Pseudomonas fluorescens phage PFX2 and use thereof

Abstract

The application discloses a fluorescent pseudomonas bacteriophage PFX2 and application thereof, and the preservation number of the fluorescent pseudomonas bacteriophage PFX2 is CCTCC NO: M 2024546. The application further provides a biological fungicide for preventing and treating fluorescent pseudomonas, wherein the biological fungicide contains the fluorescent pseudomonas bacteriophage PFX2. The bacteriophage PFX2 has good temperature and pH tolerance, higher bacterial infection capacity, faster adsorption rate and stronger lysis capacity, and has a wider applicable range and a good application prospect in actual application. The biological fungicide can effectively control the growth of the fluorescent pseudomonas in a culture medium and a food substrate, and can efficiently inhibit and remove a biofilm generated by the fluorescent pseudomonas ATCC 13525. The biological fungicide can be easily prepared into a spraying liquid or a leaching liquid to remove the biofilm in food, and does not affect the color and texture of the food itself.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of control of spoilage microorganisms in food, in particular to a Pseudomonas fluorescens bacteriophage PFX2 and its application. BACKGROUND

[0002] Spoilage microorganisms are one of the important reasons for causing food spoilage. Spoilage bacteria mainly exist in food substrates and reproduce by utilizing nutrients in food. During the bacterial reproduction and metabolism, various enzymes of spoilage bacteria will decompose the nutrients in food, thereby generating various toxic and foul-smelling metabolites, affecting food quality, and causing pollution and economic losses in the food industry.

[0003] Pseudomonas is an important spoilage bacteria, which causes significant economic losses in the processing and storage of food. Among them, Pseudomonas fluorescens is a representative spoilage bacteria in Pseudomonas. Pseudomonas fluorescens exists in various environments, has the characteristics of aerobic, acidophilic, proteolysis and fat decomposition, is often associated with the deterioration of frozen high-protein food, including raw fish, ready-to-eat fresh vegetables, dairy products and meat products, and is an important spoilage bacteria of animal-derived food (including meat, poultry, milk and fish). Pseudomonas fluorescens is prone to produce biofilm during growth. Biofilm (also known as biofilm) is a group of bacteria adhering to and coated in a self-produced mucous heterogeneous polymer matrix to form a growth mode different from planktonic cells in order to adapt to the living environment, and polysaccharide is one of the main components of biofilm. Because the formation of biofilm increases the tolerance to environmental factors by about 10-1000 times compared with planktonic bacteria, the resistance to antibiotics and food preservatives and sterilizing agents is significantly enhanced, and it is difficult to remove from the food contact surface and processing equipment, which brings serious harm to the storage and processing of food. Therefore, effective control of Pseudomonas fluorescens in food is the best way to prevent food spoilage caused by Pseudomonas fluorescens, and is of great significance to the development of food industry.

[0004] Bacterial biofilms are microorganisms attached to biological or non-biological surfaces and embedded in extracellular matrix consisting of extracellular polysaccharides, proteins and extracellular DNA, which are usually referred to as extracellular polymeric substances (EPS). In most biofilms, bacteria only account for less than 10%, while extracellular polymers account for more than 90%, which forms the scaffold of the three-dimensional structure of the biofilm, connects and fixes the cells in the biofilm, and improves the mechanical stability and adhesion to the surface. Although various physical, chemical and biological strategies have been proposed to control biofilms, such as natural substances, ultrasonic waves, UV-C irradiation, cold oxygen plasma, etc. However, the effectiveness of these methods is limited, and most of the techniques are not actually suitable for the implementation of biofilm control in the food industry. In recent years, bacteriophages are considered as a new biological method to control bacterial biofilms.

[0005] Bacteriophages are viruses that act as natural enemies of bacteria, and bacteriophages specifically target and destroy bacteria by invading their host bacterial cells and disrupting their metabolism. In the past decade, bacteriophages have shown outstanding potential in controlling bacterial contamination in food, and a variety of bacteriophages have been directly applied to the prevention and control of bacteria, for example, the bacteriophage product ListShield developed by Intralytix TM for controlling Listeria in meat and poultry products, which was approved by FDA in 2006 and is GRAS certified, and can be used in ready-to-eat meat and poultry products. The bacteriophage preparation Listex developed by EBIFood Safety TM P100 is approved for use in all food susceptible to Listeria contamination and is certified by the US Department of Agriculture as organic. In addition to the successful development and approval of bacteriophage products, many bacteriophages have been applied to the prevention and control of bacteria in laboratory environments, and bacteriophage therapy has been proven to be effective in reducing the formation of biofilms of Pseudomonas, Salmonella, Escherichia coli and Vibrio parahaemolyticus. As a bactericide, bacteriophages can control bacteria in food raw materials, clean production processing environments and processing equipment, and extend food storage time, and are less likely to cause bacterial resistance than chemical bactericides, and are considered a natural and effective alternative to bactericides. In addition, specific bacteriophages only attack target pathogens, so they are safe to use in food and clinical environments. Although research has demonstrated the potential of using bacteriophages to control bacterial infections, to date, few studies have focused on exploring bacteriophages that can be used to control the formation of biofilms of Pseudomonas fluorescens in food and on the surfaces of processing equipment and their inhibitory efficacy. SUMMARY

[0006] The present application aims at overcoming the deficiencies of the prior art, and provides a Pseudomonas fluorescens phage PFX2 and application thereof.

[0007] To achieve the above-mentioned object, the technical scheme of the present application is as follows:

[0008] The present application provides a Pseudomonas fluorescens phage PFX2, and the preservation number of the Pseudomonas fluorescens phage PFX2 is CCTCC NO: M 2024546.

[0009] The Pseudomonas fluorescens phage PFX2 is isolated and purified from a beef sample with Pseudomonas fluorescens ATCC 13525 as a host bacterium. The phage forms a transparent plaque on a double-layer plate, and the plaque is clear and transparent, and a halo appears around the plaque. Transmission electron microscopy shows that the phage belongs to the family of short-tailed phages, the head is icosahedral, the diameter is 601.23±0.84nm, and the tail length is 185.81±1.38nm. Further research shows that the phage has strict specificity and can only recognize Pseudomonas fluorescens.

[0010] The Pseudomonas fluorescens phage PFX2 has high stability under the conditions of temperature less than 60 DEG C and pH of 3-11; has strong ability to infect host bacteria, and the optimal MOI is 0.001; has the ability to quickly adsorb Pseudomonas fluorescens, and the maximum adsorption rate is 80.18% at 9min; the one-step growth curve reflects that the latent period of the phage is 10min, the lysis period is 90min, and the lysis amount reaches 67.51 PFU / CFU.

[0011] The phage has good stability under various conditions, which indicates that it has potential value for further research.

[0012] The genome of the above-mentioned Pseudomonas fluorescens phage PFX2 is obtained by a second-generation genome sequencing method, and the genome is analyzed by bioinformatics method. The whole genome size of the phage PFX2 is 38,103 bp, and the average G+C content is 61.57%. According to the ICTV virus classification, it belongs to the Duplodnaviria domain, Heunggongvirae kingdom, Uroviricota door, Caudoviricetes class, Autographiviridae family. The Pseudomonas fluorescens phage PFX2, the preservation number is: CCTCC NO: M2024546, the preservation date is March 25, 2024, and the address is: Wuhan University, Luojia Hill Road 16, Wuchang District, Wuhan City, Hubei Province, China.

[0013] The application also provides an application of the above-mentioned Pseudomonas fluorescens phage PFX2 in preventing and treating Pseudomonas fluorescens.

[0014] Further, the Pseudomonas fluorescens is Pseudomonas fluorescens ATCC 13525.

[0015] The application also provides a biological fungicide for preventing and treating Pseudomonas fluorescens, wherein the biological fungicide contains 10 9 PFU / mL of Pseudomonas fluorescens phage PFX2.

[0016] The application also provides an application of the above-mentioned biological fungicide in inhibiting the growth of Pseudomonas fluorescens in meat products.

[0017] The meat product is beef; and the biological fungicide can effectively inhibit the spoilage pollution of Pseudomonas fluorescens in beef at 4 DEG C and 25 DEG C.

[0018] The application also provides an application of the above-mentioned biological fungicide in inhibiting the growth of Pseudomonas fluorescens biofilm and removing mature biofilm formed by Pseudomonas fluorescens.

[0019] The biological fungicide is used for inhibiting the formation of Pseudomonas fluorescens ATCC 13525 biofilm, and the inhibition rate of biofilm formation reaches 81.53% after 18h of treatment of the biological fungicide.

[0020] The biological fungicide is used for removing mature biofilm formed by Pseudomonas fluorescens ATCC 13525, and the removal rate of mature biofilm reaches 83.33% after 6h of treatment of the biological fungicide.

[0021] The application also provides an application of the above-mentioned biological fungicide in removing mature biofilm formed on the surface of food and processing equipment.

[0022] Further, the food is beef, and the material of the processing equipment is selected from the group consisting of glass, stainless steel and polyethylene plastic.

[0023] The above-mentioned biological fungicide is used for removing mature biofilm of Pseudomonas fluorescens ATCC 13525 formed on the surface of food and its processing equipment, and has a significant destructive effect on the mature biofilm when treated for 2 hours, and can reduce about 2log 10 CFU / cm 2 on the surface of food and its processing equipment (the material of which is glass, stainless steel and polyethylene plastic), effectively reducing bacterial contamination and economic loss caused by Pseudomonas fluorescens in the food industry.

[0024] The application also provides a use of the above-mentioned biological fungicide in biological control of Pseudomonas fluorescens in food.

[0025] Further, the food is beef.

[0026] The above-mentioned biological fungicide has no significant effect on the color and texture of beef when treating beef, and can be used for biological control of Pseudomonas fluorescens in food.

[0027] The application also provides a kit for preventing and treating Pseudomonas fluorescens, which comprises a biological fungicide, a buffer, an activation solution 1 and an activation solution 2; wherein the titer of bacteriophage PFX2 in the biological fungicide is 10 9 PFU / mL,

[0028] The buffer is a 1×PBS buffer,

[0029] The activation solution 1 is a sterile LB broth medium,

[0030] The activation solution 2 is Pseudomonas fluorescens ATCC 13525 with a concentration of 10 9 CFU / mL.

[0031] The activation solution 1 of the kit can be stored at 4℃, the biological fungicide and the activation solution 2 can be stored at -20℃ for 2 years, and if not used for more than 1 month, the biological fungicide is activated before use, and the steps are as follows:

[0032] 1) 100 μL of the biological fungicide and 100 μL of the activation solution 2 are mixed with 10 mL of the activation solution 1, and are cultured at 28℃ on a shaking table (120 rpm) for 12 hours;

[0033] 2) After the culture is completed, centrifugation is performed at 4℃ and 8000 r / min for 20 minutes, the supernatant is filtered by a 0.22 μm sterile filter membrane, and the filtered supernatant is the activated biological fungicide.

[0034] The kit can effectively inhibit food spoilage caused by the growth of Pseudomonas fluorescens in beef, and can significantly remove biofilm on the surface of beef and food processing equipment, thereby reducing economic losses caused by spoilage bacteria in the food industry.

[0035] Principles of the application:

[0036] Phages are viruses that can specifically infect bacteria, and their main components include nucleic acids and proteins. Phages specifically bind to target bacterial surface receptors through protein shells to achieve infection of specific bacteria, thus having the characteristics of high specific recognition of host bacteria. In food environments, the use of phages has certain safety guarantees and is considered a natural and effective alternative to bactericides. Compared with traditional bactericides, phages are widely distributed, easy to obtain, low in cost and not prone to bacterial resistance. In the bactericidal process, phages not only degrade EPS through the enzyme activity of dispersin, but also improve the efficacy of bactericides by removing the diffusion boundary, so the application of phages is considered a new biological method for controlling bacterial biofilm.

[0037] Advantages of the application:

[0038] 1. The phage PFX2 of the application has good temperature and pH tolerance, high bacterial infection capacity, fast adsorption rate and strong lysis capacity, and has a wide applicable range and good application prospect in practical application.

[0039] 2. The biological bactericide of the application can effectively control the growth of Pseudomonas fluorescens in culture medium and food matrix, and can also efficiently inhibit and remove the biofilm produced by Pseudomonas fluorescens ATCC 13525. Moreover, the biological bactericide can be easily prepared into a spraying liquid or a leaching liquid to remove the biofilm in food without affecting the color and texture of the food itself.

[0040] 3. The portable kit of the application can be applied in food to quickly and efficiently control food pollution caused by Pseudomonas fluorescens and reduce economic losses caused by spoilage bacteria in the food industry. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 : Picture of phage plaques in the double-layer plate of phage PFX2,

[0042] Figure 2 : Transmission electron microscope photo of phage PFX2,

[0043] Figure 3 : Biological property graph of phage PFX2,

[0044] In the figure, A is the optimal infection multiple graph of phage PFX2.

[0045] B is a graph of the adsorption rate curve of the phage PFX2;

[0046] C is a graph of the one-step growth curve of the phage PFX2;

[0047] D is a graph of the lysis curve of the phage PFX2;

[0048] E is a graph of the temperature stability of the phage PFX2;

[0049] F is a graph of the pH stability of the phage PFX2;

[0050] Figure 4 G is a graph of the growth inhibition of Pseudomonas fluorescens ATCC 13525 by the phage PFX2,

[0051] Figure 5 H is a graph of the inhibition of biofilm formation and the removal of mature biofilm of Pseudomonas fluorescens ATCC 13525 by the phage PFX2,

[0052] A is a graph of the inhibition of biofilm formation of Pseudomonas fluorescens ATCC 13525 by the phage PFX2;

[0053] B is a graph of the removal of mature biofilm of Pseudomonas fluorescens ATCC 13525 by the phage PFX2;

[0054] Figure 6 G is a graph of the growth inhibition of Pseudomonas fluorescens ATCC 13525 by the phage PFX2,

[0055] Figure 7 H is a graph of the growth inhibition of Pseudomonas fluorescens ATCC 13525 by the phage PFX2,

[0056] Figure 8 G is a graph of the growth inhibition of Pseudomonas fluorescens ATCC 13525 by the phage PFX2, DETAILED DESCRIPTION

[0057] The present application will be further described in conjunction with specific examples so that those skilled in the art will understand.

[0058] Example 1 Isolation and purification of phage and morphological analysis

[0059] 1. Isolation and purification of phage

[0060] The beef samples were purchased from the fresh supermarket of Huazhong Agricultural University in Wuhan, Hubei Province, China, and the Pseudomonas fluorescens ATCC 13525 was used as the host bacteria to isolate and purify the Pseudomonas fluorescens bacteriophage. First, the beef samples were cultured with the Pseudomonas fluorescens ATCC 13525 in a 28°C shaking incubator (120 r / min) for 14±2 h. The culture was centrifuged at 8000 r / min for 20 min at 4°C, and the supernatant was filtered with a 0.22 μm sterile filter membrane. The obtained bacterial solution was spotted on a double-layer plate and cultured at 28°C for 4-6 h. The sample with clear spots on the double-layer plate was the bacteriophage stock solution. The purified bacteriophage was obtained by the double-layer plate method. A single bacteriophage plaque on the plate was selected and purified for 8-10 times until the size and transparency of the plaques on the double-layer plate were consistent, and the purified bacteriophage was obtained. Figure 1

[0061] 2. Morphological analysis of the bacteriophage

[0062] The phosphotungstic acid negative staining method was used to observe the morphology of the bacteriophage under a transmission electron microscope. The specific method was as follows: the bacteriophage solution was ultracentrifuged at 35000 r / min for 1 h at 4°C to precipitate the bacteriophage particles. The precipitate was resuspended with 1 mL sterilized PBS. The copper grid was first immersed in the bacteriophage suspension for 10 min, and then the excess liquid was absorbed with filter paper. The copper grid was then immersed in a 2% phosphotungstic acid dye solution (pH 7) for 10 min, and the excess liquid was then absorbed. The copper grid was naturally dried until completely dry. The prepared copper grid was observed under a transmission electron microscope, and the particle size was analyzed by using the software Digital Micrograph Demo. Figure 2

[0063] The results showed that the bacteriophage PFX2 had an icosahedral head with a diameter of 601.23±.84 nm, and a tail length of 185.81±1.38 nm, which belonged to the short-tailed bacteriophage family.

[0064] 3. Genome analysis of the bacteriophage PFX2

[0065] 3.1 Extraction of the bacteriophage PFX2 genome

[0066] 1) 1 mL of high-titer bacteriophage suspension (obtained by ultracentrifugation or solid proliferation method) was taken, 3 μL of DNase I and RNase A were added to the bacteriophage suspension, mixed well, and incubated at 37°C for 1 h to degrade the residual host bacterial DNA and RNA.

[0067] 2) After incubation, 24 μL of 0.5% EDTA (pH=8.0) was added, mixed well, and the bacteriophage suspension was placed in a 80°C water bath for 15 min to inactivate the nucleases. ​​

[0068] 3) Add 1.5 μL 20 μg / mL proteinase K, 30 μL 10% SDS solution into the enzyme-treated phage solution, mix well by slowly inverting, incubate at 56°C for 1 h, and cool naturally to room temperature.

[0069] 4) Add equal volume of equilibrated phenol solution into the obtained mixture, extract nucleic acid, centrifuge at 12000 r / min for 10 min, and carefully pipette the upper aqueous phase into a new centrifuge tube.

[0070] 5) Add equal volume of phenol / chloroform / isoamyl alcohol (volume ratio 25:24:1) into the pipetted upper solution, mix well to extract nucleic acid, centrifuge at 12000 r / min for 10 min, and carefully pipette the upper aqueous phase into a new centrifuge tube.

[0071] 6) Add equal volume of chloroform into the pipetted upper solution, mix well to remove residual phenol, centrifuge at 12000 r / min for 10 min, and carefully pipette the upper aqueous phase into a new centrifuge tube.

[0072] 7) Add equal volume of isoamyl alcohol into the pipetted upper solution, mix well, and precipitate DNA by ice-bath at -20°C for 3 h, centrifuge at 13000 r / min for 20 min at 4°C, slowly pour off the supernatant, and collect the precipitate.

[0073] 8) Add 1 mL of 75% ethanol (pre-cooled at -20°C), stand for 10 min, centrifuge at 12000 r / min for 10 min at 4°C, slowly pour off the ethanol, and place at an ultra-clean bench for 10 min to completely evaporate the ethanol.

[0074] 9) Dissolve the DNA with TE (pH 7.6) to obtain a genomic DNA sample, and store at -20°C. The sample is electrophoresed on a 0.8% agarose gel at 100 V for about 40 min, scanned and saved the image by a gel imaging instrument after electrophoresis, and observe whether a band appears.

[0075] 3.2 Analysis of phage PFX2 genome

[0076] The whole genome of bacteriophage PFX2 was sequenced by using the second-generation sequencing technology based on Illumina HiSeq sequencing platform by whole genome shotgun strategy. The raw data was quality controlled by using software Trimmomatic-0.36d, and de novo assembled by using software MicrobeTrakr plus v.0.9.1 to obtain the assembled whole genome sequence. The whole genome analysis showed that the genome of bacteriophage PFX2 was 38,108 bp, and the average G+C content was 61.57%. According to the ICTV virus classification, it belongs to Duplodnaviria domain, Heunggongvirae kingdom, Uroviricota phylum, Caudoviricetes class, Autographiviridae family.

[0077] Example 2: Analysis of biological characteristics of bacteriophage PFX2

[0078] 1. Determination of host spectrum of bacteriophage PFX2

[0079] The determination of bacteriophage host spectrum used the spotting method:

[0080] 100 μL of bacteria to be tested cultured to the logarithmic phase was added to a warm semi-solid medium, mixed, and then poured onto a pre-prepared LA plate. After solidification, 10 μL of bacteriophage liquid with a titer of 10 9 PFU / mL was added to the surface of the upper plate, and after the double-layer plate was dried, it was inverted and cultured in a 28°C incubator for 4-5 h, and the lysis was observed. The results are shown in Table 1.

[0081] Table 1 Host spectrum of bacteriophage PFX2

[0082]

[0083]

[0084] Note: a+++ indicates clear and transparent plaques, and — indicates no lysis ability.

[0085] b ATCC, American Type Culture Collection; CMCC, China Center for Type Culture Collection; CVCC, National Center for Veterinary Microbial Culture Collection; NCTC, National Collection of Type Cultures.

[0086] 2. Optimal multiplicity of infection (MOI) of bacteriophage PFX2

[0087] Multiplicity of Infection (MOI) is the ratio of the number of phages to the number of host bacteria at the initial infection. Phage suspension and host bacteria solution were mixed in 500 μL each at a certain MOI value (0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10, 100, 1000), and incubated at 28°C for 4 h on a shaker. The mixture was centrifuged at 8000 r / min for 10 min, and the phage titer of the supernatant at different MOI values was determined by double-layer plate method.

[0088] The results are shown in Figure 3 A, the phage titer of phage PFX2 reached the maximum at MOI = 0.001, i.e., the optimal multiplicity of infection of phage PFX2 was 0.001. The results showed that the phage had strong ability to infect bacteria.

[0089] 3. Adsorption rate of phage PFX2

[0090] Fresh phage solution and host bacteria suspension were mixed in 3 mL each at the optimal MOI value in a 10 mL centrifuge tube, and incubated at 28°C on a shaker. Every 3 min, 200 μL was taken and centrifuged at 8000 r / min for 2 min, and the titer of unadsorbed phage in the supernatant was determined by double-layer plate method.

[0091] Adsorption rate = 1 - (titer of unadsorbed phage / titer of phage at 0 min) x 100%.

[0092] The adsorption rate of phage PFX2 on host bacteria is shown in Figure 3 B, and the maximum adsorption rate of phage PFX2 was 80.18% at 9 min.

[0093] 4. One-step growth curve of phage PFX2

[0094] The one-step growth curve of phage reflects the growth law of phage from the recognition of host bacteria to the completion of replication and assembly in host bacteria, and the release of progeny phage. Phage and host bacteria were mixed at the optimal MOI value, and incubated at 28°C for 9 min on a shaker to allow phage to adsorb on bacteria. The mixture was centrifuged at 8000 r / min for 2 min at 4°C, and the supernatant was discarded. The bacteria were resuspended with an equal volume of LB, and the operation was repeated twice to remove unadsorbed phage. The above 1 mL resuspension was added to 9 mL LB liquid medium, and every 10 min, 200 μL was taken and centrifuged at 8000 r / min for 2 min. The titer of phage in the supernatant was determined by double-layer plate method, and the initial bacterial concentration was determined to calculate the lysis amount.

[0095] Lysis amount = titer of phage at the end of lysis / infection initial host bacteria concentration.

[0096] The results are shown inFigure 3 C, the latent period of phage PFX2 is 10 min and the lysis period is 90 min, and the lysis amount reaches 67.51 PFU / CFU. The latent period and lysis amount of the phage are key factors to consider whether the phage can be used for biological control experiments. The shorter the latent period of the phage and the larger the lysis amount, the better the effect of the phage in bacterial control.

[0097] 5. Lysis ability of phage PFX2

[0098] The phage was added to a 96-well plate at MOI = 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100, respectively, and 100 μL of host bacteria liquid was added. A negative control was set up by adding 200 μL of LB medium, and a positive control was set up by adding 100 μL of host bacteria liquid and 100 μL of LB liquid medium. The absorbance value OD 600nm at 600 nm was measured every 1 h, and the total measurement time was 16 h.

[0099] The results are shown in Figure 3 D. Under all MOI conditions, the growth of the host bacteria was obviously inhibited within 8 h, and the OD 600nm maintained at the initial value without growth. After 9 h, the host bacteria began to grow, but the OD 600nm of the phage-treated groups was significantly lower than that of the positive group without phage treatment, indicating that the phage PFX2 has strong lysis and can significantly inhibit the growth of Pseudomonas fluorescens, but cannot completely kill the host bacteria.

[0100] 6. Stability of phage PFX2

[0101] 1 mL of phage diluent with a titer of 10 6 PFU / mL was aliquoted in a 1.5 mL sterile centrifuge tube, and the centrifuge tube was placed in a constant-temperature water bath at 30°C, 40°C, 50°C, 60°C, and 70°C, respectively. The titer of the phage in each tube was measured at 0 min, 30 min, and 60 min. The results are shown in Figure 3 E. Between 30°C and 40°C, the activity of the phage remained at a level comparable to the initial level. At 50°C and above, the activity of the phage gradually decreased. The titer decreased by 2.53 ± 0.02 log 10 PFU / mL after 30 min of incubation at 60°C, and the phage PFX2 was completely inactivated after 60 min of incubation at 60°C. With the increase of temperature and the extension of time exposed to high temperature, the activity of the phage gradually decreased until complete inactivation.

[0102] 1 mL of phage diluent with a titer of 10 8The 100 μL of bacteriophage suspension with PFU / mL was added to 900 μL of PBS buffer with different pH (2-13), and the titer of bacteriophage in each centrifuge tube was determined after 2 h of shaking at 28°C. The results are shown in Fig. 1. Figure 3 As shown in Fig. 1, the bacteriophage was stable in the pH range of 3-11, and no bacteriophage was detected at pH 2 or pH greater than 12, indicating that the bacteriophage was completely inactivated in the strong acid and strong base environment at pH 2 and pH greater than 12.

[0103] Example 3: Preparation of the biological fungicide

[0104] The biological fungicide for controlling Pseudomonas fluorescens contains PFX2 bacteriophage of Pseudomonas fluorescens, wherein the titer of PFX2 bacteriophage of Pseudomonas fluorescens is 10 9 PFU / mL.

[0105] Example 4: Inhibition of the growth of Pseudomonas fluorescens by the biological fungicide

[0106] Pseudomonas fluorescens ATCC 13525 was cultured to the logarithmic growth phase and inoculated into LB broth, nutrient broth medium (10 g / L peptone, 3 g / L beef extract and 5 g / L sodium chloride) and fresh sterile beef (under sterile conditions, washed with distilled water for 2 min, cut into small pieces of 3x3 cm with scissors, and placed in a petri dish, and the beef sample was irradiated with ultraviolet light for 30 min on both sides to ensure sterility) to a final concentration of 10 5 CFU / mL. After mixing, the biological fungicide was added, and an equal volume of PBS buffer was added to the control group. The treated samples were incubated at 4°C and 25°C, respectively, and taken out at 1, 3, 6 and 9 h, and the bacterial amount of Pseudomonas fluorescens in the samples was detected by plate counting method.

[0107] The inhibition effect of the biological fungicide on the growth of Pseudomonas fluorescens is shown in Fig. 2. Figure 4 As shown in Fig. 2, the biological fungicide had a significant inhibitory effect on the growth of Pseudomonas fluorescens in LB broth. At 4°C, the bacterial number gradually decreased with the increase of the treatment time of the biological fungicide, and the bacterial number was reduced by 4.01±0.11 log 10 CFU / mL compared with the control group after 9 h of treatment at 4°C; at 25°C, the bacterial number was reduced by 4.99±0.09 log 10 CFU / mL compared with the control group after 9 h of treatment of the biological fungicide. In NB nutrient broth, the biological fungicide could inhibit the growth of Pseudomonas fluorescens to a certain extent. At 4°C, the biological fungicide had a certain inhibitory effect on the growth of bacteria after 6 h and 9 h of treatment, and the bacterial number was reduced by 1.33±0.13 log 10 CFU / mL and 1.94±0.09 log10 CFU / mL; the number of bacteria was reduced by 2.07 ± 0.06 log after 9 h of treatment with the bio- bactericide at 25°C 10 CFU / mL. In sterile beef, the number of bacteria was reduced by 2.08 ± 0.02 log after 3 h of treatment with the bio-bactericide at 4°C 10 CFU / mL; the number of bacteria was reduced by 1.66 ± 0.06 log after 9 h of treatment with the bio-bactericide at 25°C 10 CFU / mL.

[0108] In summary, the bio-bactericide has a significant inhibitory effect on the growth of P. fluorescens, indicating that the bio-bactericide has great potential for further research on the prevention and control of bacteria in food.

[0109] Example 5: Application of the bio-bactericide to control P. fluorescens biofilm

[0110] 1. Inhibitory effect of the bio-bactericide on the formation of P. fluorescens biofilm

[0111] P. fluorescens ATCC 13525 was cultured to the logarithmic growth phase, and the bacterial solution was adjusted to a concentration of 10 5 CFU / mL, 100 μL of the bio-bactericide was mixed with 100 μL of the bacterial solution in a 96-well plate. In the negative control, LB broth was used instead of the bacteriophage diluent. The 96-well plate was incubated at 28°C for 12, 18, and 24 h, and the effect of the bio-bactericide on P. fluorescens biofilm was detected by viable cell counting and crystal violet staining, respectively.

[0112] Viable cell counting method:

[0113] After incubation, the culture medium was removed, and the wells were rinsed with 200 μL of PBS buffer for 3 times to remove excess culture medium and planktonic cells. The biofilm on the surface of the wells was collected by cotton ball wiping method, and the cotton ball after wiping was placed in a centrifuge tube containing 10 mL of PBS and a certain number of glass beads, and vortexed thoroughly for 2 min to allow the bacteria on the cotton ball to fall off completely. The supernatant after vortexing was diluted by a factor of 10, and the appropriate gradient was selected for plating on a flat plate, which was incubated at 28°C for 14 h for viable cell counting.

[0114] Crystal violet staining method:

[0115] After incubation, remove the culture medium, rinse the wells 3 times with 200 μL PBS buffer; place the 96-well plate in a 50°C oven to dry; add 200 μL of 0.1% crystal violet solution to each well, and stain at room temperature for 20 min; aspirate the staining solution, and gently wash with PBS, then dry; add 200 μL of 95% ethanol to each well until complete dissolution. Use a microplate reader to measure the absorbance at 595 nm (the maximum absorption wavelength of crystal violet). The depth of purple color in the solution (absorbance) is directly proportional to the amount of biofilm produced.

[0116] Results are shown in Figure 5 As shown in Figure A, after 12-24 h of treatment with the bio-sterilizing agent, the mass of P. aeruginosa biofilm decreased significantly. When co-cultured with the bacteriophage for 18 h, the OD 595nm decreased by 2.19 ± 0.03, at which time the bio-sterilizing agent had the best inhibitory effect on the formation of P. aeruginosa biofilm, with an inhibition rate of 81.53%.

[0117] 2. Bio-sterilizing agent removal of mature biofilm formed by P. aeruginosa

[0118] P. aeruginosa (10 5 CFU / mL) was incubated in a 28°C incubator for 18 h to prepare a biofilm. After the biofilm was matured, the bacterial solution in the 96-well plate was aspirated, and the wells were rinsed twice with PBS buffer. The bio-sterilizing agent was added to each well, and an equal volume of LB broth was added to the negative control group. The samples were treated at 28°C for 2, 4, and 6 h, respectively. The removal ability of the bio-sterilizing agent on the P. aeruginosa ATCC 13525 biofilm was detected by viable cell counting and crystal violet staining.

[0119] Results are shown in Figure 5 As shown in Figure B, after 2 h of treatment of the P. aeruginosa mature biofilm with the bio-sterilizing agent, the OD 595nm decreased by 1.78 ± 0.03, and the biofilm mass decreased by 71.59%. After 6 h of treatment, the OD 595nm decreased by 2.20 ± 0.08, and the biofilm mass decreased by 83.33%. This shows that the bio-sterilizing agent has a removal effect on the biofilm formed by P. aeruginosa ATCC 13525, and can effectively eliminate the biofilm formed by P. aeruginosa ATCC 13525.

[0120] Example 6. Application of the bio-sterilizing agent to control of mature biofilm formed by P. aeruginosa in food

[0121] 1. Bio-sterilizing agent removal of mature biofilm on the surface of beef

[0122] Fresh sterile beef was placed in a sterile petri dish, sealed, and refrigerated at 4°C for preservation. Pseudomonas fluorescens ATCC 13525 was cultured to the logarithmic growth phase, and the bacterial solution was adjusted to 10 5 CFU / mL. Under sterile conditions, the beef was soaked in the bacterial solution at 10 5 CFU / mL for 10 min, then removed from the petri dish, dried in a biosafety cabinet for 20 min, and then placed in a sterile petri dish and cultured in a 28°C incubator for 18 h to form a mature biofilm; after incubation, the beef was removed from the petri dish, washed twice with PBS buffer to remove unattached bacteria; then the beef was soaked in a biocide for 10 min, transferred to a sterile petri dish, and dried for 20 min. The control group was treated with PBS buffer instead of the biocide. After 2 h of incubation at 28°C, the beef was soaked in 1 mL of PBS buffer, vortexed thoroughly, diluted, and spread on agar plates for colony counting. Other prepared beef samples were washed twice with PBS buffer, and the samples were fixed with 2.5% glutaraldehyde solution at room temperature in the dark for 4 h. Dehydration was performed with increasing ethanol gradients (15%, 30%, 40%, 50%, 70%, and 100% v / v) for 15 min. After dehydration, the samples were coated with platinum and visualized using FE-SEM (Hitachi / Baltec, S-4700).

[0123] The effect of the biocide on the removal of Pseudomonas fluorescens biofilm formed in beef is shown in Figure 6 . After 2 h of biocide treatment, the mass of the biofilm on the surface of the beef decreased significantly. The live cell counting method was used to evaluate the effect, and the number of live cells on the surface of the beef treated with the biocide for 2 h decreased by 1.61 ± 0.05 log 10 CFU / cm 2 compared to the untreated control. This indicates that the biocide can remove Pseudomonas fluorescens ATCC 13525 biofilm formed in food and effectively eliminate Pseudomonas fluorescens ATCC 13525 biofilm formed in food.

[0124] Scanning electron microscopy (SEM) analysis showed that the beef sample without biocide treatment formed a typical biofilm with a dense structure. In the beef sample treated with the biocide, the structure of the biofilm changed significantly, the number of bacteria in the biofilm decreased significantly, and the distribution was more dispersed. Compared with the control group without phage treatment, the biofilm was thinner and the structure was looser.

[0125] 2. Effect of biocide on the removal of mature biofilm on the surface of food processing equipment

[0126] The sterilization treatment of glass and stainless steel was to immerse the two materials in anhydrous ethanol for 24 h, then ultrasonic cleaning with deionized water for 15 min, and sterilization with high-pressure steam at 121 ℃ for 15 min after washing clean; the sterile treatment of polyethylene plastic was to immerse the polyethylene plastic in anhydrous ethanol for 24 h, then clean with sterile ultrapure water for several times, place in a clean bench, irradiate the material front and back with ultraviolet light for 1 h to ensure sterility, and finally dry in a 40 ℃ oven.

[0127] First, Pseudomonas fluorescens (10 5 CFU / mL) was inoculated into beef samples; the beef was attached to the treated glass, polyethylene plastic and stainless steel, and cultured at 28 ℃ for 18 h to form a biofilm in a sealed culture dish; after incubation, the treated materials were placed in a sterile 6-well plate, and a biocide was added to completely immerse the three materials, and an equal volume of PBS buffer was added to the negative control group; after incubation at 28 ℃ for 2 h, the material surface was washed twice with PBS buffer, and the biofilm on the material surface was collected with a cotton ball, the bacteria on the cotton ball were resuspended in PBS buffer, and the appropriate gradient was selected for viable cell counting on LA plates by ten-fold gradient dilution.

[0128] The removal effect of the biocide on the biofilm formed by Pseudomonas fluorescens ATCC 13525 on the surface of meat processing equipment is shown in Figure 8 . After 2 h of biocide treatment, the mass of the biofilm on the surface of the processing equipment decreased significantly. The effect was evaluated by viable cell counting, and compared with the control group without biocide treatment, the number of viable cells on the surface of glass, PE plastic and stainless steel decreased by 2.21 ± 0.08 log 10 CFU / cm 2 , 1.66 ± 0.05 log 10 CFU / cm 2 and 2.71 ± 0.10 log 10 CFU / cm 2 , respectively.

[0129] Example 7 Influence of the biocide on the quality of beef

[0130] The same size of sterile beef pieces were divided into two groups, each group having three beef samples. The first group of beef samples was placed at room temperature without any treatment, and the second group of beef samples was soaked in the biocide for 10 min and then incubated at room temperature for 2 h. The quality of the two groups of beef was detected by using a colorimeter and a texture analyzer.

[0131] The results showed that after 2 h of treatment with the biocide, the color of the beef surface did not change significantly, and the hardness, adhesiveness, elasticity, cohesiveness, gumminess, chewiness and resilience in the texture of the beef did not change significantly.

[0132] Table 2 Color determination of beef before and after treatment with the bio- bactericide

[0133]

[0134] Note: a L represents black and white brightness, a represents red and green value, and b represents yellow and blue value.

[0135] b The difference between groups was analyzed by variance analysis, and the same letter in different groups (a / b / c) indicated that the difference between two groups was not significant (P < 0.05).

[0136] Table 3 Texture characterization of beef before and after treatment with the bio- bactericide

[0137]

[0138] Note: a The texture analyzer uses a P / 36R probe, and the measurement conditions are as follows: pre-measurement speed: 1 mm / s; measurement speed: 0.5 mm / s; post-measurement speed: 0.5 mm / s; dwell time between two presses: 5 s; compression percentage: 30%.

[0139] b The difference between groups was analyzed by variance analysis, and the same letter in different groups (a / b / c / d / e) indicated that the difference between two groups was not significant (P < 0.05).

[0140] Example 8 Kit for preventing and treating Pseudomonas fluorescens

[0141] The kit comprises a bio-bactericide, a buffer, an activation solution 1, and an activation solution 2; wherein the titer of the bacteriophage PFX2 in the bio- bactericide is 10 9 PFU / mL,

[0142] The buffer is a 1×PBS buffer,

[0143] The activation solution 1 is a sterile LB broth medium,

[0144] The activation solution 2 is Pseudomonas fluorescens ATCC 13525 with a concentration of 10 9 CFU / mL.

[0145] The activation solution 1 of the kit can be stored at 4°C, and the bio- bactericide and the activation solution 2 can be stored at -20°C for 2 years. If not used within 1 month, the bio-bactericide is activated before use, and the steps are as follows:

[0146] 1) 100 μL of the bio-bactericide and 100 μL of the activation solution 2 are mixed with 10 mL of the activation solution 1, and incubated at 28°C on a shaker (120 rpm) for 12 h;

[0147] 2) After the culture is completed, centrifuge at 4℃, 8000r / min, 20min, the supernatant is filtered by 0.22μm sterile filter membrane, and the filtered supernatant is the activated bio-sterilizing agent.

[0148] The bio-sterilizing agent in the kit can effectively inhibit the food spoilage phenomenon caused by the growth of Pseudomonas fluorescens in beef, and can significantly remove the biofilm on the surface of beef and food processing equipment, reducing the economic loss caused by spoilage bacteria in the food industry.

[0149] Other parts not specifically described are prior art. Although the above examples make a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiments without creativity, which belong to the protection scope of the present application.

Claims

1. A fluorescent Pseudomonas phage PFX2, characterized in that: The fluorescent pseudomonad phage ( Pseudomonas fluorescens The accession number for PFX2 (phage) is: CCTCC NO: M 2024546.

2. The application of the fluorescent Pseudomonas phage PFX2 as described in claim 1 in the prevention and control of Fluorescent Pseudomonas, characterized in that: The fluorescent Pseudomonas is fluorescent Pseudomonas ATCC 13525.

3. A biocide for controlling *Pseudomonas fluorescens*, characterized in that: The biocidal agent contains the fluorescent Pseudomonas phage PFX2 as described in claim 1, wherein the titer of the fluorescent Pseudomonas phage PFX2 is 10. 9 PFU / mL.

4. The use of the biocide of claim 3 in inhibiting the growth of Pseudomonas fluorescens in meat products.

5. The use of the biocide of claim 3 in inhibiting the growth of *Pseudomonas fluorescens* biofilm and removing mature biofilms formed by *Pseudomonas fluorescens*.

6. The application of the biocide of claim 3 in removing mature biofilms formed by *Pseudomonas fluorescens* on the surface of food and its processing equipment, characterized in that: The food product is beef, and the processing equipment is made of glass, stainless steel, and polyethylene plastic.

7. The application of the biocide according to claim 3 in the biocontrol of Pseudomonas fluorescens in food.

8. A kit for preventing and treating Pseudomonas fluorescens, characterized in that: The kit comprises the biocide described in claim 3, a buffer solution, activation solution 1, and activation solution 2; wherein the biocide contains bacteriophage PFX2 with a titer of 10. 9 PFU / mL, The buffer solution is 1×PBS buffer. Activation solution 1 is sterile LB broth medium. Activation solution 2 has a concentration of 10. 9 CFU / mL of fluorescent Pseudomonas ATCC 13525.

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