Development of active packaging based on chitosan-gelatin blend films functionalized with Chinese hawthorn (Crataegus pinnatifida) fruit extract

International Journal of Biological Macromolecules 140 (2019) 384–392

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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac

Development of active packaging based on chitosan-gelatin blend films functionalized with Chinese hawthorn (Crataegus pinnatifida) fruit extract Juan Kan, Jing Liu, Huimin Yong, Yunpeng Liu, Yan Qin, Jun Liu ⁎ College of Food Science and Engineering, Yangzhou University, Yangzhou 225127, PR China

a r t i c l e

i n f o

Article history: Received 26 July 2019 Received in revised form 12 August 2019 Accepted 17 August 2019 Available online 21 August 2019 Keywords: Active packing Chitosan Gelatin Chinese hawthorn

a b s t r a c t The fruits of Chinese hawthorn (Crataegus pinnatifida) have been used as the functional food and folk medicine due to potent antioxidant activity. In this study, polyphenols were extracted from the fruits of Chinese hawthorn and further added into chitosan-gelatin blend films to develop active packaging. The microstructure, physical, mechanical, barrier and antioxidant properties of the films were investigated in details. Results showed epicatechin, chlorogenic acid and procyanidin B2 were the main polyphenols in the extract of hawthorn fruits. The inner microstructure of chitosan-gelatin blend films became more compact when the extract was incorporated. The intermolecular interactions between film matrix and the extract were through hydrogen bonding and electrostatic interactions. The incorporation of the extract remarkably increased the thickness, tensile strength and elongation at break of chitosan-gelatin blend films. However, the moisture content, water vapor permeability and light transmittance of chitosan-gelatin blend films were significantly reduced by the addition of the extract. Moreover, chitosan-gelatin blend films containing the extract exhibited potent free radical scavenging ability. Our results suggest Chinese hawthorn fruit extract can be used as a natural antioxidant to improve the mechanical, barrier and antioxidant properties of chitosan-gelatin blend films. © 2019 Elsevier B.V. All rights reserved.

1. Introduction In recent years, biopolymer-based packaging films have been considered as the substitutes for conventionally used petroleum-derived plastic packaging [1]. Within biopolymers, gelatin and chitosan have attracted considerable attention since they are cheap, nontoxic, biodegradable, biocompatible, renewable and edible [2]. Gelatin is an animal protein obtained by the controlled hydrolysis of fibrous insoluble collagen, which is generated as the waste during animal slaughtering and processing [3]. Gelatin is also one of the most commonly used proteins for food packaging film production due to its abundance, good film forming ability and excellent barrier properties against light, oxygen and lipids [4]. However, gelatin film exhibits poor water vapor barrier property due to the hydrophilic nature of gelatin molecules. One of the promising ways to overcome the poor water vapor barrier property of gelatin film is to blend gelatin with other biopolymers, such as agar, chitosan and starch [5–7]. Chitosan is a cationic polysaccharide derived from chitin through deacetylation in alkaline media. The unique cationic character of chitosan in acidic conditions offers the opportunity to establish electrostatic ⁎ Corresponding author. E-mail address: [email protected] (J. Liu).

https://doi.org/10.1016/j.ijbiomac.2019.08.155 0141-8130/© 2019 Elsevier B.V. All rights reserved.

interactions with other negatively charged compounds [8]. Thus, chitosan and gelatin are compatible in the film matrix due to the electrostatic interactions and hydrogen bonding, where chitosan is positively charged and gelatin is negatively charged under appropriate pH levels [9]. Importantly, gelatin-chitosan blend films show enhanced mechanical and barrier properties when compared to films made from their individual components [10–12]. In general, the properties of chitosangelatin blend films are affected by the nature of chitosan (e.g. molecular weight and degree of deacetylation) [13] and gelatin (e.g. gelatin origin) [14] as well as their proportions in the film matrix [7,10–12,15]. The antioxidant packaging is a category of active packaging that contains antioxidant substances. The antioxidants can interact with the headspace and packaged food, aiming to limit or prevent lipid oxidation and to avoid direct addition of active chemical compounds into food [16]. Plant extracts, especially those rich in polyphenols have received increasing attention due to their considerable antioxidant activity [17,18]. Till now, numerous polyphenol-rich plant extracts (e.g. cinnamon, guarana, rosemary, boldo-do-chile and red grape seed ethanolic extracts) and pure polyphenols (e.g. gallic acid, quercetin and ferulic acid) have been incorporated into chitosan-gelatin blend films to develop antioxidant packaging [19–23]. The incorporation of polyphenol-rich plant extracts and pure polyphenols into chitosangelatin blend films can remarkably improve the physical, mechanical,

J. Kan et al. / International Journal of Biological Macromolecules 140 (2019) 384–392

barrier and antioxidant properties of the resulting films. Moreover, the interactions between plant extracts and film matrix can provide the controlled release of antioxidant ingredients over the storage period. Therefore, chitosan-gelatin blend films functionalized with plant extracts have the potential to protect food products against oxidation [20,24]. Hawthorn, a member of the Crataegus genus in the Rosaceae family, is a thorny shrub that normally has green leaves, white flowers and red fruits [25]. Hawthorn is widely grown in Europe, Asia and North America, containing over 1000 species. Chinese hawthorn (Crataegus pinnatifida) has a cultivation history of over 1700 years [26]. In China, the fruits of hawthorn are used as both functional food and folk medicine to improve digestion, decrease food stasis, relieve “fullness of the stomach,” manage hyperlipidemia and treat dyspnea [27]. The pharmacological effects of Chinese hawthorn fruits are mainly attributed to the presence of abundant phenolic compounds that possess potent antioxidant activity [28–31]. Therefore, the polyphenol-rich Chinese hawthorn fruit extract can be used as a natural antioxidant to develop antioxidant packaging. However, there is no available information on the development of antioxidant packaging based on the Chinese hawthorn fruit extract. The aim of this study was to develop antioxidant packaging by adding polyphenol-rich Chinese hawthorn fruit extract into chitosangelatin blend films for the first time. The active films were characterized in terms of microstructure, intermolecular interactions, and physical, mechanical and barrier properties. To establish the potential functionality of the active films in food packaging, the antioxidant activity of the films was also evaluated. 2. Materials and methods 2.1. Materials and chemical reagents Fresh Chinese hawthorn (C. pinnatifida Bge. var. major NEBr.) fruits were collected from Yimeng Mountain (Shandong, China). Chitosan (deacetylated degree of 90%) with a molecular weight of 1.5 × 105 Da was obtained from Sangon Biotechnology Co. Ltd. (Shanghai, China). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and gelatin from cold water fish skin (molecular weight of 6 × 104 Da) were purchased from Sigma-Aldrich (MO, USA). Other chemical reagents including ethanol, acetic acid, Folin-Ciocalteu reagent and glycerol were of analytical grade and purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). 2.2. Extraction and characterization of polyphenols from hawthorn fruits Polyphenols were extracted from hawthorn fruits according to the method of Liu et al. [32] with some modifications. Briefly, the seedless hawthorn fruits (100 g) were milled and extracted twice in 500 mL of 80% aqueous ethanol solution at 4 °C overnight. All the extract solutions were combined and centrifuged at 8000 ×g for 20 min to remove debris. The obtained supernatant was rotary evaporated at 35 °C to remove ethanol and then vacuum dried to obtain the extract powder. The total phenolic content in the extract was determined by the Folin– Ciocalteu method [33]. The polyphenolic composition in the extract was analyzed by Agilent 1200 HPLC system (Agilent Technologies, CA, USA) equipped with 6460 triple quadrupole mass spectrometer according to the chromatographic method of Wang et al. [34]. Mass spectrometry analysis was performed in positive ionization mode with m/z in the range of 100–2000.

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by dissolving 3.2 g chitosan in 160 mL acetic acid solution (1%, v/v) with continuous stirring at room temperature for 6 h, and 30 wt% of glycerol on chitosan basis was then added as a plasticizer. Meanwhile, 2% (w/v) of gelatin solution was prepared by dissolving 3.2 g fish gelatin powder in 160 mL distilled water under continuous stirring at 50 °C for 1 h, and 30 wt% of glycerol on gelatin basis was then added as a plasticizer. Afterwards, the film forming solutions were prepared by mixing 80 mL of chitosan solution, 80 mL of gelatin solution and different contents of the extract powder (0, 2, 4 and 6 wt% on the basis of the total weight of chitosan and gelatin) at 20 °C for 30 min. The obtained filmforming solutions were degassed before being poured into Plexiglas plates (24 cm × 24 cm), which were dried in a ventilated climatic chamber at 30 °C with 50% relative humidity (RH) for 48 h. The developed film containing 0, 2, 4 and 6 wt% of the extract was named as CF, CFHE I, CF-HE II and CF-HE III films, respectively. All the films were stored in a desiccator with 50% RH at 20 °C for 48 h before analysis. 2.4. Structural characterization of the films 2.4.1. Scanning electron microscopy (SEM) The microstructure of the film was characterized by GeminiSEM 300 equipment (Carl Zeiss, Oberkochen, Germany). Film sample was immersed in liquid nitrogen and cryofractured. Then, the fractured film sample was fixed on a stainless steel support with double-side conductive adhesive, coated with gold palladium and analyzed on the SEM at 3 kV with a magnification of 800×. 2.4.2. Fourier transform infrared (FT-IR) spectroscopy The FT-IR spectrum of the film was recorded on Varian 670 spectrometer (Agilent Technologies, CA, USA) equipped with attenuated total reflectance (ATR) attachment. The spectrum was collected in the wavenumber range of 400–4000 cm−1 by accumulating 32 scans. 2.4.3. X-ray diffraction (XRD) The crystalline character of the film was determined by D8 Advance X-ray diffractometer (Bruker AXS GmbH, Karlsruhe, Germany). The XRD pattern of film piece (4 cm × 1 cm) was recorded from 5° to 80° with the scanning rate of 0.1°/s at 40 mA and 40 kV using Ni-filtered Cu Kα radiation. 2.5. Determination of the physical, mechanical and barrier properties of the films 2.5.1. Color The color parameters (L, a and b) of the film were recorded by SC80C colorimeter (Kangguang Instrument Co., Ltd., Bejing, China) with a white plate as the standard background. The total color difference (ΔE) was calculated by the following equation:

ΔE ¼

qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2  2 ðL −LÞ þ ða −aÞ2 þ ðb −bÞ

ð1Þ

where L* (92.60), a* (−0.92) and b* (−2.24) were the color parameters of white plate.

2.3. Preparation of the films

2.5.2. Thickness The thickness of the film was measured by a Mitutoyo No. 293–766 digital micrometer (Tester Sangyo Co., Ltd., Tokyo, Japan) with the precision of 0.001 mm. Ten random positions on the film sample were recorded and the mean value of the measurements were calculated.

Chitosan-gelatin blend films with and without hawthorn fruit extract were prepared based on the method of Hosseini et al. [11] with some modifications. First, 2% (w/v) of chitosan solution was prepared

2.5.3. Moisture content The moisture content of the film was determined by drying film piece (4 cm × 4 cm) in an oven at 105 °C to a constant weight and

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2.7. Statistical analysis

calculated according to the following equation: Moisture content ð%Þ ¼

ðM i −M t Þ  100 Mi

ð2Þ

where Mi and Mt were the initial and final weights of film piece, respectively. 2.5.4. Mechanical property The tensile stress and elongation at break of the film were determined by fixing film strip (6 cm × 1 cm) on the grips of TMS-Pro texture analyzer (Food Technology Co., VA, USA). The initial grip separation and cross-head speed were set at 4 cm and 6 cm/min, respectively. The tensile strength and elongation at break of the film were calculated as follows: Tensile strength ðMPaÞ ¼

F xW

Elongation at break ð%Þ ¼

ð3Þ

ΔL  100 L0

ð4Þ

where F was the stress for film fracture (N), x was film thickness (mm), and W was film width (mm), ΔL was the increased length when film break (mm) and L0 was the initial film length (mm). 2.5.5. Water vapor permeability (WVP) The water vapor barrier property of the film was determined by the method of Yong et al. [35]. Briefly, film piece (6 cm × 6 cm) was sealed on a test tube containing 40 g anhydrous silica gels. The tube was then stored at 20 °C in a desiccator containing distilled water (100% RH). The weight of the tube was recorded every 24 h for 7 days. The WVP of the film was calculated by the following equation: WVP ¼

W x t  A  ΔP

ð5Þ

where W was the increased weight of film sealed cup (g), x was film thickness (m), t was time (s), A was permeation area (m2), and ΔP was saturated water vapor pressure at 20 °C (Pa). 2.5.6. Light transmittance and opacity The UV–vis light barrier property of the film was measured by scanning film stripe (4 cm × 0.9 cm) on a Lambda 35 UV–Vis spectrophotometer (PerkinElmer Ltd., MA, USA) from 200 to 800 nm. The absorbance of film sample at 600 nm was used to calculate the opacity of film [34]: Opacity ¼

A600 x

ð6Þ

where A600 was the absorbance of film sample at 600 nm, and x was film thickness (mm). 2.6. Determination of the antioxidant property of the films The antioxidant property of the film was evaluated by DPPH radical scavenging assay [36]. Briefly, film sample (4–20 mg) was reacted with 4 mL of 100 μM DPPH methanol solution at 20 °C for 60 min in the dark. The absorbance of reaction solution was measured at 517 nm. The DPPH radical scavenging activity of the film was calculated as follows: DPPH radical scavenging activity ð%Þ ¼

A0 −A1  100 A0

ð7Þ

where A0 and A1 were the absorbance of the blank and reaction solutions, respectively.

Duncan test and one-way analysis of variance were carried out by SPSS 13.0 software package (SPSS Inc., IL, USA). Difference was considered as statistically significant if p b 0.05. 3. Results and discussion 3.1. Characterization of polyphenols in the extract Polyphenols were extracted from hawthorn fruits by using 80% aqueous ethanol solution, and the extraction yield was 2.56% on the basis of the fresh weight of hawthorn fruits. The total phenol content in the extract was 551 mg gallic acid equivalent/g extract. Furthermore, the polyphenolic composition in the extract was identified by HPLC-MS technique. As summarized in Table 1, six main polyphenolic compounds were identified. Notably, epicatechin, chlorogenic acid and procyanidin B2 were the main polyphenols in the extract. Similar polyphenolic compositions were also reported in the extract of hawthorn fruits by many other researchers [29–32]. Liu et al. [29] found the procyanidins from Chinese hawthorn (C. pinnatifida Bge. var. major) exhibited higher in vitro antioxidant activity than vitamin C and vitamin E. Wen et al. [30] reported procyanidin B2 was most abundant phenolic compound in three different varieties of hawthorn, including Shanlihong (C. pinnatifida Bge. var. major N.E.Br.) Shanzha (C. pinnatifida Bge) and Dajinxing (C. pinnatifida Bge var. major). Notably, the polyphenol composition and cellular antioxidant activity of hawthorn extract were closely related to the variety of hawthorn. Table 1. 3.2. Microstructure of CF-HE films The inner microstructure of the film depends on the compatibility and miscibility between different film components, which directly affect the mechanical and barrier properties of the film [37]. A continuous and compact structure without any pores and phase separation was observed in the cross-section of CF film (Fig. 1A), indicating the compatibility among chitosan, gelatin and glycerol. The compact and homogeneous microstructure of CF film was attributed to the hydrophilic nature of chitosan, gelatin and glycerol that could interact with each other through associative interactions [12]. The similar phenomena were also observed in chitosan-gelatin blend films by many researchers [38,39]. When 2 or 4 wt% of the extract was incorporated into the blend, the composite film showed a smooth cross-section as compared with CF film (Fig. 1B and C), indicating the high compatibility and miscibility between the extract and other film components. The homogeneous state of CF-HE I and CF-HE II films reflected that more compact inner microstructure was produced by the addition of the extract, which was benefit to improve the mechanical and barrier properties of the films. Rezaee et al. [21] illustrated that chitosan-gelatin blend films incorporated with gallic acid showed smooth and uniform microstructure, suggesting gallic acid incorporation performed well with good compatibility at the molecular level. Bonilla and Sobral [20] also reported that cinnamon, guarana, rosemary and boldo-do-chile Table 1 The polyphenolic composition in the extract of Chinese hawthorn fruits as analyzed by HPLC-MS. Retention time (min)

Polyphenols

[M]+ (m/z)

Relative content (%)

7.59 11.71 12.94 24.49 35.74 36.17

Epicatechin Chlorogenic acid Cyanidin-3-O-galactoside Procyanidin B2 Isoquercitrin Hyperoside

290 354 449 579 464 464

27.09 15.61 5.96 38.35 7.82 5.17

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Fig. 1. The cross-sectional morphology of CF (A), CF-HE I (B), CF-HE II (C) and CF-HE III (D) films.

ethanolic extracts were well compatible with chitosan-gelatin blend films. However, Bonilla et al. [9] found the incorporation of eugenol and ginger essential oils produced several pores inside the chitosangelatin blend films that resulted from collapsed hydrophobic oil droplets during film drying process. These suggested the microstructure of the composite films were greatly affected by the nature of addictives. When 6 wt% of the extract was incorporated, the cross-section of CFHE III film became a little rougher than CF-HE I and CF-HE II films (Fig. 1D). This was probably due to the limited solubility of the extract in the film-forming solution when the extract reached a certain high concentration [39]. However, the cross-section of CF-HE III film was still more compact and homogeneous as compared to CF film. 3.3. FT-IR spectra of CF-HE films The intermolecular interactions between different film components were revealed by ATR FT-IR technique (Fig. 2). The extract exhibited the characteristic bands of polyphenols including 3312 cm−1 (O − H stretching), 1603 and 1517 cm−1 (C_C stretching of aromatic ring), and 1441 and 1354 cm−1 (–CH3 bending) [9,35]. The spectra of CF film exhibited the characteristic bands of both chitosan and gelatin at 3284 cm−1 (amide-A, N − H and O − H stretching coupled with hydrogen bonds), 2927 and 2874 cm−1 (amide-B, antisymmetric and symmetric stretching of C − H), 1634 cm−1 (amide I, C_O stretching), 1534 cm−1 (amide II, N − H bending and C − N stretching), 1239 cm−1 (amide III, N − H and C − N in-plane bending of bound amide or − CH2 groups of glycine), and 925, 1026 and 1152 cm−1 (skeletal vibrations of the pyranose structure of CS) [37,40]. Till now, several researchers have reported significant changes occurred in the regions of amide I, amide II and amide III bands when chitosan and gelatin were

blended, which was attributed to the hydrogen bonding and electrostatic interactions formed between chitosan and gelatin [7,10–12,15,41]. When HE was incorporated, some differences in the relative intensity and the position of bands were observed in CF film. The amide-A band slightly broadened and shifted to 3271/3273 cm−1, which was caused by hydrogen bonding formed between the hydroxyl groups of polyphenols in the extract and the amino/hydroxyl groups in chitosan/gelatin [7,20,40,41]. Except for amide-A band, the amide II and amide III bands of CF film also shifted to 1535/1536 cm−1 and 1248/ 1249 cm−1, respectively. The shifts in the position of amide-II and amide-III bands were resulted from the electrostatic interactions formed between the amino and carbonyl groups in the extract, chitosan and gelatin [10,41]. Other researchers also found the amide bands of chitosan-gelatin blend films shifted after the incorporation of polyphenols, such as procyanidin, ferulic acid or quercetin [19,39]. 3.4. XRD patterns of CF-HE films The presence of intermolecular interactions between different film components was also confirmed by XRD analysis (Fig. 3). The XRD pattern of the extract exhibited a broad peak around 23.0°, demonstrating the extract was in the amorphous state. The similar XRD patterns were observed in other polyphenol-rich plant extracts, such as purple corn and mulberry extracts [42,43]. The XRD pattern of CF film showed four diffraction peaks at 8.1°, 11.3°, 18.1° and 22°, suggesting the film had a semi-crystalline property. The diffraction peak at 8.1° corresponded to the triple-helix crystalline structure of gelatin and the relatively regular crystal lattice of chitosan [44]. The diffraction peak appeared at 11.3° was related to the 020 diffraction plane of anhydrous chitosan crystal, and a broad peak centered at 22° corresponded to the

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Fig. 2. FT-IR spectra of the extract (a), and CF (b), CF-HE I (c), CF-HE II (d) and CF-HE III (e) films.

amorphous structure of chitosan and gelatin [38]. Several researchers documented the typical peaks of both chitosan and gelatin weakened after being prepared into blend films, which could be explained by the formation of strong intermolecular interactions between chitosan and gelatin that destroyed their close packing for the formation of regular crystallites [7,13,38]. In addition, the presence of glycerol in CF film could increase the mobility of polymer chains but decrease the internal cohesion of the matrix, thereby hindering long-range chain arrangements [38]. When the extract was incorporated into CF film, the diffraction peak intensity of the composite films at 8.1°, 11.3° and 18.1° first decreased and then gradually increased. This was because the incorporation of the extract hindered the formation of ordered film structure due to the interactions between the extract and chitosan/gelatin. A similar behavior was observed by Wang et al. [34] who incorporated black soybean seed coat extract into chitosan based films. However, PérezCórdoba et al. [44] found the intensity of the crystalline peak at 10° increased after incorporating nanoencapsulated plant-derived active compounds into chitosan-gelatin blend films. 3.5. Colors and thicknesses of CF-HE films The color of the film greatly affects the overall appearance of the packaged food products and consumer acceptance. As presented in Fig. 4, CF film was almost colorless whereas CF-HE films were light yellow. With the increasing of the extract content, CF-HE films became

darker and yellower; which could be attributed to the presence of polyphenols in the extract. Similar phenomena were observed when gallic acid was incorporated into chitosan-gelatin blend films [21,22]. In general, the differences in the color of the film can be reflected the color parameters, such as L, a, and b values. Meanwhile, the color parameter of ΔE indicates the degree of total color difference of the film. As listed in Table 2, the b values of CF-HE films significantly increased from 1.63 to 6.51 when the incorporation amount of the extract increased. This was in agreement with the gradually deepened yellowness of CF-HE films. However, the L values of CF-HE films decreased from 88.26 to 80.98 with the increase of the extract content, which was accompanied by the increase in the ΔE values of CF-HE films from 5.46 to 14.27. Other researchers also found the incorporation of essential oils could significantly increase the b and ΔE values of chitosan-gelatin blend films [9,20,37]. The thicknesses of CF and CF-HE films are summarized in Table 3. CF film without extract addition showed the lowest thickness (0.052 mm). Some researchers suggested that the thicknesses of chitosan-gelatin blend films were closely related with the ratio of chitosan and gelatin in the films [12,15]. The incorporation of the extract remarkably increased the thickness of CF film, which was because the extract had inserted into the network of film matrix and increased the solid content of the films. The similar enhancement in film thickness was also observed when other polyphenols (e.g. gallic acid, procyanidin, ferulic acid and quercetin) were incorporated into chitosan-gelatin blend films [19,22,39].

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Fig. 3. XRD patterns of the extract (a), and CF (b), CF-HE I (c), CF-HE II (d) and CF-HE III (e) films.

3.6. Moisture contents of CF-HE films Moisture content represents the total free volume occupied by water molecules in the film network [37]. CF film exhibited the highest moisture content (26.77%), which was attributed to the hydrophilicity of chitosan, fsh gelatin and glycerol [40]. By contrast, CF-HE films showed lower moisture contents (23.21%–25.69%) than CF film. In addition, the moisture contents of CF-HE films decreased with the increase of extract incorporation amount. The incorporation of polyphenol-rich extract could establish strong intermolecular interactions (e.g. hydrogen bonding and electrostatic interactions) between the extract and chitosan/gelatin, which greatly limited the interactions between moisture and chitosan/gelatin [34]. Thus, the moisture contents in CF-HE films decreased after the addition of the extract. Rui et al. [22] also reported the moisture contents of chitosan-gelatin blend films were slightly reduced by the incorporation of gallic acid. 3.7. Mechanical properties of CF-HE films The mechanical properties including tensile strength and elongation at break of CF and CF-HE films are shown in Fig. 5. The mechanical properties of the film represent its ability to keep the integrity and to endure

CF film

CF-HE I film

external stress during the processing, transportation, handling and storage of the packaged food products [37]. The tensile strength of CF film was 33.7 MPa, which was in accordance with the results of several previously reports [12,19,39]. In addition, some researchers also revealed the tensile strength of chitosan-gelatin blend films increased with the increase of gelatin proportion in blend films, which was because gelatin film was mechanically stronger than chitosan film [10,12,38]. As compared with CF film, CF-HE films showed significantly higher tensile strength (35.1–40.9 MPa). The enhancement in the tensile strength of CF-HE films could be related with the formation of more stable and denser film network (Fig. 1B and Fig. 1C) due to the interactions between the extract and chitosan/gelatin through electrostatic interactions and hydrogen bonding [10,37]. Similar results were previously found in chitosan-gelatin blend films incorporated with ferulic acid, quercetin or gallic acid [19,21,22]. However, the tensile strength of CFHE film slightly decreased when the extract content reached 6 wt%, which was because the undissolved extract particles in the film matrix restricted intermolecular interactions among different film components as confirmed by SEM images (Fig. 1D). Ramziia et al. [39] also found the incorporation of a high content of procyanidin caused the generation of heterogeneous structure with intermittent areas in film matrix, which decreased the tensile strength of chitosan-gelatin blend films.

CF-HE II film

Fig. 4. Physical appearances (A) and of CF and CF-HE films.

CF-HE III film

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Table 2 Color values including L, a, b and ΔE of CF and CF-HE films. Films

L

a

b

ΔE

CF film CF-HE I film CF-HE II film CF-HE III film

93.56 ± 0.15a 88.26 ± 0.68b 84.91 ± 0.64c 80.98 ± 0.83d

−0.91 ± 0.02d −0.61 ± 0.04b −0.68 ± 0.03c −0.40 ± 0.07a

0.15 ± 0.09d 1.63 ± 0.42c 4.37 ± 0.43b 6.51 ± 0.59a

1.66 ± 0.10d 5.46 ± 0.79c 9.78 ± 0.78b 14.27 ± 0.97a

Values are given as mean ± SD (n = 3). Different letters in the same column indicate significantly different (p b 0.05).

Elongation at break is the maximum change in the length of the film until it breaks compared to the initial length. Elongation at break is frequently used to determine the flexibility and stretchability of the film [37]. The elongation at break of CF film (10.1%) was comparable to that reported by Mohammadi et al. [12] and Ramziia et al. [39]. Notably, the elongation at break of CF film was increased by 49%, 171% and 117% when 2 wt%, 4 wt% and 6 wt% of the extract was incorporated, respectively. Thus, CF-HE films were generally more deformable than CF film. This result was in agreement with that of Benbettaïeb et al. [19], who reported the incorporation of ferulic acid or quercetin increased the elongation at break of chitosan-gelatin blend films. The enhancement in the elongation at break of CF-HE films was probably because the extract could penetrate into the film network and play a plasticizing effect on the films. As a result, the intermolecular forces along polymer chains were reduced and the flexibility and chain mobility of the films were improved. Similar enhancement in elongation at break was observed when essential oils were incorporated into chitosan-gelatin blend films, which was also attributed to the plasticizing effect of essential oils [44]. However, Ramziia et al. [39] found the elongation at break of chitosan-gelatin blend films was not significantly influenced by procyanidin incorporation. Thus, the elongation at break of polyphenol-rich chitosan-gelatin blend films was related with the character of polyphenols incorporated. 3.8. WVP of CF-HE films WVP is an important barrier parameter for packaging films to protect food against water-induced spoilage. WVP is controlled by the diffusivity and solubility of water molecules within the film matrix [11]. As shown in Fig. 6A, the WVP of CF film was 0.80 × 10−10 g m−1 s−1 Pa−1, which was comparable to that reported by Bonilla et al. [9], Benbettaïeb et al. [10] and Rui et al. [22]. Mohammadi et al. [12] found the WVP of chitosan-gelatin blend films decreased with the increase of chitosan proportion in the film, which was due to relatively lower WVP of chitosan film. The WVP of CF-HE films was lower than that of CF film, varying between 0.37 and 0.61× 10−10 g m−1 s−1 Pa−1. The reduction in the WVP of CF-HE films was because the addition of the extract produced a tortuous path, which hindered the passage of water molecules through film matrix. On the other hand, the free volume in the polymer matrix was greatly reduced by the extract incorporation, thereby producing a denser structure that decreased the WVP of the films to considerable extent. Benbettaïeb et al. [19] also found the incorporation of ferulic acid or quercetin reduced the WVP of chitosan-gelatin blend films, which was because polyphenols Table 3 Thicknesses, moisture contents and opacity of CF and CF-HE films. Films

Thickness (mm)

Moisture content (%)

Opacity (mm−1)

CF film CF-HE I film CF-HE II film CF-HE III film

0.052 ± 0.002c 0.057 ± 0.004b 0.060 ± 0.003ab 0.062 ± 0.003a

26.77 ± 0.23a 25.69 ± 0.15b 24.37 ± 0.01c 23.21 ± 0.11d

0.71 ± 0.01d 1.51 ± 0.03c 1.69 ± 0.02b 2.14 ± 0.05a

Values are given as mean ± SD (n = 10 for film thickness, and n = 3 for moisture content and opacity). Different letters in the same column indicate significantly different (p b 0.05).

Fig. 5. The tensile strength (A) and elongation at break (B) of CF and CF-HE films.

incorporation provided a denser structure for the film matrix. Rui et al. [22] and Ramziia et al. [39] revealed the WVP of chitosan-gelatin films could be reduced by the incorporation of gallic acid or procyanidin since polyphenols could interact with other film components through intermolecular interactions and decrease the free volume in the polymer matrix. Notably, the WVP of CF-HE films first decreased and then increased with the increase of the extract content. This was because the extract could disperse well in the film matrix at low levels (2 and 4 wt%), thereby blocking the transfer of water vapor. However, additional extract could aggregate and somewhat disrupt the compact film matrix. Thus, the WVP of CF-HE film slightly increased when the extract content reached 6 wt%. A similar effect on the behavior of WVP was observed when chitosan nanoparticles were added into gelatin films [45]. 3.9. Light transmittance and transparency of CF-HE films The UV–vis barrier property is important for food packaging films since it can retard the lipid oxidation and preserve the organoleptic properties of the packaged food. As shown in Fig. 6B, the transmission of CF film was negligible when the wavelength was lower than 240 nm, but was relatively high (N75%) when the wavelength exceeded 400 nm. This indicated CF film possessed UV–vis light barrier property to some extent. This result is in agreement with previous reports on chitosan-gelatin blend films [11,12,15]. As compared with chitosan, gelatin has better UV barrier property due to the presence of aromatic amino acid residues (e.g. tyrosine and phenylalanine) that can absorb UV light [20]. Notably, CF-HE films possess higher UV light barrier properties than CF film, since the transmittance of these films was almost zero in the ultraviolet light region (200–300 nm). The good UV barrier properties of CF-HE films could be attributed to the UV light absorption ability of film matrix as well as the polyphenols in the extract. In

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Fig. 7. The DPPH radical scavenging activity of CF and CF-HE films. Each value represents mean ± standard deviation of triplicates.

Fig. 6. The water vapor permeability (A) and light transmittance (B) of CF and CF-HE films.

addition, CF-HE films also showed lower transmittance in the visible light region (400–800 nm) as compared to CF film, indicating the addition of the extract into the film matrix reduced the transparency of the films. The good UV–vis light barrier property were also found by many other researchers in chitosan-gelatin blend films containing essential oils, eugenol, procyanidin or gallic acid [9,22,37,39,44]. Opacity is a critical property for food packaging films when the films are used as surface coating or to improve product appearance [11]. The opacity values of CF and CF-HE films are shown in Table 3. CF film had a very low opacity value (0.71 mm−1), suggesting it was very transparent. By contrast, CF-HE films exhibited higher opacity values (1.51–2.14 mm−1) than CF film. Overall, the transparency of CF film decreased with the addition of the extract due to the light scattering effect of the extract in the film network, which interfered with light transmission. The similar reduction in transparency was observed when procyanidin was added into chitosan-gelatin blend films, which was because procyanidin is capable to impede light transmission through the film matrix [39]. Likewise, Haghighi et al. [37] also found chitosangelatin blend films enriched with different essential oils were less transparent than chitosan-gelatin film. 3.10. Antioxidant property of CF-HE films Antioxidant packaging is one of the major categories of active packaging, which is promising to extend the shelf life of food products. The antioxidant property of CF and CF-HE films was evaluated by free radical scavenging assay and presented in Fig. 7. CF film exhibited a very low DPPH radical scavenging activity (3.89% at 5 mg/mL), which was ascribed to the antioxidant activity of both chitosan and gelatin. The antioxidant property of chitosan is the contribution of free amino groups, which can react with free radicals with the formation of stable

macromolecular radicals and ammonium groups [44]. Jridi et al. [15] suggested the antioxidant mechanism of chitosan might be also related to its ability to chelate metal ions and/or bond with lipids. As for gelatin, its antioxidant property is attributed to the presence of amino acids carrying unsaturated double bonds [9]. Jridi et al. [15] found the DPPH radical scavenging activity of chitosan-gelatin film decreased with the increase of chitosan proportion in the film, demonstrating gelatin possessed high free radical scavenging ability than chitosan. When the extract was added into CF film, the DPPH radical scavenging activity of the composite films were greatly elevated (33.42–84.40% at 5 mg/mL). This indicated there was a positive correlation between extract content and the antioxidant property of CF-HE film. The elevated antioxidant property of CF-HE films was mainly caused by the polyphenols released from film matrix. Polyphenols have been demonstrated as potent antioxidants due to their capacity to donate hydrogen atoms or electrons and to capture the free radicals, thereby terminating the lipid peroxidation reactions [46]. Till now, the antioxidant property of hawthorn fruit extract has been well demonstrated by in vitro and cellular assays [29,30]. Our results suggested the antioxidant property of chitosangelatin blend films could be significantly elevated by the addition of hawthorn fruit extract. Many other researchers also reported the incorporation of gallic acid, procyanidin, essential oils could greatly enhance the antioxidant property of chitosan-gelatin blend films [9,20,22,39]. 4. Conclusion In this study, active packaging films were successfully fabricated by adding polyphenol-rich Chinese hawthorn fruit extract into chitosangelatin film matrix. The addition of the extract could interact with film matrix through hydrogen bonding and electrostatic interactions, resulting in more compact inner microstructure in the films. As a result, the mechanical and water vapor barrier properties of chitosan-gelatin films were significantly improved by the extract incorporation. Due to abundant polyphenolic content, the extract also elevated the light barrier and antioxidant properties of chitosan-gelatin films. Notably, the microstructure, physical, mechanical, barrier and antioxidant properties of the films were closely related to the content of extract. In general, the films containing 2 wt% or 4 wt% of the extract showed better mechanical and water vapor barrier properties; whereas the films containing 6 wt% of the extract exhibited higher light barrier and antioxidant properties. In future, the extract incorporated chitosan-gelatin films can be further used as active packaging to extend the shelf life of food products. Acknowledgements This work was supported by Grants-in-Aid for scientific research from the National Natural Science Foundation of China (No. 31571788),

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