KR102844759B1 - Antibacterial film comprising antimicrobial peptide, manufacturing method thereof, and wrapping sheet for food - Google Patents

Abstract

The present invention discloses an antimicrobial film containing an antimicrobial peptide having high antimicrobial activity, which can block the low antimicrobial activity of organic extracts and the toxicity problems of inorganic substances, and in which the antimicrobial substance exists in a high content gradient on the film surface, a method for producing the same, and a food packaging material including the same.

Description

Antibacterial film comprising antimicrobial peptide, manufacturing method thereof, and wrapping sheet for food comprising the same

The present invention relates to an antibacterial film containing an antibacterial peptide, a method for producing the same, and a food packaging material.

Food packaging protects the food itself from external microorganisms and other contaminants. Antimicrobial agents are slowly released from the packaging to the food surface, inhibiting microorganisms that invade from outside or remain on the food surface, thereby preventing contamination and extending the food's shelf life. Antimicrobial packaging is widely used in a variety of household items, including food transport containers, kitchenware, household goods, home appliances, trash cans, building materials, wallpaper, and curtains.

Antibacterial substances used in antibacterial packaging are largely organic substances such as food additives and natural extracts, and inorganic substances such as zeolite, zinc oxide, and silver ion compounds.

Patent Document 1 uses lemongrass oil as an antibacterial agent, Patent Document 2 uses mustard essential oil and peppermint oil as an antibacterial agent, and Patent Document 3 uses silver nanomaterials as an antibacterial agent.

Natural extracts generally have low antibacterial activity per gram and are vulnerable to heat. Therefore, when used as antibacterial agents, they decompose or volatilize during the high-temperature extrusion or injection molding processes used in packaging manufacturing, preventing the desired level of antibacterial activity from being achieved. Furthermore, to ensure effectiveness, large quantities must be used, which can degrade the packaging's physical properties (strength, elongation, hardness, etc.).

Inorganic materials such as zinc oxide and silver nanoparticles are more readily applicable to packaging production. However, as noted in Non-Patent Documents 1 and 2, zinc oxide causes cytotoxicity and mitochondrial dysfunction, and silver nanoparticles are also toxic, requiring caution when directly applying them to foodstuffs.

Antibacterial packaging materials currently on the market are divided into dissolution-type, non-dissolution-type, adsorption-type, and spray-application-type depending on their method of action. Organic extracts and inorganic substances are mainly used in the manufacture of non-dissolution-type antibacterial packaging materials.

Non-dissolvable antibacterial packaging exhibits antibacterial activity only on the surface that comes into contact with the food. Antibacterial substances, such as organic extracts or inorganic substances, are physically mixed or chemically bound to the packaging, and then exert their effects during storage after packaging.

Antibacterial packaging can be produced by melt extrusion by mixing antibacterial substances and polymers into pellet form. However, this method does not effectively produce antibacterial activity on the surface because the antibacterial substances are mixed throughout the packaging rather than being located on the surface, as the antibacterial substances are evenly distributed throughout the packaging.

Accordingly, multilayer antimicrobial packaging materials have been proposed, which incorporate antimicrobial agents only in areas that come into contact with food. While these multilayer antimicrobial packaging materials offer superior antimicrobial activity, they require multiple manufacturing steps, such as laminating each film after production or forming an antimicrobial layer through additional coating on the substrate, which increases manufacturing costs.

Meanwhile, antimicrobial peptides are known to have very low human toxicity and excellent antimicrobial activity as they are composed of a linked structure of amino acids, and their application to packaging is being attempted.

Patent Document 4 discloses an antimicrobial coating composition comprising an adhesive protein coupled with an antimicrobial peptide and dopamine, while Patent Document 5 discloses an adhesive composition incorporating an acrylate group into the molecular backbone of a mussel adhesive protein. The antimicrobial substances described in these patents can be applied to the manufacture of packaging by forming an antimicrobial layer using a coating method. However, due to the aforementioned process issues, these compositions are not suitable for mass production. Furthermore, they suffer from peeling of the antimicrobial coating layer and dissolution of the antimicrobial substance from the packaging, requiring further improvement.

The problem of antimicrobial material dissolution can be solved by applying it directly to the pellets used in packaging manufacturing rather than through coating.

The manufacturing process for packaging using synthetic polymers involves melt-extruding a master batch, in pellet form, containing a specific ratio of polymer and a standard synthetic polymer pellet. Antibacterial agents are mixed with the polymer and produced in pellet form, allowing for the production of antibacterial packaging through melt-extrusion.

This manufacturing method has the effect of improving the problem of dissolution of antibacterial substances, but since the antibacterial substances are evenly distributed throughout the antibacterial packaging, the antibacterial substances are not located on the surface of the packaging but are mixed throughout, so the antibacterial activity occurring on the surface is not efficient.

1. KR Registration No. 10-2518466 2. KR Registration No. 10-1072883 3. KR Registration No. 10-0800611 4. KR Registration No. 10-2195156 5. KR Registration No. 10-2424889

1. Rob J Vandebriel et al, A review of mammalian toxicity of ZnO nanoparticles, Nanotechnology, Science and Applications 5, 61-71, 2012 2. Song Seung-yong, "A Study on the Characteristics of Surface Materials Affecting the Toxicity of Silver Nanoparticles," Graduate School of General Studies, Kyung Hee University, 2015

The present applicant has conducted various studies to solve the problem of dissolution of antibacterial substances, to ensure high antibacterial activity by having the antibacterial substance present in a high content in the part of the packaging that comes into contact with the packaging material, and to obtain a method that is easy to manufacture through an extrusion process using pellets during the manufacturing process, thereby facilitating mass production.

Accordingly, an antimicrobial peptide composed of a linked structure of amino acids was selected as a new antimicrobial substance that can block the low antimicrobial activity of existing organic extracts and the toxicity problem of inorganic substances.

In addition, in terms of the manufacturing process, an antimicrobial peptide was sprayed onto a polymer resin compound to manufacture a mixed compound, and through phase separation in the film process after melt extrusion, the antimicrobial peptide was distributed at a high concentration on the surface of the antimicrobial film, and a concentration gradient was formed in which the concentration gradually decreased toward the inside.

The purpose of the present invention is to provide an antimicrobial film containing an antimicrobial peptide, a method for producing the same, and food packaging paper having excellent antimicrobial activity even when using a small amount of the antimicrobial peptide.

The antibacterial film of the present invention has a structure in which an antibacterial peptide is included in a polymer resin in the form of a film.

The antibacterial film of the present invention may have a gradient in which the concentration of the antibacterial peptide gradually decreases from the surface to the inside in the thickness direction of the film.

The above antimicrobial peptide may be LL37 (LLGDLLRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES, sequence number).

The polymer resin may include at least one selected from the group consisting of polyvinyl alcohol, polyvinyl chloride, polyethylene terephthalate, polyethylene, polypropylene, ABS (Acrylonitrile Butadiene Styrene), polycarbonate, polystyrene, polyacrylate, polyethylene terephthalate, polymethyl methacrylate, ethylene-vinyl acetate copolymer (EVA), polyamide, and silicone resin.

The above antibacterial film may have a thickness of 0.1 ㎛ to 500 ㎛.

The present invention provides a use as a food packaging material including the above antibacterial film.

The antibacterial film of the present invention can be manufactured by including the steps of S1) preparing a mixed compound by spraying an antibacterial peptide aqueous solution onto a polymer resin compound and then drying it; S2) melt-extruding the mixed compound; and S3) forming the obtained melt-extruded product into a film.

At this time, the film forming may include a step of rolling the obtained molten extrudate; a step of cooling to room temperature; and a step of annealing.

During the cooling, the antimicrobial peptide may undergo spinodal phase separation to move to the film surface, thereby causing a gradient in which the concentration of the antimicrobial peptide gradually decreases from the surface toward the inside in the thickness direction of the film.

The antibacterial film according to the present invention has the advantage of excellent stability by using an antibacterial peptide with low toxicity to the human body.

Furthermore, the antimicrobial film of the present invention has a gradient in which the concentration of the antimicrobial peptide gradually decreases from the surface toward the inner surface along the thickness of the film. Consequently, when used as antimicrobial packaging, the concentration of the antimicrobial peptide at the contact surface with the packaging target, such as food, is high, ensuring excellent antimicrobial activity. Furthermore, the use of the antimicrobial peptide can fundamentally eliminate the low antimicrobial activity of existing organic extracts and the toxicity issues of inorganic substances.

Furthermore, the antibacterial film of the present invention exhibits excellent stability during the extrusion molding process and does not deteriorate the film's physical properties. By utilizing a compound manufacturing method rather than conventional coating or mixing methods, the film can be easily applied to mass production processes without the problem of antibacterial peptide dissolution.

Figure 1 is an image showing the results of Coomassie brilliant blue staining according to the content of antimicrobial peptide in the antimicrobial film.

The present invention provides an antimicrobial film comprising an antimicrobial peptide, a method for producing the same, and a use thereof as a food packaging material.

antibacterial film

The antibacterial film of the present invention refers to a molded product containing an antibacterial peptide in a polymer resin in the form of a film.

The antibacterial film of the present invention has a gradient in which the concentration of the antibacterial peptide gradually decreases from the surface to the inside in the thickness direction of the antibacterial film.

An antimicrobial peptide (AMP) according to one embodiment is the LL37 (AMP: cathelicidin antibacterial peptide) peptide. This antimicrobial peptide is composed of amino acids, which are components of the human body, exhibits high antibacterial efficacy at low concentrations, and has no issues with resistance or residual properties in the human body, making it applicable to a variety of fields.

The LL37 peptide is the only cathelicidin-based peptide found in the human body and is a peptide with a broad spectrum of antibacterial activity with the ability to directly destroy bacteria and viruses. The LL37 peptide can be produced by a conventional peptide synthesis method known in the art, and the production method is not particularly limited. As a method for the synthesis, it is preferable to synthesize it by a conventional peptide chemical synthesis method in the art, and specifically, it is more preferable to synthesize it by a solution-phase peptide synthesis method, a solid-phase peptide synthesis method, a fragment condensation method, and an F-moc or T-BOC chemical method, and more specifically, it is most preferable to synthesize it by a solution-phase peptide synthesis method (Merrifield, RB., J. Am. Chem. Soc., 85, 2149, 196), but is not limited thereto. The above LL37 peptide has a strong heat resistance characteristic, and in this test example, it was produced by Biobit Co., Ltd. and LLGDLLRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (sequence number) was purchased and used.

The polymer resin may be any type of synthetic resin that can be used as an antibacterial film, and is not particularly limited to a type such as a thermoplastic plastic or a thermosetting plastic. For example, the polymer resin may be at least one selected from the group consisting of polyvinyl alcohol, polyvinyl chloride, polyethylene terephthalate, polyethylene, polypropylene, ABS (Acrylonitrile Butadiene Styrene), polycarbonate, polystyrene, polyacrylate, polyethylene terephthalate, polymethyl methacrylate, ethylene-vinyl acetate copolymer (EVA), polyamide, and silicone resins, and preferably polyvinyl chloride.

The antimicrobial film of the present invention substantially does not include an "additional layer" when packaging a packaging target material (e.g., food). The additional layer may include a substrate film, a primer layer, or a separate coating layer. The antimicrobial film of the present invention, which does not include the additional layer, has a single-layer film structure.

The thickness of the antibacterial film of the present invention may vary depending on the material to be packaged, and may be 0.1 ㎛ or more, 1 ㎛ or more, 5 ㎛ or more, 10 ㎛ or more, 20 ㎛ or more, 50 ㎛ or more, 70 ㎛ or more, or 100 ㎛ or more, and may be 500 ㎛ or less, 450 ㎛ or less, 400 ㎛ or less, 350 ㎛ or less, 300 ㎛ or less, 250 ㎛ or less, or 200 ㎛ or less, but is not limited thereto.

According to one embodiment, the content of the antimicrobial peptide in the antimicrobial film of the present invention may be greater than 0.05 wt%, greater than 0.01 wt%, greater than 0.015 wt%, greater than 0.02 wt%, greater than 0.03 wt%, greater than 0.04 wt%, or less than 1.0 wt%, less than 0.7 wt%, less than 0.5 wt%, less than 0.4 wt%, less than 0.3 wt%, or less than 0.1 wt%, within 100 wt% of the antimicrobial film. Preferably, it may be greater than 0.01 wt% and less than 1.0 wt%, or greater than 0.01 wt% and less than 0.5 wt%, greater than 0.02 wt% and less than 0.3 wt%, greater than 0.02 wt% and less than 0.1 wt%, greater than 0.01 wt% and less than 0.3 wt%, or greater than 0.01 wt% and less than 0.2 wt%. The remaining residue at this time is polymer resin.

At this time, 0.01 wt% is converted to 100 ppm, and 1 wt% is converted to 10,000 ppm.

In other words, the concentration of the antimicrobial peptide in the antimicrobial film of the present invention may be greater than 50 ppm, greater than 100 ppm, greater than 150 ppm, greater than 200 ppm, greater than 300 ppm, greater than 400 ppm, or less than 10,000 ppm, less than 7,000 ppm, less than 5,000 ppm, less than 4,000 ppm, less than 3,000 ppm, or less than 1,000 ppm, and preferably greater than 100 ppm and less than 10,000 ppm, greater than 100 ppm and less than 5,000 ppm, greater than 200 ppm and less than 3,000 ppm, greater than 200 ppm and less than 1,000 ppm, greater than 100 ppm and less than 300 ppm, or greater than 100 ppm and less than 200 ppm.

The content of the antimicrobial peptides presented above may vary depending on the thickness of the antimicrobial film. As the thickness increases, the required antimicrobial peptide content may increase. The most appropriate content is one where the antimicrobial peptides are not uniformly distributed within the antimicrobial film, but rather exist in a concentration gradient.

As an example, when producing an antimicrobial film having a thickness of 100 μm, the content of the antimicrobial peptide is preferably 0.01 wt% or more and 0.2 wt% or less (100 ppm or more and 200 ppm or less). When the content of the antimicrobial peptide increases at the same thickness, a concentration gradient does not occur and it is uniformly distributed throughout the film.

The content of such antimicrobial peptides is a content that can secure sufficient antimicrobial activity. If it is below the above range, the desired level of antimicrobial activity cannot be secured due to low antimicrobial activity. If it is outside the above range, it is uneconomical as there is no significant increase in antimicrobial activity compared to the cost, so it is appropriately used within the above range.

The antibacterial film of the present invention has a characteristic in that the antibacterial peptide has a gradient in which the concentration gradually decreases from the surface to the inside in the thickness direction of the antibacterial film.

The above concentration gradient is generated throughout the thickness of the antibacterial film.

In the present invention, having a concentration gradient is not particularly limited as long as the concentrations are different at any two points in the thickness direction. In the present invention, the concentration gradient of the antimicrobial peptide has a concentration gradient such that one surface side of the antimicrobial film has a high concentration and the other surface side has a low concentration.

In the antibacterial film of the present invention, the surface of the antibacterial film in which the antibacterial peptide is present at a high concentration is in direct contact with the packaging target material (e.g., food), thereby effectively securing excellent antibacterial activity.

The antibacterial film of the present invention has a structural difference from a film in which the antibacterial peptide is present at the same concentration throughout the antibacterial film, and thus has several advantages.

As an example, when dispersing antimicrobial peptides in the same amount within an antimicrobial film, it is possible to produce an antimicrobial film having a concentration gradient, as in the present invention, and an antimicrobial film having the same concentration throughout the film. Looking at the concentration of antimicrobial peptides on the surfaces of these two antimicrobial films, the antimicrobial film of the present invention has a relatively high concentration. Therefore, the antimicrobial film of the present invention, which has a high concentration of antimicrobial peptides on its surface, is relatively advantageous in terms of its antimicrobial activity against packaging materials compared to existing antimicrobial films.

In addition, when manufacturing an antimicrobial film in which antimicrobial peptides are uniformly present, it is inevitable to use a relatively excessive amount of antimicrobial peptides in order to have the same concentration of antimicrobial peptides on the surface, which leads to an increase in cost.

As a result, the antibacterial film having a concentration gradient of the present invention has the advantage of further enhancing antibacterial activity while using a small amount of antibacterial peptide.

Furthermore, the antimicrobial film of the present invention avoids problems that arise in antimicrobial films that form a separate coating layer, as the antimicrobial peptide is present within the film. Since the coating layer is formed through various wet or dry coatings on a substrate film, the process becomes complex and costs increase. Furthermore, the formed coating layer may peel off or dissolution during distribution or use. Adding additional ingredients to the coating layer to suppress this dissolution can result in reduced antimicrobial activity, excessively thickening the coating layer for antimicrobial activity, or increased use of antimicrobial agents, which also increases costs.

Antibacterial film manufacturing method

The antibacterial film of the present invention must be manufactured to include an antibacterial peptide but have a concentration gradient, so a new method other than the existing simple coating or lamination method must be proposed.

The antibacterial film of the present invention comprises the steps of S1) preparing a mixed compound by spraying an antibacterial peptide aqueous solution onto a polymer resin compound and then drying it; S2) melt-extruding the mixed compound; and S3) forming the obtained melt-extruded product into a film.

Each step is explained below.

(S1) Mixed compound manufacturing step

First, a mixed compound is prepared by spraying an antimicrobial peptide solution onto a polymer resin compound.

A polymer resin compound is an intermediate product (dough) that can be processed through extrusion or injection molding by adding various types of additives or reinforcing agents to polymeric raw materials and then performing a compounding process.

The compounding process involves melting and kneading a mixture of additives and reinforcing agents, together with a pellet-shaped polymer, which is the raw material for the antibacterial film. During the compounding process, which involves melting and kneading the individual components contained in the compound, any known extruder with good mixing properties that is commonly used in this field can be used without limitation. Examples of such extruders include single-screw extruders, co-rotating twin-screw extruders, and bidirectional rotating twin-screw extruders. The melted mixture extruded through the extruder can be solidified and pelletized to produce a compound.

Conditions for each process, such as temperature, speed, and time, may vary depending on the type of polymer, additive, or reinforcing agent. Processing is performed according to established conditions in the field. Available additives include flame retardants, plasticizers, lubricants, stabilizers, antioxidants, colorants, curing agents, and coupling agents. Additives such as compatibilizers may also be used, along with reinforcing agents such as fillers.

The polymer resin compound of the present invention contains one or more polymer resins among the above-mentioned polymers, and can be manufactured directly or purchased and used commercially.

The mixed compound of the present invention means a compound obtained by spraying an antimicrobial peptide solution onto a polymer resin compound and then drying it.

The content of the antimicrobial peptide in the mixed compound may be more than 0.05 wt%, 0.01 wt% or more, 0.015 wt% or more, 0.02 wt% or more, 0.03 wt% or more, 0.04 wt% or more, or 1.0 wt% or less, 0.7 wt% or less, 0.5 wt% or less, 0.4 wt% or less, 0.3 wt% or less, or 0.1 wt% or less, within 100 wt% of the total weight. Preferably, it may be 0.01 wt% or more and 1.0 wt% or less, or 0.01 wt% or more and 0.5 wt% or less, 0.02 wt% or more and 0.3 wt% or less, 0.02 wt% or more and 0.1 wt% or less, 0.01 wt% or more and 0.3 wt% or less, or 0.01 wt% or more and 0.2 wt% or less. In this case, the remaining portion is a polymer resin.

An antimicrobial peptide is dissolved in water to obtain an antimicrobial peptide solution, which is sprayed onto the surface of a polymer resin compound so that it can be evenly coated on the surface of the polymer resin compound.

The concentration of the antimicrobial peptide solution of the present invention is determined in consideration of the content of the antimicrobial peptide present in the final antimicrobial film, but is set to have a range that allows easy spraying. For example, the antimicrobial peptide solution is prepared at a concentration that is 5 to 15 times, 7 to 12 times, and preferably 10 times, the concentration of the antimicrobial peptide contained in the antimicrobial film. For example, when preparing an antimicrobial film containing an antimicrobial peptide at a concentration of 0.1 wt%, the antimicrobial peptide solution is prepared at a concentration of 0.5 to 1.5 wt%, 0.7 to 1.2 wt%, and preferably 1 wt%. If necessary, the concentration may vary depending on the spraying device, and the antimicrobial peptide solution may be prepared at a concentration lower than the above range and sprayed twice or more.

The above spraying can be performed using various spraying devices known in the art. Various parameters, such as the spraying amount and spraying speed, can be appropriately selected by those skilled in the art.

After coating with an antimicrobial peptide solution, drying is performed at room temperature for 1 to 5 hours to allow the antimicrobial peptide to coat the surface of the polymer resin compound. This allows the antimicrobial peptide to be distributed primarily on the surface rather than within the polymer, and subsequently, during the cooling process, the peptide migrates to the surface due to spinodal phase separation or other phenomena.

Since the antimicrobial peptides are concentrated on the surface of the polymer resin compound, the antimicrobial activity on the surface is very strong, which is particularly advantageous in cases where external contact is required for significant antimicrobial activity.

(S2) Melt extrusion stage

Next, the mixed compound obtained in the above step is melt-extruded into a liquid state.

Melt extrusion can be performed by placing the above-mentioned mixed compound into an extruder hopper, melting it through the screw feeding zone, and then extruding it into a liquid state through a die. The melt extrusion process can vary depending on the type of polymer and can be performed at 100°C to 300°C. For example, in the case of polyvinyl chloride, melting point is around 160°C, so melt extrusion is performed at 140°C to 190°C.

A method of adding an antimicrobial peptide to a polymer resin compound during melt extrusion may be considered. In this case, the antimicrobial peptide is uniformly distributed throughout the polymer, and a concentration gradient that would result in high concentrations of the antimicrobial peptide on the surface does not occur. This may limit the antimicrobial activity concentrated on the surface.

In addition, the interaction between the antimicrobial peptide and the polymer resin compound added together is low, so the stability of the antimicrobial peptide is reduced, making it impossible to secure the desired level of antimicrobial activity.

(S3) Film forming step

Next, the obtained molten extrudate is manufactured into an antibacterial film through a film forming process.

The above molten extrudate is a molten resin in a liquid state.

The film forming process of the present invention is performed including the steps of rolling the obtained molten extrudate; cooling to room temperature; and annealing.

First, the molten extrudate is pressed flat using a pair of rolling rolls to ensure uniform thickness. This process compresses the film, ensuring a uniform concentration of antimicrobial peptides throughout the film. The temperature of the rolling rolls is maintained between 140°C and 160°C, as polyvinyl chloride, for example, has a melting point of around 160°C.

Next, the rolled film is cooled to room temperature. This cooling is preferably performed quickly to prevent the film from deforming at high temperatures, stabilize its internal structure, and stabilize the thickness and mechanical properties of the antibacterial film. During this process, the polymer within the film crystallizes, and the antibacterial peptide undergoes phase separation between the polymer and the antibacterial peptide due to their different material properties (e.g., crystallization temperature, molecular weight, surface energy).

It is generally known that mixtures spontaneously phase separate in a thermodynamically unstable state, and this is called spinodal decomposition or spinodal phase separation. ‘Spinodal phase separation’ is one of the processes in which a homogeneous substance in one state separates into two phases as conditions change. It is a method in which two phases are spontaneously formed at various points of the substance with a regular periodicity, and over time, the unique properties of the two phases approach each other. After spinodal phase separation, a coarsening phenomenon occurs in which each phase gradually grows over time. This means that a gradient is formed in the distribution of a specific component, and as a result, in order to minimize the system energy for film formation, antimicrobial peptides with low surface energy move microscopically in one direction, so that the antimicrobial peptides have a gradient in which the concentration gradually decreases from the surface of the antimicrobial film toward the inside.

According to one embodiment, when the cross-section of the antimicrobial film of the present invention is divided into three equal parts, a first layer, a second layer, and a third layer, the concentration of the antimicrobial peptide has a concentration gradient such as first layer > second layer > third layer. For example, the concentration of the antimicrobial peptide present in each layer is in the range of 60 wt% or more and 60 to 90 wt% for the first layer, less than 40 wt% and 10 to 35 wt% for the second layer, and less than 5 wt% and 0 to 5 wt% for the third layer, within 100 wt% of the total antimicrobial peptide content.

Next, annealing is performed.

Annealing is a process in which a cooled film is gradually heated at a constant temperature to remove residual stress and improve the alignment of polymer chains. This annealing improves the mechanical properties of the antimicrobial film and prevents deformation. This process optimizes the fundamental properties of the antimicrobial film, and the concentration gradient of the antimicrobial peptide is also firmly maintained.

Annealing is performed for 1 to 30 minutes, or 5 to 15 minutes, within the Tg temperature range of the polymer.

Table 1 below shows the glass transition temperatures of the polymers.

Plymer Glass transition temperature (Tg, ℃) Polyvinyl chloride (PVC)
Polyvinyl alcohol (PVA)
Polyethylene terephthalate (PET)
Polypropylene (PP)
Acrylonitrile Butadiene Styrene (ABS)
Polycarbonate (PC) 65 to 85
85
70 to 80
-30 to -20
100
145

The polyvinyl chloride described in the table above has a Tg in the range of 65°C to 85°C, and the annealing process can be performed for about 5 to 10 minutes until the final film forming while maintaining a temperature of 80°C or higher.

food packaging

The purpose of the antibacterial film of the present invention is to manufacture an antibacterial packaging material for food, and its thickness follows the above-mentioned, and it is recommended to selectively apply the thickness depending on the application target.

The antibacterial film according to the present invention has the advantage of excellent stability by using an antibacterial peptide with low toxicity to the human body.

Furthermore, the antimicrobial film of the present invention has a gradient in which the content of antimicrobial peptides gradually decreases from the surface toward the inner surface. As a result, it can be used as food packaging, and the high content of antimicrobial peptides at the contact surface with the packaging target, such as food, ensures excellent antimicrobial activity. Furthermore, the use of the antimicrobial peptides can fundamentally eliminate the low antimicrobial activity of existing organic extracts and the toxicity issues of inorganic substances.

Furthermore, the antibacterial film of the present invention exhibits excellent stability during the extrusion molding process and does not deteriorate the film's physical properties. By utilizing a compound manufacturing method rather than conventional coating or mixing methods, the film can be easily applied to mass production processes without issues with antibacterial agent dissolution.

Hereinafter, the present invention will be described in detail through examples. However, the following examples are only illustrative of the present invention, and the present invention is not limited to the following examples.

[Example 1]

An antibacterial peptide LL37 (sequence number LLGDLLRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES), manufactured by Biovit Co., Ltd. as an antibacterial substance, was dissolved in purified water to prepare an antibacterial peptide solution at a concentration of 0.05 wt%.

The above antimicrobial peptide solution was evenly sprayed onto a polyvinyl chloride compound (Changwoo Chemical) and then dried to produce a mixed polymer resin compound.

The above mixed polymer resin compound was fed into a single screw extruder. At this time, the temperature of the cylinder was set to 180°C to melt the polymer resin compound and then extruded in a liquid state using the screw extruder.

The molten extrudate was transported by conveyor and formed into a film shape through a rolling roll maintained at 140 to 160°C, then cooled to room temperature and annealed again at 80°C for 5 minutes to produce an antibacterial film with a thickness of 100 μm.

The antimicrobial peptide content in the manufactured antimicrobial film is 0.005 wt% (50 ppm).

[Example 2]

An antimicrobial film was manufactured in the same manner as in Example 1, except that an antimicrobial peptide solution was prepared at a concentration of 0.1 wt% and sprayed. The antimicrobial peptide content in the manufactured antimicrobial film was 0.01 wt% (100 ppm).

[Example 3]

An antimicrobial film was manufactured in the same manner as in Example 1, except that an antimicrobial peptide solution was prepared at a concentration of 0.2 wt% and sprayed. The antimicrobial peptide content in the manufactured antimicrobial film was 0.02 wt% (200 ppm).

[Example 4]

An antimicrobial film was manufactured in the same manner as in Example 1, except that an antimicrobial peptide solution was prepared at a concentration of 0.5 wt% and sprayed. The antimicrobial peptide content in the manufactured antimicrobial film was 0.05 wt% (500 ppm).

[Comparative Example 1]

99.9 wt% polyvinyl chloride compound (Changwoo Chemical) and 0.02 wt% antimicrobial peptide were fed into a single screw extruder. The cylinder temperature was set to 180°C to melt the polymer resin compound, which was then extruded in a liquid state using the screw extruder.

The molten extrudate was transported by conveyor and formed into a film shape through a rolling roll maintained at 140 to 160°C, then cooled to room temperature and annealed again at 80°C for 5 minutes to produce an antibacterial film with a thickness of 100 μm.

The antimicrobial peptide content in the manufactured antimicrobial film is 0.02 wt% (200 ppm).

[Comparative Example 2]

Polyvinyl chloride compound (Changwoo Chemical) was introduced into a single-screw extruder. The cylinder temperature was set to 180°C to melt the polymer resin compound, which was then extruded in a liquid state using the screw extruder.

The molten extrudate was transported by conveyor and formed into a film shape through a rolling roll maintained at 140 to 160°C, then cooled to room temperature and annealed again at 80°C for 5 minutes to produce a polyvinyl chloride film having a thickness of 100 μm.

An antimicrobial peptide solution having a concentration of 0.2 wt% was prepared and coated on the polyvinyl chloride film to produce an antimicrobial film. At this time, the content of the antimicrobial peptide in the antimicrobial film was adjusted to 0.02 wt% (200 ppm).

[Test Example 1] Identification of antimicrobial peptides in antimicrobial films

The concentration gradient of antimicrobial peptides within the antimicrobial film was confirmed through Coomassie Brilliant Blue staining. Coomassie Brilliant Blue binds to peptides and turns them blue; therefore, the darker the blue color displayed on the film, the greater the concentration of antimicrobial peptides.

Figure 1 is an image showing the results of Coomassie Brilliant Blue staining according to the content of antimicrobial peptide (% means weight%, and AMP means antimicrobial peptide).

As shown in Figure 1, the antibacterial film exhibits a blue color, indicating the presence of antibacterial peptides within the film. As the antibacterial peptide content increased, the blue color tended to become increasingly darker. Furthermore, when examining the antibacterial peptide content in the thickness direction, the upper side (surface) of the antibacterial film exhibited the deepest blue color.

Looking at the antibacterial film of Example 1, the dyeing was uneven and appeared as a cloudy blue color. This indicates that the content of antibacterial peptides in the antibacterial film was low.

The antibacterial films of Examples 2 to 4 exhibited a darker blue color compared to Example 1, and the color expression became darker as the content increased.

[Test Example 2] Antibacterial activity analysis

The antimicrobial activity test of antimicrobial films produced by varying the content of antimicrobial peptides was conducted in accordance with JIS Z 2801, an official test method for hard surfaces. In this case, Control Example 1 refers to a case in which no antimicrobial peptide was used.

1-1. Fungal culture

The test strains used in this experiment were Staphylococcus aureus , a Gram-positive bacterium, and Escherichia coli, a Gram-negative bacterium. The two strains were cultured in a 37°C shaking incubator using Soybean-Casein Digest Agar (BBL™ Trypticase™ Soy Agar, TSA) liquid medium. The solid medium used in this experiment was prepared by adding agar to the above liquid medium.

1-2. Measurement of antibacterial activity (according to JIS Z 2801)

Antibacterial activity was measured according to the JIS Z 2801 test method. That is, the control specimen used a polyethylene (PE) film, and the test specimen used an antibacterial film manufactured in the present invention. Each control specimen and test specimen was used in a size of 5 cm × 5 cm in accordance with the JIS Z 2801 test method, and was prepared by washing with sterile distilled water and drying. The control specimen and test specimen in a petri dish were inoculated with Staphylococcus aureus and Escherichia coli , respectively, and then sealed and cultured at 37°C for 24 hours. After 24 hours, dilution water was added to the specimen, and a certain amount of the bacterial solution was extracted from each specimen, spread on a solid medium, and cultured at 37°C for 24 hours. The number of colonies was measured to calculate the antibacterial activity.

At this time, the antibacterial activity value can be calculated using the following formula, and the higher the value, the better the antibacterial activity.

[Formula 1]

- Antibacterial activity value (R) = (Ut - Uo) - (At - Uo) = Ut - At

Here,

R: Antibacterial activity value

Uo: Average log value of viable cell count immediately after inoculation of Blank

Ut: Log average of the number of viable cells after 24 hours in the blank

At: Average log value of the number of viable bacteria on the antibacterial film after 24 hours.

- Antibacterial activity value: 1 or higher (antibacterial effect, 90.00% or higher)

- Antibacterial activity value: 2 or higher (antibacterial effect, 99.00% or higher)

- Antibacterial activity value: 3 or higher (antibacterial effect, 99.90% or higher)

- Antibacterial activity value: 4 or higher (antibacterial effect, 99.99% or higher)

Test items
Test results (/mL) Blank Example 1 Example 2 Example 3 Example 4 Escherichia coli
ATCC 8739 Initial bacterial count 2.8×10 ³ 2.8×10 ³ 2.8×10 ³ 2.8×10 ³ 2.8×10 ³ Antibacterial activity - 0.3 1.1 2.8 3.2 antibacterial activity - <90% >99.0% >99.0% >99.9% Staphylococcus aureus
ATCC 6538P Initial bacterial count 2.3×10 ³ 2.3×10 ³ 2.3×10 ³ 2.3×10 ³ 2.3×10 ³ Antibacterial activity - 0.6 1.2 3.0 3.8 antibacterial activity - <90% >90% >99.9% >99.9%

As can be seen from Table 2 above, the antibacterial activity of the antibacterial film containing the antibacterial peptide is superior.

[Test Example 3] Antibacterial activity analysis to confirm concentration gradient

Since the concentration gradient of the antimicrobial peptide could not be clearly determined through the dyeing test of Test Example 1, this experiment was conducted.

In this experiment, we analyzed the differences between the surface and inner surfaces of antimicrobial films, utilizing their antimicrobial activity. The antimicrobial films manufactured in Examples 1 through 4 were used to measure their antimicrobial activity against Escherichia coli. Antimicrobial activity was measured on Side A (the food-contacting side) and Side B (the other side) of each antimicrobial film.

Test items
Test results (/mL) Blank Example 1 Example 2 Example 3 Example 4 Side A Initial bacterial count 2.8×10 ³ 2.8×10 ³ 2.8×10 ³ 2.8×10 ³ 2.8×10 ³ Antibacterial activity - 0.3 1.1 2.8 3.3 antibacterial activity - <90% >99.0% >99.0% >99.9% Side B Initial bacterial count 2.3×10 ³ 2.3×10 ³ 2.3×10 ³ 2.3×10 ³ 2.3×10 ³ Antibacterial activity - 0.0 0.0 0.5 1.2 antibacterial activity - 0% 0% <90% >90%

Looking at the results in Table 3 above, it can be seen that the antibacterial activity on both sides of the antibacterial films of Examples 1 to 4 is different.

The antibacterial films of Examples 1 and 2 contained a relatively small amount of antibacterial peptide, and thus no antibacterial activity was observed on side B.

The antibacterial films of Examples 3 and 4 contained relatively large amounts of antibacterial peptides, and exhibited antibacterial activity on Side B, but exhibited lower antibacterial activity than on Side A. These results indirectly suggest that the antibacterial peptides, which are antibacterial substances, exist at different concentrations within the antibacterial films, and that the antibacterial peptides exist in a concentration gradient within the antibacterial films.

[Test Example 4] Analysis of antibacterial activity according to manufacturing method

This was performed to confirm the antibacterial activity according to the injection method of antibacterial peptides.

The antibacterial films used were those manufactured in Example 3, Comparative Example 1, and Comparative Example 2. They have the same antibacterial peptide content, but differ in their manufacturing methods.

Antibacterial activity was evaluated in the same manner as in the above test example, and the results are shown in Table 4 below.

Test items
Test results (/mL) Blank Comparative Example 1 Example 3 Comparative Example 2 Escherichia coli
ATCC 8739 Initial bacterial count 2.2×10 ³ 2.2×10 ³ 2.2×10 ³ 2.2×10 ³ Antibacterial activity - 0.1 2.9 0.9 antibacterial activity - <90% >99% <90% Staphylococcus aureus
ATCC 6538P Initial bacterial count 3.0×10 ³ 3.0×10 ³ 3.0×10 ³ 3.0×10 ³ Antibacterial activity - 0.1 3.0 1.0 antibacterial activity - <90% >99.9% >90%

Looking at Table 4 above, it can be seen that when an antibacterial peptide was simply added to a polyvinyl chloride compound as in Comparative Example 1, a certain level of antibacterial activity was exhibited, but the value was very low.

In addition, Comparative Example 2 is an antibacterial film coated with an antibacterial peptide, and its antibacterial activity increased to some extent compared to the antibacterial film of Comparative Example 1, but elution occurred in some of the test films.

Additionally, the antibacterial activity of the antibacterial films of Comparative Examples 1 and 2 on the A side (food contact side) and the B side (other side) of the antibacterial film presented in Test Example 3 was measured. As a result, the A side and the B side of the antibacterial film of Comparative Example 1 exhibited antibacterial activity similar to the above values, and the A side of the antibacterial film of Comparative Example 2 exhibited the same value as the above values, but the B side exhibited almost no antibacterial activity.

[Test Example 5] Analysis of antibacterial activity according to annealing conditions

To determine changes in annealing temperature during the film forming process, tests were conducted as shown in Table 5 below. The specific process conditions were the same as those in Example 3. The Tg of polyvinyl chloride was set at 65°C to 85°C.

Test items
Test results (/mL) Blank 50℃/ 5 minutes 60℃/ 5 minutes 80℃/ 5 minutes (Example 3) 100℃/ 5 minutes Escherichia coli
ATCC 8739 Initial bacterial count 2.4×10 ³ 2.4×10 ³ 2.4×10 ³ 2.4×10 ³ 2.4×10 ³ Antibacterial activity - 0.1 0.3 3.0 1.0 antibacterial activity - <90% <90% >99.9% >90% Staphylococcus aureus
ATCC 6538P Initial bacterial count 3.1×10 ³ 3.1×10 ³ 3.1×10 ³ 3.1×10 ³ 3.1×10 ³ Antibacterial activity - 0.2 0.7 3.1 2.0 antibacterial activity - <90% <90% >99.9% >99%

As shown in Table 5 above, antibacterial activity tends to decrease when the annealing temperature exceeds Tg, and antibacterial activity increases when performed near Tg. These results are attributed to the concentration gradient of antibacterial peptides through phase separation during annealing near Tg.

[Test Example 6] Food Preservation Test

After commercially available cherry tomatoes were wrapped with antibacterial film and stored in a constant temperature chamber at 20℃ and 30℃ for 5, 7, and 10 days, the degree of decay of the cherry tomatoes was observed with the naked eye, and the results are shown in Table 6 below. The symbols in Table 6 below indicate the degree of decay visually confirmed; "○" means good condition, "△" means slightly decayed condition, and "X" means "decayed condition."

5 days 7 days 10 days 20℃ 30℃ 20℃ 30℃ 20℃ 30℃ Example 1 O O O △ △ △ Example 2 O O O O △ △ Example 3 O O O O O O Example 4 O O O O O O Control example 1 X X X X X X Comparative Example 1 △ △ △ X X X Comparative Example 2 O △ △ X X X Comparative Example 3 O △ △ △ X X

As shown in Table 6 above, it can be seen that the freshness of cherry tomatoes is maintained when the antibacterial films of Examples 3 and 4 according to the present invention are used, indicating excellent food preservation properties.

Claims (7)

delete delete delete delete For the manufacture of antibacterial films for food packaging,
S1) A step of preparing a mixed compound by spraying an aqueous solution of LL37 (LLGDLLRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES, sequence number) antimicrobial peptide onto at least one polymer resin compound selected from the group consisting of polyvinyl alcohol, polyvinyl chloride, polyethylene terephthalate, polyethylene, polypropylene, ABS (Acrylonitrile Butadiene Styrene), polycarbonate, polystyrene, polyacrylate, polyethylene terephthalate, polymethyl methacrylate, ethylene-vinyl acetate copolymer (EVA), polyamide, and silicone resin, and then drying the mixture;
S2) A step of melt-extruding the above mixed compound; and
S3) A step of forming the obtained molten extrudate into a film; including manufacturing an antibacterial film,
The above film forming comprises the steps of rolling the obtained molten extrudate; cooling to room temperature; and annealing.
The above antibacterial film comprises an LL37 antibacterial peptide in a polymer resin in a film form in an amount of more than 0.05 wt% and less than 1.0 wt%, and the LL37 antibacterial peptide has a gradient in which the concentration gradually decreases from the surface to the inside in the thickness direction of the antibacterial film, and has a single-layer film structure that does not include an additional layer other than the antibacterial film.
Method for manufacturing an antibacterial film for food packaging. In paragraph 5,
A method for manufacturing an antibacterial film for food packaging, wherein the antibacterial film has a thickness of 0.1 ㎛ to 500 ㎛. In paragraph 5,
A method for manufacturing an antimicrobial film for food packaging, wherein the antimicrobial peptide undergoes a spinodal phase separation during cooling, in which the antimicrobial peptide moves to the surface of the antimicrobial film, thereby causing a gradient in which the concentration of the antimicrobial peptide gradually decreases from the surface to the inside in the thickness direction of the antimicrobial film.