coatings Effects of Pine Bark Extract on Physicochemical Properties and Biological Activity of Active Chitosan Film by Bionic Structure of Dragonﬂy Wing

: Bionic and active ﬁlms based on chitosan were developed with the bionic structure of dragonﬂy wings incorporating pine bark extract (PBE). Physicochemical properties of the ﬁlms, including thickness, opacity, moisture content, color, mechanical properties, and water vapor permeability were measured. Antioxidant activity of the ﬁlms was characterized by DPPH free radical scavenging activity. The interaction between chitosan and PBE was explored by attenuated total reﬂectance Fourier transform infrared spectrometry, X-ray diffraction, and differential scanning calorimetry. The results indicated that the addition of PBE gave rise to the ﬁlms greater opacity, redness, and darker appearance. Compared with pure chitosan ﬁlm, the thickness, opacity, mechanical properties, and oxidation resistance of the bionic chitosan–PBE ﬁlm increased, and the water vapor permeability decreased. The ﬁlms based on chitosan incorporated PBE and with the bionic structure of dragonﬂy wings can potentially be applied to food packaging. of the B-0%PBE ﬁlm had a structure similar to the micronano structure on the surface of the dragonﬂy’s wings, which indicated that the ﬁlm had successfully bionic dragonﬂy wings. After incorporating PBE into a chitosan ﬁlm, ﬁlms only replicate the macroscopic structure of dragonﬂy wings. White spots on bionic chitosan–PBE ﬁlms surface may be insoluble particles from PBE. At the same time, these small spots might be the reason that hinders the replication of the microstructure of the dragonﬂy’s wings by the ﬁlm. stretching [21]. The results suggested that the addition of PBE in chitosan ﬁlms leaded to interactions between chitosan and PBE.


Introduction
Plastic food packaging materials are mostly non-degradable, resulting in serious environmental pollution. The World Health Organization has reported that about 8 million tons of plastic waste enter the ocean every year, which has a serious impact on the ocean and marine life and potential impact on human health [1]. Meanwhile, microbial contamination and oxidation are the main problems affecting food quality and safety [2]. Therefore, it is necessary to develop biodegradable food packaging films with antibacterial and antioxidant properties [3].
Chitosan ( Figure 1A) is a natural polysaccharide, which is yielded by partial deacetylation of chitin [4]. Chitosan possesses various features, such as biodegradability, filmforming properties, and biocompatibility, and becomes one of the research hotspots in food packaging materials [5][6][7]. However, pure chitosan has the disadvantage of poor antibacterial and antioxidant properties [8,9], so natural active ingredients are often incorporated to improve the antibacterial or antioxidant properties of chitosan, such as pomegranate peel extract and Thymus kotschyanus essential oil [10], chestnut extract [11], and litchi peel extract [12]. aging materials with PBE.
In this study, active films from chitosan incorporated PBE and with bionic structur of dragonfly wings were developed. The physiochemical, structural, and biological prop erties of the bionic chitosan-PBE film was evaluated.

Preparation of Negative Replica of Dragonfly Wings
Bio-template method was used to prepare negative replica of dragonfly wing Firstly, dragonfly wings were put in an ultrasonic cleaner for 30 min, then remover int an oven at 60 °C for 30 min. After that, dragonfly wings were fastened with double-side adhesive tape on the glass plate and arrange the dragonfly wings as closely as possible Pine bark extract (PBE) has a long history of use in Europe and North America [13]. PBE contains a variety of active ingredients, mainly including proanthocyanidins ( Figure 1B) composed of catechins and epicatechin subunits. These ingredients have a wide range of biological and pharmacological activities, such as antioxidant, promoting wound healing, anti-inflammatory and so on [13][14][15]. Nevertheless, there are fewer reports on food packaging materials with PBE.
In this study, active films from chitosan incorporated PBE and with bionic structure of dragonfly wings were developed. The physiochemical, structural, and biological properties of the bionic chitosan-PBE film was evaluated.

Preparation of Negative Replica of Dragonfly Wings
Bio-template method was used to prepare negative replica of dragonfly wings. Firstly, dragonfly wings were put in an ultrasonic cleaner for 30 min, then remover into an oven at 60 • C for 30 min. After that, dragonfly wings were fastened with double-sided adhesive tape on the glass plate and arrange the dragonfly wings as closely as possible. PDMS Coatings 2021, 11, 1077 3 of 11 solution (200 g) was poured on the plate (diameter 16 cm), then the plate was place in a vacuum drying oven for 20 min until all bubbles are discharged. Finally, the plate was placed in an oven at 70 • C for 5 h. After the PDMS solidified, it was carefully peeled off from the plate, then the negative replica of the dragonfly wings was obtained.

Preparation of Bionic Chitosan-PBE Film
Chitosan solution (4 wt %) was prepared by dissolving chitosan in 2% (V/V) acetic acid aqueous solution and stirring at 600 rpm for 30 min at 60 • C. Glycerin with a concentration of 30% by weight of chitosan was added to the chitosan solution as a plasticizer. Different concentrations of PBE solutions were prepared by mixing PBE and distilled water at 50 • C and stirred at 200 rpm for 15 min. Chitosan solution containing PBE with the concentration (weight percentage) of 30%, 40%, and 50% was prepared by mixing chitosan solution and PBE solution in a 1:1 weight ratio and stirred at 400 rpm for 15 min. After removing bubbles by ultrasonic wave for 15 min, film solution (50 g) was distributed into the negative replica of the dragonfly wings for casting and fix it with a stainless-steel ring with a diameter of 9 cm and soft glue. After drying at 60 • C and 30% RH, the film was peeled off and stored in a 75% relative humidity and room temperature environment for 48 h. The chitosan film without bionic structure and PBE was used as the control. The bionic chitosan-PBE films with concentration of 0%, 30%, 40%, and 50% named B-0%PBE, B-30%PBE, B-40%PBE, and B-50%PBE, respectively.

Scanning Electron Microscopy (SEM)
The microstructure of film samples was observed by Zeiss-evo18 SEM (Carl Zeiss Co., Ltd., Shanghai, China) with 10 kV of acceleration voltage.

Attenuated Total Reflectance-Fourier Transform Infrared Analysis (ATR-FTIR)
ATR-FTIR spectra of film samples was determined by using a Nicolet iS50 FTIR Spectrometer couple with an ATR attachment (Nicolet, Waltham, MA, USA). The scans were carried out in range of 4000-400 cm −1 with 64 scans and a resolution of 4 cm −1 .

X-ray Diffraction (XRD)
The XRD analysis was carried out in a SmartLab 3 KW diffractometer (Rigaku, Tokyo Japan), using Cu-X radiation at a voltage of 40 kV and a current of 40 mA. Film samples were measured at a scan rate of 2 • /min within 2θ range (5-40 • ) according to the method of Wang et al. [16].

DPPH Free Radical Scavenging Activity
The DPPH free radical scavenging activity of film samples was determined according the method of Wang et al. [17]. With some alteration, 9 mL of film samples extract solution was mixed with 3 mL of DPPH 10 −3 mol/L ethanol solution, then shaken in an oscillator for 30 s, incubated in dark room for 30 min at room temperature and measured at 517 nm using UV spectrophotometer. DPPH scavenging activity was calculated based on the following equation: where A DPPH is the absorbance value of the DPPH methanol solution and A S is the absorbance value of the DPPH assay solution.

Water Vapor Permeability (WVP)
The WVP of film samples was measured using the method of Bai et al. [18]. The thickness of the films was determined by using a hand-held digital micrometer (EVERTE, Henan, China) at five random positions, then the average values were taken.
Opacity of the films was determined by measuring the absorbance of film samples at 600 nm using a UV spectrophotometer (Model Lambda 365, PerkinElmer, Waltham, MA, USA) according to the method of wang et al. [19]. Films were cut into slices (1 × 4 cm), then placed in a spectrophotometer test cell. An empty test cell was used as the reference. The opacity was calculated based on the following Equation: where O is the opacity, Abs 600 is the value of absorbance at 600 nm, and d is the films thickness (mm). The moisture content of the film samples was measured by placing the films (1 × 4 cm) in an oven at 105 • C for 12 h. Then the moisture content of each sample was calculated according to the following Equation: Moisture where M w is the weight of the films in 75% RH and M d is dry weight of the films.

Mechanical Properties
The tensile strength (TS) and elongation at break (EAB) of the film samples (10 × 40 mm) were measured using a texture analyzer machine (WDW-200H, Jinan HengXu Testing Machine Technology Co., Ltd., Jinan, China) at a crosshead speed of 50 mm/min.

Color Properties
The color values of the film samples were determined by using a chromometer with a 14 mm aperture (HunterLab ColorFlex, Shanghai, China), a D65 illuminant and a 10 • angle. Prior to analysis, the chromometer was calibrated with a standard plate CX2064. Then film samples were subjected to chromometer for lightness (L*-value), redness (a*-value), and yellowness (b*-value). ∆E and C were calculated as follows:

Statistical Analysis
The difference among samples was evaluated by one-way analysis of variance (ANOVA). Duncan's multiple range tests were used to compare the means to identify which groups were significantly different from other groups (p < 0.05). Each experiment was repeated in three times and all data are presented as mean ± standard deviation.

Scanning Electron Microscopy (SEM)
Scanning electron microscope can effectively observe the surface morphology and structure of films. Figure 2 presented the SEM image of the film samples surface under 3000 times magnification. The results suggested that the pure chitosan film (control) was smooth. Figure 2B 1 showed that B-0%PBE successfully replicated the microstructure of dragonfly wings with a rough and uneven appearance. Figure 2B 2 showed that the surface of the B-0%PBE film had a structure similar to the micronano structure on the surface of the dragonfly's wings, which indicated that the film had successfully bionic dragonfly wings. After incorporating PBE into a chitosan film, films only replicate the macroscopic structure of dragonfly wings. White spots on bionic chitosan-PBE films surface may be insoluble particles from PBE. At the same time, these small spots might be the reason that hinders the replication of the microstructure of the dragonfly's wings by the film.
Scanning electron microscope can effectively observe the surface morphology and structure of films. Figure 2 presented the SEM image of the film samples surface under 3000 times magnification. The results suggested that the pure chitosan film (control) was smooth. Figure 2B1 showed that B-0%PBE successfully replicated the microstructure of dragonfly wings with a rough and uneven appearance. Figure 2B2 showed that the surface of the B-0%PBE film had a structure similar to the micronano structure on the surface of the dragonfly's wings, which indicated that the film had successfully bionic dragonfly wings. After incorporating PBE into a chitosan film, films only replicate the macroscopic structure of dragonfly wings. White spots on bionic chitosan-PBE films surface may be insoluble particles from PBE. At the same time, these small spots might be the reason that hinders the replication of the microstructure of the dragonfly's wings by the film.

Attenuated Total Reflectance-Fourier Transform Infrared Analysis (ATR-FTIR)
ATR-FTIR spectroscopy was carried out to observe the variation of transmission peaks in the bionic chitosan-PBE films, which could explain the interactions between chitosan and PBE. ATR-FTIR spectra of chitosan-PBE films were shown in Figure 3. There was no significant (p < 0.05) difference between the control and B-0%PBE, because the two samples possessed the same composition. In addition, the ATR-FTIR spectra of bionic chitosan-PBE films exhibited the same main peaks, however, the amplitudes of the peaks differed depending on the concentration of PBE. With the PBE concentration increasing, the peak at 3620, 1540, 1070, and 1030 cm −1 became more and more small. When the concentrate of PBE was 50%, the peak at 1070 cm −1 became more flattened and less discernible. Similar results were found by Wang et al. who prepared chitosan film with Herba

Attenuated Total Reflectance-Fourier Transform Infrared Analysis (ATR-FTIR)
ATR-FTIR spectroscopy was carried out to observe the variation of transmission peaks in the bionic chitosan-PBE films, which could explain the interactions between chitosan and PBE. ATR-FTIR spectra of chitosan-PBE films were shown in Figure 3. There was no significant (p < 0.05) difference between the control and B-0%PBE, because the two samples possessed the same composition. In addition, the ATR-FTIR spectra of bionic chitosan-PBE films exhibited the same main peaks, however, the amplitudes of the peaks differed depending on the concentration of PBE. With the PBE concentration increasing, the peak at 3620, 1540, 1070, and 1030 cm −1 became more and more small. When the concentrate of PBE was 50%, the peak at 1070 cm −1 became more flattened and less discernible. Similar results were found by Wang et al. who prepared chitosan film with Herba Lophatheri extract [17]. In the literature, the peak at 3620 cm −1 belongs to multiple absorption peaks superimposed on the O-H stretching vibration absorption peak and the N-H stretching vibration absorption peak [20], the peak at 1070 cm −1 was related to C-O-C symmetric stretching [21]. The results suggested that the addition of PBE in chitosan films leaded to interactions between chitosan and PBE.
Lophatheri extract [17]. In the literature, the peak at 3620 cm −1 belongs to multiple absorption peaks superimposed on the O-H stretching vibration absorption peak and the N-H stretching vibration absorption peak [20], the peak at 1070 cm −1 was related to C-O-C symmetric stretching [21]. The results suggested that the addition of PBE in chitosan films leaded to interactions between chitosan and PBE.

X-Ray Diffraction (XRD)
The XRD diffractograms of film samples were shown in Figure 4. As seen in Figure 4, we find that the chitosan film was in crystalline state, and there were four main diffraction peaks at around 8°, 11°, 18°, and 23°. Similar observations were also made by Sun et al. who incorporated thinned young apple polyphenols in chitosan films [22]. The diffraction peaks at near 8° and 18° were ascribed to the hydrated crystalline structures [23], while the around 11° peak denoted the anhydrous structure [24]. There was no significant change in the four main diffraction peaks between the control and B-0%PBE, indicating that the bionic structure had no effect on the crystallization properties of chitosan films. After adding PBE, the peaks at around 18° and 23° became flatter and peak at around 8° disappeared, suggesting that the crystallinity of chitosan decreased. The peak at around 23° was a typical fingerprint for chitosan, and incorporated PBE in chitosan films had no changes in this peak. Similar observations were also made by Liu et al. who incorporated protocatechuic acid in chitosan films [25]. Moreover, when incorporated 50% content in chitosan films, two small peaks at around 28° and 29° were appeared. It may be because the interactions between chitosan and PBE.

X-ray Diffraction (XRD)
The XRD diffractograms of film samples were shown in Figure 4. As seen in Figure 4, we find that the chitosan film was in crystalline state, and there were four main diffraction peaks at around 8 • , 11 • , 18 • , and 23 • . Similar observations were also made by Sun et al. who incorporated thinned young apple polyphenols in chitosan films [22]. The diffraction peaks at near 8 • and 18 • were ascribed to the hydrated crystalline structures [23], while the around 11 • peak denoted the anhydrous structure [24]. There was no significant change in the four main diffraction peaks between the control and B-0%PBE, indicating that the bionic structure had no effect on the crystallization properties of chitosan films. After adding PBE, the peaks at around 18 • and 23 • became flatter and peak at around 8 • disappeared, suggesting that the crystallinity of chitosan decreased. The peak at around 23 • was a typical fingerprint for chitosan, and incorporated PBE in chitosan films had no changes in this peak. Similar observations were also made by Liu et al. who incorporated protocatechuic acid in chitosan films [25]. Moreover, when incorporated 50% content in chitosan films, two small peaks at around 28 • and 29 • were appeared. It may be because the interactions between chitosan and PBE.

DPPH Free Radical Scavenging Activity
Due to the harmful effects of free radicals on foods, antioxidant activity packaging can remove free radicals and extend the shelf life of foods [26]. The DPPH free radical scavenging activity of bionic chitosan-PBE films are shown in Table 1. The free scavenging rate of pure chitosan films was weak, which was similar to the results reported by Kadam et al. [27]. There was no significant (p < 0.05) difference in the scavenging rate of free radicals between control and B-0%PBE. The addition of PBE significantly (p < 0.05) improved the free radical scavenging activity of bionic chitosan-PBE films, and the free radical scavenging activity was enhanced with the increase of PBE concentration. When the concentration of PBE was 50%, the free radical scavenging rate was the highest, reaching 60.56%, which was about 14 times higher than that of pure chitosan film. According to the report by Romani et al., commercial packaging PE film does not possess free radical scavenging activity [28]. Therefore, compared with PE film, the bionic chitosan-PBE film in this study showed significantly (p < 0.05) higher free radical scavenging activity. This is because the PBE added to the film has strong antioxidant activity [14]. In addition, the DPPH free radical scavenging activity of the bionic chitosan-PBE was higher than those of chitosan films incorporated with peanut skin extract [29] and Chinese chive (Allium tuberosum) root extract [30], also indicating that PBE has good antioxidant activity and can be used for enhancing the antioxidant activity of chitosan film.

DPPH Free Radical Scavenging Activity
Due to the harmful effects of free radicals on foods, antioxidant activity packaging can remove free radicals and extend the shelf life of foods [26]. The DPPH free radical scavenging activity of bionic chitosan-PBE films are shown in Table 1. The free scavenging rate of pure chitosan films was weak, which was similar to the results reported by Kadam et al. [27]. There was no significant (p < 0.05) difference in the scavenging rate of free radicals between control and B-0%PBE. The addition of PBE significantly (p < 0.05) improved the free radical scavenging activity of bionic chitosan-PBE films, and the free radical scavenging activity was enhanced with the increase of PBE concentration. When the concentration of PBE was 50%, the free radical scavenging rate was the highest, reaching 60.56%, which was about 14 times higher than that of pure chitosan film. According to the report by Romani et al., commercial packaging PE film does not possess free radical scavenging activity [28]. Therefore, compared with PE film, the bionic chitosan-PBE film in this study showed significantly (p < 0.05) higher free radical scavenging activity. This is because the PBE added to the film has strong antioxidant activity [14]. In addition, the DPPH free radical scavenging activity of the bionic chitosan-PBE was higher than those of chitosan films incorporated with peanut skin extract [29] and Chinese chive (Allium tuberosum) root extract [30], also indicating that PBE has good antioxidant activity and can be used for enhancing the antioxidant activity of chitosan film. Values are given as mean ± standard error. Different letters in the same row indicate significant differences (p < 0.05).

Water Vapor Permeability (WVP)
One of the most important functions of food packaging film is to hinder the exchange of moisture between the atmosphere and the food [31]. The WVP values of bionic chitosan-PBE films were shown in Table 1. The WVP of B-0%PBE was lower (8.38%) than that of control, indicating that the bionic structure decreased the WVP of the chitosan film, because the bionic structure increased the thickness of films and hindered the permeation of moisture. The WVP of the bionic chitosan-PBE films decreased significantly (p < 0.05) with the increasing PBE concentration. Compared with B-0%PBE, WVP of B-30%PBE, B-40%PBE, and B-50%PBE decreased 28.63%, 44.54%, and 59.49%. This downward trend was beneficial to the preservation of food, which was consistent with the reported by Wang et al. [32]. In addition, the WVP values of bionic chitosan-PBE were lower than those of chitosan film incorporated with protocatechuic acid [25] and carboxymethyl chitosan film incorporated with quercetin [18].
Generally, WVP depends on the diffusivity and solubility of water molecules in the film matrix [15]. As shown in Table 2, the addition of PBE increased the thickness of the film and filled the structural gap in the polysaccharide chain, so it reduced the gap space in the chitosan matrix, blocked the transmission channel of water in the film and reduced the diffusion and fusion rate of water molecules in the films [33]. Meanwhile, ATR-FTIR spectra showed that the interaction between chitosan and PBE may be one of the reasons for the decrease of WVP of bionic chitosan-PBE film, which may reduce the effectiveness of the hydrophilic groups in chitosan and their interaction with water.

Thickness, Opacity and Moisture Content
The thickness, opacity, and moisture content of the bionic chitosan-PBE films was displayed in Table 2. The thickness of B-0%PBE was higher than the control, since there were a lot of small humps on the surface of the bionic film. With the increase of the PBE content, the thickness of bionic chitosan-PBE films was increased due to the addition of PBE. Similarly, Kadam et al. reported that the thickness of chitosan film samples with the addition of pine needles extract was higher than that of the pure chitosan films [27].
The bionic design and the addition of PBE significantly (p < 0.05) increased the opacity of the film samples; one reason for this was that the bionic structure increased the thickness of the film, another reason was that the PBE had brownish red color. In the literature, Riaz et al. report that apple peel polyphenols also improved the opacity of the chitosan film [34]. According to Equation (2), larger O values indicate higher opacity and lower transparency. Therefore, the control (without PBE and bionic structure) had the highest transparency, chitosan film with bionic structure had lower transparency, and bionic chitosan-PBE film had the lowest transparency.
As shown in Table 2, we can find that the bionic structure and PBE content had a significant (p < 0.05) impact on the moisture content of the film. Compared with the control, the moisture content of B-0%PBE decreased up to 8.52%, and the moisture content of B-50%PBE decreased up to 40.12%. This result indicated that the bionic design could reduce the moisture content of the chitosan film. One reason was that the bionic structure increased the surface area of the films. Another reason is that the interactions between chitosan and PBE could reduce the availability of hydroxyl and amino groups, thus limiting the interaction between chitosan and water through hydrogen bonding [15].

Mechanical Properties
The mechanical properties of bionic chitosan-PBE film samples were displayed in Table 3. The tensile strength of B-0%PBE was lower than the control, indicating that the bionic structure reduced the tensile strength of chitosan films. With the increase of PBE content, the tensile strength of films significantly (p < 0.05) improved, while there was no significant difference (p > 0.05) among all the films expect B-0%PBE. Regarding the elongation at break, there were no significant differences (p > 0.05) between the control and the bionic chitosan-PBE films. B-40%PBE possessed the highest value that increased up to 26.83% in comparison with B-0%PBE. These results suggested that PBE improved tensile strength and elongation at break of the bionic films. Similar results were found by Yan et al. [35], who found that adding butterfly pudding extract to the chitosan film also improved the tensile strength and elongation at break of the film. Mechanical properties are mainly related to the type and content of active compounds, the type of polymer matrix, and the specific interaction between different components [36]. Values are given as mean ± standard error. Different letters in the same row indicate significant differences (p < 0.05).

Color Properties
The result of color values of bionic chitosan-PBE films with different PBE concentrations were showed in Table 4. There was no significant (p < 0.05) difference between the control and B-0%PBE in all color parameters, which indicated that the bionic design does not affect the color of the chitosan films. With the increase of PBE concentration, the value of L*, b*, and C* decreased significantly (p < 0.05), which indicated that the film tended to be dark and blue. Moreover, incorporation of PBE significantly (p < 0.05) increased the a* and ∆E values, which indicating that the film has a tendency toward redness. Compared with the carboxymethyl chitosan film incorporated with corn peptide, the bionic chitosan-PBE films in this study showed darker, redder, and bluer color [25]. Values are given as mean ± standard error. Different letters in the same row indicate significant differences (p < 0.05).

Conclusions
The results of this study suggested that the bionic structure of dragonfly wings and PBE has a considerable influence on the physicochemical properties and biological activity of the bionic chitosan-PBE film. The bionic structure increased the thickness and opacity of the film and decreased the moisture content, mechanical properties, and water vapor permeability of the film. Moreover, the addition of PBE in chitosan films with bionic structure improved the thickness, opacity, mechanical properties, and antioxidant activity of the film, but reduced the WVP of the film. Compared with the control film, the thickness, opacity, tensile strength, and DPPH free scavenging activity of bionic chitosan-PBE film increased up to 68.25%, 9.31 times, 21.77%, and 13.11 times, respectively, and WVP and moisture content decreased by 59.49% and 40.12%, respectively. Since the antioxidant activity of the film has increased and the water vapor permeability has decreased, this is beneficial to the application of the film in active food packaging. Further research is still needed, such as the inhibition of some microorganisms and the analysis of food shelf life.