A METHOD OF OBTAINING A PACKAGING FILM AND A PACKAGING FILM OBTAINED BY THE SAID METHOD

Technical Field

The present invention relates to a method of obtaining a packaging film which has temperature buffering properties so as to take an active role in keeping the temperature of a food/cosmetic/medicine within desired limits, combines antioxidant and antimicrobial properties with capabilities of being effective in enhancing quality and safety as well as shelf life, and is also biodegradable; and a packaging film obtained by using the said method.

Background of the Invention

Food packaging takes a significant role in preservation and transport of foodstuffs without affecting their flavour or quality. Packaging preserves a content against toxins and moisture; ensures that food products are protected from physical damage, external contamination and spoilage and helps to maintain their shape and quality. Packaging also serves as a modem marketing tool for sellers to attract consumers’ attention. It is stated that the global food packaging market has reached a value of 345.3 Billion US Dollar in 2021. The market is expected to reach $483.4 Billion Dollar by 2027. Along with the food retail sector expanding worldwide, the rapid change in food preferences of consumers is one of the most significant factors enabling market growth. The increasing working population have led to increased disposable income and active lifestyles, and increase of consumption of convenience food and ready-to-eat products. Therefore, research groups and leading companies research and develop novel packaging materials in order to extend shelf life of products. Additionally, upon increasing environmental concerns, producers adopt environmentally friendly food packaging by using biodegradable or easily recyclable, reusable and renewable materials.

Therefore, there is need for new options for flexible packaging films which are cost- effective and can be easily applied to industrial production processes; have temperature buffering properties so as to take an active role in keeping the temperature of a food/cosmetic/medicine within desired limits; and will be effective in enhancing quality and safety as well as shelf life, in the packaging industry.

The Chinese patent document no. CN106750580, an application in the state of the art, discloses preparing a chitosan solution and a gelatin solution, respectively; adding glycerin plasticizing agents for mixing, respectively; adding cinnamon essential oil as a natural antibacterial agent; preparing a food packaging film by means of film pouring method by adding Tween 80 as a surfactant and emulsifying it in a mechanical stirrer at 13000 rpm. However, the Chinese patent document no. CN106750580 does not disclose adding pure oleic acid at high ratios as a phase change material so as to provide temperature buffering function to chitosan-gelatin mixture; adding lavender oil in order to strengthen its antioxidant and antimicrobial properties as well; also dispersing lavender oil within a hydrophilic chitosan-gelatin base by creating a nanoemulsion, by utilizing the hydrophilic property of oleic acid; and ensuring that the film does not “vomit” the lavender oil after drying. In addition, it is not also disclosed to obtain chitosan films by means of pouring method by adding lavender essential oil and oleic acid into chitosan-gelatin mixture and then preparing nanoemulsion via ultrasonication.

Summary of the Invention

An objective of the present invention is to realize a packaging film which has temperature buffering properties so as to take an active role in keeping the temperature of a food/cosmetic/medicine within desired limits, combines antioxidant and antimicrobial properties with capabilities of being effective in enhancing quality and safety as well as shelf life, and is also biodegradable; and a method of obtaining a packaging film.

Another objective of the present invention is to realize a chitosan-based packaging film with a thin, flexible structure which has heat absorption-emission capacity and thermal cycling property in a low temperature range, has antioxidant and antimicrobial functions, is thermally and chemically stable, has water barrier property; and an obtaining method thereof.

Detailed Description of the Invention

“A Method of Obtaining a Packaging Film and a Packaging Film Obtained by the Said Method” realized to fulfil the objectives of the present invention is shown in the figures attached, in which:

Figure l is a flowchart of the inventive method.

Figure 2 shows SEM images of chitosan film samples (x2000): a) CH; b) CH-L013; c) CH-LO23; d) CH-LO24; e) CH-LO25

Figure 3 shows DSC curves of a) pure oleic acid and b) the sample code CH-LO25 at the 2 ^nd and 10 ^th heating-cooling cycle.

The components illustrated in the figures are individually numbered, where the numbers refer to the following:

100. Method

The inventive method (100) of obtaining a packaging film which has temperature buffering properties so as to take an active role in keeping the temperature of a food/cosmetic/medicine within desired limits, combines antioxidant and antimicrobial properties with capabilities of being effective in enhancing quality and safety as well as shelf life, and is also biodegradable; comprises steps of preparing a chitosan solution in acetic acid solution (101); preparing a gelatin solution with purified water (102); adding oleic acid and lavender oil into the mixture, by mixing chitosan and gelatin solutions (103); subjecting the mixture, wherein oleic acid and lavender oil are added, to ultrasonication in order to transform it from coarse emulsion into nanoemulsion (104); and obtaining chitosan films upon the ultrasonicated mixture is poured into plastic plate, left to gelation and drying and then kept in an oven at (105).

At the step of preparing a chitosan solution in acetic acid solution (101) of the inventive method (100); an acetic acid (aq) solution of 1% (0.17 M) is prepared by using 99,7% concentrated acetic acid and distilled water at first, in order to prepare a chitosan solution of 2% ([C ₆ HuNO ₄ ]n; CAS No: 9012-76-4; MW: 100,000 - 300,000 gmol ^-1 ) (CH) in acetic acid (aq) solution. Then, 10-20 grams of chitosan is added into 250-500 mL of 1% acetic acid solution. A chitosan solution is obtained by mixing the obtained mixture for 8-12 hours in a mechanical stirrer at 1500-2000 rpm, in a water bath at 70-75°C, as sealed.

At the step of preparing a gelatin solution with distilled water (102) of the inventive method (100); 100-200 g of gelatin is added into distilled water of 500-1000 mL in a beaker and it is mixed in a water bath at 70-75°C with a mechanical stirrer at 300-500 rpm for 120-180 min and then stored in a sealed beaker, in order to prepare a gelatin solution of 10% (CAS 9000-70-8; protein ratio 70-90%, gel strength-Bloom No- 275-325; powder) (GE).

At the step of adding oleic acid and lavender oil into the mixture, by mixing chitosan and gelatin solutions (103) of the inventive method (100); 53.5-107 mL 2% CH (aq), 10.70-21.40 mL 10% GE, 0.5-1.0 mL glycerol are added into a vessel in a hot water bath of 70-75°C and then mixed at 5000-5500 rpm for 60-120 h by means of ultrathorax mixing device (IKA). Mixing is performed at 5000-5500 rpm at the end of 60-120 h and meanwhile a mixture of 1-2 ml of lavender oil (LO) (d=0.879 g/cm ³ ) and 0-4.5 ml of oleic acid (OA) (d=0.89 g/cm ³ ; HLB: 1.0), that is prepared in a separate vessel and functions as both an organogelator and a phase change material (PCM), is added into the vessel in a water bath. Then, 1 -2 mL CaCh (aq) of %5 is added into the vessel in a water bath dropwise. It is terminated at the end of 120-180 h.

At the step of subjecting the mixture, wherein oleic acid and lavender oil are added, to ultrasonication in order to transform it from coarse emulsion into nanoemulsion (104) of the inventive method (100); an ultrasound apparatus (operating frequency and power of the device is 45-50 kHz, 500-550W) probe with a diameter of 5.5:0.0, 5.6 mm and a length of 60 mm is immersed in a vessel containing the emulsion mixture wherein oleic acid and lavender oil are added, and nanoemulsion mixtures are obtained by applying ultrasonication at an amplitude of 70-80% for 20-30 minutes at 40-55°C and antimicrobial and antioxidant activity is developed by using active lipophilic essential oil components at low doses by increasing the stability of these mixtures.

Composition ratios of a sample from the prepared chitosan nanoemulsions by volume are given in the Table la. pH, relative viscosity, conductivity and temperature values of the prepared mixtures are measured and given in the Table lb. In order that complexation occurs between chitosan and gelatin, pH is kept in the range of 4.67-3.87 and it is ensured that the selected pH value is kept over the isoelectric point (pH = 3.7-5.2) of gelatin wherein all gelatin chains are negatively charged and below pH 6.2 so as to prevent chitosan from precipitating from the solution. Table l.a. Composition ratios of chitosan nanoemulsions by volume (CH: Chitosan; GE: Gelatine; Lavender oil:LO; Oleic acid: OA)

5%

2% CH 10% GE CaC12

Nanoemulsion (w:v aq) (w:v aq) (w:v aq) Glycerol samples (mL) (mL) LO (mL) OA (mL) (mL) (mL)

CH 53.50 10.70 0.0 0.0 1.0 0.5

CH-LO10 53 50 10.70 1.0 0.0 1.0 0.5

CH-LO11 53 50 10.70 1.0 1.0 1.0 0.5

CH-LO12 53 50 10.70 1.0 2.0 1.0 0.5

CH-LO13 53 50 10.70 1.0 2.5 1.0 0.5

CH-LO20 53 50 10.70 2.0 0.0 1.0 0.5

CH-LO21 53 50 10.70 2.0 1.0 1.0 0.5

CH-LO22 53 50 10.70 2.0 2.0 1.0 0.5

CH-LO23 53 50 10.70 2.0 2.5 1.0 0.5

CH-LO24 53 50 10.70 2.0 3.5 1.0 0.5

CH-LO25 53 50 10.70 2.0 4.5 1.0 0.5

Table l.b. Composition ratios by mass percent, pH, relative viscosity and conductivity values of chitosan nanoemulsions (CH: Chitosan; GE: Gelatine;

Lavender oil:LO; Oleic acid: OA)

GE

(by LO OA

CH mass (by mass (by mass Relative Conductivity

Sample (by mass %) %) %) %) pH viscosity (mS) CH 37.9% 37.9% 0.0% 0.0% 5.00 62.47 5.70 CH-LO10 28.9% 28.9% 23.8% 0.0% 4.08 37.54 4.44 CH-LO11 23.3% 23.3% 19.2% 19.4% 3.99 17.20 3.89 CH-LO12 19.5% 19.5% 16.0% 32.5% 3.93 18.43 3.77 CH-LO13 18.1% 18.1% 14.8% 37.6% 3.87 19.01 3.73 CH-LO20 23.4% 23.4% 38.4% 0.0% 4.09 19.83 3.60 CH-LO21 19.6% 19.6% 32.2% 16.3% 4.08 20.90 3.46 CH-LO22 16.8% 16.8% 27.7% 28.0% 4.06 20.39 3.43 CH-LO23 15.7% 15.7% 25.8% 32.7% 4.05 24.75 3.40 CH-LO24 13.9% 13.9% 22.9% 40.5% 4.67 39.79 3.28 CH-LO25 12.5% 12.5% 20.5% 46.7% 4.64 43.13 3.38

At the step of obtaining chitosan films upon the ultrasonicated mixture is poured into vessels, left to gel and dry and then kept in an oven (105) of the inventive method (100); the nanoemulsion mixture obtained is poured into tared Petri dishes (2R=11,5 cm) and then left to gel and dry for ten to twelve days at +18°C in a well- ventilated environment. Subsequent to the gelling and drying process, the air-dried nanoemulsion mixture is kept in an oven at 50-55°C for two days and chitosan films are obtained. When the images of the film samples containing LO and OA in different mass ratios within their structure are compared with the CH film, which is a control group; it is observed that film structures of nanoemulsions containing a mixture of lavender essential oil, oleic acid and film base are ensured to be homogeneous, and lavender essential oil disperses uniformly in chitosan film.

The packaging film, in other words chitosan film, obtained with the inventive method (100) is a flexible product which prevents products sensitive to temperature changes such as fresh and/or processed foods, food supplements, cosmetics from being affected by sudden temperature changes in environments where they are stored, by means of thermal properties of oleic acid that is added into the structure thereof at different mixing ratios; has temperature buffering properties so as to take an active role in keeping the temperature within desired limits; slows down oxidation by means of synergistic interaction of chitosan, lavender oil and oleic acid; avoids bacterial growth; preserves the unique taste/odour and colour of foods/cosmetics/etc.; is thermally stable and has very low water vapour permeability; is suitable for packaging, cost-effective and easily applicable to industrial production processes; and is also effective in enhancing quality and safety as well as shelf life. The said packaging film comprises 28.9-12.5% chitosan by mass, 28.9-12.5% gelatine by mass, 23.8-20.5% lavender oil by mass and 0-46.7% oleic acid by mass.

The light transmittance and opacity of the obtained chitosan films were examined. Light transmission of films to ultraviolet (UV) and visible light was determined in an absorption (A) mode at selected wavelengths (800-200 nm) by using a UV- Visible spectrophotometer (SOIF UV-5100 UV/VIS 200-1000 nm, Shanghai Metash Instruments, China). The films were cut into samples of 10 * 40 mm and placed directly on the inner wall of the sample cell of the spectrometer. Zero adjustment was performed in the range of 800-200 nm with an empty sample cell and then the samples were measured. Absorption values were measured at = 600 nm in order to determine the opacity of the film samples. Each sample was tested 3 times. The opacity value for each film was calculated by using equation 1 : Opacity value = (-logic T600)/x . (1) wherein T600 is visible light transmittance at 600 nm and x is film thickness (mm). The light absorption, transmittance and opacity values of chitosan nanocomposite films at 600 nm in the visible region are given in the Table 2.

Table 2. Absorption, permeability, average thickness and opacity values of chitosan films at 600 nm

Average thickness

Film samples Absorption Permeability (cm) Opacity (-logio Tsoo)/x)

CH 0.27 0.543 0.034 7.8

CH-LO10 0.26 0.544 0.039 6.8

CH-LO11 1.13 0.075 0.042 26.8

CH-LO12 1.65 0.023 0.036 45.7

CH-LO13 1.83 0.015 0.042 43.5

CH-LO20 0.75 0.177 0.025 24.7

CH-LO21 0.99 0.102 0.052 18.2

CH-LO22 1.43 0.037 0.046 30.9

CH-LO23 1.79 0.016 0.042 42.1

CH-LO24 1.40 0.040 0.099 14.8

CH-LO25 2.43 0.004 0.093 25.8

As seen in the Table 2, the opacity value of all film samples increased in comparison to the CH control group. The opacity value varied between 14.8 - 45.7, depending on the change of the mass ratio of lavender oil and oleic acid. While the opacity value of the control group (CH) was 7.8, an increase was observed in the opacity value thereof upon the addition of LO and OA. The opacity value of the sample code CH-LO24 wherein LO and OA concentrations were 22.9% and 40.5%, respectively, was calculated as the lowest -namely 14.8- among the experimental groups.

The colour changes of chitosan films were examined. The color change of the films was measured with a precision colorimeter (3nh Co. Ltd., NR.100, Shenzhen, Chain). Upon the Hunter color (L*, a*, b*) values of the standard white plate was measured as L* = 91.71, a* = 0.49, b* = -3.42, a triplicate colour measurement was performed at different positions by placing each film sample on a white plate. The total colour difference (AE) was calculated by using equation 2:

> (2) wherein AL*, Aa* and Ab* indicates the difference of colour parameters of the film samples according to the white standard; namely lightness/darkness (L), a (red- green) and b (yellow-blue).

The effects of addition of LO and OA on the film colour are shown in the Table 3. Addition of LO and OA into chitosan films affected the L (lightness/darkness), a (redness/greenness) and b (yellowness/blueness) values of the film surface significantly. The results are compatible with the opacity values. While the control group CH was lighter in color (L*= 73.04), the L* value of the sample code CH- LO21 (containing 32.2% LO and 16.3% OA) decreased to 57.64 and its darkness increased in comparison to the control group. In CH-LO21, the a* value -which is the indicator of tendency to redness- increased to 15.55 by growing; whereas the b* value -which is the indicator of tendency to yellowness- decreased slightly, namely 48.70, in comparison to the control group. L*=65.19, a*= 13.68, b*= 51.65 values of the sample code CH-LO24 -wherein LO concentration is 22.9% and OA concentration is 40.5%, and opacity value is calculated as 14.8- were measured as (CH) (L*=73.04, a*= 7.66, b*= 49.87), AL*=-7.85 (darkened), Aa*= 6.02 (redshifted), Ab*= 1.78 (light yellow) in comparison to the control group and determined as the sample with lightest colour among the experimental groups. Table 3. Colour values of chitosan films

LO (by mass OA (by

Film samples %) mass % L* a* b* AE*

CH 0.0 0.0 73.04 7.66 49.87 56.92

CH-LO10 23 8 0.0 66.69 9.87 53.00 62.42

CH-LO11 19 2 19.4 60.59 15.03 55.79 68.28

CH-LO12 16 0 32.5 55.46 18.91 47.19 64.92

CH-LO13 14 8 37.6 61.64 13.24 49.66 62.33

CH-LO20 38 4 0.0 60.59 15.41 55.08 67.92

CH-LO21 32 2 16.3 57.64 15.55 48.70 64.06

CH-LO22 27 7 28.0 57.73 17.07 42.78 59.2

CH-LO23 25 8 32.7 61.79 14.01 54.96 66.98

CH-LO24 22 9 40.5 65.19 13.68 51.65 62.53

CH-LO25 20 5 46.7 61.54 14.13 45.31 58.73

SEM images of the chitosan films were examined. In the Figure 2 (a-e), SEM images of the film samples code CH-LO13 ( LO:37.6% and OA: 18.1%), CH- LO23 (LO:25.8% and OA: 32.7%), CH-LO24 ( LO:22.9% and OA: 40.5%), CH- LO25 (LO :20.5% and OA: 46.7%) in different ratios and the sample code CH (LO:0% and OA: 0%) -which is the control group- are included at ^z 2000 magnification. The surface of the control group (CH) is essentially smooth, it does not include pores and cracks, and this indicates that matrix formation has occurred successfully. The distribution of Ca+2(aq) ions added into the matrix mixture as a binder is seen within the chitosan structure in white dots. The EDS results support this observation. The surface of the sample code CH-LO13 containing LO at the highest ratio and OA at the lowest ratio by mass exhibits a relatively rough and fibrous structure. It is observed that this roughness and fibrous structure decrease in samples code CH-LO23 and CH-LO24 wherein the LO ratio gradually decreases and the OA ratio increases, and the emulsion of hydrophobic lavender oil prepared with oleic acid improves the distribution in the hydrophilic chitosan matrix. Slight shrinkage was observed on the surface of the sample code CH-LO25 wherein the LO ratio was LO rate as 20.5% and the OA rate was 46.7%. And this indicates the presence of long chains of oleic acid on the surface, based on the high concentration of oleic acid within the nanoemulsion prepared. However, the surface is not porous and this indicates that the nanoemulsion formation is successful and the lavender oil does not move away from the structure during drying. These observations are compatible with the thickness measurements of films. Thicknesses of the film samples code CH-LO24 and CH-LO25 were measured as 0.099 cm and 0.093 cm, respectively, on average and these values were found out to be quite high compared to the control group (CH) and film thicknesses of the samples (CH-LO10 and CH- LO20) comprising only LO.

Thermal properties of chitosan films were examined. As an example of the DSC behaviours of chitosan films, thermal behaviour of five different chitosan films at the 10 ^th heating-cooling cycle is given in Table 4 and as an example of the thermal cycle property, thermal behaviours of pure oleic acid and the sample code CH- LO25 at the 10 ^th heating-cooling cycle are given in Table 3.

Table 4. Phase transformation properties of chitosan film samples measured by DSC at the 10 ^th heating and cooling cycles, in the temperature range of -55 °C - +40 °C (10 °Cmin ^-1 )

10 ^th Cycle heating 10 ^th Cycle cooling

Sample

Oleic acid -29 -17 133 -4- -38 117

CH-LO21 -32—3 4 -20- -33 2

CH-LO22 -10- 1 2 -18- -29 2

CH-LO23 -26 -20 21 -20—25 2

CH-LO24 -26-20 29 0 - -39 16

CH-LO25 -26-20 36 0 - -39 43

At the 10 ^th heating-cooling cycle of the samples code CH-LO23 (LO: 25.8% and OA: 32.7% by mass), CH-LO24 (LO: 22.9% and OA: 40.5% by mass), CH-LO25 (LO: 20.5% and OA: 46.7% by mass) -which are quite successful in terms of film formation- measured by means of DSC, the temperature range of solid-liquid phase change was measured as -26-20°C and the corresponding heat capacities were determined as 21 Jg ^-1 , 29 Jg ^-1 and 36 Jg ^-1 , respectively. The observed leftward shifts observed at the cooling cycle coincide with the over-cooling tendency of oleic acid. The fact that no change occurs at the 10 ^th heating-cooling cycle, the temperature range of phase change and the heat capacities indicates that the samples are thermally stable and suitable for repetitive usage.

Heat capacity of the chitosan film sample code CH-LO25 corresponds to 27% of 133 Jg' ¹ value of pure oleic acid, which is the heat absorption capacity thereof. The sample code CH-LO25 wherein 46.7% oleic acid by mass is used distinguish by its film properties, antioxidant and antimicrobial structure and heat capacities. Properties of the samples produced in experiments carried out repetitively are similar.

Dynamic mechanical analysis results of chitosan films were examined. The film samples were subjected to dynamic mechanical analysis (DMA) in a single cantilever bending mode by using Perkin-Elmer DMA 8000 according to ASTM D4065 standard. The samples were cut into rectangular plates of 12x12 mm in size. DMA tests were carried out in fixed displacement of 0,02, in the range of 20-60°C, at heating rate of 5°C/min, under nitrogen atmosphere, by using 1 Hz oscillation frequency. As an example, a comparative summary of the DMA results of five different samples is given in the Table 5.

Table 5. Dynamic mechanical analysis results of chitosan films (25 °C)

Stiffness of an object is a measure of the resistance of an elastic body to deformation. A decrease in E’ and stiffness values is a measure of the decrease in resistance to elastic deformation (stiffness, rigidity). Tan 6 values, which are described as the ratio of the loss modulus (E”) and the elastic modulus (E ¹ ) to each other, increased approximately 2.2 times compared to the control group. The CH ratio decreases, whereas a change occurs in the film samples towards viscoelastic behavior as the OA ratio increases.

Water vapour permeabilities of chitosan films were examined. Water vapour permeability test was carried out according to ASTM E96-00 (ASTM. 2004) with some changes. Eppendorf tubes (V=1.5 mL) were filled with anhydrous silica (0% RHc), the rim (2R=9.47 mm) was tightly closed with the film sample and weighed (Wi). A saturated NaCl(aq) solution (75% RHd) of 100 mL was added into a desiccator, the tubes were placed in the desiccator vertically. Experiments were carried out at 20°C. Water vapour permeability ratios of chitosan films at the end of seven days are seen in the Table 6. The addition of LO and OA into the chitosan film caused the obtained films to be denser and too have less water vapour permeability. The water vapour permeability of the control group was 42.6%, whereas the water vapor permeability of the samples code CH-LO23, CH-LO24 and CH-LO25 -which were found out to have the highest thermal buffering performance- decreased significantly and found out to be 11.4%, 9.7% and 6.9%, respectively.

Table 6. Density, average thickness and water vapour permeability ratios of chitosan films after seven days ry Water film vapour

Film samples m; (g) m _t (g) (m _t -m;) g CH 2.4456 2.8000 0.35436

5 1

CH-LOIO 2.5890 2.9233 0.33435 0.13 1.10 0.39 33.1

2 7

CH-LO11 2.4159 2.5636 0.14771 0.06 1.05 0.42 14.6

2 3

CH-LO12 2.5070 2.6742 0.16721 0.07 1.19 0.36 18.5

7 8

CH-LO13 2.4140 2.6061 0.19202 0.08 1.14 0.42 18.9

9 1

CH-LO20 2.2500 2.4474 0.19747 0.09 1.54 0.25 35.1

0 7

CH-LO21 2.5188 2.6864 0.16753 0.07 0.84 0.52 12.8

8 1

CH-LO22 2.3669 2.4536 0.08670 0.04 1.15 0.46 7.9

0 0

CH-LO23 2.3553 2.4678 0.11247 0.05 1.22 0.42 11.4

4 1

CH-LO24 2.3390 2.5637 0.22470 0.10 0.70 0.99 9.7

5 5

CH-LO25 2.4153 2.5720 0.15669 0.06 0.89 0.93 6.9

4 3

Swelling properties of chitosan films based on pH and their solubility at different pHs were examined.

Buffer solutions with pH 3.0 and 7.0 were used for swelling (S) measurements at different pHs. The film samples were cut in 10 x 20 mm size, weighed in tared Petri dishes (minitiai), kept at room temperature for 60 minutes by dropping 2 mL from the buffer solution, and weighed (mrmai) by removing the excess of the solution phase on the sample by means of filter paper. Swelling at different pHs was calculated by using equation 3 :

S=[(mt-minitiat)/minitial] ^X 100 . (3) wherein S= Swelling in water (t=60 ^th minute); minitiai= initial dry mass (g); m _t = Wet mass (g) (t= 60 ^th minute). Upon the above-stated experiment, a buffer solution of 2 mL was added onto each sample and it was kept for 24 hours. Together with the sample petri dish, each film was dried in an oven at 50°C for 24 hours and the final amount of dry substance (mdiy) was determined by weighing. Solubility in water was calculated by equation 4.

Water solubility (WS) ={l-[( ui itiat-uifinai/ni itiain xlOO . (4)

Swelling ratios of chitosan films in buffer solutions with pH 3.0 and pH 7.0 at the end of 60 minutes are given in the Table 7, and their solubility in solutions at the end of 24 hours is given in the Table 8.

Table 7. Swelling ratios of chitosan films in buffer solutions with pH 3.0 and pH

7.0 (t=60 min.)

Film samples m; (g) S (%) CH 0.06440 0.11039 71.4 0.05828 0.08275 42.0 CH-LO10 0.11330 0.25406 124.2 0.11841 0.22506 90.1 CH-LO13 0.18855 0.26942 42.9 0.18065 0.24783 37.2 CH-LO20 0.08970 0.12275 36.8 0.09377 0.12476 33.0 CH-LO23 0.14207 0.19592 37.9 0.14337 0.18765 30.9 CH-LO24 0.16843 0.24408 44.9 0.16021 0.21323 33.1 CH-LO25 0.18126 0.25673 41.6 0.19651 0.25327 28.9

It was observed that the swelling capacity of the films changed slightly depending on the pH and the film swelling capacity, which was relatively higher in acidic medium, decreased in neutral medium. Addition of LO and OA into chitosan film caused the swelling values of films at pH 3.0 and 7.0 to decrease. The swelling values of the control group at pH 3.0 and 7.0 were 71.4% and 42.0%, respectively; whereas the swelling values of the samples code CH-LO23, CH-LO24 and CH- LO25, which had the highest thermal buffering performance, were found out to be 37.9-44.9-41.6 % at 3.0 and 30.9-33.1-28.9% at pH 7.0, respectively, by decreasing significantly at pH 3.0 and 7.0. On the other hand, solubility of LO and OA-doped films reached the ratios of 83-88% at pH 3.0 and 81-84% at pH 7.0 at the end of 24 hours. Table 8. Dissolution ratios of chitosan films in buffer solutions with pH 3.0 and pH 7.0 (t=24 hours)

Antioxidant properties of chitosan films were examined. At this stage, the total amount of phenol was determined. The antioxidant activity of phenolic compounds is based on their capabilities of providing H+ ion (hydroxyl group) and free electron delocalization. The total phenolic content of the films was determined by using Folin-Ciocalteu method (Dou et al., 2018) with some changes. The steps are as follows: The film sample (30 mg) was mixed with purified water of 10 mL in a glass tube at 45°C for 10 minutes at 500 rpm. In order to separate the liquid phase, it was centrifuged at 2500 rpm for 5 minutes upon it was transferred to the centrifuge tube. A liquid phase of 1 mL volume was added into 0.2 M Folin- Ciocalteu reagent of 5.0 mL at room temperature. A mixture was prepared with purified water in the same volume as the control group. After five minutes, 7.5% ISfeCCL of 4 mL was added and then the tube was agitated well. The samples were kept in the dark at room temperature for 60 minutes. The absorbance value of the extracts obtained from the film samples was measured at 760 nm by means of a UV-Visible spectrometer. The concentration of total phenolic compounds in the samples was determined as gallic acid equivalents (GAE). Results reflecting the phenolic content are given in the Table 9 as milligram gallic acid equivalents per gram film (mg GAE/g film). The total phenolic content in GAE (mgGAE/g film) of the samples containing LO and OA increased 2.5-3.9 times compared to the CH control group.

Table 9. Total phenolic content of chitosan films in GAE (mg GAE/g film)

C= c x Vfilm extract

I mfilm (mg GAE/g

Film samples LO% OA% ABS760 CGAE (mg/L) film)

CH 0.0 0.0 0.0250 62.20 2.48

CH-LO10 23 8 0.0 0.1440 300.20 8.65

CH-LO-OA11 19 2 19.4 0.1163 244.80 9.60

CH-LO-OA12 16 0 32.5 0.0880 188.20 6.16

CH-LO-OA13 14 8 37.6 0.0848 181.80 6.48

CH-LO20 38 4 0.0 0.1008 213.80 7.62

CH-LO-OA21 32 2 16.3 0.0886 189.44 6.75

CH-LO-OA22 27 7 28.0 0.0918 195.80 6.51

CH-LO-OA23 25 8 32.7 0.1024 217.00 6.90

CH-LO-OA24 22 9 40.5 0.1440 300.20 8.65

CH-LO-OA25 20 5 46.7 0.1163 244.80 9.60

The antioxidant activity was determined by means of dpph method. The antioxidant activity of the film samples was evaluated by performing the DPPH (2,2-diphenyl- 1-picrylhydrazyl) (Molar mass: 394.32 gmol-1) free radical scavenging test, which is a simple and rapid test, (Wangcharoen & Morasuk, 2007). 10 mg film and 10 mL ethanol were mixed in a plastic centrifuge tube, the mixture was kept at room temperature for 30 min. in the dark by agitating occasionally. Then, it was centrifuged at 500 rpm for 2 minutes. A supernatant 1.5 of mL was taken from the centrifuge tube, mixed with 0.06 mM DPPH(ethanol) solution of 1.5 mL and kept at room temperature for 1 minute. Thereafter, the absorbance at 516 nm was measured. The percentage of DPPH free radical scavenging activity was determined by using equation 5:

DPPH scavenging activity %=[(AbsDPPH-Abssam _P ie)/AbsDPPH] x 100 . (5) wherein: the absorbance value of Abs DPPH = 0.06 mM DPPH (ethanol) solution at 516 nm is the absorbance value of Abs Sample = Sample extracts at 516 nm. Table 10. DPPH scavenging activity of chitosan films (%g-l%)

DPPH DPPH scavenging scavenging

Film samples LO% OA% activity (*> activity /mass (%g-i)

CH 0.0 0.0 52.2 5.7

CH-LO10 23 8 0.0 105.1 9.9

CH-LO11 19 2 19.4 107.1 10.2

CH-LO12 16 0 32.5 120.3 11.4

CH-LO13 14 8 37.6 126.2 12.1

CH-LO20 38 4 0.0 123.5 12.3

CH-LO21 32 2 16.3 119.9 11.3

CH-LO22 27 7 28.0 115.0 11.4

CH-LO23 25 8 32.7 116.6 12.3

CH-LO24 22 9 40.5 83.2 7.9

CH-LO25 20 5 46.7 82.5 7.1

ABTS free radical scavenging activity was carried out. The method of determining ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) free radical scavenging activity of film samples is based on the capability of the antioxidant component released from the films to scavenge the ABTS radical cation. As the concentration of the antioxidant component increases, the oxidation of ABTS is delayed. The ABTS free radical scavenging activity given in the Table 11 showed an increase in parallel with the increase in the phenolic content and the increase in the DPPH scavenging activity.

Table 11. ABTS free radical scavenging activity of chitosan films (TROLOX equivalent mg/L.g film)

ABTS free radical ABTS free radical scavenging scavenging

(TROLOX equivalent (TROLOX equivalent

Film samples LO% OA% mg/L) mg/L.g film)

CH 0.0 0.0 1.32 0.12

CH-LO10 23.8 0.0 1.71 0.16

CH-LO11 19.2 19.4 1.30 0.14

CH-LO12 16.0 32.5 1.37 0.13

CH-LO13 14.8 37.6 1.32 0.13

CH-LO20 38.4 0.0 1.69 0.15

CH-LO21 32 2 16 3 1.32 0.12

CH-LO22 27.7 28.0 1.31 0.12

CH-LO23 25 8 32 7 1.30 0.14

CH-LO24 22.9 40.5 1.71 0.16

CH-LO25 20.5 46.7 1.72 0.16 The antimicrobial activity of films is determined. The antimicrobial activity of films was carried out through an accredited food laboratory by means of detection and enumeration of Total Coliform Bacteria, common foodborne pathogenic bacteria, which is one of the standard tests related to substances and materials in contact with food by using agar diffusion method; detection and enumeration of coagulasepositive staphylococci (Staphylococcus aureus and other species), (TS EN ISO 6888-1); detection and enumeration of Pseudomonas aeruginosa (TS EN ISO 13720); Listeria 1 monocytogenes (SWAP) (TS EN ISO 11290-1) tests and the results are given in the Table 12.

Table 12. Antimicrobial test results of chitosan films

Coagulase

Coliform positive Pseudomonas Listeria 1 bacterium Staphylococcus aeruginosa monocytogenes Film samples (cfu/g) (cfu/g) (cfu/g) (cfu/25 g) he plate. n the plate.

According to the analysis results, formation of coliform bacteria, coagulase positive staphylococci, pseudomonas aeruginosa and listeria monocytogenes -which are common foodborne pathogenic bacteria- were not observed in any sample, including the control group.

Within these basic concepts; it is possible to develop various embodiments of the inventive “A Method (100) of Obtaining a Packaging Film and a Packaging Film Obtained by the Said Method (100), the invention cannot be limited to examples disclosed herein and it is essentially according to claims.