Phage microgel as well as preparation method and application thereof

Abstract

The invention relates to bacteriophage microgel as well as a preparation method and application thereof. Trehalose dihydrate and whey protein are combined with bacteriophage to form a mixed solution, the mixed solution is gelatinized on a microporous template to form the bacteriophage microgel, and the bacteriophage microgel is stripped from the microporous template to prepare a sterile aqueous suspension; the bacteriophage microgel suspension can be used for killing bacteria. According to the hydration characteristic of the bacteriophage microgel, the bacteriophage is protected from being influenced by drying, so that the biological activity of the bacteriophage is reserved, and the bacteriophage microgel has a relatively good bacterium killing function.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a bacteriophage microgel and a preparation method and application thereof. Background Art

[0002] Phages are natural bacterial predators that infect bacteria in a highly targeted manner, meaning they are able to specifically infect and lyse certain types of bacteria without adversely affecting other non-target microorganisms. This property makes phages particularly important in food, agriculture, and human treatment. Unlike most antimicrobial agents, phages do not affect the taste, texture, and nutritional quality of food. Phages can play an important role in the entire supply chain from "farm to table" and effectively inhibit pathogenic bacteria to ensure the quality and safety of agricultural products. In essence, phages are protein nanoparticles that encapsulate genomes and can propagate wild-type virus particles into a suspension of identical and monodisperse nanoparticles. In addition, the chemical composition of the phage surface can be customized with atomic precision through chemical coupling, making phage virus particles a powerful component of multifunctional antimicrobial materials. Existing phage gels have a short activity retention time, and extending the activity and storage time of phages is one of the main challenges of current research. Summary of the invention

[0003] In order to solve the above technical problems, the present invention provides a bacteriophage microgel and a preparation method and application thereof.

[0004] The technical scheme adopted by the present invention is: a method for preparing bacteriophage microgel, mixing bacteriophage with trehalose dihydrate and whey protein, loading them onto a microporous membrane template for gelation, and separating the formed gel from the microporous membrane template to obtain the bacteriophage microgel.

[0005] Preferably, the specific steps are as follows:

[0006] Step 1: In a closed humid environment, drop the polystyrene solution onto a glass slide. The white film formed after solidification is the microporous honeycomb membrane template.

[0007] Adding trehalose dihydrate to a whey protein aqueous solution, wherein the ratio of whey protein to trehalose dihydrate is 2:1; adding bacteriophage to the mixture to form a bacteriophage mixed solution;

[0008] Step 2: Apply the phage mixture droplets on the microporous honeycomb membrane template and let it stand at room temperature for gelation;

[0009] Step 3: After removing the microporous honeycomb membrane template, phage microgel is obtained.

[0010] Preferably, in step 1, after the Salmonella phages are enriched, they are centrifuged to obtain a phage suspension, which is then filtered using a 0.22 μm filter membrane to obtain a pure phage solution, which is then titered and confirmed to be free of contamination before use.

[0011] Preferably, in step 2, the phage mixture is dropped onto the surface of the honeycomb membrane and then placed in a vacuum environment, so that the mixture solution fills the micropores of the honeycomb membrane;

[0012] Preferably, a petri dish filled with water is placed in the plasma treatment machine to create a humid environment, the honeycomb membrane with the phage mixture on the surface is placed in the plasma treatment machine, and the vacuum pump is turned on for 1 minute.

[0013] Preferably, in step three, the top layer of the gelled phage microgel honeycomb membrane is peeled off, and the bottom layer is immersed in sterile water and ultrasonicated for 10 minutes, so that the phage microgel falls off from the template and disperses in the sterile water to obtain a phage microgel suspension.

[0014] Preferably, the bacteriophage is Salmonella ATCC14028 phage.

[0015] The phage microgel prepared by the phage microgel preparation method.

[0016] Preferably, it has autofluorescence.

[0017] A bacteriophage microgel spray is provided, wherein a suspension comprising bacteriophage microgel is loaded into a spray bottle.

[0018] Includes the application of phage microgel suspension or phage microgel spray in biological control.

[0019] The advantages and positive effects of the present invention are: the method does not require heat treatment and organic solvents, and retains the biological functions of viruses, proteins and nucleic acids, and has better biological friendliness;

[0020] Phage microgels exhibit complex and highly ordered nanostructures with larger surface area, thus more contact points between phage and contaminating bacteria, and enhanced fluidity in suspension, allowing for administration via spray or injection, properties that make them a more versatile option for biocontrol in environmental, food, and medical applications;

[0021] The preparation method does not require any large-scale equipment, is simple in preparation, and significantly reduces labor costs and the cost of establishing a production line. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Morphology of honeycomb membranes prepared from polystyrene;

[0023] Figure 2Morphology of honeycomb membrane loaded with phage microgels;

[0024] Figure 3 Bright field and fluorescence images of phage microgel honeycomb membranes;

[0025] Figure 4 Phage microgel spray;

[0026] Figure 5 Results of antibacterial activity of phage microgel patches;

[0027] Figure 6 Antimicrobial results of phage microgel spray;

[0028] Figure 7 Activity of phage microgel spray after storage at different temperatures;

[0029] Figure 8 Activity of phage microgel suspension / spray after storage for different lengths of time; (a) Changes in activity of phage microgel spray after storage at different temperatures for 2 h, 24 h, and 48 h; (b) Changes in activity of free phage suspension after storage at different temperatures for 2 h, 24 h, and 48 h. DETAILED DESCRIPTION

[0030] The embodiments of the present invention are described below with reference to the accompanying drawings.

[0031] The present invention relates to a bacteriophage microgel and a preparation method and application thereof. The bacteriophage microgel is formed by combining trehalose dihydrate and whey protein with bacteriophage. The gel form has a larger surface area. When it is used to kill bacteria, it can increase the contact site between the bacteriophage and the contaminating bacteria, thereby improving the sterilization effect. The bacteriophage microgel is prepared in a spray form, the fluidity of the microgel in the suspension is enhanced, and the bacteriophage can be protected from the influence of drying, thereby retaining the biological activity of the bacteriophage.

[0032] In certain embodiments of the present invention, a polystyrene honeycomb membrane is provided, and the honeycomb membrane is prepared by a breathing diagram method using polystyrene as a raw material. The method is simple, easy and fast, and does not require any large-scale equipment. First, a chloroform solution of polystyrene is prepared, and it is fully stirred and dissolved at room temperature; in a closed and humid environment, the chloroform solution of polystyrene is dripped on a glass slide, and the polystyrene solution is spread in the form of a circle; the transparent polystyrene solution becomes turbid due to condensation of water vapor in a short time, and after a period of time, it can solidify into a white film on the glass slide. 100 μL of polystyrene solution dripped on a glass slide can form a honeycomb membrane with a diameter of about 1 cm, which is flexible and can be stored in a dust-proof container at room temperature for at least 6 months. Observation of its microstructure shows that the honeycomb membrane is regular and uniform, and is a template with a single layer, close stacking and uniform micropores.

[0033] The purified phage is loaded onto a honeycomb membrane, and phage microgel is obtained after separation. First, the phage is amplified and purified to form a phage solution; trehalose dihydrate and whey protein are mixed, added to the phage solution and mixed to form a phage mixture. The phage microgel can show significant autofluorescence, which is mainly due to the n-π* electron transition of C=N in the Schiff base produced by the cross-linking reaction between the phage and trehalose dihydrate. The hydration property of the phage microgel can protect and preserve phages that are sensitive to drying. In certain embodiments of the present invention, the phage microgel can use other different cross-linking agents to reduce the impact on the activity of the phage.

[0034] The phage mixture mixed with a cross-linking agent is loaded onto a honeycomb membrane for the preparation of phage microgels, and its size is controlled to be 35.77±1.84μm. Specifically, a trace amount of phage mixture solution can be dropped on the surface of the honeycomb membrane so that the solution covers most of the film. Furthermore, a petri dish filled with water can be placed in the plasma treatment machine to create a humid environment, and the honeycomb membrane containing the phage mixture on the surface is placed in the plasma treatment machine, and the vacuum pump is turned on for 1 minute to create a low-pressure environment to help the mixture solution fill into the micropores of the honeycomb membrane. Wait for the phage mixture to gel on the honeycomb membrane at room temperature. After gelation, a flexible composite membrane is obtained in which the microgel is encapsulated in the template pores. It is separated from the honeycomb membrane and dispersed in sterile water to obtain a phage microgel suspension, which can be sealed and stored. The preparation efficiency of phage microgel is 2.04x10 5 Phage particles, a single microgel can contain more phages. When used, it can be placed in a spray bottle, and a spray is formed under the action of the spray bottle, which improves its convenience of use.

[0035] Compared with other microgel preparation methods, the prepared phage microgels have many advantages. Heat treatment and organic solvents are usually involved in microgel preparation and separation, which will irreversibly damage and destroy the biological functions of the building blocks; this method does not require heat treatment and organic solvents, and retains the biological functions of viruses, proteins and nucleic acids, making phage microgels more biofriendly. Simple workflow: Existing template methods, emulsification methods and microfluidics require large equipment and specialized infrastructure, and this preparation method does not require any large equipment. And this method is suitable for phages, but it is also applicable to other biological products such as protein or DNA microgels. Phage microgels exhibit complex and highly ordered nanostructures with larger specific surface areas, so phages have more contact points with contaminating bacteria, and enhanced fluidity in suspensions allows administration by spray or injection. These properties make them a more versatile choice for biological control in environmental, food and medical applications.

[0036] The scheme of the present invention is described below in conjunction with the accompanying drawings, wherein the experimental methods without specific operating steps are all carried out in accordance with the corresponding product instructions, and the instruments, reagents, and consumables used in the examples can all be purchased from commercial companies unless otherwise specified.

[0037] Example 1: Amplification and purification of bacteriophage

[0038] Prepare phage solution with Salmonella ATCC14028 phage as a representative. Culture of host bacteria: Spread the preserved host bacteria on LB solid culture medium, place the culture dish in a 37℃ constant temperature incubator for overnight culture. Transfer 5mL LB liquid culture medium into a sterile tube, pick a single colony with an inoculation loop, immerse it in the culture solution and gently shake the inoculation loop to disperse the bacteria to be inoculated in the culture solution. Cover the test tube and culture it in a 37℃ shaking incubator overnight.

[0039] Detect the titer of the original phage and dilute the original phage tenfold (10 -1 -10 -11 ), take 100μL of the cultured Salmonella suspension, add it to the 55℃ upper culture medium, mix well and pour it onto the prepared LB solid culture medium. After the upper semi-solid culture medium solidifies, divide the plate into sections, take 5μL of phage liquid and drop it onto the upper agar by the drop method, and culture it at 37℃ after the phage liquid is air-dried.

[0040] For the microgel constructed by phage, the initial phage suspension must be concentrated enough to prevent the concentration of the phage microgel spray from being too low. The formula for calculating the phage titer is: phage titer (pfu / ml) = plaque number / sample volume (ml) × dilution multiple; select 30-300 plaques for calculation, and the calculation results are as follows;

[0041] Original phage titer = 23 × 10 7 ×200=4.6×10 10 pfu / mL.

[0042] The specific steps for enriching phages are as follows:

[0043] a Take 1 mL of the cultured Salmonella suspension and add it to LB liquid medium. Incubate in a shaking incubator at 37°C for about 2 hours. OD≈0.3, 1x10 9 CFU / mL was used as the host bacterial solution.

[0044] b. Dilute the original phage 10 times to a concentration of 4.6 x 10 9 pfu / mL.

[0045] c. Take 217 μL of the phage in b and put it into LB solution. Incubate with shaking at 37°C for 2 h to enrich the phage.

[0046] d. Mix the phage solution in c with chloroform at a volume ratio of 200:1 and let stand at room temperature for 15 minutes.

[0047] e. Take the supernatant of the sample in d and centrifuge it at 5000r / min and 4℃ for 20min, then take the supernatant.

[0048] f. Centrifuge the supernatant in e at 12000 r / min at 4°C for 2 h to obtain the phage precipitate, and soak the precipitate in 2.5 mL of SM buffer solution at 4°C for 24 h.

[0049] g. After resuspending the phage in f, filter it with a 0.22 μm filter membrane. The filtrate is the phage solution. The phage titer is determined by the double-layer plate method. The results are as follows;

[0050] The phage titer after enrichment was 121x10 9 x10=1.21x10 12 pfu / mL.

[0051] Example 2 Preparation of bacteriophage microgel spray

[0052] 2.1 Preparation of honeycomb membrane

[0053] Add 1.0g of polystyrene to a glass bottle containing 12.75mL of chloroform and stir magnetically at room temperature for 30 minutes to dissolve the polystyrene. Place a glass slide in a square petri dish and add an appropriate amount of sterile water around it to maintain the humidity of the environment. Use a pipette to drop 100μL of polystyrene solution on the glass slide, and the polystyrene will spread out in the form of a circle. Quickly cover the lid of the square petri dish. The transparent polystyrene solution becomes turbid due to condensation of water vapor within 5 minutes, and solidifies into a white film on the glass slide in about 20 minutes. The finished product is as follows Figure 1 As shown, the test found that the honeycomb membrane is regular and uniform, with an overall diameter of about 1 cm. It is a template with a single layer, close stacking and uniform micropores.

[0054] 2.2 Preparation of phage-loaded microgels

[0055] Whey protein was hydrated in sterile water, and the prepared whey protein solution was kept at 4°C overnight to unfold the protein structure. Trehalose dihydrate was directly added to the whey protein solution to reach a total solid content of 10% (w / v). The ratio of whey protein to trehalose dihydrate was 2:1. Bacteriophage was added to the above mixture to reach a volume percentage concentration of 10% (v / v).

[0056] Drop 10 μL of the phage mixture solution on the surface of the honeycomb membrane so that the solution covers most of the film. Place a petri dish filled with water in the plasma treatment machine to create a humid environment, place the honeycomb membrane with the phage mixture on the surface into the plasma treatment machine, turn on the vacuum pump for 1 minute to create a low-pressure environment to help the mixture solution fill the micropores of the honeycomb membrane. Take out the sample and place it at room temperature to wait for gelation. The finished honeycomb membrane loaded with phage microgel is as follows: Figure 2 The bright field and fluorescence images of the phage microgel honeycomb membrane were taken using an inverted microscope. Figure 3 As shown in the figure, the honeycomb membrane presents an ordered microporous structure, and the phage microgels cross-linked with trehalose dihydrate and whey protein show obvious autofluorescence in the blue, green, and red channels, which is due to the electronic transition of C=N in the cross-linking reaction.

[0057] After gelation, a flexible composite film is obtained in which the microgels are encapsulated in the template holes. Use scissors to cut off the circular edges of the film to cut the film into squares. Then, a transparent tape is attached to the film and the tape is torn off in a continuous motion. In this way, the honeycomb membrane is divided into two layers, the top layer is attached to the tape, and the microgels on the bottom layer are exposed. The bottom film is immersed in sterile water and ultrasonically treated for 10 minutes, and the microgels fall off the template and disperse in the water. The film is removed, and the resulting suspension contains the microgels loaded with bacteriophages. The suspension can be directly added to a spray bottle, and the finished product is as follows Figure 4 As shown, it can be used for further antibacterial characterization and application of phage microgel spray.

[0058] Example 3: Preparation efficiency of phage microgels

[0059] Before microcapsule separation, we measured the size of the honeycomb film. The microgels separated from the template were collected in 1 mL of sterile water. To count the number of microgels in 1 mL of suspension, we drop-cast 5 μL of the sample on a glass slide and took an image covering the entire droplet using an inverted microscope. Subsequently, we manually counted the number of microgels in the droplet and calculated the microgel preparation efficiency according to formula (1):

[0060] η= N microgel X200 / S template (1)

[0061] Where η is the microgel preparation efficiency, N microgel is the number of microgels in the droplet, S template is the size of the honeycomb membrane. The preparation efficiency of phage microgel is calculated to be 2.04x10 5 Phage particles.

[0062] Example 4: Antibacterial experiment of phage microgel patch

[0063] The gelled phage microgels prepared in Example 2 were gelled, and the composite film was washed with sterile water for 20 minutes to remove free phages. 100 μL of Salmonella suspension was mixed with 3 mL of molten LB top agar in a sterile tube at 55°C. Vortex at medium speed for 5-10 seconds to ensure uniformity, and then the mixture was gently poured onto the prepared solid culture medium. Wait for 20 minutes until the top agar solidifies. Gently place each washed microgel patch on the solid culture medium containing Salmonella and culture it in a constant temperature incubator at 37°C overnight. The results are shown in the figure. Figure 5 As shown, bacteriolytic zones appeared around the plaques on the bacterial layer, indicating that the microgels could kill the bacteria.

[0064] Example 5: Antibacterial experiment of phage microgel spray

[0065] Mix 100 μL of the Salmonella suspension with 3 mL of molten LB top agar at 55°C in a sterile tube. Vortex at medium speed for 5-10 seconds to ensure homogeneity, and then gently pour the mixture onto the prepared solid medium. Wait for 20 minutes until the top agar solidifies. Take 50 μL of the suspension prepared in Example 2 and spray it onto the solid medium containing Salmonella; culture it in a 37°C constant temperature incubator overnight. The results are as follows: Figure 6 As shown, visible spots appeared on the bacterial layer, indicating that the microgel spray can kill bacteria.

[0066] Example 6: Preservation experiment of phage microgel spray

[0067] The free phage suspension and phage microgel spray were stored at 4°C, 25°C, 37°C and 50°C for 2h, 4h, 6h and up to 48h. 200μL of the Salmonella suspension was pipetted into a single well of a sterile 96-well plate. 10μL of each group of free phage suspension and phage microgel spray was added to each well containing bacterial suspension. The plate was placed on an oscillator for incubation, and the optical density at 600nm was measured using a microplate reader.

[0068] The stability of the phage microgel spray at different temperatures was compared. The phage microgel spray was stored at 4°C, 25°C, 37°C and 50°C for 48 hours, and the OD600 value was tested every two hours. The test results are as follows: Figure 7 As shown, the phage microgel spray can have better antibacterial activity when stored at 25°C.

[0069] Compare the effects of different storage time on the stability of phage microgel suspension or spray. Store the phage microgel suspension or spray at 4℃, 25℃, 37℃ and 50℃ respectively, and test the OD600 at 2h, 24h and 48h. The results are as follows Figure 8 As shown, the activity of the phage microgel spray after storage at different temperatures for 2h, 24h and 48h was more stable than that of the free phage suspension, and the surface phage microgel spray had a strong preservation property.

[0070] The embodiments of the present invention are described in detail above, but the contents described are only preferred embodiments of the present invention and cannot be considered to limit the scope of implementation of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A method for preparing bacteriophage microgel, characterized in that: The bacteriophage is mixed with trehalose dihydrate and whey protein, loaded onto a microporous membrane template for gelation, and the formed gel is separated from the microporous membrane template to obtain the bacteriophage microgel.

2. The method for preparing bacteriophage microgel according to claim 1, characterized in that: The specific steps are as follows: Step 1: In a closed humid environment, drop the polystyrene solution onto a glass slide. The white film formed after solidification is the microporous honeycomb membrane template. Adding trehalose dihydrate to a whey protein aqueous solution, wherein the ratio of whey protein to trehalose dihydrate is 2:1; adding bacteriophage to the mixture to form a bacteriophage mixed solution; Step 2: Apply the phage mixture droplets on the microporous honeycomb membrane template and let it stand at room temperature for gelation; Step 3: After removing the microporous honeycomb membrane template, phage microgel is obtained.

3. The method for preparing bacteriophage microgel according to claim 2, characterized in that: In step 1, after the Salmonella phage is enriched, the phage suspension is obtained by centrifugation, and then filtered through a 0.22 μm filter membrane to obtain a pure phage solution, which is then titered and confirmed to be free of contamination before use.

4. The method for preparing bacteriophage microgel according to claim 2, characterized in that: In step 2, the phage mixture is dropped onto the surface of the honeycomb membrane and then placed in a vacuum environment to allow the mixture solution to fill the micropores of the honeycomb membrane; preferably, a culture dish filled with water is placed in the plasma processor to create a humid environment, the honeycomb membrane with the phage mixture on the surface is placed in the plasma processor, and the vacuum pump is turned on for 1 minute.

5. The method for preparing bacteriophage microgel according to claim 2, characterized in that: In step three, the top layer of the gelled phage microgel honeycomb membrane is peeled off, and the bottom layer is immersed in sterile water and ultrasonicated for 10 minutes, so that the phage microgel falls off from the template and disperses in the sterile water to obtain a phage microgel suspension.

6. The method for preparing bacteriophage microgel according to any one of claims 1 to 5, characterized in that: The bacteriophage is Salmonella ATCC14028 phage.

7. The phage microgel prepared by the method for preparing phage microgel according to any one of claims 1 to 6.

8. The bacteriophage microgel according to claim 7, characterized in that: It has autofluorescence.

9. A bacteriophage microgel spray, characterized in that: The suspension comprising the bacteriophage microgel of claim 7 is loaded into a spray bottle.

10. Use of a suspension comprising the bacteriophage microgel according to claim 7 or 8 or a spray comprising the bacteriophage microgel according to claim 9 in biological control.

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