Coating composition containing bacteriophage and antibacterial film produced using the same

Abstract

The present invention relates to coating compositions containing bacteriophages and antimicrobial films produced therewith. Since the coating composition of the present invention contains a bacteriophage capable of killing Salmonella, it is possible to produce a coating having antibacterial activity by using the coating composition, and the bacteriophage can stably survive even after the coating is formed, so that excellent antibacterial activity can be continuously maintained. When the present invention is applied to a coating or film for food packaging, it is possible to effectively prevent food contamination by Salmonella and improve the safety and shelf life of food.

Description

Technical Field

[0001] The present invention relates to a coating composition containing bacteriophage and an antibacterial film produced using the same. More specifically, the present invention relates to a coating composition containing bacteriophage having a killing ability against Salmonella, capable of producing a coating excellent in the stability and antibacterial activity of the bacteriophage, and an antibacterial film produced using the composition.

Background Art

[0002] Food is highly likely to be contaminated by pathogens during the processes of production, distribution, and storage. When contaminated with bacteria, not only does the quality deteriorate, but it may also cause food poisoning upon ingestion. According to the statistical data of the Ministry of Food and Drug Safety, during the period from 2017 to 2020, the number of food poisoning patients due to Salmonella infection was reported to account for 33.5% of the total food poisoning patients. Therefore, it is important to prevent food contamination by pathogenic bacteria such as Salmonella.

[0003] As a general technique for preventing food contamination by pathogenic bacteria and ensuring the freshness and safety of food, there is a method of killing pathogenic bacteria using antibiotics. However, in the case of antibiotics, it is difficult to show a long-term effect due to the problem of the emergence of resistant bacteria, so research on antibacterial substances to replace antibiotics has been necessary.

[0004] As an alternative to antibiotics, techniques using natural antibacterial materials such as natural extracts and essential oils have been developed. For example, Korean Registered Patent Publication No. 10-1072883 describes an antibacterial coating and packaging material using mustard essential oil. However, when using natural materials, it is difficult to ensure stable antibacterial activity, which is disadvantageous for preserving the sensory properties of food, and there is a possibility of removing beneficial bacteria and disrupting the balance of the microbiome.

[0005] Techniques using bacteriophages as substances that can replace existing antibacterial substances such as antibiotics and natural materials have attracted attention. A bacteriophage is a virus that uses bacteria as its host and is an antibacterial substance that binds to the host bacteria and induces death. In particular, bacteriophages have the property of killing specific categories of bacteria and not affecting other bacteria. As an example, Korean Patent Publication No. 10-2018-0100533 describes a bacteriophage that has the ability to specifically kill Pseudomonas aeruginosa. Due to such bacteria-specific properties, using bacteriophages can kill only the target pathogenic bacteria, which has the advantage that the problem of killing beneficial bacteria does not occur.

[0006] Since 2006, bacteriophages have been recognized by the US Food and Drug Administration (FDA) as generally recognized as safe (GRAS) biological materials and are applied to food additives to prevent food contamination by pathogenic bacteria. However, when applying bacteriophages to a coating film, the survival rate of bacteriophages in the coating decreases depending on the coating formation process and the substances used in the coating, so there is a limit in that it cannot exhibit excellent antibacterial activity as a coating form. As a result, the use of bacteriophages is mainly limited to solution or powder form, so there is a need to develop a technology that can ensure the stability of bacteriophages even after coating formation and maintain excellent antibacterial activity against Salmonella bacteria.

Summary of the Invention

Problems to be Solved by the Invention

[0007] An object of the present invention is to provide a coating composition capable of producing an antibacterial film excellent in the survival rate and stability of bacteriophages.

[0008] Another object of the present invention is to provide an antibacterial film produced using the coating composition.

[0009] Another object of the present invention is to provide a bacteriophage having a killing ability specific to Salmonella bacteria.

Means for Solving the Problems

[0010] To achieve the above object, the present invention provides a coating composition containing a bacteriophage having a bactericidal ability against Salmonella sp. bacteria, a polymer compound, and a plasticizer.

[0011] In the present invention, the Salmonella bacteria may include Salmonella enterica.

[0012] In the present invention, the Salmonella bacteria may include at least one Salmonella enterica serotype selected from the group consisting of S. Enteritidis, S. Typhimurium, S. Paratyphi, S. Salamae, S. Diarizonae, and S. Dublin.

[0013] In the present invention, the bacteriophage may belong to the family Siphoviridae.

[0014] In the present invention, the bacteriophage may be the bacteriophage with the deposit number KCTC14929BP having a specific killing ability against Salmonella sp. bacteria.

[0015] In the present invention, the polymer compound can include at least one selected from the group consisting of polyvinyl alcohol (PVA), polylactic acid (PLA), polycaprolactone (PCL), polybutylene succinate (PBS), polyethylene terephthalate (PET), polybutylene terephthalate (PBT), polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC), polyamide (PA), and polyurethane (PU).

[0016] In the present invention, the polymer compound can include a biodegradable polymer.

[0017] In the present invention, the plasticizer can include at least one selected from the group consisting of sorbitol, glycerol, trehalose, fructose, sucrose, mannitol, propylene glycol, and polyethylene glycol.

[0018] In the present invention, the plasticizer can be included in an amount of 10 to 30% by weight based on the weight of the polymer compound.

[0019] In the present invention, the coating composition can further include a solvent.

[0020] In the present invention, based on the total volume of the coating composition, the bacteriophage is 1×108 ~1×10 12 It can contain PFU / mL.

[0021] In the present invention, based on the total volume of the coating composition, the polymer compound can be contained in an amount of 5 to 20 g / 100 mL.

[0022] In the present invention, based on the total volume of the coating composition, the plasticizer can be contained in an amount of 1 to 5 g / 100 mL.

[0023] The present invention also provides an antibacterial film produced using the coating composition.

[0024] In the present invention, the antibacterial film can be produced by coating a substrate with the coating composition and then drying it at a temperature of 20 to 30 °C for 10 to 20 hours.

[0025] In the present invention, the antibacterial film can be produced by coating a coating object with the coating composition and then drying it at a temperature of 20 to 30 °C for 10 to 180 minutes.

[0026] In the present invention, the antibacterial film can be a coating for food packaging.

[0027] The present invention also provides a bacteriophage with the accession number KCTC14929BP that has a specific killing ability against Salmonella sp.

Effects of the Invention

[0028] The coating composition of the present invention can exhibit antibacterial activity including bacteriophage having a killing ability against Salmonella bacteria, and the bacteriophage can stably survive even after coating formation, and excellent antibacterial activity can be continuously maintained. Thereby, when the present invention is applied to a food packaging coating or film, contamination of food by Salmonella bacteria can be effectively prevented, and the safety and shelf life of food can be improved.

Brief Description of Drawings

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[0030] Hereinafter, specific embodiments of the present invention will be described in more detail. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

[0031] The present invention relates to a bacteriophage, a coating composition containing the same, and an antibacterial film produced using the same.

[0032] The coating composition of the present invention can exhibit antibacterial activity including bacteriophage, and the bacteriophage can stably survive and exhibit persistent antibacterial activity even after forming a coating or film using the same. Further, in the present invention, by using a bacteriophage that is excellent in the ability to kill Salmonella sp., which is a food pathogenic bacterium, and has high stability against heat and pH, an antibacterial film that can be usefully applied to food coatings and packaging materials can be provided.

[0033] A bacteriophage is a virus that uses bacteria as a host and can be abbreviated as "phage". A bacteriophage kills a host bacterium through a lytic cycle and / or a lysogenic cycle. For example, according to the lytic cycle, after infecting a bacterium, a bacteriophage can grow inside the bacterial cell, and after growth, it can be released while destroying the cell wall of the host bacterium to kill the bacterium. Since one type of bacteriophage has the ability to kill only specific categories of host bacteria, the bacteriophage can be selected according to the type of bacterium to be killed, or a new bacteriophage can be excavated and used.

[0034] The bacteriophage used in the present invention can have the ability to kill Salmonella sp., which is a typical food pathogenic bacterium. Thereby, when the bacteriophage is applied to a food packaging material, it exhibits antibacterial activity to kill Salmonella bacteria and suppress their growth, and can prevent food from being contaminated by Salmonella bacteria.

[0035] Specifically, the bacteriophage used in the present invention can have the ability to specifically kill Salmonella enterica, and among them, in particular, it can exhibit the ability to kill at least one serotype selected from the group consisting of S. Enteritidis, S. Typhimurium, S. Paratyphi, S. Salamae, S. Diarizonae, and S. Dublin.

[0036] In one embodiment of the present invention, the bacteriophage can be bacteriophage PBSE191 (hereinafter referred to as "phage PBSE191"). The phage PBSE191 has been deposited with the Korean Collection for Type Culture under the accession number KCTC14929BP (date of deposit: March 31, 2022).

[0037] The phage PBSE191 belongs to the family Siphoviridae. In the examples of the present invention, it was confirmed that phage PBSE191 specifically exhibits antibacterial activity against Salmonella sp. bacteria, particularly Salmonella enterica, is excellent in thermal stability and pH stability, and can be applied under various temperature and pH conditions. Thus, if phage PBSE191 is applied to food packaging materials, it can exhibit excellent killing ability against Salmonella bacteria, which are food pathogens, and improve the safety and shelf life of food.

[0038] Therefore, the present invention can provide a coating composition containing bacteriophage, more specifically, an antibacterial coating composition for food packaging containing bacteriophage. When the coating composition of the present invention is used, a film with high survival rate and stability of bacteriophage and excellent antibacterial activity can be produced even after coating formation.

[0039] The coating composition of the present invention can contain bacteriophage, a polymer compound, and a plasticizer.

[0040] Since the bacteriophage in the coating composition exhibits a killing ability against bacteria as described above, a film having antibacterial activity can be produced using the same.

[0041] In the present invention, the polymer compound serves as a matrix of the coating, and can be polyvinyl alcohol (PVA), polylactic acid (PLA), polycaprolactone (PCL), polybutylene succinate (PBS), polyethylene terephthalate (PET), polybutylene terephthalate (PBT), polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC), polyamide (PA), polyurethane (PU), etc. Among them, biodegradable polymers such as polyvinyl alcohol, polylactic acid, polycaprolactone, and polybutylene succinate can be used. In particular, in the case of polyvinyl alcohol, it is harmless to the human body and biodegradable, and is excellent in film-forming ability and oxygen barrier property, so it can be preferably used for the production of environmentally friendly food packaging materials.

[0042] In the present invention, as the polyvinyl alcohol, those having a weight average molecular weight (Mw) of 5,000 to 50,000, specifically 10,000 to 30,000, for example 13,000 to 23,000 can be used, and those having a saponification degree of 80 to 95%, preferably 82 to 92%, for example 87 to 89% can be used.

[0043] In the present invention, the plasticizer means an additive that is blended with a polymer compound to adjust the physical properties of the film. Generally, when bacteriophage is applied to a polymer coating, there is a problem that the bacteriophage is inactivated depending on the type of polymer and the coating process, resulting in a decrease in the antibacterial ability of the coating. In such a situation, the inventors of the present invention have found that when bacteriophage is applied to a coating film, the plasticizer can not only simply adjust the physical properties of the film, but also has an important influence on the survival rate of bacteriophage, and thus completed the present invention. According to the present invention, by using a plasticizer together with bacteriophage and a polymer compound and adjusting its type and content, a coating film having extremely excellent antibacterial ability can be provided.

[0044] The plasticizer used in the present invention can include sorbitol, glycerol, trehalose, fructose, sucrose, mannitol, propylene glycol, polyethylene glycol, etc., and preferably can include sorbitol. When sorbitol is used, the survival rate of bacteriophage is higher after film formation compared to other plasticizers, it can exhibit excellent antibacterial activity, long-term stability can be ensured, and antibacterial activity can be maintained continuously.

[0045] In the present invention, the plasticizer can be contained in an amount of 10 to 30% by weight, preferably 15 to 25% by weight, more preferably 18 to 22% by weight based on the weight of the polymer compound. In this content range, bacteriophages can stably survive even after coating formation and exhibit excellent antibacterial activity. If the content of the plasticizer becomes lower or higher, the amount of bacteriophages that die during the coating formation process or after coating formation increases, and the antibacterial activity of the coating may decrease. In addition, if the content of the plasticizer is too low, the mechanical properties and oxygen barrier properties of the coating may decrease. If the content of the plasticizer is too high, there is a risk of a decrease in the strength and discoloration of the coating, and the solubility and moisture permeability may become too high, making it unsuitable for use as a food packaging material.

[0046] In the case of general polymer coatings, the type and content of the plasticizer are determined based on the physical properties of the target coating. However, in the present invention, when introducing bacteriophages into the coating, it has been clarified that the type and content of the plasticizer contribute to the survival rate and stability of bacteriophages, and it has an excellent technical significance in that an optimal composition that can maximize the activity and stability of bacteriophages has been discovered.

[0047] According to a preferred embodiment of the present invention, polyvinyl alcohol can be used as the polymer compound in the coating composition containing bacteriophages, and sorbitol can be used as the plasticizer. In this case, optimal activity can be exhibited in terms of the survival rate, long-term stability, and antibacterial activity of bacteriophages after coating formation.

[0048] From the viewpoints of the survival rate and stability of bacteriophages, the content of the polymer compound can be 5 to 20 g / 100 mL, preferably 8 to 15 g / 100 mL based on the volume of the entire coating composition of the present invention, and the content of the plasticizer can be 1 to 5 g / 100 mL, preferably 1.5 to 2.5 g / 100 mL. At this time, the bacteriophage is based on plaque forming unit (PFU) standard 1×10 8~1×10 12 PFU / mL, for example 1×10 9 ~1×10 10 PFU / mL, and specifically may be contained at 2×10 9 ~8×10 9 PFU / mL.

[0049] The coating composition of the present invention can further contain additives such as wetting agents and preservatives as necessary. Further, it can be used in the form of a solution by adding a solvent for coating, and at this time, the volume of the composition can be based on the volume of the entire solution. As the solvent, water or an organic solvent can be used, and an appropriate one can be used according to the type of the polymer compound. For example, when using polyvinyl alcohol, water can be used as the solvent to produce a coating solution.

[0050] Therefore, the present invention can also provide an antibacterial film formed using the above coating composition.

[0051] In the present invention, the antibacterial film can be produced using the above coating composition, that is, a coating composition containing bacteriophage, a polymer compound, and a plasticizer. At this time, the coating composition can be used in the form of a solution containing a solvent for coating properties.

[0052] In the present invention, the film can be interpreted to include all forms of films directly coated on a coating object such as a food like an egg or a food container and single film forms.

[0053] Specifically, the antibacterial film can be formed by adding bacteriophage to a solution containing a polymer compound and a plasticizer, then coating the solution on an object or a substrate and drying it. At this time, the solution can be diluted and used as necessary.

[0054] In the present invention, when the antibacterial film is directly formed on food or a food container, it can be formed by spraying a solution onto the object or immersing the object in the solution. For example, after coating the object with the coating composition, a film can be formed by drying at a temperature of 20 to 30°C for 10 to 180 minutes.

[0055] Alternatively, when the antibacterial film is manufactured in the form of a single film, it can be manufactured using a method of coating a solution onto a substrate by a method such as casting. For example, after coating the substrate with the coating composition under a relative humidity condition of 30 to 70 RH%, a film can be formed by drying at a temperature of 20 to 30°C for 10 to 20 hours.

[0056] When the present invention is used, bacteriophage can stably survive even in the form of a film and exhibit excellent antibacterial activity. Therefore, when the present invention is applied to food packaging materials, it can effectively prevent food from being contaminated by pathogens and improve the safety and storage stability of food.

Examples

[0057] The present invention will be described in more detail through the following examples. However, these examples show some experimental methods and configurations for illustrative purposes of the present invention, and the scope of the present invention is not limited to such examples.

[0058] Experimental method In the experiment, Salmonella Enteritidis ATCC 13076 was used as the host bacterium, and LB broth (MB-L4488; MB cell, Seoul, Korea), 0.5% (w / v) LB molten agar, and 1.5% (w / v) LB agar medium (MB-L4487, MB cell) were used as the culture media.

[0059] The phage titer was measured using a double-layer agar plate with 0.5% (w / v) LB molten agar and 1.5% (w / v) LB agar as the upper and lower layers, respectively.

[0060] Production Example 1: Purification, Propagation, and Stock Production of Bacteriophage Bacteriophage (hereinafter referred to as "phage") was obtained from a domestic sewage sample and purified through a double-layer agar assay and a plaque assay. After resuspending one plaque in phosphate-buffered saline (PBS), it was centrifuged at 15,000×g for 1 minute at 4°C, and then filtered through a sterile Whatman TM PVDF membrane filter with a pore size of 0.22 μm. The above filtration step was repeated 5 times.

[0061] To propagate the isolated phage, the phage was cultured in LB broth using S. Enteritidis ATCC 13076 as the host. Specifically, after subinoculating S. Enteritidis ATCC 13076 at 1%, it was cultured at 37°C for 1.5 hours. Then, the phage was cultured under aerobic conditions at 37°C for 4 hours. The sample was centrifuged at 15,000×g for 10 minutes at 4°C, and the supernatant was filtered through a sterile Whatman TM PVDF membrane filter with a pore size of 0.45 μm. The above steps were continuously carried out for three volume conditions (3, 50, and 300 mL of the cultured bacteria) to obtain a sufficient amount of phage lysate.

[0062] To obtain a phage stock with a higher titer, the purified phage lysate was centrifuged at 30,000×g for 30 minutes at 4°C to obtain a pellet. The phage concentration (PFU / mL) was measured using the double-layer agar assay. The purified phage was amplified to 10 10A lysate having a titer of 10⁶ PFU / ml or higher was obtained and stored at 4°C until use. When stored for a long period, it was stored in 35% glycerol at -80°C.

[0063] The phage isolated and purified by the above method was named "Phage PBSE191" and deposited with the Korean Collection for Type Culture, and was assigned the accession number KCTC14929BP as of March 31, 2022.

[0064] Experimental Example 1: Transmission Electron Microscope (TEM) Analysis of Phage For phage PBSE191, the morphology was analyzed using a transmission electron microscope (TEM). A 200-mesh copper grid coated with formvar / carbon was pretreated with an electrical discharge machine (US / 91000, USA). After loading the phage onto the copper grid, it was negatively stained with 2% (v / v) uranyl acetate (pH 4.5). The sample was analyzed with an energy-filtering Libra 120 transmission electron microscope (Carl Zeiss, Germany), and the results are shown in Figure 1.

[0065] Referring to the TEM image in Figure 1, it can be confirmed that phage PBSE191 has an icosahedral head and a non-contractile flexible tail. Specifically, the head of the phage has an icosahedral shape with a particle size of 58.84 ± 1.78 nm (n = 13), the length of the tail is 115.06 ± 5.64 nm (n = 13), and the width is 10.13 ± 0.91 nm (n = 13).

[0066] The phage of phage PBSE191 was similar to phages LPST94, BSPM4, and CGG3-1 in terms of structure, but had a shorter tail compared to the said phages. From the above results, it was found that phage PBSE191 belongs to the family Siphoviridae of the order Caudovirales.

[0067] Experimental Example 2: Bacterial challenge assay using phage Bacterial challenge assay was performed using S. Enteritidis ATCC13076 and phage PBSE191. The subcultured S. Enteritidis was cultured at 37°C for 1.5 hours under aerobic conditions. Then, phage infection was carried out on the cultures under the conditions of multiplicity of infection (MOI) of 0.01, 0.1, 1, 10, and 100, respectively. While growing the host under aerobic conditions at 37°C for 9 hours, the absorbance at 600 nm was measured using a UV-visible spectrophotometer (SP-UV 300, Spectrum Instruments, Perkin Elmer, UK) to confirm the lysis activity, and the said experiment was repeated 3 times.

[0068] Figure 2 is a diagram showing the results of measuring the growth inhibitory activity of Salmonella bacteria in the presence of the said phage. From the growth inhibitory activity in Figure 2, it was confirmed that the said phage can rapidly inhibit the growth of S. Enteritidis. Compared with the negative control group, the results showed that the phage rapidly inhibited the growth of host bacterial cells within 1 hour under the conditions of MOI values of 100, 10, 1, 0.1, and 0.01. Furthermore, such growth inhibition persisted for 6 hours in all experimental groups, and the phage showed higher lysis activity at high density, rapidly lysing the host bacteria and inhibiting continuous growth.

[0069] From the above results, it was confirmed that phage PBSE191 is excellent and exhibits long-term persistent bacteriolytic properties.

[0070] Experimental Example 3: Host Range Determination of Phage A spot test was performed on the bacteria in Table 1 to confirm the host range of phage PBSE191. Among the test bacteria, lawns of the test bacteria excluding Pectobacterium caratovorum and Staphylococcus aureus were prepared using LB medium, and lawns of P. caratovorum and S. aureus were prepared using TSA medium.

[0071] Phage lysate (2×10 8 PFU) was spotted on the lawn of each strain and then cultured at 37 °C for 24 hours. However, in the case of P. caratovorum KACC 21701, it was cultured at 30 °C for 24 hours. The plaque formation efficiency of the phage was measured for Salmonella strains and several Gram-positive and Gram-negative strains, and the results are shown in Table 1 below. The efficiency of plating (EOP) was calculated according to the following formula. Based on the EOP criteria, +++ indicates greater than 1, ++ indicates 0.001 - 1, + indicates less than 0.001, and - means no sensitivity to the phage.

[0072]

Number

[0073]

Table 1

[0074] Referring to the results in Table 1, phage PBSE191 specifically infected Salmonella enterica and did not cause infection in other strains.

[0075] Specifically, it was found that the phage was active against a wide range of Salmonella including six serotypes: S. Enteritidis, S. Typhimurium, S. Paratyphi, S. Salamae, S. Diarizonae, and S. Dublin.

[0076] When compared with the existing phages SS3e and BSP101 that are active not only against Salmonella but also against Shigella or E. coli, phage PBSE191 has the characteristic of specifically infecting Salmonella and can kill various types of Salmonella. Therefore, phage PBSE191 can be expected to be usefully used in the food industry where control of Salmonella is required.

[0077] Experimental Example 4: Phage adsorption analysis The adsorption ability of phage PBSE191 was evaluated using the time it takes for the phage to adsorb to the surface of the host bacterium. An overnight culture of strain S. Enteritidis ATCC 13076 was diluted 1:100 in LB broth and cultured under aerobic conditions at 37°C for 3 hours. The cultured bacteria (4.7×10 8 CFU) were centrifuged at 15,000×g for 1 minute, and the pellet was immediately resuspended in 10 mL of fresh LB broth.

[0078] After infecting the cells with the phage under the condition of MOI 0.001, 1 mL of the suspension was taken as a sample and cultured statically at 37°C respectively. Samples were taken after 0, 5, 10, 15, 20, 25, and 30 minutes respectively. Each sample was immediately centrifuged at 15,000×g for 1 minute, filtered, and then plated using the double-layer agar assay method to determine the phage titer. The above experiment was repeated three times, and the results are shown in Figure 3. The phage adsorption ability can be calculated according to the following formula.

[0079]

Equation

[0080] Referring to Fig. 3, the results show that 92.03% and 99.85% of the initial phage population adsorbed to the surface of the host bacteria after 10 minutes and 25 minutes, respectively, thereby confirming the rapid adsorption ability of the phage.

[0081] Experimental Example 5: One-step growth analysis of phage To measure the latent period and burst size of phage PBSE191, one-step growth analysis was performed. The phage and the suspension were cultured at 37°C for 25 minutes in a static state to allow the phage to adsorb to the bacterial surface. After culturing, the suspension was centrifuged at 15,000×g for 1 minute, and the plaque assay method was performed on the supernatant to measure the titer of the unadsorbed phage.

[0082] The pellet containing the phage-infected host bacteria was immediately resuspended in 10 mL of LB broth, then cultured at 37°C, and 100 μl of samples were collected every 10 minutes for 2 hours. The collected samples were plated on LB agar and used for phage counting through the double-layer agar technique.

[0083] The latent period (min) was confirmed by the time it took for a significant increase in phage titer and for the infected bacteria to lyse, and the burst size can be calculated using the following formula.

[0084]

Number

[0085] Figure 4 shows the one-step growth curve of the phage. Referring to this figure, it was confirmed that the latent period when infected with S. Enteritidis was short at 20 minutes, and the first and second release time points were 30 minutes and 50 minutes, respectively. Also, the initial release amount was 265 PFU / infected cell, and the second release amount was 127 PFU / infected cell. Considering that the average release amount of previously reported Salmonella phages was 112 ± 48 PFU / infected cell (n = 15), it can be confirmed from the above results that the phage has an excellent release amount.

[0086] Experimental Example 6: Measurement of Thermal Stability and pH Stability of Phage The survival rate of phage PBSE191 was measured and its stability was evaluated in a wide temperature range from -18 to 80 °C and a pH range from 1 to 9. For the measurement of thermal stability, phage lysate (10 8 PFU) was cultured at different temperatures in the range of -18 to 80 °C for 30 minutes. For the measurement of pH stability, phage lysate (2×10 8 PFU) was cultured in buffer solutions of various pH values (pH 2 - 9) for 30 minutes. The remaining phages were calculated by plating, and after repeating the experiment three times, the experimental results are shown in Figures 5 and 6 respectively. The stability of the phage can be calculated according to the following formula.

[0087]

Equation

[0088] Referring to the results of the thermal stability test in Figure 5, it was confirmed that the survival rate of the phage was not significantly affected after culturing at -18 to 60 °C for 30 minutes. On the other hand, although there was an impact at temperatures of 70 °C and 80 °C, it was confirmed that more than 5 log PFU / mL of the phage remained after 30 minutes of treatment. These results are at the same level as phages LSE7621, LPST10, and vB_SalP_TR2, and the survival rate is better than that of phages SE-P3, SE-P16, SE-P37, and SE-P47 at 80 °C.

[0089] According to the pH stability test results in Figure 6, the phage survived stably even after being cultured for 30 minutes in the pH range of 5 to 7. Optimal stability was observed with a phage reduction of less than 1 log PFU / mL in the pH range of 4 to 9. The phage was inactivated at pH 1, but more than 3.5 log PFU / mL survived after 30 minutes of treatment at pH 3.

[0090] These results are comparable to those of phage SS3e, and it was confirmed that phage PBSE191 showed stability under a wide range of temperature and pH conditions and could be usefully used in the food and food manufacturing industries from now on.

[0091] Experimental Example 7: Host Bacterium Receptor Analysis of Phage Using S. Typhimurium LT2C as the host bacterium, receptor analysis of phage PBSE191 was performed.

[0092] The △rfbP / LT2C knock-out mutant and its complemented strain (△rfbP complemented with pUHE::rfbP / LT2C plasmid) were provided by a laboratory at Seoul National University and used. After wild-type bacteria and the knock-out mutant were cultured overnight in LB broth, the complemented strain was cultured at 37°C under aerobic conditions in LB broth containing carbenicillin. A spotting assay was performed with wild-type, △rfbP / LT2C mutant, and △rfbP complemented strain to confirm the phage receptor, and the results are shown in Figure 7.

[0093] Referring to Figure 7, the results showed that the S. Typhimurium △rfbP / LT2C mutant was resistant to the phage, and after complementing the strain with rfbP, the sensitivity to the phage was restored.

[0094] These results mean that phage PBSE191 recognizes the O-antigen of Salmonella lipopolysaccharide (LPS) as the receptor of the host bacterium.

[0095] Experimental Example 8: DNA Analysis of Phage Purification of Phage DNA The DNA of phage PBSE191 was purified using the standard phenol-chloroform extraction method. Before purification, 500 μl of the phage lysate was treated with 1 μl / mL of DNase I and 1 μl / mL of RNase I at room temperature for 30 minutes to remove bacterial DNA and RNA contaminants. Next, the phage lysate was treated with a lysis buffer containing 0.5% sodium dodecyl sulfate (SDS), 0.5 M EDTA (pH 8.0), and 50 μl / mL proteinase K, and the mixture was incubated at 65°C for 15 minutes.

[0096] Thereafter, phenol was added to extract the phage DNA, and the mixture was centrifuged at 5,000 rpm for 5 minutes at room temperature. Next, the aqueous layer was carefully mixed with a phenol-chloroform-isoamyl alcohol (25:24:1) solution and then centrifuged at 5,000 rpm for 5 minutes to remove unnecessary components such as polysaccharide and protein components. Thereafter, the same step was repeated with chloroform.

[0097] The aqueous layer was collected with 3 M sodium acetate (NaOAc, pH 5.2), followed by ethanol precipitation. Finally, the purified phage genomic DNA was stored in TE buffer and used for the experiment.

[0098] Genetic Sequence Analysis and Biological Information Analysis The open reading frames (ORFs) of the phage genome were investigated using the RAST (https: / / rast.nmpdr.org / ), GeneMarkS (http: / / exon.gatech.edu / GeneMark / genemarks.cgi), and FgenesV (trained Pattern Markov chain-based viral gene prediction software) programs. Unknown ORFs were referenced for ORF inference using the non-overlapping protein NCBI database (http: / / blast.ncbi.nlm.nih.gov / ) with BLASTP and the homologous ORFs of other existing bacteriophages.

[0099] Based on the above analysis results, a genome map was created using Genescene software (DNAstar, Madison, WI) and is shown in Figure 8. The phage genome sequence was registered in GenBank with the accession number OM291373 (https: / / www.ncbi.nlm.nih.gov / nuccore / OM291373). As a result of the analysis, the genome of phage PBSE191 was composed of 41,332 bp, with a GC content of 49.84%, and was inferred to encode 43 ORFs.

[0100] As a result of ORF confirmation, it was confirmed that the phage did not have lysogeny module genes such as cro, cI, integrase, or toxic genes, thereby confirming the safety of the phage.

[0101] For the phylogenetic confirmation of the phage, phylogenetic analysis based on the major capsid protein (ORF29) was performed using the neighbor-joining method with 2,000 bootstrap repetitions using Molecular Evolutionary Genetics Analysis 11 (MEGA 11) software, and the phylogenetic tree is shown in Fig. 9. In Fig. 9, close relationships on the phylogenetic tree are indicated by *.

[0102] As a result of the phylogenetic analysis, the major capsid protein of phage PBSE191 was similar to the major capsid proteins of phages L13, SS3e, and TS3, thereby confirming that it belongs to the family Salmonellaphage.

[0103] Production Example 2: Production of PVA Film Using Phage Using phage PBSE191, a polyvinyl alcohol (PVA) film containing the phage was produced. As the PVA, that purchased from Sigma-Aldrich was used, and the weight average molecular weight of the PVA was 13,000 to 23,000, and the saponification degree was 87 to 89%.

[0104] After preparing an 11 g / 100 mL PVA solution using distilled water, sorbitol (D-sorbitol 97%), glycerol (99%), or trehalose (D-(+)-trehalose dihydrate), used as a plasticizer and a moisturizer, was added to the 11% PVA solution at concentrations of 0%, 10%, and 20% (w / w) based on the weight of the PVA, followed by stirring for 60 minutes and heating to 80°C.

[0105] When completely dissolved, the solution was autoclaved at 121°C for 15 minutes for sterilization. The autoclaved solution was cooled to room temperature, and a PBS-based phage lysate (10 10After adding to the prepared solution of PFU) at a volume ratio of 9:1, it was uniformly mixed and degassed. The film solution of the control group was prepared by mixing 11% autoclaved PVA solution with sterilized PBS buffer at a volume ratio of 9:1.

[0106] 1 mL of each solution was poured into a Petri dish and dried at 25 °C and 50 RH% for 15 hours using a hygro-thermostat. The dried film was peeled off from the casting surface and used for the experiment.

[0107] Figure 10 is a diagram showing photographs of a phage-free film (left side) and a phage-containing film (right side) for a film containing 10% PVA and 2% sorbitol (w / w). Referring to the drawing, it can be confirmed that there is no significant difference in appearance depending on whether or not it contains phage.

[0108] Experimental Example 9: Analysis of the viability of phage in a film To confirm the survival rate of phage in a PVA film containing glycerol (G), sorbitol (S), or trehalose (T) as a plasticizer, according to the method of Production Example 2, the substance was added at 10 or 20% by weight based on the weight of PVA to produce a PVA film. The initial phage titer of each film solution was 4×10 9 PFU / mL was set.

[0109] The produced film was dissolved in 10 mL of PBS buffer at 20 °C and 200 rpm for 30 minutes, and the survival rate of phage was evaluated using the double-layer plaque assay method. The surviving phage were counted, and the results of measuring the viability of phage in the phage-containing PVA film containing each plasticizer are shown in Figure 11.

[0110] According to the results of Figure 11, in the 10% (w / v) PVA film without plasticizer, phages of more than 2 log PFU were inactivated, while in the films containing sorbitol, glycerol, or trehalose, it was confirmed that the viability of phages was improved. In particular, sorbitol showed excellent activity from the viewpoint of phage protection compared to other wetting agents.

[0111] Specifically, in 20% sorbitol, most phage particles survived, and the inactivated phages were less than 0.5 log PFU. On the other hand, in 10% sorbitol, 20% glycerol, 10% glycerol, 20% trehalose, and 10% trehalose, phages of 1.1, 1.1, 1.3, 1.1, and 1.2 log PFU were inactivated, respectively. From this, it was confirmed that the phage survival rate in the film (PVAS20) using 20% sorbitol in the 10% (w / v) PVA film was the most excellent.

[0112] Experimental Example 10: Confirmation of Phage Stability in PVA Film The stability of phages in the phage-containing PVAS20 film was confirmed for 30 days. The phage-containing PVAS20 film was dissolved in 10 mL of PBS buffer at 20 °C and 200 rpm for 30 minutes, and the viability of phages was evaluated using the double-layer plaque assay method. By the above method, the stability of phages in the film was tested every 1, 3, 10, 20, and 30 days for 30 days. The phages that survived in plating were counted, and the experiment was repeated 3 times.

[0113] Figure 12 is a diagram showing the measurement results of phage stability in the PVA film. Referring to Figure 12, it was confirmed that phages showed a surprising excellent survival rate for 30 days under dry conditions, and from this, it was found that phages were successfully protected and preserved in the PVA polymer matrix.

[0114] Experimental Example 11: Confirmation of Antibacterial Activity of PVA Film Containing Phage The antibacterial activity against Salmonella bacteria was tested on the phage-containing PVAS20 film. To measure the antibacterial activity, 10 mL of an S. Enteritidis ATCC 13076 cell suspension (about 10 5 CFU / mL, early-exponential phase) in LB broth was prepared. Then, the film was immersed in the suspension while shaking at 200 rpm at 37 °C for 4 hours. The amount of phage in the film was about 10 8 PFU / film, and a phage-free film was used as a control group. The antibacterial activity was measured at 0.5, 1, 2, and 4 hours, and the results are shown in Fig. 13.

[0115] According to Fig. 13, the results showed that phage particles in the film maintained host lysis activity against S. Enteritidis over 4 hours. Thus, it was confirmed that the phage-containing PVAS20 film can continuously exhibit antibacterial activity.

[0116] Experimental Example 12: Measurement of Antibacterial Activity of Phage-Containing PVA Coating To evaluate the antibacterial activity of the phage-containing coating, eggshell samples with a particle size of 2.5 cm (0.46 ± 0.05 g, n = 125) were prepared using an egg opener (Guangzhou Le Tian Pen Co., Ltd., China) and sterilized by autoclaving at 121 °C for 15 minutes. Next, 54 clean eggshells were randomly divided into three groups (control group, phage-free PVAS20 coating group, and phage-containing PVAS20 coating group).

[0117] Subcultured S. Enteritidis was cultured at 37 °C for 1.5 hours (early exponential phase) under aerobic conditions. The culture was centrifuged at 15,000 × g for 1 minute, and the bacterial pellet was resuspended in 100 μl of LB broth. 2.4 × 10 810 μl of bacterial cells at CFU / mL were spot inoculated onto the eggshell surface and dried in air at room temperature for 30 minutes.

[0118] In the case of the phage coating group, the inoculated eggshells were immersed in a phage-containing PVAS20 coating solution (4.0×10 9 PFU / mL) for 3 seconds and then dried at room temperature for 40 minutes. In the control group, the eggshells were either not coated or immersed in a PVAS20 coating solution without phage and dried for 40 minutes. Six eggshell samples were selected per group, stored at 5 °C and 50% relative humidity for 24 hours, and then tested for antibacterial activity against Salmonella.

[0119] For the antibacterial activity test, the samples were homogenized with 10 ml of sterile PBS buffer for 30 seconds using a Pulsifier II (Microgen Bioproducts Ltd., UK). All samples were diluted to 10 -2 and plated on XLD agar (MB-X1060; MB cell, Seoul, Korea), and then cultured at 37 °C for 24 hours. The number of black colonies was counted on plating, and the titer of phage remaining on the coated eggshells was measured by the double-layer agar assay.

[0120] Figure 14 is a diagram showing the measurement results of S. Enteritidis cells before coating, immediately after coating, and 24 hours after coating. Referring to Figure 14, it can be confirmed that a significant amount of Salmonella bacteria (about 1 log CFU) die at room temperature immediately after forming a PVAS20 coating containing phage on the eggshell. In the case of the phage-containing PVAS20 coating, it induced a cell reduction of about 2 log CFU compared to the initial inoculum after 24 hours, while in the control group, it was confirmed that the reduction was about 1 log CFU.

[0121] From the above results, it was confirmed that in the case of the phage-containing PVAS20 coating, the phage can come into contact with bacteria during the drying stage (40 minutes) after coating and induce death. Also, it was confirmed that the decrease in the number of bacteria persists even after 24 hours because the phage survives for a long time and continuously shows an effect with the PVAS20 coating.

[0122] Thus, it was confirmed that when the PVA coating containing phage is applied to the eggshell, it exhibits excellent stability and antibacterial activity.

[0123] As described above, some embodiments of the present invention have been explained. However, the present invention is not limited only to the embodiments as described above, and can be implemented with modifications and variations within the scope not departing from the gist of the present invention. It must be understood that the forms added with such modifications and variations also belong to the technical idea of the present invention. (Supplementary Note) The invention of the present disclosure includes the following aspects. <Item 1> A coating composition comprising a bacteriophage having a killing ability against Salmonella sp. bacteria, a polymer compound, and a plasticizer. <Item 2> The coating composition according to <Item 1>, wherein the Salmonella bacteria include Salmonella enterica. <Item 3> The coating composition according to <Item 1>, wherein the Salmonella bacteria include at least one Salmonella enterica serotype selected from the group consisting of Salmonella Enteritidis, Salmonella Typhimurium, Salmonella Paratyphi, Salmonella Salamae, Salmonella Diarizonae, and Salmonella Dublin. <Item 4> The coating composition according to <Item 1>, wherein the bacteriophage belongs to the family Siphoviridae. <Item 5> The coating composition according to <Item 1>, wherein the bacteriophage is the bacteriophage with the accession number KCTC14929BP having a specific killing ability against Salmonella sp. bacteria. <Item 6> The coating composition according to <Item 1>, wherein the polymer compound contains at least one selected from the group consisting of polyvinyl alcohol (PVA), polylactic acid (PLA), polycaprolactone (PCL), polybutylene succinate (PBS), polyethylene terephthalate (PET), polybutylene terephthalate (PBT), polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC), polyamide (PA), and polyurethane (PU). <Item 7> The coating composition according to <Item 1>, wherein the polymer compound contains a biodegradable polymer. <Item 8> The coating composition according to <Item 1>, wherein the plasticizer contains at least one selected from the group consisting of sorbitol, glycerol, trehalose, fructose, sucrose, mannitol, propylene glycol, and polyethylene glycol. <Item 9> The coating composition according to <Item 1>, wherein the plasticizer is contained in an amount of 10 to 30% by weight based on the weight of the polymer compound. <Item 10> The coating composition according to <Item 1>, wherein the coating composition further contains a solvent. <Item 11> Based on the total volume of the coating composition, the bacteriophage is contained at 1×10 8 ~1×10 12 PFU / mL. The coating composition according to <Item 1>. <Item 12> The coating composition according to <Item 1>, wherein the polymer compound is contained in an amount of 5 to 20 g / 100 mL based on the total volume of the coating composition. <Item 13> The coating composition according to <Item 1>, wherein the plasticizer is contained in an amount of 1 to 5 g / 100 mL based on the total volume of the coating composition. <Item 14> An antibacterial film produced using a coating composition containing a bacteriophage having a killing ability against Salmonella sp. bacteria, a polymer compound, and a plasticizer. <Item 15> The antibacterial film according to <Item 14>, which is produced by coating the substrate with the coating composition and then drying it at a temperature of 20 to 30°C for 10 to 20 hours. <Item 16> The antibacterial film according to <Item 14>, which is produced by coating the object to be coated with the coating composition and then drying it at a temperature of 20 to 30°C for 10 to 180 minutes. <Item 17> The antibacterial film according to <Item 14>, wherein the antibacterial film is a film for food packaging. <Item 18> A bacteriophage with the accession number KCTC14929BP having a specific killing ability against Salmonella sp. bacteria.

[0124] JPEG0007699854000006.jpg223162

Claims

**Claim 1** A coating composition comprising a bacteriophage having a killing ability against Salmonella sp. bacteria, a polymer compound, and a plasticizer, wherein the plasticizer contains sorbitol, the plasticizer is contained in an amount of 18 to 22% by weight based on the weight of the polymer compound, and the polymer compound contains polyvinyl alcohol (PVA). **Claim 2** The coating composition according to claim 1, wherein the Salmonella bacteria include Salmonella enterica. **Claim 3** The coating composition according to claim 1, wherein the Salmonella bacteria include at least one Salmonella enterica serotype selected from the group consisting of S. Enteritidis, S. Typhimurium, S. Paratyphi, S. Salamae, S. Diarizonae, and S. Dublin. **Claim 4** The coating composition according to claim 1, wherein the bacteriophage belongs to the family Siphoviridae. **Claim 5** The coating composition according to claim 1, wherein the bacteriophage is the bacteriophage with the accession number KCTC14929BP having a specific killing ability against Salmonella sp. bacteria. **Claim 6** The coating composition according to claim 1, wherein the plasticizer further contains at least one selected from the group consisting of glycerol, trehalose, fructose, sucrose, mannitol, propylene glycol, and polyethylene glycol. **Claim 7** The coating composition according to claim 1, wherein the coating composition further contains a solvent. **Claim 8** Based on the volume of the entire coating composition, the bacteriophage is contained at 1×10 ８ to 1×10 １２ PFU / mL. The coating composition according to claim 1. **Claim 9** The coating composition according to claim 1, wherein the polymer compound is contained in an amount of 5 to 20 g / 100 mL based on the total volume of the coating composition.

10. The coating composition according to claim 1, wherein the plasticizer is contained in an amount of 1 to 5 g / 100 mL based on the total volume of the coating composition.

11. A method for producing an antibacterial film, comprising: producing an antibacterial film using a coating composition, wherein the coating composition contains a bacteriophage having a killing ability against Salmonella sp. bacteria, a polymer compound, and a plasticizer, the plasticizer contains sorbitol, the plasticizer is contained in an amount of 18 to 22% by weight based on the weight of the polymer compound, and the polymer compound contains polyvinyl alcohol (PVA), the method for producing the antibacterial film.

12. The method for producing an antibacterial film according to claim 11, comprising drying at a temperature of 20 to 30°C for 10 to 20 hours after coating the substrate with the coating composition.

13. The method for producing an antibacterial film according to claim 11, comprising drying at a temperature of 20 to 30°C for 10 to 180 minutes after coating the object to be coated with the coating composition.

14. The method for producing an antibacterial film according to claim 11, wherein the antibacterial film is a film for food packaging.

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