WO2019046981A1

WO2019046981A1 - Antimicrobial composition comprising polyvinylpyrrolidone, polyethylene glycol, polyacrylic acid and copper and use of same; coating for solid surfaces comprising said composition and use thereof

Info

Publication number: WO2019046981A1
Application number: PCT/CL2018/000037
Authority: WO
Inventors: Rodrigo MUÑOZ GONZALEZ
Original assignee: Protevid Spa
Priority date: 2017-09-08
Filing date: 2018-11-08
Publication date: 2019-03-14
Also published as: CL2017002277A1

Abstract

The invention relates to: a new antimicrobial composition including a mixture of polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), polyacrylic acid (PAA) and copper in specific proportions; the use of this composition, which adheres to solid surfaces, thereby providing protection against pathogenic attack; a coating for solid surfaces, formed by the composition comprising PVP, PEG, PAA and copper, the composition adhering to a solid support, which can be paper or film; and the use of the coating to allow water to be partially repelled and UV rays and infrared radiation to be selectively filtered, to control ripening and improve the organoleptic properties of fruit.

Description

ANTIMICROBIAL COMPOSITION COMPRISING POLYINYL PYRROLIDONE, POLYETHYLENGLICOL, POLYACRYLIC ACID AND COPPER, AND THEIR USE, COATING OF SOLID SURFACES COMPRISING SUCH COMPOSITION AND USE

TECHNICAL FIELD

The present disclosure relates to the development of a product for the coating of grape bunches, whose design promotes ventilation and the arrival of phytosanitary products to the fruit; but that also has antimicrobial properties, is easily biodegradable, and presents the ability to partially repel water and selectively filter UV and infrared radiation, in order to control maturation and improve the organoleptic properties of grapes.

BACKGROUND

The export market for table grapes is the largest in the country, with $ 1,530,707 thousand FOB dollars in 2013, representing 10% of total Chilean forestry and agricultural exports to the world (PASO, 2014). In quantity of table grape exported, in the 2014/15 season, Chile exported

748.35 tons (Decofrut, 2015), which represents 52.6% of the total exported by the Southern Hemisphere. The varieties that concentrate the greatest amount of total exports are Red Globe (27.7%), Thompson Seedless (24.5%) and Crimpson Seedless (21%) (Odepa, 2013). The Thompson Seedless (the only white grape on the list) has been experimenting for several years a sustained drop in exports, in fact, emblematic Thompson Seedless businesses in the US were recently faced with problems in the quality of the fruit, since a high proportion of this variety was found with varying degrees of rot in February and something in March, which directly affected sales prices (Red Agrícola, 2015).

There are several factors that can affect the quality of this variety of table grapes, and among the main ones is rotting by Botrytis cinerea, as well as the high radiation of the sun to which they are exposed, which causes loss of color and browning. the shoulders in the grape cluster. To address these problems, producers have considered various alternatives. The simplest and which has given better results is the protection of the grapes through the use of umbrellas or bags that are installed in each cluster. This is an artisanal and totally manual technique, which consists of covering the bunches until they are harvested, protecting them from external factors such as insects and birds, and climatic factors (excessive sun, rain, etc.). However, these protectors present various disadvantages such as:

Slow installation: For the installation of these protectors temporary equipment must be contracted using moorings and / or brackets to position them in the clusters, making the process very slow. It is necessary to consider that per hectare there is an average of 45,000 bunches, and if we consider a plot of 5 hectares of white table grapes, it means an expense for this concept of at least 625 man hours only in the installation of protectors (considering 10 seconds per cluster), which generates a significant increase in production costs.

Problems with the geometry and / or design of the protector: The protectors that currently in the market are either of the "skirt" or "umbrella" type. The skirt type protectors (very popular in Spain (Uva Doce, 2016)) have the great disadvantage that they generate a barrier against phytosanitary products (like fungicides) and prevent the horizontal growth of the cluster, as well as these products are paper, water it slides on the outside, but by capillarity it re-enters, generating humidity around the cluster, favoring the growth of fungi. Additionally, these protectors do not optimize the use of material, which greatly increases the cost of each device. With respect to the protectors type "paragua", these have the disadvantage that they easily lose their geometry product of the effect of capillary water (wrinkle), losing the protection of the cluster. Due to the lack of optimization of its design, an approximate 25% of the raw material is lost in the assembly of the protector, and they do not adequately protect the cluster.

Material: In the market you can find various materials for the preparation of the protectors: plastic, satin paper on the outside, recycled paper, even mineral paper. When evaluating this variety of materials, we have realized that none of these has been evaluated in hydrophobic properties, capacity of filtration of UV rays or infrared radiation, antimicrobial properties, etc., so that no paper is certified and it is unknown which It has the right characteristics for the cultivation of export table grapes. If this information were available, an intelligent use could be made of the protectors, to delay the ripening of the fruit, to increase its concentration of sugars and to improve in general the organoleptic properties of the grape, improving its price and quality.

Additionally, this research could optimize a formulation for the fruit crop, allowing the generation of a portfolio with new related initiatives in the fruit export industry.

Availability of the protector: Nowadays, all table grape producers use some kind of protection of their bunches (especially the white grape producers), however due to the high demand of material, some are in need of protection. cover their clusters with kraft paper or newspaper, those that get wet when watering the parronales, lose their shape easily, favor the proliferation of fungi like Botrytis cinerea and do not let pass the UV rays or infrared radiation that is beneficial for the cultivation of grape.

BRIEF DESCRIPTION OF THE FIGURES.

Figure 1. Protector of grape clusters. In A the protector installed on a bunch of grapes in primary state is observed. In B, a scheme with the format of the protector is observed. In C, the assembly mechanism is observed.

Figure 2. Support produced for transmittance measurement in

spectrophotometer Three structures were made that function as support for the analysis of each material. The dimensions of each component correspond to: Large hollow cylinder: External diameter (DE) 37mm, Internal diameter (DI) 32mm; Small hollow cylinder DE: 29mm, DI: 24mm; Parallelepiped dimensions 28x24x32mm whose roof is made complementary to the large cylinder.

Figure 3. Inoculation scheme for analysis of antimicrobial surfaces. 1: Polyethylene film. 2: 0.4 mL inoculum. 3: Analysis surface. 4: Petri dish. 5: Petri dish lid. DETAILED DESCRIPTION OF THE INVENTION.

Taking into account the development of the protectors seen in the market, which must be assembled using a bracket, there is a need to design an easy and quick assembly system in the field installation. For this purpose, a foldable laminar protector was designed, with a wedge itself to protect the table grape (figure 1).

The format of the protector (figure 1 B) is made to protect the bunch during the whole growth process, its size allows to protect the bunch in its final size, even before the harvest, but it is thought so that it can be installed when the bunch It is still small. Its use, on the other hand, makes its installation fast and easy, without the need for brackets or extra fixings. It is made in such a way that no matter how the operator takes it, or if the person is right or left handed, it can be installed anyway, a detail that becomes relevant when installing large quantities for each hectare planted. The assembly procedure involves the hooking of a corner with arrowhead, which is folded up to the insertion within a draft positioned at the other end of the shaft of the protector (Figure 1C).

In turn, a novel antimicrobial composition is presented that includes a mixture of polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), polyacrylic acid (PAA) and copper in certain proportions, which have been shown to confer a surprising and effective effect on their antimicrobial effect , different from what was tested for each component separately. This surprising effect was found after testing 1100 different formulations. The use of this composition is presented, which adheres to solid surfaces, thus providing protection against the attack of pathogens. It presents a coating of solid surfaces formed by the composition of PVP, PEG, PAA and copper, which is adhered on a solid support that can correspond to a paper or a film. Also, the use of the coating, whose object has the ability to partially repel water and selectively filter UV and infrared radiation, in order to control maturation and improve the organoleptic properties of the fruit.

The coating adheres on a solid support that can be a paper or a film, which is selected from the group comprised of mineral paper, newspaper or newspaper, kraft paper, greaseproof paper, butter paper, film low density polyethylene (LDPE), high density polyethylene film, polyethylene terephthalate film, polypropylene film and polystyrene fil.

EXAMPLES OF APPLICATION Example 1. Antimicrobial activity on the surface

The antimicrobial capacity of the surfaces of each material used as protector in the agricultural market was evaluated. This characteristic is relevant, since a material that presents antimicrobial activity is immediately excluded as a source of microbial contamination for the fruit. If not, this point against becomes an opportunity to innovate and improve the materials of interest.

Example 4.1. Culture media

Culture media prepared in the following way were used:

Liquid medium: 3 grams of yeast extract were dissolved, 10 grams of peptone and 5 grams of sodium chloride in 1000 mL of nanopure water. The pH is adjusted to values between 6.8 and 7.2 using sodium hydroxide or hydrochloric acid. The culture medium is sterilized by the autoclave process under the Conditions of 0.18 MPa, 121 ° C for 25 minutes. Subsequently the medium is stored at room temperature or in refrigeration between 5 to 10 ° C. Solid medium: 5 grams of yeast extract, 10 grams of peptone, 5 grams of sodium chloride and 15 grams of agar agar are dissolved in 1000 ml of nanopure water. Heat with a magnetic / thermal stirrer for a few minutes until the agar dissolves completely. PH is adjusted to values between 7 and 7.2 (at 25 ° C) with sodium hydroxide and hydrochloric acid. It is sterilized by autoclaving under the conditions of 0.8 MPa, 121 ° C

for 25 minutes. It is then stored at room temperature or refrigerated at 5 to 10 ° C.

For the preparation of the bacterial cultures, using a sterile tube, a stock of culture medium of 5 mL was transferred and the bacterium was inoculated from a storage stock of -80 ° C on the culture medium. The mixture was incubated at 35 ° C for 16hr to 24hr. Subsequently, this culture saturated with bacteria was removed 100 uL to inoculate it in a new stock of 5 mL of culture medium to incubate again at 35 ° C for 16 to 24 hours.

Example 4.2. Preparation of the tests.

For the development of the test, it will be carried out in at least three samples of each treated test material. At least six specimens of the untreated material are required. Half of the untreated test samples are used to measure viable cells immediately after inoculation and half are used to measure viable cells after incubation for 24 h. The use of more than three replicate copies of the treated test material can help reduce variability, especially for materials that show minor antimicrobial effects.

When testing a series of antibacterial treatments for a single polymer, each antibacterial treatment can be compared to a single set of untreated samples if all assays are performed at the same time using the same test inoculum.

Prepare flat samples (50 ± 2) mm χ (50 ± 2) mm from the treated and untreated test materials. When preparing the samples, care was taken to avoid contamination with microorganisms or foreign organic waste.

In the same way, the specimens were not allowed to come in contact with each other. A metallic device is used to avoid cross contamination, which has no antibacterial effect. The test samples were UV sterilized for 20 minutes before testing.

Example 4.3. Preparation of the inoculum for the test.

Regarding the inoculum preparation for the assay, using a microbiological loop, the pre-incubated test bacteria are transferred as set forth above in a small amount of liquid medium. It should be ensured that the test bacteria are uniformly dispersed and the number of bacteria is estimated using direct microscopic observation and a counting chamber or other appropriate method (eg, spectrophotometrically). To subsequently dilute this suspension with 1/500 in liquid medium, as appropriate for the estimated bacterial concentration, to obtain a bacterial concentration that is between 2.5 * 10 ⁵ cells / mL and 10 χ 10 ⁵ cells / mL, with a target concentration of 6 x 10 ⁵ cells / mL.

Example 4.4. Inoculation of the test samples.

For the inoculation of the samples, the surface tested is the exposed outer surface of the product. They were not analyzed in cross sections of the product. Each prepared test sample was placed in a separate sterile Petri dish with the test surface above. 0.4 ml of the prepared test inoculum was pipetted onto the test surface. The test inoculum was covered with a piece of film (polyethylene) measuring 40 mm ^χ 40 mm, which was gently pressed on the film so that the test inoculum extended to the edges. It was observed that the test inoculum does not drip past the edges of the film. After the sample was inoculated and the cover film applied, the lid of the Petri dish was replaced (see Figure 3).

It is essential that the test inoculum does not escape beyond the edges of the cover film. For some surfaces (for example, those that are very hydrophilic), it can be difficult to avoid such leakage. In the case of the papers analyzed, it was resorted to decrease the volume of test inoculum applied to the test surface, taking the safeguard of not using less than 0.1 test inoculum. When the volume of the test inoculum decreases, the concentration of the bacterial cells in the inoculum is increased to provide the same number of bacterial cells as when the normal volume of the test inoculum is applied. For other cases, the viscosity of the test inoculum was increased by adding an inert thickener such as agar to ensure that no leakage occurs. Example 4.5. Incubation of the inoculated samples.

The Petri dishes were incubated at a temperature of 35 ± 1 ° C and a relative humidity of not less than 90% for 24 ± 1 hours. The antimicrobial efficacy of a product was evaluated based on the value of the antimicrobial activity obtained from the assay at the specified incubation temperature.

Example 4.6. Recovery of bacteria from the test samples.

Immediately after inoculation, half of the untreated test specimens were processed by adding 10 ml of LB medium or a suitable and validated neutralizer to the Petri dish containing the test sample. This value was used to determine the rate of recovery of the bacteria from the specimens under investigation. It is important to make sure that the neutralizer completely washes the specimens using a pipette to collect and release the LB medium at least four times.

When working with medium supplemented with agar to increase viscosity, mechanical agitation, such as vortexing or sonication, may be required. If they show a recovery rate equivalent to or greater than that obtained using the above method, such methods can be used. In case it is difficult to recover the test bacteria with 10 ml of the neutralizer due to the size and characteristics of the test sample, then the volume of solution can be increased. If the volume of the neutralizer used is different from 10 ml, the actual volume used will be included in the test report and will be taken into account in the calculation of the antibacterial effect.

After incubation, the remaining samples are processed by counting the viable bacteria recovered from the test sample. Example 4.7. Determination of the viable bacteria count by means of the culture method of pouring plate.

Additionally, a count of the viable bacteria was developed by means of the culture method of pouring plate. For this the viable bacteria were enumerated by carrying out serial dilutions of the culture medium in phosphate-buffered saline. 1 ml of each dilution, as well as 1 mL of the culture medium recovered from the test sample, was placed in separate sterile Petri dishes. 15 ml of LB agar medium was transferred into each Petri dish and stirred gently to disperse the bacteria. All the coating will be done in duplicate. Invert the Petri dishes and incubate them at (35 ± 1) ° C for 40 h at 48 h.

After incubation, the number of colonies in Petri dishes containing 30 to 300 colonies was counted. For each dilution series, the number of colonies recovered was recorded at two significant figures, as well as the dilution factor for the plates used for counting.

Example 4.8. Determination of the number of viable microorganisms.

For the determination of the number of viable microorganisms recovered, equation (1) was used:

^ _ (100x C * Z} xI /) Where N is the number of viable bacteria recovered per cm ² per test sample; C is the average plate count for duplicate plates; Give the dilution factor for the counted plates; V is the volume, in me, of Culture medium added to the sample; A is the surface, in mm ² , of the cover film. Calculate the geometric mean of the number of viable bacteria recovered for each set of test samples and express this value at two significant figures.

Example 4.9. Conditions that must be met to validate the trial.

To consider the validity of the antimicrobial activity test on surfaces, three Conditions must be met:

I. - The logarithmic value of the number of viable bacteria recovered immediately after inoculation of the untreated test samples shall satisfy the following requirement:

( ^α □□□ ~ P □ α □) _< Q 2

^ □□□□

where:

Lmax is the common logarithm (ie, base 10 logarithm) of the maximum number of viable bacteria found in a sample;

Lmin is the common logarithm of the minimum number of viable bacteria found in a sample;

Lmean is the common logarithm of the average number of viable bacteria found in the specimens.

II. - The average number of viable bacteria recovered immediately after inoculation of the untreated test they are within the range of 6.2 x 10 ³ cells / cm ² to 2.5 * 10 ⁴ cells / cm ² .

III.- The number of viable bacteria recovered from each untreated test sample after incubation for 24 h, should not be less than 6.2 χ 10 ¹ cells / cm ²

Example 4.10. Calculation of antibacterial activity

When the test is considered valid, the antibacterial activity is calculated using the equation, recording the result with a decimal.

α = (α _η - □ ") - (t = ¡ _D - c = i ₀ ) = α _α - a _D

R is the antibacterial activity;

Uo is the average of the common logarithm of the number of viable bacteria, in cells / cm ² , recovered from the untreated test samples immediately after inoculation;

Ut is the average of the common logarithm of the number of viable bacteria, in cells / cm ² , recovered from untreated test samples after 24 h;

At is the average of the common logarithm of the number of viable bacteria, in cells / cm ² , recovered from the treated test samples after 24 h.

Example 4.11. Efficacy of the antibacterial agent

The value of the antibacterial activity can be used to characterize the effectiveness of an antibacterial agent. The values of the antibacterial activity used to define the efficacy will be agreed upon by all interested parties. Example 4.12. Activity tests

The experiments were developed using two model microorganisms: Escherichia coli and Streptococcus thermophilus. These microorganisms are representatives of physiological characteristics rooted in their type of cell membrane, being Gram - and Gram +, respectively.

The results are shown below:

Number of colonies per cm ²

Material £ coli S. thermophilus

Diary paper (56g) 840 ± 43 736 ± 34

Kraft paper (80gr) 838 ± 54 735 ± 23

Kraft paper (100gr) 836 ± 32 740 ± 52

Greaseproff paper (35gr) 839 ± 44 732 ± 33

Paper Butter (40gr) 840 ± 32 737 ± 34

Mineral paper (140gr) 835 ± 54 730 ± 53

LDPE film 842 ± 33 738 ± 45

PET (control) 837 ± 33 745 ± 52

The results show that all materials do not have antimicrobial characteristics on their surface, which reinforces their quality as a container focus of phytopathogenic microorganisms.

This experimental background allows us to demonstrate that there are problems that can be counteracted through the use of other materials (in the case of wettability and the effect of radiation absorption) and the research and development of a coating that provides antimicrobial properties to the surfaces of these materials.

Example 2. Antimicrobial additive for surfaces.

A coating of grape bunches was designed, whose design favors ventilation and the arrival of phytosanitary products to the fruit; but which is also easily biodegradable, and has the ability to partially repel water and selectively filter UV and infrared radiation; however, none of the analyzed materials has antimicrobial capacity, so the generation of a coating that is applied on said materials in order to grant this property is required.

In the state of the art, the antimicrobial capacity of spheroidal copper is known, with a broad spectrum of activity on bacteria or fungi; However, to be used to cover the materials analyzed above, two major technical problems must be corrected. The first is to adhere to the surface of the same, and also to look for the conditions that favor their solubilization under the conditions of development of the experiment. how much is a reagent of high insolubility.

With respect to achieving the adhesion of spheroidal copper to the materials, it was decided to use polyacrylic acid (PAA). To this end, 7 mL of a 2% stock of polyacrylic acid (PAA) was prepared in nanopure-grade water at room temperature. The solubility of PAA is slow in time, vortexing was applied at maximum speed for 10 seconds. A consistent foam is formed that easily adheres to the walls of a P1000 tip, which was used to perform the mixing process by pipetting. The stock with foam was left at rest for 12 hours, to evaluate if it presents a dissolution kinetics over time. The results showed the complete solubilization of PAA 2% in water, where no particles were observed in suspension that showed the partial solubility of the reagent. Additionally, a change in viscosity was observed in comparison with the 15 mL falcon tube. The pH of the solution was evaluated, which indicated a value of 1.85 indicating its acid characteristic. The reagent was neutralized by applying 4 aliquots of 3M sodium hydroxide to raise its pH ± 7.

To achieve solubilization of the spheroidal copper, 0.9 g of polyvinylpyrrolidone (PVP) were massed in a 15 ml mini-tube, which was then added with 1.8 g of polyethylene glycol (PEG) 400. The mixture was hydrated with nano-water and mixed by stirring for 10 seconds. It was left to rest for 5 minutes and mixed again by inversion. They achieved complete solubilization of the compound, which changed its viscosity. Subsequently, 0.6 g of spheroidal copper was massed and added to the previous formulation. The result showed insolubility of the copper in the solution which, by means of incubation processes at temperatures above 60 ° C, did not prove to solubilize the copper. However, if the formulation is maintained under agitation, a complete re-suspension of the copper can be obtained, which is aspersible and homogeneously applied on the surfaces.

In this way, a formulation was evaluated that allows to grant antimicrobial capacities on surfaces equivalent to paper.

To this end, a test matrix was prepared that considers a concentration sweep for each selected reagent, establishing the conditions where the lowest possible formulation cost is established, generating the same effect. 1100 different formulations were tested.

The explored conditions were performed in triplicate in papers whose area was 1 cm ² and the results were evaluated based on the removal of the film by abrasion with the fingertips.

The results showed that the use of the formula at concentrations higher than 2% (p / V) of PÁA, generate a film that is not removed by abrasion, maintaining a stable film. It should be mentioned that in concentrations of up to 1% spheroidal copper, antimicrobial activity is observed. Next, it shows the table with the result of colony count, on the surface of mineral paper treated with the formulation whose production complies with the quality / price ratio and its variation of spheroidal copper between 0.5% and 4%.

Count of viable colonies

Formulation (2% PAA) Escherichia coli Streptococcus thermophilus

141 (2% PAA) 436 + 21 371 ± 17

142 (2% PAA) 123 ± 1 1 109 ± 21

143 (2% PAA) 46 ± 10 44 ± 5

144 (2% PAA) 20 ± 4 23 ± 1

145 (2% PAA) 3 ± 1 4 ± 2

Control 898 ± 55 763 ± 43

As shown in the table, the results demonstrate the effect of coating on the viability of bacteria during exposure for 24 hours at 35 ° C. The effect of the formulations in the presence of 0.5%, 1%, 2%, 3% and 4% spheroidal copper, reduces the viability of the growth of £ coli by 48.5%, 13.7%, 5.1%, 2% and 0.3% respectively. On the other hand, for S thermophilus there is a reduction of 48.6%, 14.3%, 5.7%, 3% and 0.5% respectively.

The same exercise was carried out, but applying the coating on a LDPE film, working with the same formulations. The results are shown below: Count of viable colonies

Formulation (2% PAA) Escherichia coli Streptococcus thermophilus

141 (2% PAA) 587 ± 45 389 ± 24

142 (2% PAA) 153 ± 30 122 ± 13 43 (2% PAA) 61 ± 9 52 ± 11

144 (2% PAA) 16 ± 2 14 + 1

145 (2% PAA) 5 + 2 2 ± 0.5

Control 921 + 42 732 ± 31

As shown in the table, the results demonstrate the effect of coating on the viability of bacteria during exposure for 24 hours at 35 ° C. The effect of the formulations in the presence of 0.5%, 1%, 2%, 3% and 4% spheroidal copper, reduces the viability of E coli growth by 63.7%, 16.6%, 6.6 %, 1, 7% and 0.5% respectively. On the other hand, for S thermophilus there is a reduction of 53.1%, 16.6%, 7.1%, 1, 9% and 0.27% respectively.

Based on these results, it is obtained that the coating inhibits growth and bacterial viability.

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Claims

SUBMISSION OF SUBMISSIONS

1. ANTIMICROBIAL COMPOSITION CHARACTERIZED because it comprises polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), polyacrylic acid (PAA) and copper.

2. Use of the composition according to that indicated in claim 1, CHARACTERIZED because it adheres to solid surfaces, thereby providing protection against the attack of pathogens.

3. Coating solid surfaces CHARACTERIZED because it comprises a composition containing polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), polyacrylic acid (PAA) and copper, which is adhered on a solid support that can correspond to a paper or a film.

4. Use of the coating according to that indicated in claim 3 characterized in that it has the ability to partially repel water and selectively filter UV and infrared radiation, in order to control maturation and improve the organoleptic properties of the fruit.

5. Coating according to that indicated in claim 3 CHARACTERIZED because the paper is mineral paper.

6. Coating according to that indicated in claim 3 CHARACTERIZED because the paper is newspaper or newspaper.

7. Coating according to that indicated in claim 3 CHARACTERIZED because the paper is brown paper or kraft.

8. Coating according to that indicated in claim 3 CHARACTERIZED because the paper is greaseproof or greaseproof paper.

9. Coating according to that indicated in claim 3 CHARACTERIZED because the paper is butter paper.

10. Coating according to that indicated in claim 3 CHARACTERIZED because the film is low density polyethylene (LDPE).

11. Coating according to that indicated in claim 3 CHARACTERIZED because the film is high density polyethylene.

12. Coating according to that indicated in claim 3 CHARACTERIZED because the film is polyethylene terephthalate.

13. Coating according to that indicated in claim 3 CHARACTERIZED because the film is polypropylene.

14. Coating according to that indicated in claim 3 CHARACTERIZED because the film is polystyrene.