The adenine-modified edible chitosan films containing choline chloride and citric acid mixture

A series of biopolymeric chitosan-based (Ch) films were prepared with choline chloride and citric acid plasticizer (deep eutectic solvent, DES). An effect of adenine (A, vitamin B4) addition on the functional properties of these films was evaluated. Several physicochemical and mechanical properties were tested: Fourier-transformed infrared spectra proved DES's plasticizing and crosslinking effect, while scanning electron microscopy and atomic force microscopy techniques confirmed the possible phase separation after adenine addition. These changes affected the mechanical characteristics and the water vapor and oxygen permeability. The prepared materials are not water soluble because the CA acts as a crosslinker. The adenine addition on antioxidative and antimicrobial properties was also checked. It was found that Ch-DES materials with A exhibit improved antioxidative properties (55.8–66.1% of H2O2 scavenging activity) in contrast to the pristine chitosan-DES material (51.1% of H2O2 scavenging activity), while the material is still non-mutagenic (lack of growth of Salmonella typhimurium) and possesses antimicrobial features (no E. coli observed for all the tested films and inhibition zones noted for S. aureus). The mentioned properties, reduced oxygen transmission (1.6–2.1 g m−2 h−1), and mechanical characteristics within the range of typical food packaging plastics proved the potential of Ch-DES-A films in the packaging sector. Moreover, the antioxidative properties, usage of substrates being allowed as food additives, and the presence of adenine create the advantage of the Ch-DES-A materials as edible coatings, being also a source of Vitamin B4.


Materials and methods
Materials and solutions.The chitosan powder (Ch) with a degree of deacetylation (DD) equal to 83.4 ± 2.4% applied for films' formation was acquired from BioLog Heppe GmbH (Landsberg (Germany)).Choline chloride (ChCl) (Acros Organics, Poland, 139.62 g•mol -1 ) and citric acid (CA) (Sigma Aldrich, Poland, 192.12 g mol −1 ) were used as received.An acetic acid solution of 2% (w/v) was prepared using concentrated acetic acid (Avantor Performance Materials Poland S.A, purity > 99%).Adenine (A) powder of 135.13 g·mol -1 molecular weight was purchased from Sigma-Aldrich and used as received.Deionized water was used throughout the entire experiment.The chemical structures of Ch, ChCl, CA, and A are given in Figure S1 (Supplementary Materials).
DES was prepared according to the following experimental procedure: choline chloride, previously dried at 70 ºC, acting as the hydrogen bond acceptor (HBA), was mixed with the appropriate amount of citric acid (hydrogen bond donor, HBD) at a 1:1 molar ratio, then the mixture was heated at 95 ºC under stirring until a clear liquid of DES (ChCl-CA) was reached.Afterward, the resulting DES was maintained at room temperature for 2 h before use.
Chitosan stock solution was prepared by dispersing chitosan in 2%(w/v) acetic acid at room temperature.The obtained solution was filtered and degassed.
Adenine stock solution was prepared by dissolving adenine in 2% (w/v) acetic acid to reach 0.01 g·ml -1 .

Film characterization.
Most applied characterization methods were performed according to the previously developed and used for other chitosan-based materials [18][19][20] .
Fourier-transformed infrared spectroscopy.FTIR spectra of all neat components and polymeric films were recorded using FT-IR Vertex 70 V (Bruker Optik) with the diamond crystal in ATR mode.The following recording parameters were used: resolution 4 cm −1 , number of scans 32, wavelength range 400-4000 cm −1 .
SEM and AFM imaging.Scanning electron microscopy (SEM) surface and cross-section imaging was performed using the LEO1430 VP microscope (Leo Electron Microscopy Ltd., Cambridge, UK).Dry samples were immersed in liquid nitrogen and cracked into small pieces before testing.A thin layer of gold was sputtered to improve surface layer conductivity.Surface roughness and morphology of thin films were analyzed at room temperature in the air using a microscope with a scanning SPM probe of the NanoScope MultiMode type (Veeco Metrology, Inc., Santa Barbara, CA, USA), which operated in a tapping mode.Film samples of 1 × 1 cm 2 were cut and subjected to analysis.Surface roughness was determined by measuring the root-mean-square (Rq) roughness and the arithmetic mean (Ra) roughness parameters within the Nanoscope v6.11 software (Bruker Optic GmbH, Ettlingen, Germany).
Mechanical properties.Elongation at break (E b ), tensile strength (TS), and Young's modulus (YM) were determined using an Instron 1193 testing machine equipped with adequate tensile test grips according to the PN-C-89034:1981 with the crosshead speed 1 cm·min -1 .There were at least 10 repetitions per sample.
The thickness of the films (h) was measured in ten different film zones using a micrometer (Absolute Digimatic Indicator, Sylvac S229 swiss, Switzerland) with a precision of 0.001 mm.
The thickness values were used to determine the materials' density.For this purpose, several round-shaped samples were prepared using a circle cutter of known radius (r).The film density was determined by the sample weight and volume π • r 2 • h .or Opacity.Opacity was determined according to the Siripatrawan and Harte 33 method.The films were cut into rectangular pieces and placed in a spectrophotometer test cell.An empty test cell was used as the reference.The film absorbance at 600 nm was measured using a UV-Vis spectrophotometer (Ruili Analytical Instrument Company, Beijing, China).Finally, the opacity of the films was calculated with the following Equation: where: A 600 is the absorbance at 600 nm, and h is the film thickness [mm].According to this Equation, the high values of absorbance result in more opaque and less transparent materials.

Water vapor transmission rate (WVTR) and water vapor permeation (WVP).
The WVTR of films was investigated using the method described by Souza et al. 34 with slight modifications.The tested films were sealed on the top of 29 mm diameter plastic containers containing a known mass of dried calcium chloride (0% relative humidity, RH) and placed at 30 °C in a desiccator containing a saturated NaCl solution (RH = 75%).The desiccant was prepared by drying at 110 °C under reduced pressure for 24 h before use.Five independent samples (1) As the films exhibited different thicknesses, also the WVP was calculated Pv represents the partial pressure difference of the water vapor at test temperature between the two sides of the film (P container = 0 Pa, P saturated NaCl = 3218 Pa, Pv = 3218 Pa at 30 °C).

Oxygen transmission rate (OTR).
The oxygen transmission rate (OTR) of polymer films was carried out by measuring the changes in oxygen content in the distilled water as a recipient using Winkler's method 35 .Deionized water was boiled for 15 min to remove dissolved oxygen, and then 50 ml was transferred to a plastic container (40 mm in diameter), finally, covered with polymer films.The open container, allowing oxygen to enter the flask and dissolve in the water freely, was used as a control.The flasks were placed in an open environment at room temperature for 24 h.The results were expressed as the amount of dissolved oxygen (DO).All studies were carried out in triplicate.The oxygen transmission rate (OTR) was then calculated: where DO-is the dissolved oxygen [g dm −3 ], V-the volume of the water used [dm 3 ], t-time [h], and A-the area of the film exposed for oxygen permeation [m 2 ].
Antioxidant activity.DPPH radical scavenging assay.The antioxidant activity of the film samples was evaluated using DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging assay according to Siripatrawan and Harte 33 with slight modification.Briefly, 500 μl of methanolic extracts of chitosan films (2 g of solid/30 mL of MeOH) was introduced into the test tubes, followed by 2 mL of methanolic solution of DPPH (C = 304 μmol•dm -3 ).The test tubes were kept in the dark at room temperature, and the absorbance was measured at 517 nm.When the DPPH solution was mixed with the sample mixture, a stable non-radical form of DPPH was formed with a simultaneous change of the violet color to pale yellow.The percentage scavenging activity was calculated using the following Equation: where: Abs DPPH is the absorbance of the methanolic solution of DPPH, and Abs extract is the absorbance of the sample extracts.Each sample was assayed at least five times.
Hydrogen peroxide radical scavenging assay.The hydrogen peroxide (H 2 O 2 ) radical scavenging activity of chitosan films was determined using the method reported by Hafsa et al. 36 Film extract was prepared by adding 500 mg film pieces into 15 ml methanol and ultrasonication of the mixture for 3 h, followed by 30 min centrifugation to collect the supernatant.3 mL of fresh 40 mM H 2 O 2 solution (in phosphate buffer pH 7.4) was mixed with 500 μL of different methanolic extracts, incubated at 37 °C for 10 min, and absorbance was measured at 230 nm.A phosphate buffer without H 2 O 2 was taken as a blank.For each concentration, a separate blank sample was used for background subtraction.Antioxidant activity was determined using the Equation: where: A sample is the absorbance of a mixture containing film extract and H 2 O 2 solution, A control is the absorbance of H 2 O 2 solution without film extract.
Biological activity.Antibacterial properties.Antibacterial activity was determined based on the standard: ISO 20645, 2006 "Flat textile products.Determination of antibacterial activity.Diffusion method on an agar plate" 37 Two bacterial reference strains were used in the study: Escherichia coli (ATCC 8739) and Staphylococcus aureus (ATCC 6538P).
The agar medium was inoculated with microorganisms, and a circular sample with a 25 ± 5 mm diameter was applied.Incubation was carried out for 20 h at 37 ± 1 °C temperature, and after that time, the zones of growth inhibition were measured and calculated using the following formula: The assessment criteria described in standard 37 were used to assess the effectiveness of the antibacterial effect of the films (Table S1 in Supplementary Materials).Ames test.A mutagenicity test was performed to determine the direct effect of the test substance or its metabolites on the cell's genotype.The potential mutagenicity of the test samples was analyzed using the in vitro method according to the Ames test.M9 minimal medium ( Na 2 HPO 4 × 12 H 2 O, KH 2 PO 4 , NaCl, NH 4 Cl, agar, MgSO 4 , CaCl 2 , glucose, ampicillin, biotin, histidine) was inoculated with Salmonella typhimurium strain.Then, the samples were placed on the prepared pans.The controls constituted Petri dishes containing only the medium with the Salmonella typhimurium inoculated strain.All plates were incubated upside down and wrapped in aluminum foil for two days at 37 °C.The lack of growth of a significant amount of bacterial cells around the sample indicates that the film is not mutagenic.

Statistical analysis.
All values are presented as mean ± standard deviation.To evaluate the significance of adenine addition on the changes of all the evaluated parameters, the analyses of variance (one-way ANOVA) were performed using SPSS/PS Imago Pro (version 8.0, Predictive Solutions, Poland).Differences were considered significant if the p < 0.05.

Results and discussion
FTIR spectroscopy.FTIR spectra of adenine (A), citric acid (CA), choline chloride (ChCl), and choline chloride-citric acid (1:1 mol/mol) deep eutectic mixture are provided in Supplementary Materials (Figures S2  and S3).In the adenine spectrum (Figure S2) following characteristic bands were noticed 38 . The 3600-2800 cm −1 bands represent symmetrical (3284 cm −1 ) and asymmetrical (3106 cm −1 ) stretching vibrations in the -NH 2 group forming H-bonds.The FTIR spectra of CA, ChCl, and their equimolar deep eutectic mixture (Figure S3) have been discussed in detail by us earlier 20 .It was stated that the formation of hydrogen bonds between both DES components results in the shifting of stretching vibration bands at ca. 3300 cm −1 and vibrational bands of C=O and C-O (in the carboxylic group) characteristic of CA structure.
The analysis of FTIR spectra recorded for Ch and Ch-DES films indicated several differences (Fig. 1).In the Ch-DES spectrum, new intense absorption bands, characteristic of the DES structure, were noticed 39 : (a) 1714 cm −1 (C=O vibration in carboxyl group), (b) 1478 cm −1 (CH 2 bending), (c) 1191 cm -1 (C-O stretching).It can not be excluded that the band at 1714 cm −1 represents not only the vibration characteristic for CA but also the ester bonds formed in the reaction of -COOH groups in CA and -OH functionals of chitosan.As described by Wu et al. 40 , adding citric acid (CA) into the starch/chitosan (PS/CS) mixture resulted in a new peak at ca. 1724 cm −1 in the PS/CS/CA film spectrum.The authors stated that it could be attributed to esterification between CA and both polymers.Similar findings have been made by Seligra et al. 41 for starch-based materials and Priyadarshi et al. 42 for chitosan films plasticized with glycerol and crosslinked with citric acid.
Substantial changes also occurred in the spectral 1500-1650 cm −1 region, where the band at 1540 cm −1 corresponding to N-H bending in the amide group (amide II vibration) and the band at 1631 cm −1 representing C=O stretching in the amide group (amide I vibration) of chitosan were found 43 .After the DES addition, the broad vibrational band with two distinct submaxima (1570 and 1533 cm −1 ) and a small shoulder (1626 cm -1 ) in the spectrum of Ch-DES can be noticed.It is well known that amino groups of chitosan are protonated under acidic conditions and thus can ionically interact with anionic species.The existence of -NH 3 + functionals in Ch-based materials can be confirmed by a new band in the FTIR spectrum located in higher frequencies than those of -NH 2 .Based on the obtained results, it can be assumed that within the Ch-ChCl-CA matrix, the new ionic crosslinking interactions between protonated amino groups of chitosan and -COO -groups of citric acid occur.The changes in the vibrations corresponding to the amide II as a confirmation of ionic crosslinking were previously described by others 44,45 .The simple 24 h solubility test was also performed to prove the crosslinking process's occurrence.The neat Ch-film cast from the acetic acid solution disintegrated in water, while the Ch-DES and Ch-DES-A films retained their solid state.The effect of the ChCl-CA mixture on chitosan film solubility was already shown by us earlier 20 .
The differences between Ch and Ch-DES were also noticed in the 3000-3500 cm −1 region, which corresponds mainly to the vibrations of -NH 2 and -OH.The bands observed in the Ch spectrum are in the spectrum of Ch-DES shifted into the higher frequencies.That indicated the changes in the hydrogen bonding after adding the ChCl-CA mixture to chitosan.It can be assumed that some H-bonds in chitosan are destroyed, but new ones between Ch, ChCl, and CA are formed.
In turn, there are no distinct differences between the Ch-DES and Ch-DES-A3 spectra.It is probably due to the low amount of adenine added concerning the amounts of other film components.Only slight shifting of some bands' positions can be noted.Thus, it can be assumed that in the applied preparation condition, the only interaction between adenine and other film constituents is the hydrogen bonding between -NH 2 groups of A and -NH 2 /-OH groups of chitosan.It should also be mentioned that N atoms in the adenine ring structure, based on the calculated Mulliken partial charges 46 , can also act as H-bonds acceptors.
Surface structure.AFM images show essential information about surface characteristics, including 3D topography images and quantitative data analysis (surface roughness, grain size, step height, etc.).At the same time, SEM imaging gives general specimen property information (compositional microstructure, topography, www.nature.com/scientificreports/shape, etc.). Figure 2 represents three-dimensional and phase AMF images of neat and ChCl-CA modified chitosan-films with different adenine content.Simultaneously, the surface roughness parameters (Ra and Rq) are given.The SEM surface and cross-section images of these same materials are shown in Fig. 3.
The AFM and SEM images of the surface and fracture proved that all tested films are dense and non-porous.The most uniform and smooth surface was noticed for neat chitosan film.The effect of different DESs addition on the chitosan film surface morphology has been observed earlier by us [18][19][20] and others 21,47,48 , especially for those materials where DES content exceeded 60 wt.%.The current findings, an increase in the Rq parameter from 1.38 ± 0.66 nm for neat chitosan film to 21.95 ± 2.10 nm for Ch-DES film, stay in agreement with the literature and can be explained the phase separation.As shown, for lower DES contents, DES penetrates the polymeric matrix and forms a continuous phase with the polymer.When DES content increases, the number of polymer functional groups is insufficient to interact with DES components.Thus, in the case of the Ch-DES film in the current study, the formation of micro intrusions can be suggested.Adding a low amount of adenine does not counteract the Ch-DES separation (Fig. 3, phase images).The AFM phase images revealed that with the increasing adenine content, the surface topography represents the larger nodules.As the size of the nodules increases, the apparent chitosan film roughness decreases, from 17.70 ± 1.80 nm for Ch-DES-A1 to 12.17 ± 1.87 nm for Ch-DES-A3.The higher size of nodules can be related to the chemical structure of adenine (Figure S1, Supplementary Materials).As the upper and lower surfaces of the adenine rings are hydrophobic, thus the addition of the nucleic acid acts synergistically with the DES addition on the chitosan-films surface structure, causing the progressive increase in phase separation phenomenon.Analogous changes in the surface morphology can be observed in the high-resolution SEM images (Figure S4a-e, Supplementary Materials).

Mechanical properties.
The mechanical properties (Young's modulus, tensile strength, and elongation at break) of neat Ch-DES film and Ch-DES films with various amounts of adenine were compared (Fig. 4).The results showed that adenine significantly affected the mechanical resistance and extensibility of the chitosanbased films.
Elongation at the break (E b ) is an indication of the films' flexibility and stretchability (extensibility), which is determined at the point when the film breaks under tensile testing and is expressed as the percentage of change of the original length of the specimen between the grips of a film to stretch (extend) 32 .The E b values of the chitosan films decreased from 146.4 ± 1.5% for neat Ch-DES film to a minimum of 35.8 ± 1.8% when the adenine content reached 3 wt.%(Fig. 4).It can be assumed that this behavior is associated with the interactions of adenine with the Ch and DES components discussed in the FTIR section.As stated by Huang et al. 49 , hydrogen bonds, ionic, metal-ligand, host-guest, and hydrophobic interactions affect the mechanical properties of polymeric materials; however, this impact can be opposed.It was also found that this impact depends on the binding energy in the case of H-bonds.Namely, H-bonds of moderate binding energies can presumably improve the stretchability by delaying fracture or increasing the rigidity while high energy H-bonding (-COOH groups) is formed.In the case of multicomponent films being an object of this study, the overall changes (disrupting of existing H-bonds in Ch-DES, formation of new H-bonds with adenine) cause the formation of less flexible materials.Similar findings have been made by Ojagh et al. 50for chitosan films plasticized with glycerol containing a cinnamon essential oil (CEO).They noticed a substantial reduction in E b after adding a low amount of hydrophobic CEO.Even if a significant reduction of elasticity was detected, the E b values for Ch-DES-A films are still higher than this of pristine chitosan film 51 .
The tensile strength (TS) of the chitosan-based composite films behaved similarly to the E b value, i.e., decreased with the increasing content of adenine.The tensile strength of a material is the maximum tensile stress sustained by the sample during the tension test, expressed as the maximum amount of tensile stress that it can take before failure (breaking or permanent deformation).Pure Ch-DES film exhibited a TS of 18.7 ± 0.5 MPa, while the TS of Ch-DES composite films with 1, 2, and 3 wt.%adenine decreased to 16.4 ± 0.8, 13.4 ± 0.6, and 10.2 ± 1.2 MPa, respectively, and are also lower than TS of neat chitosan film 51 .The observed trend indicates that the adenine causes the reduction of force needed to break the plasticized film.Thus it can be assumed that Ch-DES-A has a less ordered structure than Ch-DES film 52 .The literature data indicate that polymers most commonly used in the food packaging sector exhibit different mechanical properties, depending on the exact application area, especially the type of packaged food.Some packages are expected to be flexible, i.e., films, thin layer laminates, or rigid, i.e., thick plastics.As presented by Robertson 53 , there is considerable differentiation in mechanical characteristics between packages made with different polymers like PLA, PHA, PHB, PET, PS, PP, or LDPE.The TS values of the Ch-DES and Ch-DES-A films are within the range characteristic of polymers most commonly used in food packaging 53,54

Opacity and color.
Color properties are essential for film appearance, influencing consumer acceptance of the packaged products.The results of the measurements performed on the chitosan film's color were expressed following the CIELab system, and the rectangular coordinates (L, a, and b), the total color difference (ΔE), hue angle (Hue), and chroma were calculated (Figs. 5 and 6).The introduction of adenine into chitosan-DES films significantly affected L (lightness/darkness), a (redness/greenness), and b (yellowness/blueness) values of the film surface.It was found that with the higher adenine content, the darker films were obtained as indicated by L values (Fig. 5A), changing from 91.43 ± 2.11 (for Ch-DES film) to 86.37 ± 1.22 (for Ch-DES-A3).Moreover, also other color parameters change continuously: a decreased from -1.61 ± 0.11 to -4.67 ± 0.31 (an indicator of the tendency towards redness), while b values increased from 4.52 ± 0.20 to 8.84 ± 0.32 (an indicator of the tendency towards yellowness), as the adenine concentrations increased from 0 to 3 wt.%(Fig. 5B, C).According to  the ANOVA analysis (Figs. 5 and 6, Table S2 in Supplementary Materials), all observed changes are statistically significant.Moreover, the high values of ΔE (Fig. 6A), calculated concerning the neat chitosan film, indicate that human eyes can notice the color difference.As Hue and C* are computed using a and b, they are indexed somewhat analogous to color saturation or intensity.The C* values increased when the adenine content also increased (Fig. 6B).It can be attributed to the characteristic of pure adenine provided by supplier 55 , who indicated the color of adenine as a light yellow.It was found that the values of Hue (Fig. 6C, Table S2 in Supplementary Materials) do not differ significantly within the applied adenine content.
Based on the opacity values calculated with Eq. 6 (Table 1), it can be stated that chitosan films without adenine were more transparent (lower opacity value) than those incorporated with adenine.The opacity of the film samples significantly increased with increasing adenine concentration.Ch-DES-A3 film has the highest opacity (0.583 ± 0.015) value, followed by Ch-DES-A2 (0.223 ± 0.019), Ch-DES-A1 (0.160 ± 0.013) and Ch-DES (0.039 ± 0.008) films.The increase in the opacity stays in agreement with the AFM analysis and can be explained by the light scattering occurring when different phases are present in the polymeric material.

Water vapor (WVTR) and oxygen transmission rate (OTR). Water vapor and oxygen transmission
properties are essential to food packaging materials.The permeation of oxygen and water from the environment to food has a crucial effect on food quality and shelf life.Oxygen causes food deterioration relying on lipid and vitamin oxidation, leading to sensory and nutrient changes.Thus, one of the main functions of a film for food packaging is to impede moisture and O 2 transfer between food and the surrounding atmosphere, so the WVTR and OTR of the film should be as low as possible.As it is well known, the permeability of a film depends on its chemical structure and morphology, the nature of the permeant, and the temperature of the environment 56 .
The WVP of the chitosan films incorporated with different concentrations of adenine was examined at 30 °C (RH = 75%).The effect of adding ChCl-CA mixture into a neat chitosan matrix was discussed by us earlier 20 and showed that DES addition increases WVTR by 75% in relation to neat Ch.It was ascribed to the hydrophilic characteristic of DES and its plasticizing role, resulting in improved segmental polymer chain mobility.The results of water transport properties through adenine-incorporated Ch-DES (Table 1, Table S2 in Supplementary Materials) showed that adenine also significantly affects the WVTR and WVP of the films.It was found that the addition of adenine substantially increases the WVTR and WVP parameters, but when the adenine content in Ch-DES film increases from 1 to 3 wt.%, the WVTR, and WVP slightly decrease from 37.1 ± 2.9 to 30.9 ± 1.1 g m −2 h −1 , and from 4.1 10 -6 to 3.6 10 -6 g m m -2 Pa −1 h −1 , respectively.Thus it can be stated that undesirable changes in WVTR and WVP values after DES addition are enhanced.The substantial increase in both water vapor transport parameters after adding adenine stands in opposition to the increase in density.However, Table 1.Opacity, thickness, density, water vapor transmission rate, and oxygen permeability of the Ch-DES and Ch-DES-A films.Values with asterisks * represent significant differences (p < 0.05) relative to the Ch-DES.it should be emphasized that in the case of films being an object of this study, adding adenine noticeably changed the internal structure of the chitosan-DES-based films discussed in the SEM and AFM section.Thus, it can be assumed that the enhancement of water vapor transport relates to the phase separation phenomenon.The slight decrease in WVTR and WVP with the increase in adenine content suggests smaller interstitial spaces between the polymeric chains (i.e., higher free volume) that correspond to the increasing film density and the chitosan-DES-adenine interactions (Table 1).When adenine content increases, the formation of a denser network prevents the chitosan from swelling, reducing water vapor transmission through the film 57 .Moreover, when hydrophilic water transport is considered, adenines's hydrophobic characteristics can not be neglected.The changes in WVTR and WVP after the addition of hydrophobic substance were previously discussed also by Bai et al. 58 , and Huang et al. 59 .Bai et al. 58 reported antioxidative active packaging based on carboxymethyl chitosan (CMCS) with different amounts of quercetin.They noted that the addition of hydrophobic quercetin caused an increase in WVP from 15.6 10 -11 g m m −2 Pa −1 h −1 for CMCS to 18.18 10 -11 g m m −2 Pa −1 h −1 for CMCS-quercetin III samples with 7.5 wt.% quercetin content.Huang et al. 59 prepared kafirin(KF)-quercetin (KQ) films for packaging cod (Gadus morhua) fillets during cold storage at 4 °C.The addition of quercetin significantly decreased the WVP of the film from 1.84 ± 0.05 (KF film) to 1.46 ± 0.07 g mm m -2 h −1 KPa −1 .However, the observed reduction was assumed to be very low.The Authors explained this phenomenon as an effect of possible interactions between hydroxyl and carbonyl groups in quercetin with the kafirin resulting in reduced water absorption sites.The polysaccharide-based films' poor water barrier properties (especially under high humidity conditions) are well-known.WVTRs of tested films are 3-100 times greater than those of conventional films made from synthetic polymers 53 , e.g., poly(L-lactic acid) (WVTR = 8.75 g m -2 h -1 , 90% RH, T = 25 °C, 0.25 mm thickness) or LDPE (WVTR = 0.32 g m −2 h −1 , 100% RH, T = 38 °C, 0.76 mm thickness).Thus, further modification is needed to reduce the tested films' water vapor transport properties.

Adenine content [wt.%] Opacity
It was experimentally proved that the oxygen transport represented by OTR values (Table 1) was reduced after adding adenine to the Ch-DES film.Generally, chitosan-based films constitute a good barrier against the permeation of oxygen.According to the previous findings 60 , the chitosan-based films exhibit oxygen permeability (1.6 10 -5 cm 3 m −2 day −1 atm −1 ) similar to the commercially available ethylene vinyl alcohol copolymer films or polyvinylidene chloride (PVDC).In contrast, films based on synthetic polymers (PET, polyethylene, etc.) exhibit higher OTR values than chitosan films 54,60,61 .As DES addition reduces the OTR values 20 , the adenine incorporation exacerbates this effect.Introducing up to 3 wt.% of adenine to the chitosan-DES film induced a significant decrease in the OTR values up to ca. 70%.It can be suggested that the OTR of the Ch-DES-A films decreased with an increasing adenine concentration due to the microstructural changes in the film.This result could be caused by the decrease in the chitosan matrix's free volume by adding the adenine, as mentioned earlier.The results are in accordance with the other literature reports 62,63 .Cerqueira et al. 62 prepared several chitosan-based films plasticized with glycerol or sorbitol, and hydrophobic corn oil.They found that in each case addition of oil reduces the OTR values.The Authors attributed the observed changes mainly to the differences in the internal film structure.
Similarly, Zhong et al. 63 proved that oxygen permeability strongly depends on the interactions between the polymer matrix and the permeating gas.The improvement of oxygen barrier properties (OTR reduction) of chitosan/cassava starch/gelatin blends plasticized with glycerol increased with the increase of cassava starch and gelatin due to the formation of intermolecular hydrogen bonds between NH 4 + of chitosan and gelatin backbone and OH -of cassava starch.The enhanced molecular interaction resulted in a compact structure and low permeable film.
Antioxidant activity.Antioxidant properties, especially radical scavenging activities, and hydrogen peroxide radical scavenging, are significant due to free radicals' deleterious role in foods and biological systems.The oxidation of food makes it unsuitable for consumption.For this reason, novel packages are developed, representing both classic package features and preventing food spoilage.It is especially crucial when the packaging material can be consumed together with food, thus bringing added antioxidative value to the other food components.
The possible antioxidant activity of the films was measured by two methods, DPPH and H 2 O 2 .The application of two methods to evaluate the antioxidant activity has been recommended to better understand the new materials' antioxidant properties.
DPPH radical scavenging assay.DPPH radical scavenging assay has been widely used to test the ability of compounds to act as free radical scavengers and, thus, to evaluate the antioxidant activity of chitosan films.This assay is based on the ability of DPPH, a stable free radical, to be quenched and decolorized in the presence of antioxidants, resulting in reduced absorbance values 65 .The radical scavenging activity of chitosan-DES films with and without incorporated adenine was determined and is given in Fig. 7A.The Ch-DES control film showed a free radical scavenging activity of 48.9 ± 1.6%.It may be because that free radicals can react with the residual free amino (NH 2 ) groups of chitosan to form stable macromolecule radicals, and the NH 2 groups can form ammonium ( NH + 3 ) groups by absorbing a hydrogen ion from the solution 66,67 .It was found that citric acid can also act as an antioxidant and is very effective in retarding the oxidative deterioration of lipids in foods 67 .Incorporating adenine into Ch-DES only slightly changes the free radical scavenging activity.The free radical scavenging activity slightly decreases when 1 wt.% of adenine is reached and then negligibly increases with adenine content.Ch-DES films incorporated with 1, 2, and 3 wt.% of adenine exhibited 38.5 ± 2.8%, 39.8 ± 2.0, and 42.2 ± 3.1% DPPH scavenging activity, respectively.
Hydrogen peroxide radical scavenging assay.Although H 2 O 2 is not a free radical, H 2 O 2 is very harmful to cells because it may cross biological membranes and can be a substrate of the highly reactive hydroxyl radical by a Fenton reaction 36,68 .Avoiding H 2 O 2 from food contact becomes an essential part of food packaging.The inhibition of hydrogen peroxide radical by Ch-DES films incorporated with adenine at varying concentrations is shown in Fig. 7B.The chitosan film with choline chloride-citric acid mixture showed a hydrogen peroxide radical scavenging activity of 51.1 ± 2.1%, which was higher than that reported by Priyadarshi et al. 42 for citric acid crosslinked and glycerol plasticized chitosan film (29.80 ± 0.57%).The scavenging capacities of chitosan films incorporated with adenine increased with increasing adenine concentration, and the results showed that inhibition of H 2 O 2 varied from 55.8 ± 3.2% to 66.1 ± 2.5%.
Thus, even if adenine is not known as an antioxidant, it slightly enhances the antioxidative properties of chitosan-DES films.The ANOVA analysis proved that the differences in scavenging activity between the Ch-DES and Ch-DES-A films are statistically significant (p < 0.05).

Biological activity. Antimicrobial properties.
The results of the bactericidal properties of chitosan membranes with adenine are given in Table 2 and Fig. 8.
A good effect is required for antimicrobial treatment, obtained for gram-negative and gram-positive bacteria described in the method.There was no E. coli observed for all the tested films, while different sizes of inhibition   zones were noted for S. aureus (in the range between 15 and 20 mm) (Fig. 8, Table 2).The results indicated that adding DES to chitosan films results in a film of bactericidal properties.The effectiveness of DES addition to the antimicrobial properties of chitosan materials, was already shown by others.Yu and coworkers 28 found excellent antibacterial properties against E. coli and S. aureus of chitosan films plasticized with choline-based DESs containing acetylsalicylic acid, malonic acid, and lactic acid.Similarly, Zhang et al. 47 showed good antibacterial activity against S. aureus and E. coli of chitosan-lignin containing citric acid, adipic acid, and betaine.The effectiveness of DES as an antimicrobial agent was already discussed by Rachmaniah et al. 69 , Radosevic et al. 70 , and others 71 .It was indicated that the factors affecting the bacterial membrane are still unknown.Adding adenine only slightly reduces the antimicrobial activity, and  within the tested adenine content, the antimicrobial effectiveness is comparable.The observed effect is probably due to the low antimicrobial characteristics of adenine itself 72 .Ames test.Figure 9 shows the testing results providing information about polymeric films' mutagenicity.
The obtained results prove the lack of mutagenicity of the tested samples.In addition, this analysis indicates that the tested samples show bactericidal properties against the Salmonella typhimurium strain.
The results indicated that DES and adenine added to chitosan biopolymeric film do not cause biopolymer mutagenicity.It is a positive finding as DES exhibits negligible mutagenic character 73 , ascribed to the hydrogen bonds formed during its formation and the delocalized charges.

Conclusions
The plasticized chitosan-DES films with adenine have been developed and formed into flat polymeric films.It has been proved that the materials are still dense and non-porous after adenine addition to Ch-DES.The progressive phase separation increased the water vapor permeability by 215%, which is lower when the adenine content reaches higher values (increases of 175%).Oppositely, oxygen transmission is reduced to 2.1-1.6 gm −2 h −1 , and the OTR is lower than commercial HDPE and LDPE packages.Thus, Ch-DES-A films reduce the possible unfavorable food oxidation processes.The DPPH and H 2 O 2 radical scavenging test proved the stated hypothesis that the presence of adenine and its content affects the antioxidative properties of the pristine chitosan-DES material, even if the final effect is not substantial.The changes in physicochemical and antimicrobial properties allow the classifying of the Ch-DES-A films to the active packaging.What is crucial, adenine addition unfavorably changes the mechanical properties, an essential parameter of package material.However, the measured values are still within the typical food packaging plastics range.Even if less elastic than Ch-DES (E b = 146.4%), the three-component films are still more elastic (E b = 35.8%)than neat chitosan.Finally, it can be stated that the developed Ch-DES-A films are an example of a material with very good biological and functional properties that are currently in demand in the packaging industry.However, these materials must still be tested in the "real-use" application conditions.The improved antioxidative properties, usage of substrates being allowed as food additives, and the presence of adenine create the advantage of the Ch-DES-A materials as edible coatings, being also a source of Vitamin B4.

Figure 2 .
Figure 2. AFM 2D and phase images of chitosan films' surface and surface roughness parameters (Ra and Rq).
, i.e., ca. 10 MPa for LDPE, through ca.20 for PHA and 40 for PHB, up to ca. 70 for PET.Moreover, the Ch-DES and Ch-DES-A films

Figure 3 .
Figure 3. SEM images (500 × magnitude) of the surfaces and cross-section of the films.

Figure 4 .
Figure 4. Evolution of Young's modulus (YM), tensile strength (TS), and elongation at break (E b ) of Ch-DES films with different amounts of adenine.

Figure 5 .
Figure 5.Effect of adenine content on (A) L, (B) a, and (C) b values of the Ch-DES films (values with asterisks * represent significant differences (p < 0.05) relative to the Ch-DES).

Figure 6 .
Figure 6.Effect of adenine content on (A) ΔE and (B) chroma values of the Ch-DES films (values with asterisks * represent significant differences (p < 0.05) while ** represents no significant differences (p > 0.05) relative to the Ch-DES).

Figure 7 .
Figure 7. DPPH scavenging activity (A) and hydrogen peroxide scavenging activity (B) of chitosan-DES films incorporated with adenine (values with asterisks * represent significant differences (p < 0.05) relative to the Ch-DES).

Figure 8 .
Figure 8. Bacterial growth and size of the inhibition zone in the presence of samples and after sample removal.

Figure 9 .
Figure 9. Ames test: (a) control-a culture of Salmonella typhimurium (reverse control), (b) tested Ch-DES-A samples in the presence of the Salmonella typhimurium strain (induction of mutation with tested polymer materials).Numbers refer to the adenine content in Ch-DES films.
were prepared for each film.The amount of water vapor permeated was evaluated based on the changes in the weight of the container with the film, and CaCl 2 was determined each 1 h.It was found that 24 h was enough to reach an equilibrium state.The values of WVTR were calculated according to the following Equation:where w-is the weight gained [g], t-time [h], and A-the area of the film exposed to water vapor permeation [m 2 ].

Table 2 .
Size of the growth inhibition zones [mm] of E. coli and S. aureus.