Bio-Based pH Indicator Films for Intelligent Food Packaging Applications

The widespread concerns about the environmental problems caused by conventional plastic food packaging and food waste led to a growing effort to develop active and intelligent systems produced from renewable biodegradable polymers for food packaging applications. Among intelligent systems, the most widely used are pH indicators, which are generally based on a pH-sensitive dye incorporated into a solid support. The objective of this study was to develop new intelligent systems based on renewable biodegradable polymers and a new bio-inspired pH-sensitive dye. The structure of the dye was elucidated through FT-IR and 1D and 2D NMR spectroscopic analyses. UV-VIS measurements of the dye solutions at various pH values proved their halochromic properties. Their toxicity was evaluated through theoretical calculations, and no toxicity risks were found. The new anthocyanidin was used for the development of biodegradable intelligent systems based on chitosan blends. The obtained polymeric films were characterized through UV-VIS and FT-IR spectroscopy. Their thermal properties were assessed through a thermogravimetric analysis, which showed a better stability of chitosan–PVA–dye and chitosan–starch–dye films compared to those of chitosan–cellulose–dye films and the dye itself. The films’ sensitivity to pH variations was evaluated through immersion in buffer solutions with pH values ranging from 2 to 12, and visible color changes were observed.


Introduction
The food industry is confronted with two major problems that have raised concerns at the global level: the accumulation of synthetic plastic and food waste. Petroleum-based materials' availability is limited, and their waste disposal is problematic due to poor degradability, leading to the accumulation of plastic materials and plastic pollution [1,2]. Food waste, which accounts for 20 to 40% of the global production depending on the type of food, is gaining attention not only because of its ethical and economic aspects, but also because it depletes the environment of limited natural resources, which is even more worrying in view of the projected increase in the worldwide population [3,4]. Advances in food packaging materials seem to be the solution to both problems. Other significant aspects that strongly influence food packaging development are food safety regulations, which require constant upgrades and updates, as well as consumers' demands for safe, functional, and convenient packaging to respond to their modern lifestyles [5][6][7]. The above-mentioned reasons led to an increased research interest from both the scientific and industrial communities to develop more advanced types of packaging: active and intelligent systems produced from renewable biodegradable polymers [8,9].
Intelligent food packaging systems are designed to monitor and inform on the condition of food and/or the environment surrounding the food during transport and storage. These systems have the ability to track and record critical parameters for food quality,
All chemicals, reagents, and solvents were used as purchased, without further purification, for the synthesis and preparation of samples.

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Chitosan-cellulose films A 2% (w/v) chitosan solution was prepared by dissolving the appropriate amount of chitosan in 2% (v/v) aqueous acetic acid. To 100 mL of the obtained solution, 0.2 g and 10 mg of the flavylium dye were added. The solution was magnetically stirred at room temperature until all components were dissolved. The mixture was carefully poured into Petri dishes with a 15 cm diameter, which were placed for 5 min in an exicator saturated in NH 3 vapors [39]. Afterwards, the Petri dishes were dried in an oven for 48 h at 40 • C. Following the same steps, a film without the dye was prepared. The polymeric films were carefully peeled off and kept in closed containers at room temperature in dark conditions until characterization.

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Chitosan-starch films The second blend was obtained using a 2% (w/v) chitosan solution prepared as above and a 5% (w/v) starch aqueous solution by dissolving the appropriate amount of starch in distilled water. The ratio between the two solutions was 1:1 (v/v). To 100 mL of this mixture, 0.4 g of glycerol and 10 mg of the flavylium dye were added [40]. The solution was magnetically stirred until the solid was dissolved; then, it was placed into an ultrasonic bath for CO 2 removal. The films were obtained by pouring the solution into Petri dishes with a 15 cm diameter, which were dried in an oven for 48 h at 40 • C. A film without the dye was prepared according to the same procedure. The polymeric films were carefully peeled off and kept in closed containers at room temperature in dark conditions until characterization.

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Chitosan-PVA films The third blend was prepared by mixing a 2% (w/v) chitosan solution and a 10% (w/v) PVA aqueous solution at a ratio of 3:7 (v/v) [41]. To 100 mL of the obtained solution, 10 mg of the pH-sensitive dye was added. The solution was magnetically stirred until the solid was dissolved; then, it was placed into an ultrasonic bath for CO 2 removal. The films were obtained by pouring the solution into Petri dishes with a 15 cm diameter, which were dried in an oven for 48 h at 40 • C. A film without the dye was prepared by following the same procedure. The polymeric films were carefully peeled off and kept in closed containers at room temperature in dark conditions until characterization.

Study of the Halochromic Properties
The investigation of the halochromic properties of the flavylium dye involved measurements of the UV-VIS spectra of the flavylium dye solutions (7 × 10 −5 M in methanol:water 1:14) over time at pH values ranging from 2 to 12. The buffer solutions were prepared by following a previously described procedure [42], and the pH values were measured with a Mettler Toledo Seven Compact S210-K (Mettler Toledo, Columbus, OH, USA) at 25 • C.
The colorimetric response to pH changes was assessed by recording the UV-VIS spectra of 1 × 1 cm polymeric films after they had been immersed for 1 h in buffer solutions ranging from 2 to 12, then washed with distilled water and dried at 40 • C for 4 h.

Infrared Spectroscopy (ATR FT-IR)
FT-IR spectra were registered on a Bruker Vertex 70 (Bruker Daltonik GmbH, Bremen, Germany) spectrometer equipped with a Platinium ATR, Bruker Diamond Type A225/Q, in attenuated total reflectance (ATR) mode. The spectra of the flavylium dye and the polymeric films were obtained through the co-addition of 64 scans in the 4000-400 cm −1 spectral domain with a resolution of 4 cm −1 .

UV-VIS Spectroscopy
The UV-VIS absorption spectra were recorded on an Agilent Cary 60 spectrophotometer (Agilent Technologies, Waldbronn, Germany) at 25 • C.

pH Measurements
The pH of the solutions was measured with a Mettler Toledo Seven Compact S210-K (Mettler Toledo, Columbus, OH, USA) at 25 • C.

NMR Analysis
For the NMR analysis, the samples were dissolved in DMSO-d 6 . The NMR spectra (1D NMR: 1 H, 13

Thermogravimetric Analysis
The thermograms of the films and dye were recorded by using a TG 209 F1 Libra thermogravimetric analyzer (NETZSCH-Gerätebau GmbH, Selb, Germany). The analyses were carried out in a nitrogen atmosphere from 20 to 600 • C, with a heating rate of 10 • K/min. All data were processed with the Netzsch Proteus Thermal Analysis software version 6.1.0. (NETZSCH-Gerätebau GmbH, Selb, Germany).
The melting point of the dye was determined on a Carl Zeiss melting point apparatus (Carl Zeiss, Oberkochen, Germany) and is uncorrected.

Results and Discussion
The development of bio-inspired synthetic pH-sensitive dyes could be the solution to the poor chemical and color stability of natural dyes with applications in intelligent packaging materials for the food industry. Synthetic anthocyanidins can be easily obtained through a sustainable chemical route involving acid-catalyzed condensation between acetophenones and substituted salicylaldehydes.

Synthesis and Characterization of the pH-Sensitive Dye
In this study, we report the synthesis and characterization of a new bio-inspired flavylium dye with possible applications as a pH-sensitive dye for intelligent packaging systems. The reaction scheme is presented in Figure 1.
The pH of the solutions was measured with a Mettler Toledo Seven Compact S210-K (Mettler Toledo, Columbus, OH, USA) at 25 °C.

NMR Analysis
For the NMR analysis, the samples were dissolved in DMSO-d6. The NMR spectra (1D NMR: 1 H, 13 C, DEPT 135 and 2D NMR: COSY, HQSC, HMBC) were recorded on a Bruker AVANCE III spectrometer (Bruker Daltonik GmbH, Bremen, Germany) operating at 500.0 MHz ( 1 H) and 125.0 MHz ( 13 C) at 298 K. The chemical shifts δ are reported in ppm versus tetramethylsilane (TMS), and the coupling constants are reported in Hz. For the splitting patterns, the following abbreviations are used: s (singlet), d (doublet), dd (doublet of doublets), t (triplet), and q (quartet).

Thermogravimetric Analysis
The thermograms of the films and dye were recorded by using a TG 209 F1 Libra thermogravimetric analyzer (NETZSCH-Gerätebau GmbH, Selb, Germany). The analyses were carried out in a nitrogen atmosphere from 20 to 600 °C, with a heating rate of 10 °K/min. All data were processed with the Netzsch Proteus Thermal Analysis software version 6.1.0. (NETZSCH-Gerätebau GmbH, Selb, Germany).
The melting point of the dye was determined on a Carl Zeiss melting point apparatus (Carl Zeiss, Oberkochen, Germany) and is uncorrected.

Results and Discussion
The development of bio-inspired synthetic pH-sensitive dyes could be the solution to the poor chemical and color stability of natural dyes with applications in intelligent packaging materials for the food industry. Synthetic anthocyanidins can be easily obtained through a sustainable chemical route involving acid-catalyzed condensation between acetophenones and substituted salicylaldehydes.

Synthesis and Characterization of the pH-Sensitive Dye
In this study, we report the synthesis and characterization of a new bio-inspired flavylium dye with possible applications as a pH-sensitive dye for intelligent packaging systems. The reaction scheme is presented in Figure 1. The synthetic anthocyanidin was characterized with UV-VIS, FT-IR, and NMR spectroscopy methods. Its thermal stability was evaluated through thermogravimetric analysis.
The FT-IR analysis revealed the presence of the main functional groups and structural elements in the synthesized dye, which were evidenced by the following The synthetic anthocyanidin was characterized with UV-VIS, FT-IR, and NMR spectroscopy methods. Its thermal stability was evaluated through thermogravimetric analysis.
The FT-IR analysis revealed the presence of the main functional groups and structural elements in the synthesized dye, which were evidenced by the following characteristic absorption bands:  The full NMR analysis is presented in the Supplementary Materials. The UV-VIS spectra of the dye showed an absorption maximum at 539 nm (Supplementary Materials, Figure S6).
The thermogram of the dye revealed a single degradation step with an inflexion point at 193.8 • C (Supplementary Materials, Figure S7).

Halochromic Properties of the Dye
The pH-dependent photochromic properties of the synthesized dye were evaluated through an UV-VIS spectroscopy study. The UV-VIS spectra of the dye solutions at pH values ranging from 2 to 12 were registered over time. The overlaid spectra presented in Figure 2a show the existence of multiple species upon pH changes, as evidenced by the shifting of the absorption maximum and by the color modification in time (Figure 2b).
The full NMR analysis is presented in the Supplementary Materials. The UV-VIS spectra of the dye showed an absorption maximum at 539 nm (Supplementary Materials, Figure S6).
The thermogram of the dye revealed a single degradation step with an inflexion point at 193.8 °C (Supplementary Materials, Figure S7).

Halochromic Properties of the Dye
The pH-dependent photochromic properties of the synthesized dye were evaluated through an UV-VIS spectroscopy study. The UV-VIS spectra of the dye solutions at pH values ranging from 2 to 12 were registered over time. The overlaid spectra presented in Figure 2a show the existence of multiple species upon pH changes, as evidenced by the shifting of the absorption maximum and by the color modification in time (Figure 2b). The synthetic anthocyanidins followed the same well-known network of reversible chemical reactions upon pH changes as the natural ones. Figure 3 presents the main species of the synthesized dye involved in the reversible transformations that occurred under different acidic and basic conditions. The synthetic anthocyanidins followed the same well-known network of reversible chemical reactions upon pH changes as the natural ones. Figure 3 presents the main species of the synthesized dye involved in the reversible transformations that occurred under different acidic and basic conditions.  The dye was obtained in its flavylium cation form AH + (λ = 539 nm). This species w stable at pH values below 5. Once the pH rise, there were two compet transformations-a very fast deprotonation of the AH + , which led to the quinoidal bas (λ = 586 nm), and a much slower hydration process, which resulted in the color hemiketal B. In time, the hemiketal was transformed through tautomerization into a chalcone Cc, which, after an isomerization process, gave a trans-chalcone Ct. The chalc species were yellow to orange in color (λ = 418 nm, λ = 456 nm). In very basic conditio the trans-chalcone was fully deprotonated, resulting in the red-colored Ct 2-species ( 500 nm).
All registered UV-VIS spectra are presented in the Supplementary Materials, Fig  S8. The dye was obtained in its flavylium cation form AH + (λ = 539 nm). This species was stable at pH values below 5. Once the pH rise, there were two competing transformationsa very fast deprotonation of the AH + , which led to the quinoidal base A (λ = 586 nm), and a much slower hydration process, which resulted in the colorless hemiketal B. In time, the hemiketal was transformed through tautomerization into a cis-chalcone Cc, which, after an isomerization process, gave a trans-chalcone Ct. The chalcone species were yellow to orange in color (λ = 418 nm, λ = 456 nm). In very basic conditions, the trans-chalcone was fully deprotonated, resulting in the red-colored Ct 2species (λ = 500 nm).
All registered UV-VIS spectra are presented in the Supplementary Materials, Figure S8.

Theoretical Toxicity Evaluation
The toxicity of organic compounds used in food packaging is essential in food security and health. To evaluate the toxicity risk of these compounds, the OSIRIS Property Explorer was used [43]. This is a system that evaluates the risk of mutagenic capacity, tumor generation, and irritating and reproductive effects of tested organic compounds by comparing them with a large set of known structures that emphasize these unwanted properties. Other interesting molecular descriptors calculated are molecular weight, octanol/water partition coefficient (cLogP), solubility (logS), topological polar surface area (TPSA), drug-likeness, and, finally, the drug score, which is a cumulative computed property based on all others.
The above descriptors for malvidin structure (natural compound) and the synthesized dye were calculated using the OSIRIS Property Explorer. The results are presented in Table 1. Based on these results, there is no toxicity risk for either the natural compound or the synthesized one. On the other hand, none of these compounds can be classified as drug-similar (the drug-likeness was negative, and the overall drug score was very small).

Film Characterization
The bio-based films were characterized with spectroscopic methods (FT-IR and UV-VIS) and thermogravimetric methods.

FT-IR
FT-IR spectroscopy was employed to characterize the presence of the main functional groups of chitosan, cellulose, starch, and PVA in the prepared bio-based polymeric films and to evidence the inclusion of the dye in their matrices.

UV-VIS
UV-VIS spectroscopy evidenced the incorporation of the dye in the polymeric matrices. The control films had no significant absorption in the visible region, as shown in Figure 5, whereas the bio-based polymeric films with added dye showed specific absorption maxima.

UV-VIS
UV-VIS spectroscopy evidenced the incorporation of the dye in the polymeric matrices. The control films had no significant absorption in the visible region, as shown in Figure 5, whereas the bio-based polymeric films with added dye showed specific absorption maxima.

Thermal Analysis
The thermal properties of the bio-based polymeric films were investigated with thermogravimetric methods to determine the stability and decomposition temperature. The chitosan-PVA-dye and the chitosan-starch-dye films (Figure 5a,c) had an absorption maximum at about 460 nm, while for the chitosan-cellulose-dye film (Figure 5b), the maximum was evidenced at 470 nm. In all of the UV-VIS spectra of the bio-based-dyecontaining film, two shoulders could be observed around 575 and 650 nm.

Thermal Analysis
The thermal properties of the bio-based polymeric films were investigated with thermogravimetric methods to determine the stability and decomposition temperature.
In Figure 6, the thermograms of all obtained polymeric blends of the chitosan-cellulosedye film, chitosan-cellulose film, and dye are presented. It can be observed that the polymeric films showed three stages of weight loss. In the temperature range of 25-200 • C, there were two weight losses attributed to the moisture loss from the surface water of the films and to the intrinsic water loss, with onsets at 81.6 • C (chitosan-cellulose film) and 177.9 • C (chitosan-cellulose-dye film). The film containing the dye exhibited similar thermal behavior, but the onsets were shifted to 81.1 and 175.6 • C. The most important mass loss occurred in the third stage, in the temperature range of 200-400 • C, with onsets at 256.5 • C for the chitosan-cellulose film and 265.3 • C for the chitosan-cellulose-dye film. Thermal degradation and decomposition occurred in this stage. The chitosan-PVA-dye and the chitosan-starch-dye films (Figure 5a,c) had an absorption maximum at about 460 nm, while for the chitosan-cellulose-dye film (Figure  5b), the maximum was evidenced at 470 nm. In all of the UV-VIS spectra of the bio-baseddye-containing film, two shoulders could be observed around 575 and 650 nm.

Thermal Analysis
The thermal properties of the bio-based polymeric films were investigated with thermogravimetric methods to determine the stability and decomposition temperature.
In Figure 6, the thermograms of all obtained polymeric blends of the chitosancellulose-dye film, chitosan-cellulose film, and dye are presented. It can be observed that the polymeric films showed three stages of weight loss. In the temperature range of 25-200 °C, there were two weight losses attributed to the moisture loss from the surface water of the films and to the intrinsic water loss, with onsets at 81.6 °C (chitosan-cellulose film) and 177.9 °C (chitosan-cellulose-dye film). The film containing the dye exhibited similar thermal behavior, but the onsets were shifted to 81.1 and 175.6 °C. The most important mass loss occurred in the third stage, in the temperature range of 200-400 °C, with onsets at 256.5 °C for the chitosan-cellulose film and 265.3 °C for the chitosan-cellulose-dye film. Thermal degradation and decomposition occurred in this stage. The mass losses of all obtained polymeric blends that occurred in different temperature ranges are presented in the Supplementary Materials, Tables S1-S3.
Comparing the thermograms of the chitosan-starch films with and without the dye with the thermogram of the bio-inspired dye, a better thermal stability of films up to 270 • C can be observed, while the degradation of the dye started after 200 • C. The thermograms of the polymeric films show two weight loss stages, which are indicated by the onset points at 83.7 and 280.6 • C for the chitosan-starch-dye film and at 71.7 and 260.2 • C for the chitosan-starch film. The first stage corresponded to the loss of surface moisture, while the second stage could be correlated to a degradation process of the films.
The chitosan-PVA films presented a thermal behavior similar to that of the chitosancellulose films due to the existence of three weight loss stages, but in this case, the third stage took place at higher temperature: 414.3 • C for the chitosan-PVA-dye film and 419.5 • C for the chitosan-PVA film. The first stage, which had its onset at 112.4 • C for the chitosan-PVA-dye film and at 111.7 • C for the chitosan-PVA film, corresponded to surface water vaporization, while the second stage, which had onsets at 278.5 • C for the chitosan-PVA-dye film and 276.9 • C for the chitosan-PVA film, was attributed to the degradation of chitosan and the dihydroxylation of PVA [41,47].

Films' Sensitivity to pH Changes
The sensitivity of the bio-based-dye-containing polymeric films to pH changes was evaluated through immersion in buffer solutions with pH values ranging from 2 to 12. Visible color changes were observed for all the tested samples after one hour of immersion, as shown in Figure 7.
The polymer matrix appeared to influence the color changes of the films due to the different intra-and intermolecular interactions with the dye. degradation of chitosan and the dihydroxylation of PVA [41,47].

Films' Sensitivity to pH Changes
The sensitivity of the bio-based-dye-containing polymeric films to pH changes was evaluated through immersion in buffer solutions with pH values ranging from 2 to 12. Visible color changes were observed for all the tested samples after one hour of immersion, as shown in Figure 7. The polymer matrix appeared to influence the color changes of the films due to the different intra-and intermolecular interactions with the dye.
The UV-VIS spectra after 1 h of immersion were recorded for both the polymeric films and the buffer solutions to evaluate desorption of the dye (Supplementary Materials, Figures S11-S13). For the chitosan-cellulose-dye, no desorption was shown, whereas for the chitosan-starch and chitosan-PVA dye-containing films, the UV-VIS spectra of the buffer solutions showed that the dye migrated from the polymeric matrix to the solution to some extent. In the pH ranges from 2 to 4 and from 10 to 12, greater desorption was observed than in the pH interval of 4 to 10. One possible cause could be the solubility of chitosan in acidic media, so the dye was forced out of the matrix. The desorption at high pH values could be explained by the dye's greater solubility in alkaline media and/or because, at these pH values, the dye remained without protons, so it could not bind to the polymer matrix through H-bonds. In the 4 to 10 pH range, there was only a small desorption of the dye, which is a promising aspect because most of the pH changes caused The UV-VIS spectra after 1 h of immersion were recorded for both the polymeric films and the buffer solutions to evaluate desorption of the dye (Supplementary Materials, Figures S11-S13). For the chitosan-cellulose-dye, no desorption was shown, whereas for the chitosan-starch and chitosan-PVA dye-containing films, the UV-VIS spectra of the buffer solutions showed that the dye migrated from the polymeric matrix to the solution to some extent. In the pH ranges from 2 to 4 and from 10 to 12, greater desorption was observed than in the pH interval of 4 to 10. One possible cause could be the solubility of chitosan in acidic media, so the dye was forced out of the matrix. The desorption at high pH values could be explained by the dye's greater solubility in alkaline media and/or because, at these pH values, the dye remained without protons, so it could not bind to the polymer matrix through H-bonds. In the 4 to 10 pH range, there was only a small desorption of the dye, which is a promising aspect because most of the pH changes caused by food spoilage occur in this interval. Based on the observed color changes, upon pH modification, the most suitable food products for this to be tested on could be milk, pork and chicken meat, and fish or shrimp. The pH indicators could be tested as freshness indicators for intelligent food packaging in different scenarios: they could be incorporated into conventional food packaging that has direct contact with the food (e.g., minced meat, chicken, and milk) and for the monitoring of the environment around the food (fish or shrimp).
For food packaging, the storage stability is one of the most important aspects to be studied. However, intelligent food packaging with freshness indicators can only be used for short-term storage due to the limited shelf life of the food products that these indicators monitor.

Conclusions
In this study, a new bio-inspired pH-sensitive dye was synthesized through acidcatalyzed condensation. The structural identity of the compound was elucidated with FT-IR and 1D and 2D NMR spectroscopic methods. The toxicity evaluation of the synthesized dye and its natural analogue, malvidin, which was carried out using theoretical methods, showed no toxicity risks, which is an essential requirement for food safety. Of course, experimental toxicity studies are mandatory and must be performed, but these are promising results that can lead to the creation of reproducible films that are satisfactory for industrial applications and that cannot be obtained with natural dye extracts or would be too expensive with pure anthocyanins. The bio-inspired dye exhibited pH-dependent photochromic properties, which were evidenced by the presence of multiple species at different pH values. A network of reversible chemical reactions for transformations upon pH changes was proposed. The dye was successfully incorporated into biodegradable intelligent systems based on chitosan blends, namely, chitosan-PVA, chitosan-cellulose, and chitosan-starch, respectively, which was proven by the absorption maxima corresponding to the dye in the UV-VIS spectra of the polymeric matrices. The thermal analysis of the pH indicator films showed that the chitosan-PVA-dye film exhibited the most thermal stability up to 300 • C. The sensitivity to pH variations was evaluated in buffer solutions over time, and visible color changes were observed. The perceived color variation of films in the pH range of 4 to 8 is essential for their application as freshness indicators. Moreover, in this pH domain, a minimal desorption of the dye was determined. Further studies will require testing of the films on food matrices; however, the key factor was for the films to exhibit pH-responsive behavior, since pH indicators based on these types of blends are widely tested on food with promising results as to their mechanical, thermal, and barrier properties, along with processability.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/polym14173622/s1, Figure S1: FT-IR spectrum of the synthesized dye; Figure S2: 1 H-NMR spectrum of the bio-inspired dye; Figure S3: 13 C-NMR spectrum of the bio-inspired dye; Figure S4: DEPT 135 spectrum of the synthetized dye; Figure S5: HMBC spectrum of the synthesized dye; Figure S6: UV-VIS spectrum of the dye; Figure S7: Thermogram of the dye; Figure S8: UV-VIS spectra of dye solutions (7·10 -5 M methanol: water 1:14) at pH values ranging from 2 to 12 over time; Figure S9: FT-IR spectra of chitosan-starch-dye (green), chitosan-starch (blue), and dye (red); Figure S10: FT-IR spectra of chitosan-PVA-dye (green), chitosan-PVA (blue), and dye (red); Figure S11: UV-VIS spectra of the chitosan-starch-dye films after 1 h in buffer solutions a) and of the buffer solutions b); Figure S12: UV-VIS spectra of the chitosan-PVA-dye film after 1 h in buffer solutions a) and those of the buffer solutions b); Figure S13: UV-VIS spectra of the chitosan-cellulosedye film after 1 h in buffer solutions; Table S1: Weight losses of the chitosan-cellulose-dye film, chitosan-cellulose film, and the dye; Table S2: Weight losses of the chitosan-starch-dye film, chitosanstarch film, and the dye; Table S3: Weight losses of the chitosan-PVA-dye film, chitosan-PVA film, and the dye.