Cronobacter phage, liquid formulation thereof, and use thereof in prevention and control of cronobacter sakazakii

Abstract

Provided are a Cronobacter phage, a liquid formulation thereof, and use thereof in the prevention and control of Cronobacter sakazakii. The provided phage is Cronobacter phage vB_CsaM_2375H2, which is a novel phage belonging to Pseudotevenvirus and has an accession number of CCTCC NO: M 2024570. The phage vB_CsaM_2375H2 has good stability at pH 3-11 and a temperature of 25-70 °C and does not carry any virulence or antibiotic resistance gene, meeting the safety requirements in actual applications. The phage vB_CsaM_2375H2 specifically lyses Cronobacter sakazakii and can remove the biofilm of Cronobacter sakazakii, with a biofilm removal rate of up to 62.55%.

Description

Cronobacter bacteriophage, liquid preparation thereof and application thereof in preventing and controlling Cronobacter sakazakii

[0001] The present application claims priority to the Chinese patent application No. CN202410527669.X filed on April 29, 2024, and entitled "Preparation method of bacteriophage and bacteriophage preparation for precisely preventing and controlling Cronobacter sakazakii and biofilm", the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present application belongs to the field of microbial technology, and particularly relates to a novel bacteriophage for precisely preventing and controlling Cronobacter sakazakii and application thereof. BACKGROUND

[0003] Cronobacter is a kind of foodborne pathogenic bacteria isolated from various foods, which is a facultative anaerobic gram-negative bacteria consisting of 7 species: Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter turicensis, Cronobacter muytjensii, Cronobacter condimenti, Cronobacter universalis and Cronobacter dublinensis. Among them, Cronobacter sakazakii and Cronobacter malonaticus are the main pathogenic bacteria in clinic. The bacteria can cause meningitis, necrotizing enterocolitis and sepsis in newborns and infants, and can cause serious sequelae of the nervous system. Once newborns are infected, the mortality rate is as high as 40% to 80%. Due to the strong virulence of Cronobacter sakazakii to children with immature immunity, it has become a focus of attention in recent years.

[0004] Bacteria can colonize and form biofilms on various biological or non-biological surfaces, such as natural and synthetic material surfaces. Bacterial biofilms are an important factor that cannot be ignored in causing repeated bacterial infections in hospitals, food spoilage, industrial pipeline pollution, plant diseases, etc. Many foodborne pathogens can directly or indirectly enter the food supply chain, causing biological harm to humans and animals, and the formation of biofilms protects the internal bacteria from the effects of disinfectants and antibiotics. Among them, Cronobacter sakazakii has strong resistance to environmental stress, and the bacteria can survive in infant formula milk powder for up to 2.5 years. This strong dry resistance is related to the formation of biofilm, which helps Cronobacter sakazakii to persist in the infant formula milk powder production environment. Therefore, the effective prevention and control of biofilm is still a difficult problem to be solved in the food industry.

[0005] Phages are a kind of viruses that infect bacteria but not human cells, harmless to humans, animals and plants. Compared with antibiotics, phage therapy has high specificity and effectiveness for target bacteria. Phages have the advantages of rich sources, strong specificity, high safety, and are an excellent alternative in the prevention and control of biological pollution in food. Phages have good bactericidal effect, which can greatly reduce the formation of target bacterial biofilm. In order to effectively control Cronobacter sakazakii pollution and biofilm in food, it is particularly important to screen new Cronobacter phages that can specifically prevent and control Cronobacter sakazakii and its biofilm.

[0006] SUMMARY

[0007] In order to effectively inhibit Cronobacter sakazakii, without lysis effect on non-Cronobacter sakazakii, without drug resistance genes and virulence genes, and safe and reliable, the application provides a phage for precisely preventing and controlling Cronobacter sakazakii and biofilm, and a preparation method of the phage preparation.

[0008] The application provides a phage for precisely preventing and controlling Cronobacter sakazakii and biofilm, the preservation number of which is CCTCC NO: M 2024570, the classification name is Cronobacter phage, the preservation unit is China Center for Type Culture Collection, and the preservation date is March 27, 2024.

[0009] The phage provided by the application can specifically lyse Cronobacter sakazakii or effectively prevent and control the biofilm of Cronobacter sakazakii; and the titer is stable in the range of 25-70 DEG C and pH 3-11.

[0010] The preparation operation steps of the phage preparation based on the phage are as follows:

[0011] (1) The logarithmic phase Cronobacter sakazakii, Cronobacter phage and LB broth medium are uniformly mixed at a mass ratio of 1:1:50, and are cultured at 37 DEG C for 4h to obtain a culture solution;

[0012] The logarithmic phase Cronobacter sakazakii and Cronobacter phage are both obtained by culturing in LB broth medium;

[0013] The titer of the culture solution is 10 10 PFU / mL or more;

[0014] (2) The culture solution is centrifuged, and the supernatant is taken and filtered to obtain a Cronobacter phage proliferation solution;

[0015] (3) 0.5M sodium chloride (NaCl) with a final concentration and 10% (w / v) polyethylene glycol 8000 with a final concentration are added to 100mL of the Cronobacter phage proliferation solution to obtain a mixture, the mixture is uniformly mixed, and centrifugation is performed to obtain a precipitate;

[0016] (4) 1 mL SM buffer was added into the precipitate of 30 mL mixture and mixed evenly to obtain the Cronobacter bacteriophage concentrated solution, i.e. the bacteriophage liquid preparation;

[0017] The working titer of Cronobacter bacteriophage in the bacteriophage liquid preparation is ≥ 1 x 10 4 PFU / mL, 10 5 ~ 10 9 PFU / mL; and the time for lysing Cronobacter sakazakii or removing the biofilm of Cronobacter sakazakii is 2 ~ 12 h.

[0018] Further, in step (2), the centrifugal condition is: speed 4000 g, time 15 min.

[0019] Further, in step (3), the centrifugal condition is: speed 12000 g, time 15 min.

[0020] Further, the working titer of Cronobacter bacteriophage in the bacteriophage liquid preparation is 10 6 ~ 10 7 PFU / mL; and the time for lysing Cronobacter sakazakii or removing the biofilm of Cronobacter sakazakii is 6 ~ 8 h.

[0021] The beneficial technical effects of the present application are embodied in the following aspects:

[0022] 1. The Cronobacter bacteriophage vB_CsaM_2375H2 is screened by using the pathogenic Cronobacter sakazakii cro2375w as the host bacteria, and the bacteriophage specifically lysing Cronobacter sakazakii only and has no lytic effect on non-Cronobacter sakazakii. The Cronobacter bacteriophage vB_CsaM_2375H2 provided in the present application has an average nucleotide identity of 93.8% and 93.7% with Cronobacter bacteriophage vB_EkoM5VN (LC589952) and Enterobacteriophage EC-W1 (MN508621) respectively (Figure 3), and is a novel bacteriophage.

[0023] 2. The Cronobacter bacteriophage vB_CsaM_2375H2 in the present application is a virulent bacteriophage isolated from nature, and can stably survive under different conditions of temperature 25 ~ 70℃ and pH value 3 ~ 11. The bacteriophage genome does not contain virulence genes and drug resistance genes, and the bacteriophage is not genetically modified in the present application, and can be safely applied to the prevention and control of Cronobacter in food.

[0024] 3. The Cronobacter phage vB_CsaM_2375H2 of the present application can remove the biofilm produced by Cronobacter sakazakii, and the biofilm removal rate can reach 62.55%. The results show that the phage can effectively kill Cronobacter sakazakii on the surface of food, reduce the formation of harmful metabolites, and provide a new strategy for preventing and controlling Cronobacter sakazakii in food processing and environment. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below.

[0026] Fig. 1 is a plaque map of the phage of the present application;

[0027] Fig. 2 is an electron microscope map of the phage of the present application;

[0028] Fig. 3 is a genomic analysis heat map of the phage of the present application;

[0029] Fig. 4 is a main capsid protein phylogenetic tree analysis map of the phage of the present application;

[0030] Fig. 5 is a visual analysis map of the whole genome of the phage vB_CsaM_2375H2, EC-W1 and vB_EkoM5VN of the present application;

[0031] Fig. 6 is a schematic diagram of the optimal infection multiple of the phage of the present application;

[0032] Fig. 7 is a one-step growth curve schematic diagram of the phage of the present application;

[0033] Fig. 8 is a temperature tolerance schematic diagram of the phage of the present application;

[0034] Fig. 9 is a pH tolerance schematic diagram of the phage of the present application;

[0035] Fig. 10 is a schematic diagram of the ability of the phage of the present application to remove mature biofilm of C. sakazakii cro2375w;

[0036] Fig. 11 is a schematic diagram of the removal rate of the phage of the present application to remove mature biofilm of C. sakazakii cro2375w.

[0037] Biological preservation information

[0038] The Cronobacter phage vB_CsaM_2375H2 is classified as Cronobacter phage, and was preserved in the China Center for Type Culture Collection (CCTCC) on March 27, 2024, located in Wuhan, Wuhan University, China, with a postal code of 430072, and the preservation number is CCTCC NO: M 2024570. DETAILED DESCRIPTION

[0039] The present application provides a bacteriophage vB_CsaM_2375H2 which can lyse Cronobacter sakazakii, and the preservation number of the bacteriophage vB_CsaM_2375H2 is CCTCC NO: M 2024570.

[0040] The Cronobacter sakazakii bacteriophage vB_CsaM_2375H2 of the present application is isolated from sewage samples collected from the Hudaqing Water Purification Plant in Hefei, Anhui Province. Transmission electron microscopy imaging results show that the head of the bacteriophage is in the shape of a regular icosahedron, and a retractable tail is also observed. The tail structure of the bacteriophage includes a sheath, a base plate, and a tail fiber. The head of the bacteriophage vB_CsaM_2375H2 is about 120 nm long and about 86 nm wide, and the retractable tail is about 111 nm long.

[0041] The full-length genome of the Cronobacter sakazakii bacteriophage vB_CsaM_2375H2 of the present application is 179363 bp, and the GC content is 44.78%. The bacteriophage does not contain virulence genes and drug resistance genes. The average nucleotide identity of the bacteriophage with Cronobacter sakazakii bacteriophage vB_EkoM5VN (LC589952) and Enterobacteriaceae bacteriophage EC-W1 (MN508621) is 93.8% and 93.7%, respectively. The bacteriophage is a new type of bacteriophage belonging to Pseudotevenvirus.

[0042] The Cronobacter sakazakii bacteriophage vB_CsaM_2375H2 of the present application can specifically lyse Cronobacter sakazakii, and has no lytic effect on non-Cronobacter sakazakii. The multiplicity of infection of the bacteriophage of the present application is preferably 0.001-1, and the optimal multiplicity of infection is 0.01. The bacteriophage has good temperature tolerance and pH stability. The titer is stable at a pH of 3-11 and a temperature of 25-70°C.

[0043] The present application also provides a preparation method of a bacteriophage liquid preparation based on the bacteriophage for precision prevention and control of Cronobacter sakazakii and biofilm. The bacteriophage is cultured in a culture medium containing host bacteria, and the host bacteria are removed to obtain a bacteriophage lysate. The removal of the host bacteria is preferably by filtration or centrifugation. The host bacteria are Cronobacter sakazakii, and the culture medium is preferably LB broth.

[0044] The preparation operation steps of the bacteriophage preparation based on the bacteriophage vB_CsaM_2375H2 of the present application are as follows:

[0045] (1) The logarithmic phase Cronobacter sakazakii, Cronobacter sakazakii bacteriophage, and LB broth are mixed in a mass ratio of 1:1:50, and cultured at 37°C for 4 h to obtain a culture solution;

[0046] The Cronobacter and the Cronobacter phage in the logarithmic phase are obtained by culturing in LB broth medium;

[0047] The titer of the culture solution is 10 10 PFU / mL or more;

[0048] (2) The culture solution is centrifuged at a speed of 4000g for 15 min. The supernatant is taken and filtered to obtain a Cronobacter phage propagation solution;

[0049] (3) 0.5M sodium chloride (NaCl) and 10% (w / v) polyethylene glycol 8000 are added to 100 mL of the Cronobacter phage propagation solution to obtain a mixture, which is mixed uniformly and centrifuged at a speed of 12000g for 15 min to obtain a precipitate;

[0050] (4) 1 mL of SM buffer is added to the precipitate of the 30 mL mixture to obtain a Cronobacter phage concentrated solution, i.e., a phage liquid preparation;

[0051] The working titer of the Cronobacter phage in the phage liquid preparation is ≥1×10 4 PFU / mL, 10 5 ~ 10 9 PFU / mL.

[0052] The application provides an application of the preparation in the above technical solution in preventing and removing Cronobacter sakazakii biofilm.

[0053] When used for removing the biofilm produced by mature Cronobacter sakazakii, the application for removing the biofilm produced by mature Cronobacter sakazakii preferably comprises the following step: adding the phage vB_CsaM_2375H2 to the biofilm that has been formed after the Cronobacter sakazakii is cultured for 48 h to remove the biofilm. The working titer of the phage vB_CsaM_2375H2 used for removing is preferably 10 5 ~ 10 9 PFU / mL, further preferably 10 6 ~ 10 8 PFU / mL, and more preferably 10 6 ~ 10 7PFU / mL. The time for clearance according to the present application is preferably 2-12 h, further preferably 4-10 h, and more preferably 6-8 h. The results of the examples according to the present application show that the bacteriophage vB_CsaM_2375H2 can clear the biofilm of Cronobacter sakazakii cro2375w that has already formed, and the clearance effect on the biofilm that has already formed is best at 8 h, with a clearance rate of 62.55%.

[0054] The bacteriophage, lysate or preparation according to the present application, when contacting Cronobacter sakazakii for prevention and control, preferably has a pH value of 3-11 in the contacting medium. Within the above pH range, the bacteriophage can maintain high activity against Cronobacter sakazakii. Since the tolerance temperature of the bacteriophage is 25-70°C, the temperature of the bacteriophage, lysate or preparation according to the present application should be less than 70°C during storage or use.

[0055] The technical solutions provided by the present application will be described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present application.

[0056] Example 1 Isolation and purification of bacteriophage

[0057] (1) Treatment of water sample

[0058] The water sample according to the present application was collected in Hedaqing Water Plant, Hefei City, Anhui Province in 2022.

[0059] The water sample was centrifuged at 10000g for 10 min to remove most of the solid particles and part of the bacteria, and then the mixture was filtered with a 0.45μm water microporous filter under the action of a circulating water vacuum pump to remove most of the bacteria. To the filtered water sample, MgSO4with a final concentration of 50mM was added, and the mixture was dissolved thoroughly and left to stand for 20 min. Then the mixture was vacuum filtered with a 0.22μm water microporous filter, the collected filter was cut into pieces, and then placed in an appropriate amount of eluent. The bacteriophage particles on the filter were eluted by an ultrasonic cleaner for 5 min. The bacteria were removed by filtration with a 0.45μm water filter head, and the liquid was the bacteriophage mixture, which was stored at 4°C for later use.

[0060] (2) Isolation and purification of bacteriophage

[0061] 500μL of the bacteriophage mixture, 500μL of double LB broth containing CaCl2with a final concentration of 2mM, and 50μL of cro2375w logarithmic phase bacterial solution were mixed uniformly and cultured at 37°C for 8h. The culture was centrifuged at 4000g for 15 min, and the supernatant was filtered with a 0.45μm filter head into a sterile centrifuge tube, which was the bacteriophage lysate. The bacteriophage lysate was stored at 4°C for later use.

[0062] First, LB solid medium was poured into a sterile culture dish. 100 μL of cro2375w bacterial solution and 100 μL of phage lysate were added to 5 mL of 0.4% LB-Ca semi-solid medium, mixed well, spread on the dried lower medium, and then incubated at 37°C for 4 h with inversion. The presence or absence of clear phage plaques was observed. A single transparent plaque was picked and mixed well in SM buffer, and then incubated at 4°C overnight. The SM buffer containing the phage after overnight incubation was centrifuged at 4000g for 15 min. The supernatant was filtered with a 0.45 μm water filter to remove bacteria, and then the resulting solution was the initial purified phage solution. The phage was purified 3-4 times until the plaques were single and transparent (Fig. 1).

[0063] (3) Propagation and concentration of phage

[0064] The 100 mL LB-Ca broth medium was added with 2 mL of cro2375w bacterial solution and 2 mL of phage purified solution, and then incubated at 37°C for 4 h. The supernatant was filtered with a 0.45 μm filter to remove bacteria, and then the resulting solution was the phage propagation solution. The final concentration of 0.5 M NaCl and 10% polyethylene glycol 8000 was added to the resulting phage propagation solution, mixed well, and then incubated at 4°C overnight to precipitate the phage particles. The supernatant was discarded by centrifugation at 12000g for 15 min, and then the precipitate was dissolved in an appropriate amount of SM buffer to obtain the phage concentrated solution, which was stored at 4°C for later use.

[0065] Example 2: Observation of phage morphology

[0066] The purified phage suspension was dropped on a copper mesh, and then the excess liquid was absorbed with filter paper after 5 min. A drop of phosphotungstic acid was added, and then the excess liquid was absorbed after 3 min of staining. The copper mesh was dried in air, and then observed with a field emission transmission electron microscope. The phage vB_CsaM_2375H2 belongs to the Pseudotevenvirus family, and has a head length of about 120 nm, a head width of about 86 nm, and a contractile tail of about 111 nm (Fig. 2).

[0067] Example 3: Sequencing and analysis of phage genome

[0068] The phage genomic DNA was extracted by the phenol-chloroform-isoamyl alcohol method, and then sequenced by Illumina.

[0069] The sequencing results show that the bacteriophage vB_CsaM_2375H2 is linear and dsDNA bacteriophage. The complete genome sequence of the bacteriophage vB_CsaM_2375H2 is 179363 bp in length, with a GC content of 44.78%, and contains 279 coding sequences. By comparison with the virulence database and antibiotic resistance gene database, the bacteriophage vB_CsaM_2375H2 does not contain virulence genes and antibiotic genes, indicating that it can be safely used for the prevention and control of Cronobacter. The bacteriophage genome analysis heat map shows that the average nucleotide identity of the bacteriophage with Cronobacter bacteriophage vB_EkoM5VN (LC589952) and Enterobacter bacteriophage EC-W1 (MN508621) is 93.8% and 93.7% respectively (Figure 3), so the bacteriophage vB_CsaM_2375H2 is a new type of bacteriophage belonging to Pseudotevenvirus. The phylogenetic tree analysis diagram of the major capsid protein also supports this conclusion (Figure 4).

[0070] The bacteriophage vB_CsaM_2375H2 is predicted to contain 279 ORFs, without tRNA. Among them, 152 ORFs are annotated as functional proteins, and the rest are annotated as hypothetical proteins. The functional proteins of the bacteriophage are divided into five modules: DNA metabolism module, lysis module, packaging module, structural module, and other functional module (Figure 5).

[0071] Example 4 Determination of the optimal multiplicity of infection of the bacteriophage

[0072] The bacteria were cultured to the early logarithmic phase, so that the bacterial concentration was 10 8 CFU / mL, the bacteriophage solution and the host bacteria solution were added in the ratio of multiplicity of infection of 10, 1, 0.1, 0.01, 0.001, 0.0001, and cultured at 37°C, 200 rpm for 4h, centrifuged at 12000g for 15min, filtered with a 0.45μm filter head, and the titer was measured by double-layer agar plate method. The multiplicity of infection that produces the highest titer is the optimal multiplicity of infection. The results show that when MOI = 0.01, the titer is 1.33×10 10 PFU / mL, which is the highest titer among the six multiplicities of infection measured, that is, the optimal MOI of the bacteriophage vB_CsaM_2375H2 is 0.01 (Figure 6).

[0073] Example 5 Determination of the one-step growth curve of the bacteriophage

[0074] The bacteria were cultured to the early logarithmic phase, so that the bacterial concentration was 10 8CFU / mL, host bacteria cro2375w and phage vB_CsaM_2375H2 were added at the ratio of 0.01 of the optimal multiplicity of infection, incubated at 37°C for 5 min, centrifuged at 12000g for 30 s, and the supernatant was discarded. The precipitate was washed twice with LB medium. 30 mL of 37°C preheated LB medium was added, and the culture was shaken at 37°C. The sample was taken every 5 min in the first 20 min, and then every 10 min. The sample was centrifuged at 12000g for 30 s, filtered with a 0.45 μm filter, and the phage titer at each time point was determined. The one-step growth curve was plotted with the infection time as the abscissa and the phage titer as the ordinate. The results showed that the latent period of phage vB_CsaM_2375H2 was 0-20 min, 20-100 min was the lysis period of phage vB_CsaM_2375H2, and 100 min entered the plateau phase (Figure 7).

[0075] Example 6 Determination of phage temperature stability

[0076] The titer of phage vB_CsaM_2375H2 was 10 10 PFU / mL. The phage vB_CsaM_2375H2 was incubated at 25°C, 30°C, 37°C, 40°C, 50°C, 60°C, 65°C, 70°C, 75°C, and 80°C for 1 h, and the sample was cooled to room temperature. The phage titer was determined by double-layer agar plate method. The results showed that the titer of phage vB_CsaM_2375H2 remained above 10 9 PFU / mL after 1 h at 25-60°C, and the titer decreased by 2.36 and 2.75 log PFU / mL after 1 h at 65°C and 70°C, respectively. The phage vB_CsaM_2375H2 lost activity at temperatures above 70°C (Figure 8).

[0077] Example 7 Determination of phage pH stability

[0078] 1010PFU / mL of phage vB_CsaM_2375H2 was added to LB broth with pH values of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, respectively, and the final concentration of phage was 10 9 PFU / mL. After 1 h of water bath at 37°C, the phage titer under different conditions was determined by double-layer agar plate method. The results showed that the phage vB_CsaM_2375H2 could still maintain a titer of 10 8 PFU / mL in the environment of pH 4-11, but the phage vB_CsaM_2375H2 could not survive at pH 2 and 12 (Figure 9).

[0079] Example 8 Phage removal of biofilm

[0080] First, the host bacteria C. sakazakii cro2375w was cultured to the early logarithmic phase, and then 2% of the bacterial solution was inoculated into LB broth medium, mixed, and then added to a 96-well plate and incubated at 37°C for 48 h. The excess liquid was aspirated, and the 96-well plate was washed twice with sterile normal saline to remove free bacteria. The treated 96-well plate was used as the 96-well plate with the biofilm formed. Then, 200 μL of bacteriophage vB_CsaM_2375H2 with a titer of 10 10 , 10 9 , 10 8 , 10 7 , 10 6 , 10 5 , 10 4 PFU / mL was added, and the control group was added with 200 μL of SM buffer. The 96-well plate was incubated at 37°C for 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h, respectively. After the solution in the 96-well plate was discarded, 200 μL of sterile normal saline was added and washed three times, and the biofilm was dried and fixed. After staining with 0.1% crystal violet at room temperature for 30 min, the crystal violet was discarded, and the plate was washed twice with distilled water and dried. After adding 200 μL of 33% acetic acid solution to dissolve the crystal violet at room temperature for 30 min, the OD 590 of the plate was measured with a microplate reader. The amount of biofilm removal rate was calculated according to the following formula, and the results are shown in FIGS. 10 and 11.

[0081] Biofilm amount removal rate = (control group biofilm amount - experimental group biofilm amount) / control group biofilm amount x 100%.

[0082] As can be seen from FIGS. 10 and 11, for the removal of the biofilm that has been formed, the titer of the bacteriophage vB_CsaM_2375H2 was 10 6 and 10 7PFU / mL of the phage vB_CsaM_2375H2 had the best effect on removing the formed biofilm, and the effect was better with the increase of time, and the biofilm removal rate was the highest at 8h, which could reach 61.11% and 62.55% respectively. Until 10h, the removal rate of the phage vB_CsaM_2375H2 on the formed biofilm began to decrease. Although the removal rate of the phage vB_CsaM_2375H2 on the biofilm began to decrease, it did not mean that the removal effect of the phage vB_CsaM_2375H2 on the biofilm began to decrease, but the mature biofilm began to fall off from the surface of the 96-well plate, thereby leading to the decrease of the removal rate. Comparing the amount of the biofilm at 12h and the highest removal rate at 8h, the amount of the biofilm at 12h was still lower than that at 8h, which indicated that the removal effect of the phage vB_CsaM_2375H2 on the biofilm did not decrease. For the formed biofilm, the removal effect was the most obvious when the phage was added for 6-8h, and the P value was less than 0.001. From the addition of 10 10 、10 9 、10 8 、10 7 、10 6 、10 5 、10 4 PFU / mL of the phage vB_CsaM_2375H2 had the best effect on removing the formed biofilm, and the effect was better with the increase of time, and the biofilm removal rate was the highest at 8h, which could reach 61.11% and 62.55% respectively. Until 10h, the removal rate of the phage vB_CsaM_2375H2 on the formed biofilm began to decrease. Although the removal rate of the phage vB_CsaM_2375H2 on the biofilm began to decrease, it did not mean that the removal effect of the phage vB_CsaM_2375H2 on the biofilm began to decrease, but the mature biofilm began to fall off from the surface of the 96-well plate, thereby leading to the decrease of the removal rate. Comparing the amount of the biofilm at 12h and the highest removal rate at 8h, the amount of the biofilm at 12h was still lower than that at 8h, which indicated that the removal effect of the phage vB_CsaM_2375H2 on the biofilm did not decrease. For the formed biofilm, the removal effect was the most obvious when the phage was added for 6-8h, and the P value was less than 0.001. From the addition of 10 6 and 10 7 PFU / mL of the phage vB_CsaM_2375H2 had the best effect on removing the formed biofilm, which indicated that the higher the concentration of the phage was, the better the removal effect on the biofilm was not. In summary, the phage vB_CsaM_2375H2 had a good removal effect on the formed biofilm, which indicated that the phage vB_CsaM_2375H2 had the prospect of being applied to food to remove Cronobacter biofilm.

[0083] Example 9 Determination of the host spectrum of the phage

[0084] 1.5% LB solid medium was spread on a dry sterile petri dish, and then dried. 100 μL of bacterial liquid cultured to the logarithmic phase was added to 5 mL of 0.4% LB agar, mixed, spread on the dried plate, and then naturally dried to solidify the soft agar. 2 μL of phage culture was added to the soft agar by spotting, naturally dried, and then cultured at 37°C for 4h, and the results were observed.

[0085] The results are shown in Table 1. The phage vB_CsaM_2375H2 only specifically lysed Cronobacter sakazakii, did not lyse other bacteria in the Cronobacter genus, and did not lyse non-Cronobacter bacteria.

[0086] Table 1 Host range of phage vB_CsaM_2375H2

[0087] Note: The results were divided into clear spotting area (+) and no plaque in spotting area (-).

[0088] The above only is the preferred embodiment of the present application, it should be pointed out that, for the ordinary skilled in the art, without departing from the principles of the present application, can make a number of improvements and refinements, these improvements and refinements should also be regarded as the protection scope of the present application.

Claims

1. A Cronobacter phage vB_CsaM_2375H2, with the preservation number of CCTCC NO: M 2024570.

2. Use of the Cronobacter phage vB_CsaM_2375H2 in the preparation of a preparation for preventing and controlling Cronobacter sakazakii.

3. A liquid formulation of bacteriophage, characterized in that, The working titer of Cronobacter phage vB_CsaM_2375H2 in the phage liquid preparation is ≥ 1 x 10 4 PFU / mL; The preservation number of the Cronobacter phage vB_CsaM_2375H2 is CCTCC NO: M 2024570.

4. The phage liquid formulation according to claim 3, characterized in that, The working titer of Cronobacter phage vB_CsaM_2375H2 in the phage liquid preparation is 1 x 10 5 ~ 1 x 10 9 PFU / mL.

5. The phage liquid formulation according to claim 4, characterized in that, The working titer of Cronobacter phage vB_CsaM_2375H2 in the phage liquid preparation is 1 x 10 6 ~ 1 x 10 7 PFU / mL.

6. A preparation method of the phage liquid preparation according to any one of claims 3-5, comprising the following steps: (1) mixing logarithmic phase Cronobacter, Cronobacter phage vB_CsaM_2375H2 and LB broth medium in a mass ratio of 1:1:50, and culturing at 37°C for 4h to obtain a culture solution; The logarithmic phase Cronobacter and the Cronobacter phage vB_CsaM_2375H2 are both obtained by culturing in LB broth medium; The titer of the culture solution is 10 10 PFU / mL or more; (2) centrifuging the culture solution to obtain a supernatant, and filtering the supernatant to obtain a Cronobacter phage proliferation solution; (3) adding sodium chloride with a final concentration of 0.5M and polyethylene glycol 8000 with a final concentration of 10w / v% to 100mL of the Cronobacter phage proliferation solution to obtain a mixture, mixing the mixture uniformly, and centrifuging to obtain a precipitate; (4) adding 1mL of SM buffer to 30mL of the precipitate to obtain a Cronobacter phage concentrated solution, i.e. the phage liquid preparation.

7. The preparation method according to claim 6, characterized in that, The Cronobacter is Cronobacter sakazakii.

8. The preparation method according to claim 6, characterized in that, In step (2), the centrifugation is performed at a speed of 4000g for 15min.

9. The preparation method according to claim 6, characterized in that, In step (3), the centrifugation is performed at a speed of 12000g for 15min.

10. Use of the Cronobacter phage vB_CsaM_2375H2 according to claim 1 or the phage liquid preparation according to any one of claims 3-5 or the phage liquid preparation obtained by the preparation method according to any one of claims 6-9 in preventing and controlling Cronobacter sakazakii.

11. Use according to claim 10, characterized in that, The prevention and control of Cronobacter sakazakii includes prevention and control of Cronobacter sakazakii in food and / or environment.

12. The use according to claim 10, characterized in that, The Cronobacter phage or the phage liquid preparation prevents and controls Cronobacter sakazakii by lysing Cronobacter sakazakii and / or removing the biofilm of Cronobacter sakazakii.

13. Use according to any one of claims 10 to 12, characterized in that, The Cronobacter sakazakii is Cronobacter sakazakii cro2375w.

14. A method of clearing a biofilm of Klebsiella oxytoca, characterized in that, comprising the following steps: mixing Cronobacter sakazakii forming a biofilm and the phage liquid preparation according to any one of claims 3-5, and culturing at 25-70°C for 2-12h.

15. The method of claim 14, wherein, The culturing time is 4-10h.

16. The method of claim 15, wherein, The culturing time is 6-8h.

17. The method of claim 14, wherein, The culturing temperature is 37°C.

18. The method of claim 14, wherein, The pH value of the culturing is 3-11.

19. The method of claim 18, wherein, The pH value of the culturing is 4-11.

20. The method according to any one of claims 14 to 19, characterized in that, The Cronobacter sakazakii is Cronobacter sakazakii cro2375w.

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