with one or two other Vibrio alginolyticus phages, such as the existing Vibrio alginolyticus phage vB_ValP_PJ115 (see patent publication number CN113201505B) and Vibrio alginolyticus phage vB_ValP_PJ32 (see patent publication number CN111718907B), to broaden the bactericidal spectrum and kill as many Vibrio alginolyticus pathogens in the environment as possible, for the prevention and control of Vibrio alginolyticus disease.

[0017] Optionally, the phage composition further includes: Vibrio parahaemolyticus phage and / or Vibrio harveyi phage. The above-mentioned Vibrio alginolyticus phage PJ644 can also be combined with other different types of existing phages (such as existing Vibrio parahaemolyticus phage and / or Vibrio harveyi phage) for the prevention and control of diseases caused by Vibrio infection (such as aquatic Vibrio disease).

[0018] Thirdly, this application also provides the use of the aforementioned Vibrio alginolyticus phage or the aforementioned phage composition in the preparation of drugs for preventing and treating diseases caused by Vibrio infection, aquatic feed additives, water disinfectants, and aquatic product preservatives.

[0019] The prevention and treatment include both prevention and treatment. The term "prevention" as used herein refers to all behaviors that suppress or delay the disease by administering the composition. The term "treatment" as used herein refers to all behaviors that improve or alleviate the disease by administering the composition.

[0020] The diseases caused by Vibrio infection include various diseases caused by Vibrio alginolyticus, Vibrio parahaemolyticus and / or Vibrio harveyi infection. Optionally, the diseases include aquatic diseases caused by Vibrio alginolyticus, Vibrio parahaemolyticus and Vibrio harveyi infection, such as acute hepatopancreatic necrosis, ulcerative syndrome, etc.

[0021] Fourthly, this application also provides a phage drug formulation, the active ingredient of which includes the aforementioned Vibrio alginolyticus phage or the aforementioned phage composition.

[0022] Preferably, the phage drug formulation further includes other antibacterial or bactericidal active ingredients.

[0023] Optionally, the drug formulation may be an oral dosage form, a topical dosage form, or a parenteral dosage form.

[0024] Optionally, the phage drug formulation may further comprise a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not cause significant irritation to the organism and does not eliminate the biological activity and properties of the administered active component. For the pharmaceutical composition to be formulated into a liquid formulation, the pharmaceutically acceptable carrier must be suitable for sterility and biocompatibility. Examples include saline, sterile water, Ringer's solution, buffered physiological saline, albumin infusion, glucose solution, maltodextrin solution, glycerol, ethanol, various culture media, etc. They can be used alone or in any combination thereof. Other conventional additives, such as antioxidants, buffers, and antibacterial agents, may be added if desired. When combined with diluents, dispersants, surfactants, binders, and / or lubricants, the compositions of the present invention can also be formulated into injectable and oral dosage forms (e.g., aqueous solutions, suspensions and emulsions, pills, capsules, granules) and other intermediate dosage forms, such as lyophilized formulations.

[0025] Optionally, the dosage forms of the bacteriophage drug formulation include solid dosage forms and liquid dosage forms, wherein solid dosage forms include powders, tablets, granules and microcapsules; and liquid dosage forms include solutions and injections.

[0026] Optionally, the phage drug formulation is in powder form, and is prepared from the following components in parts by weight: 90-110 parts of phage proliferation solution, 1-5 parts of gelatin, 1-5 parts of sodium glutamate, and 1-5 parts of mannitol. Preferably, the potency of the phage proliferation solution is 10. 9 PFU / mL or higher.

[0027] Preferably, the phage drug formulation is prepared from the following components in parts by weight: 100 parts stock solution, 4 parts gelatin, 2 parts sodium glutamate, and 3 parts mannitol.

[0028] More preferably, the phage drug preparation is prepared by the following method: after mixing all raw materials, spray drying is performed, and then the spray-dried particles are collected to obtain the phage drug preparation.

[0029] Preferably, the spray drying conditions are: inlet temperature of 80°C, airflow velocity of 120 L / min, spray rate of 100%, and spray drying for about 2 hours.

[0030] The application method of the bacteriophage drug preparation is as follows: bacteriophage or its combination is added to water or feed as a therapeutic drug. In this way, diseases caused by Vibrio infection can be prevented and treated, and animal survival rate can be improved.

[0031] Fifthly, this application provides an aquatic feed additive whose active ingredient includes the aforementioned Vibrio alginolyticus phage or the aforementioned phage composition.

[0032] Preferably, the concentration of bacteriophage is 10. 7 PFU / mL or higher. By mixing the above-mentioned aquatic feed additives into the feed and feeding them to aquatic animals, vibrio infection can be effectively prevented and controlled.

[0033] Optionally, the aquatic feed additive may also contain other active ingredients for inhibiting or eliminating bacteria in the water. The aquatic feed additive may be in liquid or solid form (including powder), but is not limited to these forms.

[0034] Sixthly, this application also provides a water disinfectant, the active ingredient of which includes the aforementioned Vibrio alginolyticus phage or the aforementioned phage composition. Preferably, the concentration of the phage in the disinfectant is 10. 8 PFU / mL or higher.

[0035] Optionally, to further improve the disinfection effect of the disinfectant, the disinfectant may also contain other active ingredients for inhibiting or eliminating bacteria in the aquaculture environment. Preferably, the targets of the environmental disinfectant include: feed, water bodies, and the aquaculture environment, wherein the aquaculture environment includes ponds, feeding platforms, feeding equipment, and recirculating aquaculture systems.

[0036] Optionally, the water disinfectant is applied by spraying or soaking to disinfect the aquaculture environment and feeding equipment with Vibrio, wherein the aquaculture environment is a pond, feeding environment, feeding platform, feeding equipment or recirculating aquaculture system.

[0037] The application methods of the water disinfectant include, but are not limited to, disinfecting and decontaminating the recirculating aquaculture system, aquaculture facilities, feeding equipment, pools or other environmental surfaces in the aquaculture environment by means of liquid immersion, spraying, or combined use with water-containing carriers, and disinfecting and preserving feed. This disinfectant can be used to replace antibiotics or traditional disinfection products, and it will not cause harm to humans or aquatic animals.

[0038] Seventhly, this application provides a preservative for aquatic products, which includes the aforementioned Vibrio alginolyticus phage or the aforementioned phage composition.

[0039] The application method of the aquatic product preservative is as follows: spray it on the surface or inside of the aquatic product, or soak duck meat products in a solvent containing the aquatic product preservative, thereby inhibiting the reproduction of various Vibrio species (Vibrio alginolyticus, Vibrio parahaemolyticus and Vibrio harveyi) in the aquatic product and achieving the preservation effect.

[0040] The present invention has the following beneficial effects: 1. This invention provides a self-isolated, highly thermoresistant Vibrio alginolyticus phage, PJ644. This phage not only has excellent thermoresistance and can adapt to high temperature environments, but also maintains higher activity during the high-temperature processing of phage products. Furthermore, it exhibits broad-spectrum lytic activity against various Vibrio species, showing specific lytic advantages against Vibrio alginolyticus, Vibrio parahaemolyticus, and Vibrio harveyi, and can be used for the prevention and treatment of diseases caused by Vibrio infections.

[0041] 2. The Vibrio alginolyticus phage PJ644 can be used as an active ingredient to prepare drugs for preventing and treating diseases caused by Vibrio infection, aquatic feed additives, water disinfectants, and aquatic product preservatives. This allows it to play a role in the prevention and treatment of Vibrio diseases at all stages of aquatic production, solving Vibrio infection while avoiding the problems of antibiotic residues and pathogen resistance caused by the use of antibiotics.

[0042] 3. This application also provides a powdered phage drug formulation, which has a simple formulation and preparation method, and its high temperature stability, acid and alkali stability and long-term storage stability at room temperature are significantly improved, which is conducive to the promotion and application of the phage product.

[0043] 4. The bacteriophage PJ644 is obtained from nature and is easy to produce industrially. Drugs or disinfectants prepared from the above bacteriophage can not only reduce costs, but also have the advantages of being green and environmentally friendly. Attached Figure Description

[0044] Figure 1 Image of a phage plaque for Vibrio alginolyticus phage PJ644; Figure 2 Electron micrograph of Vibrio alginolyticus phage PJ644; Figure 3 The results of pH stability test for Vibrio alginolyticus phage PJ644; Figure 4 The temperature stability test results for Vibrio alginolyticus phage PJ644; Figure 5 The first-step growth curve of Vibrio alginolyticus phage PJ644. Detailed Implementation

[0045] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. 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. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0046] Example 1: Isolation and purification of Vibrio alginolyticus phage PJ644 1. Experimental Methods (1) Resuscitation and culture of host bacteria Ten strains of Vibrio alginolyticus preserved in our laboratory were selected. Streaking was performed on TCBS medium with a sterile inoculating tube dipped in the cryopreservation solution. The culture was then incubated at 37°C for 18-24 h to obtain single colonies. A single colony was picked and inoculated into 5 mL of 2216E broth. The culture was then incubated at 37°C with shaking at 170 rpm / min for 16 h to obtain fresh Vibrio alginolyticus bacterial solution.

[0047] (2) Isolation of bacteriophages Fifteen water samples from shrimp farms in Guangxi were placed in a blister bottle. An appropriate amount of 2216E broth medium was added, along with eight strains of Vibrio alginolyticus. The mixture was incubated at 37°C with shaking at 170 rpm / min for 12 hours, centrifuged at 11,000 rpm for 5 minutes, and then filtered through a 0.22 μm sterile microporous membrane to obtain phage proliferation solution. The phage stock solution was diluted 10-fold, and appropriate gradient phage dilutions were mixed with each of the eight strains of Vibrio alginolyticus at a 1:1 ratio. After incubation at 37°C for 5 minutes, 200 μL of the mixture was placed in the upper agar (0.7% concentration), mixed well, and quickly poured onto a 2216E lower agar (1.5% concentration) plate. The plate was shaken well and allowed to solidify. After incubation at 37°C for 4-6 hours, double-layer plates with phage plaques were obtained.

[0048] (3) Purification of bacteriophages A single phage plaque was picked from the plaque-forming bilayer agar medium and placed in 1 mL of PBS. The plate was incubated at 37°C and 170 rpm for approximately 30 min to obtain phage extract. The phage extract was mixed 1:1 with the corresponding Vibrio alginolyticus (hereinafter referred to as the host bacterium) proliferation solution, incubated at 37°C for 5 min, and then 200 μL of the mixture was transferred to the upper agar layer and mixed thoroughly before being quickly poured onto a 2216e lower agar plate. The plate was shaken well and allowed to solidify. It was then incubated upside down at 37°C for 4–6 h to obtain another bilayer agar plate with plaque formation. A single phage plaque was picked from the plaque-forming bilayer agar plate using sterile forceps and placed in 1 mL of PBS. The plate was incubated at 37°C and 170 rpm for approximately 30 min to obtain phage extract. This process was repeated three times to obtain the purified phage extract.

[0049] (4) Phage proliferation and titer determination Equal volumes of purified phage extract and host bacterial proliferation broth were placed in 5 mL of liquid 2216e medium and cultured at 37°C with shaking at 170 rpm until the liquid became clear. The clear liquid was centrifuged at 11000 rpm for 10 min, and the supernatant was collected and filtered through a 0.22 μm sterile microporous membrane to obtain the phage proliferation broth. The titer of the newly isolated phage was determined using the double-layer plate method.

[0050] 2. Experimental Results and Analysis Following the experimental method described above, ten Vibrio alginolyticus isolates were used to screen for ten Vibrio alginolyticus phages, which were sequentially numbered PJ640 to PJ649. All ten Vibrio alginolyticus phages formed clear plaques on double-layer agar plates, without surrounding halos, with clearly visible edges, and a diameter of approximately 0.5 mm to 1.2 mm. The results in Table 1 show that the titers of these ten phages were around 2.7 × 10⁻⁶. 8 PFU / mL 8.4×10 10 PFU / mL, among which phage PJ644 had the highest titer, and it was used as the experimental subject for various experiments in subsequent embodiments.

[0051] Table 1. Phage titers (PFU / mL) of 10 newly isolated Vibrio alginolyticus strains.

[0052] Example 2: Morphological observation and identification of Vibrio alginolyticus phage PJ644 1. Copper mesh fabrication and electron microscopic observation Take 20 µL of phage sample and drop it onto a copper grid with a carbon-coated film. Allow it to settle naturally for 15 min. After slightly blotting it with filter paper, stain it with 2% (w / v) phosphotungstic acid (PTA) for 1-2 min. After slightly blotting it with filter paper and allowing it to dry, observe and photograph it under a transmission electron microscope.

[0053] 2. Appraisal Results like Figure 2 As shown, bacteriophage PJ644 has a polyhedral head structure and a contractile tail. The head is 74-86 nm wide and 110-120 nm long, and the tail is about 110-122 nm long. According to the classification method of the International Committee on Taxonomy of Viruses (ICTV), the morphology of the bacteriophage in this application conforms to the characteristics of the Myotail Phagesidae family and belongs to Myotail Phage.

[0054] Example 3: Whole genome analysis of Vibrio alginolyticus phage PJ644 The genome of bacteriophage PJ644 was extracted and subjected to whole-genome sequencing and sequence analysis. The results are as follows: (1) The genome is 81,502 bp in length, with a G+C content of 38.95%, and the base contents of C, G, A, and T are 23.31%, 21.82%, 23.87%, and 30.98%, respectively. Online RAST annotation of the whole genome showed that it contains 120 open reading frames (ORFs). Among these 120 ORFs, various structural proteins were found, mainly including phage structural and packaging proteins (tail proteins, binding proteins, neck proteins, head proteins, capsid proteins, etc.), phage-like proteins, regulatory proteins, and other functional proteins. Meanwhile, analysis using tRNAscan-SE software showed that the genome does not contain tRNA genes. Analysis using the online tool CGE server showed that the genome does not contain drug resistance genes or virulence genes. Analysis using PHASTER showed that the genome does not contain lysogenic genes.

[0055] (2) The conserved genes in the phage genome include: the tail protein gene sequence, which is shown in Sequence 1 of the sequence listing. Based on the detection of these conserved genes, phage PJ644 can be rapidly identified. The specific sequence information is shown in Table 2 below.

[0056] Table 2 Gene Sequence Information

[0057] Example 3: Determination of the lysis spectrum of Vibrio alginolyticus phage PJ644 1. Experimental Method: Forty pathogenic Vibrio strains of different types isolated from different regions and preserved in the laboratory were selected, including 18 Vibrio alginolyticus strains, 12 Vibrio parahaemolyticus strains, and 10 Vibrio harveyi strains. The lysis rate of PJ644 against these 40 Vibrio strains was determined by the double-layer plate method. Detailed information on the strains and lysis profiles is shown in Table 3 below.

[0058] 2. Experimental Results and Analysis The results of the cleavage spectrum experiments in Table 3 show that: (1) The Vibrio alginolyticus phage PJ644 was able to lyse 35 out of 40 different types of Vibrio strains, with a lysis rate as high as 87.5%. This phage could lyse 17 out of 18 Vibrio alginolyticus strains, with a lysis rate of 94.44%, 83.33% for 12 Vibrio parahaemolyticus strains, and 90% for 10 Vibrio harveyi strains. These results indicate that phage PJ644 has a specific lysis advantage against different types of Vibrio.

[0059] Table 3. Lysis profiles of Vibrio alginolyticus PJ644 against 40 Vibrio strains.

[0060] Note: "++" indicates a clear and bright spot, "++" indicates a slightly blurred spot, "+" indicates a relatively blurred spot, and "-" indicates no lysis. Example 4: Lysis test of Vibrio alginolyticus phage PJ644 against non-host bacteria 1. Experimental Methods Twenty-five non-host bacteria of different types were selected, including ten strains of Escherichia coli, five strains of Staphylococcus aureus, five strains of Proteus vulgaris, and five strains of Clostridium perfringens. The lysis spectrum of Vibrio alginolyticus phage PJ644 was determined according to the lysis spectrum determination method in Example 3.

[0061] 2. Experimental Results and Analysis No clear plaques were found in the double-layer plates of Vibrio alginolyticus phage PJ644 and the above 25 non-host bacteria, indicating that the phage could not recognize these 10 Escherichia coli strains, 5 Staphylococcus strains, 5 Proteus strains, and 5 Clostridium perfringens strains. This shows that the tested phage PJ644 has extremely strong host specificity and does not damage the microbial community, and its specificity can be used to prepare detection kits.

[0062] Example 5: pH tolerance of Vibrio alginolyticus phage PJ644 1. Experimental Methods Add 4.5 mL of PBS at different pH values ​​(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) to three sterile test tubes. Then place the test tubes in a 37°C water bath. After the temperature stabilizes, add 500 μL of 3×10⁻⁶ PBS to each tube. 10 Pfu / mL phage proliferation solution was mixed and incubated in a water bath at 37°C for 1 h, 2 h, and 3 h. Immediately after incubation, an appropriate amount of 1 mol / L HCl or NaOH was added to the mixture to adjust the pH to approximately 7. The mixture was then serially diluted 10-fold, and the titer was determined using appropriate dilution gradients. Two replicates were set up for each pH value. A pH stability curve of the phage was plotted with pH value on the x-axis and the logarithm of the phage titer on the y-axis.

[0063] 2. Experimental Results and Analysis from Figure 3 It can be seen that the titer of bacteriophage PJ644 remains at 10 within the pH range of 5.0–10.0. 9 The activity was stable at pfu / mL. After 2 hours of treatment at pH 2.0, the potency remained at 10. 5 The phage has a pfu / mL concentration, enabling it to resist certain strongly acidic environments; after 3 hours of treatment at pH 11, the titer of this phage remains at 10. 7 PFU / mL, at pH 13, for 3 hours, with a potency not lower than 10. 3With a concentration of pfu / mL, phage PJ644 exhibits strong stability in acidic environments and can adapt to a certain range of strongly acidic environments.

[0064] Example 6: Survival stability test of Vibrio alginolyticus phage PJ644 1. Experimental Methods 3×10 10 The PFU / mL phage PJ644 proliferation solution was stored at 4℃, 18℃, and 30℃. The phage titer was determined periodically at different temperatures using the double-layer plate method after 1 month, 3 months, 6 months, and 12 months of storage.

[0065] 2. Experimental Results and Analysis The results are shown in Table 4. The results show that the titer of Vibrio alginolyticus phage PJ644 did not change significantly after 12 months of storage at 4℃, decreased by one gradient after 12 months of storage at 18℃, and decreased by two gradients after 12 months of storage at 30℃, indicating that Vibrio alginolyticus phage PJ644 has good storage stability.

[0066] Table 4. Survival stability of Vibrio alginolyticus phage PJ644

[0067] Example 7 Temperature tolerance of Vibrio alginolyticus phage PJ644 (1) Experimental methods 3×10 10 PFU / mL phage PJ644 proliferation solution was incubated at 65℃, 70℃, 75℃, 80℃, and 85℃ (three replicates at each temperature) for 20 min, 40 min, and 60 min respectively. After incubation, the samples were immediately cooled in an ice bath, and the phage titer at different temperatures was determined using the double-layer plate method. A thermostability curve of phage PJ644 was plotted with temperature on the x-axis and the logarithm of the phage titer on the y-axis.

[0068] (2) Experimental results and analysis The results are as follows Figure 4 It is evident that bacteriophage PJ644 retained its original activity after being exposed to temperatures below 70℃ for 60 minutes; and after exposure to temperatures of 75℃ for 40 minutes, the bacteriophage still contained 10... 9 PFU / mL live phage, incubated at 80℃ for 20 min, yielded a phage titer of approximately 3.4 × 10⁻⁶. 8 PFU / mL, incubated at 80℃ for 60 min, potency 2×10 8 After incubation at 85℃ for 60 min, phage PJ644 still retained some activity, with a titer of approximately 1.08 × 10⁻⁶ PFU / mL. 7PFU / mL. This indicates that Salmonella phage PJ644 exhibits good temperature stability and can adapt to relatively high temperature environments.

[0069] Example 8: One-step growth curve of Vibrio alginolyticus phage PJ644 1. Experimental Methods 1 mL each of the proliferation broth of phage PJ644 (multiple of infection 10) and fresh proliferation broth of the host bacteria were thoroughly mixed (timing started at this point), incubated at 37°C for 5 min, centrifuged at 13000 rpm for 30 s, and the supernatant was removed as much as possible using a micropipette. The sample was then washed once with 5 mL of PBS (centrifuged at 13000 rpm for 30 s), and the supernatant was discarded. The precipitate (total volume 5 mL) was resuspended in preheated PBS and thoroughly mixed. The sample was then quickly placed in a shaker at 37°C and 170 rpm for incubation. 150 μL was taken at time 0 and every 5 min, and centrifuged at 10000 rpm for 1 min. After a 10-fold serial dilution with PBS, the phage titer was determined using the double-layer plate method. Three replicates were performed, and the average value was taken. A one-step growth curve was plotted with infection time as the x-axis and the phage titer in the infection system as the y-axis to obtain the phage latency period and outbreak period, and the outbreak quantity was calculated.

[0070] Outbreak size = Total number of bacteriophages at the end of the outbreak / Total number of bacteria at the beginning of the outbreak 2. Experimental Results and Analysis from Figure 5 The results showed that after phage PJ644 infected the host bacteria, the phage lysis cycle lasted approximately 75 minutes, the latency period was approximately 10 minutes, and the phage outbreak period was approximately 60 minutes. After 75 minutes, the phage number remained essentially unchanged, entering a stationary phase, at which point the titer could reach 10. 10 The pfu / mL concentration of phage PJ644 was 132.

[0071] Example 9: Determination of the optimal multiple of infection (MOI) of Vibrio alginolyticus phage PJ644 1. Experimental Methods Colonies of *Vibrio alginolyticus* were picked and inoculated into 5 ml of 2216e medium and incubated at 37°C and 170 rpm for 12–16 h to obtain a bacterial suspension. The bacterial concentration was determined using the pour method, and the suspension concentration was adjusted to 1 × 10⁻⁶ based on the results. 9 cfu / mL, 1×10 8 cfu / mL, 1×10 7 cfu / mL, 1×10 6 cfu / mL, 1×10 5 cfu / mL. The concentration of Vibrio alginolyticus phage PJ644 was adjusted to 1×10⁻⁶. 5 PFU / mL, 1×10 6PFU / mL, 1×10 7 PFU / mL. Phages and bacterial culture were added to 2216e medium according to the phage-to-bacterial ratio in Table 5 and incubated at 37°C and 170 rpm in a shaker until the liquid became clear. The incubation time was recorded. An appropriate amount of the clear liquid was centrifuged at 11000 rpm for 10 min. The supernatant was collected and filtered through a 0.22 μm sterile microporous membrane. The phage titer in the filtrate was determined using the double-layer plate method. The MOI (phage count / bacterial count) with the highest phage titer was the optimal multiple of infection for that phage.

[0072] 2. Experimental Results The results are shown in Table 5. The titer of phage PJ644 reached its highest value (1.2 × 10⁻⁶) at an MOI of 0.01:1. 11 At a concentration of PFU / mL, the initial amount of phage introduced is relatively small, yet the highest reproduction yield can be achieved, which is beneficial for large-scale industrial production.

[0073] Table 5. Titer of Salmonella phage PJ644 at different multiplicity of infection.

[0074] Example 10: Safety test of Vibrio alginolyticus phage PJ644 1. Experimental Methods Healthy whiteleg shrimp weighing approximately 5g were selected and acclimated overnight on an empty stomach. Experimental and control groups were established. Bacteriophage PJ644 was uniformly mixed with the shrimp feed at a volume-to-mass ratio of 5%, resulting in a phage titer of 1×10⁻⁶ after mixing. 8 The feed was air-dried and then fed to the experimental group shrimp at a dose of 5% of the shrimp's body weight. The control group was fed the same dose of feed soaked in TSB medium. The phage content in the shrimp intestine, hepatopancreas, and normal shrimp in the blank control group was measured at 0.5, 6, 24, 48, 72, 96, 120, and 144 hours after feeding. 2. Experimental Results and Analysis After feeding shrimp with feed soaked in bacteriophage PJ644 for 0.5 hours, the bacteriophage was detected in the intestines and hepatopancreas of the shrimp in the experimental group, with a titer of up to 8.5 × 10⁻⁶. 6 The PFU / g concentration of bacteriophages allows them to enter shrimp bodies through feeding and persist for more than 2 days. This provides guidance for the use of bacteriophages as aquatic feed additives and can be used for the prevention and treatment of vibriosis. Experimental results also show that bacteriophages have no impact on the health of Litopenaeus vannamei and do not affect the normal activity of shrimp, indicating that the bacteriophages have excellent safety.

[0075] Example 11 Preparation and performance determination of dried phage powder of Vibrio alginolyticus phage PJ644 1. Experimental Methods (1) Preparation of phage spray-dried powder: Using gelatin, monosodium glutamate, and mannitol as excipients, the ratio of gelatin, monosodium glutamate, and mannitol in the raw materials of the phage dry powder is 100:4:2:3. PJ644 proliferation solution (final concentration 1×10⁻⁶) is added. 10 After mixing PFU / g evenly, spray drying is performed.

[0076] Specifically, it adopts the Swiss BUCHIB The phage was dried using a Type 90 nano-spray dryer with the following process parameters: inlet temperature 80℃, airflow velocity 120L / min, spray rate 100%, and nozzle cap size 4.0μm. After approximately 2 hours of spray drying, the spray-dried particles were collected. Ultimately, phage PJ644 was formulated into a spray-dried powder, thus obtaining the phage drug formulation with a phage concentration of 1×10⁻⁶. 10 PFU / g.

[0077] (2) The temperature stability and acid-base stability of the phage dry powder were tested.

[0078] ① The potency prepared in this embodiment is 3×10 10 Phage drug preparations of PFU / mL phage PJ644 and phage proliferation solutions were treated at pH 4 and pH 10 for 3 h, respectively, and then the phage titer and acid-base stability were determined.

[0079] ②The phage drug preparation and phage proliferation solution were placed at 30℃ for 6 months and their stability at room temperature was tested.

[0080] ③ The phage drug preparation and phage proliferation solution were placed at 85℃ for 1 hour and their potency was determined to test their high temperature stability.

[0081] 2. Experimental Results and Analysis The results are shown in Table 6. After the Vibrio alginolyticus phage PJ644 proliferation solution was prepared into a phage drug formulation, its high temperature stability, acid-base stability and long-term storage stability at room temperature were significantly improved.

[0082] Table 6 Performance determination of PJ644 after preparation into pharmaceutical formulations

[0083] Example 12 Application of Vibrio alginolyticus phage PJ644 phage dry powder This embodiment uses the phage dry powder prepared in Example 11 as the experimental subject to test its application in the prevention and treatment of vibrio infection in shrimp.

[0084] 1. Experimental Methods Healthy whiteleg shrimp weighing approximately 5g were prepared and accumulating them overnight on an empty stomach. Each group consisted of 50 shrimp. The experiment included a prevention group, a treatment group, a challenge group, and a blank control group. The treatments for each group were as follows: Prevention group: Phage spray-dried powder was added to shrimp feed at a dosage of 5% and mixed thoroughly. The feed was administered at a dosage of 3% of the shrimp's body weight, with a phage content of 1×10⁻⁶. 8 PFU / g; Treatment group: All shrimp were injected with Vibrio alginolyticus at a dose of 3.25 × 10⁻⁶. 6 CFU / shrimp, 1 hour after challenge, mix the bacteriophage with the shrimp feed at a volume-to-mass ratio of 5%, and feed at a dose of 3% of the shrimp's body weight. Challenge group: All shrimp were fed the same amount of shrimp feed, and 1 hour later, all shrimp were injected with Vibrio alginolyticus at a dose of 3 × 10⁻⁶. 6 CFU / each; Blank control group: fed the same dose of conventional feed as usual.

[0085] Record the number of dead shrimp in each group within 48 hours and calculate the phage protection rate. After 15 days, take samples from the water, test for Vibrio counts, and record the data.

[0086] 2. Experimental Results and Analysis The experimental results are shown in Table 7. The results show that the survival rate of shrimp in the prevention group was as high as 100%, and the survival rate of shrimp in the treatment group reached 92%, both significantly higher than that of the challenge group. The mortality rate was significantly reduced, and the Vibrio content in the dead shrimp was also much lower than that in the challenge group. This indicates that feed containing bacteriophage dry powder can significantly reduce the number of Vibrio in the water, effectively preventing Vibrio infection in shrimp and improving survival rate. It can also effectively treat shrimp already infected with Vibrio, further improving shrimp survival rate.

[0087] Therefore, the results of this experiment show that the phage powder of this embodiment, as a novel phage drug preparation, can also be used as a feed additive. It has the potential to reduce the number of Vibrio in water and protect shrimp production, and can provide a new biological control method for aquaculture.

[0088] Table 7. Preventive and curative effects of Vibrio alginolyticus phage PJ644 mixed in feed against Vibrio.

[0089] Example 13 Optimization of the formulation of Vibrio alginolyticus phage PJ644 phage dry powder 1. Experimental Methods According to the proportions of the protectants in Table 8, prepare the dry powder of Vibrio alginolyticus phage PJ644. After mixing, spray dry using Swiss BUCHIB. The phage was dried using a Type 90 nano-spray dryer with the following process parameters: inlet temperature (80℃), airflow velocity (120L / min), spray rate (100%), and nozzle cap size (4.0μm). After approximately 2 hours of spray drying, the dried particles were collected. Ultimately, phage PJ644 was formulated into a spray-dried powder, thus obtaining the phage drug formulation.

[0090] The phage titer retention rate, moisture content, particle size, reconstitution time, and stability of the phage dry powders prepared in each group were tested.

[0091] (1) Determination of reconstitution time: Add 1 mL of physiological saline to a vial containing 50 mg of phage powder to dissolve it. Record the reconstitution time by observing the dissolution state of the powder. While starting the vortex mixer, add all the powder into the vial and start timing immediately. Carefully observe the suspension state. Stop timing when the solution becomes homogeneous and transparent, and no particles or clumps are visible to the naked eye. This time period is the reconstitution time (unit: seconds).

[0092] (2) Stability determination: The reconstituted phage suspension was serially diluted 10-fold and stored at 4°C for 30 days. Its potency was determined by the double-layer plate method, and its stability was calculated.

[0093] (3) Calculation method of valence retention rate Potency retention rate (%) = (Total activity after reconstitution / Total activity before drying) × 100% Table 8 Formulation optimization of phage protectants

[0094] 2. Experimental Results and Analysis The test results are shown in Table 9. The phage titer in each group of protectants was tested. The results showed that Group 1 had the highest phage retention rate, lowest moisture content, smallest particle diameter, shortest reconstitution time, and better stability than other groups, making it the superior group. Therefore, the preferred formulation for phage dry powder is: 100 parts phage stock solution, 4 parts gelatin, 2 parts monosodium glutamate, and 3 parts mannitol.

[0095] Compared to group 1, the potency retention rates of groups 2 and 4, which had their components replaced, were significantly reduced. This indicates that the components in the aforementioned phage dry powder work synergistically as a whole, and that modifying or replacing them with other existing components would lead to a deterioration in their technical effectiveness.

[0096] Table 9 Comparison of key indicators after spray drying

[0097] It is understood that those skilled in the art can make equivalent substitutions or modifications to the technical solutions and concepts of this invention, and all such substitutions or modifications should fall within the protection scope of the appended claims.

Claims

1. A thermoresistant Vibrio alginolyticus bacteriophage ( Vibrio alginolyticus phage ), characterized in that, It was named Vibrio alginolyticus phage PJ644, and its accession number is CGMCC No. 46165.

2. A bacteriophage composition, characterized in that, Includes the Vibrio alginolyticus phage as described in claim 1.

3. The application of the Vibrio alginolyticus phage as described in claim 1 or the phage composition as described in claim 2 in the preparation of drugs for preventing and treating diseases caused by Vibrio infection, aquatic feed additives, water disinfectants, and aquatic product preservatives.

4. A bacteriophage drug formulation, characterized in that, Its active ingredients include Vibrio alginolyticus phage as described in claim 1 or phage composition as described in claim 2.

5. The phage drug formulation according to claim 4, characterized in that the dosage form... include: Solid dosage forms and liquid dosage forms, wherein solid dosage forms include powders, tablets, granules and microcapsules; Liquid dosage forms include solutions and injections.

6. The phage drug formulation according to claim 5, characterized in that, The dosage form is a powder, and the phage drug preparation is prepared from the following components in parts by weight: 90-110 parts of phage proliferation solution, 1-5 parts of gelatin, 1-5 parts of sodium glutamate, and 1-5 parts of mannitol.

7. The phage drug formulation according to claim 6, characterized in that, The phage drug preparation was prepared by mixing all raw materials, spray drying, and collecting the spray-dried particles.

8. An aquatic feed additive, characterized in that, The active ingredient includes Vibrio alginolyticus phage as described in claim 1 or the phage composition as described in claim 2.

9. A water disinfectant, characterized in that, The active ingredient includes Vibrio alginolyticus phage as described in claim 1 or the phage composition as described in claim 2.

10. A preservative for aquatic products, characterized in that, Includes the Vibrio alginolyticus phage as described in claim 1 or the phage composition as described in claim 2.

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