GB2631119A

GB2631119A - Anti-bacterial article

Info

Publication number: GB2631119A
Application number: GB2309286.9A
Authority: GB
Inventors: Tatlock Elizabeth; Zdziarska Marzena
Original assignee: Innovia Films Ltd
Current assignee: Innovia Films Ltd
Priority date: 2023-06-20
Filing date: 2023-06-20
Publication date: 2024-12-25
Also published as: WO2024261173A1

Abstract

Anti-bacterial article comprising a substrate having a layer of immobilised phage thereon, wherein the phage are immobilised on the substrate by ionic bonds and wherein said layer of immobilised bacteriophage further comprises a non-ionic surfactant (e.g. alcohol ethoxylate). The ionic bonds may arise due to corona treatment of, or a primer layer upon, the substrate. The primer layer may comprise a polyurethane. The substrate may be a polyolefin film. Film may have a clarity of more than 90 %, haze of less than 10 %, transmission of more than 90 % or a gloss of more than 60 %. A method of creating the anti-bacterial article comprising the step of coating a substrate with the solution of phage and non-ionic surfactant. Coating step may be gravure coating. Solution may be dried on substrate at above room temperature, preferably > 40 °C. Also claimed is a packaging made from the anti-bacterial article.

Description

ANTI-BACTERIAL ARTICLE

The present invention concerns an anti-bacterial article and methods of making such an article.

A bacteriophage (phage) is a virus that specifically infects and replicates within bacteria. The application of phage in a wide variety of industries, from food preservation to wound dressings, is known in the art, as they provide a natural, readily available anti-bacterial material, that is not harmful to humans.

W02019/150121 discloses a solution of phage, which includes a buffer and one or more salts. This document discloses a variety of different phage species and mixtures thereof, which are formulated for application to food, to plants and/or to seeds prior to packaging, in order to decontaminate them and prevent rot.

However, the associated increase in moisture in the packaging following said spray was found to promote spoilage or contamination of foods, as well as increasing fogging within the package which is aesthetically detrimental. Free phage also have reduced activity against bacteria and a reduced reproductive rate. Thus, ways of immobilising phage on a surface were investigated, as discussed in W02019/063810, which discusses a packaging containing phage covalently attached to a surface.

This is further discussed in US2005/220770, which discusses the covalent bonding of the head region of a phage to a substrate, thereby leaving the tail region, which is necessary for bacteria-specific recognition, free to infect bacteria. Various coupling agents are disclosed that can form a covalent bond between the phage and the substrate, including carbodiimide and glutaraldehyde.

This is in line with the general understanding in the art that covalent bonding was responsible for adhering the phage to a substrate.

US2009/053789 discloses a further mechanism for immobilising phage on a substrate. This document discloses a method of binding phage to particles by exposing the particles to an electrical discharge and subsequently mixing the particles with the phage. However, some phage will be bound in such a manner as to obscure the site at which they bind to bacteria. Additionally, this method involves specialist machinery and is relatively energy-intensive.

Phage can also be destabilised in solution, thereby reducing their efficacy in the resulting product. Thus, a cheaper and simpler mechanism for immobilising bacteriophage on a substrate in a manner that retains their activity against bacteria is required.

According to a first aspect of the present invention, there is provided an anti-bacterial article comprising a substrate having a layer of immobilised phage thereon, wherein the phage are immobilised on the substrate by ionic bonds and wherein said layer of immobilised phage further comprises a non-ionic surfactant.

It was surprisingly found that phage can be adhered to the substrate using ionic bonding, rather than covalent bonding, and that the use of a non-ionic surfactant surprisingly retained phage activity levels and created a film with improved optical properties, at least in part because it did not interact with the ionic bonds between the phage and the substrate. Thus, it was found that the use of a non-ionic surfactant both sufficiently immobilised the phage on the substrate and ensured that the anti-bacterial activity was retained.

Additionally, without wishing to be bound by theory, the non-ionic surfactant appears to protect the activity of the phage, particularly during the application process and the subsequent drying process. This may be through the formation of micellular orientation of both the phage and the non-ionic surfactant in solution. Thus, the present invention provides a substrate with an improved phage activity. At least 25%, preferably at least 40% of the substrate, may have 1x104 PFU/cm3 or more after the coating has been dried.

The inclusion of a non-ionic surfactant when applying the phage also surprisingly improved the distribution of the phage across the surface of the substrate. Thus, there may be less than 25%, preferably less than 10%, of the substrate that is absent phage after the coating has been dried. There may be less than 25%, preferably less than 10%, of the substrate that has 10 PFU/cm2 or less after the coating has been dried. There may be less than 25%, preferably less than 10%, of the substrate that has 5 PFU/cm2 or less after the coating has been dried.

Non-ionic surfactants are known in the art to reduce the fogging of a film. Thus, any of the conventionally known anti-fogging non-ionic surfactants could be used in the present invention.

Alcohol alkoxylates, particularly alcohol ethoxylates (such as Lutensol"), are preferred. The term alcohol alkoxylates as used herein includes embodiments in which the alkoxylates are aliphatic or aromatic hydrocarbon units. Thus, alkyl phenol alkoxylates are also included. It has surprisingly been found that alcohol ethoxylates improve phage activity retention compared to other non-ionic surfactants.

The substrate may comprise a primer layer, which may form ionic bonds with the phage. This can be any primer layer that increases the surface energy of the substrate by increasing the polar groups present on the surface (as determined by contact angle measurement that quantifies polar and disperse components of surface energy). The primer layer may be a polyurethane primer layer. By "polyurethane primer layer", it is meant a primer layer that contains polyurethane. Preferably, the primer layer contains more than 80% polyurethane, more preferably more than 90% polyurethane.

Primer layers have been used in other applications to solve reticulation, as the primer layer can create a surface tension that is more compatible with water-based solutions. However, the inclusion of such a primer layer would be expected to make the covalent bonding of the phage to the substrate more difficult as instead, primer layers tend to favour the creation of other bonds such as ionic bonds. Thus, the conventional understanding in the art that the phage was attached to the substrate using covalent bonding would suggest that a primer layer should not be included.

The inclusion of a primer layer has surprisingly been found to both improve immobilisation of the phage on the substrate and create a long activity life. It was previously thought that covalent bonds between the phage and the substrate were required for both immobilisation of the phage and activity retention. However, the use of a primer layer, particularly a polyurethane primer layer, that creates ionic bonds with the phage rather than covalent bonds has surprisingly been found to work, and even to create a long phage activity life.

The inclusion of a primer layer has surprisingly been found to further improve the distribution and activity of the phage immobilised on the substrate. This is thought to be due to the increased surface energy resulting from the polar groups on the primer layer. This also means that the use of a non-ionic surfactant does not interfere with the ionic bonds between the phage and the primer layer.

Additionally or alternatively to a primer layer, the surface of the substrate onto which the layer of immobilised phage is applied may have been corona treated. This also increases the polar groups present on the substrate and therefore its ability to create ionic bonds with the phage. The corona treatment may be conducted at a level of between 10 and 50 Wimin/m2, preferably between 15 and 45 W/min/m2. Thus, the ionic bonds between the phage and the substrate can be due to corona treatment and so corona treating a film can increase the number of phage adhered thereto.

As with the inclusion of a primer layer, the increase of phage adhesion resulting from corona treatment in combination with the use of a non-ionic surfactant surprisingly results in an improved immobilisation of the phage on the substrate, as well as an improvement in the phage activity.

The substrate may be a film. The substrate may be a polyolefin film, such as a polypropylene or a polyethylene film. This film may be monoaxially or biaxially oriented. The film may be a monolayer film or a multilayer film. The film may be heat sealable. This provides a flexible anti-bacterial article that can be used in applications such as packaging.

The film may have a clarity of more than 90%, preferably more than 93%. The clarity of the film comprising the phage may be less than 10%, preferably less than 5% different to an identical film that does not comprise the phage and the non-ionic surfactant. The identical film in this instance may be identical to that of the invention but without any coating, or a buffer such as Phosphate Buffered Saline (PBS) may have been applied instead of a coating containing phage and a non-ionic surfactant.

The film may have a haze of less than 10%. The haze of the film comprising the phage may be less than 10%, preferably less than 5% different to an identical film that does not comprise the phage and the non-ionic surfactant. The identical film in this instance may be identical to that of the invention but without any coating, or a buffer such as PBS may have been applied instead of a coating containing phage and a non-ionic surfactant.

The film may have a transmission of more than 90%, preferably more than 93%. The transmission of the film comprising the phage may be less than 10%, preferably less than 5% different to an identical film that does not comprise the phage and the non-ionic surfactant. The identical film in this instance may be identical to that of the invention but without any coating, or a buffer such as PBS may have been applied instead of a coating containing phage and a non-ionic surfactant.

The film may have a gloss of more than 60%, preferably more than 70%. The gloss of the film comprising the phage may be less than 10%, preferably less than 5% different to an identical film that does not comprise the phage and the non-ionic surfactant. The identical film in this instance may be identical to that of the invention but without any coating, or a buffer such as PBS may have been applied instead of a coating containing phage and a non-ionic surfactant.

The substrate may also be particles or other materials, such as those for wound dressing.

The substrate may retain phage activity for at least 5 months, preferably at least 7 months. The phage activity retained is preferably sufficient to significantly reduce the spoilage of food in contact with the substrate.

According to a second aspect of the present invention, there is provided a method of creating the antibacterial article discussed above, comprising the step of coating a substrate with the solution of phage and a non-ionic surfactant. The phage attach to the substrate by ionic bonds.

The solution may be an aqueous solution. The solution may further comprise a pH buffer in order to stabilise the pH of the solution. The buffer may be, for example, phosphate buffered saline. The solution may further comprise one or more salts, such as chloride salts.

The substrate may be corona treated before the solution is coated on the corona treated surface. The corona treatment may be to a level of between 10 and 50 W/min/m2, preferably between 15 and 45 W/min/m2. This can increase the number of ionic bonds between the substrate and the phage.

Additionally or alternatively, the substrate may comprise a primer layer, optionally a polyurethane primer layer. As with corona treatment, this increases the number of ionic bonds between the substrate and the phage.

The coating step may comprise any known coating method, including but not limited to direct gravure coating (forward and reverse), offset gravure coating, slot die coating, wire rod coating, dip coating, spray coating or kiss coating. It was surprisingly found that higher shear/turbidity coating methods such as reverse gravure coating did not detriment the activity of the phage in the present invention, as would be expected due to phage lysis or other physical damage.

The coating may be applied at a wet coat thickness of between 5 and 30 pm, preferably between 7 and 20 pm.

The solution may be dried on the substrate at above room temperature (20°C), preferably at above 40°C, in order to create an anti-bacterial article. The solution is also preferably dried at a temperature below that which would be detrimental to the phage and/or the substrate. For example, the solution is preferably dried at a temperature below 100°C. An elevated temperature may be created by an oven, which may run at a speed of between 5 and 20 m/min and may have a length of between 1 and 3 m.

Alternatively, the solution may be dried at or around room temperature (20°C).

It was surprisingly found that the non-ionic surfactant had a greater effect on phage stability when the solution was dried at elevated temperature rather than at room temperatures. Without wishing to be bound by theory, this was thought to be due to the prolonged drying process and the transition state being extended if drying is conducted at room temperature.

The presence of a non-ionic surfactant in a phage solution has surprisingly been found to improve the stability of the phage once it has been incorporated onto a substrate. Additionally, the presence of a non-ionic surfactant has surprisingly been found to protect the phage during the coating and drying process, particularly at high temperatures.

The non-ionic surfactant may be present at 0.1 to 2% of the solution, preferably 0.2 to 1.5% of the solution.

According to a third aspect of the present invention, there is provided a packaging made from the anti-bacterial article discussed above.

This packaging may demonstrate improved phage adhesion and activity, as well as improved optical properties.

The anti-bacterial article may be a film, which can then be wrapped around an item to create a packaging. The film may be shaped to form a pouch. The film may be heat sealable in order to create a packaging.

The packaging can be used to improve the shelf life of items, such as fresh produce, by killing the bacteria responsible for the degradation of said item. Preferably, the packaging is air-tight.

Any feature discussed in reference to one of the aspects of the present invention applies equally to all of the other aspects discussed herein.

The invention will now be more particularly described with reference to the following examples and figures, which are not intended to be limiting to the scope of protection and in which; Figure 1 illustrates the optical properties of films containing a non-ionic surfactant and those without a non-ionic surfactant; Figure 2 illustrates the effect of different additives on phage activity; Figure 3 illustrates the effect of a non-ionic surfactant on phage distribution after application; Figure 4 illustrates the effect of different non-ionic surfactants on phage activity; and Figure 5 illustrates the effect of a substrate of the present invention on colour, texture and buyability of spinach leaves over time.

Example 1

T4 phage, a bacteriophage for Escherichia coli (E-coli), were immobilised onto the surface of a corona treated or polyurethane primed 30 pm thick biaxially oriented polypropylene film. The phage was applied using either a Phosphate Buffered Saline (PBS) solution, or a solution of PBS with 0.7% Lutensol TO89. The coating was applied using an RK coater at a wet coat thickness of 12 p.m and left to air-dry at room temperature (20T) and pressure before visual assessment and photography.

Figure 1 illustrates the resulting images of the coated surface against a black background. Sample A illustrates phage applied onto a polyurethane primed surface from a PBS solution containing 0.7% Lutensol TO89, while Sample B illustrates phage applied onto a polyurethane primed surface from a PBS solution. Sample C illustrates phage applied onto a corona treated surface from a PBS solution, while Sample D illustrates phage applied onto a corona treated surface from a PBS solution containing 0.7% Lutensol TO89.

As demonstrated in Figure 1, Sample A demonstrates no reticulation or visible salt lines, while Sample B demonstrates very fine reticulation across the entire surface. Sample C demonstrates severe reticulation across the surface, while Sample D demonstrates minimum reticulation or tide marks on the surface.

Thus, the inclusion of Lutensol (a non-ionic surfactant, specifically an alcohol ethoxylate) improves the optical properties of both corona treated and polyurethane primed surfaces to which a PBS solution containing phage has been applied.

Example 2

To assess the effect on phage activity of non-ionic surfactants, T4 phage in a variety of different solutions was applied onto 30 tm thick biaxially oriented polypropylene film using the drawdown technique, with a red kbar. The liquid coating was dried at room temperature (20°C) and pressure.

The following solutions were used, each containing the same amount of phage: Sample No. Solution 1 Phosphate buffer solution (PBS) 2 PBS and 0.7% Lutensol TO89 3 50: 50 Isopropylalcohol: deionised water 4 PBS and Trehalose PBS is frequently used as a stabilising agent for phage in solution and so was selected as a baseline sample. Isopropylalcohol (IPA) is known to reduce reticulation and phage have been viable in solutions with high concentrations of IPA, so this composition provides a solution to compare the effect of non-ionic surfactants against. Trehalose was selected due to its similar chemistry to the non-ionic surfactant, thereby providing another solution to compare the effect of non-ionic surfactants against.

T4 phage activity across an 8 cm by 12 cm sheet of 30 pm thick biaxially oriented polypropylene film was assessed, by splitting the sheet into a grid and measuring phage activity in each section of said grid. This was achieved by measuring plaque forming units (PFU) present per cm2 in each grid section using a plaque well plate assay. The resulting activity levels are shown in Figure 2.

As demonstrated in Figure 2, the incorporation of Lutensol T089 improves both the distribution of phage across the surface and also improves the concentration of active phage across the surface. For example, the use of PBS alone or IPA with the phage resulted in various grid sections that did not contain phage, while both Lutensol and trehalose created an even distribution of phage over the grid.

Further, the inclusion of Lutensol resulted in a much higher concentration of active phage than any of the other solutions, thereby demonstrating that non-ionic surfactants can act to improve the phage activity on a substrate. While trehalose did prove to be a good carrier medium and allowed for distribution of the phage across the surface, the number of PFU (plaque-forming units)/cm2 was one log lower than for the sample with Lutensol across the majority of the surface.

This experiment also showed that IPA reduced the amount of reticulation on the surface, but this did not lead to an increase in phage activity across the surface. Instead, upon drying, the phage were not active. Thus, an improvement in distribution and/or optical properties does not necessarily result in an improvement in phage activity.

Example 3

A series of samples were produced on an RK pilot coater using either reverse gravure or kiss coating, with T4 phage in either PBS or PBS and 0.7% Lutensol. A 120 trihelical gravure was used, and the substrate was a 300 mm wide, 30 tm thick biaxially oriented polypropylene film that had been corona treated at a level of 32.7 W/min/m2.

The samples were dried at ambient temperatures, 50°C and 70°C, using an oven length of 1.8 m and a running speed of 10 m/min. These samples were then assessed for phage activity by identifying number of PFU/cm2.

It was expected that kiss coating would lead to more viable phage as this is a less aggressive technique with reduced shear/turbidity at the coating head. In contrast, the aggressive nature of reverse gravure was expected to cause phage lysis, or physical damage to the phage. However, as demonstrated in Figure 3, it was surprisingly found that kiss-coated samples had fewer viable phage compared to reverse gravure coating.

Further, as illustrated in Figure 3, it was also found that there was a higher degree of phage activity for samples that contained Lutensol TO89 compared to the phage in PBS only. This improvement was significantly greater where the phage solution was dried using oven heat, at both 50T and 70T, with a 1 x 103 PFU/cm2 improvement due to the presence of Lutensol at both drying temperatures.

It appears that Lutensol T089 may have provided protection of phage during the drying process.

Without wishing to be bound by theory, it is postulated that this could be via the formation of micellular orientation of both the phage and Lutensol TO89 component in solution.

Additionally, it can be noted that there is an improvement in phage activity when a phage solution containing Lutensol is dried at an elevated temperature compared to ambient temperature. The protection provided by Lutensol may be less effective at room temperature due to the prolonged drying process and the transition state being extended, as phage typically destabilise in liquid over a prolonged period of time if not stored correctly.

Example 4

Polyurethane primer was applied onto 30 1.1m thick biaxially oriented polypropylene film, before the application of a T4 phage solution in PBS containing one of three commercially available surfactants, namely Radiasurf 7147 (glycerol stearate), Lutensol T089 (alcohol ethoxylate) and Borchi Gol LA150 (polyether modified polysiloxane), at a variety of concentrations.

It was surprisingly found that the polyurethane primer layer enabled efficient immobilisation of bacteriophage, with phage retaining their activity. Further, as demonstrated in Figure 4, Lutensol TO89 showed the smallest drop in bacteriophage population over a given time period. While the other agents trialled were also non-ionic surfactants, they did not allow for the same phage stability with time.

Example 5

The effect of the substrate discussed herein on shelf-life was assessed using a PBS-coated biaxially oriented polypropylene film and a phage-coated biaxially oriented polypropylene film according to the present invention. In all samples, the biaxially oriented polypropylene film was corona treated with a treatment level of 32.7 W/min/m2. A 120 trihelical gravure was used to apply 12.tm wet coating, via reverse gravure technique, onto the treated polypropylene film. The coating containing PBS, and optionally also 0.7% Lutensol TO89 and active phage material, was then dried onto the polypropylene film surface at a temperature of 50°C.

The optical properties of the films are reproduced in the table below and compared to a control corona treated biaxially oriented polypropylene film, without a coating: Small bags of spinach were produced by producing pre-formed bags on a Vertical Fill Form Seal machine. The pre-formed bags were slit just below the top-seal, before 10 g of spinach was added to each bag. The bags were then sealed using an RDM Heatsealer with crimped jaws. Seal settings were 130°C/130°C, 10 psi and 2 second dwell time. A total of 40 bags were produced for assessment.

The bags were then kept at room temperature (20°C) and pressure, with a panel of 10 people visually assessing salad bags over time. The assessors were split into 2 groups and asked to track the changes of 20 bags until the point they would no longer purchase the bags, based on both colour and texture.

Buyability was also assessed. Each group assessed 10 bags coated with PBS (PBS bags) and 10 bags coated with PBS + 0.7% Lutensol TO89 + active phage material (phage bags).

The assessors were asked to provide a grading for the spinach leaves out of 5, for both colour and texture. Thresholds of 3 and 3.5 were selected for both colour and texture. The amount of days following sealing of the bags until these thresholds were reached was measured. As demonstrated in Figure 5a, the number of days to reach both of these thresholds for colour was higher for the phage Sample Control ettg,gg__ 96.7, 93.45 I§11 4L8L, bags compared to the PBS bags. Further, as demonstrated in Figure 5b, the number of days to reach both of these threshold for texture was higher for the phage bags compared to the PBS bags.

The assessors were also asked to provide a grading for the buyability of the spinach out of 1. The buyability threshold was set to 0.5 and 0.8 and the amount of days until these thresholds were reached was measured. As demonstrated in Figure 5c, the number of days to reach these thresholds was higher for the phage bags compared to the PBS bags.

Overall, bags according to the present invention had significantly longer shelf-lives than bags formed from the substrate and PBS. The shelf-life extension ranged between 0.5 and 1 days, depending on the parameter being assessed. Thus, the substrate according to the present invention has a significant effect on the shelf-life of spinach.

Claims

CLAIMS1. An anti-bacterial article comprising a substrate having a layer of immobilised phage thereon, wherein the phage are immobilised on the substrate by ionic bonds and wherein said layer of immobilised phage further comprises a non-ionic surfactant.
2. The anti-bacterial article of Claim 1, wherein the ionic bonds between the phage and the substrate are due to corona treatment of the substrate and/or a primer layer on the substrate.
3. The anti-bacterial article of Claim 2, wherein the primer layer comprises polyurethane.
4. The anti-bacterial article of Claim 2, wherein the corona treatment is conducted at a level of between 10 and 50 W/min/m2.
5. The anti-bacterial article of any one of Claims 1 to 4, wherein the non-ionic surfactant is an alcohol alkoxylate, preferably an alcohol ethoxylate.
6. The anti-bacterial article of any one of Claims 1 to 5, wherein the substrate is a film, preferably a polyolefin film.
7. The anti-bacterial article of Claim 6, wherein the film has one or more of the following: a. a clarity of more than 90%, preferably more than 93%, b. a haze of less than 10%, c. a transmission of more than 90%, preferably more than 93%, d. a gloss of more than 60%, preferably more than 70%.
8. A method of creating the anti-bacterial article of any one of Claims 1 to 7 comprising the step of coating a substrate with the solution of phage and a non-ionic surfactant.
9. The method of Claim 8, wherein the substrate has been corona treated before coating.
10. The method of Claim 8 or 9, wherein the substrate comprises a primer layer, optionally wherein said primer layer is a polyurethane layer.
11. The method of any one of Claims 8 to 10, wherein the coating step is direct gravure coating (forward and reverse), offset gravure coating, slot die coating, wire rod coating, dip coating, spray coating or kiss coating, preferably gravure coating.
12. The method of any one of Claims 8 to 11, wherein the solution is dried on the substrate at above room temperature, preferably at above 40°C.
13. The method of any one of Claims 8 to 12, wherein the non-ionic surfactant is present at 0.12%, preferably 0.2 to 1.5%.
14. The method of any one of Claims 8 to 13, further comprising a pH buffer and/or salts.
15. The method of any one of Claims 8 to 14, wherein the coating is applied at a wet coat thickness of between 5 and 30 p.m, preferably between 7 and 20 p.m.
16. A packaging made from the anti-bacterial article of any one of Claims 1 to 7.