A broad-spectrum bacteriophage and its probiotic preparation and application

Abstract

The present invention belongs to the field of environmental protection and green and healthy breeding, and specifically relates to a broad-spectrum phage and its probiotic preparation and application. The broad-spectrum phage is phage YTLJ-VBN1, which was deposited in the Guangdong Provincial Microbial Culture Collection Center on January 22, 2024, with a deposit number of GDMCC No: 64300-B1, classified as Vibrio phage, and the deposit address is Guangzhou, China. Broad-spectrum phage can simultaneously and efficiently kill a variety of highly pathogenic Vibrio, Aeromonas, Edwardsiella and other pathogens, which can overcome the problem of the narrow bactericidal spectrum of traditional phages and can solve the problem of drug resistance that is easy to develop during the process of antibiotic prevention and treatment of pathogens in an ecologically friendly way.

Description

Technical Field

[0001] The present invention belongs to the field of environmental protection, green and healthy breeding, and specifically relates to a broad-spectrum bacteriophage and its probiotic preparation and application. Background Art

[0002] Large-scale aquaculture activities have led to a series of challenges, including frequent disease outbreaks and bacterial resistance. Traditional antibiotics are generally effective against pathogens, but their overuse can easily lead to the accumulation of drug-resistant pathogens and disrupt or even collapse recirculating water biological treatment systems in aquaculture, especially high-density factory aquaculture.

[0003] Green and ecological aquaculture has become a hot topic in the aquaculture industry. In recent years, bacteriophages have begun to gain traction as a new technology for killing pathogens. This technology can rapidly eliminate pathogens, purifying the environment and ensuring healthy aquaculture. However, bacteriophages have a narrow bactericidal spectrum and cannot simultaneously control multiple pathogens. Summary of the Invention

[0004] The purpose of the present invention is to provide a broad-spectrum bacteriophage and a probiotic preparation and application thereof.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] A broad-spectrum phage, the broad-spectrum phage is phage YTLJ-VBN1, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on January 22, 2024, with the deposit number GDMCC No: 64300-B1, the classification name is Vibrio phage, and the deposit address is Guangzhou, China.

[0007] An application of the broad-spectrum bacteriophage, and an application of the bacteriophage YTLJ-VBN1 in aquaculture.

[0008] The bacteriophage YTLJ-VBN1 is used to inhibit pathogenic Vibrio in aquaculture.

[0009] The pathogenic Vibrio is one or more of Vibrio harveyi, Vibrio parahaemolyticus, Vibrio alginolyticus, Aeromonas, Edwardsiella, Vibrio Campbellii, Vibrio cholerae, Vibrio penaeocidalis, Vibrio vulnificus, and Vibrio anguillarum.

[0010] A broad-spectrum phage probiotic preparation, comprising the broad-spectrum phage, or the broad-spectrum phage and Bacillus brevis YTLJ-N-SXH2.

[0011] The Brevibacillus sp. YTLJ-N-SXH2 was deposited in the Guangdong Provincial Microbial Culture Collection Center on January 17, 2022, with the deposit number GDMCC No: 62215, the classification name is Brevibacillus sp., and the deposit address is Guangzhou, China.

[0012] A method for preparing the broad-spectrum phage probiotic preparation, wherein the broad-spectrum phage probiotic preparation comprises broad-spectrum phage and Bacillus brevis YTLJ-N-SXH2, comprises the following steps:

[0013] 1) Phage and bacterial strain propagation: 1-10 wt% of phage YTLJ-VBN1 and Vibrio are sequentially inoculated into an enrichment medium, and the culture is maintained at a constant temperature of 28-35°C for 12-48 hours. The Vibrio is then filtered to remove the Vibrio from the culture medium to obtain a Vibrio phage YTLJ-VBN1 enrichment culture medium; 1-10 wt% of Bacillus brevis YTLJ-N-SXH2 is inoculated into an enrichment medium, and the culture is maintained at a temperature of 28-35°C for 24-72 hours to obtain a Bacillus brevis YTLJ-N-SXH2 enrichment culture medium;

[0014] The Vibrio species are Vibrio parahaemolyticus, Vibrio anguillarum, Vibrio alginolyticus, Vibrio cholerae, Vibrio harveyi, and Vibrio Campbellii;

[0015] 2) Assembly and mixing of phage and probiotics: freeze-drying the culture fluids of the expanded Vibrio phage YTLJ-VBN1 and Bacillus brevis YTLJ-N-SXH2 obtained above to obtain their respective freeze-dried powders, and then mixing the freeze-dried powders of Vibrio phage YTLJ-VBN1 and Bacillus brevis YTLJ-N-SXH2 at a mass ratio of 1 to 10:1 to obtain a mixed freeze-dried powder;

[0016] 3) Preparation of auxiliary agent: polyglutamic acid, brevicaulis antimicrobial peptide-montmorillonite complex, inulin, rhubarb, and eucommia polysaccharide are mixed at a mass ratio of 0.01-0.1:1:0.001-0.01:0.001-0.01:0.0001-0.001 to prepare the auxiliary agent; wherein the brevicaulis antimicrobial peptide-montmorillonite complex is prepared by ball-milling brevicaulis antimicrobial peptide and montmorillonite at a mass ratio of 1:10-100 and then setting aside;

[0017] 4) Preparation of probiotic preparation: The mixed lyophilized powder obtained in step 2) is mixed with the auxiliary agent obtained in step 3) at a mass ratio of 1:1-100 to prepare a probiotic preparation; wherein the phage titer of the probiotic preparation is ≥1.0×10 9 / mL, total effective viable count ≥1.0×10 9 pieces / mL.

[0018] The brevis antimicrobial peptide is prepared by adding 70-90% by weight of ammonium sulfate to the culture solution of the brevis YTLJ-N-SXH2 expanded in step 1), adjusting the pH of the system to 9.0-10.0 after mixing, and then allowing the solution to stand for 6-24 hours. After standing, the brevis antimicrobial peptide is freeze-dried to obtain the brevis antimicrobial peptide, and stored in a dry place for use.

[0019] An application of the broad-spectrum phage probiotic preparation, and an application of the broad-spectrum phage probiotic preparation in aquaculture.

[0020] The broad-spectrum phage probiotic preparation is used in aquaculture to inhibit multiple pathogens and reduce mortality.

[0021] The addition amount of the broad-spectrum bacteriophage probiotic preparation is 0.1-1 wt% of the water mass in the aquaculture system.

[0022] The advantages of the present invention are:

[0023] 1) The obtained phage is a broad-spectrum phage that can simultaneously and efficiently kill a variety of highly pathogenic Vibrio, Aeromonas, Edwardsiella and other pathogens. It can overcome the problem of the narrow bactericidal spectrum of traditional phages and can solve the problem of drug resistance that is easy to develop during the process of antibiotic prevention and treatment of pathogens in an ecologically friendly way.

[0024] 2) The broad-spectrum bacteriophage YTLJ-VBN1 of this invention is highly complementary to the probiotic Bacillus brevis YTLJ-N-SXH2. Bacteriophage YTLJ-VBN1 is capable of killing a variety of highly pathogenic bacteria, including Vibrio harveyi, Vibrio parahaemolyticus, Aeromonas, Edwardsiella, Vibrio alginolyticus, Vibrio Campbellii, Vibrio cholerae, Vibrio penetropicis, Vibrio vulnificus, and Vibrio anguillarum. Bacillus brevis YTLJ-N-SXH2, on the other hand, is capable of inhibiting pathogens such as Acinetobacter, Pseudomonas, Nocardia, Saprolegnia, Gill mold, and Fusarium. The combination of these two phages offers complementary advantages, providing a new approach for the combined ecological control and removal of multiple pathogens, further addressing the narrow host spectrum of traditional phage therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is an electron microscopic image of the bacteriophage YTLJ-VBN1 provided in an embodiment of the present invention. Measurements show that the phage head is 76.55±3.05 nm wide and the tail is 108.52±1.98 nm long, exhibiting a typical myoviral morphology.

[0026] Figure 2 A proteome tree is generated based on the similarity of the whole genome sequence of bacteriophage YTLJ-VBN1 provided in the embodiment of the present invention.

[0027] Figure 3This is a growth curve diagram of bacteriophage YTLJ-VBN1 provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0028] The present invention is further described below by way of examples, however the present invention is not limited to the following examples.

[0029] The Brevibacillus sp. YTLJ-N-SXH2 described in the following examples was deposited in the Guangdong Provincial Microbial Culture Collection Center on January 17, 2022, with a deposit number of GDMCC No: 62215, and is classified as Brevibacillus sp., with a deposit address of Guangzhou, China.

[0030] The enrichment medium is composed of, by mass ratio, 0.05% NH4NO3, 0.05% NH4NO2, 0.2% sodium acetate, 2% K2HPO4, 1% MgSO4, 1% NaCl, 0.01% FeSO4, 0.01% MnSO4 and 0.01% La(NO3)3, with the balance being water.

[0031] The Vibrio parahaemolyticus is Vibrio parahaemolyticus CICC 24174;

[0032] The Aeromonas is Aeromonas salmonicida CICC 23566;

[0033] The Edwardsiella is Edwardsiella tarda CICC 10497;

[0034] The Vibrio anguillarum is Vibrio anguillarum CICC 24712;

[0035] The Vibrio alginolyticus is Vibrio alginolyticus CICC 10889;

[0036] The Vibrio cholerae is Vibrio cholerae CICC 25296;

[0037] The Vibrio harvey is Vibrio harvey SHBCC D10490;

[0038] The Vibrio Campbellii is Vibrio campbellii BNCC 337376. Example 1

[0039] Isolation of broad-spectrum phage YTLJ-VBN1:

[0040] Bacteriophage YTLJ-VBN1 was isolated from a coastal environment. The specific isolation process was as follows: 10 g of seawater sample (without filtration) was placed in 50 mL of sterile seawater and shaken at 30°C for 48 h. The bacterial solution was centrifuged at 8000 rpm for 10 min, and the supernatant was collected. Vibrio parahaemolyticus, Aeromonas, and Edwardsiella were added to the supernatant (the amount of Vibrio parahaemolyticus, Aeromonas, and Edwardsiella added was 10% and 10% respectively). 9 , 10 8 , 10 8 CFU / mL), enriched and cultured at 30℃ for 24h. The enriched phage suspension was mixed with Vibrio parahaemolyticus in 2216E semi-solid medium and cultured in a 30℃ constant temperature incubator. The formation of transparent spots was observed after 3-8h. A single plaque was picked into 2216E semi-solid medium and Vibrio parahaemolyticus (V. parahaemolyticus, 10 9 CFU / mL), placed in a constant temperature shaker (30℃, 220r / min) and shaken for 24h. Then pick a single plaque to expand the culture, and repeat this step 5-10 times. Repeat the plaque picking and culture many times to obtain a single phage, complete the phage purification, and obtain phage YTLJ-VBN1 (see Figure 1 ). Tests have shown that this phage is a broad-spectrum phage that can kill a variety of highly pathogenic bacteria, including Vibrio harveyi, Vibrio parahaemolyticus, Aeromonas, Edwardsiella, Vibrio alginolyticus, Vibrio Campbellii, Vibrio cholerae, Vibrio penetropicis, Vibrio vulnificus, and Vibrio anguillarum.

[0041] The broad-spectrum phage YTLJ-VBN1 was deposited in the Guangdong Provincial Microbial Culture Collection Center on January 22, 2024, with the deposit number GDMCC No: 64300-B1, the classification name Vibrio phage, and the deposit address is Guangzhou, China.

[0042] Blast analysis of the broad-spectrum phage YTLJ-VBN1 revealed a similar phage sequence with a 96.7% similarity. The full length was 129,160 bp. Of the 228 annotated ORFs, 74 mapped to known functional genes, and no drug resistance genes or virulence factors were detected. This confirmed that phage YTLJ-VBN1 is a newly discovered phage.

[0043] The full sequence of the most similar Vibrio phage qdvp001 was downloaded from NCBI and visualized using DiGAlign and VBN genome-wide similarity. The two phages were not completely similar, confirming that phage YTLJ-VBN1 is a newly discovered phage.

[0044] A proteome tree generated by calculating whole-genome sequence similarity using tBLASTx revealed that phage YTLJ-VBN1 could not be clustered into a specific viral family, further confirming that phage YTLJ-VBN1 was a newly discovered phage.

[0045] Application of the broad-spectrum phage YTLJ-VBN1 obtained above in the control of Vibrio: phage YTLJ-VBN1 was mixed with Vibrio parahaemolyticus and cultured in 2216e medium (the phage inoculation amount was 10 7 PFU / mL, Vibrio parahaemolyticus inoculation volume 10 8 CFU / mL), treated at 30℃ for 12h. The OD value of the control group without phage addition was 600 The bacterial density of the phage-treated group gradually decreased, reaching 0.08 at 3 h, and then slowly increased until 12 h, when the OD value of the phage-treated group was 0.39, and the culture concentration was significantly lower than that of the control group (see Figure 3 ).

[0046] like Figure 3 As shown, the incubation period of bacteriophage YTLJ-VBN1 is 20 minutes, the duration of the burst period is 40 minutes, and the burst volume is 10 8 PFU / cell is a virulent phage with a short incubation period and a large lysis capacity. Example 2

[0047] Probiotic preparations are used in eel factory farming systems:

[0048] 1) Bacteriophage and bacterial strains were propagated separately: 5 wt% of bacteriophage YTLJ-VBN1 and Vibrio anguillarum were inoculated into the propagation medium, cultured at 30°C for 24 h, and then filtered with a 0.22 μm filter membrane to remove Vibrio anguillarum from the culture medium to obtain the bacteriophage YTLJ-VBN1 propagation medium; 5 wt% of Bacillus brevis YTLJ-N-SXH2 was inoculated into the propagation medium, cultured at 30°C for 48 h, and the Bacillus brevis YTLJ-N-SXH2 propagation medium was obtained.

[0049] 2) Assembly and mixing of phage and probiotics: The culture solutions of the expanded Vibrio phage YTLJ-VBN1 and Bacillus brevis YTLJ-N-SXH2 were freeze-dried (pre-freezing temperature -80°C, pre-freezing for 3 hours, freeze-drying for 6 hours) to obtain their respective freeze-dried powders. The freeze-dried powders of Vibrio phage YTLJ-VBN1 and Bacillus brevis YTLJ-N-SXH2 were then mixed at a mass ratio of 5:1 to obtain a mixed freeze-dried powder.

[0050] 3) Preparation of auxiliary agent: polyglutamic acid, brevicaulis antimicrobial peptide-montmorillonite complex, inulin, rhubarb, and eucommia polysaccharide were mixed at a mass ratio of 0.01:1:0.001:0.001:0.0001 to prepare the auxiliary agent; wherein, the brevicaulis antimicrobial peptide-montmorillonite complex was prepared by ball-milling brevicaulis antimicrobial peptide and montmorillonite at a mass ratio of 1:10.

[0051] The brevis antimicrobial peptide is prepared by adding 70% by weight of ammonium sulfate to the brevis YTLJ-N-SXH2 culture medium obtained by propagation in step 1), adjusting the pH of the system to 9.0 after mixing, and then standing for 6 hours. After standing, the brevis antimicrobial peptide is freeze-dried (pre-freezing temperature -80°C, pre-freezing for 3 hours, freeze-drying for 4 hours) and stored in a dry place for use.

[0052] 4) Preparation of probiotic preparation: The mixed lyophilized powder obtained in step 2) and the auxiliary agent obtained in step 3) are mixed at a mass ratio of 1:10 to prepare a probiotic preparation; wherein the phage titer of the probiotic preparation is ≥1.0×10 9 / mL, total effective viable count ≥1.0×10 9 pieces / mL.

[0053] 5) The probiotic preparation obtained above was added to the eel culture system at an addition rate of 0.5wt% (the eel culture density of the culture system before the probiotic preparation was added was 80kg / m 3 , nitrate nitrogen concentration was 25.81 mg / L, and Vibrio concentration reached 3×10 4 CFU / mL), and the culture system without adding probiotic preparations was used as a control; after 5 days of culture with the addition of probiotic preparations, the water quality in the system was good (nitrate nitrogen concentration dropped to 15.75 mg / L), and the water was relatively clear. At the same time, compared with the control culture system without adding probiotic preparations, the Vibrio concentration in the culture system with probiotics was reduced to 2×10 3 CFU / mL, the removal rate exceeded 90%, and the eel mortality rate was reduced by 80%. Example 3

[0054] Probiotic preparations are used in grouper factory farming systems:

[0055] 1) Bacteriophages and bacterial strains were propagated separately: 1 wt% of bacteriophage YTLJ-VBN1 and Vibrio alginolyticus were inoculated into the propagation medium, cultured at 35°C for 48 h, and then filtered with a 0.22 μm filter membrane to remove Vibrio from the culture medium to obtain the bacteriophage YTLJ-VBN1 propagation medium; 1 wt% of Bacillus brevis YTLJ-N-SXH2 was inoculated into the propagation medium, cultured at 35°C for 72 h, and the Bacillus brevis YTLJ-N-SXH2 propagation medium was obtained.

[0056] 2) Assembly and mixing of bacteriophage and probiotics: The culture fluids of the expanded bacteriophage YTLJ-VBN1 and Bacillus brevis YTLJ-N-SXH2 were freeze-dried (pre-freezing temperature -80°C, pre-freezing for 3 hours, freeze-drying for 5 hours) to obtain their respective freeze-dried powders. The freeze-dried powders of bacteriophage YTLJ-VBN1 and Bacillus brevis YTLJ-N-SXH2 were then mixed at a mass ratio of 3:1 to obtain a mixed freeze-dried powder.

[0057] 3) Preparation of auxiliary agent: polyglutamic acid, brevicaulis antimicrobial peptide-montmorillonite complex, inulin, rhubarb, and eucommia polysaccharide were mixed at a mass ratio of 0.05:1:0.005:0.005:0.0005 to prepare the auxiliary agent; wherein, the brevicaulis antimicrobial peptide-montmorillonite complex was prepared by ball-milling brevicaulis antimicrobial peptide and montmorillonite at a mass ratio of 1:50.

[0058] The brevis antimicrobial peptide is prepared by adding 75% by weight of ammonium sulfate to the brevis YTLJ-N-SXH2 culture medium obtained by propagation in step 1), adjusting the pH of the system to 9.5 after mixing, and then standing for 12 hours. After standing, the brevis antimicrobial peptide is freeze-dried (pre-freezing temperature -80°C, pre-freezing for 4 hours, and freeze-drying for 4 hours) and stored in a dry place for use.

[0059] 4) Preparation of probiotic preparation: The mixed lyophilized powder obtained in step 2) is mixed with the auxiliary agent obtained in step 3) at a mass ratio of 1:10 to prepare a probiotic preparation; wherein the phage titer of the probiotic preparation is ≥1.0×10 9 / mL, total effective viable count ≥1.0×10 9 pieces / mL.

[0060] 5) The probiotic preparation obtained above was added to the grouper culture system at an addition rate of 1 wt% (the grouper culture density in the culture system before the probiotic preparation was added was 80 kg / m 3 , the nitrate nitrogen concentration was about 19.55 mg / L, and the Vibrio concentration was 4×10 4 CFU / mL), and the culture system without adding probiotic preparations was used as a control. After 4 days of culture with the addition of probiotic preparations, the water in the system was clearer and the nitrate nitrogen concentration dropped to 13.37 mg / L. In addition, compared with the control system without adding probiotic preparations, the Vibrio concentration in the culture system with probiotic preparations dropped to 7×10 3 CFU / mL, the removal rate exceeded 80%, and the mortality rate of grouper was reduced by 70%. Example 4

[0061] Probiotic preparations are used in factory shrimp farming systems:

[0062] 1) Phage and bacterial strains were propagated separately: phage YTLJ-VBN1 and Vibrio cholerae were inoculated into a propagation medium at 6 wt %, and the mixture was cultured at 32°C for 36 h. The culture medium was then filtered through a 0.22 μm filter membrane to remove the Vibrio from the culture medium to obtain a phage YTLJ-VBN1 propagation medium; Bacillus brevis YTLJ-N-SXH2 was inoculated into a propagation medium at 6 wt %, and the mixture was cultured at 32°C for 36 h to obtain a Bacillus brevis YTLJ-N-SXH2 propagation medium.

[0063] 2) Assembly and mixing of phage and probiotics: The culture fluids of the expanded phage YTLJ-VBN1 and Bacillus brevis YTLJ-N-SXH2 were freeze-dried (pre-freezing temperature -80°C, pre-freezing for 4 hours, and freeze-drying for 6 hours) to obtain their respective freeze-dried powders. The freeze-dried powders of phage YTLJ-VBN1 and Bacillus brevis YTLJ-N-SXH2 were then mixed at a mass ratio of 8:1 to obtain a mixed freeze-dried powder.

[0064] 3) Preparation of auxiliary agent: polyglutamic acid, brevicaulis antimicrobial peptide-montmorillonite complex, inulin, rhubarb, and eucommia polysaccharide were mixed at a mass ratio of 0.06:1:0.008:0.006:0.0006 to prepare the auxiliary agent. The brevicaulis antimicrobial peptide-montmorillonite complex was prepared by ball-milling brevicaulis antimicrobial peptide and montmorillonite at a mass ratio of 1:60.

[0065] The brevis antimicrobial peptide is prepared by adding 80% by weight of ammonium sulfate to the brevis YTLJ-N-SXH2 culture medium obtained by propagation in step 1), adjusting the pH of the system to 9.6 after mixing, and then standing for 16 hours. After standing, the brevis antimicrobial peptide is freeze-dried (pre-freezing temperature -80°C, pre-freezing for 3 hours, and freeze-drying for 5 hours) and stored in a dry place for use.

[0066] 4) Preparation of probiotic preparation: The mixed lyophilized powder obtained in step 2) is mixed with the auxiliary agent obtained in step 3) at a mass ratio of 1:50 to prepare a probiotic preparation; wherein the phage titer of the probiotic preparation is ≥1.0×10 9 / mL, total effective viable count ≥1.0×10 9 pieces / mL.

[0067] 5) The probiotic preparation obtained above was added to the shrimp culture system at an addition rate of 0.8 wt%. (Before adding the probiotic preparation, the shrimp culture density of the culture system reached 18.8 kg / m 3 , nitrate nitrogen concentration was 6.75 mg / L, and Vibrio concentration reached 5×10 5CFU / mL), and the culture system without adding probiotic preparations was used as a control; after 6 days of culture with the addition of probiotic preparations, the water quality in the system was good, and the nitrate nitrogen concentration dropped to 1.55 mg / L. At the same time, compared with the control system without adding probiotic preparations, the concentration of Vibrio in the culture system dropped to 1×10 4 CFU / mL, the removal rate exceeded 95%, and the shrimp mortality rate was reduced by 90%. Example 5

[0068] Probiotic preparations are used in sea bass farming systems:

[0069] 1) Phage and bacterial strains were propagated separately: phage YTLJ-VBN1 and Vibrio harveyi were inoculated into the propagation medium at 8 wt %, cultured at 29°C for 15 h, and then filtered through a 0.22 μm filter membrane to remove Vibrio from the culture medium to obtain the phage YTLJ-VBN1 propagation medium; Bacillus brevis YTLJ-N-SXH2 was inoculated into the propagation medium at 9 wt %, cultured at 28°C for 20 h, and the Bacillus brevis YTLJ-N-SXH2 propagation medium was obtained.

[0070] 2) Assembly and mixing of phage and probiotics: The culture fluids of the expanded phage YTLJ-VBN1 and Bacillus brevis YTLJ-N-SXH2 were freeze-dried (pre-freezing temperature -80°C, pre-freezing for 3 hours, freeze-drying for 4 hours) to obtain their respective freeze-dried powders. The freeze-dried powders of phage YTLJ-VBN1 and Bacillus brevis YTLJ-N-SXH2 were then mixed at a mass ratio of 2:1 to obtain a mixed freeze-dried powder.

[0071] 3) Preparation of auxiliary agent: polyglutamic acid, brevicaulis antimicrobial peptide-montmorillonite complex, inulin, rhubarb, and eucommia polysaccharide were mixed at a mass ratio of 0.08:1:0.007:0.0008:0.0005 to prepare the auxiliary agent; wherein, the brevicaulis antimicrobial peptide-montmorillonite complex was prepared by ball-milling brevicaulis antimicrobial peptide and montmorillonite at a mass ratio of 1:100.

[0072] The brevis antimicrobial peptide is prepared by adding 90% by weight of ammonium sulfate to the brevis YTLJ-N-SXH2 culture medium obtained by propagation in step 1), adjusting the pH of the system to 10.0 after mixing, and then standing for 24 hours. After standing, the brevis antimicrobial peptide is freeze-dried (pre-freezing temperature -80°C, pre-freezing for 2 hours, and freeze-drying for 5 hours) and stored in a dry place for use.

[0073] 4) Preparation of probiotic preparation: The mixed lyophilized powder obtained in step 2) is mixed with the auxiliary agent obtained in step 3) at a mass ratio of 1:100 to prepare a probiotic preparation; wherein the phage titer of the probiotic preparation is ≥1.0×10 9 / mL, total effective viable count ≥1.0×10 9pieces / mL.

[0074] 5) The above-obtained probiotic preparation was added to the seabass culture system at an addition rate of 0.1 wt% (the seabass culture density in the culture system before the addition of the probiotic preparation was 55 kg / m 3 , nitrate nitrogen concentration was 13.78 mg / L). At the same time, the culture system without adding probiotic preparations was used as a control. After adding probiotic preparations, the water quality in the system was good and clear, and the nitrate nitrogen concentration dropped to 2.31 mg / L. At the same time, compared with the control system without adding probiotic preparations, the removal rate of Nocardia was more than 80% (the concentration was increased from 6×10 5 The CFU / mL decreased to 9×10 4 CFU / mL), sea bass mortality was reduced by 80%. Example 6

[0075] Probiotic preparations are used in factory-scale crucian carp farming systems:

[0076] 1) Phage and bacterial strains were propagated separately: 10 wt% of phage YTLJ-VBN1 and Vibrio Campbellii were inoculated into the propagation medium, cultured at 28°C for 12 h, and then filtered with a 0.22 μm filter to remove the Vibrio from the culture medium to obtain the phage YTLJ-VBN1 propagation medium; 10 wt% of Bacillus brevis YTLJ-N-SXH2 was inoculated into the propagation medium and cultured at 28°C for 24 h to obtain the Bacillus brevis YTLJ-N-SXH2 propagation medium.

[0077] 2) Assembly and mixing of phage and probiotics: The expanded phage YTLJ-VBN1 and Bacillus brevis YTLJ-N-SXH2 culture fluids were then freeze-dried (pre-freezing temperature -80°C, pre-freezing for 3 hours, freeze-drying for 4 hours) to obtain their respective freeze-dried powders. The freeze-dried powders of phage YTLJ-VBN1 and Bacillus brevis YTLJ-N-SXH2 were then mixed in a mass ratio of 1:1 to obtain a mixed freeze-dried powder.

[0078] 3) Preparation of auxiliary agent: polyglutamic acid, brevicaulis antimicrobial peptide-montmorillonite complex, inulin, rhubarb, and eucommia polysaccharide were mixed at a mass ratio of 0.1:1:0.01:0.01:0.001 to prepare the auxiliary agent; wherein, the brevicaulis antimicrobial peptide-montmorillonite complex was prepared by ball-milling brevicaulis antimicrobial peptide and montmorillonite at a mass ratio of 1:100.

[0079] The brevis antimicrobial peptide is prepared by adding 90% by weight of ammonium sulfate to the brevis YTLJ-N-SXH2 culture fluid obtained by propagation in step 1), adjusting the pH of the system to 10.0 after mixing, and then standing for 24 hours. After standing, the brevis antimicrobial peptide is freeze-dried (pre-freezing temperature -80°C, pre-freezing for 4 hours, and freeze-drying for 4 hours) and stored in a dry place for use.

[0080] 4) Preparation of probiotic preparation: The mixed lyophilized powder obtained in step 2) is mixed with the auxiliary agent obtained in step 3) at a mass ratio of 1:100 to prepare a probiotic preparation; wherein the phage titer of the probiotic preparation is ≥1.0×10 9 / mL, total effective viable count ≥1.0×10 9 pieces / mL.

[0081] 5) The probiotic preparation obtained above was added to the crucian carp culture system at an addition rate of 0.2 wt% (the crucian carp culture density in the culture system before the probiotic preparation was added was 60 kg / m 3 , nitrate nitrogen concentration was 14.95 mg / L, and Aeromonas concentration reached 8×10 5 CFU / mL), and the culture system without adding probiotic preparations was used as a control; after 5 days of culture with the addition of probiotic preparations, the water quality in the system was good, and the nitrate nitrogen concentration dropped to 4.60 mg / L. At the same time, compared with the control system without adding probiotic preparations, the Aeromonas concentration in the culture system with the addition of probiotic preparations dropped to 1×10 5 CFU / mL, the removal rate exceeded 80%, and the mortality rate of crucian carp was reduced by 85%.

Claims

1. A bacteriophage, characterized in that: The phage is phage YTLJ-VBN1, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on January 22, 2024, with the deposit number GDMCC No: 64300-B1, the classification name is Vibrio phage, and the deposit address is Guangzhou, China.

2. A use of the bacteriophage according to claim 1, characterized in that: The bacteriophage YTLJ-VBN1 is used in preparing a Vibrio parahaemolyticus inhibitory preparation in aquaculture.

3. A bacteriophage probiotic preparation, characterized in that: The phage probiotic preparation contains the phage according to claim 1.

4. A bacteriophage probiotic preparation, characterized in that: The phage probiotic preparation contains the phage according to claim 1 and Bacillus brevis YTLJ-N-SXH2; The Brevibacillus sp. YTLJ-N-SXH2 was deposited in the Guangdong Provincial Microbial Culture Collection Center on January 17, 2022, with the deposit number GDMCC No: 62215, the classification name is Brevibacillus sp., and the deposit address is Guangzhou, China.

5. A method for preparing the bacteriophage probiotic preparation according to claim 4, characterized in that: Here are the steps: 1) Phage and bacterial strain propagation: 1-10 wt% of phage YTLJ-VBN1 and Vibrio are sequentially inoculated into an enrichment medium, and the culture is maintained at a constant temperature of 28-35°C for 12-48 hours. The Vibrio is then filtered to remove the Vibrio from the culture medium to obtain a Vibrio phage YTLJ-VBN1 enrichment culture medium; 1-10 wt% of Bacillus brevis YTLJ-N-SXH2 is inoculated into an enrichment medium, and the culture is maintained at a temperature of 28-35°C for 24-72 hours to obtain a Bacillus brevis YTLJ-N-SXH2 enrichment culture medium; 2) Assembly and mixing of phage and probiotics: freeze-drying the culture fluids of the expanded Vibrio phage YTLJ-VBN1 and Bacillus brevis YTLJ-N-SXH2 obtained above to obtain their respective freeze-dried powders, and then mixing the freeze-dried powders of Vibrio phage YTLJ-VBN1 and Bacillus brevis YTLJ-N-SXH2 at a mass ratio of 1 to 10:1 to obtain a mixed freeze-dried powder; 3) Preparation of auxiliary agent: polyglutamic acid, brevicaulis antimicrobial peptide-montmorillonite complex, inulin, rhubarb, and eucommia polysaccharide are mixed at a mass ratio of 0.01-0.1:1:0.001-0.01:0.001-0.01:0.0001-0.001 to prepare the auxiliary agent; wherein the brevicaulis antimicrobial peptide-montmorillonite complex is prepared by ball-milling brevicaulis antimicrobial peptide and montmorillonite at a mass ratio of 1:10-100 and then setting aside; 4) Preparation of probiotic preparation: The mixed lyophilized powder obtained in step 2) is mixed with the auxiliary agent obtained in step 3) at a mass ratio of 1:1-100 to prepare a probiotic preparation; wherein the phage titer of the probiotic preparation is ≥1.0×10 9 / mL, total effective viable count ≥1.0×10 9 pcs / mL; The preparation method of the brevis antimicrobial peptide comprises adding 70-90% by weight of ammonium sulfate to the culture solution of the brevis YTLJ-N-SXH2 obtained by the expansion in step 1), adjusting the pH of the system to 9.0-10.0 after mixing, and then allowing the solution to stand for 6-24 hours. After standing, the brevis antimicrobial peptide is freeze-dried to obtain the brevis antimicrobial peptide, and stored in a dry place for use.

6. Use of the bacteriophage probiotic preparation according to claim 3 or 4, characterized in that: The bacteriophage probiotic preparation is used for non-disease diagnosis and treatment purposes in aquaculture.

7. The use of the bacteriophage probiotic preparation according to claim 6, characterized in that: The amount of the bacteriophage probiotic preparation added is 0.1-1 wt% of the water mass in the aquaculture system.

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